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Loss of heterozygosity (LOH) occurring on various chromosomes has been described in the majority of human tumors and its targets are believed to be tumor suppressor genes. Although allelic deletion of the p53 gene (over 60%) has been frequently observed in gastric cancer, as well as in other human malignancies, LOH of other tumor suppressor genes is still discrepant in gastric cancer. To our knowledge, simultaneous analysis of LOH using PCR in adenomatous polyposis coli (APC), deleted in colon cancer (DCC), and retinoblastoma susceptibility (Rb) genes has not yet been reported in sporadic gastric cancer. We examined 21 advanced gastric cancers (12 intestinal type and 9 diffuse type) for LOH at the APC, DCC, and Rb loci using PCR. Inclusion of these tumor suppressor genes in the allelic deletions was directly ascertained by performing PCR at polymorphic sites within the genes. LOH occurred in 30% of informative cases at APC, in 27% of informative cases at DCC, and in 30% of informative cases at Rb. Thirty-three percent of tumors informative at all loci (fully informative) lost heterozygosity at all three loci. There were no significant differences among histologic types in the prevalence of LOH at any locus and no correlations between losses involving APC, DCC, and Rb genes. These data suggest that inactivation of APC, DCC, and Rb is involved in the development and progression of some human gastric cancers regardless of histologic type.

<h4>Background</h4>The mortality of patients with colorectal carcinoma depends mainly upon subsequent liver metastasis even after curative operation. However, it is very difficult to predict the incidence of liver metastasis by analyzing conventional tumor markers or pathologic findings. In the current study, the authors examined the genetic alterations of p53/DCC loci and Ki-ras in colorectal carcinoma in relation to liver metastasis, and investigated whether these gene alterations could be prognostic markers for liver metastasis.<h4>Methods</h4>Primary tumor tissue samples were collected at surgery from 30 patients with no liver metastasis and 24 patients with liver metastasis. Among the 30 patients with no liver metastasis at surgery, 9 developed liver metastasis after surgery. After the extraction of DNA, we investigated the loss of heterozygosity at p53/DCC loci and mutations of Ki-ras codon 12 in the colorectal carcinoma tissue from these patients.<h4>Results</h4>The incidence of allelic loss of the DCC locus was significantly greater for patients with liver metastasis that for patients who had no liver metastasis for more than 2 years (19/20:95% vs 2/5:40%, P < 0.05). However, mutations of Ki-ras codon 12 were significantly less in patients with liver metastasis than in patients with no liver metastasis for more than 2 years (6/33:18% vs 6/11:55%, P < 0.05).<h4>Conclusion</h4>The current study indicated that detection of allelic loss of DCC and absence of Ki-ras codon 12 mutations are associated with the metastatic potential of colorectal carcinoma in the liver. These results suggested that these gene alterations might be reliable biologic markers for assessing the potential of liver metastasis after colorectal resection.

The expression of the constituent alpha 1 chain of laminin-1, a major component of basement membranes, is markedly regulated during development and differentiation. We have designed an antisense RNA strategy to analyze the direct involvement of the alpha 1 chain in laminin assembly, basement membrane formation, and cell differentiation. We report that the absence of alpha 1-chain expression, resulting from the stable transfection of the human colonic cancer Caco2 cells with an eukaryotic expression vector comprising a cDNA fragment of the alpha 1 chain inserted in an antisense orientation, led to (a) an incorrect secretion of the two other constituent chains of laminin-1, the beta 1/gamma 1 chains, (b) the lack of basement membrane assembly when Caco2-deficient cells were cultured on top of fibroblasts, assessed by the absence of collagen IV and nidogen deposition, and (c) changes in the structural polarity of cells accompanied by the inhibition of an apical digestive enzyme, sucrase-isomaltase. The results demonstrate that the alpha 1 chain is required for secretion of laminin-1 and for the assembly of basement membrane network. Furthermore, expression of the laminin alpha 1-chain gene may be a regulatory element in determining cell differentiation.

Fluorescent polymerase chain reaction (PCR) was used to assay 12 microsatellite markers (APC x 2, DCC, P53 x 2, RB1, NM23, WT1, D6S260, D6S262, D6S281 and TNFa) to look for evidence of microsatellite instability in 40 cases of follicle centre cell lymphoma (FCC). Evidence of novel alleles seen in the tumour tissue but not the normal uninvolved tissue was seen in seven cases (17%). In only two of these cases (5%) was more than one locus involved but in these cases multiple affected loci were seen (4/12 and 7/12 respectively). The detection of microsatellite instability indicates a DNA repair defect such as that which would be predicted to occur in cells with mutated mismatch repair genes, a novel finding in FCC lymphoma.

Iodine-123-labelled 3beta-(4-iodophenyl)tropane-2beta-carboxylic acid ([123I]beta-CIT) labels both the dopamine transporter (DAT) and the serotonin transporter (5-HTT) and this ligand is able to clarify pathological changes in both dopaminergic and serotonergic systems. However, the differential kinetics of beta-CIT binding to DAT and 5-HTT has not been clarified fully. In this study we examined time-activity curves of [123I]beta-CIT in individual regions in the rat brain. Using cerebellum as the reference region, k3 and k4 values were estimated by a two-compartment kinetic analysis. In the striatum, the kinetics was slowest among all brain areas. In this area specific binding reached its peak 4 h after the injection. In the hypothalamus, specific binding reached its peak 1 h after the injection and its amount did not change until 4 h after the injection. In the occipital cortex, the binding and washout of the ligand were fastest among all brain regions. Estimated k3 values were 0.040+/-0.003 in the striatum, 0.019+/-0.002 in the hypothalamus and 0.082+/-0.011 in the occipital cortex (min-1, mean +/-SD). Estimated k4 values were 0.0034+/-0.0005 in the striatum, 0.0071+/-0.0009 in the hypothalamus and 0.083+/-0.013 in the occipital cortex (min-1, mean +/-SD). Therefore binding kinetics of [123i]beta-cit in the region rich in dat is apparently different from that in the region rich in 5-HTT. These results will provide fundamental data to image both DAT and 5-HTT in one series of examinations with [123I]beta-CIT.

Recent findings have firmly established the existence of circular exons in vivo. We were interested in the possible splicing mechanism by which these unusual mRNA molecules could be created in vitro, though no biological relevance has been attached to their existence as yet. In this report we demonstrate that a modified synthetic linear yeast ACT1 transcript whose sequence begins with the 3'-part of its original intron, is continued by 247 nt of exon sequence and terminates with the 5'-part of its intron will generate a circular exon when introduced to standard in vitro splicing reactions in whole cell splice extracts from Saccharomyces cerevisiae. The formation of a circular exon was found to be independent of specific circular or secondary structures of the pre-mRNA transcript. We hypothesize that circular exons which are found in vivo may be generated from pre-mRNAs which derive from rare events of transcription initiation within an intron.

Thrombin is a multifunctional protein that has both proteinase and growth factor-like activities. Its regulation is largely mediated by interaction with a host of inhibitors including antithrombin III (ATIII), heparin cofactor II (HCII), alpha2-macroglobulin (alpha2-M), protease nexin I, and plasminogen activator inhibitor-1 (PAI-1). ATIII, HCII, and alpha2-M are all abundant in blood and can inactivate blood-borne thrombin leading to rapid hepatic clearance of the thrombin-inhibitor complex. PAI-1 alone, a poor solution phase inhibitor of thrombin, can efficiently inhibit thrombin in the presence of native vitronectin (VN). In this study, active thrombin was found to be efficiently endocytosed and degraded by cultured pre-type II pneumocyte cells, and both processes could be blocked by polyclonal antibodies to PAI-1. When the relative efficiency of cellular endocytosis of thrombin in complex with a number of inhibitors was examined, 125I-thrombin-PAI-1 complexes were most efficiently cleared compared to 125I-thrombin in complex with the serpins ATIII, HCII, alpha1-proteinase inhibitor, or d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone. Low density lipoprotein receptor-related proteins 1 (LRP) and 2 (gp330/megalin) mediate the endocytosis of thrombin-PAI-1, since antagonists of receptor function such as LRP-1 and LRP-2 antibodies and the 39-kDa receptor-associated protein blocked 125I-thrombin-PAI-1 endocytosis and degradation. The LRP-mediated clearance of exogenously added 125I-thrombin by cultured cells was found to be enhanced 5-fold by inclusion of wild-type PAI-1 but by only 2-fold when a mutant form of PAI-1 that is unable to bind VN was included. This wild-type PAI-1 enhancement of 125I-thrombin clearance was found to occur only in the presence of native VN and not with its conformationally altered form. The results highlight a novel mechanism for cellular clearance of thrombin involving native VN promoting the interaction of thrombin and PAI-1 and the subsequent endocytosis of the complex by LRP-1 or LRP-2. This pathway is potentially important for the regulation of the potent biological activities of thrombin, particularly at sites of vascular injury.

<h4>Purpose</h4>Allelic losses within several tumor suppressor genes have been detected frequently in various types of human cancer. We investigated the roles and possible interactions of the tumor suppressor genes p53, Rb, DCC and APC in bladder cancer.<h4>Materials and methods</h4>Loss of heterozygosity (LOH) of these 4 genes was examined in 45 human bladder cancers by polymerase chain reaction and restriction fragment length polymorphism assay.<h4>Results</h4>Of the evaluated cases, LOH was seen at P53 in 38%, at Rb in 22%, at DCC in 36% and at APC in 6% of tumors. Loss of heterozygosity at p53 and Rb was predominantly observed in high grade (grade 3) and/or invasive (T2 or greater) tumors, whereas LOH at DCC was present irrespective of tumor grade and stage. Allelic losses at either p53, Rb, DCC or APC were seen in 82% of high grade tumors, but in only 21% of low grade (grade 1 and 2) tumors (p<0.005). Similarly, 71% of invasive tumors had LOH at one or more loci compared with 20% of superficial (Ta and T1) tumors (p<0.005). Interestingly, p53-LOH and Rb-LOH were often observed simultaneously in the same tumor.<h4>Conclusions</h4>These results suggest that loss of the p53, Rb and/or DCC genes is involved in most of the late and some of the early steps of bladder carcinogenesis.

We report the isolation of 10 differentially expressed cDNAs in the process of apoptosis induced by the p53 tamor suppressor. As a global analytical method, we performed a differential display of mRNA between mouse M1 myeloid leukemia cells and derived clone LTR6 cells, which contain a stably transfected temperature-sensitive mutant of p53. At 32 degrees C wild-type p53 function is activated in LTR6 cells, resulting in programmed cell death. Eight genes are activated (TSAP; tumor suppressor activated pathway), and two are inhibited (TSIP, tumor suppressor inhibited pathway) in their expression. None of the 10 sequences has hitherto been recognized as part of the p53 signaling pathway. Three TSAPs are homologous to known genes. TSAP1 corresponds to phospholipase C beta 4. TSAP2 has a conserved domain homologous to a multiple endocrine neoplasia I (ZFM1) candidate gene. TSAP3 is the mouse homologue of the Drosophila seven in absentia gene. These data provide novel molecules involved in the pathway of wild-type p53 activation. They establish a functional link between a homologue of a conserved developmental Drosophila gene and signal transduction in tumor suppression leading to programmed cell death.

Hereditary mixed polyposis syndrome (HMPS) is characterized by atypical juvenile polyps, colonic adenomas, and colorectal carcinomas. HMPS appears to be inherited in an autosomal dominant manner. Genetic linkage analysis has been performed on a large family with HMPS. Data did not support linkage to the APC locus or to any of the loci for hereditary nonpolyposis colorectal cancer. Evidence that the HMPS locus lies on chromosome 6q was, however, provided by significant two-point LOD scores for linkage between HMPS and the D6S283 locus. Analysis of recombinants and multipoint linkage analysis suggested that the HMPS locus lies in a 4-cM interval containing the D6S283 locus and flanked by markers D6S468 and D6S301.

Soft tissue sarcomas represent a heterogeneous group of mesenchymal malignancies, and the majority of the previous scientific studies that have analyzed the occurrence of cell cycle regulators aberrations within soft tissue sarcomas have dealt with broad categories of different tumors. As a consequence, data concerning single classes of sarcomas are very limited. The authors analyze herein a histologically homogeneous series of 23 cases of leiomyosarcoma of the deep soft tissue. The p53 pathway was studied by investigating the p53 gene and protein, MDM2 protein, and p21waf1 protein. The Rb-cyclin D pathway was analyzed by studying the Rb gene and protein, p16MTS1/INK4A gene and protein, cyclin D1Prad1/bcl1 and cyclin D3 proteins. Aberrations of the p53 pathway were observed in about 16 percent of cases and were limited to the p53 gene. Such a finding contrasts with the higher rates of p53/MDM2 abnormalities reported in other types of sarcomas such as liposarcoma. Interestingly, abnormalities involving the Rb-cyclin D pathway were detected in about 90 percent of cases. The Rb-cyclin D pathway therefore emerges as the preferred target for molecular abnormalities in this subset of soft tissue sarcomas.

<h4>Aims</h4>To investigate whether colonic cancer cells exfoliated into the lumen of the organ can be detected by identification of their abnormal CD44 gene products.<h4>Methods</h4>Exfoliated cells were obtained by centrifugation of saline wash-outs of 27 surgically resected colon specimens obtained from 15 patients with carcinoma, seven with ulcerative colitis and five with Crohn's disease. After extracting cellular mRNA, amplification by the reverse transcription-polymerase chain reaction (RT-PCR) technique and analysis by Southern blot hybridisation was carried out to examine the levels and patterns of transcription of exons 11(v6), and 12(v7) and intron 9 of the CD44 gene. The transcription of these CD44 components was also examined by RT-PCR of snap-frozen solid tissue specimens from 11 of the above patients with colorectal carcinoma, seven with ulcerative colitis and five with Crohn's disease.<h4>Results</h4>Abnormal expression of exons 11(v6) and 12(v7) was detected in exfoliated cells from 11 (73%) of 15 patients with carcinoma, but not in any patients with inflammatory bowel disease (IBD). The retention of intron 9 in CD44 mRNA transcripts was detected in washings from four (27%) carcinoma specimens but not in washings from non-malignant specimens. It was confirmed that in solid tissue samples from the same carcinomas there was abnormal over-expression of numerous alternatively spliced CD44 species containing transcripts of exons 11 and 12 and retention of intron 9. Low level expression of these exons was detected in tissue from inflammatory lesions from five of seven patients with ulcerative colitis and four of five with Crohn's disease. The retention of intron 9 was not seen in normal mucosa nor IBD.<h4>Conclusion</h4>Abnormal expression of the variant exons and of intron 9 of the CD44 gene in tumour cells exfoliated into the colonic lumen may be helpful markers for the early, non-invasive, diagnosis of colorectal cancer.

We have constructed a yeast artificial chromosome (YAC) contig over the candidate hemochromatosis gene region. This contig comprises 16 YACs from the CEPH, Washington University, and ICI YAC libraries and covers 4.5 Mb at 6p21.3-6p22. The complete contig has been restriction mapped, enabling the precise relationship between the YACs to be determined and the mapping of a total of 12 STSs. Nine of these are highly polymorphic STSs that are closely linked to hemochromatosis; this series includes D6S265 and D6S1260, which comprise the most proximal and distal markers linked to HC. This is the first YAC contig that spans the hemochromatosis candidate region, and it provides valuable resource material for the cloning of this and other genes in the region distal to the MHC class I complex.

Neonatal exposure to antigen is believed to result in T cell clonal inactivation or deletion. Here we report that, contrary to this notion, neonatal injection of BALB/c mice with a hen egg lysozyme peptide 106-116 in putative "tolergenic" doses induced a T cell proliferative and an immunoglobulin G (IgG) antibody (Ab) response of both T helper cell 1 (Th1)- (IgG2a, IgG2b, and IgG 3) and Th2-dependent (IgG1) isotopes. Upon subsequent challenge with the peptide in complete Freund's adjuvant in adult life, although this neonatal regimen suppressed proliferation and the production of Th1 cytokines (interleukin[IL]-2 and interferon gamma), Th2 cytokine (IL-5, IL-4, and IL-10) secretion was increased, and the serum levels of Th1- and Th2-dependent isotypes of peptide-specific Ab remained elevated. The in vitro proliferative unresponsiveness in Th1 cells could be reversed by Abs to Th2 cytokines (IL-4 and IL-10). Thus, neonatal treatment with a peptide antigen induces T cell priming including production of IgG Abs of both Th1- and Th2-dependent isotypes. Upon subsequent peptide exposure, the peptide-specific T cell responses undergo an effective class switch in the direction of Th2, resulting in T cell proliferative unresponsiveness. Accordingly, this shift towards increased Ab production to autoantigen could be deleterious in individuals prone to antibody-mediated diseases. Indeed, neonatal treatment with a self-autoantigenic peptide from an anti-DNA monoclonal Ab (A6H 58-69) significantly increased the IgG anti-double-stranded DNA Ab levels in lupus-prone NZB/NZW F1 mice, despite suppressing peptide-specific T cell proliferation. This adverse clinical response is in sharp contrast to the beneficial outcome of neonatal treatment with autoantigens in Th1-mediated autoimmune diseases, such as autoimmune encephalomyelitis, as reported by others. A Th1 to Th2 immune deviation can explain the discordant biological responses after the presumed induction of neonatal tolerance in autoantibody- vs. Th-1 mediated autoimmune diseases.

Site-specific recombination of immunoglobulin and T cell receptor gene segments in B and T lymphocytes is dependent on the expression of two recombinant activation genes, Rag-1 and Rag-2. Here, we show that RAG-1 protein turnover in pre-B cells depends on the expression of RAG-2. The apparent half-life of RAG-1 protein is increased when RAG-2 is not expressed in differentiating pre-B cells.

Regularly spaced nucleosomal arrays equilibrate between unfolded and highly folded conformations in <2 mM MgCl2, and self-associate above 2 mM MgCl2 [Schwarz, P. M., & Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. Here we use analytical and differential sedimentation techniques to characterize the molecular mechanism and determinants of oligonucleosome self-association. Divalent cations induce self-association of intact nucleosomal arrays by binding to oligonucleosomal DNA and neutralizing its negative charge. Neither linker histones nor H2A/H2B dimers are required for Mg2+ - dependent self-association. However, divalent cations are unable to induce self-association of trypsinized nucleosomal arrays lacking their N- and C-terminal core histone tail domains. This suggests that the H3/H4 tail domains directly mediate oligonucleosome self-association through a non-Coulombic-based mechanism. Self-association occurs independently of whether the oligonucleosome monomers are folded or unfolded. The first step in the self-association pathway is strongly cooperative and produces a soluble association intermediate that sediments approximately 10 times faster than the oligonucleosome monomers. The size of the oligonucleosome polymers increases rapidly as a consequence of small increases in the divalent cation concentration, eventually producing polymeric species that sediment at >> 10 000 S. Importantly, all steps in the self-association pathway are freely reversible upon removal of the divalent cations. Taken together, these data indicate that short oligonucleosome fragments composed of only core histone octamers and DNA possess all of the structural features required to achieve chromosome-level DNA compaction. These findings provide a molecular basis for explaining many of the recently uncovered structural features of interphase and metaphase chromosomal fibers.

The scenario of multistep of stomach carcinogenesis differs depending on the two histological types, well differentiated adenocarcinoma and poorly differentiated adenocarcinoma, because the two types may have different genetic pathways. Genetic instability, reactivation of telomerase and abnormal transcript of CD44 including intron 9 are common events of both well and poorly differentiated type carcinomas. These occur at early stage of carcinogenesis, even in precancerous lesions such as intestinal metaplasia and adenoma. Inactivation of APC, activation of K-ras, amplification of c-erbB2, and allelic loss of DCC locus are associated with well differentiated type, while amplification of K-sam and functional loss of cadherin/catenin are characteristics of poorly differentiated type. HGF/c-met system plays a pivotal role in morphogenesis of both histological types through interaction with cell-cell adhesion molecules. Reactivation of telomerase or genetic instability may be an initial event for accumulation of multiple genetic alterations during the progression of stomach carcinogenesis.

We described a clinicopathological study of primary hepatoma associated with alcoholic liver diseases without viral liver diseases. In 150 patients with primary hepatoma, 6 patients (4%) have hepatoma associated with pure alcoholic liver disease, although 143 hepatoma were associated with chronic viral liver diseases and one was with primary biliary cirrhosis. All patients were male. The diagnosis of hepatoma was obtained at the age of 54 to 67 years old, and the duration of ethanol intake was 33 to 40 years. Three cases had a history of temperance. As an underlying liver disease, liver fibrosis was found in 3 cases and liver cirrhosis was in 3 cases. Chronic infections of hepatitis B and C viruses were ruled out by assaying serum virus markers. Autoimmune hepatitis and primary biliary cirrhosis were neglected by serum autoantibody. Hemochromatosis and Wilson's disease were also excluded. Hepatocellular carcinoma was diagnosed histologically in all the cases. Serum alpha-fetoprotein and PIVKA-II were positive in patients with advanced hepatocellular carcinoma. In cases with small hepatoma, the tumor was resected surgically in two cases and percutaneous ethanol injection against hepatoma was performed in one case. In these cases with small hepatoma, the patients were alive without tumor recurrence during observation period. In advanced hepatoma, transcatheter arterial infusion of anticancer agent was performed in two cases and no therapy was performed due to poor general condition in one case. One case was alive with recurrent hepatoma for 27 months, during which a therapy was repeated five times. Other 2 cases were died. The clinicopathological features of hepatoma associated with alcoholic liver disease were essentially same as those associated with chronic viral infection, although the incidence of hepatoma in alcoholic liver disease was lower than in viral liver disease. The mechanism of hepatocarcinogenesis in alcoholic liver disease was unclear and, therefore, further study of molecular biology and biochemistry was necessary.

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. The majority of patients who develop HCC have underlying cirrhosis, which suggests that cirrhosis itself represents a preneoplastic condition. Nevertheless, whereas patients with cirrhosis of any origin are at increased risk of developing HCC, those with chronic hepatitis B or C infection seem to be at greatest risk. Patients with cirrhosis resulting from chronic alcohol use, hemochromatosis, autoimmune hepatitis, or alpha-1 antitrypsin deficiency have less risk of developing this cancer, and some hepatic diseases, such as primary biliary cirrhosis and Wilson's disease, do not predispose affected persons to an appreciable risk of developing HCC. Certain histological features, such as liver cell dysplasia and macroregenerative nodules, may represent preneoplastic alterations of hepatocytes, but these changes do not seem to be a necessary step in the evolution of liver cancer. The pathogenesis of HCC is unclear, but seems to involve several steps. Hepatitis B virus infection may result in the malignant transformation of hepatocytes by some directly oncogenic mechanism, whereas other necroinflammatory conditions probably predispose to the development of HCC through the introduction of genetic alterations coupled with a reduction of genetic repair functions. Screening patients at risk for the development of HCC using alpha fetoprotein measurements and ultrasonography is widely practiced despite inconclusive evidence of efficacy. If screening is performed, the program used should be tailored to the perceived risk for a particular patient.

<h4>Objectives</h4>To evaluate new ELISA for measurement of thrombin-antithrombin III (TAT) concentration, and to correlate the values to other tests of hemostasis in horses with colic.<h4>Design</h4>Plasma TAT concentration and 8 other hemostasis analytes were measured in horses with colic at hospital admission and during the next 4 days. Retrospectively, data were analyzed by outcome, broad-category diagnosis, and clinical management, and for correlation between TAT and other assays.<h4>Animals</h4>100 horses with colic.<h4>Procedure</h4>Plasma samples were evaluated for TAT, fibrinogen, and fibrin degradation products concentrations; antithrombin III (ATIII), protein C, alpha 2-antiplasmin, and plasminogen activities; prothrombin time (PT); and activated partial thromboplastin time.<h4>Results</h4>Changes were indicative of a hypercoagulable state, most severe in nonsurviving horses, characterized by increased TAT concentration; decreased ATIII, protein C, and plasminogen activities; and increased PT. Nonsurvivors had significantly increased TAT concentration compared with that in survivors, without regard to sample collection time; however, compared over time, TAT was significantly increased only at admission. Highest TAT concentration was in nonsurvivors with inflammatory intestinal lesions. There was significant negative correlation between TAT and ATIII, protein C, alpha 2-antiplasmin, and plasminogen values, and significant positive correlation between TAT and PT, and fibrin degradation products values.<h4>Conclusions</h4>Plasma TAT reflects the current state of coagulation system activation and is a good assay for early diagnosis of the hypercoagulable state in horses with the most severe forms of colic.<h4>Clinical relevance</h4>Measurement of equine TAT provides further information to characterize the hypercoagulable state in horses to aid in case management.

The serotonin transporter (HTT) is an important candidate gene for the genetic transmission of bipolar disorder. It is the site of action of many antidepressants, and plays a key role in the regulation of serotonin neurotransmission. Many studies of affectively ill patients have found abnormalities in serotonin metabolism, and dysregulation of the transporter itself. The human serotonin transporter has been recently cloned and mapped to chromosome 17. We have identified a PstI RFLP at the HTT locus, and here report our examination of this polymorphism for possible linkage to bipolar disorder. Eighteen families were examined from three populations: the Old Order Amish, Iceland, and the general North American population. In addition to HTT, three other microsatellite markers were examined, which span an interval known to contain HTT. Linkage analyses were conducted under both dominant and recessive models, as well as both narrow (bipolar only) and broad (bipolar + recurrent unipolar) diagnostic models. Linkage could be excluded to HTT under all models examined. Linkage to the interval spanned by the microsatellites was similarly excluded under the dominant models. In two individual families, maximum lod scores of 1.02 and 0.84 were obtained at D17S798 and HTT, respectively. However, these data overall do not support the presence of a susceptibility locus for bipolar disorder near the serotonin transporter.

We evaluated the changes over time in hemostatic factors during ongoing arterial thrombosis in rats, as induced by a loop-shaped aortic prosthesis. Moreover, we investigated this condition by inspecting in parallel local thrombus growth, systemic vascular prostacyclin and t-PA production. One minute after loop insertion, activated platelets spread on the internal surface of the prosthesis and 24 hrs later numerous platelet aggregates supported by a fibrin network could be observed. However, no evidence for platelet activation could be concomitantly found in peripheral blood. A sustained increased in PGI2 formation was detected together with a progressive increase in plasma fibrinolytic activity during thrombus growth. The levels of fibrinogen as well as antithrombin III (ATIII) and heparin cofactor II (HCII) activities were steadily increased in loop-bearing animals. In conclusion, the dynamic phases of thrombus formation, in an aortic prosthesis, produce changes in vascular function and in hemostatic factors at the level of systemic blood.

A simple, rapid, and highly efficient method for intramolecular disulfide formation in tryptophan-containing peptides using hydrogen peroxide was elaborated. Solid phase synthesis of the peptide fragment corresponding to 601-617 sequence of transmembrane gp41 glycoprotein of HIV-1 was performed by Fmoc-technique. Coupling of Fmoc-Asn-OH by DCC-HOBt method was shown to be accompanied by a side reaction of dehydration of asparagine amide function with the formation of side product (22%) containing 3-cyanoalanine residue. This side reaction was not observed, when Fmoc-Asn-OH was coupled in the form of its p-nitrophenyl ester and with HOBt as a catalyst.

The motor pattern that drives each crayfish swimmeret consists of alternating bursts of impulses in power-stroke (PS) and return-stroke (RS) motor neurons. A model of the neural circuit that generates this pattern focused on connections between motor neurons themselves (Heitler, 1978, 1981). The model predicts that synergist motor neurons are electrically coupled, whereas antagonists make mostly inhibitory synapses. We tested this model by observing the responses of motor neurons to pressure ejection of GABA and glutamate, transmitters that crayfish motor neurons release at neuromuscular junctions, and by measuring the strengths and delays of synapses between pairs of motor neurons. Both GABA and glutamate inhibited motor neurons. This inhibition persisted when synaptic transmitter release was blocked by high Mg2+. The effects of GABA were mimicked by muscimol, but not by baclofen or the GABAc receptor agonist cis-4-aminocrotonic acid, and they were not blocked by bicuculline. The effects of glutamate were mimicked by ibotenic acid. Picrotoxin partially blocked glutamate's inhibition of the motor pattern, but did not affect GABA responses. Most (87%) pairs of synergist motor neurons tested made weak, noninverting connections. Approximately half of these had synaptic delays of <2 msec, consistent with direct electrical or chemical synapses. Individual motor neurons were dye-coupled to between one and three other motor neurons, and to interneurons. Less than half (44%) of the pairs of antagonist motor neurons tested made synaptic connections. These connections were weak, had long latencies (>4 msec), and therefore were probably polysynaptic. We conclude that direct synapses between swimmeret motor neurons cannot account for alternation of PS and RS bursts.

The effects of chronic novel stressors, for 21 days, on the behavior and the serotoninergic (5-HT) system in Sprague-Dawley (SD) and Wistar Kyoto (WKY) rats were studied. Open-field and forced-swim tests revealed a significantly greater behavioral depression in the WKY strain. SD rats showed a decrease in 3H-DPAT binding to 5-HT1A receptors in the hippocampus, whereas WKY rats revealed an increase in 3H-DPAT binding in the hippocampus and hypothalamus. Stress did not appear to alter the binding of 3H-DPAT to 5-HT1A sites in the dorsal raphe or median raphe in either strains. SD rats revealed a modest increase in 5-HT transporter (5-HTT) sites in the cortex; WKY rats revealed a decrease in 5-HTT sites in the cortex and the hippocampus. Stress caused an increase in 3H-CNIMI binding to 5-HTT sites in the dorsal and median raphe nuclei in both strains. The results suggest that the greater susceptibility to behavioral depression in WKY rats may account for the differential effects on 5HT1A sites as well as 5-HTT sites in limbic regions and cell body area as compared to SD rats.

Hemochromatosis (HC) is an inherited disorder of iron metabolism and is frequently seen in Caucasians. The biochemical defect and the responsible gene are unknown, but the HC locus is closely linked to HLA-A on human chromosome 6 in the region 6p21.3. Although extensive studies have been performed in several populations, the precise location of the gene is still undefined. Linkage disequilibrium with HC has been detected for loci that are 3 cM apart: HLA class I and D6S105, which is located on the telomeric side of HLA-A. We have analyzed the inheritance of several multi-allele polymorphisms that map to 6p (D6S265, Y52, HLA-F, D6S306, D6S105, D6S464, D6S299) in 34 Italian HC families and in 17 unrelated patients. Significant association with HC was shown for alleles of multiple markers in the HLA-A region, for the distant marker D6S105, but not for the D6S299 marker at 4 cM from HLA-A on the telomeric side. HC status was unambiguously assigned to 70 affected and 63 unaffected chromosomes from family studies. Thirty five different haplotypes were found in 70 HC chromosomes when considering four markers most tightly associated with the disease. A predominant haplotype comprising alleles 1-3-1-8 (marker order D6S265, HLA-A, Y52, D6S105) accounted for 30% of the HC chromosomes and was absent in normals. A minority of other HC haplotypes could be related to the major haplotype by assuming single crossover events. Results of haplotype studies suggest a founder effect in the Italian population, as previously shown in Australian patients, and a possible common mutation shared with affected individuals of Celtic origin.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by the expansion of a CAG repeat in a gene coding for a protein of unknown function. We have raised a polyclonal antibody against a 12 amino acid peptide (residues 2110-2121 of human huntingtin) which specifically recognises huntingtin on Western blots of human, rat and mouse brain. We have characterised huntingtin expression in the mouse. The protein was detected on Western blots of all mouse tissues examined, with the highest expression seen in brain. Human, mouse and rat brain were fractionated by differential centrifugation and discontinuous Percoll gradients. The fractions were analysed by Western blotting for huntingtin and synaptophysin (a synaptic vesicle localised protein). In mouse brain, huntingtin was localised in the soluble S3 fraction; in rat brain it was localised in the soluble S3 fraction and also in the membrane P2 and P3 fractions; in both normal and HD-affected human brain, huntingtin was membrane bound with a distribution essentially the same as that of synaptophysin. These observed differences in the subcellular localisation of huntingtin between mouse and human brain are important in the context of mouse models for HD.

The ability of heparin to interact with plasma proteins, in particular antithrombin III (ATIII) and thrombin, is its primary mechanism as an anticoagulant drug. Research efforts have focused on the biological activity of heparin under three conditions: in solution as a free molecule, chemically coupled directly onto a polymer surface, and coupled onto a polymer surface using hydrophilic spacer groups. Each of these conditions yields altered biological activity, presumably a result of differing binding interactions with ATIII and thrombin. In this report, insights into binding interaction of direct versus polyethylene oxide space immobilized heparin with ATIII, thrombin, and the generation of the thrombin-antithrombin complex will be presented.

The white rot fungus Pycnoporus cinnabarinus was characterized with respect to its set of extracellular phenoloxidases. Laccase was produced as the predominant extracellular phenoloxidase in conjunction with low amounts of an unusual peroxidase. Neither lignin peroxidase nor manganese peroxidase was detected. Laccase was produced constitutively during primary metabolism. Addition of the most effective inducer, 2,5-xylidine, enhanced laccase production ninefold without altering the isoenzyme pattern of the enzyme. Laccase purified to apparent homogeneity was a single polypeptide having a molecular mass of approximately 81,000 Da, as determined by calibrated gel filtration chromatography, and a carbohydrate content of 9%. The enzyme displayed an unusual behavior on isoelectric focusing gels; the activity was split into one major band (pI, 3.7) and several minor bands of decreasing intensity which appeared at regular, closely spaced intervals toward the alkaline end of the gel. Repeated electrophoresis of the major band under identical conditions produced the same pattern, suggesting that the laccase was secreted as a single acidic isoform with a pI of about 3.7 and that the multiband pattern was an artifact produced by electrophoresis. This appeared to be confirmed by N-terminal amino acid sequencing of the purified enzyme, which yielded a single sequence for the first 21 residues. Spectroscopic analysis indicated a typical laccase active site in the P. cinnabarinus enzyme since all three typical Cu(II)-type centers were identified. Substrate specificity and inhibitor studies also indicated the enzyme to be a typical fungal laccase. The N-terminal amino acid sequence of the P. cinnabarinus laccase showed close homology to the N-terminal sequences determined for laccases from Trametes versicolor, Coriolus hirsutus, and an unidentified basidiomycete, PM1. The principal features of the P. cinnabarinus enzyme system, a single predominant laccase and a lack of lignin- or manganese-type peroxidase, make this organism an interesting model for further studies of possible alternative pathways of lignin degradation by white rot fungi.

The DCC gene is one of several genes altered during tumorigenesis. Recent studies demonstrate the existence of 29 exons and it spans 1.4 Mb. DCC gene is a candidate tumor-suppressor gene encoding a protein with a sequence similarity to cell adhesion molecule. Although the mutation of DCC gene was few, high incidence of LOH and decreased mRNA expression in colorectal cancer especially with hepatic metastasis was observed. These DCC gene abnormalities possibly be related to predictive marker for liver metastasis.

Genetic instability, alterations of tumor suppressor genes as well as activation of oncogenes and aberrant expression of growth factor/receptor system found in human stomach carcinogenesis are overviewed. Aberrant expression and amplification of the c-met gene, inactivation of the p53 gene and amplification of the cyclin E gene are common events of both well differentiated and poorly differentiated gastric carcinomas. K-ras mutations, c-erbB2 gene amplification, loss of heterozygosity (LOH) and mutations of the APC, LOH of the bcl-2 gene and LOH at DCC locus are preferentially associated with well differentiated gastric cancer. On the other hand, microsatellite instability, reduction or loss of cadherin and catenins, K-sam and c-met gene amplification confer the development and progression of poorly differentiated or scirrhous gastric carcinomas. Interaction between cell-adhesion molecules in the c-met expressed cancer cells and hepatocyte growth factor from stromal cells is involved in morphogenesis of gastric cancer.

Though colorectal tumorigenesis has long been thought to be a multistep mechanisms, recently has it become possible to identify the molecular events that underlie the initiation and progression of colorectal carcinoma. Though the analysis of mutations in colorectal tumors at various stages of their development allows definition of a model for colorectal tumorigenesis, because the progression is the result of a series of genetic changes that accumulate activation of oncogene (K-ras), inactivation of tumor-suppressor gene (two-hit mutation of APC, Pla2s, p53, suppressor gene on chromosome 8p22 locus, NF2 and DCC) and mismatch repair gene (hMSH2, hMLH1 hPMS family and TGF beta II receptor linked DNA repair). These accumulation of genetic alterations contribute to tumor development and/or progression in primary colorectal carcinoma.