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Radiolabeled antithrombin III (ATIII) was incubated at 37 degrees C with purified vitronectin (VN) or fibrinogen-deficient plasma before thrombin was added to initiate complex formation. Incorporation of radiolabeled ATIII was detected using polyacrylamide gel electrophoresis (PAGE) and autoradiography. The PAGE conditions appeared to be crucial for the detection of VN.TAT complexes. In the absence of SDS, ternary complexes formed instantaneously, whereas in the presence of SDS, only 50% of the TAT was associated with VN after a 60-min incubation. Formation of ternary complexes could be confirmed by gel filtration of the plasma to which thrombin was added. Furthermore, TAT in patient plasmas (disseminated intravascular coagulation and sepsis) was found to bind to heparin-Sepharose, indicating that this endogenously formed TAT was also associated with VN. The amino-terminal region of VN and the thrombin moiety of the TAT complex were found to be responsible for their interaction, which was stabilized by disulfide bridges. These results indicate that in normal plasma all TAT is complexed with VN. This association alters the conformational state of plasma VN, which appears to be responsible for the clearance of thrombin complexes from the circulation.

Activation of the tyrosine kinase of the c-src gene product, pp60c-src, has been shown to occur in nearly every primary colorectal carcinoma, and is found as early as in polyps of high malignant potential. However, no studies have addressed potential pp60c-src changes which occur during progression. To examine this question, we have studied kinase activity and protein levels in 7 colonic polyps, 19 primary lesions, and 19 liver metastases relative to normal colonic mucosa. Significant increases in tyrosine kinase activity were seen as early as in colonic polyps of high malignant potential. Further increases were observed in activity and level in primary tumors. However, the greatest increases in activity and protein levels were observed in liver metastases. Additionally, six metastatic lesions were obtained in which synchronous primary tumor was resected. In each of these liver metastases, pp60c-src activity and level were significantly increased relative to the corresponding primary tumor, as well as to normal colonic mucosa. Our results demonstrate that progression of colon primary tumors to liver metastases correlates with increased pp60c-src kinase activity and protein level.

The mRNA expression levels of DCC gene, which is cloned from the deleted region of chromosome 18q in colorectal cancers and thought to be a tumor-suppressor gene, was evaluated in tissue specimens surgically resected from patients with colorectal cancer by RT-PCR. This method was chosen as the expression level of DCC mRNA is below the detectable level for Northern-blot analysis. Semi-quantitative measurements of DCC mRNA were performed based on a standard curve defined by serial dilution of DCC cDNA. As a result, the expression level of DCC mRNA was found to be lower in 17 out of 30 colorectal cancers than in adjacent non-cancerous tissues. Inclusion of smooth muscle in tissue specimens was observed to have little disturbing effect on comparisons between cancerous and non-cancerous regions. In addition, all 4 specimens of colorectal cancer with liver metastasis showed the decreased expression level of DCC mRNA, suggesting that functional loss of DCC in cancerous tissues may play an important role in metastatic events.

In this report the structure of the novel polypeptide h3, isolated from subsets of tissues of a single species, and the structural similarity between interspecies h3 were investigated by peptide mapping of enzymatic and chemical cleavage fragments of h3 in one-dimensional SDS-PAGE; the peptide maps were commented on in comparison with the known sequence of 21 kDa protein, a h3-like ox brain protein. The following results were obtained: peptides generated by chymotrypsin, protease XX, BNPS-skatole and CNBr cleavage of different tissues in a single species were strikingly identical, whereas peptide maps obtained from analogue tissues in different species revealed slight structural differences. Possible ligand-h3 binding was studied by comparing the c.d. spectra of native h3, and h3 incubated with several phospholipids. Given the presence of h3 or h3-like protein in rat and human platelets, h3 was also assessed in platelet aggregation in the presence of h3 and specific anti-h3 antiserum. So far, the results emphasize the unique intra- and interspecies molecular form of h3, allow us to assign to known amino acid sequence of 21 kDa to a large extent to human h3, but do not identify h3 as a phospholipid binding protein.

Twenty-two patients on regular hemodialysis treatment suffering from renal anemia were treated with intravenous recombinant human erythropoietin (rhEPO) for more than 8 weeks. Before and 4 and 8 weeks after the start of rhEPO administration, we measured prothrombin time, activated partial thromboplastin time, fibrinogen (FBG), antithrombin III activity (ATIII), plasminogen activity (PLG), alpha 2-plasmin inhibitor activity (alpha 2 PI), alpha 2-plasmin inhibitor-plasmin complex (alpha 2 PIC), and cross-linked fibrin degradation products (XL-FDP) in citrated plasma to determine whether rhEPO treatment enhances coagulation and fibrinolytic activity. The pretreatment values of FBG, alpha 2 PIC, and XL-FDP were significantly higher than the normal control values. The pretreatment values of ATIII, PLG, and alpha 2 PI were significantly lower than the normal control values. Platelet count and FBG were significantly increased 4 and 8 weeks after treatment with rhE-PO. The prothrombin time was significantly shortened 8 weeks after rhEPO treatment, but the activated partial thromboplastin time did not change. PLG was significantly decreased 4 and 8 weeks after rhEPO treatment, and ATIII and alpha 2 PI were significantly decreased 8 weeks after rhEPO treatment. alpha 2 PIC was significantly increased 8 weeks after rhEPO treatment, and XL-FDP was significantly increased 4 and 8 weeks after rhEPO treatment. These data suggest that in patients on regular hemodialysis treatment coagulation and fibrinolysis are already enhanced before the start of rhEPO treatment and that rhEPO administration further enhances these disorders.

It has previously been shown that the hepatitis B virus (HBV) X gene product, HBx, transactivates homologous and heterologous transcriptional regulatory sequences of viruses, including the human immunodeficiency virus type 1 (HIV1) long terminal repeat (LTR), and various cellular genes in vitro. To evaluate the transactivating function of HBx in vivo, we generated transgenic mice carrying the X open reading frame under the control of the human antithrombin III (ATIII) gene regulatory sequences. These mice express the 16 Kd HBx protein in the liver, as demonstrated by immunoprecipitation studies. Crossbreeding of HBx mice with transgenics carrying either the chloramphenicol acetyl transferase (CAT) bacterial or the lacZ reporter gene driven by the HIV1-LTR allowed us to demonstrate, for the first time, the in vivo transactivating function of HBx protein.

The influence of the continuous-flow automated blood cell separator. Fenwal CS-3000, on blood coagulation and the fibrinolytic system in blood donors was studied. Blood samples were taken from the collection line of donors undergoing extracorporeal circulation, before and after platelet pheresis. Of the molecular markers, prothrombin fragment-F1 + 2 (PF1 + 2) markedly increased from 0.8 +/- 0.3 to 2.9 +/- 2.0 nM/ml (P < .004), thrombin antithrombin III complex (TAT) also markedly increased from 2.6 +/- 1.3 to 56.0 +/- 24.0 micrograms/L (P < .001), fibrinopeptide A (FPA) increased slightly from 0.8 +/- 0.9 to 3.8 +/- 4.2 micrograms/L (P < .05), and alpha 2-plasmin inhibitor (alpha 2-PI) decreased slightly from 95 +/- 8 to 91 +/- 9% (P < .05). In one donor with the highest level of PF1 + 2, TAT, FPA, and plasmin inhibitor complex after platelet pheresis, protein C, protein S, C4b-binding protein, ATIII, plasminogen, alpha 2-PI, and coagulation factors were decreased. In blood donors undergoing platelet pheresis using the continuous-flow automated blood cell separator, Fenwal CS-3000, a hypercoagulable state was observed. Changing the materials of the plastic disposables to a more thromboresistant material may prevent the hypercoagulable state in donors induced by platelet pheresis using the blood cell separator.

The use of heparin rather than citrate as primary anticoagulant has been shown to significantly improve the initial activity, stability and recovery of factor VIII:C from human plasma, cryoprecipitates or factor VIII concentrates if the plasma was initially frozen at -80 degrees C and subsequently stored at this temperature. If frozen and stored at progressively warmer temperatures however, increasing amounts of insoluble protein aggregates, termed storage precipitates (SPs), were recovered in the thawed plasma and cryoprecipitate fractions. Plasma recovery by centrifugation at 7,000 g for 7 min [Method I (MI)], 2 x 10 min (MII) or 15 min (MIII) had little effect on SP formation after 1 month at any storage temperature. After 4 months at -20 degrees C, more SP was recovered from MIII plasma whereas at -40 degrees C, more SP was recovered from MI plasma. Also, the preparation method had little or no effect on factor VIII:C activity at equivalent storage times or temperatures. A trend towards improved factor VIII recoveries was noted at lower freezing and storage temperatures however. SP formation was associated with reduced fibrinogen levels in the recovered plasma without loss of antithrombin-III or increased fibrinopeptide-A. Western blots showed polymerization of A alpha or gamma-chains of fibrinogen. SP formation was reduced or eliminated with factor XIII inhibitors, antibody to the active factor XIII a subunit or adjustment of heparinized plasma to 5-10 mM sodium citrate before initial freezing and storage. Although plasma factor VIII:C recoveries were only slightly affected at these citrate concentrations under most conditions, its recovery in cryoprecipitates was substantially improved owing to the reduction or absence of SPs.

In pre-eclampsia (PE), reduced levels of plasma urokinase-like plasminogen activator (u-PA) and plasminogen activator inhibitor-2 (PAI-2), and increased levels of plasma tissue-type plasminogen activator (t-PA) antigen were seen. The majority of moderate and severe pre-eclamptic women (7 out of 10) ended up with pre-term delivery as compared with 2 out of 11 who went on to term. Patients with moderate and severe PE had significantly lower levels (mean +/- SD, ng/ml) of PAI-2 (58.4 +/- 34.9) and u-PA antigen (1.61 +/- 0.62) as compared to those with mild PE (95.6 +/- 39.3 and 1.61 +/- 0.62 and 2.12 +/- 0.61, respectively). Significantly raised t-PA antigen (14.6 +/- 5.7 ng/ml) was seen in moderate and severe PE as compared with mild PE (9.9 +/- 3.4 ng/ml). PAI-1 activity was significantly raised only in moderate and severe PE as compared with normal pregnancy. There were no significant differences in thrombin-antithrombin-III complexes, D-dimer and beta-thromboglobulin levels between the PE group and normal pregnancy, although these parameters were above the non-pregnant levels. Platelets in PE were within the range found in normal pregnancy. It appears that measurements of plasma u-PA and PAI-2 levels in patients with PE may have prognostic value in determining the outcome of pregnancy in this pregnancy disorder.

Quarter samples of foremilk and residual milk were taken approximately every second week from 2 days post partum (pp) throughout lactation month 9, from 5 dairy cows in their second lactation period. Bacteriologically positive milk samples were excluded. The aim was to study the variation in total and differential leukocyte counts, N-acetyl-beta-D-glucosaminidase (NAGase), antitrypsin (ATR) and serum albumin (BSA) in milk during the lactation period and different stages of oestrous cycle. Also the between milkings variation was studied from lactation month 4 to 9. At 2 days pp, each fraction of milk contained significantly higher numbers of leukocytes and had a higher activity of NAGase and ATR than later in the lactation period. In foremilk the highest content of BSA was also recorded at 2 days pp. From lactation month 2 to 9, stage of lactation had, in general, a slight effect on the variation in the variables measured. The total leukocyte count in residual milk tended to increase as lactation proceeded. The proportion of monocyte-macrophages in foremilk was significantly decreased during the last 4 months. NAGase and BSA in both fractions and ATR in residual milk increased significantly towards the end of the lactation period. From lactation month 4 to 9 the highest recorded ranges of variation between milkings, within quarter and stage of lactation, in the total leukocyte count, proportions of neutrophils, lymphocytes, monocyte-macrophages, NAGase, ATR and BSA in foremilk were 215 x 10(3)/ml, 42%, 34%, 54%, 6.68 units, 0.36 units and 0.14 mg/ml respectively. The corresponding figures in residual milk were higher except for the variation in BSA which was slightly lower in residual milk than in foremilk. In residual milk there was a positive correlation between the proportion of neutrophils and the total leukocyte count, when calculated on data from all cows and the entire experimental period. During the oestrous periods, the proportion of neutrophils in residual milk was higher than during the dioestrous periods. Foremilk and residual milk differed in the total as well as the differential leukocyte counts in all the various stages of lactation, whereas the contents of NAGase, ATR and BSA were equal in both fractions. The exception was 2 days pp when the proportions of lymphocytes were equal in both fractions and BSA-significantly higher in foremilk than in residual milk.

The nonsteroidal antiandrogen casodex has been described as a peripherally selective drug for the treatment of prostatic cancer. In this study we determined its activity in various models of hormone-dependent malignancies including those of the prostate and the breast. Analysis of endocrine effects in rats after 15 days of treatment revealed a strong reduction of the weights of prostates and seminal vesicles and a significant rise of testosterone serum levels as a result of the interference with central feedback mechanisms. The growth of androgen-sensitive human LNCaP/FGC prostate cancer cells was strongly inhibited by casodex. Unlike hydroxyflutamide, casodex was also active in hormone-depleted medium. The inhibitory effect was overcome by addition of testosterone propionate, which indicates an androgen-receptor-mediated mode of action. In rats bearing Dunning R3327-G prostate carcinomas casodex exerted a strong antitumour effect at the beginning of therapy. However, after 4 weeks of treatment tumours resumed growth whereas diethylstilboestrol-treated tumours remained static. In MXT-M3.2 mouse mammary tumours with significant quantities of androgen receptors casodex was also effective in inhibiting tumour growth. After 6 weeks of treatment, tumour weights were reduced by 69% whereas uterine weights were significantly increased, possibly because of a progestin-like activity of the drug. Csodex is very active in various models of hormone-dependent carcinomas. However, the limited duration of action in prostatic tumours and the incomplete growth inhibition in mammary tumours suggest that it should be used only combination with other endocrine therapies.

Extensive transfusion of blood components as a typical feature of OLT has been described by many authors. The perioperative requirement for transfusion, however, follows a downward trend, although the indications for OLT have been extended. Functional disturbances such as hyperfibrinolysis or platelet dysfunction, demonstrated by laboratory tests, such as platelet counts, PT, aPTT, TT, fibrinogen, and ATIII are often used to direct the transfusion of blood components, although preoperative data give insight only into the insufficient function of the old liver, which will be explanted and replaced by the donor graft. We have described a retrospective statistical analysis of laboratory data, clinical data, and perioperative blood requirements from 250 liver transplantations performed in our hospital from 1988 to 1992. The OLT was performed according to the usual surgical methods using a venovenous bypass system. Intraoperatively, volume was restored with packed RBC and FFP according to hemodynamic data, hemoglobin, and diuresis; clotting data were not used as indications for blood replacement. This analysis demonstrated that neither preoperative nor intraoperative clotting parameters were able to allow a prediction of the intraoperative requirement for transfusion of blood components; these findings parallel those of previous reports. With respect to other studies showing that perioperative transfusion rates correlated with postoperative infections and mortality, we strongly suggest that perioperative clotting measures as indicators for transfusion requirement should be analyzed with caution. Whether other diagnostic methods such as TEG are useful alternatives has to be determined.

The serpin-enzyme complex (SEC) receptor recognizes a pentapeptide neo-domain of alpha 1-antitrypsin (alpha 1 AT)-elastase complexes and, in so doing, mediates internalization and intracellular catabolism of the macromolecular complex, mediates an increase in synthesis of alpha 1 AT, and elicits neutrophil chemotactic activity. In previous studies we have shown that this pentapeptide domain is highly conserved among members of the serpin family and that binding of a synthetic peptide corresponding to this region (125I-peptide 105Y, SIP-PEVKFNKPFVYLI, based on alpha 1 AT sequence 359-374) to HepG2 cells is blocked by several serpin-enzyme complexes. To determine whether the SEC receptor is the primary HepG2 cell surface binding site for these serpin-enzyme complexes, we examined the capacity for serpin-enzyme complexes to compete with each other for binding to the SEC receptor. The results indicate that binding of 125I-elastase-alpha 1 AT complexes is blocked by thrombin-antithrombin III (ATIII), thrombin-heparin cofactor II, and cathepsin G-alpha 1-antichymotrypsin (alpha 1 ACT) complexes. Moreover, unlabeled elastase-alpha 1 AT complexes compete for binding of 125I-thrombin-ATIII, 125I-thrombin-heparin cofactor II, and 125I-cathepsin G-alpha 1 ACT complexes. Preformed soluble tissue plasminogen activator-plasminogen activator inhibitor 1 complexes also compete for binding of elastase-alpha 1 AT complexes to the SEC receptor but do so to a less effective extent, probably because of a less favorable pentapeptide sequence for binding to the SEC receptor. Under conditions in which these serpin-enzyme complexes would be expected to bind to the SEC receptor there is an increase in synthesis of alpha 1 AT but not in synthesis of ATIII or alpha 1 ACT. Proteolytically modified alpha 1 AT also competes for binding of 125I-elastase-alpha 1 AT complexes to the SEC receptor and vice versa. The purified 51-kDa amino-terminal fragment of alpha 1 AT does not compete for binding of 125I-elastase-alpha 1 AT complexes, indicating that the pentapeptide neodomain in the 4-kDa carboxyl-terminal fragment is sufficient for binding to the SEC receptor.

A human papillomavirus type 18 (HPV-18)-immortalized human keratinocyte cell line (1811) has been transformed to tumorigenicity in nude mice by treatment with the carcinogen nitrosomethylurea (NMU). The NMU transformants (1811-NMU-T) showed additional chromosome alterations as compared with parental 1811 cells, including 18q deletion in two of two 1811-NMU-T lines analysed. Restriction fragment length polymorphism (RFLP) analysis indicated that both 1811-NMU-T lines had lost one allele of the 18q deleted in colon cancer (DCC) tumor-suppressor gene. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that DCC expression was absent or barely detectable in the 1811-NMU-T cells as compared with 1811 or normal keratinocytes, suggesting that the remaining DCC allele in the 1811-NMU-T cells was also altered. These studies indicate that reduction or loss of DCC expression may be an important step in NMU transformation of HPV-immortalized cells to tumorigenicity.

The mucosa of the pelvic ileal reservoir undergoes adaptive changes--inflammatory, architectural, and metaplastic--on exposure to the faecal stream. Twenty three quadruple loop ileal pouches constructed for ulcerative colitis (20 patients) and familial adenomatous polyposis (FAP) (three patients) were studied. No patient fulfilled clinical, endoscopic, or histopathological criteria for pouchitis. Standard duplicate biopsy specimens were taken from the proximal limb, the anterior wall, the posterior wall, and the body of the reservoir. An established scoring system was used and showed a highly significant increase in inflammatory scores in posterior wall biopsy specimens compared with those from the anterior wall. These results suggest that the adaptive changes are the direct result of contact with static faecal contents. One patient only showed significant inflammation in the proximal limb. There was no evidence of mucosal prolapse in any anterior wall biopsy specimen. Patients with colitis showed substantially more inflammatory and architectural changes than those with FAP. Ninety six per cent of pouches showed some colonic phenotypic expression as defined by mucin histochemical and PR 3A5 immunohistochemical studies. Our results suggest, however, that there may not be complete colonic metaplasia and that the mucin changes and other phenotypic alterations may represent a non-specific response to pouch inflammation and not a prerequisite for the development of pouchitis. The focal nature of the inflammatory and architectural changes, which may be the result of direct contact with static faecal residue, are clearly shown. A single random biopsy specimen of pouch mucosa is of limited value in assessing pathological changes and screening for potential neoplastic change within the reservoir.

An experimental model of secondary hemochromatosis is described. Saccharated iron was administered i.v. to rats for 7 months in total doses in the range 1.0-1.7 g per kg body weight. After the completion of iron loading, the biochemical measurements revealed elevation of alanine aminotransferase (ALT), slight reduction of plasma glucose concentration, and significant reduction of both plasma and liver ascorbic-acid levels. The mean liver iron concentration was 50 times higher in iron-loaded animals than in controls. High concentrations of inorganic iron were also observed in spleen, pancreas, and heart. Histologic analysis revealed marked hepatic fibrosis in most animals in the experimental group. These results demonstrate this animal model presents some pathologic findings observed in human transfusional hemochromatosis. Additionally, hydroxyl free radicals were detected by electron paramagnetic resonance (EPR) spectroscopy in the iron-overloaded liver tissue processed at pH 5.0. No free radicals were detected at pH 7.4. These results suggest the possible participation of hydroxyl free radicals in the cellular toxicity of iron overload.

The relationship between tissue-type plasminogen activator (t-PA) antigen content and receptor status was investigated in 200 human breast cancer cytosols to evaluate the efficacy of t-PA as a marker of functional steroid receptors. t-PA level was measured by an enzyme-linked immunoassay (ELISA) and estrogen (ER) and progesterone (PgR) receptors were assayed by the DCC method. A highly significant correlation was found between t-PA levels and receptor status. The mean +/- SE enzyme content was 13.5 +/- 2.9 in ER+ tumors and 1.8 +/- 0.3 ng/mg protein in ER- tumors; the enzyme content in PgR+ tumors was 14.2 +/- 3.3 and 3.4 +/- 1.4 ng/mg protein in PgR- tumors. When tumors were divided into four subgroups according to receptor content (ER+PgR+, ER+PgR-, ER-PgR+, and ER-PgR-), t-PA concentration was able to differentiate these groups. Also, t-PA level was compared to several clinical variables; it was not correlated with menopausal status or lymph node involvement. However, t-PA content varied according to tumor size. Our data shows that t-PA content in tumor cytosols is statistically related to receptor status and that determination of t-PA levels in breast cancer might furnish additional information as to the functional state of the receptors which may be necessary for planning hormone therapy.

A recently described splice variant of CD44 expressed in metastasizing cell lines of rat tumors has been shown to confer metastatic potential to a non-metastasizing rat pancreatic carcinoma cell line and to non-metastasizing sarcoma cells. Homologues of this variant as well as several other CD44 splice variants are also expressed at the RNA level in human carcinoma cell lines from lung, breast, and colon, and in immortalized keratinocytes. Using antibodies raised against a bacterial fusion protein encoded by variant CD44 sequences, we studied the expression of variant CD44 glycoproteins in normal human tissues and in colorectal neoplasia. Expression of CD44 variant proteins in normal human tissues was readily found on several epithelial tissues including the squamous epithelia of the epidermis, tonsils, and pharynx, and the glandular epithelium of the pancreatic ducts, but was largely absent from other epithelia and from most non-epithelial cells and tissues. In human colorectal neoplasia CD44 variant proteins, including homologues of those which confer metastatic ability to rat tumors, were found on all invasive carcinomas and carcinoma metastases. Interestingly, focal expression was also observed in adenomatous polyps, expression being related to areas of dysplasia. The distribution of the CD44 variants in human tissues suggests that they play a role in a few restricted differentiation pathways and that in colorectal tumors one of these pathways has been reactivated. The finding that metastasis-related variants are already expressed at a relatively early stage in colorectal carcinogenesis and tumor progression, i.e., in adenomatous polyps, suggests the existence of a yet unknown selective advantage linked to CD44 variant expression. The continued expression in metastases would be compatible with a role in the metastatic process.

The preferential usage of certain T cell receptor (TCR) V beta genes has been well established in several major histocompatibility complex (MHC)-restricted immune responses. However, V beta usage among allogeneic responses remains unclear. Because recent findings of ours and others indicate that V beta 8 predominates in certain Ld-restricted, peptide-specific responses, we examined the V beta 8 usage in allogeneic responses to Ld. To selectively recognize the Ld molecule, cells from BALB/c-H-2dm2 (dm2), the Ld-loss mutant mouse, were stimulated in vitro or in vivo with wild-type BALB/c cells. We report here that after the intraperitoneal administration of the anti-V beta 8 monoclonal antibody (mAb) F23.1, peripheral V beta 8 T cells were depleted from dm2 mice. This in vivo depletion abrogated the ability of dm2 splenocytes to mount a primary response to Ld molecules. This abrogation was specific, since the response of V beta 8-depleted dm2 cells to Kb/Db antigens was the same as that of control nondepleted dm2 cells. Furthermore, in vivo depletion of V beta 8 cells was found to cause a dramatic prolongation of Ld-disparate skin grafts (mean survival time [MST] 22.1 +/- 2.1 vs. 10.3 +/- 1.1 d for saline-treated controls, or 10.9 +/- 1.7 d for controls treated with mAb KJ23 to V beta 17). By contrast, V beta 8 depletion had no effect on recipients grafted with haplotype-mismatched skin or single Dk-locus-disparate skin. These findings demonstrate that V beta 8+ T cells predominate in allogeneic response to Ld but not other alloantigens. The effect of V beta 8 depletion was found to be even more dramatic on recipients grafted with Ld-disparate vascularized heart transplants (MST > 100 vs. 8.6 +/- 0.5 d for controls). In total, these findings establish the efficacy of using mAb to the V beta gene family to specifically and significantly enhance the survival of allografts. The implications of detecting V beta 8 usage in both alloreactive or MHC-restricted TCR responses to the same class I molecule are discussed.

We evaluated the chronic effect of angiotensin-III (AIII) in the promotion of drinking behavior in spontaneously hypertensive (SH) and normotensive Wistar-Kyoto (WKY) rats, using conscious, freely moving, male, adult animals that had been instrumented with an intracerebroventricular (icv) cannula connected to an osmotic mini-pump for 7-day infusion. Chronic icv infusion of AIII (5 or 10 pmol/min) elicited robust, dose-dependent, and Ile7-AIII (100 pmol/min; as specific antagonist)-reversible dipsogenesis in both SH and WKY rats, with higher water intake in the former strain. However, the drinking response in the SHRs exhibited a sharp drop after 3 days of AIII infusion, during which acute AIII (80 pmol, icv) challenges also failed to induce dipsogenesis. Chronic icv infusion of bestatin (150 pmol/min), an aminopeptidase-B inhibitor, did not by itself discernibly affect basal drinking. When combined with AIII (5 or 10 pmol/min), however, bestatin, respectively, suppressed and augmented the dipsogenic response of SH and WKY rats to the heptapeptide. These results suggest that chronic administration of AIII did not produce sustained drinking behavior in SHRs, possibly because of the development of early desensitization of the angiotensin receptors.

Heterozygous loss of the TP53 gene on chromosome arm 17p in colorectal carcinomas was strongly associated with DNA aneuploidy (P < 0.0001). This association was seen only in tumours with loss on both 17p and 17q (P < 0.001), but not for loss on 17p only. DNA near diploid (ND) carcinomas and DNA aneuploid (AN) tumours with DNA index > or = 1.1 and < 1.3 had similar frequencies of TP53 gene loss (49% and 42%, respectively), whereas AN tumours with DNA index > or = 1.3 had a significantly higher frequency of TP53 gene loss (85%) (P < 0.0001 and P < 0.0001, respectively). There was a significant association between loss of the TP53 gene and histological grade (P < 0.01), and there tended to be an association between loss of the TP53 gene and degree of cellular atypia (P < 0.05), with TP53 gene loss being most frequent in moderately differentiated carcinomas, and in carcinomas with severe cellular atypia, respectively. The proportion of tumours with loss of the TP53 gene increased significantly towards the distal part of the large bowel (P < 0.0001). These results indicate that different genetic mechanisms may be involved in the carcinogenesis in colon and rectum carcinomas, and in the two subsets of DNA aneuploid carcinomas. Furthermore, the data may suggest a role for the TP53 gene in the aneuploidisation process, possibly as a 'target' for a whole chromosome loss.

Direct measurements were made of the Ca distribution within sarcomeres of glycerinated rabbit psoas muscle fibers in rigor using electron probe x-ray microanalysis. Both analogue raster analysis and digital x-ray imaging were used to quantitate the Ca distribution along thick and thin filaments as a function of the concentration of free Ca2+. Even when corrected for the estimated contribution of Ca bound to thick filaments, the Ca measured in the region of overlap between thick and thin filaments significantly exceeded the Ca in the I-band at subsaturating concentrations of free Ca2+. At saturating levels of free Ca2+, the excess Ca in the overlap region was diminished but still statistically significant. The data thus suggest that the formation of rigor linkages exerts multiple effects on the binding of Ca2+ to thin filaments in the overlap region by increasing the affinity of troponin C for Ca2+ and possibly by unmasking additional Ca2+ binding sites. The data also show that the cooperativity invested in the thin filaments is insufficient to permit the effects of rigor cross-bridge formation on Ca2+ binding to propagate far along the thin filaments into the I-band.

Mononuclear cells from 5 normal men and 5 patients homozygous for hereditary haemochromatosis (HFE) have been incubated for 18 h with or without the addition of sheep red blood cells coated with antibody (SRBC). In the absence of SRBC mean H type ferritin concentrations were greater than L type (normals: mean L type 11.6 ng/10(6) cells, H type 15.5; patients, L type 23.5 ng/10(6) cells, H type 41.6). In the presence of SRBC, monocyte L type ferritin concentrations increased considerably (76 ng/10(6) cells in normals and 141 ng/10(6) cells in patients) but H type ferritin concentrations were the same or decreased compared with incubation in medium only. Incubation with additional iron (ferric ammonium citrate, 2.5 micrograms Fe/ml) increased both H and L type ferritin concentrations. Erythrophagocytosis thus appears to cause differential regulation of H and L ferritin subunit synthesis or breakdown. Normal subjects and patients do not differ in this response to erythrophagocytosis.

Gene changes in multiple oncogenes, multiple growth factors and multiple tumor-suppressor genes are observed in stomach cancer. Among them, those most commonly implicated in both well-differentiated adenocarcinoma and poorly differentiated adenocarcinoma are inactivation (mutations and allele loss) of the p53 gene, and activation (abnormal expression and amplification) of the c-met gene. Moreover, they occur at an early stage of stomach carcinogenesis. In addition, loss of heterozygosity (LOH) on chromosome 5q (APC locus) is frequently associated with well-differentiated adenocarcinoma. LOH on chromosome 18q (DCC locus) and LOH of the bcl-2 gene also are common events of well-differentiated adenocarcinoma. LOH on chromosomes 1q and 7q may be involved in the progression of well-differentiated adenocarcinoma. Conversely, the development of poorly differentiated adenocarcinoma, in addition to changes in p53 and c-met genes, requires reduction or dysfunction of cadherin. Overexpression of bcl-2 protein is observed in poorly differentiated adenocarcinoma or signet-ring cell carcinoma. Moreover, the K-sam gene is amplified preferentially in poorly differentiated adenocarcinoma of scirrhous carcinoma. K-sam amplification in scirrhous carcinoma often occurs independently of c-met gene amplification. LOH on chromosome 1p also is relatively common in poorly differentiated adenocarcinoma. Exceptionally, signet-ring cell carcinoma shares APC mutations. There are some differences in expression of the growth-factor/receptor system between well-differentiated adenocarcinoma and poorly differentiated adenocarcinoma. Moreover, interaction between cell-adhesion molecules in tumor cells expressing c-met and hepatocyte growth factor (HGF) from stromal cells is linked with morphogenesis of two histological types of stomach cancer. Intestinal metaplasia and adenoma of the stomach also contain p53 mutations and K-ras mutations or tpr-met rearrangement. Taken together, different genetic pathways of stomach carcinogenesis may exist for poorly differentiated and well-differentiated stomach cancers. Some of the latter may develop by a cumulative series of gene alterations similar to those of colorectal cancer.

<h4>Unlabelled</h4>It is still not clear whether disseminated intravascular coagulation (DIC) contributes to the hemostatic disturbances in orthotopic liver transplantation (OLT). Theoretically the lack of hepatic clearance of procoagulant factors during the anhepatic period and the release of thromboplastic material from the graft might trigger DIC. During heterotopic liver transplantation (HLT) the host liver is left in situ and procoagulant factors may still be cleared; DIC, if present, may not occur until after reperfusion. The aim of the present study was to gain more insight into the underlying mechanism of the coagulation changes during liver transplantation by comparison of OLT and HLT. Thrombin-antithrombin-III complexes (TAT), and indicator of thrombin generation, fibrin degradation products (FbDP) and routine clotting times were assayed in 12 OLTs, 18 HLTs and in a control group of 10 partial hepatic resections (PHR). TAT increased dramatically after reperfusion to 136 micrograms/l in OLT and to 94 micrograms/l in HLT (p n.s.). In contrast, FbDP levels increased only in OLT, to a maximum of 13.8 micrograms/ml. Routine clotting times changed mildly and similarly in both OLT and HLT.<h4>Conclusions</h4>Graft reperfusion triggers excessive thrombin formation, but there are no other signs of subsequent DIC. Any thrombin formed is probably rapidly inhibited by antithrombin-III. The rise in FbDP during OLT is the result of increased fibrinolysis, which occurred only in OLT and not in HLT.

A cDNA encoding a serotonin transporter (5-HTT) in the human dorsal raphe nucleus was isolated and sequenced using cross-species amplification of human 5-HTT partial cDNA by the polymerase chain reaction (PCR) and the RACE-PCR procedure, designed for rapid amplification of 3' and 5' cDNA ends. The cDNA contains an open reading frame encoding a hydrophobic polypeptide of 630 amino acids with a calculated molecular weight of approximately 70 kDa. The human 5-HTT is approximately 92% homologous to the rat protein but contains an additional consensus phosphorylation site for cAMP-dependent protein kinase recognition located in the cytoplasmic N-terminal region, while a potential protein kinase C phosphorylation site identified in the rat homolog is not conserved in the human 5-HTT. Hydropathicity analysis revealed twelve membrane spanning segments, a topology proposed for other cloned sodium-dependent transporters.

Genetic alterations of Ki-ras gene, p53 gene, and DCC gene were analyzed in human colon cancer cell lines (HCCLs). On the basis of these analyses, a HCCL (HCT116)-human chromosome 18 hybrids, and targeted cell lines that were disrupted at the activated Ki-ras gene in HCCLs (HCT116 and DLD-1), were established. Tumorigenicity and expression of c-myc gene were investigated in these cell lines, respectively. 1. Point mutations of Ki-ras gene, p53 gene, and insertion mutations of DCC gene were detected in 10 out of 18 HCCLs, 8 out of 15 HCCLs, and 3 out of 16 HCCLs, respectively. 2. HCT116-chromosome 18 hybrids were morphologically similar to the parental line, and were not suppressed for tumorigenicity in vitro, but they produced slowly growing tumors in nude mice compared with the growth of the parental line. 3. The targeted cell lines that were disrupted at the activated Ki-ras gene were morphologically altered and lost neoplastic phenotypes, including tumorigenicity in nude mice and anchorage-independent growth. Furthermore, expression of c-myc gene in these clones was much reduced compared with findings in the parental line, regardless of their growth rates.

The aim of this study was to evaluate the diagnostic usefulness of percutaneous liver biopsy and screening for hepatitis C virus antibodies with 1st and 2nd generation ELISA in asymptomatic blood donors with persistent (> 1 year) and moderate elevation (> 1.5 times the upper limit of normal) of serum alanine aminotransferase. The diagnosis was established from clinical, biological and ultrasound data before biopsies were obtained, then compared to the histological diagnosis. Thirty one of 56 blood donors who satisfied the preceding criteria accepted liver biopsy and were subsequently included in the study. An accurate diagnosis was proposed before biopsy in 20 cases. This was in agreement with the histological results in 19 cases but in 2 of these, unexpected lobular hepatitis was associated with the expected steatosis. Positive hepatitis C virus tests corresponded to chronic hepatitis in all cases (n = 5). No accurate diagnosis could be proposed in the 11 remaining cases owing to the lack of evidence of any etiology (n = 4) or because several potential etiologies were possible for the same subject (n = 7). Histological diagnoses were: isolated steatosis (n = 12), steatosis associated with lobular hepatitis (n = 7) or with chronic persistent hepatitis (n = 1), chronic active (n = 2) or chronic persistent hepatitis (n = 3), alcoholic hepatitis (n = 2), hemochromatosis (n = 1), and normal liver (n = 3). Liver biopsy is essential to the accurate etiological diagnosis of persistent and moderate elevation of aminotransferases despite hepatitis virus C tests which are associated with the correct diagnosis of chronic hepatitis in 16% of cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin complex (TAT) have been measured in the different surgical phases in 40 patients undergoing 43 orthotopic liver transplantations (OLT), in order to study thrombin generation throughout the surgical procedure. F1 + 2 and TAT progressively rose from preoperative values (1.47 +/- 0.92 nmol/l and 22.41 +/- 19.15 ng/ml, respectively) to the end of anhepatic phase (5.97 +/- 2.20 nmol/l, 64.24 +/- 11.30 ng/ml), whereas changes in circulating antithrombin III (ATIII) were negligible in this period. Thrombin generation continued to slightly but significantly increase immediately after liver graft reperfusion (F1 + 2 6.87 +/- 1.65 nmol/l, TAT 89.32 +/- 7.54 ng/ml), and ATIII levels decreased from 70.61 +/- 12.28 to 57.16 +/- 9.57%. Higher preoperative values of F1 + 2 were associated with larger requirements of both packed red blood cells and fresh frozen plasma (FFP) in the host liver explantation phase, whereas the lowest basal levels of ATIII were related to a higher plasma expense in the whole OLT procedure. The activation of circulating prothrombin begins early in OLT, and adequate FFP infusion is capable of maintaining appropriate circulating thrombin-inhibitory activity.

A yeast artificial chromosome (YAC B30) with a 320 kb insert of genomic DNA which includes the HLA-A gene was used to screen a cDNA library of human duodenal mucosa. Seven cDNA clones were isolated which correspond to seven new non-HLA class I structural genes. These new genes are located within a region that may well contain the gene responsible for hemochromatosis and have therefore been named HCG I-VII (Hemochromatosis Candidate Gene). HCG I, III, V and VI are probably single copy genes, situated at 180, 155, 140 and 230 kb centromeric to HLA-A, respectively. HCG II, IV and VII present several copies: one copy of HCG II, one of HCG IV and one of HCG VII are centromeric to HLA-A (at 30, 70 and 100 kb respectively). Another copy of HCG IV is 20 kb telomeric to HLA-A. Each of the genes localized on the YAC B30 is associated with an CpG/HTF island.

The exact knowledge of hormone receptor status is critical for therapeutic strategies in hormone-dependent tumors. The influence of tamoxifen on estrogen receptor concentration has to be taken into account when evaluating results in tamoxifen-treated patients. We studied the receptor modulation of tumors xenotransplanted into nude mice (one breast and one endometrial carcinoma) after injection of 50 micrograms tamoxifen/mouse. To differentiate between unoccupied and occupied receptors, determinations were done by an enzyme immunoassay for the estrogen receptor under low- and high-salt extraction. With low-salt extraction we found a temporary decrease of the estrogen receptor concentration within the first hours after tamoxifen treatment. This decrease lasted for several days before recovery to pretreatment levels occurred. The hormone-receptor complexes, tightly bound to acceptor sites of the DNA, increased more than 15 times within 24 h. These values remained at increased levels for 2-7 days, after which a decrease to initial level was observed.

Prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin-III-complex (TAT) levels were compared in 31 orally anticoagulated patients with inferior vena caval filters and a control group of 31 orally anticoagulated patients without caval filters and the incidence of markers of thrombophilia (deficiency of antithrombin-III, protein C, protein S and factor XII, presence of lupus anticoagulants) was determined. 8 of 31 patients (26%) from the group of caval filter carriers showed markers of thrombophilia (3 protein S deficiencies, 1 protein C deficiency, 2 factor XII deficiencies and 2 patients with lupus anticoagulants). In all orally anticoagulated patients a significant interdependence (p < 0.05) between F1 + 2- and TAT-levels and intensity (INR) of the oral anticoagulation could be observed. Comparison of F1 + 2- and TAT-levels of caval filter carriers and controls revealed no significant difference which leads to the conclusion that inferior vena caval filters do not induce detectable systemic activation of prothrombin under adequate oral anticoagulation therapy.

No abstract available.