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The c-myb gene is implicated in the differentiation and proliferation of hematopoietic cells. Truncations of the N and/or C terminus of c-Myb, found in v-Myb, can potentiate its transforming ability. Two negative regulatory subregions, located in the C terminus, were mapped previously by using GAL4-c-Myb fusion proteins in transient transfection assays for the transcriptional activation of a GAL4-responsive reporter gene. To dissect the C terminus of c-Myb in terms of its involvement in transcriptional activation and oncogenic transformation, a series of C-terminal deletion mutants of c-Myb were analyzed. In addition, linker insertion mutants within the transactivation domain and/or heptad leucine repeat of c-Myb were examined along with those deletion mutants. In this study, we demonstrated that the removal of both of the two previously mapped negative regulatory subregions from the native form of c-Myb not only supertransactivates a Myb-responsive reporter gene but also potentiates its transforming ability in culture. However, in contrast to previous results, cells transformed by all of the mutants analyzed here except v-Myb itself exhibited the same phenotype as those transformed by c-Myb. The proliferating cells were bipotenial and differentiated into both the granulocytic and monocytic lineages. This result implies that the C terminus of c-Myb alone has no effect on the lineage determination. Finally, the transactivation activities of these mutants correlated with their transforming activities when a mim-1 reporter gene was used but not when a model promoter containing five tandem Myb-binding sites was used. In particular, a very weakly transforming mutant with a linker insertion in the heptad leucine repeat superactivated the model promoter but not the mim-1 reporter gene.

<h4>Background/aims</h4>Common variable immunodeficiency (CVID) is an immunological disorder characterised by defective antibody production. Patients with CVID have a high risk of gastric cancer. It has been suggested that gastric cancer results from an interaction between environmental factors and a genetic predisposition. The role of Helicobacter pylori as an environmental factor in gastric carcinogenesis is of current interest. Moreover, p53 gene mutations have been reported in gastric cancer. This study focuses on the gastric pathology of patients with CVID and correlation with H pylori infection.<h4>Methods</h4>Thirty four consecutive dyspeptic patients with CVID (mean age 49.6 years, range 14-72; 17 men) were included in the study. An upper gastrointestinal endoscopy was performed and biopsy specimens were taken from the antrum, incisura angularis, and gastric body. Biopsies were used for histological assessment, to identify the presence of H pylori, and to evaluate p53 overexpression.<h4>Results</h4>H pylori infection was detected in 14/34 (41%) patients. Chronic active gastritis involving both antrum and body was observed more frequently in H pylori positive (79%) than H pylori negative (20%) patients (p = 0.001). Similarly, a histological feature of multifocal atrophic gastritis was found more frequently in infected (50%) than uninfected patients (10%) (p = 0.012). In addition, one case of gastric adenocarcinoma and another of notable dysplasia were observed in the H pylori positive group. Overexpression of p53 was found in six (18%) patients, including one with normal gastric mucosa.<h4>Conclusions</h4>It can be hypothesised that both H pylori and p53 alterations play a role in the gastric carcinogenesis of patients with CVID.

How aggregates of polyglutamine proteins are involved in the neurological symptoms of glutamine repeat diseases is unknown. We show that huntingtin aggregates are present in the neuronal processes of transgenic mice that express exon 1 of the Huntington's disease (HD) gene. Unlike aggregates in the nucleus, these neuropil aggregates are usually smaller and are not ubiquitinated. Electron microscopy reveals many neuropil aggregates in axons and axon terminals. Huntingtin aggregates in the axon terminal are co-localized with some synaptic vesicles, implying that they may affect synaptic transmission and neuronal communication. The formation of neuropil aggregates is highly correlated with the development of neurological symptoms. The present study raises the possibility that neuropil aggregates may cause a dysfunction in neuronal communication and con-tribute to the neurological symptoms of HD.

The transferrin receptor contains a highly conserved Arg-Gly-Asp (RGD) sequence in the C-terminal region where transferrin is thought to bind. RGD sequences are commonly involved in cell adhesion. This sequence is crucial for transferrin binding, suggesting possible evolutionary links between molecules mediating iron uptake and cell adhesion.

Expanded polyglutamine tracts cause huntingtin and other proteins to accumulate and aggregate in neuronal nuclei. Whether the intranuclear aggregation or localization of a polyglutamine protein initiates cellular pathology remains controversial. We established stably transfected pheochromocytoma PC12 cells that express the N-terminal fragment of huntingtin containing 20 (20Q) or 150 (150Q) glutamine residues. The 150Q protein is predominantly present in the nuclei, whereas the 20Q protein is distributed throughout the cytoplasm. Electron microscopic examination confirmed that most of the 150Q protein is diffuse in the nucleus with very few microscopic aggregates observed. Compared with parental PC12 cells and cells expressing 20Q, cells expressing 150Q display abnormal morphology, lack normal neurite development, die more rapidly, and are more susceptible to apoptotic stimulation. The extent of these cellular defects in 150Q cells is correlated with the expression level of the 150Q protein. Differential display PCR and expression studies show that cells expressing 150Q have altered expression of multiple genes, including those that are important for neurite outgrowth. Our study suggests that mutant huntingtin in the nucleus is able to induce multiple cellular defects by interfering with gene expression even in the absence of aggregation.

Distinct motor patterns are selected from a multifunctional neuronal network by activation of different modulatory projection neurons. Subsets of these projection neurons can contain the same neuromodulator(s), yet little is known about the relative influence of such neurons on network activity. We have addressed this issue in the stomatogastric nervous system of the crab Cancer borealis. Within this system, there is a neuronal network in the stomatogastric ganglion (STG) that produces many versions of the pyloric and gastric mill rhythms. These different rhythms result from activation of different projection neurons that innervate the STG from neighboring ganglia and modulate STG network activity. Three pairs of these projection neurons contain the neuropeptide proctolin. These include the previously identified modulatory proctolin neuron and modulatory commissural neuron 1 (MCN1) and the newly identified modulatory commissural neuron 7 (MCN7). We document here that each of these neurons contains a unique complement of cotransmitters and that each of these neurons elicits a distinct version of the pyloric motor pattern. Moreover, only one of them (MCN1) also elicits a gastric mill rhythm. The MCN7-elicited pyloric rhythm includes a pivotal switch by one STG network neuron from playing a minor to a major role in motor pattern generation. Therefore, modulatory neurons that share a peptide transmitter can elicit distinct motor patterns from a common target network.

In a study of a group of elderly athletes we observed an unexpected association between serum cholesterol levels and the HTTLPR insertion/deletion polymorphism of the promoter region of the serotonin transporter gene (HTT, SLC6A4). As a follow-up we examined the potential association of this polymorphism with cholesterol and triglyceride levels, or heart disease, in two other groups of subjects. We examined the possible association between cholesterol levels and heart disease and genotypes of the HTTLPR insertion/deletion polymorphism of the promoter region of the HTT gene, in three independent study populations ranging from 42 to 90 years of age. For subjects 55 to 70 years of age in Group 1, cholesterol levels were significantly greater in the LS heterozygotes than either LL or SS homozygotes, indicating a heterosis effect (P </= 0.0001). This was replicated in Group 2 (P </= 0.015). Triglyceride levels were also significantly elevated in the LS subjects (P </= 0.001). In Groups 1 and 3 there was a significant association between LS heterozygosity and heart disease, angina, and heart attacks in subjects 70 years of age or less. All of these associations were absent in subjects >70 years of age. While these studies are preliminary and exploratory, they are consistent with a relationship of the HTT gene in cholesterol levels and a risk for heart disease. Replication of these findings in independent, epidemiologically based studies is required.

Targeted mutagenesis was used to produce two mutations in the murine hemochromatosis gene (Hfe) locus. The first mutation deletes a large portion of the coding sequence, generating a null allele. The second mutation introduces a missense mutation (C282Y) into the Hfe locus, but otherwise leaves the gene intact. This mutation is identical to the disease-causing mutation in patients with hereditary hemochromatosis. Mice carrying each of the two mutations were bred and analyzed. Homozygosity for either mutation results in postnatal iron loading. The effects of the null mutation are more severe than the effects of the C282Y mutation. Mice heterozygous for either mutation accumulate more iron than normal controls. Interestingly, although liver iron stores are greatly increased, splenic iron is decreased. We conclude that the C282Y mutation does not result in a null allele.

A number of genes are involved in iron metabolism, including the transferrin receptor (TFR) and haemochromatosis (HFE) genes. In previous investigations an increased risk for neoplastic disease has been observed in individuals homo- and heterozygous for hereditary haemochromatosis. The HFE wild-type gene product complexes with the transferrin receptor (TF) and two different HFE mutations (Cys282Tyr and His63Asp) have been found to increase the affinity of TFR for TF and increase cellular iron uptake. In a recent study we found no associations for HFE and TFR separately, but an interaction between HFE and TFR genotypes in multiple myeloma. Individuals carrying the HFE Tyr282 allele (homo- and heterozygotes) in combination with homozygosity for the TFR Ser142 allele had an increased risk. In the present study the same association was found in breast and colorectal cancer. The odds ratio for all three neoplasms combined was 2.0 (95% CI 1.0-3.8). The risk for neoplastic disease was further increased (OR 7.7, 95% CI = 1.0-59.9) when the analysis was restricted to HFE Tyr homozygotes and compound heterozygotes in combination with TFR Ser homozygosity. Thus, an interaction between HFE and TFR alleles may increase the risk for different neoplastic disorders.

The rotational symmetry of the Caenorhabditis elegans gonad arms is generated by the symmetrical migration of two distal tip cells (DTCs), located on the anterior and posterior ends of the gonad primordium. Mutations that cause asymmetrical migration of the two DTCs were isolated. All seven mutations were recessive and assigned to six different complementation groups. vab-3(k121) and vab-3(k143) affected anterior DTC migration more frequently than posterior, although null mutants showed no bias. The other five mutations, mig-14(k124), mig-17(k113), mig-18(k140), mig-19(k142), and mig-20(k148), affected posterior DTC migration more frequently than anterior. These observations imply that the migration of each DTC is regulated differently. mig-14 and mig-19 also affected the migration of other cells in the posterior body region. Four distinct types of DTC migration abnormalities were defined on the basis of the mutant phenotypes. vab-3; mig-14 double mutants exhibited the types of DTC migration defects seen for vab-3 single mutants. Combination of mig-17 and mig-18 or mig-19, which are characterized by the same types of posterior DTC migration defects, exhibited strong enhancement of anterior DTC migration defects, suggesting that they affect the same or parallel pathways regulating anterior DTC migration.

Escape from transforming growth factor-beta (TGF-beta)-induced inhibition of proliferation has been observed in many tumor cells and may contribute to loss of growth control. Smad proteins have been identified as major components in the intracellular signaling of TGF-beta family members. In this study, we examined the expression of receptor-activated, common-mediator and inhibitory Smads by immunohistochemistry in human colorectal cancers. We found increased expression of receptor-activated Smads in a fraction of the tumor cells, while no immunostaining for Smad2, Smad3 or Smad5 and only occasional staining for Smad1/8 was found in epithelial mucosa of normal colon. No or only weak staining for receptor-activated Smads, common-mediator Smad4 and inhibitory Smads was observed in the tumor stroma. Common-mediator Smad4 and inhibitory Smads were detected in cells of both tumor and normal tissues. We observed a distinct pattern of Smad4 immunostaining of epithelial cells along colon crypts, with high expression in zones of terminal differentiation. Our data show selective up-regulation of receptor-activated Smad proteins in human colorectal cancers and suggest involvement of Smad4 in differentiation and apoptosis of surface epithelial cells of normal crypts.

We have previously described biological model systems for studying tumor suppression in which, by using H-1 parvovirus as a selective agent, cells with a strongly suppressed malignant phenotype (KS or US) were derived from malignant cell lines (K562 or U937). By using cDNA display on the K562/KS cells, 15 cDNAs were now isolated, corresponding to genes differentially regulated in tumor suppression. Of these, TSAP9 corresponds to a TCP-1 chaperonin, TSAP13 to a regulatory proteasome subunit, and TSAP21 to syntaxin 11, a vesicular trafficking molecule. The 15 cDNAs were used as a molecular fingerprint in different tumor-suppression models. We found that a similar pattern of differential regulation is shared by activation of p53, p21(Waf1), and the human homologue of Drosophila seven in absentia, SIAH-1. Because SIAH-1 is differentially expressed in the various models, we characterized it at the protein and functional levels. The 32-kDa, mainly nuclear protein encoded by SIAH-1, can induce apoptosis and promote tumor suppression. These results suggest the existence of a common mechanism of tumor suppression and apoptosis shared by p53, p21(Waf1), and SIAH-1 and involving regulation of the cellular machinery responsible for protein folding, unfolding, and trafficking.

Dystrophic cardiac calcinosis (DCC) occurs in certain inbred strains of mice, including DBA/2 and C3H/He, and is generally found as an incidental lesion in adult animals at necropsy. Preliminary genetic studies into the cause of DCC have been performed in DBA/2 mice and suggest that DCC is inherited as an autosomal recessive trait involving three or four unlinked genes. To investigate the genetics of DCC further, we produced myocardial cell death by freeze-thaw injury to induce DCC. Experiments were conducted with three F1 hybrids made using three inbred strains of mice (DBA/2J and C3H/HeJ, DCC-susceptible strains; C57BL/6J, DCC-resistant strain) to compare the genetic factors in the development of DCC. We found that DBA/2 and C3H/He mice share the same gene pattern(s) that is responsible for DCC. We determined by backcross linkage analysis in DBA/2 and C57BL/6 mice that at least one recessive locus is responsible for DCC. A haplotype analysis of the backcross data demonstrated that the recessive locus, designated dyscalc1, is located on Chromosome 7, 20.5 cM distal to the centromere. The likely candidate genes for dyscalc1 are discussed. Further understanding of the structure and function of these mutant genes will be beneficial in explaining the molecular pathogenesis of DCC.

<h4>Background</h4>Hemochromatosis reportedly affects 3 to 8 persons per 1,000 and is associated with an elevated risk of morbidity and mortality. We sought to ascertain its prevalence in a community and to assess the association between phenotype and genotype.<h4>Methods</h4>All health maintenance organization employees were invited to participate in hemochromatosis screening using a repeated elevation of the transferrin saturation test as the case definition (> or = 50% in women and > or = 60% in men with no other cause). Iron overload from hemochromatosis was defined as serum ferritin concentration > or = 95th percentile and mobilizable iron > or = 99th percentile for age and sex, or hepatic iron index > or = 1.9. The HFE gene was analyzed for mutations.<h4>Results</h4>Participation among employees was 28% (1,653 of 6,000); 83% were women. The prevalence of hemochromatosis was 8 per 1,000 (13 of 1,653), and the prevalence of iron overload from hemochromatosis was 4 per 1,000 (5 of 1,653). Compared with those who had no HFE mutation, the relative risk (RR) for hemochromatosis was greatest for C282Y homozygotes (RR = 147), compound heterozygotes (RR = 19), and H63D homozygotes (RR = 9). Overall, 38% of participants had at least one HFE mutation. Screening based on an initial elevated transferrin saturation test had the best sensitivity, whereas DNA testing offered the best specificity and predictive value positive for iron overload disease.<h4>Conclusions</h4>In this population, we found a greater than expected prevalence of hemochromatosis and demonstrated a clear association with the HFE genotype. Promotion of screening is complicated by controversies in case definition and the large number of persons who will be detected before they have clinically significant iron loading, in whom the risk of clinical disease is unknown. Larger screening studies in more diverse populations are necessary to characterize the burden of disease and to follow those at risk (based on HFE or iron status measures) to establish the natural history of hemochromatosis.

Targeted mutagenesis in mice demonstrates that the POU-domain gene Brn4/Pou3f4 plays a crucial role in the patterning of the mesenchymal compartment of the inner ear. Brn4 is expressed extensively throughout the condensing mesenchyme of the developing inner ear. Mutant animals displayed behavioral anomalies that resulted from functional deficits in both the auditory and vestibular systems, including vertical head bobbing, changes in gait, and hearing loss. Anatomical analyses of the temporal bone, which is derived in part from the otic mesenchyme, demonstrated several dysplastic features in the mutant animals, including enlargement of the internal auditory meatus. Many phenotypic features of the mutant animals resulted from the reduction or thinning of the bony compartment of the inner ear. Histological analyses demonstrated a hypoplasia of those regions of the cochlea derived from otic mesenchyme, including the spiral limbus, the scala tympani, and strial fibrocytes. Interestingly, we observed a reduction in the coiling of the cochlea, which suggests that Brn-4 plays a role in the epithelial-mesenchymal communication necessary for the cochlear anlage to develop correctly. Finally, the stapes demonstrated several malformations, including changes in the size and morphology of its footplate. Because the stapes anlage does not express the Brn4 gene, stapes malformations suggest that the Brn4 gene also plays a role in mesenchymal-mesenchymal signaling. On the basis of these data, we suggest that Brn-4 enhances the survival of mesodermal cells during the mesenchymal remodeling that forms the mature bony labyrinth and regulates inductive signaling mechanisms in the otic mesenchyme.

Aberrant methylation of promoter region CpG islands is associated with transcriptional inactivation of tumor-suppressor genes in neoplasia. To understand global patterns of CpG island methylation in colorectal cancer, we have used a recently developed technique called methylated CpG island amplification to examine 30 newly cloned differentially methylated DNA sequences. Of these 30 clones, 19 (63%) were progressively methylated in an age-dependent manner in normal colon, 7 (23%) were methylated in a cancer-specific manner, and 4 (13%) were methylated only in cell lines. Thus, a majority of CpG islands methylated in colon cancer are also methylated in a subset of normal colonic cells during the process of aging. In contrast, methylation of the cancer-specific clones was found exclusively in a subset of colorectal cancers, which appear to display a CpG island methylator phenotype (CIMP). CIMP+ tumors also have a high incidence of p16 and THBS1 methylation, and they include the majority of sporadic colorectal cancers with microsatellite instability related to hMLH1 methylation. We thus define a pathway in colorectal cancer that appears to be responsible for the majority of sporadic tumors with mismatch repair deficiency.

The role of hereditary antithrombotic protein defects in juvenile deep vein thrombosis (DVT) was evaluated. Fifty six young patients (age <45 yr) with doppler-proven DVT were investigated for the presence of resistance to activated protein C (APC-R), lupus anticoagulant (LA), anticardiolipin antibodies and deficiencies of protein C, protein S, ATIII activities. Fifty nine normal healthy individuals served as controls. APC-R was observed to be the commonest defect underlying the Indian DVT as seen in 39.2% of patients followed by elevated ACA (5.3%), PAI (2.8%), presence of LA (2.8%) and reduced ATIII levels (2.8%). None of the subjects had protein C or S deficiency. APC-R was associated with ATIII deficiency in one case, and elevated ACA in two cases. In two subjects, APC-R was associated with elevated PAI levels. Patients with more than one prothrombotic factor had a higher prevalence of pulmonary thromboembolism, suggesting that the thrombogenic potential of APC-R is enhanced by the presence of coexisting hereditary or acquired prothrombotic defect.

We report here the identification and characterization of human and mouse PECI, a novel gene that encodes a monofunctional peroxisomal Delta(3),Delta(2)-enoyl-CoA isomerase. Human and mouse PECI were identified on the basis of their sequence similarity to Eci1p, a recently characterized peroxisomal Delta(3),Delta(2)-enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae. Cloning and sequencing of the human PECI cDNA revealed the presence of a 1077-base pair open reading frame predicted to encode a 359-amino acid protein with a mass of 39.6 kDa. The corresponding mouse cDNA contains a 1074-base pair open reading frame that encodes a 358-amino acid-long protein with a deduced mass of 39.4 kDa. Northern blot analysis demonstrated human PECI mRNA is expressed in all tissues. A bacterially expressed form of human PECI catalyzed the isomerization of 3-cis-octenoyl-CoA to 2-trans-octenoyl-CoA with a specific activity of 27 units/mg of protein. The human and mouse PECI proteins contain type-1 peroxisomal targeting signals, and human PECI was localized to peroxisomes by both subcellular fractionation and immunofluorescence microscopy techniques. The potential roles for this monofunctional Delta(3),Delta(2)-enoyl-CoA isomerase in peroxisomal metabolism are discussed.

In response to stress, adrenocorticotropin (ACTH) is secreted from anterior pituitary corticotropes. Corticotropin-releasing hormone (CRH) is a potent stimulator of ACTH secretion. The CRH stimulation of secretion is mediated by cAMP and is largely dependent on Ca(2+) influx through voltage-gated L-type Ca(2+) channels. This study was designed to investigate whether the expression of L-type Ca(2+) channels in the rat anterior pituitary and in corticotropes is regulated by acute stress and CRH. RNase protection assays were used to quantify alpha(1C) mRNA of the L-type Ca(2+) channel. The alpha(1C) mRNA levels from stressed rats increased by 31% in anterior pituitaries of rats after 30 min of exposure to cold stress. Neither 60 min cold stress nor 30 min restraint stress had an effect on alpha(1C) mRNA levels. When alpha(1C) mRNA was detected by in situ hybridization in a population of corticotropes enriched to 90%, 0.5 nM CRH (3 h) stimulated a 36% increase in the average area of label/cell and a 10% increase in the average density of label. Our results suggest that (1) the expression of alpha(1C) subunit mRNA of L-type Ca(2+) channels is increased in the rat anterior pituitary with a stress-specific response that might reflect an increase both in thyrotropes and corticotropes (both are known to be stimulated by cold stress), and (2) the CRH-mediated increase in alpha(1C) mRNA expression in individual rat corticotropes, in vitro, supports the hypothesis that some of the increase in vivo is due to changes in corticotropes.

Modulation of Ca(2+) channels has been shown to alter cellular functions. It can play an important role in the amplification of signals in the endocrine system, including the pleiotropically regulated pituitary lactotropes. Prolactin (PRL) secretion is tonically inhibited by dopamine (DA), the escape from which triggers acute episodes of hormone secretion. The magnitude of these episodes is explained by a potentiation of the PRL-releasing action of secretagogues such as thyrotropin-releasing hormone (TRH). While the mechanisms of this potentiation are not fully understood, it is known to be mimicked by the dihydropyridine, L-type Ca(2+) channel agonist Bay K 8644 and blocked by nifedipine and methoxyverapamil. The potentiation is also blocked by inhibitors of cyclic AMP-dependent protein kinase and protein kinase C. We recently described that the escape from tonic actions of DA results in increased macroscopic Ca(2+) currents in GH(4)C(1) lactotropic clonal cells transfected with a cDNA encoding the long form of the human D(2)-DA receptor. Here we show that the withdrawal from DA potentiates the PRL-releasing action of TRH in GH(4)C(1)/D(2)-DAR cells to the same extent as in enriched lactotropes in primary culture. In both experimental models a low density of dihydropyridine receptors was shown by (+)-[(3)H]PN200-110 binding. Photoaffinity labelling with the dihydropyridine [(3)H]azidopine revealed a protein consistent with the alpha(1) subunit of L-type Ca(2+) channels of 165-170 kDa. In both experimental models, the facilitation of TRH action triggered by the escape from DA was correlated with an enhanced rate of phosphorylation of this putative alpha(1) subunit, the nature of which was further supported by immunoprecipitation with selective antibodies directed against the alpha(1C) and alpha(1D) subunit of voltage-gated calcium channels. We propose that PKA- and PKC-dependent phosphorylation of the alpha(1) subunit of high voltage activated, L-type Ca(2+) channels is responsible for the facilitation of Ca(2+) currents in lactotropes, and hence for the potentiation of secretagogue-mediated PRL secretion. Thus, phosphorylation of Ca(2+) channels in endocrine cells may be a mechanism for the regulation of various functions including amplification of hormone secretion.

Beta2-microglobulin was investigated in atypical haemochromatosis patients not homozygous for the C282Y mutation of HFE (OMIM *235200), because the HFE protein binds beta2-microglobulin, and in mice beta2-microglobulin gene knockout causes hepatic iron overload. Six unrelated patients with atypical haemochromatosis were studied. Five patients had normal HFE coding sequence and the sixth was heterozygous for C282Y. We show that the spectrum of atypical haemochromatosis includes two distinct familial forms: juvenile haemochromatosis (OMIM *602390) and a novel form of familial iron overload, with apparently autosomal dominant inheritance, predominant Kupffer cell siderosis, and possible minimal dyserythropoiesis on bone marrow examination. Serial serum beta2-microglobulin estimation showed normal levels in all patients. Southern blot analysis showed normal beta2-microglobulin gene structure, excluding major gene rearrangement. Several corrections to the published beta2-microglobulin sequence were identified, but all six patients had normal beta2-microglobulin sequence. Western blot analysis of serum showed beta2-microglobulin protein of normal size. In conclusion, we found no evidence to implicate beta2-microglobulin mutation in atypical haemochromatosis. Two forms of familial iron overload appear unrelated to either HFE or beta2-microglobulin. Linkage studies are required to identify the genes involved, which may encode novel proteins crucial to the regulation of iron metabolism. Identification of these loci will aid the diagnosis, counselling, and treatment of iron overload disorders.

Matrix metalloproteinases (MMPs) are invariably upregulated in the stromal compartment of epithelial cancers and appear to promote invasion and metastasis. Here we report that phenotypically normal mammary epithelial cells with tetracycline-regulated expression of MMP3/stromelysin-1 (Str1) form epithelial glandular structures in vivo without Str1 but form invasive mesenchymal-like tumors with Str1. Once initiated, the tumors become independent of continued Str1 expression. Str1 also promotes spontaneous premalignant changes and malignant conversion in mammary glands of transgenic mice. These changes are blocked by coexpression of a TIMP1 transgene. The premalignant and malignant lesions have stereotyped genomic changes unlike those seen in other murine mammary cancer models. These data indicate that Str1 influences tumor initiation and alters neoplastic risk.

Xenopus 13S condensin converts interphase chromatin into mitotic-like chromosomes, and, in the presence of ATP and a type I topoisomerase, introduces (+) supercoils into DNA. The specific production of (+) trefoil knots in the presence of condensin and a type II topoisomerase shows that condensin reconfigures DNA by introducing an ordered, global, (+) writhe. Knotting required ATP hydrolysis and cell cycle-specific phosphorylation of condensin. Condensin bound preferentially to (+) supercoiled DNA in the presence of ATP but not in its absence. Our results suggest a mechanism for the compaction of chromatin by condensin during mitosis.

The synthesis of a novel antithrombotic consisting of a heparin pentasaccharide conjugated to the active site inhibitor N-(2-naphtalenesulfonyl)-glycyl-(D)-4-aminophenyl-alanyl-piperidin e (NAPAP) (i.e. compound I) is reported. This conjugate shows a unique pharmacological profile both in vitro and in vivo having direct anti-thrombin and ATIII-mediated anti-Xa activity. Furthermore, conjugate I has a prolonged in vivo half-life compared to NAPAP (1.5 h vs 9 min.).

Several positron emission tomography (PET) radioligands based on the aryl tropane structure have been used for studies on monoamine reuptake sites. RTI-364, RTI-330, and RTI-357 (3-beta-(4'-n-propyl-,4'-iso-propyl-, and 4'-iso-propenyl-phenyl)nortropane-2-beta-carboxylic acid methyl ester) are three recently synthesized cocaine analogues with higher affinity for the serotonin (5-HTT) than the dopamine transporter (DAT). Unlabelled RTI-364 and RTI-330 were prepared in a two-step synthesis. The key step was the addition of the appropriate propyl Grignard reagent to anhydroecgonine methyl ester. RTI-357 was prepared in a three-step synthesis with a palladium-catalyzed coupling reaction of beta-CIT and isopropenylzinc bromide as key step. Hydrolysis of the ester functions gave the carboxylic acid analogues of RTI-364, RTI-330, and RTI-357, which were labelled with 11C using [11C]methyl iodide in dimethyl formamide (DMF) and tetrabutylammonium hydroxide (TBAH) as base. All three compounds entered the monkey brain in a high degree (approximately 5-10%). There was a low uptake of [11C]RTI-364 in serotonin-rich brain areas, whereas [11C]RTI-330 and [11C]RTI-357 showed a marked uptake of radioactivity in the thalamus and the brainstem, regions known to contain serotonin transporters. Transient equilibrium was reached at 15 and 40 min for [11C]RTI-330 and [11C]RTI-357, respectively. After pretreatment with citalopram, the ratio of radioactivity in the thalamus and the brainstem to the cerebellum were markedly reduced for [11C]RTI-357 but not for [11C]RTI-330. The results indicate that [11C]RTI-357 is a potential PET radioligand for quantitation of the serotonin reuptake site.

A common 44-base pair insertion/deletion polymorphism in the promoter region of the human serotonin transporter (5-HTT) gene has been observed to be associated with affective illness and anxiety-related traits. This biallelic functional polymorphism, designated long (L) and short (S), affects 5-HTT gene expression since the S promoter is less active than the L promoter. Since there is strong evidence of a disturbance in brain serotonergic transmission among antisocial, impulsive, and violent type 2 alcoholic subjects, we decided to test the hypothesis that the frequency of the S allele, which is associated with reduced 5-HTT gene expression, is higher among habitually violent type 2 alcoholics when compared with race and gender-matched healthy controls and non-violent late-onset (type 1) alcoholics. The 5-HTT promoter genotype was determined by a PCR-based method in 114 late onset (type 1) non-violent alcoholics, 51 impulsive violent recidivistic offenders with early onset alcoholism (type 2), and 54 healthy controls. All index subjects and controls were white Caucasian males of Finnish origin. The S allele frequency was higher among type 2 alcoholics compared with type 1 alcoholics (chi2 = 4.86, P = 0.028) and healthy controls (chi2 = 8.24, P = 0.004). The odds ratio for SS genotype vs LL genotype was 3.90, 95% Cl 1.37-11.11, P = 0.011 when type 2 alcoholics were compared with healthy controls. The results suggest that the 5-HTT 'S' promoter polymorphism is associated with an increased risk for early onset alcoholism associated with antisocial personality disorder and impulsive, habitually violent behavior.

Plasma sex hormone-binding globulin (SHBG or SBP), the specific carrier for estradiol and androgens, after binding to its membrane receptor (SHBG-R), causes a significant increase of cAMP in the presence of estradiol, in both breast (MCF-7) and prostate (LNCaP) cancer cells maintained in serum-free medium. On the other hand, it has been proposed that estrogens, in addition to the well-known nuclear receptor pathway, exert their biological effect inducing cAMP, as a consequence of a direct membrane action, in breast cancer and uterine cells. The aim of the present study was to clarify this controversial issue by verifying if the cAMP increase in MCF-7 cells was a direct effect of estradiol, or if it was mediated by FCS proteins, such as bovine sex hormone-binding globulin; and to reevaluate the effect of human SHBG on cAMP induction in the presence of FCS. MCF-7 cells were maintained in DCC-FCS (treated with DCC to remove steroids), in SHBG-FREE/DCC-FCS (treated with DCC and with a specific affinity chromatography to remove bovine sex hormone-binding globulin), or in serum-free medium (SFM). It was observed that estradiol determined a significant time-dependent increase of cAMP only in MCF-7 cells maintained in 10% DCC-FCS. When cells were maintained in 10% SHBG-FREE/DCC-FCS, estradiol had no detectable effect. However, its ability to increase cAMP was observed again after the addition of human SHBG, in doses ranging from 5 to 50 nM. Moreover, in the presence of 10% SHBG-FREE/DCC-FCS, SHBG, even in the absence of estradiol, caused a significant increase of cAMP. In conclusion, the data reported in the present study suggest that the ability of estradiol to induce cAMP in MCF-7 cells is not due to a direct membrane effect of the hormone, but rather it is mediated by FCS. SHBG is one of the serum factors mediating estradiol action. Lastly, it was proven that SHBG triggers the cAMP pathway in MCF-7 cells in a physiologic culture condition and at physiologic concentrations.

<h4>Aim</h4>To investigate the role of DNA microsatellite instability (MSI) in gastric carcinogenesis by studying associations between MSI status, clinicopathological features, and loss of genetic loci.<h4>Methods</h4>Six microsatellite loci and loss of heterozygosity at APC, DCC, and MCC were analysed by polymerase chain reaction based methods in 53 cases of advanced gastric cancer.<h4>Results</h4>MSI was observed in 32.1% of gastric carcinomas (17/53) and 20% of foci of intestinal metaplasia (3/15). Seven gastric carcinomas (13.7%) were MSI-high (MSI-H) (three loci or more) and 10 (18.9%) were MSI-low (MSI-L) (one or two loci). The frequency of MSI-H was higher in intestinal (25.0%) than in diffuse carcinomas (3.7%) (p < 0.05). None of the MSI-H tumours showed loss of heterozygosity at APC, MCC, or DCC loci.<h4>Conclusions</h4>MSI may have an important and early role in a subset of gastric cancers, particularly the intestinal type. The MSI-H subset of gastric cancer has features in common with its colorectal counterpart, whereas MSI-L and microsatellite stable cancers appear to develop through the loss of heterozygosity pathway.

A 27-year-old man was admitted to our hospital with the complaints of swelling of his face and lower limbs. Echocardiography showed minimal pericardial effusion accompanied by disordered diastolic function. Cardiac catheterization was performed to rule out constrictive pericarditis. Normal pressure tracings of the right heart rule out constrictive pericarditis, however, a narrowing of the inferior vena cava was observed. Venographies of the inferior and superior vena cavae showed extensive thrombotic involvement of these great veins. Protein C, protein S, anticardiolipin antibodies, fibrinogen, antithrombin-III, activated protein C resistance, and factor V levels were in normal limits. Heterozygosity for factor V Leiden mutation was detected. We conclude that factor V Leiden mutation can cause extensive thrombotic involvement of major veins and should be considered in idiopathic thrombosis of them.

Cancers that arise from astrocytes in the adult CNS present as either anaplastic astrocytomas (AAs) or as more aggressive glioblastomas multiforme (GBMs). GBMs either form de novo or progress from AAs. We proposed to examine the molecular genetic relationship between these CNS tumors by conducting a genome-wide allelic imbalance analysis that included 70 loci on examples of AA and GBM. We found significant loss of heterozygosity (LOH) at 13 discrete chromosomal loci in both AAs and GBMs. Loss was significant in both AAs and GBMs at 9 of these loci. AAs show the highest rates of LOH at chromosomes 1p, 4q, 6p, 9p, 11p, 11q, 13q, 14q, 15p, 17p, 17q, and 19q. GBMs showed the greatest losses at 1p, 6q, 8p, 9p, 10p, 10q, 11p, 13q, 17p, 17q, 18p, 18q, and 19q. GBMs also demonstrated significant amplification at the epidermal growth factor receptor locus (7p12). These data suggest that there are three classes of loci involved in glioma evolution. First are loci that are likely involved in early events in the evolution of both AAs and GBMs. The second class consists of AA-specific loci, typified by higher LOH frequency than observed in GBMs (4q, 6p, 17p, 17q, 19q). The third class consists of GBM-specific loci (6q, 8p, 10, 18q). Damage at these loci may either lead to de novo GBMs or permit existing AAs to progress to GBMs. Glioma-related LOH profiles may have prognostic implications that could lead to better diagnosis and treatment of brain cancer patients.

Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called 'embryoid bodies', are able to differentiate in vitro into derivatives of all three primary germ layers, the endoderm, ectoderm and mesoderm. We established differentiation protocols for the in vitro development of undifferentiated embryonic stem cells into differentiated cardiomyocytes, skeletal muscle, neuronal, epithelial and vascular smooth muscle cells. During differentiation, tissue-specific genes, proteins, ion channels, receptors and action potentials were expressed in a developmentally controlled pattern. This pattern closely recapitulates the developmental pattern during embryogenesis in the living organism. In vitro, the controlled developmental pattern was found to be influenced by differentiation and growth factor molecules or by xenobiotics. Furthermore, the differentiation system has been used for genetic analyses by 'gain of function' and 'loss of function' approaches in vitro.