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In order to assess the thrombin and plasmin generation in vivo in disseminated intravascular coagulation (DIC), plasma levels of thrombin-antithrombin III (ATIII) complex (TAT) and plasmin-alpha 2-antiplasmin (a2AP) complex (PAP) were measured together with standard coagulation and fibrinolytic parameters in 80 patients with DIC. Both TAT and PAP were markedly elevated in patients with DIC. When plotted by the underlying disease categories, differences in the magnitude of the elevations of these complexes were recognized among groups. Patients with acute promyelocytic leukemia (APL) had the highest PAP, the lowest TAT/PAP ratio, low a2AP, and low fibrinogen, indicating that the most excessive fibrinolysis can occur in APL. Similar profiles, although less marked, were observed in patients with other leukemias and vascular diseases. Patients with sepsis showed the highest TAT/PAP ratio and the lowest PAP with no decrease in a2AP or fibrinogen, demonstrating a relatively impaired fibrinolysis. Patients with cancer had a relatively high TAT and high TAT/PAP ratio. In addition, both TAT and PAP were markedly elevated in patients with shock. From these, it was suggested that, although laboratory manifestations in DIC are extremely variable from patient to patient, underlying disorders are, at least in part, responsible for the observed variations. Recognition of this variable activation of coagulation and fibrinolysis would be helpful for the proper management of patients with DIC.

An immunohistochemical investigation of 62 human breast Cancers has been conducted using two different monoclonal antibodies, ER-ICA and ER-D 5, for the demonstration of the estrogen receptor. ER-ICA was found to be positive in 28 cases (45.9%) whereas ER-D 5 was positive in 41 cases (68.3%). In 34 cases, the results were compared to results of a biochemical assay that employed dextran-coated charcoal. The overall concordance between the ER-ICA and the DCC was 78.8%, whereas ER-D 5 and DCC was 60.6%. We thus have concluded that ER-ICA is the more sensitive antibody for evaluating the estrogen receptor status in human breast cancer cases by this immunohistochemical analysis.

We have identified the Xenopus cognates for the RNA-binding protein A1. This protein has previously been shown to be one of the components of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex in rat and human cells. We have isolated several Xenopus clones from oocyte, tailbud embryo, and leg muscle cDNA libraries and determined their nucleotide sequences. Potentially, two different A1 isoforms are expressed in Xenopus; they have been termed XA1a and XA1b. Besides insertions and deletions, the XA1a protein sequence is 92% identical to the rat protein and suggests very similar secondary structures. There are two segments in the COOH-terminal domain where deletions or insertions are apparent: the rat protein does not have a 48-residue sequence that is present in the frog protein, and the frog protein does not have a 12-residue sequence that is present in the rat protein. We have confirmed that the XA1a protein is larger than rat A1 by in vitro transcription, translation, and gel electrophoresis. The second isoform, XA1b, is very similar to the XA1a isoform, except it has a different COOH terminus due to the absence of a 73-nucleotide region from its cDNA clones. Transcripts representing both isoforms have been detected in various Xenopus RNA preparations by polymerase chain reaction experiments with A1-specific oligonucleotides. Our findings suggest that the isoforms are encoded by one or two genes and are the result of alternative splicing. We discuss the biological implications of having two forms of the A1 component of hnRNP particles.

Protein binding characteristics including percentage of total binding, total binding capacity (pmol/mg protein), degree of specific binding, competition with dihydrotestosterone (DHT) and estradiol (E2) binding sites and dissociation constants (Kd) of low and high affinity binding sites were investigated for the progestins cyproterone acetate (CPA), gestodene (G), norethisterone (NET) and levonorgestrel (LN) in serum or plasma pools from man and four laboratory animal species (rat, rabbit, dog and monkey). Serum pools from animals were constructed from samples obtained either prior to or 1 day after pretreatment with ethinyl estradiol (EE2) (5 micrograms/kg/day for 7 days). Human plasma pools differed by SHBG levels (normal/induced). All serum pools were characterized by protein content and distribution. Equilibrium dialysis or dextran-coated charcoal (DCC) methods were used to separate bound and free steroids labelled with tritium. All progestins were highly (greater than 80%) bound to proteins in all undiluted samples. Total binding capacity was highest in rat and lowest in monkey. Human plasma showed a capacity of 1.5-2.1 microgram steroid/ml. In man, monkey and rabbit LN and G were specifically bound to the same degree as DHT, whereas NET binding was 50% lower. Specific binding of CPA to dog serum was 2-3 times higher than for other steroids. Two (high and low affinity) binding sites were found for LN, G and NET in man, monkey and rabbit and in dog for LN. Kd values for high affinity binding ranged from 3.5 (G in man) to 23 (NET in man) x 10(-9)M. Kd values of low affinity binding varied from 0.5 (CPA in dog) to 4 (NET in man) x 10(-6)M. E2 and DHT competition experiments confirmed the concept of SHBG as a carrier protein of 19-nor-progestins and DHT and its occurrence in man, monkey and rabbit. A sex hormone binding protein (SBP) in the dog seems to be responsible for the relatively high specific binding of CPA. SHBG is inducible by means of EE2 in man and monkey, but not in rabbit. EE2 may induce SBP in the dog. Comparison of in vitro Kds (high affinity binding) and in vivo metabolic clearance rates showed the same rankings for LN, G and NET in man, monkey and rabbit.

A functionalized congener approach was used to design ligands for muscarinic cholinergic receptors (mAChRs). A series of omega-functionalized alkyl amides of N-methyl-4-(1-pyrrolidinyl)-2-butynamine (22) were prepared as functionalized analogues of UH 5 [N-methyl-N-[4-(1-pyrrolidinyl)-2-butynyl]acetamide], a muscarinic agonist related to oxotremorine. Intermediate 22 was coupled to a series of Boc-protected omega-amino acids, and the resulting amides were deprotected and acylated. Intermediate 22 was also acylated with succinic anhydride and derivatized. The synthetic intermediates and final compounds were evaluated in vitro for their effects on the turnover of phosphatidylinositides in SK-N-SH human neuroblastoma cells that express m3AChRs, and on the production of cyclic AMP in NG108-15 neuroblastoma x glioma cells that express only m4AChRs. The displacement of [3H]-N-methylscopolamine was also measured in membrane preparations from each of these cell lines. Conjugates of glycine and beta-alanine were agonists at m4AChRs, having little or no activity at m3AChRs. The potency in displacement of [3H]-N-methylscopolamine from both m3- and m4AChRs generally increased with increasing chain lengths of the omega-aminoalkyl congeners. The amides of 7-aminoheptanoic acid and 8-aminooctanoic acid, and their Boc-protected derivatives, had comparable affinities to UH 5 (Ki = 5.0 and 4.5 microM at m3AChRs and at m4AChRs, respectively) at both receptors but lacked any agonist effects.

Delta 3,delta 2-Enoyl-CoA isomerase (EC 5.3.3.8), an obligatory auxiliary enzyme for the metabolism of double bonds at odd-numbered positions of fatty acids during their beta-oxidation, was studied in hearts and livers of normal and clofibrate-treated rats. Hepatic peroxisomal and mitochondrial isoenzymes were separable by dye-ligand chromatography. The mitochondrial one was further purified to apparent homogeneity. An isomerase was also purified from heart muscle, a peroxisome-poor tissue. These enzymes were dimeric basic proteins (pI 9.5) with a subunit molecular weight of 30,000. Both cis- and trans-enoyl-CoA served as substrates for the hepatic enzyme studied. The velocity ratio for the C6-, C10-, and C12-trans-3-enoyl substrates was 9:2.5:1. By immunoelectron microscopy the enzyme protein selected for purification was found to be mitochondrial both in liver and heart. Chromatographic evidence, immunoelectron microscopy, and immunoblotting indicated that in the liver but not in the heart, the enzyme underwent an induction of 1 order of magnitude during clofibrate treatment. Antibodies towards the rat isomerases detected cross-reactive proteins in bovine and pig liver and heart and human placenta. The estimated subunit sizes varied from species to species, being 31,000 in bovine liver and heart, 29,000 in pig liver and heart, and 30,000 in human placenta. The data are in accord with the notion of a dual location of the delta 3,delta 2-enoyl-CoA isomerase. Mitochondrial origin of one of the isoenzymes and its tissue-specific induction by clofibrate were verified by immunochemistry and the identity of the peroxisomal one revealed by the chromatographic behavior of the proteins.

The methylation of the human oestrogen receptor (ER) gene was analysed by restriction enzymes in normal and neoplastic human breast tissues and cell lines. CCGG sequences in regions inside the gene, which are methylated both in normal breast and in tissues that are not the target of the oestrogen, are hypomethylated in 30% of tumours, both ER+ and ER- carcinomas. Moreover, 5' sequences of the gene, which are hypomethylated in normal breast and not in tissues not the target of oestrogen, are methylated to a lower degree in ER+ carcinomas, whereas they are methylated to a greater degree in ER- carcinomas. However, the same region is equally hypomethylated in both ER+ and ER- cancer cell lines. Our results indicate that in breast carcinomas ER DNA methylation is deranged, and in cancer cell lines is different from that observed in primary tumours. Furthermore, the abnormal methylation in the 5' end seems to be related to abnormal expression, namely diffuse hypomethylation in carcinomas with high ER content and hypermethylation in carcinomas without ER. These findings support our previous hypothesis that DNA methylation could be involved in the control of ER gene expression and demonstrate that abnormal ER gene methylation is a typical feature of breast cancers.

The rotavirus gene segment coding for the major outer capsid glycoprotein vp7 was amplified directly from stool specimens by the polymerase chain reaction (PCR). Double-stranded RNA extracted from stool samples was used as the template for reverse transcription, which was followed immediately and in the same reaction mix with amplification, using the Taq polymerase. Various conditions were examined to optimize the yield of the amplified gene. The concentrations of MgCl2, dimethyl sulfoxide, and template RNA were critical. The choice of primer pairs allowed amplification of the entire segment or specific portions. By using type-specific primers derived from distinct regions on the gene, we devised a PCR typing method in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels. The PCR typing method was applied to 10 rotavirus reference strains, including all 6 known human serotypes (serotypes 1, 2, 3, 4, 8, and 9), and to 34 stool specimens previously serotyped by an enzyme immunoassay with monoclonal antibodies. An absolute correlation was found between the molecular and serologic methods. In addition, 14 stool specimens nonserotypable by an enzyme immunoassay with monoclonal antibodies could be typed by the PCR method. Besides the application for rotavirus detection and typing directly from stools, the PCR method provides a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.

We have analyzed the binding of lipoprotein lipase (LPL) to the subendothelial extracellular matrix produced by cultured endothelial cells. Binding was linear up to a concentration of 0.5 microgram/ml (10 nM) enzyme used in this study, and equilibrium was achieved after 2 h of incubation with bovine 125I-LPL at 4 degrees C. Heparin and heparan sulfate effectively inhibited the binding of LPL to extracellular-matrix-coated plates; chondroitin sulfate had no effect, while high concentrations of dermatan sulfate or keratan sulfate inhibited binding of LPL to extracellular matrix by only 40%. Basic fibroblast growth factor (bFGF) did not affect LPL binding, while antithrombin-III (AT-III) caused up to a 50% inhibition of enzyme binding to extracellular matrix. alpha-Thrombin. 5.10(-6) M, and its esterolytically inactive derivative, DIP-alpha-thrombin, effectively inhibited binding of LPL to extracellular-matrix-coated plates. alpha-Thrombin was also able to release the extracellular-matrix-bound LPL in an active form. Extracellular-matrix-bound LPL detached into medium containing triolein emulsion and/or serum, and was catalytically active after being released. Extracellular-matrix-bound LPL lost 30% of its activity following incubation at 37 degrees C for 4 h. in contrast to soluble LPL which lost 75% of its activity. It is plausible to conclude from these data that in vivo the subendothelial basement membrane, similarly to extracellular matrix, sequesters and stabilizers LPL secreted into the subendothelial space by non-endothelial cells, and thus may play an important role in determining the route of LPL from its site of synthesis to its site of action.

Factor IX is the zymogen of the serine protease factor IXa involved in blood coagulation. In addition to a catalytic domain homologous to the chymotrypsin family, it has Ca2+, phospholipid, and factor VIIIa binding regions needed for full biologic activity. We isolated a nonfunctional factor IX protein designated factor IXEagle Rock (IXER) from a patient with hemophilia B. The variant protein is indistinguishable from normal factor IX (IXN) in its migration on sodium dodecyl sulfate-gel electrophoresis, isoelectric point in urea, carbohydrate content and distribution, number of gamma-carboxyglutamic acid residues, and beta-OH aspartic acid content, and in its binding to an anti-IXN monoclonal antibody which has been shown previously to inhibit the interaction of factor VIIIa with factor IXaN. Further, IXER is cleaved to yield a factor IXa-like molecule by factor XIa/Ca2+ at a rate similar to that observed for IXN. However, in contrast to IXaN, IXaER does not bind to antithrombin-III (specific inhibitor of IXaN) and does not catalyze the activation of factor X (substrate) to factor Xa. To identify the mutation in IXER, all eight exons of IXN and IXER gene were amplified by the polymerase chain reaction technique and cloned. A single point mutation (G----T) which results in the replacement of Val for Gly363 in the catalytic domain of IXER was identified. Gly363 in factor IXa corresponds to the universally conserved Gly193 in the active site sequence of the chymotrypsin serine protease family. X-ray crystallographic data in the literature demonstrate a critical role of this Gly in stabilizing the active conformation of chymotrypsin/trypsin in two major ways: 1) in the formation of the substrate binding site; and 2) in the development of the oxyanion hole. Our computer structural data support a concept that the Gly363----Val change prevents the development of the active site conformation in factor IXa such that the substrate binding site and the oxyanion hole are not formed in the mutated enzyme.

Heparin with low affinity for antithrombin III (ATIII) and devoid of anticoagulant activity was chemically oversulfated and fractionated by affinity for ATIII. The oversulfated material showed ATIII binding properties, as monitored by intrinsic fluorescence enhancement of ATIII. The fluorescence increase was comparable to that of the AT III high affinity fraction of native heparin. The estimated dissociation constants however, showed a 10-fold weaker binding of the oversulfated material to ATIII, Kd = 6.4 x 10(-8) M, as compared to native heparin, Kd = 0.63 x 10(-8) M. Concomitant with the binding-induced allosteric change in ATIII, the oversulfated material stimulated the ATIII-thrombin and ATIII-factor Xa reactions. The high affinity fractions of native heparin and the sulfated material were almost equally effective in enhancing the rate of thrombin neutralization by ATIII. However, a 3-fold faster rate of factor Xa inactivation was found with the native high affinity material.

An ELISA system, based on the novel use of a hapten-nylon conjugate as solid-phase coating antigen, has been applied in the screening of hybridoma cultures for anti-hapten monoclonal antibodies directed against the herbicide atrazine and its derivatives. Conjugation of a 2-aminocaproic acid derivative of atrazine with DCC to polyamide (Nylon 6) gave haptenated nylon which was soluble in aqueous cresol-ethanol mixtures and adsorbed efficiently on polystyrene microtitre plates. Reproducible ELISA results were obtained with culture supernatants of hybridomas derived from spleen cells of mice that had been immunized with atrazine-bovine serum albumin conjugates. Satisfactory results were also obtained with a water soluble peptide conjugated to nylon for use as a coating antigen in an ELISA. Plates coated with hapten-nylon as antigen have the added advantage that they can be stored at room temperature for at least 6 months without loss of activity. Nylon therefore appears to have general applicability as a carrier for both non-polar and polar haptens in the preparation and use of coating antigens.

Mesenteric venous thrombosis is a clinical entity, which is rarely recognized on admission. The patients are admitted with vague abdominal complaints and, eventually, abdominal sepsis might occur requiring laparotomy. Nowadays, underlying hypercoagulable states such as antithrombin-III, protein-C and protein-S deficiencies are recognized more frequently as a distinct cause of mesenteric venous thrombosis. In this paper, a case of mesenteric venous thrombosis due to protein-C deficiency is presented. The patients generally have a history of thromboembolism of the deep veins of the legs at young age. The combination of vague abdominal complaints and a history of thrombosis of the deep veins of the legs should arouse the suspicion of mesenteric venous thrombosis. In these cases, contrast-enhanced computerized tomography is a non-invasive diagnostic means which may provide the diagnosis. If infarction of the gut is present, resection is mandatory and a second-look operation should be performed. After surgery, heparinization is essential. This must be followed by administration of oral anticoagulants for an indefinite period in case of an underlying antithrombin III, protein-C or protein-S deficiency.

An automated multiple peptide synthesis method to synthesize, cleave, and purify several peptides simultaneously in a single batch has been developed. The technique is based on the synthesis of multiple peptides on a single solid phase support and is easily adapted to manual or to automated methods. The approach relies on coupling of amino acid mixtures to the resin and it has been found that DCC/HOBt gives the best coupling performance. Fast Atom Bombardment Mass Spectrometry (FAB-MS) was used to rapidly and efficiently identify the peptides in each synthetic mixture which significantly assisted the purification process by HPLC. The method has been successfully applied to the synthesis of magainin 2 and angiotensinogen peptides.

The model peptide TRH was successfully synthesized using benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent). The coupling reactions were carried out in N,N-dimethylformamide or N-methylpyrrolidone. These solvents allowed the incorporation of the N-terminal pyroglutamic acid residue into the peptide chain, without using the derivative bearing the N-benzyloxycarbonyl group, which acts as a solubility promoter. A comparative racemization study showed that Boc-His(Tos) can be coupled by means of BOP reagent with less racemization than with DCC when the amount of diisopropylethylamine (DIEA) is kept minimal (same ratio of equivalents as for Boc-His(Tos), i.e. 3 equiv.). However, with the use of a larger amount of DIEA in the coupling mixture (9 equiv.), approximately 3% of epimer was found in the crude product. Our study showed that even under low DIEA conditions, the rate of coupling of the residues with BOP remained comparable to that observed with DCC.

Antithrombin III (ATIII) deficiency is one of the few known abnormalities of the coagulation system known to predispose to venous thromboembolism but its relation to arterial disease is not established. We describe two related patients with this disorder, both of whom suffered arterial thrombotic events, at an early age. Both patients had other potential risk factors, though these would normally be considered unlikely to lead to such catastrophic events at such an age. Thrombosis due to ATIII deficiency is potentially preventable, and this diagnosis should be sought more frequently in patients with arterial thromboembolism, particularly if occurring at a young age. In addition, in patients with known ATIII deficiency, other risk factors for arterial disease should be eliminated, if possible. In particular, these patients should be counselled against smoking.