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DNA polymerase beta is known to be involved in repair of DNA damage. Frequent mutation of its gene in the segment encoding amino acids 149-297 has been reported in colorectal cancer. To investigate whether mutation in this region is available as a genetic marker for colorectal cancer, 11 primary tumors and 4 liver metastases from 11 patients were examined by fragment length analysis and single-strand conformation polymorphism (SSCP) analysis of reverse-transcription polymerase chain reaction (RT-PCR) products. Although allelic imbalance in the p53 and DCC genes were observed in ten out of eleven primary tumors and all liver metastases using a dinucleotide repeat polymorphism, mutation was not detected in the DNA polymerase beta mRNA. Neither was it detected in seven colon cancer cell lines. Present results suggest that mutation in this region is uncommon in colorectal cancers and is not a useful genetic marker for colorectal cancer.

POU domain transcription factors are required for neuropeptide expression in selected subsets of hypothalamic neuroendocrine neurons. We now report that expression of the gonadotropin-releasing hormone (GnRH) gene, which controls sexual development, is regulated by the POU protein SCIP/Oct-6/Tst-1. Reverse transcriptase PCR cloning and RNase protection assays demonstrated the presence of SCIP/Oct-6/Tst-1 mRNA in the GnRH-producing neuronal cell line GT1-7. The physiological relevance of this regulatory activity was suggested by the detection of SCIP/Oct-6/Tst-1 mRNA in a subset of GnRH neurons in the hypothalamus of prepubertal female rats. Coexpression of SCIP/Oct-6/Tst-1 in neuronal cells inhibited rat GnRH (rGnRH) promoter activity via three regions of the proximal rGnRH promoter containing SCIP/Oct-6/Tst-1 binding sites. DNase I footprinting, gel shift assays, and DNA and protein mutagenesis studies indicated that both direct DNA binding and protein-protein interactions are required for SCIP/Oct-6/Tst-1 modulation of GnRH gene expression. Activation of SCIP/Oct-6/Tst-1 expression in terminally differentiated GnRH neurons may be a factor determining the ratio of phenotypically "inactive" versus "active" GnRH neurons during postnatal life.

Four sets of cell lines (UM-SCC-14A, -14B, and -14C; UM-SCC-17A and -17B; UM-SCC-81A and -81B; and UM-SCC-83A and -83B), established from primary and metastatic or locally recurrent tumors from four patients with squamous cell carcinoma of the head and neck, were examined for loss of heterozygosity (LOH) on chromosome 18q. Metastatic or recurrent cell lines from all four exhibited 18q LOH. UM-SCC-14A, -14B, and -14C, which were derived from locally recurrent (14A and 14B) and metastatic (14C) tumors, lost all of 18q. However, in the other three cases, there was a partial loss of 18q in the recurrent or metastatic tumor cell lines but not in the primary tumor cell lines from the same patient. To determine whether the cell lines accurately reflect in vivo loss of 18q, we analyzed matched sets of normal, tumor, and tumor cell line DNA from eight patients with squamous cell carcinoma of the head and neck, including the tumor tissue corresponding to UM-SCC-81B. Three of the additional seven tumors and cell lines had 18q LOH. For all eight cases in which tumor and corresponding cell line DNAs were analyzed, there was complete concordance between allelic loss in the tumor and allelic loss in the corresponding cell line. The common region of loss established by tumors and cell lines with partial loss includes 18q21-18qter. This region contains the putative tumor suppressor gene DCC and two Mad (Mothers against dpp)-related genes, DPC4 and MADR2, which are both components in a transforming growth factor-beta-like signaling pathway. Loss of 18q in metastatic and locally recurrent tumors, but not in primary tumors from the same patients, suggests that a tumor suppressor gene in this region may be important in the progression of squamous cell carcinoma.

Recombinant human prothrombin (rII) and two mutant forms (R155A, R271A,R284A (rMZ) and R271A,R284A (rMZdesF1)) were expressed in mammalian cells. Following activation and purification, recombinant thrombin (rIIa) and stable analogues of meizothrombin (rMZa) and meizothrombin(desF1) (rMZdesF1a) were obtained. Studies of the activation of protein C in the presence of recombinant soluble thrombomodulin (TM) show TM-dependent stimulation of protein C activation by all three enzymes and, in the presence of phosphatidylserine/phosphatidylcholine phospholipid vesicles, rMZa is 6-fold more potent than rIIa. In the presence of TM, rMZa was also shown to be an effective activator of TAFI (thrombin-activatable fibrinolysis inhibitor) (Bajzar, L., Manuel, R., and Nesheim, M. E. (1995) J. Biol. Chem. 270, 14477-14484). All three enzymes were capable of inducing platelet aggregation, but 60-fold higher concentrations of rMZa and rMZdesF1a were required to achieve the effects obtained with rIIa. Second order rate constants (M-1.min-1) for inhibition by antithrombin III (AT-III) were 2.44 x 10(5) (rIIa), 6.10 x 10(4) (rMZa), and 1.05 x 10(5) (rMZdesF1a). The inhibition of rMZa and rMZdesF1a by AT-III is not affected by heparin. All three enzymes bound similarly to hirudin. The results of this and previous studies imply that full-length meizothrombin has marginal procoagulant properties compared to thrombin. However, meizothrombin has potent anticoagulant properties, expressed through TM-dependent activation of protein C, and can contribute to down-regulation of fibrinolysis through the TM-dependent activation of TAFI.

Cirrhosis is associated with compromised hemostasis and coagulopathy during orthotopic liver transplantation (OLT). It has been suggested that hemostasis is better preserved during OLT in primary biliary cirrhosis (PBC) than other cirrhotic states. The aim of this study was to compare coagulation and fibrinolysis in 15 patients with PBC with 31 patients with other liver disease before and during OLT. Preoperatively, both groups had subnormal mean levels of prekallikrein, factor XIIa, antithrombin III (ATIII), plasminogen, and alpha2-antiplasmin. C1 esterase inhibitor and kallikrein inhibition in PBC was higher than the normal range (P < .01), but not in non-PBC. Non-PBC had lower median fibrinogen levels and shorter euglobulin clot lysis times (ECLT) (P < .05). Tissue plasminogen activator (tPA) antigen levels did not differ between groups but were elevated from the normal range, as were median thrombin-antithrombin complexes (TAT). Plasminogen activator inhibitor (PAI) activity was significantly higher in PBC (0.0041). Perioperatively in the PBC group during the early anhepatic phase of OLT, there was more thrombin generation, as evidenced by higher TAT levels (P = .0455) and less hyperfibrinolysis with longer ECLTs. We hypothesize that there is a preserved capacity to generate thrombin and less fibrinolytic activation during the anhepatic phase of OLT, and we suggest that, in PBC, the use of antifibrinolytic agents may have an adverse effect.

Tenascin (TN) is an extracellular matrix glycoprotein that is expressed in a characteristic spatiotemporal pattern during development and is up-regulated in the adult during tumorigenesis, wound healing, and nerve regeneration. In previous studies, we identified a promoter within the proximal 250 bp upstream of the mouse TN gene that contains several putative regulatory elements that are conserved among vertebrate TN genes. We have identified four different DNA elements within this promoter and show that they contribute in different ways to TN gene expression in NIH 3T3 fibroblasts, C6 glioma cells, and N2A neuroblastoma cells. These elements comprise a binding site for Krox proteins, one for nuclear factor 1, an octamer motif that binds POU-homeodomain proteins, and a novel TN control element. The nuclear factor 1 and TN control element had positive effects on TN promoter activity and formed similar DNA-protein complexes with nuclear extracts from all three cell lines. The Krox element had a negative effect on TN promoter activity in N2A cells, a positive effect in C6 cells, and no effect in NIH 3T3 cells. Two DNA binding complexes, one correlated with the negative and the other with the positive activities of the Krox element, were found to contain the protein Krox24. In cotransfection experiments, the octamer motif was required for induction of TN promoter activity by the POU-homeodomain protein Brn2 in N2A cells but was inactive in C6 cells. Consistent with these findings, N2A cells transfected with Brn2 formed octamer-binding complexes containing N-Oct3, the transcriptionally active form of Brn2, whereas complexes formed in C6 cells contained only N-Oct5A and N-Oct5B. Our results provide a striking example of the diversity of regulatory mechanisms that can be called forth by combining different promoter motifs with transcriptional activators or repressors.

<h4>Objective</h4>To examine the relationship of estrogen-induced changes in lipids and lipoproteins with alterations in the coagulation system.<h4>Methods</h4>Coagulation and lipid indices were measured in 31 postmenopausal women, ages 40-60 years, after a 3-month course of 0.625-mg conjugated equine estrogen. We analyzed changes in variables from baseline to 3 months using t tests for paired samples or the Wilcoxon matched-pairs signed-rank test.<h4>Results</h4>Unopposed estrogen replacement therapy produced statistically significant decreases in antithrombin-III antigen (P = .006) and activity (P = .001) and total protein S (P = .003) and a significant increase in protein C antigen (P = .017). C4b-binding protein also decreased significantly from baseline to 3 months (P < .001). Mean fibrinogen level decreased by 18.2 mg/dL, not a statistically significant change (P = .213). Estrogen produced the expected statistically significant changes in lipids and lipoproteins. Several correlations between changes in lipids and lipoproteins and coagulation indices were statistically significant. Protein C antigen and activity changes correlated directly with high-density lipoprotein cholesterol changes (r = .52, P < or = .005; r = .38, P < or = .05; respectively), and protein C antigen also correlated directly with increases in apoprotein A-I (r = .54, P < or = .005). Triglyceride changes correlated directly with changes in protein C antigen (r = .36, P < or = .05) and activity (r = .49, P < or = .005) and inversely with C4b-binding protein (r = -.58, P < or = .01). Apoprotein B was correlated with free protein S (r = .48, P < or = .01).<h4>Conclusions</h4>Although several estrogen-induced changes may decrease atherosclerotic potential and hypercoagulability, others may promote coagulability. These divergent effects may be manipulated pharmacologically by other estrogen compounds or by the addition of various progestins.

Juvenile polyps are regarded as hamartomatous polyps and occur in sporadic and familial syndromic settings. There is increased risk of gastrointestinal neoplasia in patients with juvenile polyposis syndrome, but the molecular mechanisms are not known. We therefore studied 78 colorectal juvenile polyposis from 12 patients with juvenile polyps syndrome and 34 sporadic juvenile polyps for epithelial dysplasia and genetic changes associated with colorectal neoplasia. Dysplasia occurred in 31% of syndromic juvenile polyps but not in sporadic juvenile polyps (P < 0.0001). Topographic control of proliferation and expression of the cyclin-dependent kinase inhibitor p21(WAFI/CIP1) seen in native colorectal epithelium was lost in 79% of dysplastic juvenile polyps and in 8% of nondysplastic juvenile polyps (P < 0.000001). Somatic mutations in the adenomatous polyposis coli (APC) gene were demonstrated in 50% of dysplastic juvenile polyps (3 of 6) but not in any of 16 juvenile polyps without dysplasia (P = 0.01). Both sporadic and syndromic juvenile polyps had K-ras mutations (14%) and there was no relationship to dysplasia. p53 gene product overexpression identified by immunohistochemical staining occurred rarely in dysplastic juvenile polyps (2 of 24, 8%). Our results indicate that the multiple genetic alterations involved in usual colorectal neoplasia also play a role in neoplastic transformation of juvenile polyps, predominantly in juvenile polyposis syndrome.

Recombinant native antithrombin III (ATIII) and two genetic variants with glutamine substitutions at lysine residues 114 and 139 were expressed in insect cells using a baculovirus-driven expression system. The purified proteins were used to evaluate the potential role(s) of these residues in the pentasaccharide-mediated activation of ATIII. The second order rate constants for the inhibition of factor Xa by both of the genetic variants were nearly identical to those of recombinant native ATIII, indicating that the glutamine substitutions did not result in serious protein conformational changes. The glutamine substitution at lysine 139 had no effect on the pentasaccharide-mediated activation of ATIII toward factor Xa. In contrast, lysine 114 was found to be critical in the activation of ATIII toward factor Xa. No activation was observed, even at a pentasaccharide concentration 10 times higher than that required to activate recombinant native ATIII. These data are the first to demonstrate a pivotal role for lysine 114 in the pentasaccharide-mediated activation of ATIII.

By modulating the magnitude and duration of postsynaptic responses, carrier-facilitated serotonin (5-HT) transport into and release from the presynaptic neuron is central to the fine tuning of serotonergic neurotransmission. The 5-HT transporter (5-HTT) is the prime target for widely used antidepressants, psychostimulants, drugs of abuse and neurotoxins. We have isolated the gene encoding the murine 5-HTT and determined the sequence of all exons including adjacent intronic regions and approximately 3.6 kb of the 5'-flanking regulatory region. The murine 5-HTT gene is composed of 14 exons spanning approximately 34 kb. The single gene transcript after splicing is 2744 bp in length and it contains 186 bp of 5' untranslated region (5'-UTR) and 668 bp of 3'-UTR. A TATA-like motif and several potential binding sites for transcription factors including AP1, AP2, AP4, SP1 as well as CRE- and GRE-like motifs are present in the GC-rich 5'-flanking region. The characterization of murine 5-HTT cDNA and genomic organization will facilitate studies of 5-HT uptake function with molecular pharmacologic and transgenic strategies as well as investigations of its role in quantitative traits and psychiatric disorders.

<h4>Background/aims</h4>Hepatocellular carcinoma develops frequently in the course of genetic hemochromatosis, and a role of iron overload in hepatic carcinogenesis is strongly suggested.<h4>Methods</h4>The aim of our study was to investigate the effect of iron exposure on DNA synthesis of adult rat hepatocytes maintained in primary culture stimulated or not by EGF/pyruvate and exposed to iron-citrate complex.<h4>Results</h4>In EGF/pyruvate-stimulated cultures, the level of [3H] methyl thymidine incorporation was strongly increased as compared to unstimulated cultures. The addition of iron to stimulated cultures increased [3H] methyl thymidine incorporation. The mitotic index was also significantly higher at 72 h. However, the number of cells found in the cell layer was not significantly different from iron-citrate free culture. By flow cytometry, no difference in cell ploidy was found between iron-treated and untreated EGF/pyruvate-stimulated cultures. A significant increase in LDH leakage reflecting a toxic effect of iron was found in the cell medium 48 h after cell seeding. In addition, [3H] methyl thymidine incorporation in the presence of hydroxyurea was increased in iron-treated compared to untreated cultures.<h4>Conclusions</h4>Our results show that DNA synthesis is increased in the presence of iron in rat hepatocyte cultures stimulated by EGF/pyruvate, and they suggest that DNA synthesis is likely to be related both to cell proliferation and to DNA repair. These observations may allow better understanding of the role of iron overload in the development of hepatocellular carcinoma.

Based on the presence of multiple proline-rich motifs in the huntingtin sequence, we tested its possible association with epidermal growth factor (EGF) receptor signaling complexes through SH3 domain-containing modules. We found that huntingtin is associated with Grb2, RasGAP, and tyrosine-phosphorylated EGF receptor. These associations are regulated by activation of the EGF receptor, suggesting that they may be part of EGF receptor-mediated cellular signaling cascade. In vitro binding studies indicate that SH3 domains of Grb2 or RasGAP are required for their binding to huntingtin. Our results suggest that huntingtin may be a unique adapter protein for EGF receptor-mediated signaling and may be involved in the regulation of Ras-dependent signaling pathways.

Knockout experiments in Tetrahymena show that linker histone H1 is not essential for nuclear assembly or cell viability. These results, together with a series of biochemical and cell biological observations, challenge the existing paradigm that requires linker histones to be a key organizing component of higher-order chromatin structure. The H1 knockouts also reveal a much more subtle role for H1. Instead of acting as a general transcriptional repressor, H1 is found to regulate a limited number of specific genes. Surprisingly, H1 can both activate and repress transcription. We discuss how this architectural protein might accomplish this important regulatory role.

Recent advances in molecular biology have revealed that the alteration of multiple genes, eg., APC, K-ras, p53, DCC, are involved in multistep colorectal carcinogenesis. Some of these alterations can be used as molecular markers in genetic diagnosis. Genetic diagnoses for colorectal cancer are classified into three categories, eg., 1. identification of the career in the family of patient with hereditary disease such as FPC (Familial Polyposis Coli) or HNPCC (Hereditary Non-Polyposis Colorectal Cancer), 2. early diagnosis of colorectal cancer by identifying gene mutations in the stool, 3. assist for histopathological diagnosis, or risk assessment of the metastasis, recurrence or secondary cancer by molecular means. However, there are several problems in these genetic diagnoses. These consist of two categories, eg., 1. problems in the method of gene analyses or assay system and 2. problems in performing genetic diagnoses itself. The former includes the problem of contamination of different tissue, false positive or negative result in PCR-based analyses, heterogeneity of gene mutation in tumor tissue, and the latter includes the social, ethical or economical problems mainly related to the genetic diagnosis for hereditary colorectal cancers. In this paper, we describe the possibility of genetic diagnosis for colorectal cancers and the current problems, especially from the molecular pathological aspect, in genetic diagnosis.

The water-soluble amide to an NH2-ended monomethoxypolyethyleneglycol (MPEG-NH2, molecular mass of about 2000) of the dipyrrinone xanthobilirubic acid (XBR, 1) and the bis-amides of mesobiliverdin-XIII alpha (MBV, 2) and mesobilirubin-XIII alpha (MBR, 3) have been prepared with high yields. Contrary to what is observed with biliverdin-IX alpha, 4, the enzymatic reduction of the mesobiliverdin derivative 2-MPEGA to the corresponding mesobilirubin 3-MPEGA by the soluble biliverdin reductase/NADPH system in pH 7.4 aqueous phosphate does not occur. In contrast, thiol addition to 2-MPEGA and to 4 under similar conditions is immediate, although this equilibrium is slightly less favourable for 2-MPEGA. These results enable us to discount the intrinsically low reactivity of 2-MPEGA towards thiols as the reason for its lack of enzymatic reduction, and suggest instead that this particular mesobiliverdin cannot fit properly into the enzyme binding site, either because of steric hindrance or the lack of the two propionic acid groups.

DCC (deleted in colorectal cancer), a candidate tumor suppressor gene located in chromosome band 18q21.2, encodes a transmembrane protein of 1447 amino acids. Neogenin, a protein with nearly 50% amino acid identity to DCC, was recently identified because of its dynamic expression in the developing nervous system and gastrointestinal tract of the chicken. To explore a role for the human neogenin (NGN) gene in cancer, we have isolated cDNAs for two alternatively spliced forms of NGN, encoding proteins of 1461 and 1408 amino acids. Fluorescence in situ hybridization studies (FISH) localized NGN in chromosome band 15q22, a region infrequently affected by alterations in cancer. NGN transcripts of about 7.5 and 5.5 kb were detected in all adult tissues studied. In contrast to the frequent loss of DCC expression, no alterations in NGN expression were observed in more than 50 cancers studied, including glioblastoma, medulloblastoma, neuroblastoma, colorectal, breast, cervical and pancreatic cancer cell lines and xenografts. Based on their sequence conservation and similar expression during development, DCC and NGN may have related functions. However, the chromosomal location and ubiquitous expression of NGN in various human tumors suggest it is infrequently altered in cancer.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism that leads to excessive iron storage in the liver and other organs. Recently, between 83 and 100% of HH patients have been found to be homozygous for the same mutation in a novel major histocompatibility complex class I-like gene, called the HLA-H gene. The Cys-282 --> Tyr mutation in HH patients would be expected to disrupt the function of the HLA-H gene product by altering a critical disulfide bridge. As a first step in understanding the function of the HLA-H gene product, we generated an antibody to a C-terminal peptide and used it for immunolocalization of the HLA-H protein in the gastrointestinal tract of Finnish and American subjects presumed not to have HH. Although staining for the HLA-H protein was seen in some epithelial cells in every segment of the alimentary canal, its cellular and subcellular expression in the small intestine were quite distinct from those seen in other segments. In contrast to the stomach and colon, where staining was polarized and restricted to the basolateral surfaces, and in contrast to the epithelial cells of the esophagus and submucosal leukocytes, which showed nonpolarized staining around the entire plasma membrane, the staining in small intestine was mainly intracellular and perinuclear, limited to cells in deep crypts. Prior genetic evidence suggested that a defective HLA-H protein is the molecular basis of HH. Here we show that the HLA-H protein not only varies in its pattern of expression along the cranial/caudal axis of the gastrointestinal tract but that it has a unique subcellular localization in the crypts of the small intestine in proximity to the presumed sites of iron absorption.

Diseases resulting from defects in a single gene may be more amenable to treatment by conventional gene therapy strategies than idiopathic or polygenic disorders. We have attempted to reduce the expression in vivo of the Huntington's disease gene protein, Huntingtin, using an 18-mer fluorescein-labeled phosphorothiorated antisense oligodeoxynucleotide (ODN) targeted against the start site of the first exon of the IT15 gene. Animals were given repeated intrastriatal infusions (5 microliters of a 100 nmol/microliter solution daily over 4 days) of the antisense ODN. The treatments ended on Day 5 and the tissue was processed for immunohistochemical and Western blot analysis. The fluorescein-labeled ODN appeared to penetrate several cells and did not cause any obvious toxicity to the neurons. The average reduction in levels of Huntingtin (16.9 +/- 7.2%) did not differ significantly between striatal tissue of antisense ODN-treated animals compared to those treated with a sense ODN or vehicle. Improved methods for molecular modifications of the IT15 gene may be needed for therapeutic initiatives.

In the present study, the possible involvement of homeobox-containing genes in colorectal cancer (CRC) development was investigated. Using a stepwise screening approach and RT-PCR, we have demonstrated that the human HOXB6, B8, C8 and C9 are overexpressed at various stages of CRC. In contrast, all CRC cases exhibited a marked decrease in the homeodomain-containing Cdx1 gene expression. Recent data which suggest a regulatory link between HOXB8 and several tumor suppressor genes, such as DCC, APC, and TGF beta, sustain a possible implication of homeobox genes in colon carcinogenesis. Moreover, our data showing a decrease in Cdx1 expression are consistent with the notion that genes functioning in the establishment and maintenance of the intestinal epithelium might, upon deregulation, disturb the normal control of cellular proliferation, differentiation, and death, thus leading to cancer development.

No abstract available.

The genetic defect dentatorubral-pallidoluysian atrophy (DRPLA) is caused by expansion of a CAG trinucleotide repeat. The mutant gene is translated into protein whose electrophoretic mobility correlates to the number of expanded CAG trinucleotide repeats, indicating that the protein carries an expanded glutamine repeat. Using two polyclonal antibodies raised against the DRPLA gene product in immunoblotting, we determined the untruncated DRPLA proteins, and showed that the amounts of mutant and wild-type DRPLA proteins were similar in DRPLA brain tissues and lymphoblastoid cells, suggesting that regulation of the level of translation of the DRPLA gene is not central to the development of the disease.

We report the discovery of the huntingtin interacting protein I (HIP-I) which binds specifically to the N-terminus of human huntingtin, both in the two-hybrid screen and in in vitro binding experiments. For the interaction in vivo, a protein region downstream of the polyglutamine stretch in huntingtin is essential. The HIP1 cDNA isolated by the two-hybrid screen encodes a 55 kDa fragment of a novel protein. Using an affinity-purified polyclonal antibody raised against recombinant HIP-I, a protein of 116 kDa was detected in brain extracts by Western blot analysis. The predicted amino acid sequence of the HIP-I fragment exhibits significant similarity to cytoskeleton proteins, suggesting that HIP-I and huntingtin play a functional role in the cell filament networks. The HIP1 gene is ubiquitously expressed in different brain regions at low level. HIP-I is enriched in human brain but can also be detected in other human tissues as well as in mouse brain. HIP-I and huntingtin behave almost identically during subcellular fractionation and both proteins are enriched in the membrane containing fractions.

We studied the involvement of coagulation and fibrinolysis system in the induction and development of atherosclerosis in rabbits with hyperlipidemia induced by a high-cholesterol diet (HCD). In HCD rabbits, plasma lipids and atherogenic indices were maintained at a high level throughout the experimental period compared with those in rabbits fed on a standard diet. In the early phase, a significant increase in fibrinogen level was followed by increases in the activities of plasminogen and tissue-type plasminogen activator with a decrease in alpha 2-plasmin inhibitor activity and platelet count. In the middle and late phases, significant increases in plasminogen activator inhibitor-1 and antithrombin-III were observed in HCD rabbits. These results suggest that the early enhancement of coagulation followed by high activity of fibrinolysis is involved in the induction and development of hyperlipidemic thromboembolism and atherosclerosis in HCD rabbits.