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PubMed1988

Kinetics of proteolysis of hog gastric mucin by human neutrophil elastase and by Pseudomonas aeruginosa elastase.

Human neutrophil and Pseudomonas aeruginosa elastases were compared for their ability to degrade hog gastric mucin, which was used as a model substrate. P. aeruginosa elastase was more active than neutrophil elastase, and 2 to 10 peptide bonds were hydrolyzed within 5 min. The results demonstrate that both elastases degrade mucins actively at concentrations comparable to physiological levels of neutrophil elastase, which raises the possibility that proteolysis of mucins may be one mechanism of damage during chronic infection and inflammation of the respiratory tract.

Gastric Mucosa↗
PubMed1993

Presumptive diagnostic differentiation of hog cholera virus from bovine viral diarrhea and border disease viruses by using a cDNA nested-amplification approach.

Hog cholera virus (HCV), bovine viral diarrhea virus (BVDV), and border disease virus (BDV) are closely related pestiviruses. BVDV and BDV are found worldwide but seldom cause disease in swine. In contrast, HCV has been successfully eradicated from swine in several nations but poses a potentially devastating threat to them because of its great virulence. Rapid differential diagnosis of HCV from BVDV and BDV infections in swine is vital for detection of the possible reintroduction of HCV into national herds from which it has been eradicated. Nested polymerase chain reactions (PCRs) for each of two pestiviral genomic segments are described. Amplification of the relatively conserved 5' genomic terminus identified 59 of 61 HCV, BVDV, and BDV isolates generically as pestiviruses. Nested amplification of the second region was designed to differentiate HCV from BVDV and BDV by exploiting relatively conserved differences in the nucleotide sequences that encode the major envelope glycoprotein. This second PCR correctly identified 36 of 36 diverse HCV isolates while failing to recognize any of 25 BVDV and BDV isolates. Multiple restriction fragment length analyses confirmed the identities of both external and nested PCR products. The two sets of PCRs may help confirm the generic identity of most pestiviruses and may permit presumptive differential diagnosis of HCV from BVDV and BDV.

Animals↗
PubMed1993

Detection of hog cholera virus and differentiation from other pestiviruses by polymerase chain reaction.

Reverse transcription coupled with the polymerase chain reaction (RT-PCR) was used for the detection and differentiation of pestiviruses. For this purpose, one primer pair was selected from a highly conserved region of the genome of pestiviruses. Using these primers (PEST 1-PEST 2), DNA fragments of between 72 and 74 bp could be amplified from all pestivirus isolates tested. In order to differentiate hog cholera virus (HCV) from bovine viral diarrhea virus (BVDV) and border disease virus (BDV), we selected a primer pair from a conserved region in the genome of HCV strains that differed from that sequenced in the genome of BVDV strains. By using these primers (HCV 1-HCV 2), a DNA fragment of 478 bp could be specifically amplified from HCV isolates. By these means, viral RNA was detected in extracts of lymph node, spleen, tonsil, and lung. Such extracts were used directly for RT-PCR without prior RNA isolation. We also performed multiplex PCR by using both the PEST 1-PEST 2 and HCV 1-HCV 2 primer pairs in a single reaction. This allowed the differentiation of HCV from BVDV and BDV in one step. To assess the sensitivity of the method, RT-PCR was compared with virus propagation in tissue culture and subsequent detection by immunofluorescence staining. The results show that RT-PCR is useful for the rapid detection and differentiation of pestiviruses.

Animals↗
PubMed1991

Structural proteins of hog cholera virus expressed by vaccinia virus: further characterization and induction of protective immunity.

A cDNA fragment covering the genomic region that encodes the structural proteins of hog cholera virus (HCV) was inserted into the tk gene of vaccinia virus. Expression studies with vaccinia virus/HCV recombinants led to identification of HCV-specific proteins. The putative HCV core protein p23 was demonstrated for the first time by using an antiserum against a bacterial fusion protein. The glycoproteins expressed by vaccinia virus/HCV recombinant migrated on sodium dodecyl sulfate-gels identically to glycoproteins precipitated from HCV-infected cells. A disulfide-linked heterodimer between gp55 and gp33 previously detected in HCV-infected cells was also demonstrated after infection with the recombinant virus. The vaccinia virus system allowed us to identify, in addition to the heterodimer, a disulfide-linked homodimer of HCV gp55. The vaccinia virus/HCV recombinant that expressed all four structural proteins induced virus-neutralizing antibodies in mice and swine. After immunization of pigs with this recombinant virus, full protection against a lethal challenge with HCV was achieved. A construct that lacked most of the HCV gp55 gene failed to induce neutralizing antibodies but induced protective immunity.

Animals↗
PubMed1991

Hog cholera virus: molecular composition of virions from a pestivirus.

Virions from hog cholera virus (HCV), a member of the genus Pestivirus, were analyzed by using specific antibodies. The nucleocapsid protein was found to be a 14-kDa molecule (HCV p14). An equivalent protein could also be demonstrated for virions from another pestivirus, bovine viral diarrhea virus. The HCV envelope is composed of three glycoproteins, HCV gp44/48, gp33, and gp55. All three exist in the form of disulfide-linked dimers in virus-infected cells and in virions; HCV gp44/48 and gp55 each form homodimers, whereas gp55 is also found dimerized with gp33. Such complex covalent interactions between structural glycoproteins have not been described so far for any RNA virus.

Antigens, Viral↗
PubMed1992

A second envelope glycoprotein mediates neutralization of a pestivirus, hog cholera virus.

Several monoclonal antibodies (MAbs) raised against hog cholera virus (HCV) reacted with the HCV structural glycoprotein gp44/48 and neutralized the virus. The presence of HCV gp44/48 on the viral surface was directly demonstrated by immunogold electron microscopy. Eight anti-HCV gp44/48 MAbs were tested by immunoperoxidase assay against a panel of pestivirus strains. Each MAb showed a distinct pattern of reactivity with HCV strains. It is suggested that the MAbs are well suited for epidemiological investigations of HCV outbreaks.

Animals↗
PubMed1993

Cellular immune response to hog cholera virus (HCV): T cells of immune pigs proliferate in vitro upon stimulation with live HCV, but the E1 envelope glycoprotein is not a major T-cell antigen.

T-cell responses of pigs to hog cholera virus (HCV) have reportedly been absent or difficult to detect. Therefore, little is known about cellular immunity to HCV. In this study, we used an attenuated strain of pseudorabies virus expressing the envelope glycoprotein E1 of HCV and purified recombinant E1 to examine whether the E1 protein is a target antigen recognized by the T cells of HCV-immune pigs. We were unable to identify the E1 protein as a major target antigen recognized by the T cells of HCV-immune animals. However, such cells proliferated in vitro upon stimulation with viable HCV antigen. The lymphoproliferative response to HCV was strictly time and dose dependent and could be induced upon stimulation by live but not by UV light-inactivated HCV. Depletion studies demonstrated that lymphoproliferation depended on the presence of CD2+CD8bright+ lymphocytes, but CD2+CD4+ cells also contributed to the lymphoproliferative response. The primary lymphoproliferative response in animals inoculated with 10(7) 50% tissue culture infective doses of strain Brescia 2.1.1 was stronger than that observed in animals inoculated with 10(3) 50% tissue culture infective doses of the Cedipest strain. A remarkable finding was the increase in non-antigen-specific lymphoproliferation upon inoculation of the animals with HCV strains. This immunological phenomenon may mask a specific T-cell response to the virus.

Animals↗
PubMed1994

Antigenic structure of envelope glycoprotein E1 of hog cholera virus.

Envelope glycoprotein E1 (gp51 to gp54) is the most antigenic protein of hog cholera virus or classical swine fever virus (CSFV). Four antigenic domains, A to D, have been mapped on E1 with a panel of monoclonal antibodies (MAbs) raised against CSFV strain Brescia. The boundaries of these domains have been established by extensive studies on binding of MAbs to transiently expressed deletion mutants of E1 (P. A. van Rijn, E. J. de Meijer, H. G. P. van Gennip, and R. J. M. Moormann, J. Gen. Virol. 74:2053-2060, 1993). In this study, we used neutralizing MAbs of domains A, B, and C to isolate MAb-resistant mutants (MAR mutants) of CSFV strain Brescia and Chinese vaccine strain ("C"). The E1 genes of MAR mutants were cloned in a eukaryotic expression vector, and the effects of MAR mutations on epitopes were studied with a panel of 19 MAbs by immunostaining of COS1 cells transiently expressing these mutant E1s. Except for the MAR mutation Cys-->Arg at position 792, which abolished binding of all MAbs of domains A and D, amino acid substitutions affected only MAbs belonging to the same domain as the MAb used to select the MAR mutant. However, a MAR mutation in a particular domain did not per se abolish binding of all MAbs recognizing that domain. Furthermore, MAR mutants possessed conservative as well as nonconservative amino acid substitutions. To investigate the significance of a secondary structure for the binding of MAbs, all cysteine residues in the N-terminal antigenic part of E1 were mutated to serine. We found that the cysteines at positions 693 and 737 were essential for binding by MAbs of domains B and C, whereas those at positions 792, 818, 828, and 856 appeared to be essential for the binding of most MAbs of domains A and D. These results fully comply with the previously proposed two-unit structure of the N-terminal half of E1. One unit consists of antigenic domains B and C, whereas the other unit consists of the highly conserved domain A and domain D. We conclude that the first six cysteines are critical for the correct folding of E1. A model of the antigenic structure of E1 is presented and discussed.

Amino Acid Sequence↗
PubMed1982

Biosynthesis of CDP-diacylglycerol in hog mesenteric lymph node lymphocytes.

The synthesis of CDP-diacylglycerol by CTP: phosphatidate cytidylyltransferase (EC 2.7.7.41) has been studied in microsomes isolated from hog mesenteric lymph node lymphocytes. The properties of this enzyme were found to be similar in many respects to that described in rat liver microsomes; however, it is not stimulated by GTP or any other nucleotides under normal assay conditions. The enzyme requires that the phosphatidate be emulsified in a cationic detergent for optimal activity. The quaternary ammonium phospholipids lecithin and sphingomyelin were found to stimulate the formation of CDP-diacylglycerol even in the presence of optimal cationic detergent. Other phospholipids or detergents had no effect or were inhibitory to the reaction. Only in the presence of either lecithin or sphingomyelin did nucleotides such as ATP, GTP, UTP, and ITP stimulate the formation of CDP-diacylglycerol. The Km and Vmax for CTP were found to be 0.6 and 1.2 mM, respectively, while the apparent Km and Vmax for phosphatidate were 0.65 and 1.2 mM. Magnesium was found to be the only metal ion that stimulated the reaction, with an optimal concentration of 20 mM. Fluoride ions at 20 mM inhibited the reaction to the extent of 70%. The enzyme was found to be very unstable when the isolated microsomes were stored at -20 degrees C for 24 h, losing approximately 75% of its activity.

Animals↗
PubMed1989

Myosin dephosphorylation during rapid relaxation of hog carotid artery smooth muscle.

Changes in myosin light chain phosphorylation were measured during histamine-induced rhythmic contractions of hog carotid artery smooth muscle strips. Histamine made the muscle strips contract spontaneously every 1-5 min, and this allowed measurement of the time course of phosphorylation in relation to force development under conditions where diffusion of the agonist through tissue would not complicate the interpretation of the data. In the absence of histamine, phosphorylation was low [0.12 +/- 0.04 mol P/mol of the 20,000-Da light chain (LC 20)]. Phosphorylation was slightly (but not significantly) higher in the presence of 10 microM histamine in the relaxed state between contractions (0.20 +/- 0.03 mol P/mol LC 20). In muscle strips frozen during force development, when force had reached half of its peak value, phosphorylation was 0.38 +/- 0.06 mol P/mol LC 20. The highest levels of phosphorylation (0.49 +/- 0.04 mol P/mol LC 20) were found in strips frozen at the peak of the rhythmic contractions. Strips frozen when force had declined to half of the peak force showed low levels of phosphorylation (0.17 +/- 0.07 mol P/mol LC 20), indicating that the myosin light chain phosphatase activity was quite high. Mathematical modeling of the kinase and phosphatase reactions suggested that the apparent first-order phosphatase rate constant was at least 0.08 s-1 under these conditions. To obtain a better estimate of this rate constant, a second series of phosphorylation measurements were made early in the relaxation phase of the rhythmic contractions. The highest phosphatase rate constant obtained from these measurements was 0.23 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1977

Formation of adenosine nucleotide derivatives in isolated hog carotid artery strips.

Media strips of hog carotid artery formed hypoxanthine and inosine during incubation under conditions of normoxia (95% O2, 5% CO2). During anoxia (95% N2, 5% CO2), hypoxanthine increased fivefold and inosine twofold. Stimulation with 124 mM K+ resulted in a twofold increase in hypoxanthine and a threefold increase in inosine. Concurrent with the increase in the concentrations of purine derivatives was a decrease in tissue ATP. Although significant amounts of adenosine were not detected in the medium of incubating artery strips, the following evidence suggests adenosine was formed and rapidly deaminated to inosine: 1) Exogenous adenosine added to the medium of incubating strips was rapidly deaminated to inosine. 2) Exogenous 5'-AMP concentration decreased, whereas adenosine and, subsequently, inosine levels increased during incubation of artery strips. The reaction was specific for 5'-AMP and the data suggest that AMP is dephosphorylated to adenosine. 3) The specific activity of exogenous [U-14C]adenosine added to the medium of incubated strips decreased after 15 min. It is concluded that adenosine is formed in isolated artery strips but is rapidly deaminated to inosine.

Adenosine↗
PubMed1982

Characterization of [3H]-adenosine binding to media membranes of hog carotid arteries.

Binding of [3H]-adenosine to a microsomal fraction derived from media strips of hog carotid arteries was studied by a centrifugation technique. Specific binding was very rapid, readily and completely reversible, and temperature dependent. Under the chosen conditions of incubation, at 4 degrees C and in the presence of deoxycoformycin, about 95% of the bound radioactivity was chromatographically identified as adenosine. Scatchard analysis revealed a small number (1.7 pmol/mg protein) of high affinity (KD = 0.23 mumol/l) and a large number (21.6 pmol/mg protein) of low affinity binding sites (KD = 4.3 mumol/l). 5'-Phosphorylated adenosine compounds. 5'-deoxyadenosine, 2-chloroadenosine, and adenosine-5'-N-ethylcarboxamide, all derivatives of adenosine with pronounced vasodilatory properties, were most potent in displacing [3H]-adenosine from its binding sites with IC50 values ranging from 1.8 to 14.3 mumol/l. However, 2-chloroadenosine and (--)N6-phenylisopropyladenosine were weaker competitors of binding than adenosine, which disagrees with the pharmacological findings. Moreover, the IC50 of aminophylline was much higher than the concentration required for half-maximal inhibition of the biological actions of adenosine. This partial lack of specificity may be due to nonreceptor binding of [3H]-adenosine at the low affinity sites.

Adenosine↗
PubMed1976

Stress relaxation in hog carotid artery as related to contractile activity.

The purpose of this work was to test the hypothesis that stress relaxation (SR) in vascular smooth muscle is related to the amount of contractile activity (developed stress) and perhaps a property of the contractile element (CE). Using medial-intimal strips from the hog carotid artery, a strain (delta 1/l0, where l0 equals optimal length for force development) of 2% was applied at two different rates in the presence of various levels of developed or passive stress. Developed stress was altered either by changing the extracellular K-ion concentration with the strip length equal to l0 or by stimulating with a salt solution containing 30 mM KCl at various strip lengths. Passive stress was altered by varying strip length. At each level of developed or passive stress the magnitude of the increment in stress due to the strain (induced stress) and the SR were noted. SR varied directly with the magnitude of the developed stress, but there was a small and relatively unchanging amount of SR when passive stress was varied. At a given level of developed stress, the magnitude of SR was greater at a strain rate of 0.016 than at 0.001 l0/sec. When developed stress was altered, the magnitude of SR was essentially equal to the amount of induced stress; however, when passive stress was varied, SR was always less than induced stress. These observations are consistent with the hypothesis that, in the presence of developed stress, a large fraction of the SR represents the lengthening of the CE. This increased CE length allows a reequilibration of stress between the series elastic element and the contractile element. In the absence of developed stress, SR may represent a passive, physical property of this heterogeneous tissue.

Animals↗
PubMed1985

Effects of purified hog pancreatic kallikrein on the kinin-prostaglandin system and renin-angiotensin-aldosterone system.

In the present study, hog pancreatic kallikrein (HPK, 2,000 KU/kg body weight) was intramuscularly injected into male rabbits and several plasma hormones [kinin, prostaglandin E (PGE), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), thromboxane B2 (TXB2), plasma renin activity (PRA), aldosterone, and ACTH] were measured before and after the HPK administration, in order to clarify the role of glandular kallikrein in the blood. The plasma kinin concentrations were significantly (p less than 0.001) increased from 1 +/- 1 (mean +/- SE), the baseline level, to 230 +/- 22, 288 +/- 36 and 130 +/- 9 pg/ml at 30, 60, and 120 min, respectively, after HPK administration. The plasma level of PGE were slightly increased after HPK administration, but the change was not significant compared to the mean baseline level. The plasma levels of 6-keto-PGF1 alpha were significantly increased from 229 +/- 38, the baseline level, to 594 +/- 131 (p less than 0.05) at 30 min after the administration of HPK and tended to increase (378 +/- 67) at 60 min after the HPK administration, but fell to 278 +/- 37 pg/ml at 120 min after the administration of HPK. On the other hand, the changes in plasma TXB2, aldosterone, ACTH, and PRA before and after HPK administration were not significant. The present results showed that exogenous intramuscular administration of HPK increased the plasma levels of kinin and prostacyclin, but caused no elevation in the plasma levels of other hormones including PRA. It is concluded, therefore, that in this acute experiment, there was a close relationship between the kallikrein-kinin system and prostaglandins.

6-Ketoprostaglandin F1 alpha↗
PubMed1980

Biological activities of C5a and C5adesArg from hog serum.

The complement-derived peptides C5a and C5adesArg (highly purified from yeast-activated hog serum) were both active in the following biological assays: in aggregation of human leukocytes (potency ratio for C5a:C5adesArg 3:1), in aggregation of guinea pig platelets (10:1), in chemotaxis of peritoneal rabbit leukocytes (10:1), and in contraction of isolated guinea pig ileum (1.4:1). The fact that C5adesArg preparations have 30 and 40% of the activity of C5a in leukocyte aggregation and smooth muscle contraction but only 10% of C5a chemotactic activity, clearly indicates that aggregation and spasmogenic activity are inherent properties of C5adesArg. Evidence that C5adesArg owns all activities studied is further given by the following findings: chromatographic separation of mixtures of C5a and C5adesArg results in the appearance of two peaks of spasmogenic activity; treatment of C5adesArg with carbopeptidase B does neither lead to release of detectable arginine nor abrogate any of the biological effects; treatment of C5a with carboxypeptidase B reduces the activities quantitatively to those of C5adesArg, and liberates the theoretically expected amount of arginine.

Amino Acids↗
PubMedDate not supplied

Presence of cytochrome P-450-dependent monooxygenase in intimal cells of the hog aorta.

Cytochrome P-450-dependent mixed function oxidase activity is present in vascular tissue; however, as far as we could determine, the distribution of monooxygenase activity across the blood vessel wall has not previously been assessed. The aryl-hydrocarbon hydroxylase activity was examined by metabolism of benzo[a]pyrene in microsomes prepared from intimal and smooth muscle cell scrapings of the hog thoracic aorta. Microsomes of intimal cells comprising 95% endothelial cells showed an approximately 2.5-fold increase in aryl-hydrocarbon hydroxylase activity compared with that in microsomes prepared from medial smooth muscle cells. Michaelis-Mentin kinetics for the intimal enzyme yielded an apparent Km value of 11.11 microM and an apparent Vmax of 3-OH benzo[a]pyrene of 40 pmol/mg protein/10 min. Aryl-hydrocarbon hydroxylase activity was dependent on nicotinamide adenine dinucleotide phosphate and was inhibited by 7,8 benzoflavone, SKF 525A, and carbon monoxide. The localization of cytochrome P-450-dependent mixed function oxidase primarily to the intimal surface of the aorta may indicate a role for this enzyme system in vasoregulation and the pathogenesis of atherosclerosis.

Animals↗
PubMed1977

Large scale purification of hog renin. Physicochemical characterization.

Renin was purified from 47 kg of hog kidney to produce enough enzyme for enzymatic and physicochemical characterization. The procedure included extraction at pH 3.5 in the presence of protease inhibitors, two ammonium sulfate precipitations, ion exchange chromatography on Sepharose-hexamethylenediamino-pepstatin gel, gel filtration, and isoelectric focusing. Renin, 2.3 mg, with a specific activity of 1,100 GU/mg of protein was obtained with about 70,000-fold purification and 16% overall recovery. The purity criteria were: (1) a single band on sodium dodecyl sulfate (SDS)-gel electrophoresis, (2) same retardation factor on polyacrylamide gel electrophoresis for renin activity, protein and glycoprotein coloration. Renin was characterized by its stability at--20 degrees C, pH 6.5; its molecular weight on SDS-gel electrophoresis, 36,800; its relative mobility on polyacrylamide gel electrophoresis at pH 7.8; its isoelectric point, 5.15; its amino acid composition, which revealed that renin is a glycoprotein; and its Michaelis constant on tetradecapeptide substrate at pH 6.6, Km = 7.7 X 10(-6) M.

Amino Acids↗
PubMed1984

Histamine-induced rhythmic contraction of hog carotid artery smooth muscle.

Smooth muscle strips isolated from the hog common carotid artery can contract rhythmically, exhibiting low frequency, large amplitude oscillations in tension when stimulated with 10 microM histamine. Strips required at least 1.45 mM calcium and 2.5 mM potassium to exhibit this rhythmic activity. Rhythmic contractions could be converted to tonic contractions by removal of potassium or ouabain treatment. Relaxation by 2 mM lanthanum, 1 mM manganese, or 1 microM verapamil implies that the external medium is the source of calcium mediating the contractions. The involvement of adrenergic nerve terminals in this response was ruled out, since propranolol, phentolamine, tetrodotoxin, bretylium, or 6-hydroxydopamine treatment did not alter the oscillations. Blockade of H1 receptors with 0.1 microM diphenhydramine relaxed the muscle strips. The H2 receptor antagonist cimetidine (5 microM) had no effects. Attempts to obtain rhythmic contractions by stimulating with other vasoactive agents (norepinephrine, acetylcholine, 5-hydroxytryptamine, angiotensin II, and elevated potassium concentrations) were unsuccessful, suggesting that this is a specific histamine response mediated solely by H1 receptors. These results show that this large artery, commonly considered a multi-unit smooth muscle, can sometimes exhibit single-unit behavior.

4-Aminopyridine↗