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The concentrations of several proteases and antiproteases known to be present in ascites were tested in plasma and ascitic fluid with regard to their ability to separate ascites according to malignant or nonmalignant disease. Seventeen patients with proven malignant ascites and 37 with ascites due to liver cirrhosis were included. Activities of plasminogen, alpha 2-antiplasmin, antithrombin-III, and factor V, and the concentration of alpha 1-protease inhibitor were significantly higher in the plasma of patients with malignant ascites than in cirrhotic patients. Fibronectin, plasminogen, alpha 2-macroglobulin, alpha 1-protease inhibitor, antithrombin-III, and albumin revealed higher concentrations or activities in malignant ascites than in cirrhotic ascites. Due to a wide variation of most parameters, only fibronectin, antithrombin III, and alpha 1-protease inhibitor in ascites had a sensitivity and specificity higher than 90% for malignant ascites. When the specific protein/albumin ratio was used, only the accuracy of fibronectin was increased reaching a sensitivity and specificity of 100%. The plasma/ascites gradients of the proteins assessed differed significantly, that of fibronectin being much higher (22 +/- 7) than that of all other proteins. In malignant ascites fibronectin concentration was only correlated with alpha 1-protease inhibitor concentration but not with the concentration or activity of all other proteins, while in cirrhotic ascites most proteins revealed a positive correlation. The determination of the fibronectin concentration or the fibronectin/albumin ratio in ascites can differentiate malignant and nonmalignant ascites. All other proteases and antiproteases assessed are of lesser value for this purpose, although most are significantly increased in ascites and plasma of patients with malignant disorders.

H+-K+-exchange via the Trk-like system of K+ accumulation takes place in anaerobically grown S. typhimurium LT-2 with stable ratio of DCC-sensitive ionic fluxes, equal to 2H+ of a cell for one K+ of the medium. This exchange is now observed in the mutant S. typhimurium TH-31 with unfunctional H+-ATPase. H+-K+-exchange in aerobically grown S. typhimurium LT-2 has unstable ratio of ionic fluxes. The rate of K+ uptake in anaerobically grown bacteria is higher than that in the aerobically grown ones. Q10 is about 1.8 both for H+ transfer and K+ uptake in anaerobically grown bacteria, but it is 1.7 and 0.9 respectively in the aerobically grown ones. Delta psi is not changed by different temperatures both in anaerobically and aerobically grown bacteria. The distribution of K+ in anaerobically grown bacteria is higher than 10(3) and the potassium equilibrium potential is much higher than the measured delta psi. In aerobically grown bacteria the distribution of K+ is in good conformity with the measured delta psi. H+ and K+ transport in anaerobically grown cells is likely to proceed by the same mechanism, which includes H+-ATPase and the Trk-like system. In aerobically grown bacteria these transport systems work separately, and the Trk-like system as K+-ionophore serving for K+ uptake across the electrical field on the membrane.

cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A) tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approximately equal to 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approximately 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approximately 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.

Frozen tissue sections and cytosols from 89 specimens of breast and ovarian tumours have been assayed for the presence of estrogen receptor (ER) or related protein using four commercially available monoclonal antibody methods. These were estrogen receptor enzyme immunoassay (ER-EIA), estrogen receptor enzyme immunocytochemical assay (ER-ICA), ER D5 antigen immunoradiometric assay and ER D5 antigen immunocytochemical assay. The results have been compared with those obtained using a standard dextran coated charcoal steroid binding assay (ER-DCC). The correlation coefficient (r) between ER-DCC and ER-EIA results was 0.72 while that of both monoclonal antibody cytosol methods and their respective immunocytochemical assays was 0.66. ER-ICA gave additional valuable information concerning receptor heterogeneity in breast cancer sections. However, the correlation between ER D5 antigen assays and both ER-DCC and ER-EIA was weak (r less than 0.4). We conclude that there are a number of methodological advantages in using the kit systems including their ability to detect receptor presence in small tumour specimens (e.g., "Tru-cut" biopsies) but that their usefulness is limited by the current lack of widely available monoclonal based methods for the concurrent determination of progestogen receptor. We believe that, once these are available, immunocytochemical technology could offer an alternative method of determining the steroid receptor concentration in both ovarian and breast tumours, thus obviating the need for costly and time-consuming cytosolic methods, with their inherent difficulties of quality control.

We have investigated the sequence requirements for the cleavage-polyadenylation reaction in the human antithrombin III (ATIII) gene. A series of 5'-3' and 3'-5' deletions were produced around the AATAAA site using Bal31 nuclease. Ligation of appropriate pairs of such mutations resulted in the generation of varying sized deletions or duplications of sequences either upstream of, downstream of, or within the region encompassing the poly(A) site. Whereas a large deletion 3' to the AATAAA signal abolished cleavage and polyadenylation of ATIII transcripts, smaller deletions, all of which were subsets of the large one, did not. This indicated that the ATIII gene contains at least two independently acting poly(A)-cleavage signals 3' of AATAAA. When one of these signals was eliminated and the other was partially deleted at its 3'-end, we were able to disrupt the normal spacing between AATAAA and the cleavage site without substantially affecting the efficiency of the cleavage reaction. This suggested that the distance between AATAAA and the cleavage site is determined by the same sequence which, along with AATAAA, specifies cleavage and polyadenylation. The duplication of regions either upstream or downstream of AATAAA affected neither the efficiency nor the site of cleavage of the ATIII transcript. When a duplication included a large region containing AATAAA as well as downstream sequences, both sites were chosen for cleavage reactions. With a more delimited duplication, which included AATAAA but not the downstream cleavage signals, both sites were again used. However, two new cleavage sites were now detected. These results suggested that the distance between AATAAA sites and critical downstream cleavage-spacing sequences may also be important in determining the site of cleavage. This may in part stem from spatial constraints imposed by RNA-protein complexes which have been postulated to be critical in catalyzing the cleavage-polyadenylation reaction.

Both normal antithrombin-III (AT-III alpha) and the high heparin affinity form (AT-III beta) were isolated from pooled human plasma. AT-III beta had a lower negative charge and lower molecular mass than AT-III alpha. Sialidase and endo-F digestion indicated that the inherent difference resided in the oligosaccharide component of the molecule. CNBr fragmentation showed there was an oligosaccharide sidechain missing between residues 104 and 251, subdigestion with trypsin indicated that Asn 135 was not glycosylated in AT-III beta. Chromatography of total tryptic digests on concanavalin A-Sepharose confirmed that the high heparin affinity form of antithrombin lacked an oligosaccharide moiety at Asn 135.

In an attempt to elucidate the mechanism whereby dexamethasone could reactivate bovine herpesvirus type-1 the effect of dexamethasone on virus replication and leukocyte functions was assessed. No effect was detectable on either virus yield or in vitro replication kinetics. In contrast, dexamethasone influenced several leukocyte functions thought to be of importance in antiviral defense and maintenance of latency. In vitro exposure of peripheral blood polymorphonuclear neutrophilic granulocytes of normal animals to dexamethasone depressed their migratory and cytotoxic activities, but had no effect on Fc- and complement receptor expression. Dexamethasone also depressed lectin-induced lymphocyte proliferation and interleukin-2 generation in a dose-dependent manner. When cows were treated repeatedly with dexamethasone and their leukocytes assayed, suppression of phytohemagglutinin-induced lymphocyte proliferation, interleukin-2 generation, natural cytotoxicity of mononuclear cells and polymorphonuclear neutrophilic granulocyte functions were observed. In contrast, concanavalin A induced lymphocyte proliferation was increased following treatment.