RESUMO
Background: Protein glutaminase specifically deamidates glutamine residue in protein and therefore significantly improves protein solubility and colloidal stability of protein solution. In order to improve its preparation efficiency, we exploited the possibility for its secretory expression mediated by twin-arginine translocation (Tat) pathway in Bacillus licheniformis. Results: The B. licheniformis genome-wide twin-arginine signal peptides were analyzed. Of which, eleven candidates were cloned for construction of expression vectors to mediate the expression of Chryseobacterium proteolyticum protein glutaminase (PGA). The signal peptide of GlmU was confirmed that it significantly mediated PGA secretion into media with the maximum activity of 0.16 U/ml in Bacillus subtilis WB600. A mutant GlmU-R, being replaced the third residue aspartic acid of GlmU twin-arginine signal peptide with arginine by site-directed mutagenesis, mediated the improved secretion of PGA with about 40% increased (0.23 U/ml). In B. licheniformis CBBD302, GlmU-R mediated PGA expression in active form with the maximum yield of 6.8 U/ml in a 25-l bioreactor. Conclusions: PGA can be produced and secreted efficiently in active form via Tat pathway of B. licheniformis, an alternative expression system for the industrial-scale production of PGA.
Assuntos
Bacillus licheniformis/enzimologia , Glutaminase/metabolismo , Arginina , Plasmídeos , Prostaglandinas A/química , Bacillus subtilis , Sinais Direcionadores de Proteínas , Sequência de Bases , Mutagênese Sítio-Dirigida , Ácido Aspártico , Escherichia coli , Bacillus licheniformis/genética , Glutaminase/genéticaRESUMO
ABSTRACT Bovine viral diarrhea virus can cause acute disease in livestock, leading to economic losses. We show that Prostaglandin A1 inhibits bovine viral diarrhea virus replication in Madin-Darby bovine kidney cells (94% inhibition using 5 µg/mL). Light and electron microscopy of infected cells shows that Prostaglandin A1 also prevents virus-induced vacuolization, but at higher concentrations (10 µg/mL).
Assuntos
Animais , Bovinos , Antivirais/farmacologia , Prostaglandinas A/farmacologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Vírus da Diarreia Viral Bovina/efeitos dos fármacos , Antivirais/análise , Prostaglandinas A/análise , Replicação Viral/efeitos dos fármacos , Doença das Mucosas por Vírus da Diarreia Viral Bovina/tratamento farmacológico , Linhagem Celular , Vírus da Diarreia Viral Bovina/fisiologia , Vírus da Diarreia Viral Bovina/genética , DiarreiaRESUMO
Bovine viral diarrhea virus can cause acute disease in livestock, leading to economic losses. We show that Prostaglandin A1 inhibits bovine viral diarrhea virus replication in Madin-Darby bovine kidney cells (94% inhibition using 5 µg/mL). Light and electron microscopy of infected cells shows that Prostaglandin A1 also prevents virus-induced vacuolization, but at higher concentrations (10 µg/mL).(AU)
Assuntos
Animais , Bovinos , Vírus da Diarreia Viral Bovina , Prostaglandinas A/uso terapêutico , Replicação Viral , Vacúolos , Microscopia Eletrônica/veterináriaRESUMO
INTRODUCTION: The Mayaro virus (MAYV), which is an arbovirus closely related to the Chikungunya virus, causes a dengue-like acute illness that is endemic to Central and South America. We investigated the anti-MAYV activity of prostaglandin A1 (PGA1), a hormone which exhibits antiviral activity against both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses. Further, we examined the effects of inducting the stress protein HSP70 following PGA1 treatment. METHODS: Hep-2 cells infected with MAYV were treated with PGA1 (0.1-6µg/ml) 12h before infection and for different periods post-infection. Inhibition of viral replication inhibition was analyzed via viral titer determination, whereas the effect of PGA1 on viral morphogenesis was examined via transmission electron microscopy (TEM). Autoradiography (with 35S methionine labeling) and western blotting were used to assess the effect of PGA1 treatment on viral and cellular protein synthesis, and on HSP70 induction, respectively. RESULTS: PGA1 strongly reduced viral replication in Hep-2 cells, particularly when added during the early stages of viral replication. Although PGA1 treatment inhibited viral replication by 95% at 24 hours post-infection (hpi), viral structural protein synthesis was inhibited only by 15%. TEM analysis suggested that PGA1 inhibited replication before viral morphogenesis. Western blot and densitometry analyses showed that PGA1 treatment increased HSP70 protein levels, although this was not detectable via autoradiography. CONCLUSIONS: PGA1 inhibits MAYV replication in Hep-2 cells at early stages of viral replication, prior to production of viral structural proteins, possibly via HSP70 induction.
Assuntos
Alphavirus/efeitos dos fármacos , Células Epiteliais/virologia , Proteínas de Choque Térmico HSP70/farmacologia , Prostaglandinas A/farmacologia , Replicação Viral/efeitos dos fármacos , Alphavirus/ultraestrutura , Animais , Antivirais/farmacologia , Western Blotting , Bovinos , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Células Epiteliais/ultraestrutura , Humanos , Microscopia Eletrônica de TransmissãoRESUMO
Abstract INTRODUCTION: The Mayaro virus (MAYV), which is an arbovirus closely related to the Chikungunya virus, causes a dengue-like acute illness that is endemic to Central and South America. We investigated the anti-MAYV activity of prostaglandin A1 (PGA1), a hormone which exhibits antiviral activity against both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses. Further, we examined the effects of inducting the stress protein HSP70 following PGA1 treatment. METHODS: Hep-2 cells infected with MAYV were treated with PGA1 (0.1-6μg/ml) 12h before infection and for different periods post-infection. Inhibition of viral replication inhibition was analyzed via viral titer determination, whereas the effect of PGA1 on viral morphogenesis was examined via transmission electron microscopy (TEM). Autoradiography (with 35S methionine labeling) and western blotting were used to assess the effect of PGA1 treatment on viral and cellular protein synthesis, and on HSP70 induction, respectively. RESULTS: PGA1 strongly reduced viral replication in Hep-2 cells, particularly when added during the early stages of viral replication. Although PGA1 treatment inhibited viral replication by 95% at 24 hours post-infection (hpi), viral structural protein synthesis was inhibited only by 15%. TEM analysis suggested that PGA1 inhibited replication before viral morphogenesis. Western blot and densitometry analyses showed that PGA1 treatment increased HSP70 protein levels, although this was not detectable via autoradiography. CONCLUSIONS: PGA1 inhibits MAYV replication in Hep-2 cells at early stages of viral replication, prior to production of viral structural proteins, possibly via HSP70 induction.
Assuntos
Humanos , Animais , Bovinos , Prostaglandinas A/farmacologia , Replicação Viral/efeitos dos fármacos , Alphavirus/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/farmacologia , Células Epiteliais/virologia , Antivirais/farmacologia , Linhagem Celular , Western Blotting , Alphavirus/ultraestrutura , Microscopia Eletrônica de Transmissão , Eletroforese em Gel de Poliacrilamida , Células Epiteliais/ultraestruturaRESUMO
Bovine viral diarrhea virus can cause acute disease in livestock, leading to economic losses. We show that Prostaglandin A1 inhibits bovine viral diarrhea virus replication in Madin-Darby bovine kidney cells (94% inhibition using 5µg/mL). Light and electron microscopy of infected cells shows that Prostaglandin A1 also prevents virus-induced vacuolization, but at higher concentrations (10µg/mL).
Assuntos
Antivirais/farmacologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Vírus da Diarreia Viral Bovina/efeitos dos fármacos , Prostaglandinas A/farmacologia , Animais , Antivirais/análise , Doença das Mucosas por Vírus da Diarreia Viral Bovina/tratamento farmacológico , Bovinos , Linhagem Celular , Diarreia , Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/fisiologia , Prostaglandinas A/análise , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Ischemic stroke (IS) is a leading cause of death and disability worldwide. As genetic heritability for IS is estimated at about 35%-40%, the identification of genetic variants associated with IS risk is of great importance. The main objective of this study was to carry out a meta-analysis for polymorphisms in CRP, EPHX2, FGA, and NOTCH3 genes and the risk for IS. METHODS: Literature search for 6 candidate polymorphisms and IS was conducted using HuGE Navigator, PubMed, and Google Scholar databases. Meta-Analyst program was used to calculate pooled odds ratios (ORs) with a random effects model. RESULTS: Twenty-five published studies for 6 candidate polymorphisms were included: CRP-rs1800947 (5 studies), CRP-rs1205 (3 studies), EPHX2-rs751141 (5 studies), FGA-rs6050 (6 studies), NOTCH3-rs3815188 (3 studies), and NOTCH3-rs1043994 (3 studies), for a total number of 7,825 IS cases and 56,532 control subjects. We did not find significant pooled ORs (P values > .05) for any of the genetic variants evaluated in this work. CONCLUSIONS: Our meta-analysis results did not show significant associations between these 6 polymorphisms in 4 candidate genes and IS, despite the functional role of some of these single nucleotide polymorphisms (e.g., rs6050 in FGA gene). Future studies are needed to identify additional main genetic risk factors for IS in different populations.
Assuntos
Proteína C-Reativa/genética , Epóxido Hidrolases/genética , Predisposição Genética para Doença/genética , Variação Genética/genética , Prostaglandinas A/genética , Receptor Notch3/genética , Isquemia Encefálica/complicações , Isquemia Encefálica/genética , Bases de Dados Bibliográficas/estatística & dados numéricos , Humanos , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/genéticaRESUMO
Our present study examines, in mesenteric resistance arteries, possible vasodilation alterations, and the role of NO and COX (cyclo-oxygenase) derivatives, in cirrhosis. The vasodilator response to acetylcholine was analysed in segments from control and cirrhotic rats. The effects of the non-specific COX inhibitor indomethacin, the specific COX-1 inhibitor SC-560 and the specific COX-2 inhibitor NS-398 were analysed in segments from both groups of rats. NO release was measured, and eNOS [endothelial NOS (NO synthase)], phospho-eNOS, iNOS (inducible NOS), COX-1 and COX-2 protein expression was also analysed. The effects of the TP receptor [TXA2 (thromboxane A(2)) receptor] antagonist SQ 29548, the TXA(2) synthesis inhibitor furegrelate, the PGI(2) (prostaglandin I(2)) synthesis inhibitor TCP (tranylcypromine) or TCP+furegrelate were only determined in segments from cirrhotic rats. The vasodilator response to acetylcholine was higher in segments from cirrhotic rats. Indomethacin, SC-560 and NS-398 did not modify the vasodilator response in control rats; however, indomethacin, NS-398 and TCP+furegrelate increased, whereas SC-560 did not modify and SQ 29548, furegrelate or TCP decreased, the vasodilator response to acetylcholine in cirrhotic rats. NO release was higher in cirrhotic rats. Furegrelate decreased, whereas TCP+furegrelate increased, the NO release in segments from cirrhotic rats. eNOS and COX-1 protein expression was not modified, whereas phosho-eNOS, iNOS and COX-2 protein expression was higher in cirrhotic rats. Therefore the increase in iNOS expression and eNOS activity may mediate increases in endothelial NO release. The COX-2 derivatives TXA(2) and PGI(2) may act simultaneously, producing a compensatory effect that reduces NO release and may limit the hyperdynamic circulation.
Assuntos
Cirrose Hepática Experimental/metabolismo , Artérias Mesentéricas/metabolismo , Óxido Nítrico/metabolismo , Prostaglandinas A/fisiologia , Tromboxano A2/fisiologia , Animais , Pressão Sanguínea/fisiologia , Peso Corporal/fisiologia , Proteína C-Reativa/metabolismo , Metabolismo dos Lipídeos/fisiologia , Fígado/patologia , Cirrose Hepática Experimental/fisiopatologia , Masculino , Tamanho do Órgão/fisiologia , Prostaglandinas/metabolismo , Prostaglandinas A/biossíntese , Ratos , Ratos Sprague-Dawley , Baço/patologia , Superóxidos/metabolismo , Tromboxano A2/biossíntese , Vasodilatação/fisiologiaRESUMO
Production of Polyglutamate (PGA) biopolymer by immobilized Bacilluslicheniformis strain-R was intensively investigated. Preliminary experiments were carried out to address the most suitable immobilization methodology. Entrapment of Bacillus cells in alginate-agar led optimal PGA production (36.75 g/l), with 1.32- and 2.18-fold increase in comparison with alginate- or K-carrageenan-immobilized cells, respectively. During semicontinuous cultivation of agar-alginate gel-cell mixture, production of PGA by 10 ml mixture was increased from 2nd to 3rd run whereas, increased till the 4th run using 15ml mixture. Adsorption was the most suitable immobilization technique for production of PGA and the sponge cubes was the preferred matrix recording 43.2 g/l of PGA with the highest cell adsorption. Furthermore, no PGA was detected when B. licheniformis cells were adsorbed on wood and pumice. Although luffa pulp-adsorbed cells recorded the highest PGA production (50.4 g/l), cell adsorption was the lowest. Semicontinuous cultivation of B. licheniformis cells adsorbed on sponge led to increase of PGA production till the 3rd run and reached 55.5 g/l then slightly decreased in the 4th run. The successful use of fixed-bed bioreactor for semicontinuous cultivation of B. licheniformis cells held on sponge cubes (3 runs, 96 hours/run) provides insight for the potential biotechnological production of PGA by immobilized cells.