Gene Literature Dashboard

<h4>Background</h4>The genetic epidemiology of colorectal adenomas has not been studied prospectively in colonoscopy patients without cancer.<h4>Aims</h4>To study genetic alterations in colorectal adenomas and correlate these with patient demographics and adenoma characteristics.<h4>Methods</h4>Mutations and allelic deletions in 201 adenomas from 60 patients were compared with demographic features, adenoma characteristics, and family history.<h4>Results</h4>The most common alteration was K-ras proto-oncogene mutation, present in 35% of adenomas and 65% of patients. Patients 65 years of age and older had a decreased probability of K-ras mutations (26% versus 45%). Overexpression of p53 gene product was present in only 6% of adenomas but was more frequent in villous or tubulovillous adenomas (19% versus 3%). Allelic loss of chromosome 18q was present in only 2% of adenomas and was significantly less frequent than p53 overexpression. DNA replication errors (RER) were present in 7% of adenomas and 15% of patients, including multiple adenomas in four patients (two with hereditary non-polyposis colorectal cancer syndrome). Only 36% of RER positive adenomas had alteration of BAT-26 alleles, none had alteration of BAT-25, and only one (8%) had mutation in the transforming growth factor beta type II receptor gene. RER positive adenomas were more likely to have a K-ras mutation. In patients with multiple adenomas, there was concordance of p53 overexpression and RER but not of K-ras mutations.<h4>Conclusions</h4>Genetic progression in colorectal adenomas is heterogeneous, involving factors related to patient age and the presence of RER for the occurrence of ras mutations, but different intraindividual characteristics for the occurrence of p53 alterations and RER.

Between September 1995 and August 1998, the incidence and diversity of the main human rotavirus genotypes (G1, G2, G3, and G4 and P[8], P[4], P[6], and P[9]) among Irish children were determined by using established and adapted reverse transcriptase PCR-based genotyping methods. From a total of 193 rotavirus-positive specimens collected from nine hospitals we successfully identified the P type in 182 (94%) of the samples and the G type in 165 (85.5%) of the samples. Only four samples could not be assigned a G or P type. Two P types existed in Ireland, P[8] (78%) and P[4] (16%), and their relative incidence varied over the 3 years of this study. No P[6] or P[9] types were detected. G1 was the most predominant G type (55%), and the incidences of G2, G3, and G4 isolates were 15.5, 1, and 11%, respectively. Three percent of the samples tested had a mixed G type. A P and G type was assigned to 158 (81.8%) of samples. Of the typeable samples, G1 P[8] was the most prevalent (65%), whereas G2 P[4] (17%), G3 P[8] (1%), G4 P[8] (12%), and mixed types (all G1/ G4 P[8]) (4%) were detected less frequently. In the third year a significant genotypic shift from G1 P[8] to G2 P[4] and G4 P[8] was observed. During the study, we noticed that the inclusion of random primers during cDNA synthesis greatly increased the specificity of the PCR typing assays. No correlation was seen between the contributing hospitals and a specific genotype. In conclusion, the coverage of infection given by the recently licensed tetravalent vaccine would be significantly high in Ireland, although future monitoring of genotypic changes among Irish isolates should be encouraged.

A screening for human proteins capable of interacting with influenza virus NS1 has been carried out using the two-hybrid genetic trap in yeast. A cDNA corresponding to the human homologue of Drosophila melanogaster Staufen protein (hStaufen) was isolated that fulfilled all genetic controls of the two-hybrid protocol. Using a hStaufen cDNA isolated from a lambda human library, the interaction of hStaufen and NS1 proteins was characterised in vivo and in vitro. Co-transfection of NS1 cDNA and a partial cDNA of hStaufen led to the relocalisation of recombinant hStaufen protein from its normal accumulation site in the cytoplasm to the nuclear location of NS1 protein. NS1 and hStaufen proteins could be co-immunoprecipitated from extracts of co-transfected cells and from mixtures of extracts containing either protein, as well as from extracts of influenza virus-infected cells. Furthermore, both proteins co-localised in the ribosomal and polysomal fractions of influenza virus-infected cells. The interaction was also detected in pull-down experiments using a resin containing purified hStaufen and NS1 protein translated in vitro. Deletion mapping of the NS1 gene indicated that a mutant protein containing the N-terminal 81 amino acids is unable to interact with hStaufen, in spite of retaining full RNA-binding capacity. These results are discussed in relation to the possible mechanisms of action of hStaufen and its relevance for influenza virus infection.

Huntington's disease (HD) is one of a number of neurodegenerative disorders caused by expansion of polyglutamine-encoding CAG repeats within specific genes. Huntingtin, the protein product of the HD gene, is widely expressed in neural and nonneural human and rodent tissue. The function of the wild-type or mutated form of huntingtin is currently unknown. We have observed that relative to naive and male animals, huntingtin protein was significantly increased in the arcuate nucleus of postpartum rats. Using an oligonucleotide probe, in situ and Northern blot hybridization confirmed the expression of huntingtin mRNA. Quantification of the in situ hybridization signal in the arcuate nucleus revealed an approximate sevenfold increase in the expression of huntingtin mRNA in postpartum, lactating animals compared with naive female or male animals. Emulsion autoradiography and immunohistochemistry revealed that the cells with elevated huntingtin expression had a stellate conformation that morphologically resembled astrocytes. Dual label immunofluorescence immunohistochemistry demonstrated the colocalization of huntingtin and glial fibrillary acidic protein in these cells, confirming that they were astrocytes. Astrocytes expressing huntingtin were consistently found in close apposition to neuronal soma, suggesting interactions between these cell types. During the perinatal and postnatal period, the hypothalamus undergoes alterations in metabolic function. Our results support the idea of glia-induced metabolic changes in the hypothalamus. These results provide the first demonstration of naturally occurring changes in the expression of the Huntington's disease gene in the brain and suggest that huntingtin may play an important role in the processes that regulate neuroendocrine function.

We investigated the clinicopathological and genetic characteristics including patients' gender, age, tumour location, growth pattern, Dukes' stage, DNA ploidy, S-phase fraction, PCNA, apoptosis, c-erbB-2, bcl-2, K-ras, p53, DCC and heat shock protein in 32 mucinous carcinomas versus 261 non-mucinous carcinomas in the colorectum. Sixty percent of mucinous carcinomas were located in the right colon, 13% in the left colon and 27% in the rectum (p=0.01). More mucinous carcinomas grew in expanding pattern than non-mucinous carcinomas (66% vs 39%, p=0.005). Compared with non-mucinous carcinoma, mucinous carcinoma had more K-ras mutations (50% vs 25%, p=0.02), but less p53 expression (72% vs 49%, p=0.02) and less apoptotic activity (19% vs 51%, p=0.01). We further confirm that the mucinous carcinoma in the colorectum represents a distinct clinicopathologic and genetic features as compared to non-mucinous tumour, and may have a different biological behaviour.

The ability to sense gravity is enhanced by an extracellular structure that overlies the macular sensory epithelium. This complex consists of high density particles, otoconia, embedded within a gelatinous membrane. The tilted mouse specifically lacks otoconia, yet has no other detectable anatomic lesions. Furthermore, the penetrance of the tilted phenotype is nearly 100%. This mouse provides a model to identify genes that are involved in the development and function of vestibular otoconia. Using SSLP markers, we have mapped the tilted (tlt) gene on mouse Chromosome (Chr) 5 between D5Mit421 and D5Mit353/D5Mit128/D5Mit266/D5Mit267 by analysis of the progeny of an intersubspecific F2 intercross. We also mapped the fibroblast growth factor receptor 3 (Fgfr3) gene, a potential candidate for tlt, and the Huntington's disease homolog (Hdh) gene to D5Mit268, approximately 4.3 centiMorgans (cM) from the tilted locus. This study excludes both Fgfr3 and Hdh as candidate genes for tlt and identifies closely linked microsatellite markers that will be useful for the positional cloning of tlt.

During their circumferential migration, the nuclei of inferior olivary neurons translocate within their axons until they reach the floor plate where they stop, although their axons have already crossed the midline to project to the contralateral cerebellum. Signals released by the floor plate, including netrin-1, have been implicated in promoting axonal growth and chemoattraction during axonal pathfinding in different midline crossing systems. In the present study, we report experiments that strongly suggest that the floor plate could also be involved in the migration of inferior olivary neurons. First, we show that the pattern of expression of netrin receptors DCC (for deleted in colorectal cancer), neogenin (a DCC-related protein), and members of the Unc5 family in wild-type mice is consistent with a possible role of netrins in directing the migration of precerebellar neurons from the rhombic lips. Second, we have studied mice deficient in netrin-1 production. In these mice, the number of inferior olivary neurons is remarkably decreased. Some of them are located ectopically along the migration stream, whereas the others are located medioventrally and form an atrophic inferior olivary complex: most subnuclei are missing. However, axons of the remaining olivary cell bodies located in the vicinity of the floor plate still succeed in entering their target, the cerebellum, but they establish an ipsilateral projection instead of the normal contralateral projection. In vitro experiments involving ablations of the midline show a fusion of the two olivary masses normally located on either side of the ventral midline, suggesting that the floor plate may function as a specific stop signal for inferior olivary neurons. These results establish a requirement for netrin-1 in the migration of inferior olivary neurons and suggest that it may function as a specific guidance cue for the initial steps of the migration from the rhombic lips and then later in the development of the normal crossed projection of the inferior olivary neurons. They also establish a requirement for netrin-1, either directly or indirectly, for the survival of inferior olivary neurons.

Although hemochromatosis is characterized by dramatic morphological and functional alterations in the liver, little is known about the effects of an excess of iron on lipid metabolism. Therefore, we determined the effect of chronic iron overload on plasma lipid profile and lipoprotein composition, as well as on hepatic cholesterol metabolism and biliary sterol output. Rats administered a diet enriched with 3% iron carbonyl for 12 weeks displayed a 30-fold increase in iron (P <.0001) and a 5-fold rise in malondialdehyde (P <.001) in the liver. When compared with pair-fed controls, iron-overload rats showed a significant increase in triglycerides (P <.005), free cholesterol (P <.006), cholesteryl ester (P <.007), and high-density lipoprotein (HDL)-cholesterol (P <. 003). Triglyceride and cholesteryl ester enrichment, protein depletion, size increase, and apolipoprotein composition alterations characterized the very low density lipoprotein (VLDL) and HDL particles of iron-overload rats. Assessment of the activity of intracellular key enzymes for cholesterol homeostasis in these rats disclosed a reduction in 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (66%, P <.005) and cholesterol 7alpha-hydroxylase (58%, P <.0004) with an increment of acyl-CoA: cholesterol acyltransferase (62%, P <.002). The lack of optimal enzymatic activity may be a result of marked membrane lipid peroxidation that brings about fluidity drop (P <.029) in microsomes of iron-overload rats (5.00 +/- 0.013) versus controls (8.20 +/- 0. 03), reflected by polarization. A decline of the pool size of cholesterol and bile acids was noted in iron-overload rats during a 6-hour bile drainage. Our results show that experimental iron overload causes marked perturbations in plasma lipid transport and hepatobiliary sterol metabolism. Given the positive correlation of malondialdehyde with most of the altered parameters, iron-catalyzed lipid peroxidation may well be one of the involved mechanisms.

<h4>Background & aims</h4>Most patients with genetic hemochromatosis are homozygous for a single mutation of the HFE gene (C282Y). There is a second mutation, H63D, but its role in iron overload is less conclusive. The aim of this study was to investigate the basis of iron overload in a patient with classical hemochromatosis who was only heterozygous for C282Y and negative for H63D.<h4>Methods</h4>Genotype for the C282Y, H63D, and S65C mutations of HFE was determined in patient RFH, his family members, and 365 controls. The HFE gene was sequenced in patient RFH. Allele-specific reverse-transcription polymerase chain reaction was performed to investigate RNA splicing. Allele frequency was determined by allele-specific oligonucleotide hybridization.<h4>Results</h4>The patient is compound heterozygous for C282Y and a novel splice site mutation (IVS3 + 1G --> T). His sister has an identical genotype and elevated serum ferritin and transferrin saturation. The novel mutation functionally alters messenger RNA splicing, causing obligate skipping of exon 3. However, the IVS3 + 1G --> T mutation was found to be rare and was not detected in 630 control European chromosomes.<h4>Conclusions</h4>IVS3 + 1G --> T in the compound heterozygous state with C282Y results in iron overload that can progress to a severe phenotype of classical hemochromatosis. The demonstration of IVS3 +1G --> T highlights the possibility of other rare HFE mutations, particularly in C282Y heterozygotes with iron overload.

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28- T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.

Sox family proteins are characterized by a unique DNA-binding domain, a HMG box which shows at least 50% sequence similarity with mouse Sry, the sex-determining factor. At present almost 30 Sox genes have been identified. Members of this family have been shown to be conserved during evolution and to play key roles during animal development. Some are involved in human diseases, including sex reversal. Here we report the isolation of a novel member of the Sox gene family, Sox30, which may constitute a distinct subgroup of this family. Using a bacterially expressed DNA-binding domain of Sox30, we show that it is able to specifically recognize the ACAAT motif. Furthermore, Sox30 is capable of activating transcription from a synthetic promoter containing the ACAAT motif. The specific expression of Sox30 in normal testes, but not in maturing germ cell-deficient testes, suggests the involvement of Sox30 in differentiation of male germ cells. Mapping analyses revealed that the Sox30 gene is located on human chromosome 5 (5q33) and on mouse chromosome 11.

Ulcerative colitis (UC) is a chronic inflammatory disease of the colon associated with a high risk of colorectal cancer. This increased cancer risk is thought to result from the cellular damage induced by the inflammatory field. The aim of this study was to determine the pattern and time course of genomic instability occurring in UC-related neoplasia. Sites of cancer, dysplasia, and nondysplasia from 14 UC colectomy cases containing cancer were analyzed for chromosomal alterations by comparative genomic hybridization (CGH) and for microsatellite instability using a series of 10 microsatellite markers. Clonal chromosomal alterations were present in 85% of cancer sites, 86% of dysplasia sites, and 36% of nondysplasia sites. Losses of chromosome 18 or 18q and chromosome 5 or 5q were common in cancer and dysplasia and were occasionally detected in nondysplasia. High-level microsatellite instability was detected in the cancer and dysplasia of two cases. Samples that demonstrated high-level microsatellite instability were unlikely to have chromosomal alterations demonstrable by CGH. These studies suggest that the predominant type of genomic instability in UC-related neoplasia is associated with chromosomal alterations and that this type of genomic instability frequently occurs before the development of histologically defined dysplasia.

The physiological inhibitor of thrombin, antithrombin III (ATIII, Kybernin P) was investigated for its antiinflammatory and anticoagulant effects in a pig model of septic shock. Pigs were infused with a dose of 0.25 microgram. kg-1. h-1 of lipopolysaccharide (LPS) over a period of 3 hours. Animals developed systemic inflammation, disseminated intravascular coagulation (DIC), organ failure and cardiovascular abnormalities, namely pulmonary hypertension and systemic hypotension. Twenty septic pigs were allocated to 2 study groups, treated either with ATIII (n=10) or placebo (n=10). ATIII was administered as a 250-U/kg IV bolus infusion for 30 minutes (-60 to -30 minutes) followed by a single IV bolus of 125 U/kg (t=0) and a second 30-minute infusion of 250 U/kg (120 to 150 minutes). ATIII significantly prevented the development of a DIC; the increase in fibrin monomers (placebo, 11.4+/-9.1 reciprocal titers, at 6 hours) was completely overcome by ATIII (P<0. 05). ATIII significantly prevented the increase in thromboxane (TXB2) levels, which were 809+/-287 pg/mL in the placebo and 420+/-174 pg/mL in the verum group after 6 hours (P<0.02). On the other hand, ATIII had no influence on TNF levels. In a lethal study with an increased dose of LPS (0.5 microgram. kg-1. h-1). A significant reduction in mortality was observed in the ATIII group (0 of 7) compared with the placebo group (4 of 6) (P<0.05, chi2 test) a significant reduction of pulmonary hypertension (placebo, 42.0+/-11. 1 mm Hg; ATIII, 23.6+/-7.5 mm Hg, P<0.05), but no effect on systemic hypotension, was noted in the ATIII group. It was thus concluded that modulation of the procoagulatory state by substitution of ATIII results in a late beneficial antiinflammatory effect in this model of septic shock.

The netrins comprise a small phylogenetically conserved family of guidance cues important for guiding particular axonal growth cones to their targets. Two netrin genes, netrin-1 and netrin-2, have been described in chicken, but in mouse so far a single netrin gene, an ortholog of chick netrin-1, has been reported. We report the identification of a second mouse netrin gene, which we name netrin-3. Netrin-3 does not appear to be the ortholog of chick netrin-2 but is the ortholog of a recently identified human netrin gene termed NTN2L ("netrin-2-like"), as evidenced by a high degree of sequence conservation and by chromosomal localization. Netrin-3 is expressed in sensory ganglia, mesenchymal cells, and muscles during the time of peripheral nerve development but is largely excluded from the CNS at early stages of its development. The murine netrin-3 protein binds to netrin receptors of the DCC (deleted in colorectal cancer) family [DCC and neogenin] and the UNC5 family (UNC5H1, UNC5H2 and UNC5H3). Unlike chick netrin-1, however, murine netrin-3 binds to DCC with lower affinity than to the other four receptors. Consistent with this finding, although murine netrin-3 can mimic the outgrowth-promoting activity of netrin-1 on commissural axons, it has lower specific activity than netrin-1. Thus, like netrin-1, netrin-3 may also function in axon guidance during development but may function predominantly in the development of the peripheral nervous system and may act primarily through netrin receptors other than DCC.

HFE is a class I major histocompatibility complex (MHC)-related protein that is mutated in patients with the iron storage disease hereditary hemochromatosis. HFE binds tightly to transferrin receptor (TfR), the receptor that mediates uptake of iron-loaded transferrin. The binding affinities for TfR of HFE mutants, designed using the HFE crystal structure, were measured using biosensor assays. The results allow localization of the TfR binding site on HFE to the C-terminal portion of the alpha1 domain helix and an adjacent loop, a region distinct from the ligand binding sites on class I MHC and related proteins. A biosensor-derived pH-dependent affinity profile for the HFE-TfR interaction is discussed in terms of HFE's hypothesized role in intracellular trafficking.

Dentinogenesis imperfecta type III (DGI-III) is an autosomal-dominant disorder of dentin formation which appears in a tri-racial southern Maryland population known as the "Brandywine isolate". This disease has suggestive evidence of linkage to the long arm of human chromosome 4 (LOD score of 2.0) in a family presenting with both juvenile periodontitis and DGI-III. The purpose of this study was to screen a family presenting with only DGI-III to determine if this locus was indeed on chromosome 4q. Furthermore, we wanted to determine if DGI-III co-localized with dentinogenesis imperfecta type II (DGI-II), which has been localized to 4q21-q23. Therefore, a large kindred from the Brandywine isolate was identified, oral examination performed, and blood samples collected from 21 family members. DNA from this family was genotyped with 6 highly polymorphic markers that span the DGI-II critical region of chromosome 4q. Analysis of the data yielded a maximum two-point LOD score of 4.87 with a marker for the dentin matrix protein 1 (DMP1) locus, a gene contained in the critical region for DGI-II. Our results demonstrated that the DGI-III locus is on human chromosome 4q21 within a 6.6 cM region that overlaps the DGI-II critical region. These results are consistent with the hypothesis that DGI-II is either an allelic variant of DGI-III or the result of mutations in two tightly linked genes.

Development of databases, summarising the genetic events associated with oral squamous cell carcinoma (SCC), should increase our understanding of the molecular basis of these lesions. Additionally, databases will help establish whether different cancer subtypes show different growth characteristics, because the multistage carcinogenic process is different in the various tumour subtypes. This new knowledge may also provide new prognostic information, as these aberrations represent fundamental biological characteristics of each tumour. To assess the value of incorporating the results from loss of heterozygosity (LOH) analysis into patient-specific mutation databases, we have carried out microsatellite analysis with 52 polymorphic markers at 13 key chromosomal regions implicated in the pathogenesis of head and neck cancers. Altered expression of the Rb, p53 and DCC tumour suppressor genes has also been studied by immunohistology. Our results shed light on the different pathways that lead to cancer and reveal that a variety of different patterns of allelic imbalance (AI) were detected at all TNM stages, reflecting the different clinical behaviour that tumours classified as being of the same TNM stage may exhibit. Summarising the level of genetic damage as a fractional allelic loss (FAL) score and the presence of AI at 3p22-26, 3p14.3-12.1 and 9p21 was found to be a better predictor of outcome than the TNM system. This finding suggests that molecular data can be incorporated into conventional staging systems to provide more accurate prognostic information for this group of patients.

Hereditary hemochromatosis (HHC) represents an autosomal recessive disease in which increased iron absorption causes iron overload and irreversible tissue damage. The recently detected association between two point mutations in the HFE gene on chromosome 6p and HHC has made it possible to screen for the disease before the onset of irreversible tissue damage. Conventional genetic testing is based on restriction fragment-length polymorphisms (RFLP) using two endonuclease recognition sites in codon 63 or 282, respectively. In this study, we have adapted single-strand conformation polymorphism analysis for capillary electrophoresis (SSCP-CE) to detect homozygote or heterozygote point mutations. Two HFE gene fragments spanning codons 63 and 282 were amplified by a duplex PCR using genomic DNA from peripheral blood or from tissue sections of paraffin-embedded liver biopsies as template. Thereby, rapid genotyping of both HFE mutations was achieved with a single PCR, omitting the need of further analysis by restriction digest. Eighty-five patients with liver disease and/or suspected iron overload were genotyped using SSCP-CE, and all results were verified by conventional RFLP analysis. In summary, SSCP-CE proved to be a reliable, cost-effective, sensitive and rapid method for genotyping HFE mutations. This method will further facilitate high-throughput genetic screening using capillary array electrophoretic devices.

We compared the oestrogenic and anti-oestrogenic properties of the two well-known phyto-oestrogens, genistein and quercetin, on the oestrogen-sensitive breast cancer cell line MCF-7. Genistein exerted a biphasic effect on growth of MCF-7 cells, stimulating at low and inhibiting at high concentrations, whereas quercetin was only growth inhibitory. At doses which did not inhibit cell growth, respectively 5 and 1 microM, genistein and quercetin counteracted oestrogen- and transforming growth factor-alpha-promoted cell growth stimulation. Furthermore, genistein promoted transcription of the oestrogen-regulated genes pS2 and cathepsin-D, whereas quercetin interfered with the oestrogen-induced expression of the proteins. In in vitro binding experiments, genistein competed with oestradiol for binding to the oestrogen receptor (ER), but quercetin did not. Quercetin and genistein down-regulated cytoplasmic ER levels and promoted a tighter nuclear association of the ER, but only genistein was able to up-regulate progesterone receptor protein levels. In gel mobility assays, ER preincubation with oestradiol or with the two phyto-oestrogens led to the appearance of the same retarded band, excluding differences between the various complexes in binding to the consensus sequence. The data allowed us to conclude that quercetin acts like a pure anti-oestrogen, whereas genistein displays mixed agonist/antagonist properties, and to formulate a hypothesis on the possible mechanism of action of such phyto-oestrogens.

Increased expression of oestrogen (ER) and progesterone (PR) receptors have been reported in gastric adenocarcinoma, although results have been variable. Immunohistochemical staining methodologies, in particular in the detection of ER, have been inconsistent with many tumours being classified ER-negative. In this study we have used flow cytometry to quantify expression of ER and PR in gastric adenocarcinoma and examine their relationships with established prognostic indicators. Cytokeratin-positive cells obtained from tumour biopsies of 50 patients with gastric cancer and ten control patients were labelled with biotinylated ER or PR antibodies followed by streptavidin PE. Flow cytometry was seen to increase the detection of ER levels in gastric cancer with more receptor-positive patients in this study than in results published to date. We believe this is related to the sensitivity of the flow cytometric assay with the detection of small shifts in ER level detected using cytokeratin gating. On analysis, the data showed no significant correlations with tumour stage and grade, and no differences were seen between normal mucosa and gastric cancer samples.

The protein huntingtin (htt), aggregated in neuronal nuclear inclusions, is pathognomonic of Huntington's disease (HD). Constructs, translated in vitro from the N terminus of htt, containing either polyQ23 from a normal individual, or polyQ41 or polyQ67 from an HD patient, were all soluble. Transglutaminase (TGase) crosslinked these proteins, and the aggregations did not have the staining properties of amyloid. More TGase-catalyzed aggregates formed when the polyglutamine domain of htt exceeded the pathologic threshold of polyQ36. Furthermore, shorter htt constructs, containing 135 aa or fewer, formed more aggregates than did larger htt constructs. TGase activity in the HD brain was increased compared with the control, with notable increases in cell nuclei. The increased TGase activity was brain specific. In lymphoblastoid cells from HD patients, TGase activity was decreased. TGase-mediated crosslinking of htt may be involved in the formation of the nonamyloidogenic nuclear inclusions found in the HD brain. The staining properties of nuclear inclusions in the HD brain revealed that they were not amyloid.

<h4>Background</h4>Hereditary hemochromatosis is a common inherited disorder of iron metabolism. The gene HFE, which contains two missense mutations (C282Y and H63D), was recently identified.<h4>Objective</h4>To determine how HFE genotyping for the C282Y and H63D mutations contributes to the diagnosis of hemochromatosis and to determine the prevalence of HFE mutations in a group of patients with liver disease.<h4>Design</h4>Cross-sectional study.<h4>Setting</h4>Academic medical center.<h4>Patients</h4>66 patients with hereditary hemochromatosis and 132 referred patients with other liver diseases.<h4>Measurements</h4>At initial diagnosis, fasting transferrin saturation, ferritin level, routine chemistry panel, and complete blood count were determined. Percutaneous liver biopsy was done on all patients for histologic analysis and measurement of hepatic iron concentration and hepatic iron index. HFE genotyping for the C282Y and H63D mutations was done on all patients by using genomic DNA samples.<h4>Results</h4>Of the 66 patients with hemochromatosis diagnosed on the basis of serum iron studies and liver biopsy findings, 60 (91%) were C282Y homozygotes, 2 (3%) were compound heterozygotes, 1 (1.5%) was a C282Y heterozygote, 2 (3%) were H63D heterozygotes, and 1 (1.5%) was negative for both mutations. Of the 132 patients with liver disease, 6 (5%) were C282Y homozygotes, 8 (6%) were compound heterozygotes, 6 (5%) were C282Y heterozygotes, 5 (4%) were H63D homozygotes, 20 (15%) were H63D heterozygotes, and 87 (66%) were negative for both mutations. All 66 C282Y homozygotes had an elevated hepatic iron concentration, and 65 of the 66 patients (98%) had a transferrin saturation of at least 45%. Ten of the 66 patients (15% [95% CI, 7.5% to 26%]) had a hepatic iron index less than 1.9 mmol/kg per year; hemochromatosis was not suspected in 6 of the 10 patients before genotyping. Cirrhosis or substantial hepatic fibrosis was not seen in any (0% [CI, 0% to 18%]) of the 19 patients younger than 40 years of age who were homozygous for the C282Y mutation.<h4>Conclusions</h4>All 66 patients homozygous for the C282Y mutation of HFE had an elevated hepatic iron concentration, but approximately 15% of these patients did not meet a previous diagnostic criterion for hemochromatosis (hepatic iron index > 1.9 mmol/kg per year). Determination of HFE genotype is clinically useful in patients with liver disease and suspected iron overload and may lead to identification of otherwise unsuspected C282Y homozygotes.

<h4>Purpose</h4>To report three patients who developed heparin resistance during cardiac surgery which was successfully managed with 1000 U Antithrombin III (AT III).<h4>Clinical features</h4>We observed heparin resistance prior to cardiopulmonary bypass (CPB) in one patient and during the CPB in two patients. In the first patient who was scheduled for mitral valve replacement, although heparin was administered sequentially up to 500 U x kg(-1) prior the CPB, the ACT value was 354 sec. After 1,000 U ATIII were administered the ACT was 395 sec and CPB was initiated. The ACT remained between 496 and 599 sec throughout CPB and a total of 260 mg protamine sulfate was given. In the other two patients following 300 U x kg(-1) heparin, the ACT was up to 400 sec and CPB was initiated. During CPB, ACT were decreased 360 sec and 295 sec in patients II and III respectively. Although heparin was added 1,500 U, ACT increased to > or = 400 sec could not be achieved. In the second patient ATIII activity was found 10%. After the administration of 1,000 U ATIII, ATIII activity was found to be 67% 40 min later and ACT were increased up to 400 sec. There was no thrombosis within the extracorporeal circuit, additional heparin was not required, less protamine was administered (< or = 3 mg x kg(-1)) and no excessive postoperative bleeding was observed in all patients.<h4>Conclusion</h4>We recommend that AT III supplementation should be considered to manage heparin resistance prior or during CPB in patients undergoing open heart surgery.

Frazzled (Fra) is the DCC-like Netrin receptor in Drosophila that mediates attraction; Roundabout (Robo) is a Slit receptor that mediates repulsion. Both ligands are expressed at the midline; both receptors have related structures and are often expressed by the same neurons. To determine if attraction versus repulsion is a modular function encoded in the cytoplasmic domain of these receptors, we created chimeras carrying the ectodomain of one receptor and the cytoplasmic domain of the other and tested their function in transgenic Drosophila. Fra-Robo (Fra's ectodomain and Robo's cytoplasmic domain) functions as a repulsive Netrin receptor; neurons expressing Fra-Robo avoid the Netrin-expressing midline and muscles. Robo-Fra (Robo's ectodomain and Fra's cytoplasmic domain) is an attractive Slit receptor; neurons and muscle precursors expressing Robo-Fra are attracted to the Slit-expressing midline.

Netrins are bifunctional: they attract some axons and repel others. Netrin receptors of the Deleted in Colorectal Cancer (DCC) family are implicated in attraction and those of the UNC5 family in repulsion, but genetic evidence also suggests involvement of the DCC protein UNC-40 in some cases of repulsion. To test whether these proteins form a receptor complex for repulsion, we studied the attractive responses of Xenopus spinal axons to netrin-1, which are mediated by DCC. We show that attraction is converted to repulsion by expression of UNC5 proteins in these cells, that this repulsion requires DCC function, that the UNC5 cytoplasmic domain is sufficient to effect the conversion, and that repulsion can be initiated by netrin-1 binding to either UNC5 or DCC. The isolated cytoplasmic domains of DCC and UNC5 proteins interact directly, but this interaction is repressed in the context of the full-length proteins. We provide evidence that netrin-1 triggers the formation of a receptor complex of DCC and UNC5 proteins and simultaneously derepresses the interaction between their cytoplasmic domains, thereby converting DCC-mediated attraction to UNC5/DCC-mediated repulsion.

Two subgenomic regions within the major histocompatibility complex, the alpha and beta blocks, contain members of the multicopy gene families HLA class I, human endogenous retroviral sequence (HERV-16; previously known as P5 and PERB3), hemochromatosis candidate genes (HCG) (II, IV, VIII, IX), 3.8-1, and MIC (PERB11). In this study we show that the two blocks consist of imperfect duplicated segments, which contain linked members of the different gene families. The duplication and truncation sites of the segments are associated with retroelements. The retroelement sites appear to generate the imperfect duplications, insertions/deletions, and rearrangements, most likely via homologous recombination. Although the two blocks share several characteristics, they differ in the number and orientation of the duplicated segments. On the 62.1 haplotype, the alpha block consists of at least 10 duplicated segments that predominantly contain pseudogenes and gene fragments of the HLA class I and MIC (PERB11) gene families. In contrast, the beta block has two major duplications containing the genes HLA-B and HLA-C, and MICA (PERB11.1) and MICB (PERB11.2). Given the common origin between the blocks, we reconstructed the duplication history of the segments to understand the processes involved in producing the different organization in the two blocks. We then found that the beta block contains four distinct duplications from two separate events, whereas the alpha block is characterized by multisegment duplications. We will discuss these results in relation to the genetic content of the two blocks.

Serotonin induces vasoconstriction in the presence of atherosclerotic lesions. Platelets acquire serotonin from the extracellular space by serotonin transporter and release it following aggregation. There is a functional polymorphism in the serotonin transporter (5-HTT) gene promoter associated with transcriptional efficacy and plasma serotonin levels. To examine whether the polymorphism is associated with coronary heart disease (CHD) in the Japanese, we analyzed 144 male CHD patients with an onset age before 65 and 222 apparently healthy men. The L allele was observed significantly more frequently in the CHD patients (26%) than in the control subjects (19%); the odds ratio was 1.48 (p <0.03). A significant interaction between the polymorphism and smoking was observed for CHD (p = 0.03), suggesting that the two have a synergistic effect on CHD. Odds ratio of the combination of the L allele and smoking was 1.95 (p <0.003). The 5-HTT gene promoter polymorphism may play a role in susceptibility to CHD, particularly when it is combined with smoking.

The antithrombotic beta-D-xyloside, naroparcil, has previously been shown to induce a dose-related increase of circulating glycosaminoglycans (GAGs) together with an antithrombin activity (anti-IIa) via heparin cofactor II (HCII) in the rabbit. In order to go further in the mechanisms, the relationship between the antithrombotic activity, the HCII-mediated anti-IIa activity and the plasma GAG content was investigated. We showed that the in vitro specific activity on the inhibition of thrombin by HCII of the plasma GAG extract from naroparcil-treated rabbits was increased by a factor of 60 when compared to controls. In addition, the fractionation of the plasma GAG extract by affinity chromatography on immobilized HCII led to a more potent material whereas the low-affinity fraction was shown to be inactive in thrombin inhibition by HCII. The qualitative analysis of GAGs showed the presence of the deltaDi-4S DS disaccharide, undetectable in control, which accounted for 22% in the unfractionated GAG extract and for 60% in the high affinity fraction. In vitro experiments using immuno-depleted plasma in antithrombin III (ATIII), HCII or both, indicated that the anti-IIa activity of the plasma GAG extract from naroparcil-treated rabbits was mainly due to HCII potentialisation. The unfractionated GAG extract and the high affinity fraction were shown to be antithrombotic in a Wessler-based model in the rat, giving ED80 values of 610 UA/kg and 56 UA/kg respectively whereas the low-affinity fraction was devoid of any antithrombotic activity. These results show that the antithrombotic activity of naroparcil is dependent on modification in the plasma GAG profile which inactivates thrombin via the HCII.

Sry is the Y-chromosomal gene that acts as a trigger for male development in mammalian embryos. This gene encodes a high mobility group (HMG) box transcription factor that is known to bind to specific target sequences in DNA and to cause a bend in the chromatin. DNA bending appears to be part of the mechanism by which Sry influences transcription of genes downstream in a cascade of gene regulation leading to maleness, but the direct targets of Sry remain to be positively identified. One gene known to be downstream from Sry in this cascade is Sox9, which encodes a transcription factor related to Sry by the HMG box. Like Sry, mutations in Sox9 disrupt male development, but unlike Sry, the role of Sox9 is not limited to mammals. This review focuses on what is known about the two genes and their likely modes of action, and draws together recent data relating to how they might interconnect with the network of gene activity implicated in testis determination in mammals.

<h4>Objective</h4>To clarify the short and long term prognosis after hepatectomy and shunt ligation in patients with hepatocellular carcinoma (HCC) and spontaneous portosystemic (P-S) shunts.<h4>Design</h4>Retrospective study.<h4>Setting</h4>University hospital, Japan.<h4>Subjects</h4>Fourteen patients with HCC and a P-S shunt.<h4>Interventions</h4>Hepatic resection (radical in 12 and palliative in 2 patients) and ligation of the shunt; 12 also had prophylactic operations for oesophagogastric varices.<h4>Main outcome measures</h4>Morbidity, mortality, hepatic function, and survival.<h4>Results</h4>Serum ammonia concentrations fell after operation and stayed low throughout the postoperative period. Though prothrombin time and serum antithrombin-III concentrations deteriorated for one month after operation, they gradually returned to the preoperative values. Serum albumin did not change throughout the follow up period. Serum bilirubin concentrations were reduced postoperatively in 10 patients. Results of indocyanine green and Bromsulphalein retention tests were similar before and one month after operation. Child's grade improved in four patients within a year. At 1, 3, and 5 years 10, 5, and 3 patients were alive, and 10, 4, and 1 of these were free of disease. No patients ruptured their oesophagogastric varices during the follow up period.<h4>Conclusions</h4>Simultaneous ligation of spontaneous P-S shunts makes hepatic resection safe and produces unexpectedly good survival in patients with HCC and the severe cirrhosis associated with such shunts.

Alterations in neurotransmitter receptors are a pathological hallmark of the neurodegeneration seen in Huntington's disease (HD). However, the significance of these alterations has been uncertain, possibly reflecting simply the loss of brain cells. It is not known for certain whether the alteration of neurotransmitter receptors occurs before the onset of symptoms in human HD. Recently we developed transgenic mice that contain a portion of a human HD gene and develop a progressive abnormal neurological phenotype. Neurotransmitter receptors that are altered in HD (receptors for glutamate, dopamine, acetylcholine and adenosine) are decreased in the brain transgenic mice, in some cases before the onset of behavioural or motor symptoms. In transgenic mice, neurotransmitter receptor alterations occur before neuronal death. Further, receptor alterations are selective in that certain receptors, namely N-methyl-D-aspartate and gamma-aminobutyric acid receptors, are unaltered. Finally, receptor decreases are preceded by selective decreases in the corresponding mRNA species, suggesting the altered transcription of specific genes. These results suggest that (i) receptor decreases precede, and therefore might contribute to, the development of clinical symptoms, and (ii) altered transcription of specific genes might be a key pathological mechanism in HD.

We have shown previously by electron microscopy that the purified glutathione S-transferase (GST)-Huntington's disease (HD) exon 1 fusion protein with 51 glutamine residues (GST-HD51) is an oligomer, and that site-specific proteolytic cleavage of this fusion protein results in the formation of insoluble more highly ordered protein aggregates with a fibrillar or ribbon-like morphology (E. Scherzinger et al. (1997) Cell 90, 549-558). Here we report that a truncated GST HD exon 1 fusion protein with 51 glutamine residues, which lacks the proline-rich region C-terminal to the polyglutamine (polyQ) tract (GST-HD51 delta P) self-aggregates into high-molecular-mass protein aggregates without prior proteolytic cleavage. Electron micrographs of these protein aggregates revealed thread-like fibrils with a uniform diameter of ca. 25 nm. In contrast, proteolytic cleavage of GST-HD51 delta P resulted in the formation of numerous clusters of high-molecular-mass fibrils with a different, ribbon-like morphology. These structures were reminiscent of prion rods and beta-amyloid fibrils in Alzheimer's disease. In agreement with our previous results with full-length GST-HD exon 1, the truncated fusion proteins GST-HD20 delta P and GST-HD30 delta P did not show any tendency to form more highly ordered structures, either with or without protease treatment.

Studies of huntingtin localization in human post-mortem brain offer insights and a framework for basic experiments in the pathogenesis of Huntington's disease. In neurons of cortex and striatum, we identified changes in the cytoplasmic localization of huntingtin including a marked perinuclear accumulation of huntingtin and formation of multivesicular bodies, changes conceivably pointing to an altered handling of huntingtin in neurons. In Huntington's disease, huntingtin also accumulates in aberrant subcellular compartments such as nuclear and neuritic aggregates co-localized with ubiquitin. The site of protein aggregation is polyglutamine-dependent, both in juvenile-onset patients having more aggregates in the nucleus and in adult-onset patients presenting more neuritic aggregates. Studies in vitro reveal that the genesis of these aggregates and cell death are tied to cleavage of mutant huntingtin. However, we found that the aggregation of mutant huntingtin can be dissociated from the extent of cell death. Thus properties of mutant huntingtin more subtle than its aggregation, such as its proteolysis and protein interactions that affect vesicle trafficking and nuclear transport, might suffice to cause neurodegeneration in the striatum and cortex. We propose that mutant huntingtin engages multiple pathogenic pathways leading to neuronal death.

An increasing number of neurodegenerative disorders have been found to be caused by expanding CAG triplet repeats that code for polyglutamine. Huntington's disease (HD) is the most common of these disorders and dentatorubral-pallidoluysian atrophy (DRPLA) is very similar to HD, but is caused by mutation in a different gene, making them good models to study. In this review, we will concentrate on the roles of protein aggregation, nuclear localization and proteolytic processing in disease pathogenesis. In cell model studies of HD, we have found that truncated N-terminal portions of huntingtin (the HD gene product) with expanded repeats form more aggregates than longer or full length huntingtin polypeptides. These shorter fragments are also more prone to aggregate in the nucleus and cause more cell toxicity. Further experiments with huntingtin constructs harbouring exogenous nuclear import and nuclear export signals have implicated the nucleus in direct cell toxicity. We have made mouse models of HD and DRPLA using an N-terminal truncation of huntingtin (N171) and full-length atrophin-1 (the DRPLA gene product), respectively. In both models, diffuse neuronal nuclear staining and nuclear inclusion bodies are observed in animals expressing the expanded glutamine repeat protein, further implicating the nucleus as a primary site of neuronal dysfunction. Neuritic pathology is also observed in the HD mice. In the DRPLA mouse model, we have found that truncated fragments of atrophin-1 containing the glutamine repeat accumulate in the nucleus, suggesting that proteolysis may be critical for disease progression. Taken together, these data lead towards a model whereby proteolytic processing, nuclear localization and protein aggregation all contribute to pathogenesis.

Eight neurodegenerative diseases have been shown to be caused by the expansion of a polyglutamine stretch in specific target proteins that lead to a gain in toxic property. Most of these diseases have some features in common. A pathological threshold of 35-40 glutamine residues is observed in five of the diseases. The mutated proteins (or a polyglutamine-containing subfragment) form ubiquitinated aggregates in neurons of patients or mouse models, in most cases within the nucleus. We summarize the properties of a monoclonal antibody that recognizes specifically, in a Western blot, polyglutamine stretches longer than 35 glutamine residues with an affinity that increases with polyglutamine length. This indicates that the pathological threshold observed in five diseases corresponds to a conformational change creating a pathological epitope, most probably involved in the aggregation property of the carrier protein. We also show that a fragment of a normal protein carrying 38 glutamine residues is able to aggregate into regular fibrils in vitro. Finally, we present a cellular model in which the induced expression of a mutated full-length huntingtin protein leads to the formation of nuclear inclusions that share many characteristics with those observed in patients: those inclusions are ubiquitinated and contain only an N-terminal fragment of huntingtin. This model should thus be useful in studying a processing step that is likely to be important in the pathogenicity of mutated huntingtin.

Huntingtin was localized by using a series of antibodies that detected different areas of the protein from the immediate N-terminus to the C-terminal region of the protein. The more C-terminal antibodies gave a cytoplasmic localization in neurons of the brain in controls and cases of Huntington's disease (HD). The N-terminal antibody, however, gave a distinctive pattern of immunoreactivity in the HD brain, with marked staining of axon tracts and white matter and the detection of densely staining intranuclear inclusions. This implies some processing differences between mutated and normal huntingtin. We have also localized two interacting proteins, cystathionine beta-synthase and the nuclear receptor co-repressor (N-CoR), in brain. Cystathionine beta-synthase was not relocalized in HD brain, but the N-CoR was excluded from neuronal nuclei in HD brain, and a further protein that exists in the same repression complex, mSin3, was similarly excluded. We conclude that the co-repressor might have a part in HD pathology.

Polyglutamine (polyQ) extension in the coding sequence of mutant huntingtin causes neuronal degeneration associated with the formation of insoluble polyQ aggregates in Huntington's disease. We constructed an array of CAG/CAA triplet repeats, coding for a range of 25-300 glutamine residues, which was used to generate expression constructs with minimal flanking sequence. Normal-length (25 glutamine residues) polyQ did not aggregate when transfected alone. Remarkably, when co-transfected with extended (100-300 glutamine residues) polyQ tracts, normal-length polyQ-containing peptides were trapped in insoluble detergent-resistant aggregates. Aggregates formed in the cytoplasm but were visible in the nucleus only when a strong nuclear localization signal was present. Intermolecular interactions between polyQ tracts mediated the localization of heterogeneous aggregates into the nucleolus by nucleolin protein. Our results suggest that extended polyQ can interact with cellular polyQ-containing proteins, transport them to ectopic cellular locations, and form heterogeneous polyQ aggregates. We provide evidence for a recruitment mechanism for pathogenesis in the polyQ neurodegenerative disorders. In susceptible cells, extended polyQ tracts in huntingtin might interact with and sequester or deplete certain endogenous polyQ-containing cellular proteins.

Huntington's disease (HD) is a neurodegenerative disorder with a midlife onset. The disease is caused by expansion of a CAG (glutamine) repeat within the coding region of the HD gene. The molecular mechanism by which the mutated protein causes this disease is still unclear. To study the protein we have generated a set of rabbit polyclonal antibodies raised against different segments of the N-terminal, central and C-terminal parts of the protein. The polyclonal antibodies were affinity purified and characterized in ELISA and Western blotting experiments. All antibodies can react with mouse and human proteins. The specificity of these antibodies is underscored by their recognition of huntingtin with different repeat sizes in extracts prepared from patient-derived lymphoblasts. The antibodies were used in immunofluorescence experiments to study the subcellular localization of huntingtin in mouse neuroblastoma NIE-115 cells. The results indicate that most huntingtin is present in the cytoplasm, whereas a minor fraction is present in the nucleus. On differentiation of the NIE-115 cells in vitro, the subcellular distribution of huntingtin does not change significantly. These results suggest that full-length huntingtin with a normal repeat length can be detected in the nucleus of cycling and non-cycling cultured mammalian cells of neuronal origin. However, in HD autopsy brain the huntingtin-containing neuronal intranuclear inclusions can be detected only with antibodies raised against the N-terminus of huntingtin. Thus several forms of huntingtin display the propensity for nuclear localization, possibly with different functional consequences.

Different aspects of expanded polyglutamine tracts and of their pathogenetic role are taken into consideration here. (i) The (CAG)n length of wild-type alleles of the Huntington disease gene was analysed in instability-prone tumour tissue from colon cancer patients to test whether the process leading to the elongation of alleles towards the expansion range involves single-unit stepwise mutations or larger jumps. The analysis showed that length changes of a single unit had a relatively low frequency. (ii) The observation of an expanded spinocerebellar ataxia (SCA)1 allele with an unusual pattern of multiple CAT interruptions showed that cryptic sequence variations are critical not only for sequence length stability but also for the expression of the disease phenotype. (iii) Small expansions of the (CAG)n sequence at the CACNA1A gene have been reported as causing SCA6. The analysis of families with SCA6 and episodic ataxia type 2 showed that these phenotypes are, in fact, expressions of the same disorder caused either by point mutations or by small (CAG)n expansions. A gain of function has been hypothesized for all proteins containing an expanded polyglutamine stretch, including the alpha 1A subunit of the voltage-gated calcium channel type P/Q coded by the CACNA1A gene. Because point mutations at the same gene with similar phenotypic consequences are highly unlikely to have this effect, an alternative common pathogenetic mechanism for all these mutations, including small expansions, can be hypothesized.

A systematic search was performed for DNA sequence variation in genes regulating neurotransmitter metabolism in rhesus macaque (Macaca mulatta). These genes included dopamine and serotonin receptors and transporters, and tyrosine hydroxylase. A total of 13 single nucleotide polymorphisms in five different genes were identified, namely: DRD1 (-244T->G), q = 0.45; DRD1 (-179C->T), q = 0.19; DRD1 (-127G->A), q = 0.25; DRD1(-11T->G), q = 0.08; DRD1(-81C->T), q = 0.19; DRD3 (248G->A), q= 0.08; DRD3(341G->C), q = 0.11; DRD3(377A->G), q = 0.19; DRD3 (403C->T; A59V), q= 0.11; DRD4(2608G->A), q= 0.48; HTR1D(-506G->T), q = 0.47; HTR1D(-173C->T), q = 0.47; and HTT(340G->A), q = 0.39. The nucleotide positions listed correspond to the human homologs.

The degree of glucose intolerance and diabetes mellitus in hemochromatosis is closely associated with the stage of iron overload and thus also the stage of the accompanying liver disease. Similar to other liver diseases glucose intolerance due to insulin resistance precedes diabetes mellitus also in hemochromatosis. Insulin resistance is probably caused by alterations of insulin or glucose metabolism in the liver and potentially also in extrahepatic peripheral tissue. Diabetes mellitus is found more frequently in iron overload when compared with other forms of chronic liver disease and cirrhosis, respectively. In advanced iron overload, iron accumulation in pancreatic B-cells deteriorates pancreatic insulin secretion and leads to insulin-dependent diabetes mellitus which cannot be reversed by iron removal. In contrast, early alteration of glucose intolerance and insulin resistance may partially be improved by phlebotomies. Thus, in the future we should aim to diagnose hemochromatosis in early stages which are not associated with diabetes mellitus or liver cirrhosis.

<h4>Purpose</h4>Whether or not the deleted in colorectal cancer (DCC) gene is implicated in metastases or in predicting prognosis in patients with colorectal cancer has not previously been substantiated. Our aims were to investigate DCC expression in primary colorectal cancers and in metastases to identify any prognostic significance.<h4>Patients and methods</h4>DCC expression was examined immunohistochemically in 195 primary colorectal adenocarcinomas and in 23 paired primary tumors and lymph node metastases. DNA content and S-phase fraction were measured by flow cytometry.<h4>Results</h4>The absence of DCC expression was observed in 55 primary tumors (28%). DCC negativity was significantly related to poor prognosis in patients with DNA diploid tumors (P =.03) and those with a low S-phase fraction (< 5%, P =.02) but not in patients with nondiploid tumors or those with a higher S-phase fraction. Furthermore, DCC expression retained its prognostic significance in the diploid subgroup after adjusting for sex, age, site, stage, growth pattern, and differentiation (P =.01). DCC expression was similar in primary tumors and their metastases.<h4>Conclusion</h4>The absence of DCC predicted a poor outcome in the patients with diploid tumors and those tumors with a low S-phase fraction. Immunohistochemistry may be considered as a practical test to assess prognosis in this subgroup of patients.

<h4>Background</h4>10% of sporadic colorectal cancers are characterised by a low level of microsatellite instability (MSI-L). These are not thought to differ substantially from microsatelite-stable (MSS) cancers, but MSI-L and MSS cancers are distinguished clinicopathologically and in their spectrum of genetic alterations from cancers showing high level microsatellite instability (MSI-H).<h4>Aims</h4>To study the distribution of molecular alterations in a series of colorectal cancers stratified by DNA microsatellite instability.<h4>Methods</h4>A subset of an unselected series of colorectal cancers was grouped by the finding of DNA MSI at 0 loci (MSS) (n = 51), 1-2 loci (MSI-L) (n = 38) and 3-6 loci (MSI-H) (n = 25). The frequency of K-ras mutation, loss of heterozygosity (LOH) at 5q, 17p and 18q, and patterns of p53 and beta catenin immunohistochemistry was determined in the three groups.<h4>Results</h4>MSI-H cancers had a low frequency of K-ras mutation (7%), LOH on chromosomes 5q (0%), 17p (0%) and 18q (12.5%), and a normal pattern of immunostaining for p53 and beta catenin. MSI-L cancers differed from MSS cancers in terms of a higher frequency of K-ras mutation (54% v 27%) (p = 0.01) and lower frequency of 5q LOH (23% v 48%) (p = 0.047). Whereas aberrant beta catenin expression and 5q LOH were concordant (both present or both absent) in 57% of MSS cancers, concordance was observed in only 20% of MSI-L cancers (p = 0.01).<h4>Conclusions</h4>MSI-L colorectal cancers are distinct from both MSI-H and MSS cancers. This subset combines features of the suppressor and mutator pathways, may be more dependent on K-ras than on the APC gene in the early stages of neoplastic evolution, and a proportion may be related histogenetically to the serrated (hyperplastic) polyp.

Sequencing of HFE exons 2, 3, 4, and 5, and of portions of introns 2, 4, and 5 revealed novel mutations in four of twenty hemochromatosis probands who lacked C282Y homozygosity, C282Y/H63D compound heterozygosity, or H63D homozygosity. Probands 1 and 2 were heterozygous for previously undescribed mutations: exon 2, nt 314T-->C (314C; I105T), and exon 2, nt 277G-->(277C; G93R), respectively; these probands were also heterozygous for H63D and C282Y, respectively. Probands 3 and 4 were heterozygous for a previously described but uncommon HFE mutation: exon 2, nt 193A-->T (193T; S65C). Proband 3 was also heterozygous for C282Y and had porphyria cutanea tarda, and Proband 4 had hereditary stomatocytosis. Each of these four probands had iron overload. In each proband with an uncommon HFE coding region mutation, I105T, G93R, and S65C occurred on separate chromosomes from those with the C282Y or H63D mutations. Neither I105T, G93R, nor S65C occurred as spontaneous mutations in our probands. The I105T and G93R mutations were linked to haplotypes bearing HLA-A3,-B7 and HLA-A2,-B62, respectively. The S65C mutation was linked to a haplotype characterized by HLA-32. Sixteen other probands did not have an uncommon HFE exon mutation. In 176 normal control subjects, two were heterozygous for S65C, but I105T and G93R were not detected. Nine of twenty probands were heterozygous and two probands were homozygous for a previously described base-pair change at intron 2, nt 3671T-->C. One proband without a detectable missense mutation had a previously described intron 5 allele (nt 6700G-->A). Heterozygosity for a previously described base-pair change in intron 4 (nt 5636T-->C) was detected in all persons we studied who also had the S65C mutation. One proband was heterozygous for a previously undescribed base-pair change at intron 5 (nt 5807A-->G). We conclude that uncommon HFE exon and intron mutations may be discovered among hemochromatosis patients who have "atypical" HFE genotypes.

An increasing number of studies demonstrate a lack of phenotypic expression in subjects found to be homozygous for the common hereditary hemochromatosis (HH) mutation, C282Y. In this study the impact of possible overestimation of C282Y homozygosity, as a consequence of a MseI polymorphism identified in intron 4 of the HFE gene, was investigated in South African subjects. Utilization of a modified polymerase chain reaction (PCR)-based assay highlighted the potential implications with respect to genotype/phenotype correlation studies, particularly in the general population. Mistyping rather than lack of disease association provides a plausible explanation for the phenomenon of C282Y homozygosity without iron overload. Reassessment of C282Y mutation status in such cases may result in justified population screening in HH.

The prefrontal cortex participates in motor control and is modulated by serotonergic activity. The serotonin transporter (5-HTT) is a major regulator of serotonergic neurotransmission and may thus influence motor control. The short allele (s) of the 5-HTT linked polymorphic region (5-HTTLPR) is associated with less 5-HTT expression and function than the long variant (l). The neurophysiological parameters termed 'Go- and NoGo- centroid location' represent characteristic brain electrical substrates of the execution and inhibition of motor response elicited by the Continuous Performance Test (CPT). In the present study, the impact of the 5-HTTLPR genotype on the centroid locations was investigated in 23 healthy subjects. The NoGo-centroid, but not the Go-centroid, was located significantly more anteriorly in the short allele group (mean electrode location in s/s and s/l, 2.86+/-0.37) compared to the group with two long alleles (l/l, 3.34+/-0.49; t=2.66, p<0.05). Age, gender, and test performance did not differ between groups. The results indicate that 5-HTTLPR genotype dependent 5-HTT function is associated with the neurophysiologically assessed topography of inhibitory motor control and provides further evidence for a genetic influence on central serotonergic and motor function.

In order to assess the role of the changes of DCC and APC/MCC genes in the development and progression of gastric cancer, the loss of heterozygosity (LOH) of these genetic loci was investigated in 45 surgical specimens of gastric cancer with PCR-RFLP. The rate of LOH was 30.0% (9/30) at APC/MCC gene and 33.3% (15/45) at DCC gene. LOH was found in both intestinal and gastric types of gastric cancer and the rate of LOH of DCC gene was significantly higher in stages III to approximately IV gastric cancer (48.0%) than in stages I to approximately II (15.0%) (P<0.05). LOH of APC/MCC gene could be found in both early and advanced stages of gastric cancer. These findings suggest that changes of DCC and APC/MCC genes are involved in the development and progression of the intestinal and gastric types of gastric cancer.

No abstract available.