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We have developed yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 or YAC72) human huntingtin (htt), in a developmental- and tissue-specific manner, that is identical to endogenous htt. YAC72 mice develop selective degeneration of medium spiny projection neurons in the lateral striatum, similar to what is observed in Huntington disease. Mutant human htt expressed by YAC transgenes can compensate for the absence of endogenous htt and can rescue the embryonic lethality that characterizes mice homozygous for targeted disruption of the endogenous Hdh gene (-/-). YAC72 mice lacking endogenous htt (YAC72 -/-) manifest a novel phenotype characterized by infertility, testicular atrophy, aspermia, and massive apoptotic cell death in the testes. The testicular cell death in YAC72 -/- mice can be markedly reduced by increasing endogenous htt levels. YAC72 mice with equivalent levels of both wild-type and mutant htt (YAC72 +/+) breed normally and have no evidence of increased testicular cell death. Similar findings are seen in YAC46 -/- mice compared with YAC46 +/+ mice, in which wild-type htt can completely counteract the proapoptotic effects of mutant htt. YAC18 -/- mice display no evidence of increased cellular apoptosis, even in the complete absence of endogenous htt, demonstrating that the massive cellular apoptosis observed in YAC46 -/- mice and YAC72 -/- mice is polyglutamine-mediated toxicity from the mutant transgene. These data provide the first direct in vivo evidence of a role for wild-type htt in decreasing the cellular toxicity of mutant htt.

Decreased renal function is often a complication of hypertension. Although it has been suggested that the response of the kidney to hypertension has an underlying genetic component, there is limited information suggesting that specific genetic regions or candidate genes contribute to the variability in creatinine clearance, a commonly used measure of kidney function. As part of the Hypertension Genetic Epidemiology Network (HyperGEN) study, creatinine clearance measurements were assessed in a large biracial sample of hypertensive siblings (466 African American subjects and 634 white subjects in 215 and 265 sibships, respectively). All participants were hypertensive before the age of 60 years, and the mean age of the siblings was 52 years among the African American subjects and 61 years among the white subjects. Two residual models were created for creatinine clearance: a minimally adjusted model (which included age and age(2)) and a fully adjusted model (which included age, age(2), lean body mass, pulse rate, pulse pressure, hormone-replacement therapy, educational status, and physical activity). Standardized residuals were calculated separately for men and women in both racial groups. The heritability of the residual creatinine clearance was 17% and 18% among the African American and white subjects, respectively. We conducted multipoint variance components linkage analysis using GENEHUNTER2 and 387 anonymous markers (Cooperative Human Linkage Center screening set 8). The best evidence for linkage in African American subjects was found on chromosome 3 (LOD = 3.61 at 214.6 cM, 3q27) with the fully adjusted model, and the best evidence in white subjects was found on chromosome 3 (LOD = 3.36 at 115.1 cM) with the minimally adjusted model. Positional candidate genes that are contained in and around the region on chromosome 3 (214.6 cM) that may contribute to renal function include enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (EHHADH) and apolipoprotein D (ApoD). These findings suggest there may be genetic regions related to the variability of creatinine clearance among hypertensive individuals.

From worms to humans, recognizing and modifying a specific chromosome is essential for dosage compensation, the mechanism by which equal X-linked gene expression in males and females is achieved. Recent molecular genetic and biochemical studies have provided new insights into how regulatory factors in Drosophila are recruited and assembled on the X chromosome, leading to the essential hypertranscription of its genes.

The transferrin receptor (TfR) interacts with two proteins important for iron metabolism, transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. A second receptor for Tf, TfR2, was recently identified and found to be functional for iron uptake in transfected cells (Kawabata, H., Germain, R. S., Vuong, P. T., Nakamaki, T., Said, J. W., and Koeffler, H. P. (2000) J. Biol. Chem. 275, 16618-16625). TfR2 has a pattern of expression and regulation that is distinct from TfR, and mutations in TfR2 have been recognized as the cause of a non-HFE linked form of hemochromatosis (Camaschella, C., Roetto, A., Cali, A., De Gobbi, M., Garozzo, G., Carella, M., Majorano, N., Totaro, A., and Gasparini, P. (2000) Nat. Genet. 25, 14-15). To investigate the relationship between TfR, TfR2, Tf, and HFE, we performed a series of binding experiments using soluble forms of these proteins. We find no detectable binding between TfR2 and HFE by co-immunoprecipitation or using a surface plasmon resonance-based assay. The affinity of TfR2 for iron-loaded Tf was determined to be 27 nm, 25-fold lower than the affinity of TfR for Tf. These results imply that HFE regulates Tf-mediated iron uptake only from the classical TfR and that TfR2 does not compete for HFE binding in cells expressing both forms of TfR.

Vibrio cholerae O1, biotype El Tor, accumulates inorganic polyphosphate (poly P) principally as large clusters of granules. Poly P kinase (PPK), the enzyme that synthesizes poly P from ATP, is encoded by the ppk gene, which has been cloned from V. cholerae, overexpressed, and knocked out by insertion-deletion mutagenesis. The predicted amino acid sequence of PPK is 701 residues (81.6 kDa), with 64% identity to that of Escherichia coli, which it resembles biochemically. As in E. coli, ppk is part of an operon with ppx, the gene that encodes exopolyphosphatase (PPX). However, unlike in E. coli, PPX activity was not detected in cell extracts of wild-type V. cholerae. The ppk null mutant of V. cholerae has diminished adaptation to high concentrations of calcium in the medium as well as motility and abiotic surface attachment.

In MCF-7 breast cancer cells, hydroxytamoxifen (OH-Tam) up-regulates the estrogen receptor (ER) in a form unable to bind [(3)H]estradiol (E(2)). We show here that this property is not restricted to this antiestrogen. [(3)H]E(2) binding assays (whole cell assays, DCC assays on cell extracts) and enzyme immunoassays (Abbott) performed in parallel, establish the permanent presence of such unusual ERs in the absence of any exposure of the cells to a ligand. E(2) and the pure antiestrogen RU 58 668, which down-regulate ER, also decrease [(3)H]E(2) binding. In control cells, these ERs represent about the half of the whole receptor population; they also display a tendency to stabilize within the cell nucleus. Loss of E(2) binding ability appears irreversible, since we failed to label receptor accumulated under OH-Tam with [(3)H]E(2) or [(3)H]tamoxifen aziridine (TAZ). Cycloheximide (CHX), which blocks E(2)-induced down regulation of ER, failed to stabilize [(3)H]E(2) binding (whole cell assay) after an [(3)H]E(2) pulse (1 h), confirming that regulation of E(2) binding and peptide level are related to different regulatory mechanisms. Loss of binding ability could not be ascribed to any ER cleavage as demonstrated by Western blotting with a panel of ER antibodies raised against its various domains (67 kDa ER solely detected). We propose that loss of E(2) binding ability is related to the aging process of the receptor, i.e. it is progressively converted to a form devoted to degradation after it has accomplished its physiological role. Ligands may favor (E(2), RU 58 668) or impede (OH-Tam) this elimination process.

In the last four years there has been a major change in the approach to diagnosis of the iron overload disorder hereditary haemochromatosis (HH) following the discovery of the gene that is mutated in HH called HFE. In the first part of this review we will give a concise overview of the disease. Also the current literature on the role of HFE in iron absorption and transport at a molecular level and how mutations in HFE may lead to the break down in the regulation of iron homeostasis is reviewed. The second part of the review focuses on the molecular aspects of iron storage. Different chemical forms of storage iron deposits such as ferrihydrite and geotite are present in the iron storage proteins ferritin and haemosiderin. The type of iron storage deposits is thought to be an important factor in determining the degree of iron toxicity and tissue damage in patients with iron overload. Variations in the form of iron deposits in different types of iron overload disease e.g. HH or beta-thalessemia, the site of iron deposition and the clinical treatment used will be discussed.

The capacity of readily exchanging electrons makes iron not only essential for fundamental cell functions, but also a potential catalyst for chemical reactions involving free-radical formation and subsequent oxidative stress and cell damage. Cellular iron levels are therefore carefully regulated in order to maintain an adequate substrate while also minimizing the pool of potentially toxic 'free iron'. Iron homoeostasis is controlled through several genes, an increasing number of which have been found to contain non-coding sequences [i.e. the iron-responsive elements (IREs)] which are recognized at the mRNA level by two cytoplasmic iron-regulatory proteins (IRP-1 and IRP-2). The IRPs belong to the aconitase superfamily. By means of an Fe-S-cluster-dependent switch, IRP-1 can function as an mRNA-binding protein or as an enzyme that converts citrate into isocitrate. Although structurally and functionally similar to IRP-1, IRP-2 does not seem to assemble a cluster nor to possess aconitase activity; moreover, it has a distinct pattern of tissue expression and is modulated by means of proteasome-mediated degradation. In response to fluctuations in the level of the 'labile iron pool', IRPs act as key regulators of cellular iron homoeostasis as a result of the translational control of the expression of a number of iron metabolism-related genes. Conversely, various agents and conditions may affect IRP activity, thereby modulating iron and oxygen radical levels in different pathobiological settings. As the number of mRNAs regulated through IRE-IRP interactions keeps growing, the definition of IRPs as iron-regulatory proteins may in the near future become limiting as their role expands to other essential metabolic pathways.

Bordetella pertussis, the causative agent of whooping cough, has many well-studied virulence factors and a characteristic clinical presentation. Despite this information, it is not clear how B. pertussis interaction with host cells leads to disease. In this study, we examined the interaction of B. pertussis with a human bronchial epithelial cell line (BEAS-2B) and measured host transcriptional profiles by using high-density DNA microarrays. The early transcriptional response to this pathogen is dominated by altered expression of cytokines, DNA-binding proteins, and NFkappaB-regulated genes. This previously unrecognized response to B. pertussis was modified in similar but nonidentical fashions by the antiinflammatory agents dexamethasone and sodium salicylate. Cytokine protein expression was confirmed, as was neutrophil chemoattraction. We show that B. pertussis induces mucin gene transcription by BEAS-2B cells then counters this defense by using mucin as a binding substrate. A set of genes is described for which the catalytic activity of pertussis toxin is both necessary and sufficient to regulate transcription. Host genomic transcriptional profiling, in combination with functional assays to evaluate subsequent biological events, provides insight into the complex interaction of host and pathogen.

Screening for hereditary hemochromatosis (HHC) by means of transferrin saturation (TS) levels has been advocated and will identify many patients who are asymptomatic. The purposes of this study were (1) to determine HFE genotypes among asymptomatic HHC patients and correlate this profile with the degree of iron overload and (2) to evaluate the relationship between mobilized iron (mob Fe), age, serum ferritin (SF), and quantitative hepatic iron (QHI) in this population. One hundred twenty-three asymptomatic HHC patients were evaluated; all had quantitative phlebotomy to determine mob Fe and genotyping for C282Y and H63D mutations. Liver biopsies with QHI determinations were performed on 72 of the 123 patients. Of the entire group, 60% were homozygous for C282Y, and 13% were compound heterozygotes (C282Y/H63D). Among asymptomatic patients, the prevalence of homozygous C282Y is lower compared with previous studies that include clinically affected patients. Of those patients with more than 4 g mob Fe, 77% were homozygous C282Y. Asymptomatic patients with lower iron burdens frequently had genotypes other than homozygous C282Y. There was no correlation between age and mob Fe in these patients; however, there was a correlation between mob Fe and both SF (r = 0.68) and QHI (r = 0.75). In conclusion, asymptomatic patients with moderate iron overload had a different genotypic profile than was seen in advanced iron overload. The significance of identifying patients with modest degrees of iron loading, who may not be homozygous for C282Y, must be addressed if routine TS screening is to be implemented. (Blood. 2000;96:3707-3711)

It has been suggested that preexisting severe hepatic iron overload may adversely affect outcome after liver transplantation. The pathogenesis of iron overload in cirrhosis in the absence of hemochromatosis gene (HFE) mutations is poorly understood. The relationships between liver disease severity and etiology, degree of hepatic iron overload, and post-liver transplantation outcome were studied in 282 consecutive adult patients with cirrhosis. Thirty-seven percent of patients had stainable hepatic iron. Increased hepatic iron concentration was significantly associated with more severe liver disease (P<.001), male sex (P = .05), the presence of spur cell anemia (P<.0001), and hepatocellular liver disease (P<.0001). The HFE mutations were uncommon in patients with increased hepatic iron stores. Increased hepatic iron concentration was not associated with greater utilization of resources or a lower survival after liver transplantation. Child-Pugh score at the time of liver transplantation was the only independent variable affecting patient survival (P = .0008). In summary, our data suggest that the severity of the liver disease rather than hepatic iron concentration is the most important determinant of outcome after liver transplantation and that, in general, increasing hepatic iron concentration in cirrhosis is a surrogate marker of the severity of the underlying liver disease.

Hemochromatosis is one of the most frequent genetic diseases among the white populations, affecting one in three hundred persons. Its diagnosis has been radically transformed by the discovery of the HFE gene. In a given individual, the diagnosis can, from now on, be ascertained on the sole association of a plasma transferrin saturation (TS) over 45% and homozygosity for the C282Y mutation. Liver biopsy is only required to search for cirrhosis whenever there is hepatomegaly and/or serum ferritin >1000 ng/ml and/or elevated serum AST. Family screening is mandatory, primarily centered on the siblings. The treatment remains based on venesection therapy which improves many features of the disease (one of the most refractory, however, being the joint signs) and permits normal life expectancy provided the diagnosis is established prior to the development of cirrhosis or of insulin-dependent diabetes. In view of the prevalence, the non-invasive diagnosis, the spontaneous severity and the efficacy of a very simple therapy, hemochromatosis should benefit from population screening. This screening could be based, first, on the assessment of transferrin saturation, followed - when elevated - by the search for the C282Y mutation. The discovery of the HFE gene has also paved the road for the individualization of other types of iron overload syndromes which are not HFE-related.

The dosage compensation machinery of Caenorhabditis elegans is targeted specifically to the X chromosomes of hermaphrodites (XX) to reduce gene expression by half. Many of the trans-acting factors that direct the dosage compensation machinery to X have been identified, but none of the proposed cis-acting X chromosome-recognition elements needed to recruit dosage compensation components have been found. To study X chromosome recognition, we explored whether portions of an X chromosome attached to an autosome are competent to bind the C. elegans dosage compensation complex (DCC). To do so, we devised a three-dimensional in situ approach that allowed us to compare the volume, position, and number of chromosomal and subchromosomal bodies bound by the dosage compensation machinery in wild-type XX nuclei and XX nuclei carrying an X duplication. The dosage compensation complex was found to associate with a duplication of the right 30% of X, but the complex did not spread onto adjacent autosomal sequences. This result indicates that all the information required to specify X chromosome identity resides on the duplication and that the dosage compensation machinery can localize to a site distinct from the full-length hermaphrodite X chromosome. In contrast, smaller duplications of other regions of X appeared to not support localization of the DCC. In a separate effort to identify cis-acting X recognition elements, we used a computational approach to analyze genomic DNA sequences for the presence of short motifs that were abundant and overrepresented on X relative to autosomes. Fourteen families of X-enriched motifs were discovered and mapped onto the X chromosome.

It has been hypothesized that R-type Ca currents result from the expression of the alpha(1E) gene. To test this hypothesis we examined the properties of voltage-dependent Ca channels in mice in which the alpha(1E) Ca channel subunit had been deleted. Application of omega-conotoxin GVIA, omega-agatoxin IVA, and nimodipine to cultured cerebellar granule neurons from wild-type mice inhibited components of the whole-cell Ba current, leaving a "residual" R current with an amplitude of approximately 30% of the total Ba current. A minor portion of this R current was inhibited by the alpha(1E)-selective toxin SNX-482, indicating that it resulted from the expression of alpha(1E). However, the majority of the R current was not inhibited by SNX-482. The SNX-482-sensitive portion of the granule cell R current was absent from alpha(1E) knock-out mice. We also identified a subpopulation of dorsal root ganglion (DRG) neurons from wild-type mice that expressed an SNX-482-sensitive component of the R current. However as with granule cells, most of the DRG R current was not blocked by SNX-482. We conclude that there exists a component of the R current that results from the expression of the alpha(1E) Ca channel subunit but that the majority of R currents must result from the expression of other Ca channel alpha subunits.

Specificity in the actions of different modulatory neurons is often attributed to their having distinct cotransmitter complements. We are assessing the validity of this hypothesis with the stomatogastric nervous system of the crab Cancer borealis. In this nervous system, the stomatogastric ganglion (STG) contains a multifunctional network that generates the gastric mill and pyloric rhythms. Two identified projection neurons [modulatory proctolin neuron (MPN) and modulatory commissural neuron 1 (MCN1)] that innervate the STG and modulate these rhythms contain GABA and the pentapeptide proctolin, but only MCN1 contains Cancer borealis tachykinin-related peptide (CabTRP Ia). Selective activation of each projection neuron elicits different rhythms from the STG. MPN elicits only a pyloric rhythm, whereas MCN1 elicits a distinct pyloric rhythm as well as a gastric mill rhythm. We tested the degree to which CabTRP Ia distinguishes the actions of MCN1 and MPN. To this end, we used the tachykinin receptor antagonist Spantide I to eliminate the actions of CabTRP Ia. With Spantide I present, MCN1 no longer elicited the gastric mill rhythm and the resulting pyloric rhythm was changed. Although this rhythm was more similar to the MPN-elicited pyloric rhythm, these rhythms remained different. Thus, CabTRP Ia partially confers the differences in rhythm generation resulting from MPN versus MCN1 activation. This result suggests that different projection neurons may use the same cotransmitters differently to elicit distinct pyloric rhythms. It also supports the hypothesis that different projection neurons use a combination of strategies, including using distinct cotransmitter complements, to elicit different outputs from the same neuronal network.

The prognostic value of disseminated tumour cells derived from 353 breast cancer patients was evaluated. Disseminated tumour cells were purified from blood using a newly established method and nucleic acids were subsequently isolated. We investigated genomic imbalances (GI) such as mutation, amplification and loss of heterozygosity of 13 tumour suppressor genes and 2 proto-oncogenes using DNA from isolated minimal residual cancer cells. Significant correlations were found between genomic alterations of the DCC - and c-erbB-2 genes in disseminated breast cancer cells and actuarial relapse-free survival. Furthermore, increasing numbers of genomic imbalances measured in disseminated tumour cells were significantly associated with worse prognosis of recurrent disease. Logistic regression and Cox multivariate analysis led to the identification of genomic imbalances as an independent prognostic factor. Determination of disseminated tumour cells by genotyping of oncogenes and tumour suppressor genes seems not only to be a useful adjunct in follow up of carcinoma patients but provides also valuable additional individualized prognostic and predictive information in breast cancer patients beyond the TNM system.

We recently discovered an emerging neonatal infectious disease, neonatal toxic shock syndrome-like (TSS-like) exanthematous disease (NTED), which is induced by a superantigen, TSS toxin-1 (TSST-1), produced by methicillin-resistant Staphylococcus aureus (MRSA). Here, we analyzed the activation and the response of TSST-1-reactive Vss2(+) T cells in NTED patients during the acute and recovery phases and in asymptomatic infants exposed to MRSA. In the acute phase, Vss2(+) T cells were anergic to stimulation with TSST-1 and underwent marked expansion, but by 2 months after disease onset, their numbers had declined to about 10% of the control level. Although the percentage of Vss2(+) T cells in the ten asymptomatic neonatal MRSA carriers was within the control range, these individuals could be divided into two groups on the basis of Vss2(+) T-cell activation. Vss2(+)CD4(+) T cells from three of these infants (Group 1) highly expressed CD45RO and were anergic to TSST-1, whereas in the other seven asymptomatic neonatal MRSA carriers (Group 2), these cells expressed CD45RO at the control level and were highly responsive to stimulation with TSST-1. The serum anti-TSST-1 IgG Ab titer was negligible in the four NTED patients in the acute phase and the three asymptomatic neonatal MRSA carriers in Group 1, but it was high in the seven asymptomatic carriers in Group 2. We suggest that maternally derived anti-TSST-1 IgGs helps to suppress T-cell activation by TSST-1 and protects infants from developing NTED.

<h4>Purpose</h4>To review information on the use of laboratory tests in screening, diagnosis, and monitoring of acute and chronic hepatic injury.<h4>Data sources and study selection</h4>A MEDLINE search was performed for key words related to hepatic diseases, including acute hepatitis, chronic hepatitis, alcoholic hepatitis, cirrhosis, hepatocellular carcinoma, and etiologic causes. Abstracts were reviewed, and articles discussing use of laboratory tests selected for review. Additional articles were selected from the references. Guideline Preparation and Review: Drafts of the guidelines were posted on the Internet, presented at the AACC Annual Meeting in 1999, and reviewed by experts. Areas requiring further amplification or literature review were identified for further analysis. Specific recommendations were made based on analysis of published data and evaluated for strength of evidence and clinical impact.<h4>Recommendations</h4>Although many specific recommendations are made in the guidelines, only some summary recommendations are listed here. In acute hepatic injury, prothrombin time and, to a lesser extent, total bilirubin are the best indicators of severity of disease. Although ALT is useful for detecting acute and chronic hepatic injury, it is not related to severity of acute hepatic injury and only weakly related to severity of chronic hepatic injury. Specific tests of viral markers should be the initial differential tests in both acute and chronic hepatic injury; when positive, they are also useful for monitoring recovery from hepatitis B and C.

Genetic causes of hereditary hemochromatosis (HH) include mutations in the HFE gene, a ss2-microglobulin (ss2m)-associated major histocompatibility complex class I-like protein. Accordingly, mutant ss2m(-/-) mice have increased intestinal iron absorption and develop parenchymal iron overload in the liver. In humans, other genetic and environmental factors have been suggested to influence the pathology and severity of HH. Previously, an association has been reported between low numbers of lymphocytes and the severity of clinical expression of the iron overload in HH. In the present study, the effect of a total absence of lymphocytes on iron overload was investigated by crossing ss2m(-/-) mice (which develop iron overload resembling human disease) with mice deficient in recombinase activator gene 1 (Rag1), which is required for normal B and T lymphocyte development. Iron overload was more severe in ss2mRag1 double-deficient mice than in each of the single deficient mice, with iron accumulation in parenchymal cells of the liver, in acinar cells of the pancreas, and in heart myocytes. With increasing age ss2mRag1(-/-) mice develop extensive heart fibrosis, which could be prevented by reconstitution with normal hematopoietic cells. Thus, the development of iron-mediated cellular damage is substantially enhanced when a Rag1 mutation, which causes a lack of mature lymphocytes, is introduced into ss2m(-/-) mice. Mice deficient in ss2m and Rag1 thus offer a new experimental model of iron-related cardiomyopathy.

<h4>Background and purpose</h4>Serum ferritin and heterozygosity for the C282Y mutation of the hemochromatosis gene have both been associated with an increased risk of cardiovascular events. The purpose of the study was to test whether either is a risk predictor for asymptomatic carotid atherosclerosis.<h4>Methods</h4>We assessed carotid intima-media wall thickness (IMT) and focal plaque formation by high-resolution B-mode ultrasound, conventional risk factors, serum ferritin levels, and the C282Y mutation of the hemochromatosis gene in a randomly selected community population of 1098 subjects (545 women and 553 men) aged 27 to 77 years.<h4>Results</h4>After adjustment for conventional risk factors, serum ferritin was not associated with carotid mean IMT. Women with ferritin values over the first quartile (>34 microg/L) had an adjusted odds ratio of 2.1 (95% CI, 1. 3 to 3.4; P:=0.0016) for carotid plaque compared with the first quartile. Ferritin was not associated with carotid plaque in men. Subjects who were heterozygous for the C282Y mutation constituted 11. 4% of the population, and there was no independent association of this genotype with either carotid IMT or focal plaque formation.<h4>Conclusions</h4>We conclude that in our community population, C282Y genotype status was not a risk predictor for either carotid mean IMT or plaque formation. Serum ferritin values in women were independently associated with carotid plaque.

The enterocyte is a highly specialized cell of the duodenal epithelium that coordinates iron uptake and transport into the body. Until recently, the molecular mechanisms underlying iron absorption and iron homeostasis have remained a mystery. This review focuses on the proteins and regulatory mechanisms known to be present in the enterocyte precursor cell and in the mature enterocyte. The recent cloning of a basolateral iron transporter and investigations into its regulation provide new insights into possible mechanisms for iron transport and homeostasis. The roles of proteins such as iron regulatory proteins, the hereditary hemochromatosis protein (HFE)-transferrin receptor complex, and hephaestin in regulating this transporter and in regulating iron transport across the intestinal epithelium are discussed. A speculative, but testable, model for the maintenance of iron homeostasis, which incorporates the changes in the iron-related proteins associated with the life cycle of the enterocyte as it journeys from the crypt to the tip of the villous is proposed.

Phosphatidylcholine transfer protein (PC-TP) is a 214-amino acid cytosolic protein that promotes intermembrane transfer of phosphatidylcholines, but no other phospholipid class. To probe mechanisms for membrane interactions and phosphatidylcholine binding, we expressed recombinant human PC-TP in Escherichia coli using a synthetic gene. Optimization of codon usage for bacterial protein translation increased expression of PC-TP from trace levels to >10% of the E. coli cytosolic protein mass. On the basis of secondary structure predictions of an amphipathic alpha-helix (residues 198-212) in proximity to a hydrophobic alpha-helix (residues 184-193), we explored whether the C-terminus might interact with membranes and promote binding of phosphatidylcholines. Consistent with this possibility, truncation of five residues from the C-terminus shortened the predicted amphipathic alpha-helix and decreased PC-TP activity by 50%, whereas removal of 10 residues eliminated the alpha-helix, abolished activity, and markedly decreased the level of membrane binding. Circular dichroic spectra of synthetic peptides containing one ((196-214)PC-TP) or both ((183-214)PC-TP) predicted C-terminal alpha-helices in aqueous buffer were most consistent with random coil structures. However, both peptides adopted alpha-helical configurations in the presence of trifluoroethanol or phosphatidylcholine/phosphatidylserine small unilamellar vesicles. The helical content of (196-214)PC-TP increased in proportion to vesicle phosphatidylserine content, consistent with stabilization of the alpha-helix at the membrane surface. In contrast, the helical content of (183-214)PC-TP was not influenced by vesicle composition, implying that the more hydrophobic of the alpha-helices penetrated into the membrane bilayer. These studies suggest that tandem alpha-helices located near the C-terminus of PC-TP facilitate membrane binding and extraction of phosphatidylcholines.

Hereditary hemochromatosis is related to mutations of the HFE gene. The role of the S65C mutation of this gene was evaluated in a Spanish population, consisting of 100 controls and 41 patients who had resulted positive to screening for iron overload. Only one patient was heterozygous for the S65C mutation, so the S65C mutation is infrequent in our area. Nevertheless, it is advisable to search for this mutation in cases with iron overload and heterozygosity for the C282Y or H63D mutations of the HFE gene.

Clusters of olfactory receptor (OR) genes are found on most human chromosomes. They are one of the largest mammalian multigene families. Here, we report a systematic study of polymorphism of OR genes belonging to the largest fully sequenced OR cluster. The cluster contains 36 OR genes, of which two belong to the vomeronasal 1 (V1-OR) family. The cluster is divided into a major and a minor region at the telomeric end of the HLA complex on chromosome 6. These OR genes could be involved in MHC-related mate preferences. The polymorphism screen was carried out with 13 genes from the HLA-linked OR cluster and three genes from chromosomes 7, 17, and 19 as controls. Ten human cell lines, representing 18 different chromosome 6s, were analyzed. They were from various ethnic origins and exhibited different HLA haplotypes. All OR genes tested, including those not linked to the HLA complex, were polymorphic. These polymorphisms were dispersed along the coding region and resulted in up to seven alleles for a given OR gene. Three polymorphisms resulted either in stop codons (genes hs6M1-4P, hs6M1-17) or in a 16-bp deletion (gene hs6M1-19P), possibly leading to lack of ligand recognition by the respective receptors in the cell line donors. In total, 13 HLA-linked OR haplotypes could be defined. Therefore, allelic variation appears to be a general feature of human OR genes.

Hyalinizing trabecular tumor (HTT) of the thyroid is a neoplasm of follicular derivation with a histogenesis that is still the subject of debate. Morphologic affinities between HTT and papillary carcinoma, including nuclear pseudoinclusions and grooves, suggest that these tumors may be of similar origin. The authors investigated the relationship between these two types of tumors by assessing HTT for the presence of rearrangements of the proto-oncogene rearranged during transfection (RET) that, in thyroid tumors, are specific for papillary carcinoma. A series of 14 HTTs, including two cases associated with classic papillary carcinoma, was studied by means of immunohistochemistry and reverse transcription-polymerase chain reaction. Seven follicular adenomas with focal hyalinized trabecular areas served as control cases. Three of the 14 HTT cases under consideration displayed rearrangements of RET generating the RET/papillary thyroid carcinoma type 1 (PTC1) oncogene. In another case, RET expression was detected focally by immunohistochemistry alone. Finally, in one mixed HTT-papillary carcinoma sample, RET/PTC1 expression was detected, but only in the papillary component. None of the control follicular adenomas contained rearrangements of RET/PTC. These findings demonstrate that a comparable percentage (28.6%) of HTTs and papillary carcinomas exhibit the same RET proto-oncogene alterations. Thus, HTT may represent the "hyalinizing trabecular" variant of papillary carcinoma rather than a separate entity.

The term apoptosis often has been used interchangeably with the term programmed cell death. Here we describe a form of programmed cell death that is distinct from apoptosis by the criteria of morphology, biochemistry, and response to apoptosis inhibitors. Morphologically, this alternative form of programmed cell death appears during development and in some cases of neurodegeneration. Despite its lack of response to caspase inhibitors and Bcl-x(L), we show that this form of cell death is driven by an alternative caspase-9 activity that is Apaf-1-independent. Characterization of this alternative form of programmed cell death should lead to new insight into cell death programs and their roles in development and degeneration.

Previously we obtained modest linkage evidence implicating 17q11. 1-12 in bipolar disorder. A modified genome screen, based on gene-rich regions, on a collection of Irish sib-pair nuclear families revealed excess allele sharing at markers flanking the gene encoding the serotonin transporter (5-HTT; hSERT). Here we describe a study designed to combine the advantages of family-based association studies with the consideration of multiple polymorphic markers within a candidate gene. Ninety-two Irish families, with a total of 106 proband-parent trios, have been genotyped for 3 previously known polymorphisms within hSERT (5-HTTLPR, intron 2 VNTR, and 3' UTR G/T). Data from two and three polymorphic marker haplotypes revealed a number of marker combinations that showed evidence supportive of association; the most significant being for polymorphisms 5-HTTLPR and 3' UTR G/T (global chi(2), 12.91, df 3, P = 0.005). In addition, modest evidence of association also was observed for 5-HTTLPR alone. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:845-849, 2000.

Diagnosis of inherited diseases or cancer predispositions frequently involves determination of specific mutations or polymorphisms. The number of characterized monogenetic and polygenetic diseases is significantly rising every year. As a result, an increasing number of patient samples with a rising complexity of genetic diseases require molecular diagnostics. In order to apply genetic analyses to large groups of patients or population screening, automation of a sensitive and precise method is highly desirable. Capillary electrophoresis (CE) facilitates the development of methods which can rapidly process large number of patient samples in an automated fashion. In contrast, conventional techniques including Southern blotting, sequencing or standard gel electrophoresis are time consuming, cost ineffective and require substantial amounts of each specimen. Robustness, ease of operation, good reproducibility and low cost are the main advantages of CE. Currently, most protocols adapted to automated CE represent (i) analyses of DNA fragment length or DNA restriction patterns (RFLP), (ii) analyses of single-strand conformation polymorphism (SSCP) and (iii) microsatellite analyses. Recently, automated detection of variations in the FRAXA (CGG)n region (fragile X syndrome), LDL receptor gene, p53 gene, MTHFR (methylenetetrahydrofolate reductase) gene, HFE gene and others has been established on CE systems. These applications clearly demonstrate the suitability of CE for high throughput screening in medical applications.

Severe iron overload has been reported in patients with the beta-thalassaemia trait. Studies performed before the discovery of the haemochromatosis gene (HFE) have yielded conflicting results: some suggest that iron overload might arise from the interaction of the beta-thalassaemia trait with heterozygosity for haemochromatosis, some with homozygosity for haemochromatosis and others that it was unrelated to haemochromatosis. We have studied the clinical phenotype, iron indices and HFE genotypes of 22 unrelated patients with the beta-thalassaemia trait and haemochromatosis, the inheritance of chromosome 6p and 1q haplotypes in families of non-homozygous C282Y probands and serum measures of iron status in relatives heterozygous for C282Y with or without the beta-thalassaemia trait. We demonstrate that the beta-thalassaemia trait aggravates the clinical picture of C282Y homozygotes, favouring higher rates of iron accumulation and the development of severe iron-related complications. We suggest that the coexistence of the beta-thalassaemia trait might also increase the risk of iron overload in patients with HFE genotypes at a mild risk of haemochromatosis. Our findings do not support the hypothesis that the association of the beta-thalassaemia trait with a single C282Y or H63D allele might lead to iron overload and suggest that other non-HFE-related inherited factors are present in haemochromatosis patients with incomplete HFE genotypes.

INTRODUCTION: Systemic and topical oestrogen can provoke squamous metaplasia of epithelium. In Hereditary Haemorrhagic Telangiectasia (HTT) the underlying telangiectasia may be protected from trauma and epistaxis reduced. Oestrogens have been advocated but their efficacy is unclear.1 Recent advances have now identified two oestrogen and one progesterone receptors. The aim of this study is to analyse the sex receptor status of HHT nasal mucosa to determine if oestrogen therapy is biochemically justified. METHOD: Five HHT patients (three men, two women) and eight controls (four men, four women) underwent nasal mucosa biopsy. Samples were fixed in formalin and paraffin embedded. Alpha oestrogen (ERalpha ), beta oestrogen (Erbeta) and progesterone receptors (PgR) receptors wre identified using mouse monoclonal antibodies by the Streptavidin-biotin peroxidase method. RESULTS: Erbeta was detected in two HHT patients (one man, one woman) and two control patients. ERalpha and PgR was absent in HHT patients. CONCLUSION: This pilot study demonstrated that a subgroup of HHT patients is Erbeta positive. Oestrogen theraphy therefore has a potential therapeutic role on a biochemical basis in these patients. Erbeta status should be determined before considering oestrogen therapy.

Involvement of arachidonic acid cyclooxygenase (COX) and lipoxygenase (LOX) metabolites in platelet aggregation and coagulation induced by two varieties of cancer cells of murine transplantable tumors was studied. A lung alveolar carcinoma (LAC) and a fibrosarcoma (FS), induced platelet aggregation and plasma coagulation (P<0.05). Pretreatment of both tumor lines with a COX inhibitor did not block the tumor cell induced platelet aggregation (TCIPA). COX [12(S)-HTT] and LOX [12(S)-HETE], metabolites of washed platelets (WP), alone or co-incubated with LAC or FS cells, were analyzed. We observed higher 12(S)-HETE release with respect to 12(S)HHT when WP were co-incubated with LAC cells. With both neoplastic cell (NC) lines prothrombin time (PT) was shortened. Pretreatment of NC with iodoacetic acid, soybean trypsin inhibitor or Factor X-deficient plasma increased the PT. These results indicate that AA metabolites play a role on the procoagulation and platelet aggregation induced by mesenchymal and epithelial murine cancers.

Serotonin (5-hydroxytryptamine, 5-HT) appears to play a role in the pathophysiology of a range of neuropsychiatric disorders, and serotonergic agents are of central importance in neuropharmacology. Genes encoding various components of the 5-HT system are being studied as risk factors in depression, schizophrenia, obsessive-compulsive disorder, aggression, alcoholism, and autism. Recently, pharmacogenetic research has begun to examine possible genetic influences on therapeutic response to drugs affecting the serotonin system. Genes regulating the synthesis (TPH), storage (VMAT2), membrane uptake (HTT), and metabolism (MAOA) of 5-HT, as well as a number of 5-HT receptors (HTR1A, HTR1B, HTR2A, HTR2C, and HTR5A), have been studied and this initial research is reviewed here. After a brief introduction to serotonin neurobiology and a general discussion of appropriate genetic methodology, each of the major 5-HT-related genes and their encoded proteins are reviewed in turn. For each gene, relevant polymorphisms and research on functional variants are discussed; following brief reviews of the disorder or trait association and linkage studies, pharmacogenetic studies performed to date are covered. The critical and manifold roles of the serotonin system, the great abundance of targets within the system, the wide range of serotonergic agents-available and in development-and the promising preliminary results suggest that the serotonin system offers a particularly rich area for pharmacogenetic research.

Huntington's disease is characterised by the death of cortical and striatal neurons, and is the result of an expanded polyglutamine tract in the Huntingtin protein [1]. Huntingtin is present on both endocytic and secretory membrane organelles but its function is unclear [2,3]. Rab GTPases regulate both of these transport pathways [4]. We have previously shown that Rab8 controls polarised membrane transport by modulating cell morphogenesis [5]. To understand Rab8-mediated processes, we searched for Rab8-interacting proteins by the yeast two-hybrid system. Here, we report that Huntingtin is linked to the Rab8 protein through FIP-2, a tumour necrosis factor-alpha (TNF-alpha)-inducible coiled-coil protein related to the NEMO protein [6,7]. The activated form of Rab8 interacted with the amino-terminal region of FIP-2, whereas dominant-negative Rab8 did not. Huntingtin bound to the carboxy-terminal region of FIP-2. Coexpressed FIP-2 and Huntingtin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures, and FIP-2 promoted cell polarisation in a similar way to Rab8. We propose a model in which Huntingtin, together with FIP-2 and Rab8, are part of a protein network that regulates membrane trafficking and cellular morphogenesis.

Chronic iron (Fe) overload is associated with a marked increase in renal tissue iron content and injury. It is estimated that 10% of the American population carry the gene for hemochromatosis and 1% actually suffer from iron overload. The mechanism of iron overload-associated renal damage has not been fully elucidated. Iron can accelerate lipid peroxidation leading to organelle membrane dysfunction and subsequent cell injury/death. Iron-catalyzed generation of reactive oxygen species (ROS) is responsible for initiating the peroxidatic reaction. We investigated the possible association of oxidative stress and its impact on nitric oxide (NO) metabolism in iron-overload-associated renal injury. Rats were randomized into Fe-loaded (given 0.5 g elemental iron/kg body weight as iron dextran; i.v.), Fe-depleted (given an iron-free diet for 20 weeks), and control groups. Renal histology, tissue expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS), renal tissue expression of nitrotyrosine, plasma, and renal tissue lipid peroxidation product, malondialdehyde (MDA), and plasma and urinary NO metabolites (NOx) were examined. Iron overload was associated with mild proteinuria, tissue iron deposition together with significant glomerulosclerosis, tubular atrophy, and interstitial fibrosis. Rare focal glomerulosclerosis and tubulointerstitial changes were noted in normal controls. No renal lesions were observed in Fe-depleted rats. Iron deposits were seen in glomeruli, proximal tubules, and interstitium. The iron staining in the distal tubules was negligible. Both plasma and renal tissue MDA and renal tissue nitrotyrosine were increased significantly in Fe-loaded rats compared with control rats. In contrast, Fe-depleted animals showed a marked reduction in plasma and renal tissue MDA and nitrotyrosine together with significant elevation of urinary NOx excretion. In addition, iron-overload was associated with up-regulation of renal eNOS and iNOS expressions when compared with the control and Fe-depleted rats that showed comparable values. In conclusion, chronic iron overload resulted in iron deposition in the glomeruli and proximal tubules with various renal lesions and evidence of increased ROS activity, enhanced ROS-mediated inactivation, and sequestration of NO and compensatory up-regulation of renal eNOS and iNOS expressions. However, iron depletion was associated with reduced MDA and tissue nitrotyrosine abundance, increased urinary NOx excretion, normal nitric oxide synthase (NOS) expression, and absence of renal injury. These findings point to the possible role of ROS in chronic iron overload-induced renal injury.

HFE, the protein that is mutated in hereditary haemochromatosis, binds to the transferrin receptor (TfR). Here we show that wild-type HFE and TfR localize in endosomes and at the basolateral membrane of a polarized duodenal epithelial cell line, whereas the primary haemochromatosis HFE mutant, and another mutant with impaired TfR-binding ability accumulate in the ER/Golgi and at the basolateral membrane, respectively. Levels of the iron-storage protein ferritin are greatly reduced and those of TfR are slightly increased in cells expressing wild-type HFE, but not in cells expressing either mutant. Addition of an endosomal-targeting sequence derived from the human low-density lipoprotein receptor (LDLR) to the TfR-binding-impaired mutant restores its endosomal localization but not ferritin reduction or TfR elevation. Thus, binding to TfR is required for transport of HFE to endosomes and regulation of intracellular iron homeostasis, but not for basolateral surface expression of HFE.

Molecular analysis of hereditary nonpolyposis colorectal carcinomas (HNPCC) has identified DNA mismatch repair deficiencies with resulting microsatellite instability (MSI) as a pathway of carcinogenesis that appears to be relevant for prognosis, treatment, and possibly prevention. In this study, expression of cell cycle proteins and other known prognostic markers is correlated with the microsatellite status of colorectal cancers (CRC). One hundred consecutive cases from the CRC Registry at Thomas Jefferson University were analyzed for MSI. Immunohistochemistry was performed for the mismatch repair proteins hMLH1 and hMSH2, tumor suppressor p53, apoptosis inhibitor bcl-2, cell cycle proteins p21(WAF1/CIP1), and p27 and the proliferation markers Ki-67 and topoisomerase II. High MSI (MSI-H) is significantly correlated with loss of either hMLH1 or hMSH2, presence of bcl-2, and absence of p53. p21(WAF1/CIP1) is positive in all tumors with MSI-H. Previous findings of a lower proliferation rate were confirmed with a topoisomerase II stain. Microsatellite stable (MSS) tumors generally express both MSH2 and MLH1. Other highly significant differences are positive p53 in 56% of MSS cases and negative bcl-2 in 98% of MSS cases. p27 expression is found in approximately 50% of all CRCs irrespective of the microsatellite status. MSI-H tumors follow the mutator pathway, with loss of expression of one mismatch repair protein, wild-type p53, lower proliferation, and positivity for p21(WAF1/CIP1). MSS tumors follow the suppressor pathway, characterized by p53 overexpression, higher proliferation, and absence of bcl-2 expression; p21(WAF1/CIP1) expression can be variable. These data provide a molecular basis for the clinical observation that patients with HNPCC appear to have a more favorable prognosis. HUM PATHOL 31:1506-1514.

<h4>Objective</h4>Porphyria cutanea tarda (PCT) is commonly associated with iron overload and hepatitis C virus (HCV) infection. Association between hemochromatosis C282Y or H63D mutations and PCT has been observed, although not uniformly, and iron overload is also commonly found in chronic HCV hepatitis. The aim of the present study was to investigate the frequency of C282Y and H63D mutations and HCV infection in Brazilian patients with PCT and their relationship with iron overload.<h4>Methods</h4>Twenty-three patients (19 men) aged 39.6 +/- 11.1 yr were studied. All had dermatological lesions of PCT and high levels of urinary uroporphyrin. HCV infection and iron overload were investigated. DNA samples were analyzed for the presence of HFE mutations.<h4>Results</h4>The frequency of C282Y was significantly higher in PCT patients than in 278 healthy individuals (17.4% vs 4%, odds ratio = 5.1, 95% confidence interval 1.5-17.6, p = 0.02), whereas no difference was observed regarding the H63D mutation (30.4% vs 31%, odds ratio = 1, 95% confidence interval 0.4-2.4, p = 1). Biochemical tests in PCT patients showed iron overload with transferrin saturation = 47.3 +/- 20.7% and ferritin = 566.8 +/- 425 ng/ml. Fifteen of 23 (65.2%) patients had HCV infection and alcohol ingestion was observed in 17 of 23 (73.9%).<h4>Conclusions</h4>PCT patients exhibited evidence of iron overload, a high frequency of HCV, and an association with C282Y mutation. These data further support the notion that both acquired and inherited factors contribute to the occurrence of PCT, and indicate that screening for C282Y may be justified in PCT patients.

A synthetic pentasaccharide, containing an intact antithrombin III (ATIII) binding site that is in clinical studies a specific antifactor Xa agent, serves as a substrate for a heparin lyase (heparinase I, EC 4.2.2.7) from Flavobacterium heparinum. Heparinase I, currently being assessed as a heparin reversal agent, also reverses the antifactor Xa activity of this synthetic pentasaccharide by breaking it down to inactive disaccharide and trisaccharide products.

<h4>History and clinical findings</h4>A 24 year old yugoslavian father of two children, complained of decreased libido and impotence since seven months. He also described recurrent joint pains in the knees and wrist joints. The urological and internal examination was unremarkable.<h4>Investigations</h4>Except for slightly elevated liver enzymes and a mild thrombocytopenia the laboratory tests were normal. Testosteron, follicle stimulating hormone and luteinizing hormone concentrations were markedly decreased. Hepatosplenomegaly was demonstrated by ultrasound.<h4>Treatment and course</h4>During testosteron administration for hypogonadotrophic hypogonadism erectile dysfunction improved. 9 months later the patient became diabetic and was referred to our department. Hemochromatosis was confirmed by serum ferritin concentration of 4010 micrograms/l, transferrin saturation of 85% and hepatic iron concentration of 27,900 micrograms/g dry weight. Molecular genetics showed no mutation of the hemochromatosis gene HFE. After venesection the ferritin concentration decreased, the loss of libido and subfertility improved with testosterone administration.<h4>Conclusion</h4>In subfertility from an endocrine disorder primary hemochromatosis should be considered in the differential diagnosis. Only early diagnosis and prompt iron depletion may improve the prognosis of these patients.

Abnormalities of serotonin synthesis and metabolism may be associated with suicidality. The serotonin transporter gene (5-HTT) is one of the important genes involved in the regulation of serotonin neurotransmission. We examined the association of suicidal behavior in Chinese schizophrenic patients with a functional polymorphism of the promoter region of the 5-HTT gene (5-HTTLPR). The 5-HTTLPR genotype was determined by polymerase chain reaction for 76 suicidal and 262 non-suicidal patients with a diagnosis of schizophrenia (DSM-IV criteria). All subjects were unrelated to each other, and all were Chinese. There was no significant genotypic or allelic association of the 5-HTTLPR polymorphism with history of attempted suicide. From our results, this 5-HTTLPR polymorphism is unlikely to have a major effect on suicidal behavior in Chinese patients with schizophrenia.

<h4>Background</h4>A bioelectronic detection platform has recently been developed that facilitates the detection and characterization of nucleic acids. The DNA chip platform is compatible with homogeneous assays because separate labeling and wash steps are not required. A one-step, bioelectronic detection assay was developed to genotype patient samples with respect to the H63D polymorphism of the Hfe gene, associated with hereditary hemochromatosis.<h4>Methods and results</h4>Electrode arrays were modified with DNA capture probes that were perfectly matched to the wild-type or mutant allele of H63D. Amplicons containing the polymorphic site were hybridized with the capture probes on the electrode arrays in the presence of electronically labeled reporter (signaling) probes. Voltammetric analysis of the electrode arrays was conducted first at ambient temperature and then at elevated temperature. The electronic signal was preferentially diminished at elevated temperature from electrodes that hybridized with mismatched target amplicons.<h4>Conclusion</h4>An assay for bioelectronic genotyping of the H63D polymorphism was developed and used with six patient specimens to show the feasibility of this system as a model for point mutation detection.

Translocations involving the MLL gene on chromosome 11q23 occur in 5-10% of human leukemias, and involve fusion with more than 30 different partner genes. The MLL-AF10 fusion produced by the t(10;11)(p12;q23) or ins(10;11)(p12;q23q13) occurs in a small percentage of acute leukemias, most commonly acute myelogenous leukemia (AML) of the M5 FAB subtype. We report two cases of AML (M5a and M0) and one case of acute lymphoblastic leukemia containing MLL-AF10 fusion. Each case had varied clinical characteristics, despite expressing similar MLL-AF10 fusion transcripts. Including the three cases described in this report, we identified a total of 38 cases of leukemia with MLL-AF10 fusion. Approximately one-third of these are not M5 AML. Taken together, these findings emphasize that while the sentinel molecular event may be identical in a disease, the clinical presentation and outcome can vary widely.

<h4>Background</h4>Activated neutrophils play an important role in the pathogenesis of renal injury in humans and in experimental models of hemolytic uremic syndrome (HUS). To evaluate the clinical significance of the circulating granulocyte elastase-alpha1-proteinase inhibitor complex (GEPIC), which is a marker of neutrophil activation, we investigated the plasma concentrations of GEPIC in children with hemolytic uremic syndrome (HUS) associated with verotoxin-producing Escherichia coli (VTEC), VTEC gastroenteritis without HUS and in normal controls.<h4>Methods</h4>Of 22 children (1-19 years of age; mean age 5.5 years) with VTEC infection, nine were diagnosed with HUS. Plasma GEPIC, soluble thrombomodulin (sTM) and thrombin-antithrombin-III complex (TAT) levels were measured by ELISA.<h4>Results</h4>The number of polymorphonuclear leukocytes and the levels of plasma GEPIC in patients with HUS were significantly higher than those in non-HUS (9850+/-5091 vs. 5278+/-3327 /microL, P<0.05; 432.1+/-211.7 vs. 188.3+/-117.0 ng/mL, P<0.01) or control subjects (9850+/-5091 vs. 4728+/-1977 /microL, P<0.05; 432.1+/-211.7 vs. 105.9+/-51.1 ng/mL, P<0.001). Furthermore, plasma GEPIC levels showed a positive correlation with sTM (r = 0.522; P<0.01), a marker of endothelial cell injury, and TAT (r = 0.594; P<0.01), a marker of thrombin activity.<h4>Conclusions</h4>These results suggest that an increase in circulating GEPIC levels in patients with VTEC-associated HUS may be related to endothelial injury, which may possibly lead to a more severe episode of this disease.

Adenocarcinoma of the gastroesophageal junction is rapidly rising in incidence. It has been proposed that these tumors be classified as three different types: distal esophageal (AEG I), cardia (AEG II), and subcardia (AEG III). Using comparative genomic hybridization (CGH) analysis, one recent study reported that the 14q chromosomal arm showed a significantly higher rate of deletion in esophageal than in cardia adenocarcinoma. Using a microsatellite analysis technique, we analyzed this area and regions in the vicinity of the APC, DCC, and p53 genes. Tumor and normal tissues were microdissected from 54 cases (27 AEG I and 27 AEG III). DNA was extracted and then amplified using seven fluorescent-labeled microsatellite markers, one pair each on 5q, 18q, and 17p and four on 14q. The results were analyzed for loss of heterozygosity (LOH) and microsatellite instability (MSI). LOH varied from 20% to 30% at each locus except for the 17p locus, where it was slightly above 50% in both groups. No significant differences in LOH or MSI were found between the esophageal and gastric tumors, including the 14q chromosomal arm. These results fail to confirm the finding that abnormalities on the 14q chromosomal arm distinguish between distal esophageal and proximal gastric tumors.

In a number of recent studies, a lung tumor resistance locus designated either Par2 or Pas7 was mapped to distal chromosome 18 in crosses between susceptible A/J and more resistant BALB/c mice. This locus is important in that it accounts for as much as 60% of the difference in lung tumor susceptibility between the A/J and BALB/c mice, both of which contain the susceptible allele of Kras2, a marker and strong candidate for the major lung tumor susceptibility gene on mouse chromosome 6. We have now fine-mapped the Par2 locus by using congenic mice that were constructed by placing part of chromosome 18 from the susceptible A/J onto the genetic background of lung tumor-resistant BALB/c mice. After 7 generations of backcrossing, N7 mice that carried 28 cM of the A/J quantitative trait locus (QTL) region were crossed to the BALB/c to generate the N8 generation. Congenic strains (N8) that contain various QTL regions were generated. N9 mice, generated from N8 males x 3 BALB/c females, were genotyped in the region of the Par2 locus and treated with an initiating dose of urethane and allowed to form lung tumors over 6 months. The mice were killed and the lung tumors counted. With this cross the Par2 locus was narrowed to a 6-cM region. Potential candidate genes in this region include Smad4, Smad2, and Dcc. Previously, we excluded Smad4 and Smad2 as candidates for Par2 based on the lack of functional polymorphism(s) and differential expression in lungs from A/J and BALB/c mice. In this study, no polymorphism of the coding sequence of Dcc was observed between A/J and BALB/c mice. Further fine mapping and positional cloning are required for the identification of the Par2 gene.

We found three new variations in the OX40 ligand (OX40L, CD134L) gene and its 5' upstream region. -921 (G-->A) at 5' upstream region, -19 (C-->G) at 5' untranslated region, and 202 + 25 (G-->T) at intron 2 were identified. -921 (G-->A) and 202 + 25 (G-->T) were detected in a substantial proportion of healthy Japanese individuals and were considered to be single nucleotide polymorphisms (SNPs). -19 (C-->G) was detected in only one healthy individual. There was no association between these variations and the presence of rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE).

<h4>Background</h4>The prevalence of hereditary hemochromatosis in Norway is one of the highest reported in the world. However, the clinical presentation in patients with hemochromatosis in Norway seems to be different compared with recent studies elsewhere. The aim of this study was to investigate patients with hemochromatosis in one community hospital in Norway and to study the prevalence of the C282Y mutation.<h4>Methods</h4>One hundred and twenty patients were consecutively admitted to one medical department in Oslo. Serum transferrin and ferritin concentrations were measured in all patients, and a percutaneous liver biopsy was obtained in 108 of 120 (90%) patients. Stainable iron (Perls stain) in hepatocytes was graded from 0 to 4+ and fibrosis from 1 to 4. Genotyping for the C282Y and H63D mutation in the HFE gene was performed by PCR-RFLP.<h4>Results</h4>Forty-eight (40%) of the patients suffered from tiredness and astenia and 29 (24%) had typical arthropathy. Only 5 of 105 (4.5%) had biopsy confirmed cirrhosis and 5 had diabetes mellitus. Patients referred from a blood bank had significantly less symptoms and signs compared with other patients. Twenty-one of 120 (17.5%) patients were C282Y mutation negative. Seventeen (81%) of these patients (16 women and 1 man) had a history of extensive oral iron intake lasting from 5 to 50 years. When excluding those with extensive oral iron intake (n = 17), 92 of 103 (89%) were homozygous for the C282Y mutation, 7 (7%) were heterozygous including 3 compound heterozygous and 4 (4%) were mutation negative.<h4>Conclusions</h4>Only a minority of our patients with hemochromatosis had a far advanced disease at the time of diagnosis (less than 5% had cirrhosis) and hemochromatosis in a majority of the C282Y mutation negative patients was associated with excessive oral iron intake for several years.

<h4>Aim</h4>Hereditary hemochromatosis is the most common inherited disorder in white population (2-8 cases per 1000 habitants). Hemochromatosis is characterized by increased intestinal absorption of iron leading to its deposition into multiple organs. An early diagnosis and proper management with frequent phlebotomies are known to improve life expectancy and quality of life. Diagnosis is suggested by an elevated Transferrin saturation (TS) (more than 60%).<h4>Method</h4>Prospective study of the level of TS among 1131 healthy workers, who came to the Security and Hygiene Official Centre for their annual revision had been undertaken.<h4>Results</h4>Twenty-wo workers had high TS; in 10 of them the increase of TS was confirmed on repeated determinations. Liver biopsy was performed in six (and refused by the other four), eventually a diagnosis of hemochromatosis was confirmed in three (in-group prevalence of 2.6 per 1000 people).<h4>Conclusions</h4>In our experience, TS is the most appropriate initial screening test for detecting hereditary hemochromatosis in a normal population.

Blood coagulation parameters (thromboplastin time. PT; activated partial thromboplastin time, aPTT; fibrinogen; antithrombin III, ATIII; von Willebrand factor-concentration, vWF; factor VIII-activity, FVIII) and fibrinolytic parameters (plasminogen; (alpha-antiplasmin; euglobulin-lysis-time, Elt; tissue plasminogenactivator-antigen, tPA-antigen; plasminogenactivator-1-antigen, PAI-1-antigen) were evaluated in 34 women on low-dose oral contraceptives (OC) twice at intervals of 12 weeks each time before and after maximal exercise. During the 12 weeks, 24 women took part in an aerobic conditioning program and 10 women were requested to avoid any kind of sports activity for this period. Blood samples were taken before training and before and after maximal treadmill exercise. This procedure was repeated after the training program. After maximal exercise we found a significant reduction of aPTT and PT (increase in %), a decrease in ATIII, vWF, fibrinogen, plasminogen and alpha2-antiplasmin but an increase in fibrinolytic activity (all p<0.05). Maximal exercise is associated with an increase in blood coagulation and fibrinolysis also in women taking OC. After the physical conditioning program an increase in fibrinolytic activity at rest was noted in the training group. Opposed to that the fibrinolytic activity at rest decreased in the control group after abstinence of sports activity over this period (p<0.05, MANOVA).

Ubiquitin (UB), an intracellular protein that binds to other proteins to target them for proteolysis, is associated with Mallory hyalin (MH), which supports a biopsy diagnosis of nonalcoholic steatohepatitis (NASH). We analyzed 54 liver biopsy specimens from 49 patients with a clinical diagnosis of NASH for immunoreactive UB and multiple features of necroinflammation, fibrosis, and Prussian blue-positive iron to determine whether the presence of immunoreactive UB increases detection of MH or correlates with other features of cell injury or mutations of the HFE gene. MH and UB were graded. Analysis for HFE gene mutations was performed in 48 patients. Biopsy diagnoses were distributed as follows: NASH, 42; steatosis, 10; and nonspecific changes, 2. UB was present in 20 specimens and MH in 23. Of 31 specimens with 0 MH, 6 had UB; of 14 with 1 + (questionable) MH, 7 had 1+ or 2+ UB. UB correlated positively and significantly with the diagnosis and grade of NASH, presence of MH, cell swelling, lobular inflammation, and fibrosis. Immunostaining for UB may enhance detection of MH in questionable cases, support the diagnosis of NASH, and indicate which patients may be at risk for progression of disease.

Fourier transform infrared spectroscopy was used to study the composition and microstructure of soluble complexes produced by different initial mole ratios of bovine serum albumin(BSA) and calcium hydroxyapatite [Ca10(OH)2(PO4)6] and calcium carbonate (CaCO3) in aqueous solution. The band shifts and intensity variations suggested that calcium complexes with multiple ligands such as BSA, apatite, carbonate as well as the hydrogen bond network formed in titled system. The interactions increased the solubility of Ca10 (OH)2(PO4)6 and CaCO3 and the composition of soluble complexes is nonstoichiometric. The results provided an ideal model to investigated biomineralization process.