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Chronic hepatitis C is often associated with liver iron overload, which may affect the long-term prognosis and the response to antiviral treatment. The occurrence of hemochromatosis (HFE) mutations were studied to determine whether may contribute to the liver iron overload of chronic hepatitis C patients. The prevalence of two HFE mutations (C282Y and H63D) in 120 chronic hepatitis C patients was determined and the findings were correlated with clinical, histological and virological features. Hepatic iron was determined semiquantitatively by a histochemical hepatic iron index, defined as the ratio of a histochemical staining score to the patient's age, after correction for heterogeneous lobular iron distribution. Serum hepatitis C virus (HCV) RNA was measured by bDNA assay and typed by restriction fragment length polymorphism. Liver HCV RNA was measured by a semi-quantitative strand-specific reverse transcription-polymerase chain reaction (RT-PCR). Excess liver iron was stained in the liver of 36 patients (30%). Siderotic patients had the same geographic origin, serum and liver HCV RNA levels and H63D and C282Y mutations frequency as non-siderotic patients. However, siderotic patients were older (P = 0.015), more frequently males (P = 0.02), less frequently infected with HCV genotype 3 (P = 0.037) and had a higher liver fibrosis score (P = 0.008). The liver iron content did not correlate with the serum or liver HCV RNA titers. Ten of the 36 patients with liver siderosis had neither a history of excess alcohol intake, multiple transfusions, or HFE mutations. In conclusion, the pathogenesis of the liver iron overload in chronic hepatitis C patients cannot be fully explained by the occurrence of HFE mutations. The exact mechanism of iron accumulation in these patients therefore remains unexplained.

<h4>Background</h4>Androgen-independent growth leads to progressive prostate cancer after androgen-ablation therapy. This may be caused by altered specificity of the androgen receptor (AR), by ligand-independent stimulation of the AR, or by paracrine growth modulation by neuropeptides secreted by neuroendocrine (NE) cells.<h4>Methods</h4>We established and characterized the androgen-independent FGC-DCC from the androgen-dependent LNCaP fast growing colony (FGC) cell line. The androgen-independent DU-145, FGC-DCC, and PC-3, and the androgen-dependent LNCaP and PC-346C cell lines were used to study growth modulation of gastrin-releasing peptide (GRP), calcitonin (CT), serotonin (5-HT), and vasoactive intestinal peptide (VIP) by (3)H-thymidine incorporation. Specificity of the growth-modulating effects was tested with the anti-GRP monoclonal antibody 2A11 and induction of cAMP by neuropeptides.<h4>Results</h4>Androgen-independent growth stimulation by neuropeptides was shown in DU-145 and PC-346C. 2A11 inhibited GRP-induced (3)H-thymidine incorporation in DU-145 and PC-346C and inhibited proliferation of the FGC-DCC and PC-3 cell lines. With some exceptions, cAMP induction paralleled growth stimulation. Dideoxyadenosine (DDA) inhibited the GRP-induced growth effect in DU-145 and PC-346C, whereas oxadiazoloquinoxaline-1-one (ODQ) had no effect on (3)H-thymidine incorporation. None of the neuropeptides stimulated growth of LNCaP, FGC-DCC, or PC-3.<h4>Conclusions</h4>GRP-induced growth of DU-145 and PC-346C was specific and cAMP-mediated. Androgen-independent growth of FGC-DCC cells was mainly due to an induction of Bcl-2 expression and possibly through the activation of an autocrine and NE-like pathway, as has been shown also for the PC-3 cell line. Growth induction of non-NE cells by neuropeptides could be a possible role for NE cells in clinical prostate cancer.

Axon guidance mechanisms are crucial to the development of an integrated nervous system. One family of molecules that may be important in establishing axonal connectivity in mammals is the Netrins, and their putative receptors DCC (deleted in colorectal cancer), Neogenin, and Unc-5. Knockout and mutational analyses of some of these genes have shown that they are critically involved in the development of several specific pathways in the developing brain. However, previous expression analyses of these genes have largely been confined to the developing spinal cord. In the present study, we analyzed the expression of DCC in the developing mouse forebrain. We found that DCC protein is expressed in specific axonal populations projecting from the developing olfactory bulb, neocortex, hippocampus, and epithalamus/habenular complex. In the developing olfactory bulb and neocortex, DCC expression is particularly evident during the targeting phase of axon outgrowth and is then rapidly downregulated. As predicted from the knockout and mutational analyses of this gene, DCC is expressed in axonal commissures, in particular the corpus callosum, hippocampal commissure, and the anterior commissure. In addition, we found that DCC is expressed in the habenular commissure, the fasciculus retroflexus, and the stria medularis. Therefore, this analysis implicates a function for DCC in additional axonal guidance systems not predicted from the knockout and mutational analyses.

We developed a heteroduplex mobility assay in the gag gene (gag HMA) for the identification of group M subtypes A to H. The assay covers the region coding for amino acid 132 of p24 to amino acid 20 of p7 (according to human immunodeficiency virus type 1 [HIV-1] ELI, 460 bp). The gag HMA was compared with sequencing and phylogenetic analysis of an evaluation panel of 79 HIV-1 group M isolates isolated from infected individuals from different geographic regions. Application of gag HMA in combination with env HMA on 252 HIV-1- positive plasma samples from Bénin, Cameroon, Kenya, and Zambia revealed a high prevalence of a variety of intersubtype recombinants in Yaoundé, Cameroon (53.8%); Kisumu, Kenya (26.8%); and Cotonou, Bénin (41%); no recombinants were identified among the samples from Ndola, Zambia. The AG(IbNG) circulating recombinant form, as determined by gag HMA, was found to be the most common intersubtype recombinant in Yaoundé (39.4%) and Cotonou (38.5%). Using a one-tube reverse transcriptase PCR protocol, this gag HMA in combination with env HMA is a useful tool for rapidly monitoring the prevalence of the various genetic subtypes as well as of recombinants of HIV-1. Moreover, this technology can easily be applied in laboratories in developing countries.

The Histone Database (HDB) is an annotated and searchable collection of all full-length sequences and structures of histone and non-histone proteins containing the histone fold motif. These sequences are both eukaryotic and archaeal in origin. Several new histone fold-containing proteins have been identified, including Spt7p, and a few false positives have been removed from the earlier version of HDB. Database contents include compilations of post-translational modifications for each of the core and linker histones, as well as genomic information in the form of map loci for the human histone gene complement, with the genetic loci linked to Online Mendelian Inheritance in Man (OMIM). Conflicts between similar sequence entries from a number of source databases are also documented. Newly added to the HDB are multiple sequence alignments in which predicted functions of histone fold amino acid residues are annotated. The database is freely accessible through the WWW at http://genome.nhgri.nih.gov/histones/

We investigated the expression of p53 protein by immunohistochemistry and the expression of deleted in colorectal carcinoma (DCC) mRNA by the reverse transcription-polymerase chain reaction (RT-PCR) method in surgically resected tumors of gastric and colorectal cancers and compared these results to the clinicopathological features. Positive immunoreactions of p53 were observed in 21 of 42 gastric cancers (50%) and 25 of 37 colorectal cancers (67.6%). Decreased expression of DCC mRNA was observed in 15 of 38 gastric cancers (39.5%) and 10 of 28 colorectal cancers (35. 7%). There was a significant correlation between the immunoreaction of p53 and the depth of tumor invasion in gastric cancer, as well as between the decreased expression of DCC mRNA and nodal metastasis in colorectal cancer. In early cases without metastasis and invasion beyond muscularis propria, none of six gastric cancers showed a p53 immunoreaction, while seven of 9 colorectal cancers showed positive immunoreactions. On the other hand, two of 4 gastric cancers showed decreased expression of DCC mRNA; whereas, none of the seven colorectal cancers did. Alteration of p53 might occur at a later stage in gastric cancer than in colorectal cancer and be associated with the acquisition of an invasive character. In contrast to gastric cancer, decreased expression of DCC mRNA might be present in a later stage in colorectal cancer than in gastric cancer, and be related to the acquisition of metastatic character to the lymph nodes. In conclusion, alterations of p53 or DCC may play different roles in the progression of gastric cancers as compared to colorectal cancers, and the occurrence of both p53 and DCC genes mutations may cause these cancers to become more malignant.

This report defines a cell surface receptor (OX40) expressed on effector CD4 T cells, which when engaged in conjunction with a danger signal, rescues Ag-stimulated effector cells from activation-induced cell death in vivo. Specifically, three signals were necessary to promote optimal generation of long-lived CD4 T cell memory in vivo: Ag, a danger signal (LPS), and OX40 engagement. Mice treated with Ag or superantigen (SAg) alone produced very few SAg-specific T cells. OX40 ligation or LPS stimulation, enhanced SAg-driven clonal expansion and the survival of responding T cells. However, when SAg was administered with a danger signal at the time of OX40 ligation, a synergistic effect was observed which led to a 60-fold increase in the number of long-lived, Ag-specific CD4 memory T cells. These data lay the foundation for the provision of increased numbers of memory T cells which should enhance the efficacy of vaccine strategies for infectious diseases, or cancer, while also providing a potential target (OX40) to limit the number of auto-Ag-specific memory T cells in autoimmune disease.

During late mitosis and early G(1), a series of proteins are assembled onto replication origins, resulting in them becoming 'licensed' for replication in the subsequent S phase. Four factors have so far been identified that are required for chromatin to become functionally licensed: ORC (the origin recognition complex) and Cdc6, plus the two components of the replication licensing system RLF-M and RLF-B. Here we describe the first steps of a systematic fractionation of Xenopus egg extracts to identify all the components necessary for the assembly of licensed replication origins on Xenopus sperm nuclei (the physiological DNA substrate in this system). We have purified a new activity essential for this reaction, and have shown that it is nucleoplasmin, a previously known chromatin remodelling protein. Nucleoplasmin decondenses the sperm chromatin by removing protamines, and is required at the earliest known step in origin assembly to allow ORC to bind to the DNA. Sperm nuclei can be licensed by a combination of nucleoplasmin, RLF-M and a partially purified fraction that contains ORC, Cdc6 and RLF-B. This suggests that we are likely to have identified most of the proteins required for this assembly reaction.

Antigens of the Rh blood group system are encoded by 2 homologous genes, RHD and RHCE, that produce 2 red cell membrane proteins. The D-negative phenotype is considered to result, almost invariably, from homozygosity for a complete deletion of RHD. The basis of all PCR tests for predicting fetal D phenotype from DNA obtained from amniocytes or maternal plasma is detection of the presence of RHD. These tests are used in order to ascertain the risk of hemolytic disease of the newborn. We have identified an RHD pseudogene (RHD psi) in Rh D-negative Africans. RHDpsi contains a 37 base pair (bp) insert in exon 4, which may introduce a stop codon at position 210. The insert is a sequence duplication across the boundary of intron 3 and exon 4. RHDpsi contains another stop codon in exon 6. The frequency of RHDpsi in black South Africans is approximately 0.0714. Of 82 D-negative black Africans, 66% had RHDpsi, 15% had the RHD-CE-D hybrid gene associated with the VS+ V- phenotype, and only 18% completely lacked RHD. RHDpsi is present in about 24% of D-negative African Americans and 17% of D-negative South Africans of mixed race. No RHD transcript could be detected in D-negative individuals with RHDpsi, probably as a result of nonsense-mediated mRNA decay. Existing PCR-based methods for predicting D phenotype from DNA are not suitable for testing Africans or any population containing a substantial proportion of people with African ethnicity. Consequently, we have developed a new test that detects the 37 bp insert in exon 4 of RHDpsi. (Blood. 2000; 95:12-18)

Host proteins are incorporated both on and inside human immunodeficiency virus type 1 (HIV-1) virions. To identify cellular proteins inside HIV-1, virion preparations were treated by a protease-digestion technique that removes external host proteins, allowing for the study of the proteins inside the virus. Treated HIV-1 preparations were analyzed by immunoblot, high-pressure liquid chromatography, and protein sequence analyses. These analyses identified several cellular proteins inside HIV-1: elongation factor 1alpha, glyceraldehyde-3-phosphate dehydrogenase, HS-1, phosphatidylethanolamine-binding protein, Pin1, Lck, Nm23-H1, and the C-terminal tail of CD43. Several of these proteins were found as fragments of their full-sized proteins that appear to be generated by our protease treatment of the virions, the HIV-1 protease, or a cellular protease. Recent advances in cell biology and biochemistry have identified some of these proteins as actin-binding proteins. These results support the hypothesis that actin filaments are incorporated into the virion and may provide additional clues for the understanding of the interaction between viral and cellular proteins during assembly and budding.

Candida dubliniensis is a recently discovered yeast species principally associated with carriage and disease in the oral cavities of human immunodeficiency virus (HIV)-infected individuals. To date the majority of isolates of this species have been identified in Europe and North America. In this study, five Candida isolates recovered from separate HIV-negative hospitalized patients in Jerusalem, Israel, were presumptively identified as C. dubliniensis on the basis of their dark green coloration when grown on CHROMagar Candida medium. Their identification was confirmed by a variety of techniques, including carbohydrate assimilation profiles, absence of growth at 45 degrees C, positive reaction with C. dubliniensis-specific antibodies as determined by indirect immunofluorescence analysis, and positive amplification with C. dubliniensis-specific PCR primers. All five strains were shown to be susceptible to a range of antifungal agents, including fluconazole. One of the five isolates was recovered from urine specimens, while the remaining four were recovered from upper respiratory tract and oral samples. While none of the patients was HIV positive, all were receiving broad-spectrum antibacterials at the time isolates of C. dubliniensis were obtained from clinical specimens. This study describes the first isolates of C. dubliniensis from the Middle East and confirms that this yeast can be associated with carriage and infection in the absence of HIV infection.

In the developing vertebrate brain, growing axons establish a scaffold of axon tracts connected across the midline via commissures. We have previously identified a population of telencephalic neurons that express NOC-2, a novel glycoform of the neural cell adhesion molecule N-CAM that is involved in axon guidance in the forebrain. These axons arise from the presumptive telencephalic nucleus, course caudally along the principal longitudinal tract of the forebrain, cross the ventral midline in the midbrain, and then project to the contralateral side of the brain. In the present study we have investigated mechanisms controlling the growth of these axons across the ventral midline of the midbrain. The axon guidance receptor DCC is expressed by the NOC-2 population of axons both within the longitudinal tract and within the ventral midbrain commissure. Disruption of DCC-dependent interactions, both in vitro and in vivo, inhibited the NOC-2 axons from crossing the ventral midbrain. Instead, these axons grew along aberrant trajectories away from the midline, suggesting that DCC-dependent interactions are important for overcoming inhibitory mechanisms within the midbrain of the embryonic vertebrate brain. Thus, coordinated responsiveness of forebrain axons to both chemostimulatory and chemorepulsive cues appears to determine whether they cross the ventral midline in the midbrain.

The caspase-dependent activation of cyclin-dependent kinases (Cdks) in varied cell types in response to disparate suicidal stimuli has prompted our examination of the role of Cdks in cell death. We have tested the functional role of Cdk activity in cell death genetically, with the expression of dominant negative Cdk mutants (DN-Cdks) and Cdk inhibitory genes. Here we demonstrate that Cdk2 activity is necessary for death-associated chromatin condensation and other manifestations of apoptotic death, including cell shrinkage and the loss of adhesion to substrate. Susceptibility to the induction of the cell death pathway, including the activation of the caspase cascade, is unimpaired in cells in which Cdk2 activity is inhibited. The direct visualization of active caspase activity in these cells confirms that death-associated Cdk2 acts downstream of the caspase cascade. Cdk inhibition also does not prevent the loss of mitochondrial membrane potential and membrane phospholipid asymmetry, which may be direct consequences of caspase activity, and dissociates these events from apoptotic condensation. Our data suggest that caspase activity is necessary, but not sufficient, for the full physiological cell death program and that a requisite function of the proteolytic caspase cascade is the activation of effector Cdks.

Since 1991, 28 states have enacted laws that prohibit insurers' use of genetic information in pricing, issuing, or structuring health insurance. This article evaluates whether these laws reduce the extent of genetic discrimination by health insurers. From the data collected at multiple sites, we find that there are almost no well-documented cases of health insurers either asking for or using presymptomatic genetic test results in their underwriting decisions, either (a) before or after these laws have been enacted or (b) in states with or without these laws. By using both in-person interviews with insurers and a direct market test, we found that a person with a serious genetic condition who is presymptomatic faces little or no difficulty in obtaining health insurance. Furthermore, there are few indications that the degree of difficulty varies according to whether a state regulates the use of genetic information. Nevertheless, these laws have made it less likely that insurers will use genetic information in the future. Although insurers and agents are only vaguely aware of these laws, the laws have shaped industry norms and attitudes about the legitimacy of using this information.

Mutations in the gene for fibrillin-1 (FBN1) have been shown to cause Marfan syndrome, an autosomal dominant disorder of connective tissue characterised by pleiotropic manifestations involving primarily the ocular, skeletal, and cardiovascular systems. Fibrillin-1 is a major component of the 10-12 nm microfibrils, which are thought to play a role in tropoelastin deposition and elastic fibre formation in addition to possessing an anchoring function in some tissues. Fibrillin-1 mutations have also been found in patients who do not fulfil clinical criteria for the diagnosis of Marfan syndrome, but have related disorders of connective tissue, such as isolated ectopia lentis, familial aortic aneurysm, and Marfan-like skeletal abnormalities, so that Marfan syndrome may be regarded as one of a range of type 1 fibrillinopathies. There appear to be no particular hot spots since mutations are found throughout the entire fibrillin-1 gene. However, a clustering of mutations associated with the most severe form of Marfan syndrome, neonatal Marfan syndrome, has been noted in a region encompassing exons 24 to 32. The gene for fibrillin-2 (FBN2) is highly homologous to FBN1, and mutations in FBN2 have been shown to cause a phenotypically related disorder termed congenital contractural arachnodactyly. Since mutations in the fibrillin genes are likely to affect the global function of the microfibrils, the term microfibrillopathy may be the most appropriate to designate the spectrum of disease associated with dysfunction of these molecules. The understanding of the global and the molecular functions of the fibrillin containing microfibrils is still incomplete and, correspondingly, no comprehensive theory of the pathogenesis of Marfan syndrome has emerged to date. Many, but not all, fibrillin-1 gene mutations are expected to exert a dominant negative effect, whereby mutant fibrillin monomers impair the global function of the microfibrils. In this paper we review the molecular physiology and pathophysiology of Marfan syndrome and related microfibrillopathies.

No abstract available.

OX40 expressed on activated T cells is known to be an important costimulatory molecule on T cell activation in vitro. However, the in vivo functional significance of the interaction between OX40 and its ligand, OX40L, is still unclear. To investigate the role of OX40L during in vivo immune responses, we generated OX40L-deficient mice and a blocking anti-OX40L monoclonal antibody, MGP34. OX40L expression was demonstrated on splenic B cells after CD40 and anti-immunoglobulin (Ig)M stimulation, while only CD40 ligation was capable of inducing OX40L on dendritic cells. OX40L-deficient and MGP34-treated mice engendered apparent suppression of the recall reaction of T cells primed with both protein antigens and alloantigens and a significant reduction in keyhole limpet hemocyanin-specific IgG production. The impaired T cell priming was also accompanied by a concomitant reduction of both T helper type 1 (Th1) and Th2 cytokines. Furthermore, antigen-presenting cells (APCs) derived from the mutant mice revealed an impaired intrinsic APC function, demonstrating the importance of OX40L in both the priming and effector phases of T cell activation. Collectively, these results provide convincing evidence that OX40L, expressed on APCs, plays a critical role in antigen-specific T cell responses in vivo.

Infection of inbred mouse strains with Leishmania major is a well characterized model for analysis of T helper (Th)1 and Th2 cell development in vivo. In this study, to address the role of costimulatory molecules CD27, CD30, 4-1BB, and OX40, which belong to the tumor necrosis factor receptor superfamily, in the development of Th1 and Th2 cells in vivo, we administered monoclonal antibody (mAb) against their ligands, CD70, CD30 ligand (L), 4-1BBL, and OX40L, to mice infected with L. major. Whereas anti-CD70, anti-CD30L, and anti-4-1BBL mAb exhibited no effect in either susceptible BALB/c or resistant C57BL/6 mice, the administration of anti-OX40L mAb abrogated progressive disease in BALB/c mice. Flow cytometric analysis indicated that OX40 was expressed on CD4(+) T cells and OX40L was expressed on CD11c(+) dendritic cells in the popliteal lymph nodes of L. major-infected BALB/c mice. In vitro stimulation of these CD4(+) T cells showed that anti-OX40L mAb treatment resulted in substantially reduced production of Th2 cytokines. Moreover, this change in cytokine levels was associated with reduced levels of anti-L. major immunoglobulin (Ig)G1 and serum IgE. These results indicate that anti-OX40L mAb abrogated progressive leishmaniasis in BALB/c mice by suppressing the development of Th2 responses, substantiating a critical role of OX40-OX40L interaction in Th2 development in vivo.

We studied peripheral blood erythrocyte parameters and HFE genotypes in 94 hemochromatosis probands and 132 white, normal control subjects. Mean red blood cell counts in probands and control subjects were not significantly different. However, mean values of hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were significantly higher in C282Y/C282Y probands (n = 60) than in wild-type control subjects (n = 65). Probands with other HFE genotypes also had increased mean erythrocyte parameters (other than red blood cell count). Peripheral blood smears prepared before therapeutic phlebotomy revealed that erythrocytes in many probands had increased diameters and were well filled with hemoglobin. Erythrocyte parameters were similar in C282Y/C282Y probands with and without hepatomegaly, elevated serum concentrations of hepatic enzymes, hepatic cirrhosis, diabetes mellitus, arthropathy, or hypogonadism. Among C282Y/C282Y probands, significantly greater values of MCV (but not other erythrocyte parameters) occurred among those who had transferrin saturation values of 75% or greater or iron overload at diagnosis. After iron depletion, the mean MCV, MCH, and MCHC values of C282Y/C282Y probands decreased but remained significantly greater than values in wild-type control subjects. Mean values of prephlebotomy MCH and MCHC concentrations were lower in HLA-A3-positive than in HLA-A3-negative C282Y/C282Y probands. We conclude that increased values of mean hemoglobin, hematocrit, MCV, MCH, and MCHC in hemochromatosis probands are caused primarily by increased iron uptake and hemoglobin synthesis by immature erythroid cells. Mechanisms of iron uptake by erythrocytes that could explain these results are discussed.

HFE is related to major histocompatibility complex (MHC) class I proteins and is mutated in the iron-overload disease hereditary haemochromatosis. HFE binds to the transferrin receptor (TfR), a receptor by which cells acquire iron-loaded transferrin. The 2.8 A crystal structure of a complex between the extracellular portions of HFE and TfR shows two HFE molecules which grasp each side of a twofold symmetric TfR dimer. On a cell membrane containing both proteins, HFE would 'lie down' parallel to the membrane, such that the HFE helices that delineate the counterpart of the MHC peptide-binding groove make extensive contacts with helices in the TfR dimerization domain. The structures of TfR alone and complexed with HFE differ in their domain arrangement and dimer interfaces, providing a mechanism for communicating binding events between TfR chains. The HFE-TfR complex suggests a binding site for transferrin on TfR and sheds light upon the function of HFE in regulating iron homeostasis.

The possible role of activin in the regulation of malignant prostatic growth was studied using RNAase protection assays of activin receptors, inhibin/activin subunits and follistatin mRNAs in the human prostatic carcinoma cell lines LNCaP-FGC, -R and -LNO, in human prostatic carcinoma xenografts and in human prostatic tissue. Activin receptor types IA (ActRIA), IB (ActRIB), IIA (ActRIIA) and IIB (ActRIIB) mRNAs were generally expressed in prostate epithelial cells, with significantly lower levels of ActRIB mRNA in prostate tumour material when compared to non-malignant tissue (P < 0.05; Mann-Whitney U-test). Inhibin/activin betaA- and betaB-subunit mRNA expression was also found in prostate tissue. Androgen-independent xenografts expressed significantly lower amounts of betaB-subunit mRNA when compared to androgen-dependent xenografts (P< 0.05). While betaB-subunit mRNA was expressed by LNCaP-FGC and -LNO cells, virtually no expression was found in the androgen-independent LNCaP-R line. Inhibin alpha-subunit mRNA levels were low or undetectable in all samples investigated. Follistatin mRNA was undetectable in LNCaP-sublines, while low levels were found in prostatic tissues. In androgen-independent LNCaP-R cells, activin inhibited cell growth in a dose-dependent manner. These results suggest that prostate tumour progression is accompanied by a decrease of the inhibitory effect of locally produced activin by either a decrease in the expression of activin betaB-subunit mRNA or by a decrease of ActRIB mRNA levels.

Spinobulbar muscular atrophy and Huntington's disease are caused by polyglutamine expansion in the androgen receptor and huntingtin, respectively, and their pathogenesis has been associated with abnormal nuclear localization and aggregation of truncated forms of these proteins. Here we show, in diverse cell types, that glucocorticoids can up- or down-modulate aggregation and nuclear localization of expanded polyglutamine polypeptides derived from the androgen receptor and huntingtin through specific regulation of gene expression. Wild-type glucocorticoid receptor (GR), as well as C-terminal deletion derivatives, suppressed the aggregation and nuclear localization of these polypeptides, whereas mutations within the DNA binding domain and N terminus of GR abolished this activity. Surprisingly, deletion of a transcriptional regulatory domain within the GR N terminus markedly increased aggregation and nuclear localization of the expanded polyglutamine proteins. Thus, aggregation and nuclear localization of expanded polyglutamine proteins are regulated cellular processes that can be modulated by a well-characterized transcriptional regulator, the GR. Our findings suggest approaches to study the molecular pathogenesis and selective neuronal degeneration of polyglutamine expansion diseases.

The Genomation Laboratory in the Electrical Engineering Department at the University of Washington has been developing an automated, high-throughput, submicroliter-scale fluid-handling system for use in molecular biology, especially as part of the Human Genome Project and other high-throughput DNA sequencing endeavors. Small glass capillaries enable the preparation, handling, and monitoring of 1-microliter reaction volumes. The Genomation Laboratory, with corporate partners Orca Photonic Systems, Inc. and Engineering Arts, has developed modules for aspiration, dispensing, mixing, transport, and rapid thermal processing of biological samples contained in glass capillaries. The ACAPELLA-1K is the first integration of these modules, designed to process 1000 samples in an eight-hour day. It has served as a test bed for the technologies as well as for performing biological experiments in conjunction with the University of Washington Genome Center. This system and related results are presented in this paper. A video of the system in operation is provided at. The Genomation Laboratory is presently developing the next-stage ACAPELLA-5K system based on the results of the ACAPELLA-1K system.

To obtain functional evidence for DCC as a tumour suppressor associated with endometrial cancer, the human DCC cDNA encoding a complete open reading frame (ORF) was transfected into highly tumorigenic human endometrial carcinoma cells, HHUA and Ishikawa in which DCC expression was completely deleted. Reconstituted expression of DCC in HHUA had little effect on in vitro growth, but suppressed tumour formation in mice completely. The clones from Ishikawa had abundant DCC expression similar to that in normal endometrium. Their growth in vitro was suppressed and showed apoptotic phenotype. Lower levels of DCC expression in the prolonged passaged clones did not induce apoptosis, but still had the potential to suppress tumorigenicity. These observations imply a role of DCC in regulation of normal endometrial cell growth, and categorize DCC as the tumour suppressor gene for endometrial cancer.

<h4>Unlabelled</h4>[123I]beta-carbomethoxy-3-beta-(4-iodophenyl)-tropane (CIT) is a useful ligand for dopamine transporters (DATs) and serotonin transporters (5-HTTs). Previous SPECT studies have shown a state of sustained equilibrium in the striatum on day 2 after injection that allows quantification of striatal DATs using a simple ratio of specific-to-nondisplaceable binding. The aim of this study was to investigate the kinetics of [123I]beta-CIT uptake in the thalamus, hypothalamus, and midbrain, areas known to contain 5-HTTs in high densities.<h4>Methods</h4>SPECT with a triple-head camera was performed on 16 healthy volunteers (13 women, 3 men; mean age [+/-SD], 32 +/- 11 y) after intravenous bolus injection of 130 +/- 20 MBq (3.5 +/- 0.5 mCi) [123I]beta-CIT. Two individuals were scanned 1, 2, 4, 7, 10, 13, 16, and 24 h after injection, and the remaining 14 were scanned 4, 7, 10, 20, and 24 h after injection. Values from 19 previously examined healthy volunteers (8 women, 11 men; mean age, 52 +/- 20 y) were included in the analysis to study the age dependency of beta-CIT binding in striatal and 5-HTT-rich brain areas in a larger control sample.<h4>Results</h4>Peak uptake 4 h after injection, followed by stable uptake until 10 h and a slow decrease until 24 h, was observed in the thalamus-hypothalamus region. Activity in the midbrain-pons region peaked 2 h after injection. Because of a concomitant slow but steady decline of uptake in reference regions starting 4 h after injection, a higher stability of binding ratios for 5-HTT-rich brain areas was observed on day 2, suggesting that a state of transient equilibrium is reached between 20 and 24 h but that conditions are only close to transient equilibrium between 4 and 10 h after injection for 5-HTT-rich brain areas. In addition to an age-related decline of striatal [123I]beta-CIT binding of 6.6% per decade, a significant age-associated decrease of beta-CIT binding of 3-4% per decade was found in 5-HTT-rich brain areas. The decline of beta-CIT binding in these regions may be explained, at least in part, by a loss of monoamine transporters with age but may also be related to age-associated morphologic changes.<h4>Conclusion</h4>[123I]beta-CIT appears to be a suitable ligand for imaging serotonin transporters with SPECT. However, careful age matching is warranted for [123I]beta-CIT SPECT studies of 5-HTT changes in patients with neuropsychiatric disorders.

Serotonin transporter (5-HTT), a transmembrane protein, has been shown in adult brain to be distributed not only on synaptic terminals but to a great extent on axons as well. Here we report the ontogeny of 5-HTT and its relationship with serotonin (5-HT) neurons using established 5-HTT and 5-HT antibodies. Both 5-HTT- and 5-HT-immunostaining (-im) appear in 5-HT neurons at embryonic day 12 (E12) in rostral raphe nuclei (RRN). Soon after appearing, 5-HTT-im is highly expressed on axons, similar to adult expression. However, in contrast to adult, 5-HTT-im also outlines the soma-dendrites. Rich 5-HTT-im appears along the entire length of projecting axons, extending to the growth tip. In the next 2 days, intensive 5-HTT-im axons from RRN travel a course preferentially in the floor plate and later, the medial forebrain bundle trajectory. A group of new 5-HT-im neurons and 5-HTT-im axons appear at E13 in caudal raphe nuclei. At E16-18, taking the exact trajectory course of 5-HT axons, 5-HTT-im axons reach ganglionic eminence, olfactory bulb, and cortex and disperse into many brain regions in E18-20. No 5-HTT-im cell bodies were seen in nigral, locus ceruleus, or hypothalamus. However, the transient expression of 5-HTT on non-serotonergic system was seen in cortical and striatal neuroepithelia at E12 and sensory thalamic pathways at P0-P10. Prominent 5-HTT-im fibers in thalamocortical bundles project from sensory thalamic nuclei through reticular nucleus, internal capsule bundle and form barrels in somatosensory cortices. No 5-HTT-im was seen in glia-like cells using currently available antibody. These observations indicate that 5-HTT is: (a) associated preferentially with 5-HT neurons in brainstem, (b) temporally co-expressed with 5-HT in 5-HT neurons, (c) expressed on axons prior to synaptical sites at target neurons, which strongly indicates a volumic (extrasynaptic) transmission, (d) expressed in non-5-HT neurons within a specific window, which may affect the development of the systems "borrowing" the 5-HT. The early appearance of 5-HTT may also endow functionality as well as vulnerabilities of 5-HT, sensory thalamic, and cortical neurons to 5-HTT targeting drugs during pregnancy and after birth.

An in vitro model was developed to compare the biocompatibility of four different coating methods (three heparin and one nonheparin) under hemodynamic conditions. Fresh human donor blood (heparin 5 IU/ml) was recirculated in a standardized experimental circuit. All circuit components were either coated or remained uncoated for control purposes. The aim of the study was to investigate a wide spectrum of effects on blood; coagulation parameters (e.g., fibrinogen, ATIII, thrombin-antithrombin-complex), complement parameters (C1rsC1 Inh, C3b(Bb)P, SC5b-9, C5a), differential blood analyses, platelet activation (flow cytometric investigations), PF 4, and PMN-elastase release were examined by showing possible trends. All heparin coated systems reduced platelet stimulation in comparison to untreated biomaterials. Leukocyte activation was reduced to different degrees depending upon the coating method used. Complement activation was markedly reduced by all coated systems. The results obtained indicate that the pump driven, dynamic blood flow model is suitable to characterize the biocompatibility of surface modified biomaterials. Advantages lie in the integration of the different polymers as parts of the circuit, the low priming volume, and the generation of blood flow conditions similar to those that occur in clinical applications.

The serotonin transporter (5-HTT) gene is a promising candidate for introducing the heritability of interindividual variation in personality and the genetic susceptibility for various psychiatric diseases. Transcription of the gene is modulated by a common polymorphism in its upstream regulatory region (5-HTT gene-linked polymorphic region: 5-HTTLPR). The 5-HTTLPR consists of variation of the repetitive sequence containing GC-rich, 20-23-bp-long repeat elements. A deletion/insertion in the 5-HTTLPR was first reported to create a short (S) allele and a long (L) allele (14- and 16-repeats, respectively). Three other kinds of alleles (18-, 19- and 20-repeats) in addition to the S and L alleles in 5-HTTLPR have been reported. In the present study, we examined the 5-HTTLPR polymorphism in detail and identified ten novel sequence variants, concluding that the alleles reported as S and L are divided into four and six kinds of allelic variant, respectively. Subsequently, we developed a method for genotyping. The total number of alleles (14-A, 14-B, 14-C, 14-D, 15, 16-A, 16-B, 16-C, 16-D, 16-E, 16-F, 19, 20 and 22) in the 5-HTTLPR was 14 in our populations (Japanese: n = 131; Caucasian: n = 74) in the present study. In addition, a significant ethnic difference between Japanese and Caucasian populations was observed for distributions of alleles and genotypes (P < 0.0001 and P < 0.0001, respectively). Our results suggest that the analyses of the 5-HTTLPR should be revised by genotyping with a more complete subdivision of alleles. Molecular Psychiatry (2000) 5, 32-38.

Capillary electrophoresis in combination with fluorescence-based single-strand conformation polymorphism (SSCP) analysis was used to screen for known mutations as well as for unknown mutations. The mutations causing hemochromatosis and thrombogenetic diseases (factor V Leiden mutation and prothrombin mutation) are well defined. Familial hypercholesterolemia is caused by mutations in the low density lipoprotein (LDL) receptor gene. Because the mutations are heterogeneously localized in all 18 exons of the LDL receptor gene, effective screening procedures are necessary. The three well known mutations and 59 of 61 previously characterized mutations in the LDL receptor gene were detected by a distinct abnormal fragment pattern in capillary electrophoresis. The remaining two mutations in the LDL receptor gene showed only slight abnormalities under standard electrophoresis conditions (13 kV, 30 degrees C, 30 min). However, the abnormal pattern could be amplified by increasing the electrophoresis temperature. In all cases, heterozygous and homozygous mutations could clearly be differentiated from wild-type alleles. Because of the high efficiency of mutation detection, capillary electrophoresis in combination with fluorescence-based SSCP analysis would be attractive for the detection of well-defined mutations as well as for the screening of unknown mutations. The accuracy and the degree of automation make this technique well suited for routine genetic diagnosis.

The slit genes have recently been found to encode proteins with a conserved chemorepulsive activity for axons in invertebrates and vertebrates. We have determined the expression pattern of a slit gene in Xenopus embryos. In the neural tube, slit is expressed at the ventral and dorsal midlines, and the motor neurons. slit is also expressed in a changing pattern in the retina. The full-length Xenopus Slit protein is secreted extracellularly, whereas its receptor Roundabout can not be secreted. Using a myc-tagged secreted Slit protein, we confirmed the binding of Slit to Roundabout expressed on the cell surface. These results confirm Slit-Roundabout interactions and the biochemical properties of Slit and Roundabout proteins, and further support the idea that Slit may guide axon projections in multiple regions of the embryo.

We examined allelic polymorphisms of the serotonin transporter (5-HTT) gene and antidepressant response to 6 weeks' treatment with the selective serotonin reuptake inhibitor (SSRI) drugs fluoxetine or paroxetine. We genotyped 120 patients and 252 normal controls, using polymerase chain reaction of genomic DNA with primers flanking the second intron and promoter regions of the 5-HTT gene. Diagnosis of depression was not associated with 5-HTT polymorphisms. Patients homozygous l/l in intron 2 or homozygous s/s in the promoter region showed better responses than all others (p < 0.0001, p = 0.0074, respectively). Lack of the l/l allele form in intron 2 most powerfully predicted non-response (83.3%). Response to SSRI drugs is related to allelic variation in the 5-HTT gene in depressed Korean patients.

It has been proposed that iron overload may adversely affect liver disease outcome. The recent identification of 2 mutations in the HFE gene related to hereditary haemochromatosis (Cys282Tyr and His63Asp) provided an opportunity to test whether they are associated with hepatic iron accumulation and the activity and severity of liver disease in hepatitis C virus (HCV) infection. We investigated the prevalence of HFE mutations in 135 male patients with chronic HCV hepatitis, and correlated genotype distribution with different parameters of iron status and the activity and severity of liver disease. Of these 135 patients, 6 (4.4%) carried Cys282Tyr and 32 (23.7%) carried His63Asp, frequencies which were similar to those observed in healthy controls. Serum iron levels and transferrin saturation (but not ferritin levels or liver iron content) were significantly higher in carriers than in non-carriers of HFE mutations. No difference was observed in serum ALT, AST and GGT levels between carriers and non-carriers. Finally, scores for necroinflammatory activity and fibrosis in the liver were significantly higher in HFE carriers than in non-carriers. Patients with chronic HCV infection carrying HFE mutations tend to present more evident body iron accumulation and a higher degree of necroinflammatory activity and fibrosis in the liver. HFE gene mutations might be an additional factor to be considered among those implicated in the determination of a worse prognosis of the liver disease in chronic HCV infection.

Chromosome 18q is lost a high proportion of colorectal and pancreatic cancers. Three candidate tumor suppressor genes, DCC, Smad4 and Smad2 have been identified in this chromosome region. DCC and Smad4 aberrations have been previously identified in pancreatic and colorectal tumors. The aim of this study was to compare the presence of concurrent genetic aberrations in DCC and neighboring Smad4 and Smad2 genes during colorectal and pancreatic distal dissemination. We have used a panel of orthotopically implanted colorectal and pancreatic xenografts and corresponding metastases. We have shown that while LOH at DCC locus occurred at a similar frequency in both tumors, diminished DCC protein expression was exclusively present in colorectal tumors harboring intragenic DCC LOH. In contrast, in pancreatic xenografts loss of DCC protein and mRNA expression was restricted to metastases. Smad4 gene aberrations were detected at a similar frequency in both tumors and were selected for during distal dissemination. Acquisition of alterations in both genes occurred independently. Our results suggest that both DCC and Smad4 contribute to pancreatic and colorectal distal dissemination. However, the role of DCC may differ between both tumor types.

Estrogen receptors (ER) were determined by both the biochemical dextran-coated charcoal (DCC-ER) and the immunohistochemical Avidin biotin-peroxidase complex (IHC-ER) methods in proliferative mammary lesions collected from 37 cats: 20 malignant tumors without metastasis at first presentation, seven malignant tumors with lung and/or lymph node metastases and 10 benign tumors and dysplasias. Total number of samples analyzed by both methods was 44. The DCC-ER method was applied to frozen tissue samples and the IHC-ER method was applied to neutral buffered formalin-fixed, paraffin wax-embedded tissue samples by using the NCL-6F11 monoclonal antibody. Biochemically, 21 (47.7%) cases had equal or more than 5 fmol/mg of protein (standard positivity threshold). Immunohistochemically, 11 (25%) cases were scored positive, the percentage of positive nuclei being statistically linked to the intensity of immunostaining. Normal mammary gland tissue (13 cases) and/or dysplastic areas (5 cases) found in the surroundings of the main lesion were IHC-ER positive in 76.9% and 40% of the cases, respectively. Concordance between DCC-ER and IHC-DCC was 72.7% and the results of the DCC and the IHC-ER methods were significantly correlated (P < 0.05) by the chi2 test. Specificity (true negatives) and sensitivity (true positives) of the ICH-ER method were 95.6% and 47.6%, respectively. One out of eleven DCC-ER positive and IHC-ER negative discordant cases (9.09%) was a DCC-ER false positive, because the surrounding normal mammary gland tissue was IHC-ER positive. The remaining 10 cases had ER content values equal or lower than 23 fmol/mg of protein, a figure that could represent the sensitivity threshold of the immunohistochemical method employed.

Neuronal Cdc2-like kinase (Nclk) is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics. The active kinase consists of a catalytic subunit, Cdk5, and a 25 kDa activator protein (p25nck5a) derived from a 35 kDa neuronal-specific protein (p35nck5a). As an extension of our previous study (Qi, Z., Tang, D., Zhu, X., Fujita, D.J., Wang, J.H., 1998. Association of neurofilament proteins with neuronal Cdk5 activator. J. Biol. Chem. 270, 2329-2335), which showed that neurofilament is one of the p35nck5a-associated proteins, we now report the isolation of three other novel p35nck5a-associated proteins using the yeast two-hybrid screen. The full-length forms of these three novel proteins, designated C42, C48 and C53, have a molecular mass of 66, 24, and 57 kDa, respectively. Northern analysis indicates that these novel proteins are widely expressed in human tissues, including the heart, brain, skeletal muscle, placenta, lung, liver, kidney and pancreas. The bacterially expressed glutathione S-transferase (GST)-fusion forms of these three proteins were able to co-precipitate p35nck5a complexed with Cdk5 from insect cell lysate. Among these three proteins, only C48 and C53 can be phosphorylated by Nclk, suggesting that they may be the substrates of Nclk. Sequence homology searches have suggested that the C48 protein is marginally related to restin protein, whereas the C42 protein has homologues of unknown function in Caenorhabditis elegans and Arabidopsis thaliana.

Hereditary haemochromatosis (HHC) is a common inherited disorder of iron metabolism characterised by progressive iron loading of parenchymal cells of the liver, pancreas, heart and other organs ultimately leading to cirrhosis and organ failure. Despite HLA studies which localised the defective gene to the short arm of chromosome 6, the haemochromatosis gene remained elusive until 1996, when the gene was identified by a massive positional cloning effort. The haemochromatosis gene (HFE) encodes a novel nonclassical MHC class-1-like molecule. Two missense mutations have been identified in patients with HHC, a G to A at nucleotide 845, resulting in a substitution of tyrosine for cysteine at amino acid 282 (referred to as the C282Y mutation) and a C to G at nucleotide 187, resulting in a substitution of aspartate for histidine at amino acid 63 (H63D). An average of 85-90% of patients with typical clinical features of HHC are homozygous for the C282Y mutation. H63D is not associated with the same degree of iron loading as C282Y. Clinical expression is variable depending on environmental (dietary) iron, physiological and pathological blood loss and as yet unidentified modifying genetic factors. One recent Australian study indicates that only about 50% of homozygous subjects are fully expressing and symptomatic and that about 30% show no clinical or biochemical expression. Genetic tests for identifying mutations in the HFE gene provide precise means for diagnosis, family testing and population screening and have led to re-evaluation of the indications for liver biopsy in this disease. At the present time, however, the most practical and cost-effective method of screening is for phenotypic expression by transferrin saturation or unsaturated iron binding capacity measurement. In the future, population screening by genotype should be feasible once the relevant technical, legal and ethical issues are resolved.

Analysis of loss of heterozygosity (LOH) is very important in the study of tumor suppressor genes. However, accurate LOH analysis of tumor suppressor genes is difficult because of dilution by contaminating non-tumor DNA. Thus, enrichment of tumor DNA is required to accurately determine LOH of the tumor. We developed a new application of the fluorescent polymerase chain reaction by coupling it with crypt isolation to accurately assess the incidence of LOH of tumor suppressor genes in 45 colorectal carcinomas. LOH was observed at p53 in 26 of 37 tumors (70.3%), at APC in 13 of 35 (37.1%), at DCC in 16 of 25 (64.0%), at NF-2 in 5 of 23 (21.7%), and at nm23 H-1 in 7 of 30 (23.3%). We could clearly determine LOH of these genes because the crypt isolation technique was used. Although the incidence of LOH at each of these loci, as determined by using this technique, was similar to that obtained in previous studies using conventional methods, this method provides a simpler, more accurate way to assess LOH. In addition, the morphology of the samples can be analyzed before genetic analysis.

To clarify changes in mucus components during colorectal tumorigenesis, we developed novel monoclonal antibodies (Abs) against carbohydrate chains of human colorectal mucin (HCM) obtained from normal sigmoid and rectal mucosae. A hundred and ninety-nine cases of colorectal carcinoma and 67 cases of tubular adenoma, along with 250 normal colonic tissue samples, were investigated immunohistochemically. The results were compared with clinical stage, survival and MUC2 (core protein of the intestinal type mucin) expression, as well as with the status of the p53 and DCC (deleted in colorectal carcinomas) genes. In the normal colonic epithelium, HCM14 Ab reacted with the cytoplasmic regions of the goblet cells and enterocytes, while HCM21 Ab bound to mucous droplets in the former, suggesting a more mature carbohydrate structure. Both HCM14 and 21 scores were significantly decreased in adenomas and carcinomas. This is in line with an altered PAS-Alcian blue staining, indicating accumulation of mucins with incomplete or abnormal glycosylation in tumors. Levels of HCM14 and 21 binding tended to show a positive correlation with expression of MUC2 and DCC, and a negative association with p53 protein accumulation in carcinomas, although there was no apparent link to Duke's stage or the prognostic outcome. These findings suggest a possible involvement of alterations in mucin carbohydrate in colorectal tumor development. The observed changes may be associated with loss of MUC2 and DCC expression, as well as with p53 protein accumulation.

<h4>Objectives</h4>In order to increase our knowledge of adult haemochromatosis epidemiology and its clinical behaviour in young patients, we studied 4 patients from 3 pedigrees with idiopathic haemochromatosis.<h4>Methods</h4>Diagnostic criteria were: 1) Discarding the presence of secondary haemosiderosis. 2) Histological and histochemical confirmation of hepatic iron overload. Mean age at time of diagnosis was 25.2 years. Male/female ratio was 1.<h4>Results</h4>All of them were clinically asymptomatic, what shows the main difference with juvenile haemochromatosis, presenting earlier and more aggressively. Physical examination could not find abnormalities in any case. Regarding analytical studies, the main results were as follows: mean serum iron level was 209.5 mg/dl (s = 37.8), transferrin 206 mg/dl (s = 24.5), transferrin saturation was 77.4% (s = 9); ferritin, ASAT & ALAT were abnormal exclusively in the 2 males), the other measurements being between normal limits in all cases. Abdominal MRI showed an impaired hepatic signal in 2 cases. Liver biopsy was accomplished in 3 of them, showing massive deposits of hemosiderin in the hepatocytes in the 2 male cases and chronic hepatitis in one of them. There was no evidence of damage to other organs. Genetic studies showed that the 2 siblings were homozygous for the HFE 845GAEA (C282Y) mutation and in the other 2 cases HLA-A3,B7 was found to be associated. At present, after a year or more, all of them remain asymptomatic, liver function tests and mean serum ferritin levels are within normal limits, IST is < 65%, while Hb continues being > 11 g/dl.<h4>Conclusions</h4>To conclude, we would like to emphasize the extraordinary importance of early diagnosis and careful treatment in this common and remediable illness (otherwise lethal), to reach a normal and symptoms-free lifespan.

<h4>Background</h4>Evaluation of influence of pre-op continuous e.v. heparin infusion in patients undergoing urgent myocardial surgical revascularization, on the anticoagulation threshold needed for cardiopulmonary bypass. Analysis of the efficacy of ATIII substitutional therapy to allow best ACT values during extracorporeal circulation, and to reduce intra and post-op bleeding and need for homologus transfusion.<h4>Setting</h4>Operative room and ICU of a cardiac surgery unit in a regional hospital.<h4>Methods</h4>Two groups of coronary patients in preoperative treatment with heparin were randomized in a prospective double blind study for an intraoperative treatment with heparin and ATIII (Group A) and heparin plus placebo (Group B). An investigation was made on the influence of preoperative heparin treatment regarding extracorporeal circulation, the variation of the coagulation parameters in CEC with substitutive therapy of ATIII and the reduction of the therapeutic strength of heparin during perfusion, the problem of bleeding and the incidence of blood transfusions and lastly the economic questions of the two procedures.<h4>Results</h4>The study showed the necessity of repeated bolus of heparin during CEC and the rapid loss of its effect in the group not subjected ATIII therapy. A less incidence of bleeding in Group A was observed; for this reason the patients received significantly less packed red cells and FFP and a discrete number of patients of this group were not transfused. Surely the method of using the ATIII is much more expensive from the economic point of view, but the benefits of avoiding the problems of a blood transfusion (infections, immunodepression etc.), of the reduced stay in the Intensive Care Unit, of the riduced risk involved with problems of bleeding and the need of repeated operative procedures make this method fundamental in patients with reduced plasma levels of ATIII such as coronary patients who are under heparin treatment for several days.<h4>Conclusions</h4>Intraoperative administration of ATIII can reduce most problems due to heparinization of the extracorporeal circuit, such as onset of fibrinolysis, CID and platelets depletion or inactivation causing intra and post-op massive bleeding.

PCR amplification of the CAG repeat in exon 1 of the IT15 gene is routinely undertaken to confirm a clinical diagnosis of Huntington disease (HD) and to provide predictive testing for at-risk relatives of affected individuals. Our studies have detected null alleles on the chromosome carrying the expanded repeat in three of 91 apparently unrelated HD families. Sequence analysis of these alleles has revealed the same mutation event, leading to the juxtaposition of uninterrupted CAG and CCG repeats. These data suggest that a mutation-prone region exists in the IT15 gene bounded by the CAG and CCG repeats and that caution should be exercised in designing primers that anneal to the region bounded by these repeats. Two of the HD families segregated null alleles with expanded uninterrupted CAG repeats at the lower end of the zone of reduced penetrance. The expanded repeats are meiotically unstable in these families, although this instability is within a small range of repeat lengths. The haplotypes of the disease-causing chromosomes in these two families differ, only one of which is similar to that reported previously as being specific for new HD mutations. Finally, no apparent mitotic instability of the uninterrupted CAG repeat was observed in the brain of one of the HD individuals.

<h4>Background</h4>Many women develop stretch marks (striae gravidarum) during pregnancy. A number of creams have been used to remove these stretch marks.<h4>Objectives</h4>The objective of this review was to assess the effects of topical treatments to prevent the development of stretch marks.<h4>Search strategy</h4>We searched the Cochrane Pregnancy and Childbirth Group trials register. In addition, the Cochrane Controlled Trials Register (CENTRAL/CCTR) was searched. Date of last search: April 1999.<h4>Selection criteria</h4>Randomised trials comparing active creams with placebo for the treatment of stretch marks in pregnant women.<h4>Data collection and analysis</h4>Trial quality was assessed and data were extracted independently by two reviewers.<h4>Main results</h4>One study involving 100 women was included. Compared to placebo, treatment with a cream containing Centella asiatica extract, alpha tocopherol and collagen-elastin hydrolysates was associated with less women developing stretch marks (odds ratio 0.41, 95% confidence interval 0.17 to 0.99).<h4>Reviewer's conclusions</h4>One particular cream appears to help prevent the development of stretch marks in pregnancy, but only for women who had previously suffered stretch marks in pregnancy. There is no evidence of benefit for general use.

Alterations in the cellular genome affecting the expression or function of genes controlling cell growth and differentiation are considered to be the main cause of cancer. Genes that cause cancer are of two distinct types: oncogenes and onco-suppressor genes. The normal proto-oncogene can be converted into an active oncogene by deletion or point mutation in its coding sequence, gene amplification, and by specific chromosome rearrangements. Mutations and abnormal expression in ras, myc, c-erbB-2, and other oncogenes have been reported in several types of gynecological cancer. Onco-suppressor genes are involved in gynecological cancer, their functions are localized in different phases of the cell cycle. Structural changes and deletions of these genes can cause cancer. Mutations in the p53, BRCA1, DCC, and PTEN genes have been reported in gynecological cancers such as ovarian, cervical, and endometrial cancer. Human papillomaviruses are of major interest because specific types (HPV-16, -18, and several others) have been identified as causative agents in at least 90% of cancers of the cervix. In this study we summarize the available information regarding the implication of specific oncogenes, onco-suppressor genes, and HPV in the development of female genital malignancies.

The [3H]citalopram binding after three weeks vehicle, desipramine 10 mg/kg or citalopram 5 mg/kg treatment was studied in the cerebral cortex of normal, DSP-4-, and p-CPA-impaired rats. The DSP-4 50 mg/kg treatment decreased the affinity (Kd), but increased the maximal number of the apparent binding sites (Bmax) of the 5-hydroxytryptamine transporter (5-HTT). This effect was reversed by desipramine 10 mg/kg treatment. The p-CPA 350 mg/kg treatment decreased the Bmax value while the antidepressant treatment did not influence this parameter. In conclusion, our experiments demonstrate that the monoaminergic impairment induced by DSP-4 and p-CPA treatment evokes opposite changes in the 5-HTT binding characteristics and these changes are partially reversed by the chronic antidepressant treatment.

The neural cell adhesion molecule, L1, is thought to play a critical role in the formation and fasciculation of axon tracts during development. In the chick, the L1 cell adhesion molecule is expressed on both ipsi- and contralateral portions of commissural axons and perturbation studies produced a defasciculation of the ipsilateral commissural fibers. Yet in the rat, L1 is reported along commissural axons only after they have reached the contralateral marginal zone. When this species variation was reexamined, L1 was found to be expressed on rat commissural axons in a pattern similar to that observed in the chick. In addition, L1 is detected along commissural axons as early as embryonic day 12 in rats and maintained on both the ipsi- and contralateral surfaces during embryonic development. Other molecular markers that identify commissural axons in rats are TAG-1 (transiently expressed axonal glycoprotein) and DCC (deleted in colorectal cancer), and thus the pattern of L1 staining was compared with that of these other members of the immunoglobulin superfamily. Commissural axons emerging from dorsally located neurons are identified with TAG-1 and DCC, whereas L1 is detected only on ventrally located commissural axons. The pattern of L1 expression overlaps that of the more numerous laterally and ventromedially located GABAergic commissural axons. Furthermore, some of the GABAergic commissural axons express L1 on their surfaces. While commissural axons are often considered as a single population, differences in the combination of adhesion-type molecules on their surfaces and in their neurotransmitter phenotypes may signify distinctive neuronal subgroups.

To examine the responsiveness of the Medical Outcomes Study 36-Item Short Form (SF-36) to the development of diabetes complications over time. We studied a cohort of 331 diabetic veterans participating in a prospective study of risk factors for foot complications. Eight SF-36 scales [general health (GH), physical functioning (PF), social functioning (SF), mental health (MH), physical role (RP), emotional role (RE), bodily pain (BP), and vitality (VT)] and 25 diabetes complications characteristics (DCC) from history and symptom questionnaires and physical exam findings were compared over a mean interval of 3.1 years. The subjects were mostly married, white males with a mean age of 63.5 years, with primarily type 2 diabetes (91%) and a mean diabetes duration of 9.7 years at baseline. There was a statistically significant interval decrease in the GH scale of 6.1 points (effect size [ES] 0.24), PF decreased 9.7 (ES 0.38), SF decreased 5.8 (ES 0.19), PR decreased 14.7 (ES 0.38), BP decreased 4. 0 (ES0.14), VT decreased 4.5 (ES 0.16), total DCC increased by 1.8 (ES 0.53), and hemoglobin A(1c) declined 1.3% (ES 0.48). An increase of >1 DCC was associated with an average loss of 7.2 to 11.8 points on six SF-36 scales (GH, PF, SF, RP, BP, VT). The development of any renal complication was related to decrements in five SF-36 scales (GH, PF, SF, RP, VT) while the appearance of any neuropathy complication was associated with a decline in four SF-36 scales (GH, PF, PR, VT). These results imply that six of the SF-36 scales are responsive to the development of diabetes complications over time among elderly veterans, supporting their use in longitudinal research. Renal and neuropathy complications have the greatest effects on the SF-36.

Anaerobically grown glucose-fermenting E. coli cells produce molecular hydrogen, acidify the medium and uptake potassium ions. It was shown that the H2 release and the proton-potassium exchange with the fixed (2H+/K+) stoichiometry of the initial DCC-sensitive fluxes were lost in mutants with the deleted fdhF gene or the hycA-H operon responsible for the biosynthesis of formate dehydrogenase H (FDH,H) or hydrogenase 3 (H3), respectively, which are the main components of the formate hydrogen lyase FHL(H). However, both processes occurred in mutants with the deleted hycE, hycF or hycG genes encoding the major and minor components of H3, respectively. The K+ uptake was sensitive to the osmotic shock resulting from glucose addition to the medium and decreased significantly in the presence of valinomycin. The H2 release and the 2H+/K+ exchange were absent in the mutant with the deleted hycB gene encoding the corresponding minor component of H3. This mutant acidified the medium and uptook K+ with Km typical for TrkA, but the stoichiometry of the DCC-inhibited fluxes was variable, and the K+ gradient between the cytoplasm and the medium in this mutant was lower than in the mutants lacking other minor components of H3. The results obtained suggest that the hycB gene product, FdhF and HycE, form probably the FHL(H) complex that directly interacts with the H+-ATPase complex F0F1 and the TrkA(H) system of K+ uptake. Such a multienzyme association is responsible for the H2 production and 2H+/K+ exchange. The major and other minor components of H3 have probably no direct role in the H2 production and 2H+/K+ exchange. H2 production by precursor's or hycE mutant's protoplasts treated with toluene was shown to occur upon addition of the thiol reagent dithiothreitol to the medium containing ATP, potassium ions, NAD+, and NADH. H2 production was inhibited by DCC. The quantity of available thiol groups in membrane vesicles of the precursor or the hycE, hycF or hycG mutants, in which the H2 production and 2H+/K+ exchange were observed, was larger than in other mutants. The number of SH groups decreased in the presence of DCC. These results indicate a significance of the thiol groups for the function of the proposed association.

HFE is a non-classical major histocompatibility complex class I molecule that complexes with a beta2-microglobulin. A functional link between HFE and iron metabolism has been established by the discovery of a physical association between HFE and the transferrin receptor. By inhibiting transferrin receptor internalization, HFE functions as a negative modulator of transferrin receptor function. In addition, HFE appears to be an iron sensor that directly or indirectly communicates the body's iron status to T cells, which then use cytokines as feedback modulators to achieve iron homeostasis. A working model for the feedback regulatory mechanism between iron metabolism and immune function is proposed.

Genetic hemochromatosis (GH) is an inherited disease that results in iron overload, and, if untreated, causes irreversible organ damage. Knowledge and understanding of the early features of the condition, often nonspecific, and of the diagnostic route are necessary to detect iron overload and diagnose GH before irremedial damage has been done. Genetic testing now plays an important role in diagnosis. Management of the patient with established GH centers on venesection to return body iron levels to normal, treatment of the complications of GH, and family screening for GH. Population screening for GH, the ideal strategy to prevent any morbidity from iron overload, has not yet been accepted by public health professionals, largely because of the lack of data on the disease penetrance in genetically susceptible individuals.

Our aim was to evaluate the potential utility of mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) to detect hemochromatosis. We computed the accuracy of MCV and MCH cut-off points > or = upper reference limits using data from 94 probands and 132 white controls. Our reference ranges are MCV 80.0-97.0 fL and MCH 26.0-32.0 pg. Sensitivity of MCV was 8.6-48.3% for men and 2.8-44.4% for women (cut-off points > or = 105.0 - > or = 97.0 fL, respectively). Sensitivity of MCH was 33.9-70.7% for men and 19.6-50.0% for women (cut-off points > or = 34.0 - > or = 32.0 pg, respectively). Using MCV and a hemochromatosis frequency typical of many western Caucasian populations (0.005), positive predictive values (PV+) were 2.1-100.0% in men and 4.2-100.0% in women. Using MCH, PV+ were 1.7-8.2% in men and 1.8-6.8% in women. We also calculated PV+ using the hemochromatosis frequency 0.015, which could occur in persons receiving medical care. Using MCV cut-off points > or = 101.0 fL, PV+ were 8.9-100.0% in men and 100.0% in women with maximum sensitivities of 24.1% and 25.0%, respectively. Using MCH testing, PV+ was 21.5% in men (cut-off point > or = 34.0 pg) and 18.2% in women (cut-off point > or = 33.0 pg) with sensitivities of 33.9% and 37.0%, respectively. Using MCV or MCH, sensitivity and PV+ for the HFE genotype C282Y/C282Y were generally greater than for "nonclassical" HFE genotypes. All negative predictive values in our study were > or = 98.5%. We conclude that supranormal values of MCV or MCH could be used to detect hemochromatosis in white persons of western European descent who are receiving routine medical care. Comparisons of MCV, MCH, and transferrin saturation testing and other implications of MCV and MCH testing for hemochromatosis in medical care are discussed.

Over 90% of patients with hemochromatosis in the United Kingdom are homozygous for the C282Y mutation on the HFE gene. The Centers for Disease Control (CDC) in the United States has recommended that adults should be screened for HFE mutations to identify susceptible individuals before onset of disease. The aim of this study was to evaluate the polymerase chain reaction using sequence-specific primers (PCR-SSP) as a method of large-scale population screening for the common HFE gene mutations, H63D and C282Y. A total of 10,583 consenting blood donors were tested using nonautomated procedures. Three alleles, termed HFE-1, -2, and -3, were detected with phenotype frequencies of 94.56%, 28.33%, and 15.79%, respectively, and gene frequencies of 0.76421, 0.15342, and 0.08237, respectively. All donors identified as homozygous for the C282Y mutation or heterozygous for both the H63D and C282Y mutations were confirmed by heterduplex analysis and/or PCR-SSP. The number of technical failures that affected the identification of donors homozygous for the C282Y mutation was 390 giving an overall repeat rate 3.7%, although this fell to 1% over the last quarter of the study. This study demonstrates that PCR-SSP may be used for large-scale population screening for the C282Y genotype associated with hemochromatosis.

Hereditary hemochromatosis (HH) is a very common autosomal recessive disorder of iron metabolism and frequently associated with mutations in the HFE gene. Molecular genetic testing for HFE mutations is considered valuable for carrier identification, as well as for early diagnosis of the disease, allowing simple treatment by phlebotomy and normal survival of patients. We have developed a reverse-hybridization assay for the routine diagnosis of eight previously described and one novel (E168Q) HFE point mutations. The test is based on multiplex DNA amplification and ready-to-use membrane teststrips, which contain oligonucleotide probes for each wild-type and mutated allele immobilized as an array of parallel lines. The procedure is rapid and accessible to automation on commercially available equipment, and by adding new probes the teststrip can easily be adapted to cover an increasing number of mutations.

We describe a novel molecular screening technique for hereditary hemochromatosis through which HFE genotypes at codon positions 282 and 63 are simultaneously detected. The technique combines multiplex PCR and denaturing high-performance liquid chromatography (DHPLC) and allows automated high-throughput analysis. We used this method to genotype 43 previously characterized anonymous DNA specimens in blinded fashion and found multiplex PCR/DHPLC 100% accurate when compared with PCR/restriction enzyme digestion, yet far more efficient.

Large-scale detection of mutations at the DNA level requires the development of cost-effective methods for the screening of thousands of samples. Hemochromatosis is an appropriate testing ground for the development of such technologies, and we report the methods that we have developed to screen large numbers of samples using equipment available in most laboratories. We are able to examine DNA samples for two mutations in the HFE gene at a cost of only slightly over $8 per sample, a cost that includes overhead and the approximately 40 hr per week of technician time required to perform the studies. The technologies involved in mutation analysis are evolving rapidly and ultimately more highly automated, lower-cost technologies may become available. At present, however, we find the methodology described to be very suitable for large-scale, low-cost mutation screening.

The widespread use of the genotype assay that identifies the common C282Y mutation in the HFE gene has allowed an earlier diagnosis to be made in many subjects. A significant number of these patients may have no evidence of phenotypic disease and have a normal serum ferritin level. This phenomenon is more common when the genotype assay is used to screen populations rather than higher-risk groups such as family members of a proband with hereditary hemochromatosis. Moreover, patients with significant iron overload may be wild type for the C282Y mutation and have no other demonstrable mutation of the HFE gene. The HFE genotype assay has recently been found to give a false-positive C282Y homozygous result in half of the subjects in one population screening study due to the presence of a single nucleotide polymorphism (SNP) that interfered with primer binding in the PCR assay. The problem may be overcome by using alternate primers. A number of other groups have confirmed the finding but in a much smaller number of subjects, whereas others found that their assays were not affected by the SNP. The use of the HFE genotype assay as the sole diagnostic criterion for hereditary hemochromatosis is not recommended. The genotype assay should be used as an adjunct to the established methods of demonstrating iron overload and be viewed as a predictor of either the presence of iron overload or the subsequent development of iron overload during an individual's lifetime.

In a previous hemochromatosis screening study including a total of 65,238 individuals, 566 persons were genotyped for the C282Y and the H63D mutations. Of these, a total of 433 samples (298 homozygous C282Y and 135 homozygous wild type) were reanalyzed to investigate if the potential presence of the newly described 5569G/A polymorphism had confounded the genotyping results for the C282Y mutation. Genotyping with a polymorphism-insensitive primer pair yielded no samples that altered their genotype. By utilizing the polymorphism-sensitive primer pair and elevated annealing temperatures, 133 samples previously genotyped as heterozygous C282Y were reanalyzed to verify the presence of the polymorphism in the population studied. Out of a total of 266 chromosomes, we found the polymorphism present in 9 chromosomes, yielding an allele frequency of 0.034 in this particular subpopulation. In one of the samples, the polymorphism was present on the same DNA strand as the C282Y mutation. We conclude that in the population studied, the 5569 G/A polymorphism is present, but its presence had no implications for the outcome of the previous genotyping. Nevertheless, we recommend that C282Y genotyping by restriction endonuclease digestion of PCR products in the future should utilize a primer pair that is not influenced by the 5569G/A polymorphism.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism. Iron absorption from the gut is inappropriately high, resulting in increasing iron overload. The hemochromatosis gene (HFE) was identified in 1996 by extensive positional cloning by many groups over a period of about 20 years. Two missense mutations were identified. Homozygosity for one of these, a substitution of a tyrosine for a conserved cysteine (C282Y), has now clearly been shown to be associated with HH in 60-100% of patients. The role of the second mutation, the substitution of an aspartic acid for a histidine (H63D), is not so clear but compound heterozygotes for both these mutations have a significant risk of developing HH. Here we review other putative mutations in the HFE gene and document a number of diallelic polymorphisms in HFE introns.

The main hereditary hemochromatosis mutation C282Y in the HFE gene was recently described, and the C282Y frequencies were reported for various European populations. The aim of this synthesis is to compile the Y allele frequencies of the C282Y mutation for 40 European populations. The most elevated values are observed in residual Celtic populations in Ireland, the United Kingdom, and France, in accordance with the hypothesis of Simon et al. (1980) concerning a Celtic origin of the hereditary hemochromatosis mutation.

The C282Y mutation of the HFE gene has been reported as the main cause of hereditary hemochromatosis (HH). Another missense mutation (H63D) has also been detected at an increased frequency in a compound heterozygote state with the C282Y mutation in HH patients. However, these two mutations are not present in all of the HH patients, indicating that other mutations in the HFE gene, or in other loci, should exist. The present study reports the frequencies of the C282Y and H63D mutations in 74 Spanish HH patients and the results of the sequencing analysis of the HFE exons, intron-exon boundaries, and 588 bp of the 5' region in 5 patients negative for the C282Y mutation. We have detected a high frequency of the C282Y mutation (85.1%) in Spanish HH patients, indicating that this mutation is the most common defect associated with the disease in Spain. The screening of the HFE regions in our patients without the C282Y mutation has revealed the presence of five polymorphisms. However, no other pathological mutations have been found. Therefore, further efforts to characterize the unscreened part of the HFE gene or other loci should be taken to identify the potential genetic factors causing HH in the C282Y-negative patients.

Hereditary hemochromatosis (HH) is a disorder of iron metabolism that leads to iron overload in middle age and can be caused by homozygosity for the C282Y mutation in the HFE gene. Preliminary studies have estimated the frequency of this mutation at 0.5-1% in Italy, but this has not been verified on a large sample. We analyzed 1,331 Italian newborns for the C282Y mutation in the HFE gene using dried blood spots (DBS) from the Neonatal Screening Center in Turin, Italy. The mutation was assessed using a semi-automatable 5'-nuclease assay (TaqMan technology). We detected 55 heterozygotes and no homozygotes in our sampling, resulting in an overall frequency of 2.1% +/- 0.6 for the C282Y allele. Differences in allele frequency were observed, and ranged from 2.7% +/- 1.3 in samples from Northern Italy, to 1.7% +/- 0.9 in samples from Central-Southern Italy. The low frequency of the at-risk genotype for iron overload suggests that genetic screening for HFE in Italy would not be cost effective. The present study, in addition to defining C282Y frequency, documents detection of the major HFE mutation on routine DBS samples from neonatal screening programs using a semi-automatable, rapid, reliable, and relatively inexpensive approach.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder causing inappropriate dietary iron absorption that affects North Europeans. HH is associated with the C282Y mutation of the HFE gene, and the H63D mutation to a lesser degree. Both mutations are abundant in Europe, with H63D also appearing in North Africa, the Middle East, and Asia. Emigration from Europe over the past 500 years has introduced C282Y and H63D to America, Australia, New Zealand, and South Africa in an essentially predictable fashion. The distinctive characteristics of the population genetics of HH are the confined racial distribution and high frequency in North European peoples. C282Y frequencies in North Europeans are typically between 5% and 10%, with homozygotes accounting for between 1/100 and 1/400 of these populations. The scarcity of the C282Y mutation in other populations accounts for the lack of HH in non-Europeans.

There is interest in general population screening for hemochromatosis and other primary iron overload disorders, although not all persons are at equal risk. We developed a model to estimate the numbers of persons in national, racial, or ethnic population subgroups in Jefferson County, Alabama, who would be detected using transferrin saturation (phenotype) or HFE mutation analysis (genotype) screening. Approximately 62% are Caucasians, 37% are African Americans, and the remainder are Hispanics, Asians, or Native Americans. The predicted phenotype frequencies are greatest in a Caucasian subgroup, ethnicity unspecified, which consists predominantly of persons of Scotch and Irish descent (0.0065 men, 0.0046 women), and in African Americans (0.0089 men, 0.0085 women). Frequencies of the HFE genotype C282Y/C282Y > or = 0.0001 are predicted to occur only among Caucasians; the greatest frequency (0.0080) was predicted to occur in the ethnicity-unspecified Caucasian population. C282Y/C282Y frequency estimates were lower in Italian, Greek, and Jewish subgroups. There is excellent agreement in the numbers of the ethnicity-unspecified Caucasians who would be detected using phenotype and genotype criteria. Our model also indicates that phenotyping would identify more persons with primary iron overload than would genotyping in our Italian Caucasian, Hispanic, and African American subgroups. This is consistent with previous observations that indicate that primary iron overload disorders in persons of southern Italian descent and African Americans are largely attributable to non-HFE alleles. Because the proportions of population subgroups and their genetic constitution may differ significantly in other geographic regions, we suggest that models similar to the present one be constructed to predict optimal screening strategies for primary iron overload disorders.

Genetic hemochromatosis (GH) is a late-onset, autosomal recessive disorder. The majority of those at risk from iron overload and its clinical consequences may be detected by a simple genetic test. Furthermore, treatment by phlebotomy, if instituted early, removes excess iron and prevents the complications of iron overload which include arthralgia, diabetes, and cirrhosis of the liver. GH seems to be an obvious candidate for inclusion in national screening programs. However, important questions remain concerning the proportion of individuals with the high-risk genotype who eventually show clinical manifestations of iron overload and the significance of heterozygosity for haemochromatosis in terms of morbidity. Until these questions are resolved, the introduction of widespread genetic screening cannot be justified.

Effects of phytohormones gibberellic acid (GA) and abscisic acid (ABA) on the ATP-dependent transmembrane transport of protons were studied in plasma membrane vesicles (PMVs) from non-dormant potato tubers. The uptake of H+ into PMVs was assessed by the fluorescence quenching of acridine orange (AO) after the addition of ATP to the incubation medium. Addition of ATP to the incubation medium led to the instantaneous rise of the AO fluorescence intensity followed by its decrease. The fluorescence quenching was not observed in the presence of either protonophore CCCP or inhibitors of the membrane-bound H+-ATPase. It is concluded that the ATP-induced quenching of the AO fluorescence resulted from the accumulation of protons in PMVs due to the function of the plasma membrane-bound H+-ATPase. Depending on their concentrations, GA and ABA either inhibited or stimulated the ATP-driven H+ translocation across the vesicle membrane. The growth-stimulating hormone GA at concentrations of 10(-9)-10(-5) M increased the initial rate of the fluorescence quenching, whereas 10(-4) M GA slightly inhibited the H+ translocation. The growth inhibitor ABA at a concentration of 10(-9) M slightly increased the rate of the proton accumulation in PMVs; at higher concentrations (10(-8)-10(-4) M), ABA inhibited the H+ translocation. Acetic acid, which has pK similar to pK of GA and ABA, did not influence the ATP-dependent H+ accumulation in PMVs, suggesting the hormone-specific action of GA and ABA on the H+-ATPase activity. In the presence of DCC, which completely inhibited the accumulation of H+, GA and ABA did not affect the passive proton efflux from PMVs. It is proposed that the mechanisms of the regulatory effects of phytohormones may involve modification of H+-ATPase activity leading to changes in the electrochemical gradient of H+ across the plasma membrane.

The discovery of the HFE gene has lead to considerable improvement in the understanding and the management of genetic hemochromatosis. More than 90% of well-defined patients are homozygous for the C282Y mutation, and genetic testing has become an important diagnostic tool. The significance of the other mutations in the HFE gene remain controversial: only C282Y/H63D compound heterozygotes could present with a phenotype compatible with hemochromatosis, but with a mild expression and a low penetrance. The link between iron overload and HFE mutation is explained by the interaction between HFE protein, beta-2-microglobulin and transferrin receptor, which is abolished by the C282Y mutation, but is not yet fully understood.

Iron accumulation in the liver is frequently observed in hepatic diseases whatever their etiologies. In the majority of cases, there is no true overload, and iron accumulation corresponds to deposits in macrophages secondary to iron release from damaged hepatocytes. More rarely, namely in severe cirrhosis, there is a true overload, which is probably related to iron intestinal hyperabsorption. In such case, the site of iron excess is hepatocytic. Except for hemochromatosis, mutations of HFE gene do not play a major role in iron overload. In chronic liver diseases, iron overload could favor the development of hepatocellular carcinoma, even in the absence of cirrhosis.

Most experimental and human data support the hypothesis that iron overload is a risk factor for cancer in general and liver cancer in particular. This oncogenic effect could be explained by an overproduction of reactive oxygen species and free radicals. In cirrhosis due to genetic haemochromatosis (homozygosity for the mutation C282Y in the HFE gene) there is an increase incidence of hepatocellular carcinoma. Few cases have been reported in genetic haemochromatosis with iron overload but without cirrhosis. In hepatocellular carcinoma developed in non cirrhotic liver there is a mild iron overload in more than 50% of cases. In these patients heterozygous and compound C282Y mutations are found in 36%. In black Africans, iron overload genetically determined but not linked to mutations in the HFE gene increases also the risk of hepatocellular carcinoma. Among the many factors (viral hepatitis, alcohol, tobacco etc.) which play a role in hepatic carcinogenesis, iron overload is probably an important one and therefore should be treated.

Genetic hemochromatosis, due to its frequency (about 300,000 cases in France) and to its severity, must be considered as a public health burden. Its curability--insofar as its diagnosis has been made early--and the disposal of non invasive and reliable phenotypic (transferrin saturation) and genotypic (HFE C282Y mutation) tests make its diagnosis easy. Thus, diagnosis faced with early clinical and biological symptoms must be promulgated as screening in probant families. General screening is still hampered by ethical and socio-economic obstacles, but should be evaluated in large prospective studies. Otherwise, the recently described insulin resistance-associated liver iron overload syndrome might account for epidemiological associations that have been reported between increased body iron stores and cancerous, cardio-vascular and hepatic disorders. This might extend markedly the impact of iron overload in public health and lead to broaden screening and prevention to all iron overload conditions.

Although the genetic basis for gallbladder carcinogenesis has not been clarified, considerable evidence has shown that genetic alterations play an important role in the development and progression of human cancers. In this study, we analyzed 30 gallbladder carcinomas to investigate the role of genetic alterations in their tumorigenesis, and to study correlations with their clinicopathological features. Tissue samples were obtained from 30 patients with gallbladder carcinoma (11 men and 19 women; mean age, 62 years; age range, 38-80 years). Genomic DNAs were extracted from fresh tumor tissue. We examined loss of heterozygosity (LOH) in the p53, APC, DCC, RB, and NM23-H1 gene regions by polymerase chain reaction (PCR)-LOH assay using an automated fluorescent DNA sequencer employing four microsatellite markers (p53, APC, DCC, NM23-H1). Five additional microsatellite markers were used for the determination of microsatellite instability (MSI). LOH was found at p53 in 9 of 15 informative cases (60%), at DCC in 10 of 22 (45%), at APC in 5 of 15 (33%), at RB in 1 of 8 (13%), and at NM23-H1 in 1 of 15 (7%). MSI was observed in 5 of 30 cases (17%) in at least one chromosomal loci of these nine microsatellite markers. None of the patients with MSI-positive tumors showed lymph node metastasis, and there was an inverse correlation between MSI and the presence of LOH in gallbladder carcinoma. These results suggest that there are two independent genetic pathways in gallbladder carcinogenesis; that is, an MSI pathway and an LOH pathway.

The aim of the investigation was to test genes for predisposition to bipolar affective disorder. Therefore, we studied candidate genes in a sample of unrelated patients (n = 102) and healthy controls (n = 79) of Austrian origin, searching for a possible association between polymorphic DNA markers of 5 candidate genes (serotonin transporter, 5-HTT; serotonin 2a receptor, 5-HT2a; dopamine D2 receptor, DRD2; dopamine D3 receptor, DRD3; dopamine transporter, DAT1) and bipolar disorder. There was an association between allelic and genotypic frequencies of 5-HTT and affection status (p = 0.014 and p = 0.017, respectively). However, after correction for multiple comparisons (Bonferroni), these results did not remain significant. Nevertheless, the findings might suggest that alterations in the structure of 5-HTT are involved in the pathogenesis of bipolar disorder, which could have major implications in treatment. No association between 5-HT2a, DRD2, DRD3, DAT1 and bipolar disorder was found.

The immune response to an organ allograft involves perpetuation of T cell infiltration and activation. Advances in understanding the mechanisms of T cell activation have placed particular emphasis on the interactions between the T-cell receptor and antigen presenting cells, with little reference to the fact that in vivo activation occurs in the physical context of extracellular matrix proteins (ECM). Indeed, the possibility that ECM proteins may have a determining role in lymphocyte adhesion and tissue localization and function is now becoming more appreciated in view of growing evidence indicating that integrins and other T cell antigens bind ECM components, with some of these components exerting synergistic effects on T-cell activation. This review focuses on the importance of interactions between lymphocytes and fibronectin, a prominent ECM component, for cell migration and function in organ allograft recipients. It explores novel therapeutic approaches based on the assumption that fibronectin represents an active element in the process of T cell activation in the immune cascade triggered by organ transplantation.

Netrins are secreted proteins that direct axon extension and cell migration during neural development. They are bifunctional cues that act as an attractant for some cell types and as a repellent for others. Several lines of evidence suggest that two classes of receptors, the deleted in colorectal cancer (DCC) family and the UNC-5 family, mediate the attractant and repellent response to netrin. Although netrins were first identified as diffusible long-range cues for developing axons, recent findings provide evidence that they also function as short-range cues close to the surface of the cells that produce them. This short-range function of netrin contributes to guiding neurite outgrowth and mediating cell-cell interactions during development and perhaps also in adults.

Huntington Disease (HD) results from polyglutamine expansion within the N-terminus of huntingtin. We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) human huntingtin in a developmentally appropriate and tissue-specific manner identical to the pattern of expression of endogenous huntingtin. YAC46 and YAC72 mice show early electrophysiological abnormalities indicating neuronal cytoplasmic dysfunction prior to developing nuclear inclusions or neurodegeneration. YAC72 mice display a hyperkinetic movement disorder by 7 months of age, and have evidence for selective and specific degeneration of medium spiny neurons in the lateral striatum by 12 months of age. A key molecular feature of pathology of these YAC72 mice is cleavage of huntingtin in the cytoplasm following by translocation of the resulting huntingtin N-terminal fragments into the nucleus of striatal neurons. Increasing nuclear localization of huntingtin N-terminal fragments within medium spiny neurons of the striatum occurs concomitantly with the onset of selective neurodegeneration. Because huntingtin is a caspase substrate and truncated huntingtin fragments are toxic in vitro, inhibiting caspase cleavage of huntingtin may be of potential therapeutic benefit in HD. We show that caspase inhibitors eliminate huntingtin cleavage in cells and protects them from an apoptotic stress. We also identify caspase-6 and caspase-3 cleavage sites in huntingtin and demonstrate that neuronal and non-neuronal cells expressing a caspase-resistant huntingtin with an expanded polyglutamine tract are less susceptible to apoptosis and aggregate formation. These results suggest that caspase cleavage of huntingtin may be a crucial step in aggregate formation and neurotoxicity in HD.

Cell recognition molecules of the immunoglobulin superfamily are involved in the formation, establishment, and plasticity of neural circuits in the central nervous system (CNS). We used a polymerase chain reaction-based approach to specifically amplify molecules with conserved sequence elements of immunoglobulin-like domains. This approach enabled us to isolate Kilon, a novel immunoglobulin that has been described by Funatsu et al. (J Biol Chem 1999;274: 8224-8230) from the hippocampus. The sequence of Kilon shows a high degree of homology to that of the chicken protein neurotractin, a molecule involved in neurite outgrowth and capable of interacting with LAMP. In situ hybridization analysis was performed to analyze the Kilon mRNA distribution in the developing and adult rat brain and to compare it to that of LAMP mRNA. Kilon mRNA was found to be specifically expressed in the dentate gyrus (DG) of the adult rat, whereas LAMP transcripts were present in all regions of the hippocampal formation. These results were corroborated by RT-PCR semiquantification of gene expression in microdissected tissue prepared from the DG and the CA1 region of the hippocampus. We also performed mRNA expression analysis of both genes following hippocampal deafferentation and seizure, but neither Kilon nor LAMP gene expression showed significant alterations after lesioning on the in situ hybridization level. Our results show that the expression patterns of Kilon and LAMP during development and in the mature hippocampus are clearly distinguishable from one another, which suggests different roles for these related molecules in the hippocampus.

No abstract available.

The authors investigated the activity of procoagulants and antithrombin-III after freezing and thawing of blood plasma by different methods. It was shown that quick freezing and thawing using special apparatuses best of all preserved the coagulative properties of the plasma. The medical efficiency of quick-frozen plasma was shown to depend on the method of freezing and thawing.

In the present paper, we describe an individual, found as part of a screening study, being homozygous for the C282Y mutation and at the same time heterozygous for the H63D mutation in the HFE gene. Identical results were obtained by three different methods, i.e., by PCR-RFLP, by sequencing, and by melting curve analysis. Thus, the common conception that the C282Y and the H63D mutations are mutually exclusive is not valid. Clinical symptoms and laboratory data on the individual were similar to hemochromatosis patients homozygous for the C282Y mutation. The implications of our finding for diagnostic analytical laboratory procedures are briefly discussed.

In order to determine the molecular genetic alterations associated with tumor invasion, Barrett's adenocarcinoma and its non-invasive precursor lesions were investigated by comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and loss of heterozygosity (LOH). Along the metaplasia-dysplasia-carcinoma sequence in Barrett's adenocarcinoma gains on 7q, 8q, 20q, 2p, 10q and 17q, and losses on Y, 4q, 5q, 9p 18q and 14q became steadily more frequent. FISH demonstrated increasing DNA copy of numbers of c-erbB-2, 20q13.2 (AIB), c-myc and cyclin D1 during the development of Barrett's adenocarcinoma, and LOH confirmed DNA losses on 5q21 (APC) and 18q (DCC). Some of the chromosomal changes of the non-invasive precursor lesions were significantly different from the alterations detected in invasive carcinoma. Although molecular pathology may be used as diagnostic adjunct in future, the histopathological determination of invasion remains the most important diagnostic criterion of malignancy in Barrett's adenocarcinoma.

Passerines (songbirds) and softbills (toucans and mynahs) are increasingly presented for veterinary care as pet owners and aviculturists recognize that successful medical and surgical treatment can be performed in these often tiny patients. Even with an increasing amount of pharmacokinetic data in birds, the use of extrapolated drug regimens continues to be a common practice for these species. The extrapolation, using allometric scaling from human, mammalian, and avian drugs to passeriformes and softbills, is complicated and limited. In this article, the choice of the therapeutic approach is discussed. Once the choice for a specific drug is made, tables help calculate the dose in milligrams per kilocalorie without using complicated formulas.

Germline mutation in the APC gene is required for the initiation of the development of familial adenomatous polyposis (FAP). According to Fearon and Vogelstein model, further somatic mutations in the K-ras oncogene, DCC gene and p53 tumor suppressor gene are prerequisite for development of colon carcinoma. We have found that the germline mutations in the DNA isolated from lymphocytes of an 18 years old girl with extraordinary expressive phenotype in codons 1060-1061 of the APC gene result in truncation of the APC protein. The mutation in codons 12 and 13 of the K-ras oncogene was not detected, but another germline mutation was found in codon 210 of the p53 gene. Furthermore, no one of these germline mutations was detected in the DNA of peripheral blood lymphocytes of the patient's 21 years old healthy sister. Until now, there has been no evidence about the expressive phenotype due to mutation in codons 1060-1061 of the APC gene; the role of germline missense mutation in codon 210 of the p53 gene in the FAP malignant process remains to be elucidated too. The effect of the combination of germline mutation in two different tumor suppressor genes in the progress of disease is discussed.

The efficiency of Xenopus laevis egg extract to repair T:G and A:C mismatched base pairs in unmethylated, hemimethylated and fullymethylated heteroduplexes was investigated. Filamentous phage M13mp18 and its derivative M13mp18/MP-1 (C changed to T inside the sequence dCC*C GGG, at the position 6248) were used for heteroduplexes construction. The three origins of mismatched base-pairs in the eukaryotic DNA are mimicked by in vitro methylation: hemimethylated DNA (me-/me+) for replication errors; unmethylated (me-/me-) and fully methylated DNA (me+/me+) for recombination heteroduplexes, and fullymethylated also for locally, spontaneously deaminated 5-methylcytosine (5meC) to T, generating the exclusively T:G mismatch. The methylations were in CpG dinucleotides, mostly characteristic ofeukaryotic cells [5, 24]. In vitro methylation was done by HpaII methylase which methylate central C of dCCGG sequence in the manner of eukaryotic methylation. The position of mismatched bases was chosen so that correction of mismatched bases in any strand would create the sequence for one of the "diagnostic" restriction endonucleases, either BstNI or MspI. Correction efficiency was about 10(8) repair events per egg equivalent. Correction in favor of C:G base pair restoration occurred regardless of the T:G or C:A mispairs, with almost equal efficiency. Repair of T:G to T:A was up to 10 times less efficient comparing to C:G, and repair of C:A to T:A was in our experimental system undetectable. No significant difference in repair efficiency of mismatched bases situated in unmethylated, hemimethylated or fullymethylated heteroduplexes indicate methylation-independent repair of mismatched bases in X. laevis oocite extracts.

The molecular pathways and the timing of genetic events during human colorectal carcinogenesis are still not fully understood. We have addressed the intratumor heterogeneity of the mutational status of the k-ras oncogene and of the p53 oncosuppressor gene during the adenoma-carcinoma sequence by investigating 26 human colorectal adenomas containing early cancer. An intratumor comparative analysis was obtained among the adenomatous and carcinomatous component pairs. Additionally, we have analyzed 17 adenomas having cancer in the near vicinity. The adenomatous components of the adenomas containing early cancer and the adenomas having cancer in the near vicinity had comparable frequencies for k-ras mutations (28 and 47%) but different for p53 mutations (52 and 7%, p-value = 0.01). Interestingly, the adenomatous and carcinomatous components of the adenomas containing early cancer were rarely heterogeneous for the k-ras mutational status (only in 13% of the cases) but were characterized by heterogeneity of the p53 status in 59% of the cases (p-value < 0.01). In addition, the mutations of p53 for the adenomatous components of the adenomas containing early cancer were statistically significantly associated with severe dysplasia (p-value = 0.01). Intratumor homogeneity of k-ras status during the human colorectal adenoma-carcinoma sequence suggests that the role of k-ras is more related to tumor initiation than to tumor progression. On the contrary, intratumor heterogeneity of p53 mutations indicates that the type of the p53 mutations may also be relevant for selection and expansion of new subclones leading to tumor progression.

Soft tissue calcifications in inbred laboratory mice are frequently observed and are often associated with dystrophic cardiac calcinosis (DCC). We tested the hypothesis that an excessive intake of fluoride would inhibit pathological calcifications in DCC-susceptible mice. A diet containing either a high (200 mg F/kg added to the diet) or low fluoride content (no F added) was fed to both weanling and retired breeder DBA/2 mice. The high-fluoride diet reduced feed intake and body weight gain when given after weaning. It was found that a high fluoride intake effectively reduces soft tissue calcifications in young mice, but not in retired breeders. Because DCC in mice is a pathological finding that could interfere with certain experimental procedures, it is suggested that the optimum fluoride concentration in the diet for mice of susceptible strains should be established.

This review examines the clinical consequences for the practicing hematologist of remarkable new insights into the pathophysiology of disorders of iron and heme metabolism. The familiar proteins of iron transport and storage-transferrin, transferrin receptor, and ferritin-have recently been joined by a host of newly identified proteins that play critical roles in the molecular management of iron homeostasis. These include the iron-regulatory proteins (IRP-1 and -2), HFE (the product of the HFE gene that is mutated in most patients with hereditary hemochromatosis), the divalent metal transporter (DMT1), transferrin receptor 2, ceruloplasmin, hephaestin, the "Stimulator of Fe Transport" (SFT), frataxin, ferroportin 1 and others. The growing appreciation of the roles of these newly identified proteins has fundamental implications for the clinical understanding and laboratory evaluation of iron metabolism and its alterations with iron deficiency, iron overload, infection, and inflammation. In Section I, Dr. Brittenham summarizes current concepts of body and cellular iron supply and storage and reviews new means of evaluating the full range of body iron stores including genetic testing for mutations in the HFE gene, measurement of serum ferritin iron, transferrin receptor, reticulocyte hemoglobin content and measurement of tissue iron by computed tomography, magnetic resonance imaging and magnetic susceptometry using superconducting quantum interference device (SQUID) instrumentation. In Section II, Dr. Weiss discusses the improved understanding of the molecular mechanisms underlying alterations in iron metabolism due to chronic inflammatory disorders. The anemia of chronic disorders remains the most common form of anemia found in hospitalized patients. The network of interactions that link iron metabolism with cellular immune effector functions involving pro- and anti-inflammatory cytokines, acute phase proteins and oxidative stress is described, with an emphasis on the implications for clinical practice. In Section III, Dr. Brissot and colleagues discuss how the diagnosis and management of hereditary hemochromatosis has changed following the identification of the gene, HFE, that is mutated in most patients with hereditary hemochromatosis, and the subsequent development of a genotypic test. The current understanding of the molecular effects of HFE mutations, the usefulness of genotypic and phenotypic approaches to screening and diagnosis and recommendations for management are summarized.

The human genome contains numerous genes that encode membrane transporters and related proteins. For drug discovery, development, and targeting, one needs to know which transporters play a role in drug disposition and effects. Moreover, genetic polymorphisms in human membrane transporters may contribute to interindividual differences in the response to drugs. Pharmacogenetics, and, on a genome-wide basis, pharmacogenomics, address the effect of genetic variants on an individual's response to drugs and xenobiotics. However, our knowledge of the relevant transporters is limited at present. To facilitate the study of drug transporters on a broad scale, including the use of microarray technology, we have constructed a human membrane transporter database (HMTD). Even though it is still largely incomplete, the database contains information on more than 250 human membrane transporters, such as sequence, gene family, structure, function, substrate, tissue distribution, and genetic disorders associated with transporter polymorphisms. Readers are invited to submit additional data. Implemented as a relational database, HMTD supports complex biological queries. Accessible through a Web browser user interface via Common Gateway Interface (CGI) and Java Database Connection (JDBC), HMTD also provides useful links and references, allowing interactive searching and downloading of data. Taking advantage of the features of an electronic journal, this paper serves as an interactive tutorial for using the database, which we expect to develop into a research tool.

<h4>Introduction</h4>Human congenital hypotransferrinemia is a rare disorder characterized by the virtual absence of transferrin in the serum. No information on the causes of the disease is known.<h4>Materials and methods</h4>Here we describe the identification of a new case, its treatment and the biochemical and genetic defects underlying the disorder.<h4>Results</h4>At diagnosis the patient had serum Tf levels equal to about 1% of the normal values. The treatment with plasma infusions each month allowed a good erythropoiesis and the prevention of iron overload with no need of red blood cell transfusions or iron chelators. In order to define the genetic basis of the disease, we performed a haplotype analysis of the Tf gene region in the 26 individuals forming the proband's family, and demonstrated that the genetic defect is located in the Tf gene and that it is inherited as a recessive trait. Protein analyses indicate that the proband serum contains two transferrin forms: one of 80 kD analogous to the normal one, and a smaller one of 50 kD, which may arise from a specific degradation or be the gene product of a modified allele.<h4>Conclusion</h4>These data suggest the presence of two Tf alleles carrying genetic defects that cause two distinct abnormalities. One allele causes low expression of an apparently normal protein that probably allowed the survival of the patient in the first years of age. The other allele produces a modified Tf with different biochemical characteristics compared to the normal one.

Although erectile function is clearly androgen dependent, is it just as clear at what level of testosterone erectile dysfunction (ED) begins? Does the decline in testosterone that occurs with aging always produce ED? Are exogenous androgens the answer to ED? The answers range from clear to complex.

No abstract available.