Partial purification, characterization and application of thermoalkaliphilic proteases from Priestia endophytica, Lysinibacillus cresolivorans and Bacillus subtilis isolated from desert soil

Aims@#Thermophilic proteases are important industrial enzymes because they can be used at high temperatures in various bioprocessing schemes. The bacterial population of the Cholistan desert was explored for thermophilic proteases and their industrial applications.@*Methodology and results@#Three bacterial isolates K1, K5 and K7 were found promising protease producers. These isolates were preliminary identified as Bacillus based on morphological characteristics and biochemical tests (positive for catalase, oxidase and citrate tests, and negative for indole and urease tests). The isolates K1, K5 and K7 were further identified as Priestia endophytica, Lysinibacillus cresolivorans and Bacillus subtilis, respectively by phylogenetic analysis. The isolates grew best at 50 °C and P. endophytica (K1), L. cresolivorans (K5) and B. subtilis (K7) produced larger zones of hydrolysis at 37 °C, 45 °C and 50 °C at pH 7, respectively. The optimum temperature where protease activity was maximum was 65 °C for P. endophytica and L. cresolivorans and 55 °C for B. subtilis, and the optimum pH was 9.@*Conclusion, significance and impact of study@#The proteases produced by these isolates were found active at high temperatures (45 °C to 85 °C) and high pH (9-12), which make them industrially important thermoalkaliphilic proteases. These proteases successfully de-haired cow’s skin and de-stained blood from cotton cloth pieces, which are rarely tested applications of these proteases.

Metaloproteinases da matriz: papel no desenvolvimento das lesões periapicais e modulação após tratamento endodôntico / Matrix metalloproteinases: role in the development of apical periodontitis and modulation after endodontic treatment

Introduction: Apical periodontitis represents a local immune response directed against the progression of microorganisms from the dental pulp to the apical foramen and periapical tissues, which results in bone and dental resorption. The aim of this review is to describe the expression of this group of proteases in apical periodontitis and its modulation during the periapical healing phase following root canal treatment. Literature review: The pathogenesis of apical periodontitis involves degradation of several extracellular matrix components. Matrix metalloproteinases (MMPs) are expressed in response to specific stimuli by resident cells of connective tissue during tissue remodeling and by inflammatory cells that arrive into the surrounding tissues during inflammatory events. MMPs have been reported in apical periodontitis, either experimentally induced or obtained from humans and there is evidence that these enzymes show diff erent expression patterns in granulomas and periapical cysts. Root canal therapy is important for the reduction of periapical inflammation as well as the synthesis of MMPs, especially when using a calcium hydroxide-based dressing. Conclusion: Apical periodontitis show high expression of matrix metalloproteinases and root canal treatment results in less expression of MMPs when compared to untreated apical periodontitis.

Introdução: A lesão periapical representa a resposta imunoinflamatória devido ao aumento do número e progressão de micro-organismos advindos dos canais radiculares contaminados em direção aos tecidos apicais e periapicais, resultando em reabsorção óssea. O objetivo desta revisão será abordar a importância das metaloproteinases da matriz no desenvolvimento das lesões periapicais e sua modulação durante a fase de reparação tecidual depois de instituído o tratamento endodôntico. Revisão da literatura: A patogênese da lesão periapical envolve a degradação progressiva de diversos componentes da matriz extracelular. Dentre as proteases responsáveis pela degradação destes componentes estão as metaloproteinases da matriz (MMPs). Estas proteinases são expressas em resposta a estímulos específicos pelas células residentes do tecido conjuntivo durante o processo de remodelação tecidual e por células inflamatórias que invadem os tecidos durante eventos inflamatórios. As MMPs foram descritas em lesões periapicais experimentais e em humanos e existem evidências de que estas enzimas apresentam padrões de expressões diferentes em granulomas e cistos periapicais. A terapia endodôntica é importante para a redução da inflamação periapical assim como da síntese das MMPs, principalmente quando utilizado um curativo de demora à base de hidróxido de cálcio. Conclusão: As lesões periapicais apresentam alta expressão de metaloproteinases da matriz e o tratamento endodôntico em dentes com lesão periapical resulta em menor expressão de MMPs quando comparado às lesões periapicais não tratadas.

Correlations of plasma levels of brain natriuretic peptide and Meprin-α with the severity of coronary-artery stenosis / 中华老年医学杂志

Objective To explore the relationship of the severity of coronary-artery stenosis with plasma levels of brain natriuretic peptide (BNP) and Meprin-α.Methods Totally 237 patients in our hospital were divided into control group (CON group), stable angina group (SA group) and acute coronary syndrome group (ACS group), according to coronary artery angiography.Patients with acute coronary syndrome were divided into 3 subgroups: unstable angina (UA) group, non-ST segment elevation myocardial infarction (NSTEMI) group and ST segment elevation myocardial infarction (STEMI) group.Patients with coronary artery disease (CAD) were divided into 3 subgroups: low-score, medium-score and high-score groups, according to coronary angiography and Syntax score.BNP and Meprin-α levels were determined in patients with coronary artery disease, and the degree of coronary artery stenosis was evaluated.The differences in above indexes were analyzed and compared among the three groups.Results Plasma levels of BNP and Meprin-α were higher in ACS group than in CON group [(233.16± 78.22)ng/L vs.(33.48 ± 13.71)ng/L, (26.89 ± 6.45) nmol/L vs.(12.83±0.66)nmol/L, both P＜0.05].Compared with UA group, plasma levels of BNP and Meprin-α were increased in NSTEMI and STEMI groups (all P＜0.05).Compared with the control group, plasma levels of BNP and Meprin-α in the Syntax scores-divided subgroups were increased (all P＜ 0.05).The plasma levels of BNP and Meprin-α in CAD patients were significantly increased along with the increase of Syntax Score.Spearman correlation analysis showed that low density lipoprotein cholesterol, glucose, BNP and Meprin-α levels had positive correlations with the occurrence of coronary heart disease, while high density lipoprotein level was negatively correlated with the occurrence of coronary heart disease (all P＜0.05).Conclusions BNP and Meprin-α levels in peripheral blood are significantly elevated in patients with coronary heart disease, and they are correlated with Syntax score.The risk of ACS is increased along with the increased BNP and Meprinα levels.

The action of metaloproteinases in the atherosclerotic diseases / Ação das metaloproteinases nas doenças ateroscleróticas

Cardiovascular diseases represent the main cause of morbidity and mortality in the world and are epidemic events involving the atherosclerosis and coronary artery disease in particular. There are a wide variety of factors and markers associated with the development and aggravation of these diseases, including atherosclerosis. Subclinical Atherosclerosis can bedetermined by serum inflammatory markers present in the atherogenic process. Such markers can take a direct or indirect indicator role on atherosclerotic cardiovascular disease. The extracellular matrix metalloproteinases are biomarkers closely related into modifying and remodeling of vascular wall and other tissues and can represent predictive value patterns to support diagnosis. This review discusses the function and types of matrix metalloproteinases and its use as an indicator of support for the diagnosis of atherosclerosis.

As doenças cardiovasculares compreendem a maior prevalência de morbidade e mortalidade no mundo, sendo uma epidemia da qual se destacam a aterosclerose e a doença arterial coronária. Há grande diversidade de fatores e marcadores associados ao desenvolvimento e agravo dessas doenças, incluindo a aterosclerose, que subclinicamente pode ser evidenciada pela determinação de marcadores inflamatórios séricos participantes do processo aterogênico. Tais biomarcadores são considerados medidas diretas ou indiretas de doença cardiovascularaterosclerótica. As metaloproteinases estão relacionadas com amodificação/remodelamento da parede vascular e outros tecidos, podendo representar parâmetros com valor preditivo para o apoio diagnóstico. Esta revisão aborda o mecanismo de ação e os tipos de metaloproteinases de matriz, bem como seu uso como indicador de apoio no diagnóstico da aterosclerose.

Effect of calpain inhibitor on hyperglycemia-induced apoptosis in cultured rat cardiomyocyte / 中国医师杂志

Objective To investigate the mechanism of inhibitor of calpain on hyperglycemia-induced apoptosis in cultured rat cardiomyocyte.Methods Cardiomyocytes were randomly divided into three groups (control,high glucose,and ALLN).MTT assay was used to detect the viability of cultured cardiomyocytes.Laser confocal microscopy was used to observe the mitochondrial permeable transition and membrane potential.The change of Caspase-3 activity in cardiomyocytes was detected by western blot.Results MTT assay showed that,after 72 h of hyperglycemia,the viability of cardiomyocytes was significantly declined (55％ ± 11％),and the viability in the ALLN pretreatment group was (70％ ± 15％) (P ＜0.05).After hyperglycemia,the mitochondrial permeable transition of cardiomyocyte was increased (30％ ± 15％ vs 60％ ± 11％,P ＜0.05),and membrane potential was declined.Hyperglycemia could increase the expression of cleaved capsase-3,while with pretreatment of ALLN the expression of cleaved caspase-3 was downregulation(0.42 ± 0.11 vs 0.21 ±0.12,P ＜0.05).Conclusions The calpain inhibitor can protect cardiomyocytes from apoptosis under the high glucose condition.

What's the importance of peptidases in cancer?: [review] / Qual é a importância das peptidases em câncer?: [revisão]

A synonym for a successful tumor spread is a productive invasive cell migration, a process by which the extracellular matrix plays the role of substrate for cells to move and reach a secondary site. Peptidases participate actively in this process to degrade the extracellular matrix. The activity of these enzymes is regulated by inhibitors, activators and receptors. However, cancer occurs in a breach of the balance of proteolytic-antiproteolytic activity. The peptidases, enzymes that hydrolyze peptide bonds of proteins, can act directly by degrading the components of the extracellular matrix or indirectly by activating other peptidases, in a process that may also generate bioactive fragments, interact with cell surface receptors, and be involved in the angiogenic process. The modification and remodeling of the extracellular matrix caused by peptidases modify the anchoring mediated by integrins, focal adhesion and architecture of the cytoskeleton, and direct signaling molecules that can affect gene expression and influence some behavioral aspects, such as proliferation, survival, differentiation and mobility. Recently, some studies showed an inverse correlation between the low expression of peptidases and increased potential for tumor development. Thus, despite offering an excellent alternative of a more effective and targeted cancer treatment, protease inhibitors should be specific, administered at the correct time with the aid of biomarkers and act locally, and finally, their activity should not be prolonged to the point of interfering with the activity of peptidases when they are, for example, being used in a process of remodeling.

Um sinônimo para o sucesso da disseminação do tumor é uma produtiva migração celular invasiva, um processo pelo qual a matriz extracelular possui papel de substrato para as células se moverem e atingirem um sítio secundário. Para degradar a matriz extracelular, as peptidases participam ativamente deste processo. A atividade destas enzimas é regulada por inibidores, ativadores e receptores. Entretanto, no câncer ocorre uma quebra do balanço da atividade proteolítica-antiproteolítica. As peptidases, enzimas que clivam ligações peptídicas, podem atuar de forma direta ao degradar componentes da matriz extracelular ou de forma indireta, ao ativar outras peptidases a gerar fragmentos bioativos, interagir com receptores da superfície celular, e participar no processo angiogênico. A modificação e o remodelamento da matriz extracelular causadas por peptidases modificam a ancoragem mediada por integrinas, a adesão focal e a arquitetura do citoesqueleto direcionam moléculas de sinalização que podem afetar a expressão gênica e influenciar no comportamento como proliferação, sobrevivência, diferenciação, e mobilidade. Recentemente, alguns trabalhos demonstraram uma correlação inversa entre a baixa expressão de peptidases e o aumento do potencial do desenvolvimento do tumor. Desta forma, apesar de oferecerem uma excelente alternativa mais efetiva e direcionada para o tratamento do câncer, os inibidores de peptidases devem ser específicos, administrados no tempo correto com o auxílio de biomarcadores e atuar localizadamente.

Association between DPP6 polymorphism and the risk of sporadic amyotrophic lateral sclerosis in Chinese patients / 中华神经科杂志

Objective To investigate whether the polymorphism of rs10260404 in DPP6 gene in Chinese Han origin is associated with sporadic amyotrophic lateral sclerosis (SALS). Methods The genomic DNA was extracted from the leukocytes of whole blood samples in 58 Chinese Han patients with SALS and 52 healthy controls. The asymmetric PCR was processed in the presence of an unlabeled probe that contained the rs10260404 locus. The product was genotyped on light scanner and some was confirmed with sequencing. Results Two single nucleotide polymorphism, rs10260404 that was reportedly consistently strongly associated with susceptibility to SALS in different populations of European and American ancestry, rs10260404 were genotyped, but not strongly associated with ALS in Chinese patients(SALS:C:12.94%,T:87.06%;health controls:C:10.58%,T:89.43%;χ2=0.29,OR=1.256,95%CI 0.549-2.872, P>0.05). Conclusion The rs10260404 is not associated with ALS susceptibility in Chinese people.

The application of urinary kallidinogenase in recombinant tissue plasminogen activator intravenous thrombolytic treatment in patients with acute cerebral infartion / 中华老年医学杂志

ObjectiveTo evaluate the safety and efficacy of urinary kallidinogenase for recombinant tissue-type plasminogen activator (rt-PA) intravenous thrombolytic treatment in patients with acute cerebral infartion MethodsA randomized control study was applied. All 44 patients with acute cerebral infartion were randomized 1:1 to the experimental group (22 cases) and the control group (22 cases). Patients were administrated rt-PA(0. 9 mg/kg)in control group, and patients were given urinary kallidinogenase by intravenous drip (0.15 PNAU/d, for 7 days) after rt-PA intravenous thrombolytic treatment (0.9 mg/kg)in experimental group. The main evaluation index was the incidence of symptomatic intraeerebral hemorrhage within 24 hours, and the secondary assessing items were NIHSS and BI. ResultsThere was 1 case (4.6%) with symptomatic intracerebral hemorrhage in the experimental group and 2 (9.1%) in the control group (X2 =0.00, P= 1.000),and reinfarction rate showed a decreasing tendency in experimental group (18.2% vs. 31.8%, X2=1.091,P=0.296). Compared with the control group, the NIHSS scores were significantly lower 1,21,90 days after thrombolytic therapy (t=2.119, 2.913, 2.187);P=0.041, 0.0 06, 0.042),and the BI scores were obviously higher at 90 days after thrombolytic therapy in experimental group(t= 2.39,P= 0.012). ConclusionsWithout increasing the risk of intracerebral hemorrhage, urinary kallidinogenase may improve the curative effect for rt-PA intravenous thrombolytic treatment in patients with acute cerebral infartion

Expression of COP9, JAK2, HSP and NADH in ovarian carcinoma tissues after taxol-chemotherapy and their significance / 中华妇产科杂志

Objective To study the expression of COP9,JAK2,HSP and NADH genes in ovarian carcinoma tissues after taxol-chemotherapy and their significance. Methods The up-regulated genes of JAK2, HSP, NADH and the down-regulated gune of COP9, which were revealed by micro-array from our previous study were examined by RT-PCR and real-time-PCR in 33 cases of ovarian cancer who previously received taxol-based chemotherapy (group 1 ), and 21 cases of ovarian cancer who never received chemotherapy before operation ( group 2 ) . Results The expression rate of COP9 gene in group 1 was detected markedly lower than that in group 2 (39% vs 95%, P < 0. 01 ) ; whereas the expression rates of JAK2, HSP and NADH in group 1 were significantly higher that those in group 2 (91%, 97% , 94% vs 29%, 48%, 43% ; all P < 0. 05). And the expression of COP9, HSP and NADH genes had no significant differences among histological grades. However, a significantly higher expression of JAK2 gene was seen in grade 3 than in grade 1 -2 (P <0. 01 ). No significant difference in the expression rates of the 4 genes was seen among various tumor types or chemotherapy courses ( P > 0. 05 ). Real-time PCR showed that the level of COP9 gene copies of group 1 was significantly lower than that of group 2 (568, 1866 respectively; P <0. 05 ). However, HSP, JAK2 and NADH genes had significantly higher copy numbers in group 1 than in group 2 (5766,7653,3200 in group 1 and 3341,3094,1522 in group 2, respectively; all P < 0. 05 ). In the subgroup that received 6 - 10 chemotherapy courses, the copy concentrations of JAK2, HSP, NADH genes were higher than those in the subgroup that received 2 - 4 chemotherapy courses ( all P < 0. 05 ). In addition,we found a higher copy concentrations of JAK2, HSP, NADH genes in grade 3 than in grade 1 - 2 ( all P <0. 05 ). Though no significant differences in gene copy concentrations of the 4 genes were seen among variable tumor types. In stage Ⅳ, the copy concentrations of HSP and NADH genes were higher than those in stage Ⅲ ( P < 0. 01, P < 0. 05 respectively ), but the copy concentrations of COP9, JAK2 genes had no significant differences( both P > 0. 05 ). There were positive correlations among JAK.2, HSP and NADH genes ( r =0. 56,0. 44,0. 57 respectively ,all P <0. 01 ). COP9 gene was found to have a negative correlation with JAK2 gene ( r = - 0. 48 ; P < 0. 01 ), but not with HSP and NADH genes ( r = - 0. 18, - 0. 06, respectively;both P > 0. 05 ). Conclusion The down-regulation of COP9 gene and up-regulation of JAK2, HSP, and NADH genes are related to the mechanism of drug-resistance in ovarian cancer.

Effects of ulinastatin on plasma ?-glucuronidase, granulocyte elasta.se and fibronectin during cardiopulmonary bypass / 中华麻醉学杂志

Objective To investigate the effects of ulinastatin on plasma ?-glucuronidase ?-GCD) activity, granulocyte elastase (Gel) and fibronectin (Fn) concentrations and the dose-effect relationships of ulinastatin with them during cardiopulmonary bypass (CPB) .Methods Thirty ASA Ⅱ - Ⅲ patients (19 male, 11 female) aged 3-29 yr, weighing 12-61 kg undergoing elective open heart surgery with CPB were randomly divided into two groups : ulinastatin group (U n - 15) and control group (C n = 12) . In group U patients received ulinastatin 200 000 U in 20 ml of normal saline (NS) infused iv over 10 min after induction of anesthesia and before CPB. An additional 200 000 U of ulinastatin was given iv if CPB lasted for more than 4 h. In control group NS was given iv instead of ulinastatin. Blood samples were taken before anesthesia and after CPB for determination of plasma ?-GCD activity and Gel and Fn levels. The differences in ?-GCD, Gel and Fn between baseline and post-CPB values were calculated (△?-GCD, △Gel, △Fn). In group C the correlation between △?-GCD and AGel;△?-GCD and △Fn; △Gel and △Fn and in group U the correlation of the dose of ulinastatin (U?kg-1 body weight) with △?-GCD /△Gel /△Fn were studied.Results (1) There was no significant difference in plasma ?-GCD activity, Gel and Fn levels between the two groups before anesthesia ( P