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Hydrolysis of GTP is known to accompany microtubule assembly. Here we show that hydrolysis of GTP is also associated with the formation of linear oligomers of tubulin, which are precursors (prenuclei) in microtubule assembly. The hydrolysis of GTP on these linear oligomers inhibits the lateral association of GTP-tubulin that leads to the formation of a bidimensional lattice. Therefore GTP hydrolysis interferes with the nucleation of microtubules. Linear oligomers are also formed in mixtures of GTP-tubulin and GDP-tubulin. The hydrolysis of GTP associated with heterologous interactions between GTP-tubulin and GDP-tubulin in the cooligomer takes place at a threefold faster rate than upon homologous interactions between GTP-tubulins. The implication of these results in a model of vectorial GTP hydrolysis in microtubule assembly is discussed.

Netrin-1 has profound in vitro effects on the growth properties of vertebrate embryonic axons. In addition, netrin-1 mRNA is found in the floor plate of the embryonic nervous system, an intermediate target of many axons, including commissural axons that are affected by netrin-1 in vitro. Moreover, genetic studies of netrin-1 homologs in Caenorhabditis elegans and Drosophila implicate these proteins in commissure formation. We raised polyclonal antisera that recognize chick netrin-1 in fixed tissue sections. The antisera were used to immunohistochemically map netrin-1 in the embryonic spinal cord, brain, and retina. The relationship between netrin-1 localization and the growth of pioneering axons suggests roles for netrin-1 in the regulation of circumferential, commissural, and longitudinal axon growth in the spinal cord and brain. The data also suggest that the primary or sole effect of netrin-1 on pioneering spinal cord commissural axons is haptotactic. Furthermore, the pattern of netrin-1 localization raises the possibility that this protein helps mediate neuronal migration in the spinal cord, brain, and retina.

<h4>Background & aims</h4>Small intestinal carcinomas are rare but occur with increased incidence in Crohn's disease. The aim of this study was to elucidate the genetic alterations.<h4>Methods</h4>Mutations and deletions involved in colorectal carcinoma were studied in sporadic and Crohn's-associated intestinal carcinomas and precursors.<h4>Results</h4>c-K-ras mutations were present in all four sporadic carcinomas with contiguous adenomas, in only 18% without adenomas (P = 0.01), in 43% of Crohn's-associated carcinomas, and in 14% of dysplasias. Overexpression of p53 gene product and/or 17p allelic loss were present in 47% of sporadic carcinomas and 33% of contiguous adenomas and in 71% of Crohn's-associated carcinomas and 43% of dysplasias. In contrast, allelic losses of 5q (adenomatous polyposis coli [APC] gene region) and 18q (deleted in colorectal cancer [DCC] gene region) were rare. DNA replication errors (RERs) were present in 13% of sporadic carcinomas and in the carcinoma and dysplasias of 1 patient with Crohn's disease (14%), but mutations in the transforming growth factor beta type II receptor (TGFbeta RII) gene were absent.<h4>Conclusions</h4>Accumulation of ras and p53 alterations occurs during the adenoma/dysplasia-carcinoma sequence in small intestinal carcinogenesis, but a ras-independent pathway may also exist. The infrequent loss of the APC and DCC regions and the absence of TGFbeta RII gene mutation in RER-positive neoplasms contrast with colorectal carcinogenesis.

The binding of [125I]-factor Xa to human umbilical vein endothelial cell (HUVEC) monolayers was studied. At 7 degrees C, [125I]-factor Xa bound to a single class of binding sites with a dissociation constant value of 6.6 +/- 0.8 nM and a binding site density of 57,460 +/- 5,200 sites/cell (n = 3). Association and dissociation kinetics were of a pseudo-first order and gave association and dissociation rate constant values of 0.15 x 10(6) M-1 s-1 and 4.0 x 10(-4) s-1, respectively. [125I]-factor Xa binding was inhibited by factor Xa but was not affected by factor X, thrombin or monoclonal antibodies against factor V, antithrombin-III or tissue factor pathway inhibitor (TFPI) but was inhibited by an antibody specific for the effector cell protease receptor-1 (EPR-1), a well-known receptor of factor Xa on various cell types. [125I]-factor Xa binding to HUVEC was not affected by various inhibitors of factor Xa such as DX 9065, pentasaccharide-antithrombin-III or TFPI. Factor Xa increased intracellular free calcium levels and phosphoinositide turnover in endothelial cells and, when added to HUVEC in culture, factor Xa was a potent mitogen, stimulating an increase in cell number at a 0.3 to 100 nM concentration. HUVEC-bound factor Xa promoted prothrombin activation in the presence of factor Va only. This effect was inhibited by both indirect and direct inhibitors of factor Xa. These findings indicate that HUVEC express functional high affinity receptors for factor Xa, related to EPR-1, which may be of importance in the regulation of coagulation and homeostasis of the vascular wall.

Cell adhesion molecules of the Ig superfamily are implicated in a wide variety of biological processes, including cell migration, axon guidance and fasciculation, and growth control and tumorigenesis. Expression of these proteins can be highly dynamic and cell type specific, but little is known of the signals that regulate such specificity. Reported here is the molecular cloning and characterization of rat CDO, a novel cell surface glycoprotein of the Ig superfamily that contains five Ig-like repeats, followed by three fibronectin type III-like repeats in its extracellular region, and a 256-amino acid intracellular region that does not resemble other known proteins. In rat embryo fibroblasts, cdo mRNA expression is maximal in confluent, quiescent cells. It is rapidly and transiently down-regulated by serum stimulation of such cells, and is constitutively down-regulated in oncogene-transformed derivatives of these cells. CDO protein levels are also dramatically regulated by cell-substratum adhesion, via a mechanism that is independent of cdo mRNA expression. The amount of CDO produced at the surface of a cell may therefore be governed by a complex balance of signals, including mitogenic stimuli that regulate cdo mRNA levels, and substratum-derived signals that regulate CDO protein production. cdo mRNA is expressed at low levels in most adult rat tissues. A closely related human gene maps to chromosome 11q23-24, a region that displays frequent loss of heterozygosity in human lung, breast, and ovarian tumors. Taken together, these data suggest that loss of CDO function could play a role in oncogenesis.

Lipid peroxidation is an autocatalytic mechanism leading to oxidative destruction of cellular membranes. The deleterious consequences of this mechanism are related in part to the formation of reactive aldehydic products that bind to intra- or extracellular molecules to form adducts. Specific antibodies directed against malondialdehyde (MDA) and 4-hydroxynonenal (HNE) adducts, major aldehydic metabolites of lipid peroxidation, allowed us to investigate in situ, with an immunohistochemical procedure, the occurrence of lipid peroxidation in a panel of different chronic liver diseases. Intracellular HNE and MDA adducts were detected respectively in 24 of 39 cases (62%) and in 12 of 34 cases investigated (35%). They were localized mainly in the cytoplasm of hepatocytes, with the strongest staining observed in hemochromatosis, Wilson's disease, and in areas of acute alcoholic hepatitis in cases of alcoholic liver diseases. A peculiar pattern of immunostaining was observed in primary biliary cirrhosis where biliary cells of destroyed but also intact bile ducts strongly expressed HNE adducts. The liver extracellular matrix also displayed MDA adducts (30 of 34 cases, 88%) and HNE adducts (23 of 39 cases, 59%). While HNE adducts were specifically localized on large bundles of collagen fibers, MDA adducts were detected in a thin reticular network and in sinusoidal cells around portal tracts or fibrous septa. In conclusion, lipid peroxidation by-products are detectable in chronic liver diseases. Immunohistochemical results suggest that this mechanism is implicated very early in the pathogenesis of some of these diseases.

Huntingtin, the protein product of the Huntington's disease gene, associates with vesicle membranes and microtubules in neurons. Analysis of axonal transport with a stop-flow, double crush ligation approach in rat sciatic nerve showed that full length huntingtin (350 kDa) and an N-terminal cleavage product (50 kD) were increased within 6-12 h on both the proximal and distal sides of the crush site when compared with normal unligated nerve. The huntingtin associated protein HAP 1 and the retrograde motor protein dynein also accumulated on both sides of the crush, whereas the vesicle docking protein SNAP-25 was elevated only proximally. The cytoskeletal protein alpha-tubulin was unaffected. The rapid anterograde accumulation of huntingtin and HAP 1 is compatible with their axonal transport on vesicular membranes. Retrograde movement of both proteins, as seen by accumulation distal to the nerve crush, may be necessary for their degradation at the soma or for a function in retrograde membrane trafficking.

Analysis of 784 informative meioses in the CEPH pedigrees revealed a total of 22 recombination events having occurred in the 6-Mb region between D6S265 (70 kb centromeric of HLA-A) and D6S276. These 22 breakpoints were localized with respect to anonymous polymorphic markers, leading to a detailed genetic map of the region telomeric to the human major histocompatibility complex. A nonrandom pattern of recombination was observed throughout this region: the low recombination rate of 0.19% within the 4-Mb interval centromeric to the HLA class I-like candidate gene for hemochromatosis indeed contrasts with the approximate 1% rate observed within the most telomeric two megabases. This reduced rate of recombination may be due to selective constraints depending on environmental factors related to immunity and iron status or to structural variations hampering proper meiotic pairing of homologous sequences. Population data from other human genome segments are now needed to determine whether linkage disequilibrium extending over 4 Mb is unique to this region.

<h4>Background</h4>Alterations in blood coagulation and fibrinolysis are believed to play an important role in the pathogenesis of preeclampsia. Hypercoagulability may be associated with features seen in preeclampsia, such as fibrin deposition in various organs, consumptive thrombocytopenia, and placental hypoperfusion, insufficiency and infarction.<h4>Methods</h4>In this cohort study, we compared the plasma levels of markers of blood coagulation and fibrinolysis in preeclamptic women to normotensive, pregnant controls. We also studied the association between these markers and intrauterine growth retardation (IUGR).<h4>Results</h4>In both mild and severe preeclampsia, the mean plasma concentrations of thrombin-antithrombin III complex (TAT) and plasminogen activator inhibitor type 1 (PAI-1) activity were significantly increased, while fibrinogen, antithrombin III (ATIII) and plasminogen activator inhibitor type-2 (PAI-2) antigen levels were significantly reduced compared to controls. Plasma D-dimer concentration was significantly higher in severe, but not in mild preeclampsia compared to the controls. The plasma level of fibrin monomer was similar in patients and controls. Patients with preeclampsia delivering growth retarded infants had significantly lower PAI-1 activity and PAI-2 antigen concentrations in plasma than the remaining preeclamptic women.<h4>Conclusion</h4>We found evidence of increased intravascular coagulation and fibrin turnover in preeclampsia. Low PAI-2 antigen plasma levels were associated with severe preeclampsia and IUGR.

The effects of acute haemodilution, during prostaglandin E1 (PGE1)-induced hypotension, on the blood coagulation-fibrinolysis system were studied in 40 patients undergoing hip surgery. The patients were randomly divided into four groups of 10 patients each; Group A (control) received no induced hypotension or haemodilution, group B received hypotension alone, group C received haemodilution alone and group D received the combination of induced hypotension and haemodilution. Haemodilution in groups C and D was produced by drawing approximately 1000 mL of blood and replacing it with the same amount of 6% hydroxyethyl starch. Induced hypotension in groups B and D was conducted with PGE1 and mean blood pressure was maintained at 55 mmHg. The mean dosage of PGE1 was 648 micrograms in group B and 661 micrograms in group D. In the control and PGE1-induced hypotension groups there was no significant change in platelet count (PLT), prothrombin time (PT), activated partial thromoplastin time (aPTT), fibrinogen (FIB), antithrombin-III (AT-III) or plasminogen (PLG). Haemodilution alone caused significant decreases in PLT (-43%), PT (+21%), FIB (-33%), AT-III (-21%) and PLG (-27%), and a significant increase in aPTT (+26%), whereas the combination of PGE1-induced hypotension did not cause any further change in these parameters. Serum-fibrin degradation products (FDP) significantly increased (+300%) and PLG significantly decreased (-30%) after surgery in all groups. It can be concluded that acute haemodilution to a haematocrit value of 22 +/- 2% causes a slight coagulopathy, which is not enhanced when combined with PGE1-induced hypotension.

Oat spelts xylan (OSX), fucoidan, kappa carrageenan and chondroitin sulfates A and C were sulfated using chlorosulfonic acid-pyridine complex while the first three were phosphorylated using methane sulfonic acid-phosphorous pentoxide mixture. The compounds were isolated as the sodium salts and their in vitro anticoagulant properties were determined by measuring the concentration of each compound required to double prothrombin time of pooled normal human plasma. The results of 31P-nmr spectroscopy showed that phosphorylation significantly increased the molecular weights of the polysaccharides by forming phosphodiester and diphosphodiester bonds. In general the anticoagulant properties of the sulfated polysaccharides were related to the % sulfate while the phosphorylated polysaccharides showed increases in anticoagulant properties which were related to the increase in the molecular weight and inversely related to the % phosphate. The mechanism of action of oat spelts xylan phosphate (OSXP) was studied using 125I-thrombin and normal human plasma. The results showed that at lower concentration of the OSXP, the complexation of 125I-thrombin with heparin cofactor-II(HC-II) was enhanced, while at higher concentration of the compound, the complexation with both antithrombin-III(AT-III) and HC-II was enhanced.

A 42-year-old man was admitted to the hospital because of sudden left-lateral chest pain and dyspnea. The chest roentgenogram on admission showed a string-like shadow and pleural effusion, and analysis of arterial blood gases revealed hypoxemia. Many bilateral segmental defects were seen on a radioisotope perfusion scan of the lungs, and the chest CT scan showed a low-density area in the trunk of the pulmonary artery. From these findings, pulmonary thromboembolism was diagnosed. The serum antithrombin III (ATIII) activity and the antigen value were low, and the patient's son's serum ATIII level was also low. Both the patient and his son were given the diagnosis of congenital ATIII deficiency. Gene analysis revealed two point mutations in exon 6 of the ATIII gene, so we classified this case as type IIc. The patient was treated with intravenous urokinase and heparin, and was then given warfarin. The arterial blood gas tensions improved, and the low-density area of the pulmonary artery on the CT scan disappeared, as did the pulmonary perfusion defect on the radioisotope scan. The clinical manifestations of members of 28 families with congenital ATIII deficiency reported in Japan were reviewed. Patients in whom many point mutations were detected in the ATIII gene were rare.

End-stage liver disease secondary to cryptogenic cirrhosis is the indication for orthotopic liver transplantation (OLT) in 7% to 14% of recipients. However, there are no reports documenting the outcome of OLT for this indication. The aim of this study was to determine (1) survival and (2) the incidence of histological recurrence of cryptogenic cirrhosis after OLT. Between March 1985 and December 1994, 560 OLTs were performed at our institution. Of these, 39 transplants for cryptogenic cirrhosis were in patients who met the following criteria: antinuclear antibody < 1:40; negative anti-smooth muscle antibody, antimitochondrial antibody, polymerase chain reaction for hepatitis C virus, and hepatitis B surface antigen results; normal ceruloplasmin and alpha-1 antitrypsin phenotype; transferrin saturation < 65%; and liver biopsy specimen not suggestive of hemochromatosis or other known disorders. Histological recurrence was assessed with protocol liver biopsies in all patients who survived longer than 6 months. The mean age of cryptogenic recipients at the time of transplantation was significantly lower (40.6 years; range, 3 to 63 years) than that of noncryptogenic recipients (48.5 years; range, 1-70; P < .03). Median modified Child's-Pugh score was slightly higher for cryptogenic recipients at the time of transplantation (10.0 + 0.08 standard error of mean [SEM]), than for the noncryptogenic recipients (9.0 + 0.03 SEM; P < .02). Actuarial survival was 72% (+ 0.07 SEM) at 1 and 58% (+ 0.08 SEM) at 5 years for cryptogenic recipients compared with 89% at 1 and 80% at 5 years for noncryptogenic recipients. The difference in survival was significant (P < .001) at both 1 and 5 years. Among the 27 cryptogenic recipients surviving more than 6 months (mean follow-up, 5.5 years), 6 have persistent hepatitis histologically without apparent infectious, vascular, biliary, or drug origins. Four patients (15%) had chronic active hepatitis, and 2 (7%) had steatohepatitis. No cases of recurrent cryptogenic cirrhosis were seen. OLT for cryptogenic cirrhosis is associated with a poor outcome compared with other indications, hepatitis of uncertain origin occurred in 22% of cryptogenic recipients surviving longer than 6 months, and no evidence of recurrence of cryptogenic cirrhosis was seen thus far in follow-up.

<h4>Unlabelled</h4>The purpose of this study was an attempt to extrapolate favorable observations on the effects of magnesium on platelets and haemostasis from animal models to humans. Intravenous magnesium in the treatment of acute myocardial infarction has been tested in several large clinical trials and remains controversial. The mechanism for the cardioprotective properties of magnesium is unknown. Based on experimental studies, several different hypotheses have been advanced to explain the benefits of magnesium including it's effects on platelets and haemostasis. However, few studies have analysed such a relationship in humans. This project was designed to assess the effect of bolus magnesium infusion on ex vivo platelet aggregation and certain haemostatic parameters in healthy volunteers. One gram of magnesium was diluted in 50 ml of D5W and infused over a period of 15 min. The changes of platelet aggregability, plasma antithrombin-III, Protein C, total Protein S, fibronectin, endothelin-1, as well as the metabolites of thromboxane and prostacyclin were determined prior to and immediately after magnesium infusion.<h4>Results</h4>Serum magnesium concentration increased from 2.10 +/- 0.08 at baseline to 3.12 +/- 0.13 mg dl-1 (P = 0.0002) post-infusion. Magnesium infusion was associated with a significant elevation in ADP (+19.3%); ristocetin (+13.6%); and collagen-induced platelet aggregation (+14.2%); an increase in plasma antithrombin-III (+10.3%) and thromboxane (+49.4%) when compared to pre-infusion levels. Against this, total Protein S (-20.7%), Protein C (-11.2%) and ET-1 (-26.3%) plasma concentrations were markedly decreased. There were no significant differences in plasma fibronectin and prostacyclin levels. Contrary to expectations, magnesium infusion in human volunteers is associated with platelet activation and mostly small unfavourable changes in certain haemostatic factors. However, the observed changes were small and for the most part remained within the normal physiologic range.

Hereditary haemochromatosis (HHC), a condition of abnormal iron absorption, is among the commonest diseases in humans. Early diagnosis is vital, because the disease is easily treated by phlebotomy to remove iron. Recently, mutations were identified in a HLA-class I-like gene, designated HFE and possibly associated with HHC. It was found that 83% of unselected HHC patients and more than 90% of patients with a clear family history of HHC, were homozygous for a single point mutation resulting in an amino acid substitution (Cysteine282Tyrosine) in the HFE protein. Although these data look compelling, they are not definitive proof that HFE is the haemochromatosis gene, but identification of these mutations will be of value in early diagnosis of haemochromatosis. However, the questions when to use the assay for detection of the Cys282Tyr mutation and how to interpret the results are not fully clear yet. Therefore, caution is still advocated when using molecular testing.

<h4>Background</h4>Patients with severe hepatic failure present acquired deficiency of antithrombin III (ATIII) owing to reduced synthesis associated with intravascular activation of blood coagulation, which may be corrected by ATIII infusion.<h4>Objective</h4>The aim of this uncontrolled trial was to verify the effect of a standard dose of ATIII concentrate (Kybernin), that is, 50 U/kg of body weight per day, every 2 days, on ATIII levels in patients with severe hepatic failure and hemostatic imbalance.<h4>Patients and methods</h4>Six cirrhotic patients were studied: mean age of 44 years (14 to 63 years), who presented at least 2 abnormal coagulation tests (PT > 1.40, APTT > 1.25, Fibrinogen < 1.5 g/dL, Platelet count < 80,000/mm3). Mean serum albumin was 2.6 g/dL (1.9 to 3.8 g/dL). Blood was drawn before infusion, 4 h after the first infusion, and just before the next infusion. ATIII levels were measured by amidolytic method.<h4>Results</h4>Mean ATIII levels were: initial = 35.8%, 4th h = 56.2%*, 2nd d = 48.7%*, 4th d = 45.7%*, and 8th d = 42.3%. ATIII levels increased significantly after infusion of this standard dose in all patients, although they have not been fully corrected (Friedman test, * p < 0.02), which has been sustained till the 4th day. There was no improvement on the clinical outcome.<h4>Conclusions</h4>These findings suggest that doses of ATIII concentrate higher than 50 U/kg/infusion must be administered to patients with severe hepatic failure, to guarantee normal levels of the inhibitor, in order to verify its influence on the hemostatic mechanism.

<h4>Objective</h4>To study the relationship between estrogen receptor (ER) and progesterone receptor (PR) and the clinico-pathologic features in endometrial carcinoma.<h4>Methods</h4>ER and PR contents of fresh tumor tissues taken from 70 cases of primary endometrial carcinoma were measured by biochemical (DCC) method and 30 paraffin-embedded archival specimens of the 70 cases also by immunohistochemical (IHC) method.<h4>Results</h4>Both ER and PR positive rates were 77.1% by DCC assay and 83.3% by IHC method. Excellent correspondency of one method to the other was observed (ER 83.3%, PR 86.7%). And the IHC method displayed further the origin of the tissue examined. The ER and PR levels correlated negatively with the grades of tumor (P < 0.01). ER and PR positive rates in adenocarcinoma (including papillary adenocarcinoma) and adenoacanthoma were higher than those in the other histological types (ER, P < 0.01; PR, P < 0.005). There was a positive correlation between the ER levels and obesity.<h4>Conclusions</h4>The levels of ER and PR and the histopathological classification and grading of the tumor tissue reflect biologic behaviors of endometrial carcinoma. ER and PR assays are important for endocrinotherapy and in predicting prognosis.