The marine bacterium Alteromonas piscicida--a producer of enzymes with thrombolytic action

The ability of marine bacteria A. piscicida to produce exoproteases that were able to lyse human blood clots has been studied. Optimal conditions for biosynthesis of these enzymes have been found. The enzyme has been partially purified. In concentration of 1 mg/ml it has activity corresponding to that of 500 micrograms/l plasmine and 100 micrograms/ml trypsine. The enzyme activity was completely inhibited after incubation in human blood plasma.     

Modulation of latent protein phosphatase activity from vascular smooth muscle by histone-H1 and polylysine

An apparently latent phosphatase which migrated as a protein of Mr 130,000 during sucrose density centrifugation, and a spontaneously active phosphatase (Mr 68,000) were isolated from bovine aortic smooth muscle. Basal phosphorylase phosphatase activity of the latent preparations was stimulated 12 fold by low concentrations of lysine-rich histone-H1 (30 micrograms/ml) and 6 fold by polylysine (Mr 17,000; 12 micrograms/ml), whereas the spontaneously active enzyme was only slightly affected. The enzymatic activity of the spontaneously active preparation was completely destroyed by beta-mercaptoethanol. In contrast, the apparently latent enzyme was converted to a more active form of lower molecular weight (Mr 86,000) following treatment with beta-mercaptoethanol and this form of the enzyme was still stimulateable by histone-H1. These findings show that the aortic spontaneous and apparently latent phosphatase actives are ascribable to separate enzymes and they suggest that the activity of latent phosphatase in living cells may be modulated by cationic proteins such as histones or similar effector molecules.     

Comparative study of the properties of serine proteases of lower and higher vertebrates

Results of the comparative study of trypsin- and chymotrypsin-like serine proteases from pyloric caeca of salmon fishes and trypsin and chymotrypsin of bulls are presented in the paper. The hydrolytic activity of salmon proteases with respect to methyl ethers of N-benzoyl-L-leucine is 2.4 times higher than that of bull chymotrypsin, but with respect to methyl esters of N-benzoyl-L-tyrosine and N-benzoyl-L-arginine the activity of salmon proteases is 6.5 and 80 times lower than that of bull chymotrypsin and trypsin. Salmon proteases in contrast to bull trypsin and chymotrypsin hydrolyze but slightly N-glutaryl-L-phenylalanine para-nitroanilide. It shown that fish proteases are not absolutely specific to synthetic substrates, which is a result of their less pronounced (than in case of bull trypsin and chymotrypsin) differences in structures of binding centres. The study of the salmon protease interaction with some immobilized ligands has confirmed the higher affinity of enzymes to reagents with two space-separated aromatic rings in their composition. It is supposed that salmon proteases interact with such reagents through two sites: hydrophobic "pockets" and probably additional binding site of the active centre. The salmon protease preparation demonstrates higher resistance to inactivating action of formaldehyde within the range of concentrations 2-16% than bull chymotrypsin does.     

The mechanical and electrical effects of rhinoceros viper (Bitis nasicornis) venom on the isolated perfused guinea pig heart and atrial preparations

The mechanical and electrical effects of the venom of Bitis nasicornis were studied on the guinea-pig Langendorff and left atrial myocardium preparations. While Langendorff preparations were treated with individual doses of 0.1, 0.6 and 1.4 mg, isolated left atria were treated using concentrations of 2.0, 20 and 200 micrograms/ml of venom in the perfusion solution. In the Langendorff preparation, transient increases in left ventricular systolic pressure (LVSP) and heart rate (HR) were seen after 0.1 mg of venom. When 0.6 mg of venom was given, the increases were followed by decreases, while 1.4 mg doses simply induced decreases in LVSP and HR. After both 0.6 and 1.4 mg doses the decreases were accompanied by increases in left ventricular diastolic pressure. In addition to these mechanical effects, transient increases in HR with atrio-ventricular blocks, ventricular extrasystoles and tachycardia were observed after each dose. In the left atrium the 2 micrograms/ml venom concentration produced an increase, followed by a decrease, in the maximum tension developed, which was only seen to decrease with higher concentrations of 20 and 200 micrograms/ml of venom. A dose dependent significant reduction in the action potential duration was observed for the doses of 0.6 and 1.4 mg in the ventricle and for all three concentrations in the atrium.     

Effects of cycloheximide on inverse regulation of phenolic and nonphenolic ring deiodinases in monkey hepatocarcinoma cells

Both phenolic and nonphenolic ring deiodinase activities in monkey hepatocarcinoma cells (NCLP-6E) were increased by addition of serum in a concentration-dependent manner: the stimulatory effect of serum was evident at a concentration as low as 1.5%, and was maximal at 5%. Lineweaver-Burk analysis showed that the increases in the deiodinase activities are due to the increase in Vmax, but not in Km. The addition of cycloheximide at concentrations ranging from 0.1 to 50 micrograms/ml inhibited the stimulatory effect of serum on phenolic ring deiodinase activity progressively. On the other hand, nonphenolic ring deiodinase activity was increased as much as 4-fold by the addition of 0.5-5 micrograms/ml cycloheximide together with 0.5% serum; a high concentration of the drug, 50 micrograms/ml, however, did not elicit such an increase. Actinomycin D at 5 micrograms/ml completely abolished the increase in nonphenolic ring deiodinase activity by serum or cycloheximide. In addition, actinomycin D inhibited the increase in phenolic ring deiodinase activity by serum in a dose-dependent manner at concentrations ranging from 0.05 to 5 micrograms/ml. It is concluded that phenolic and nonphenolic ring deiodinases are regulated by different mechanisms in monkey hepatocarcinoma cells (NCLP-6E).     

Reversal of suppression of lymphokine-activated killer cells by transforming growth factor-beta in ovarian carcinoma ascitic fluid requires interleukin-2 combined with anti-CD3 antibody.

Ascitic fluid from human ovarian carcinoma (AF) has been shown to inhibit IL-2-induced lymphokine-activated killer (LAK) cell generation from peripheral blood mononuclear cells (PBMC) resulting from the presence of biologically active transforming growth factor-beta (TGF-beta). A 50% concentration of AF completely suppressed the LAK response to 100 units IL-2/ml and only partial reversal (less than 50%) could be achieved by increasing the IL-2 concentration to 1000 units/ml. We evaluated the ability of tumor necrosis factor-alpha (TNF-alpha, 1-1000 ng/ml) and anti-CD3 antibody (alpha-CD3, 1-100 ng/ml) to reverse AF-mediated suppression of IL-2-stimulated LAK generation. TNF-alpha alone did not generate significant LAK activity, but in the presence of suboptimal concentrations of IL-2 (10 and 100 units/ml), TNF-alpha significantly boosted the generation of LAK, but was unable to significantly reverse AF-mediated suppression of the IL-2 response (even at 1000 units/ml). In contrast, alpha-CD3 alone generated LAK activity at concentrations as low as 1 ng/ml and markedly enhanced generation of LAK activity when added to suboptimal concentrations of IL-2. alpha-CD3 combined with IL-2 significantly reversed AF suppression at 100 units IL-2/ml and at 1000 units/ml completely reversed suppression by two of three highly suppressive samples of AF. Significant reversal occurred with the third AF sample. It may be possible to overcome TGF-beta-mediated suppression by measures other than by increasing the IL-2 concentration.     

Differential effects of 4-hydroperoxycyclophosphamide on limb development in vitro

Cyclophosphamide must be metabolically activated to produce malformations in limbs developing in culture; 4-hydroperoxycyclophosphamide is an analog of the active metabolite of cyclophosphamide, 4-hydroxycyclophosphamide, that breaks down spontaneously in solution to form 4-hydroxycyclophosphamide. To study the mechanism by which metabolites of cyclophosphamide produce limb malformations in vitro we determined the effects of exposure of cultured limb buds to 4-hydroperoxycyclophosphamide. Fore- and hindlimbs were excised from ICR mice on day 12 of gestation and cultured in roller bottles for 6 days. Limbs were exposed to 4-hydroperoxycyclophosphamide for the first 20 hours of the culture period. Addition of 10 micrograms/ml of 4-hydroperoxycyclophosphamide to forelimb or to hindlimb buds in culture produced limb reduction malformations. A dramatic decrease in total limb bone area in fore- and hindlimbs was observed with 10 micrograms/ml of 4-hydroperoxycyclophosphamide. In forelimbs, the long bone area decreased and the paw area remained constant so that the relative contribution of the long bone area to total limb bone area was decreased. In hindlimbs treated with 10 micrograms/ml of 4-hydroperoxycyclophosphamide, no paw skeleton was observed. The DNA, RNA, and protein contents of the limbs were not affected by exposure to 1 microgram/ml of 4-hydroperoxycyclophosphamide, but were decreased by exposure to 10 micrograms/ml of this compound. Exposure to the higher concentration of 4-hydroperoxycyclophosphamide also decreased alkaline phosphatase activity, a marker for osteogenesis, in both fore- and hindlimbs; in contrast, neither concentration of 4-hydroperoxycyclophosphamide had an effect on creatine phosphokinase activity, a marker for myogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

RNA synthesis in starfish embryos: developmental consequences of its inhibition by formycin

Embryos of the starfish Asterina pectinifera were examined with regard to their ability to undergo the early events of embryonic development in the presence of formycin, an analogue of adenosine and a reported inhibitor of RNA synthesis. It was shown that in normal embryos the pool of ribonucleoside 5'-triphosphates increased during the period of blastula formation. The increase of the UTP pool was blocked nearly completely by 25 micrograms/ml formycin, and that of the CTP pool was inhibited partially by the same concentration of the drug. On the other hand, the pools of ATP and GTP were the same for both control and formycin-treated embryos. The development of embryos cultured in the presence of 25 micrograms/ml formycin stopped at the early blastula stage. Addition of 100 micrograms/ml each of uridine and cytidine to cultures of embryos that had been placed in 25 micrograms/ml formycin at the onset of blastulation allowed gastrulation to occur, suggesting that the developmental arrest produced by formycin is due primarily to the inhibition of pyrimidine nucleotide biosynthesis.     

Effects of PGI2 on the inactivation of thrombin, factor Xa, and plasmin by antithrombin-III and heparin.

The influence of PGI2 on the activity and on the inactivation of enzymes participating in blood coagulation (thrombin and Factor Xa) and fibrinolysis (plasmin) were investigated. According to the results PGI2 has no effect on the activity of Factor Xa and plasmin nor on the inactivation of these enzymes by antithrombin-III in the absence and presence of heparin at a concentration of PGI2 up to 400 micrograms/ml. An acceleration of the inactivation of thrombin by antithormbin-III was found in the presence of PGI2 within a concentration of 100-400 micrograms/ml without any effect on the heparin-accelerated inactivation of thrombin by antithrombin. We got similar results using clotting tests for the assay and the application of synthetic substrate for thrombin. This inactivation-accelerating effect of PGI2 on thrombin was only demonstratable at a concentration five magnitudes higher than that of the anti-aggregation effect on platelets.     

Differential methicillin susceptibilities of peptidoglycan syntheses in methicillin-resistant Staphylococcus aureus.

The mechanism of staphylococcal resistance to methicillin is unknown. Peptidoglycan synthesis was studied in a methicillin-resistant and a derived methicillin-sensitive Staphylococcus aureus strain. Although the methicillin minimum inhibitory concentration for growth of the methicillin-resistant strain was 1,600 micrograms/ml, peptidoglycan synthesis by the organism incubated in a wall synthesis solution was inhibited about 90% by 5 micrograms of methicillin per ml. In contrast, high concentrations of methicillin added to actively growing cultures of the methicillin-resistant strain had little effect on growth or peptidoglycan synthesis. Peptidoglycan synthesis in chloramphenicol-treated cultures was more susceptible to methicillin than it was in actively growing cultures of the methicillin-resistant strain. It is proposed that in this strain cell wall thickening peptidoglycan synthesis which predominates in cell wall synthesis solution and chloramphenicol-treated cultures is methicillin sensitive, whereas peptidoglycan synthesis involved in cell division, primarily in the region of the septum, which predominates in actively growing cultures is methicillin resistant. Both cell wall thickening and septal peptidoglycan syntheses are methicillin sensitive in the methicillin-sensitive strain.     

Studies on the antiviral activity of silver sulfathiazole

Silver sulfathiazole shows strong antibacterial activity and good tolerance after topical application. The aim of the study was to determine the antiviral activity of silver sulfathiazole in tissue culture after incubation of drug and virus. The antiviral activity was measured after various periods of exposure and at different drug concentrations. The results obtained indicate the activity of silver sulfathiazole against Herpesvirus type 1 and type 2. This drug suppresses or completely inactivates the infectivity of virus. The antiviral effect is directly related to concentration of the drug and duration of exposure. At concentration of 10 micrograms/ml it has the highest activity after 30 minutes of exposure, however at a concentration of 20 micrograms/ml it induces a similar effect after 10 minutes. Silver sulfathiazole had antiviral activity similar to that of silver nitrate, while sulfathiazole alone was ineffective.     

Stabilization of proteolytic enzymes in solution

The stability of the neutral and alkaline proteases in a Bacillus subtilis enzyme mixture was studied in aqueous solutions at room temperature. Stabilization of the proteases in solution for periods up to 25 days was achieved by the addition of various protein preparations including casein and soya protein. The degree of stabilization by casein was concentration dependent to about 2% protein. The instability of the neutral protease in solutions of the B. subtilis enzyme mixture was shown to be due primarily to proteolysis by the alkaline protease since the diisopropylfluorophosphate-treated enzyme was quite stable. Formulation of such enzyme solutions at low pH gave greater stability as did solutions containing an alkaline protease inhibitor from potatoes. A Conceptual approach to the formulation of enzyme solutions containing proteolytic enzyme to ensure maximum stability is proposed.     

Fibrinolytic properties of protease complex isolated from the culture fluid of Nocardia minima

The protease complex isolated form the Nocardia minima culture liquid was studied in vitro. The preparation had two different activities (fibrinolytic and activating), i.e. it was able to convert plasminogen into plasmin. At a concentration of 250 micrograms/ml and above the preparation lysed experimental thrombi. The fibrinolytic activity of the preparation was completely inhibited with normal human plasma.     

Absence of induction of IL-1 production in human monocytes by complement fragments

The ability of C fragments to induce IL-1 production in human monocytes was examined by using various approaches to carefully exclude the role of contaminating endotoxin. The presence of IL-1 activity in monocyte supernatants and lysates was assayed by the augmentation of PHA-induced proliferation of murine thymocytes. SRBC were opsonized with IgM rabbit antibodies and various human C components to prepare EAC reagents that contained less than 25 pg LPS/ml of EAC at 5 x 10(8) cells/ml. EAC1q, EAC4b, EAC4b2aoxy, EAC4b2aoxy C3b, EAC4b2aoxyC3bi, and EAC4b2aoxyC3d all failed to induce IL-1 production when incubated at 10- to 100-fold excess with adherent human monocytes. Similarly, LPS-free purified C3a, C5a, and C5a des Arg all showed no IL-1-inducing activities at concentrations up to 25 micrograms/ml. However, the same C5a preparations were active on human monocytes in the induction of chemotaxis, and C3a and C5a both induced skin-blueing in guinea pigs. Fragment Ba and Bb preparations purified by gel filtration chromatography contained approximately 100 pg LPS/micrograms Ba or Bb. These Ba and Bb preparations at 10 and 50 micrograms/ml, respectively, induced IL-1 production in the presence of 5 micrograms/ml polymyxin B (PMB). However, Ba and Bb preparations purified by affinity chromatography and HPLC contained lower levels of endotoxin contamination and displayed IL-1-inducing activities at Ba and Bb concentrations of 50 and 100 micrograms/ml, respectively, that were almost completely inhibited by PMB. To explore further the role of contaminating endotoxin, a Bb preparation was adsorbed with PMB-4B in the presence of a dialyzable detergent to remove LPS bound to the Bb. This LPS-free Bb preparation failed to induce IL-1 production while maintaining intact enzymatic activities. These results indicate that various solid phase or soluble C fragments, including C3b, iC3b, C3d, C3a, C5a, Ba or Bb do not induce IL-1 production in human monocytes in the absence of contaminating endotoxin.     

Penetration of zona-free hamster eggs by liposome-treated sperm from the bull, ram, stallion, and boar

Spermatozoa from each of four rams, four stallions, and three boars (six semen samples) were treated with dilauroylphosphatidylcholine (PC12) liposomes and compared with control bull sperm to induce the acrosome reaction (AR) and study possible penetration of the sperm into zona-free hamster eggs. Diluted sperm were incubated with several concentrations of PC12 for 7 min at 39 degrees C prior to insemination of the hamster eggs in vitro. The sperm from the bull were diluted to 10(6) cells/ml, as previously studied. Sperm from the ram, stallion, and boar were diluted to 6 X 10(6) and 20 X 10(6) cells/ml. After addition to the eggs, the sperm concentration was reduced by 75 percent. Inseminated eggs were incubated with sperm for 3 h at 39 degrees C prior to being fixed, stained, and observed for sperm penetration. At an initial concentration of 6 X 10(6) cells/ml, bull sperm treated with 36.7 microM PC12 achieved an egg penetration rate of 92%, whereas under nearly identical conditions stallion spermatozoa achieved only 54% egg penetration. Under similar conditions, ram spermatozoa failed to penetrate eggs, but when the initial sperm concentration was increased to 20 X 10(6) cells/ml, sperm incubated with 51.1 microM PC12 achieved 52% egg penetration. Boar spermatozoa treated with PC12 at either sperm concentration failed to exhibit an AR or penetrate hamster eggs. In general, as PC12 concentration increased the percentage of sperm with an AR increased and sperm motility decreased. It is concluded that 1) PC12 liposomes are effective in inducing the AR in sperm from the bull, ram, and stallion, but under conditions tested are ineffective with boar sperm;(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of endogenous modulators of chemoreactivity in the regulation of coronary blood flow

32 circulatory bands of coronary arteries of 5 pigs were studied. It has been revealed that the bands develop long-term tonic contraction activity in Krebs's solution (30 mM of KCl). In this case adrenalin in concentration of 10(-7) g/ml causes mild relaxation. In concentration of 10(-6) g/ml, it causes obvious relaxation. Trimetazidin and mildronat do not affect tonic contraction of the bands. They can quickly and reversibly increase relaxation effects of adrenaline. The preparations can increase ten-fold beta-adrenoreactivity of smooth muscles. This shows an important role of endogenic sensibilizers of beta-adrenoreceptors in regulation of coronary blood flow in humans.     

Fungicidal activity of cellobiose lipids from cultural fluid of yeast Cryptococcus humicola and Pseudozyma fusiformata

Cellobiose lipids of yeast fungi Cryptococcus humicola and Pseudozyma fusiformata have similar fungicidal activities against different yeast, including pathogenic Cryptococcus and Candida species. Basidiomycetic yeast reveals maximum sensitivity to these preparations; e.g., cells of cryptococcus Filobasidiella neoformans almost completely die after 30-min incubation in a glycolipid solution at a concentration of 0.02 mg/ml. The same effect toward ascomycetous yeast, including pathogenic Candida species, is achieved only at five to eight times higher concentrations of glycolipids. The cellobiose lipid from P. fusiformata, which, unlike glycolipid from Cr. humicola, has hydroxycaproic acid residue as O-subtituent of cellobiose and additional 15-hydroxy group in aglycone, inhibits the growth of the studied mycelial fungi more efficiently than the cellobiose lipid from Cr. humicola.     

Influence of nucleic acids and polysaccharides on phosphotransferase activity of preparations of secretory immunoglobulin A from human milk

The influence of nucleic acids (DNA, tRNA), synthetic oligonucleotides, and polysaccharides (lipopolysaccharides from Escherichia coli, heparin) on protein kinase and lipid kinase activities of preparations of human secretory immunoglobulin A (sIgA) has been studied. The preparations of sIgA were isolated from human milk by chromatography on the column with Protein A-Sepharose and DEAE-sorbent (sIgA1), by affinity chromatography of sIgA1 on DNA-cellulose (sIgA2), and by gel-filtration of sIgA1 in buffer containing 5% dioxane (sIgA3). Two 32P-labeled products with high and low electrophoretic mobility in polyacrylamide gel containing SDS were found after incubation of sIgA1 and sIgA2 with [gamma-32P]ATP. The product with low electrophoretic mobility was degraded in 10% trichloroacetic acid giving a radioactive background in lanes of the polyacrylamide gel. 32P-Labeled phospholipids were found among the phosphorylation products. Soluble and immobilized DNA increase lipid kinase activity of preparations of sIgA. In this case the secretory component and H-chains of sIgA were degraded. Fractions possessing lipid kinase activity were precipitated in the presence of heparin (1 mg/ml), and lipid kinase activity was separated from sIgA by gel-filtration in buffer containing 5% dioxane. 32P-Labeled products were formed in the presence of [gamma-32P]ATP as well as [32P]ortho-phosphoric acid. The influence of heparin and synthetic deoxy- and ribooligonucleotides on casein kinase activity of sIgA3 was studied. It was observed that deoxyribooligonucleotides in micromolar concentrations increased the rate of casein phosphorylation in the presence of sIgA3 and [gamma-32P]ATP. It has been proposed that catalytically active sIgA have an affinity to DNA (anti-DNA sIgA) and can be present in human milk as a part of lipoprotein complexes.     

Differential interaction of Xenopus embryonic cells with fibronectin in vitro

Two distinct cell types from the amphibian gastrula were compared with regard to their interactions in vitro with fibronectin (FN). Xenopus embryonic endoderm cells attach to FN substrates in a way characteristic of most cell types studied so far; that is, adhesion increases abruptly at a certain threshold concentration of FN, and maximal binding of cells already occurs at low FN concentrations (10 micrograms/ml). In contrast, embryonic ectodermal cells bind maximally to FN substrates only at unusually high concentrations of FN (200 micrograms/ml). This peculiar mode of attachment to FN has been characterized more closely. It is shown that the adhesion of ectodermal cells is modified by their interaction with a heparin-binding domain of the FN molecule. Furthermore, ectodermal cell adhesion increases very slowly with increasing FN concentrations. Despite these characteristic differences, both ectodermal and endodermal cells attach to the normal RGD cell-binding site of FN, as can be shown by competitive inhibition of adhesion by a hexapeptide containing the RGD sequence of amino acids.     

Effect of cyclosporin A on human lymphocyte responses in vitro. III. CsA inhibits the production of T lymphocyte growth factors in secondary mixed lymphocyte responses but does not inhibit the response of primed lymphocytes to TCGF

The mechanism whereby Cyclosporin A (CsA) inhibits secondary mixed lymphocyte responses was assessed. CsA added to secondary MLR cultures inhibited proliferation and induction of cytolytic lymphocyte activity. This inhibition was found to be associated with the inhibition of T lymphocyte stimulating growth factor(s) (TCGF) production in the supernatants of secondary MLR cultures. As little as 1.0 micrograms/ml of CsA added to secondary MLR cultures resulted in no measurable TCGF activity. In contrast, moderate doses of CsA (1.0, 2.5 micrograms/ml), which completely inhibited the secondary MLR response to alloantigen, did not inhibit the proliferative and CML response of alloantigen-primed lymphocytes to these stimulating growth factors. Even at high doses of CsA (20 micrograms/ml), substantial levels of proliferation (50% of control response) and CML induction (60% of control response) were observed when the primed cells were exposed to secondary MLR supernatants containing TCGF activity. It was concluded that inhibition of secondary mixed lymphocyte responses by CsA may be due in part to the inhibition of TCGF production rather than the inhibition of the effect of TCGF on mature cytotoxic T lymphocytes.