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Thrombin, besides being a potent coagulation factor, exerts influence on endothelial and leukocyte functions and may thus be involved in the regulation of inflammatory reactions. The present study investigated whether thrombin stimulates the production of growth-related cytokine/melanoma growth-stimulatory activity (GRO alpha/MGSA) in endothelial cells. Human umbilical vein endothelial cells (HUVEC) stimulated with thrombin were found to product GRO alpha/MGSA in a dose- and time-dependent manner. This action of thrombin was completely suppressed by preincubation with either hirudin or antithrombin-III (AT-III)-heparin. Interestingly, the thrombin receptor-activating peptide SFLLRN mimicked the action of thrombin. In addition, staurosporine, a protein kinase C (PKC) inhibitor, attenuated the production of GRO alpha/MGSA by thrombin, SFLLRN and phorbol 12-myristate 13-acetate (PMA), but left the action of interleukin-1 beta (IL-1 beta) unchanged. These results suggest that catalytic activation of thrombin receptor by thrombin results in GRO alpha/MGSA production, at least in part, via a pathway involving PKC in HUVEC.

The MOG locus, located on chromosomal bands 6p21.3-p22 and mapped about 100 kb telomeric to HLA-F, was isolated from cosmid ICRFc109A2434 and shown to contain three microsatellites. These CA-repeat polymorphic markers were characterized in a sample of 173 healthy unrelated individuals and 84 DNAs from the HLA Workshop reference panel, by a method combining fluorescence labeling of PCR products and use of an automated DNA sequencer. For the three markers, frequencies of heterozygotes are well predicted from allele frequencies by the Hardy-Weinberg rule, which suggests that problems of allele nonamplification are unlikely. Typing of cell lines homozygous in the HLA region allowed unambiguous definition of 81 HLA-MOG haplotypes and showed that several HLA ancestral haplotypes extended to the MOG region. The high degree of polymorphism (59%, 51%, and 81% at the three loci, respectively, and 87% at the haplotype level) makes these new markers informative for association or linkage studies with diseases such as hemochromatosis or multiple sclerosis, and for studies aimed at precisely delineating the site of crossover in chromosomes in which recombination occurred in the distal part of the HLA class I region.

Pseudorabies virus (PrV), an alphaherpesvirus of swine, uses cellular heparan sulfate residues as a receptor for attachment. Interaction of the virus with its receptor is mediated by the envelope glycoprotein C (PrV-gC), a protein with heparin-binding properties. We have previously shown that a region of this protein shows structural similarities to the high-affinity heparin-binding site of the serum protease-inhibitor antithrombin III (ATII). In this publication, we describe the effect of ATIII on interaction of PrV with its cellular receptor. ATIII bound specifically to heparan sulfate residues on the surface of herpesvirus-permissive RK13 cells. Binding of ATIII to RK13 cells interfered with adsorption of radioactively labelled PrV to these cells. Enzymatic treatment using heparinase I (E.C. 4.2.2.7) removed the receptor for PrV as well as the receptor for ATIII. Since amino acids 130-137 of the high affinity heparin-binding site of ATIII show structural similarities to amino acids 134-141 of PrV-gC, both sequences were synthesized as synthetic peptides. Although interaction of the peptide derived from ATIII with heparin was significantly stronger, both peptides interacted specifically with heparin in assays in vitro. These results suggest that PrV and ATIII interact with the same structure on the cellular surface.

In order to detect regions of DNA containing tumor suppressor genes involved in the development of gastric cancer, we performed an allelotype study on 78 gastric adenocarcinomas from a population composed largely of Texan Hispanics and Anglos, two ethnic groups that have a ratio of incidence rates of gastric cancer of approximately 2:1. In total, 42 microsatellite markers were employed, which detected at least one site per arm of each autosome in the human genome. These included several markers linked to known tumor suppressor genes (TP53, APC, DCC, RB1, and BRCA1). Sites showing quantitative allelic imbalance (AI) greater than 30% were located on 3p (36%), 11q (31%), 12q (38%), 13q (33%), 17p near TP53 (74%), and 17q near BRCAI (32%). Among the 22% of cases showing microsatellite instability (MI), a subset (4 of 17) showed instability at 59% or more of sites tested. No ethnic bias was detected in cases showing MI or in cases with AI at sites with rates of AI above 30%. Tumors of the intestinal subtype were significantly more likely than diffuse tumors to show AI at DI3S170 (P = 0.01). A deletion map of chromosome arm 3p was prepared for tumors with AI at D3S1478. These data indicate that a tumor suppressor gene on chromosome arm 3p is involved in the development of a subset of gastric cancers.

The blood coagulation and fibrinolysis of 33 patients with compensated liver cirrhosis and 31 patients with hepatocellular carcinoma were examined using several markers, namely thrombin-antithrombin III complex (TAT), plasmin-alpha 2 plasmin inhibitor complex (PIC), antithrombin-III (AT-III) and prothrombin time, and the relationship between these markers, endotoxemia, and TNF-alpha was examined. These patients had no complications due to hepatic failure, such as infections, encephalopathy, ascites, G-I bleeding and clinical DIC. PIC was not elevated, but TAT tended to be elevated in LC and significantly elevated in HCC. AT-III was decreased in LC and HCC, and the blood endotoxin was partly positive in LC and HCC, but was not correlated with AT-III or PT. The TAT level in the blood-endotoxin-positive patients measured by endospecy methods was higher than that in the negative patients, and was significantly correlated with the blood endotoxin level in the LC and HCC patients (r = 0.57, r = 0.88, p < 0.01). No relationship was observed between TNF-alpha and blood endotoxin. In conclusion, (1) blood coagulability was activated already in compensated LC and HCC, but was not connected with fibrinolysis, (2) the activation of coagulability was closely related with endotoxemia, and (3) TNF-alpha was not correlated with blood endotoxin or TAT.

Huntington's disease (HD) is an inherited, neurodegenerative disorder caused by expansion of a CAG repeat in the IT15 gene, leading to an expanded glutamine repeat in the HD protein. The mechanism by which the expanded repeat causes expression of the disease is not known, though there do not appear to be changes in the mRNA levels. We have conducted quantitative Western blot analyses of HD patients and controls. Expression of the IT15 protein is essentially equal in control and HD frontal cortex. In caudate from HD patients, IT15 protein is decreased in parallel with the decrease in a neuronal marker, suggesting that loss of IT15 protein is secondary to neuronal loss. In order to determine expression of the two alleles of the IT15 protein we used Western blots of 4% polyacrylamide gels. Both alleles of the IT15 protein were expressed at similar levels in HD lymphoblastoid cell lines and HD post-mortem hippocampus and cerebellum (regions relatively spared in HD), indicating that even very long CAG repeats can be translated into polyglutamine. In contrast, in cerebral cortex and caudate (regions severely affected in HD), in the longer expanded repeat cases the expanded allele of the IT15 protein was present at a significantly lower level (compared with the normal length allele), often with a smear of more slowly migrating reactivity above it. These data suggest the possibility of altered structure, abnormal processing or abnormality of protein-protein interactions involving the IT15 protein with the expanded glutamine repeat.

The primary objective of this study was to apply a sophisticated coagulation monitoring system including estimation of the concentration of prothrombin fragment 1 + 2 (PTF 1 + 2) and the activity of prothrombin (coagulation factor II or FII) to cases of stent implantation and to compare the results with those of standard coagulation tests. The secondary objective was to detect the incidence after stenting of subacute thrombosis (SAT) and bleeding complications in these patients and to compare the results with those of a group of patients with stent implantation in whom coagulation was monitored exclusively by standard tests. SAT several days after coronary stenting occurs in up to 20% despite aggressive intravenous and overlapping oral anticoagulation. According to a prospective study protocol 120 consecutive patients with implantation of 155 Palmaz-Schatz stents underwent coagulation monitoring including single daily estimation of the concentration of PTF1 + 2 (target range < 0.5 nmol/L) and of FII activity (15% to 35%) in addition to the standard tests of thrombin time (TT), partial thromboplastin time (aPTT), international normalized ratio (INR), antithrombin III (ATIII), and fibrinogen. Adjustment of heparin and phenprocoumon dosages in this study group was based only on the results of PTF1 + 2 and FII measurements. A control group consisted of 53 patients with implantation of 64 stents who were matched for baseline, angiographic, and procedure-related characteristics. After stenting, anticoagulation was monitored by estimation of TT (target range > 70 seconds), aPTT (> 70 seconds), INR (3.0 to 4.5), AT III (> 80%), and fibrinogen (< 450 mg/dl) in this control group. There was a weak correlation between PTF1 + 2 and aPTT (r = 0.337; PTF1 + 2 = -0.00169aPTT + 0.491) and PTF1 + 2 and TT (r = 0.328; PTF1 + 2 = -0.00142TT + 0.494). A better correlation was found between FII and INR (r = 0.983; FII = -23.8 INR + 134). Stable oral anticoagulation was maintained 2.8 +/- 0.9 days later according to an FII concentration of < 35% compared with an INR > 3. The incidence of SAT was 3.3% with 3.0% for elective versus 3.8% for nonelective stenting. The sensitivity, specificity, and accuracy of the PTF1 + 2 test were 100%, 88%, and 88%, respectively. In the control group the incidence of SAT was 17%, with 16.1% for elective versus 18% for nonelective stenting. Major bleeding complications occurred in 10% (study group) and in 11.3% (control group) of patients (no statistical difference).(ABSTRACT TRUNCATED AT 400 WORDS)

The expression of epidermal growth factor receptor (EGFR), oestrogen receptor (ER) and progesterone receptor (PR) was assayed by a radioreceptor method in 117 primary ovarian cancers. EGFR was not significantly related to any of the clinicopathological parameters examined. In patients with stage II-IV disease who underwent second-look surgery after primary chemotherapy, a significant correlation between high EGFR levels and poor response to chemotherapy was demonstrated (P = 0.031). Moreover, post-operative residual tumour showed an independent role in predicting chemotherapy response (P = 0.0007) and EGFR status showed a borderline significance (P = 0.052) in the multivariate analysis. No correlation between steroid hormone receptors and clinicopathological parameters was observed. Whereas a significant relationship was shown between EGFR positivity and a shorter overall survival (OS) (P = 0.0022) and progression-free survival (PFS) (P = 0.0033), patient survival was not related to steroid hormone receptor status. Among the parameters tested only stage, ascites and EGFR status retained an independent prognostic value in the multivariate analysis.

A cytosolic 21-23 kDa protein isolated from bovine brain was demonstrated to bind hydrophobic ligands, particularly phosphatidylethanolamine. The protein was encountered in numerous tissues of several species. High expression of the mRNA encoding the 21-23 kDa protein was found in rat testes. Immunohistochemical studies showed the presence of the 21-23 kDa protein in the elongated spermatids and epididymal fluid of rat testis and in brain oligodendrocytes of developing rats. As the bovine, human and rat brain 21-23 kDa proteins had only few sequence homologies with already know proteins, ti was concluded that they belong to a new protein family. In order to get additional information on the structural features of the 21-23 kDa protein, we built a molecular model which displayed a nucleotide binding site. The affinity of the bovine brain 21-23 kDa protein towards nucleotides as well as its association with cytosolic proteins and small GTP-binding proteins were demonstrated. Recently, significant sequence homologies were found with an antigen from Onchocerca volvulus, a fruit fly odorant-binding protein and the yeast protein TFS1 which is a dosage-dependent suppressor of CDC25 mutations. A positive regulation of RAS is carried out by CDC25 product which facilitates the GDP/GTP exchange on RAS proteins. These results imply that 21-23 kDa proteins function in oxidoreduction reactions and signal mechanisms during cell growth and maturation.

The aim of the present was to define prognosis and life expectancy in patients with chronic liver disease of different etiologies and to relate them to an age- and sex-matched normal population. After a follow-up of 15 years, life expectancy of 620 patients with chronic liver disease was retrospectively calculated and compared with an age- and sex-matched normal population. Among patients with cirrhosis, prognosis was dependent upon Child classification (P = 0.001). Patients with alcoholic cirrhosis and fatty liver disease were younger (P = 0.01) and had a lower life expectancy than patients with other causes of chronic liver disease (P = 0.004). Patients with hepatitis B and hepatitis C cirrhosis showed a comparable prognosis and a significantly lower life expectancy than the age- and sex-matched population. Cryptogenic and autoimmune liver diseases showed a comparable life expectancy but a significantly shorter life expectancy than the normal population. In patients with alpha 1-antitrypsin deficiency-associated cirrhosis, a high viral coinfection rate was found (P = 0.01). For patients with noncirrhotic hemochromatosis, prognosis was poorer than that for the age- and sex-matched population. In patients with asymptomatic primary biliary cirrhosis, chronic persistent hepatitis B, and alpha 1-antitrypsin deficiency without cirrhosis, life expectancy was equal to that of the normal population. Prognosis and life expectancy in chronic liver disease depend on stage, cause, and symptoms of chronic liver disease; age; and possibilities of treatment. In patients with hereditary liver disease, additional viral infection of alcohol abuse lead to a significant deterioration of life expectancy. Patients with alcoholic chronic liver disease have the poorest prognosis.

We have analyzed 12 microsatellite markers on chromosome 9p in 54 paired cutaneous malignant melanoma (CMM) tumors and normal tissues. Forty-six percent of the tumors, including two in situ CMMs, showed loss of heterozygosity (LOH) at 9p. Only one tumor was homozygously deleted for 9p markers. The smallest deleted region was defined by five tumors and included markers D9S126 to D9S259. Loss of eight or more markers correlated significantly with worse prognosis (P < .002). Among the primary tumors, 87.5% of those with large deletions have a high risk of metastasis, as compared with only 18% of those without deletions or with loss of fewer than 8 markers (P < .001). It was not possible to demonstrate homozygous deletions of p16 in any of the CMM tumors. In four tumors, the LOH for 9p markers did not involve p16. The reported data suggest the existence of several tumor suppressor genes at 9p that are involved in the predisposition to and/or progression of CMM and exclude p16 from involvement in the early development of some melanoma tumors.

Serotonin transporter (5-HTT) mRNA and 5-HTT sites were measured 3 days after treatment with p-chlorophenylalanine, a tryptophan hydroxylase inhibitor. While 5-HTT mRNA levels decreased (P < 0.001) in the dorsal raphe nucleus, 5-HTT binding sites remained unchanged, suggesting that an acute depletion of 5-HT may induce an increase in the turnover of 5-HTT mRNA without affecting protein levels at 3 days.

Crossbred lambs (47.3 kg BW) were used to study the effects of restraint and isolation stress on endocrine status and blood metabolites, antemortem glycogenolysis, and incidence of the dark-cutting condition (DCC) in the longissimus muscle (LM) and to determine the role of muscle contraction in the formation of the DCC in sheep. Lambs were assigned randomly to three treatments: unstressed controls (C); a single 6-h period of restraint and isolation stress (RIS); and a single 6-h period of RIS following epidural blockade (RISEB) with lidocaine. Blood was collected immediately before lambs were subjected to RIS and RISEB and at 12-min intervals during the 6-h period. Serum concentrations of glucose, lactate, and insulin were higher (P < .01) in RIS and RISEB lambs than in C lambs. Serum free fatty acid concentrations were higher (P < .01) in stressed lambs only during the first 4 h of stress. Plasma epinephrine and cortisol concentrations also were higher (P < .01) in RIS and RISEB lambs than in C lambs. Lambs were slaughtered within 30 min after completion of stress. Immediately after stunning and at .75, 3, 6, 12, and 24 h postmortem, samples were removed from the LM in the hindsaddle and foresaddle for glycogen, lactate, and pH determinations. Muscle pH was elevated (P < .01) by RIS and RISEB; ultimate pH exceeded 6.0. The LM from carcasses of RIS and RISEB lambs had lower (P < .01) glycogen and lactate concentrations in both regions than the LM of C lambs. Subjecting sheep to a single 6-h period of RIS was an effective animal model to induce the DCC. Failure of the epidural blockade to inhibit antemortem glycogen metabolism and formation of the DCC indicates that muscle contraction was not requisite to those processes in sheep.

Underlying disorders of the coagulation system such as inhibitor deficiencies or decreased fibrinolysis are common in patients suffering from venous thrombosis. They may lead to the necessity of a lifelong prophylaxis. Prompt diagnosis is obviously to the patients benefit. We investigated 22 patients suffering from venous thromboses for the inhibitors antithrombin III (ATIII), protein C, and protein S during the first 8 to 12 days after admission to hospital and in addition after withdrawal from anticoagulant treatment after several months. At the day of admission ATIII and protein C levels were comparable to those several months later, but after 2 days they shifted downward or upward, respectively. Protein S did not shift during the period of hospitalisation, but was initially slightly lower than several months later. For inhibitors the day of admission to hospital is most suitable to take the samples. About 50% of the patients still had elevated activation markers (prothrombin fragments F1+2, thrombin-antithrombin complex TAT, and D-dimers) after several months.