Effects of flunixin meglumine, meloxicam, and firocoxib on the acute-phase proteins in horses following standing castration

ABSTRACT: Excessive infection and inflammation are the most common complications associated with castration. The objective of this study was to compare the efficacy of flunixin meglumine (FM), meloxicam (MX), or firocoxib (FX) for inflammation control after castration in horses using acute-phase proteins (APP) as markers of inflammation. Thirty healthy, unbroken, mixed-breed horses (body weight 358.62±45.57kg and age 4.99±2.63 years) were randomly (n=10 animals/group) allocated to receive one of three different post-castration anti-inflammatory medicines: Group 1 (FM 1.1mg/kg bwt, IV, s.i.d for 5 days); Group 2 (MX 0.6mg/kg bwt, IV, s.i.d for 5 days); and Group 3 (FX 0.1mg/kg bwt, IV, s.i.d for 5 days). All horses were castrated in standing position, using the open technique. Serum and peritoneal APP concentrations were measured by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and determined before castration (0), and 3, 5, 24, 48, 72, 120 and 168 hours after castration. The results were submitted to analysis of variance using the SAS statistical program, and means were compared by the Student-Newman-Keuls test (p 0.05). Three animals from the MX group developed hyperthermia (with rectal temperatures of 39.8, 39.3 and 38.9°C on day 4, 5 and 6, respectively) and showed local clinical signs of inflammation (inguinal and excessive scrotal edema) and reluctance to walk, as well as a rigid gait of the hind limbs. The same complications were observed in one FX horse. No complications were observed among the FM animals. The castration resulted in significant changes in serum and peritoneal values of total proteins, ceruloplasmin (Cp), transferrin (Tf), albumin (Alb), haptoglobin (Hp) and 1-acid glycoprotein (Gp) in animals of all experimental groups. However, the animals of the MX and FX groups presented more intense acute phase response compared to the animals of the FM group. Changes in the APP were associated with the surgical trauma of castration, but the differences between groups were associated with the ability of the nonsteroidal anti-inflammatory drug to control the inflammation. In conclusion, and based on the findings of acute phase proteins, flunixin is more efficient to control the magnitude of inflammation following castration as compared to meloxicam and firocoxib.

RESUMO: Infecção e inflamação excessivas são as complicações mais comuns associadas à castração. O objetivo deste estudo foi comparar a eficácia do flunixin meglumine (FM), meloxicam (MX) ou firocoxib (FX) no controle da inflamação após a castração em cavalos usando proteínas da fase aguda (APP) como marcadores de inflamação. Trinta equinos saudáveis (358,62±45,57kg; 4,99±2,63 anos) foram em função dos anti-inflamatórios utilizados após as castrações aleatoriamente (n= 10 animais/grupo) alocados em três diferentes grupos: Grupo 1 (FM 1,1mg/kg de peso, IV, sid por 5 dias); Grupo 2 (MX 0,6mg/kg de peso, IV, s.i.d por 5 dias); e Grupo 3 (FX 0,1mg/kg de peso, IV, s.i.d por 5 dias). Todos os cavalos foram castrados em posição quadrupedal, utilizando a técnica aberta. As concentrações de APP sérica e peritoneal foram separadas por eletroforese em gel de poliacrilamida (PAGE) com dodecil-sulfato de sódio (SDS) e determinadas no momento 0 (antes da castração) e com 3, 5, 24, 48, 72, 120 e 168 horas após a castração. Os resultados foram submetidos à análise de variância pelo programa estatístico SAS e as médias foram comparadas pelo teste de Student-Newman-Keuls (p 0,05). Três animais do grupo MX desenvolveram hipertermia (com temperatura retal de 39,8, 39,3 e 38,9° C nos dias 4, 5 e 6, respectivamente) e mostraram sinais clínicos locais de inflamação (edema inguinal e escrotal excessivo) e relutância em andar, bem como marcha rígida dos membros posteriores. As mesmas complicações foram observadas em um cavalo do FX. Não foram observadas complicações entre os animais do FM. Independente do grupo, a castração resultou em alterações significativas nos valores séricos e peritoneais de proteínas totais, ceruloplasmina (Cp), transferrina (Tf), albumina (Alb), haptoglobina (Hp) e glicoproteína ácida 1 (Gp). No entanto, os animais dos grupos MX e FX apresentaram resposta de fase aguda mais intensa quando comparados aos animais do FM. Alterações na resposta de fase aguda deveram-se ao trauma cirúrgico da castração, mas as diferenças entre os grupos foram associadas à capacidade do anti-inflamatório em controlar a inflamação. Em conclusão, baseado da resposta de fase aguda, o flunixin em comparação com o meloxicam e o firocoxib é mais eficiente no controle da inflamação após a castração em equinos.

Effects of flunixin meglumine, meloxicam, and firocoxib on the acute-phase proteins in horses following standing castration / Efeitos do flunixin meglumine, meloxicam e firocoxib na concentração de proteínas da fase aguda após em equinos após castração

Excessive infection and inflammation are the most common complications associated with castration. The objective of this study was to compare the efficacy of flunixin meglumine (FM), meloxicam (MX), or firocoxib (FX) for inflammation control after castration in horses using acute-phase proteins (APP) as markers of inflammation. Thirty healthy, unbroken, mixed-breed horses (body weight 358.62±45.57kg and age 4.99±2.63 years) were randomly (n=10 animals/group) allocated to receive one of three different post-castration anti-inflammatory medicines: Group 1 (FM 1.1mg/kg bwt, IV, s.i.d for 5 days); Group 2 (MX 0.6mg/kg bwt, IV, s.i.d for 5 days); and Group 3 (FX 0.1mg/kg bwt, IV, s.i.d for 5 days). All horses were castrated in standing position, using the open technique. Serum and peritoneal APP concentrations were measured by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and determined before castration (0), and 3, 5, 24, 48, 72, 120 and 168 hours after castration. The results were submitted to analysis of variance using the SAS statistical program, and means were compared by the Student-Newman-Keuls test (p<0.05). Three animals from the MX group developed hyperthermia (with rectal temperatures of 39.8, 39.3 and 38.9°C on day 4, 5 and 6, respectively) and showed local clinical signs of inflammation (inguinal and excessive scrotal edema) and reluctance to walk, as well as a rigid gait of the hind limbs. The same complications were observed in one FX horse. No complications were observed among the FM animals. The castration resulted in significant changes in serum and peritoneal values of total proteins, ceruloplasmin (Cp), transferrin (Tf), albumin (Alb), haptoglobin (Hp) and α1-acid glycoprotein (Gp) in animals of all experimental groups. However, the animals of the MX and FX groups presented more intense acute phase response compared to the animals of the FM group. Changes in the APP were associated with the surgical trauma of castration, but the differences between groups were associated with the ability of the nonsteroidal anti-inflammatory drug to control the inflammation. In conclusion, and based on the findings of acute phase proteins, flunixin is more efficient to control the magnitude of inflammation following castration as compared to meloxicam and firocoxib.(AU)

Infecção e inflamação excessivas são as complicações mais comuns associadas à castração. O objetivo deste estudo foi comparar a eficácia do flunixin meglumine (FM), meloxicam (MX) ou firocoxib (FX) no controle da inflamação após a castração em cavalos usando proteínas da fase aguda (APP) como marcadores de inflamação. Trinta equinos saudáveis (358,62±45,57kg; 4,99±2,63 anos) foram em função dos anti-inflamatórios utilizados após as castrações aleatoriamente (n= 10 animais/grupo) alocados em três diferentes grupos: Grupo 1 (FM 1,1mg/kg de peso, IV, sid por 5 dias); Grupo 2 (MX 0,6mg/kg de peso, IV, s.i.d por 5 dias); e Grupo 3 (FX 0,1mg/kg de peso, IV, s.i.d por 5 dias). Todos os cavalos foram castrados em posição quadrupedal, utilizando a técnica aberta. As concentrações de APP sérica e peritoneal foram separadas por eletroforese em gel de poliacrilamida (PAGE) com dodecil-sulfato de sódio (SDS) e determinadas no momento 0 (antes da castração) e com 3, 5, 24, 48, 72, 120 e 168 horas após a castração. Os resultados foram submetidos à análise de variância pelo programa estatístico SAS e as médias foram comparadas pelo teste de Student-Newman-Keuls (p<0,05). Três animais do grupo MX desenvolveram hipertermia (com temperatura retal de 39,8, 39,3 e 38,9° C nos dias 4, 5 e 6, respectivamente) e mostraram sinais clínicos locais de inflamação (edema inguinal e escrotal excessivo) e relutância em andar, bem como marcha rígida dos membros posteriores. As mesmas complicações foram observadas em um cavalo do FX. Não foram observadas complicações entre os animais do FM. Independente do grupo, a castração resultou em alterações significativas nos valores séricos e peritoneais de proteínas totais, ceruloplasmina (Cp), transferrina (Tf), albumina (Alb), haptoglobina (Hp) e glicoproteína ácida α1 (Gp). No entanto, os animais dos grupos MX e FX apresentaram resposta de fase aguda mais intensa quando comparados aos animais do FM. Alterações na resposta de fase aguda deveram-se ao trauma cirúrgico da castração, mas as diferenças entre os grupos foram associadas à capacidade do anti-inflamatório em controlar a inflamação. Em conclusão, baseado da resposta de fase aguda, o flunixin em comparação com o meloxicam e o firocoxib é mais eficiente no controle da inflamação após a castração em equinos.(AU)

Purification and characterization of extracellular alkaline protease from Streptomyces sp. LCJ12A isolated from Pichavaram mangroves

Extracellular protease from culture supernatants of Streptomyces sp. LCJ12A was purified and characterized in thisstudy. The collected supernatant of Streptomyces sp. LCJ12A was purified by ammonium sulfate precipitation anddiethylaminoethyl cellulose chromatography. The molecular weight of the enzyme was 20.1 kDa. Sodium dodecylsulfate-polyacrylamide gel electrophoresis was used to determine the molecular weight of the enzyme. The culture filtrate,partially purified enzyme, and ammonium sulfate precipitate displayed catalysis and stability over pH 3–11.5 and showedoptimal activity at pH 10. The culture filtrate, ammonium sulfate precipitate, and partially purified enzyme were goodbetween 30 and 40°C and the optimum temperature was 35°C. The purified protease exhibited high catalytic activityand stability under different pH and temperature. The partially purified protease from Streptomyces sp. LCJ12A showeda substantial relative activity of 78% with bovine serum albumin (BSA), 43% with gelatin, and 96% relative activitywith casein. Lineweaver–Burk plot was used to calculate the Km and Vmax values for the partially purified protease.The Km values of Streptomyces sp. LCJ12A were 73.5 mM for casein, 44.54 mM for BSA, and 43.45 mM for gelatin.The Vmax values were 500 mM min−1 for casein, 303.03 mM min−1 for BSA, and 270.27 mM min−1 for gelatin fromStreptomyces sp. LCJ12A. The statistical analysis confirmed that compared to the other substrates, casein was significant.

ISSR Analysis of Genetic Relationship and Population Structure of Turpinia arguta Fresh Leaves in Jiangxi Province / 中国实验方剂学杂志

Objective:To explore genetic relationship and population structure of Turpinia arguta in six locations of Jiangxi province by inter-simple sequence repeat （ISSR） molecular marker technique， and to provide theoretical basis for the protection and utilization of this medicinal material resource. Method:A total of 22 samples from six locations in four counties in Jiangxi province were collected， and genomic DNA was extracted by kit method. Polymerase chain reaction （PCR） amplification was performed using sixty-four universal ISSR molecular marker primers， and the products were detected with polyacrylamide gel electrophoresis （PAGE）. NTsys 2.10e software was selected to calculate the genetic similarity coefficient by unweighted pair group method with arithmetic mean （UPGMA） and cluster analysis. Population genetic structure was analyzed by Structure 2.1 software. Result:A total of forty-eight ISSR primers were amplified to obtain the product， the percent of polymorphic bands ranged from 45.45% to 100%. UPGMA cluster analysis showed that these plant individuals could not be clustered according to their respective executive locations. Analysis of population genetic structure showed that 22 samples of T. arguta could be divided into three populations. Conclusion:There is gene exchange among the populations of T. arguta in Jiangxi province， and it can affect the genetic structure of germplasm resources from different geographical sources.

Discrimination of Rana dybowskii,Its Analogues and Counterfeits Based on Polyacrylamide Gel Electrophoretograms Combined with Cluster Analysis and Multivariate Statistical Analysis / 中国实验方剂学杂志

Objective:To establish an effective classification and identification method for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) maps of Rana dybowskii,its analogues and counterfeits based on cluster analysis and multivariate statistical analysis. Method:SDS-PAGE maps of 18 batches of R. dybowskii,its analogues and 2 counterfeits were obtained by SDS-PAGE method. SDS-PAGE maps were transformed into data matrix. NTSYSpc 2.10e statistical analysis software was used for cluster analysis,and SMICA-P 14.1 software was used for multivariate statistical analysis. Unsupervised Principal Component Analysis (PCA),Supervised Partial Least Squares Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) were performed for multivariate analysis and evaluation. Result:SDS-PAGE maps technology combined with cluster analysis and multivariate statistical analysis could accurately classify and identify R. dybowskii,its analogues and counterfeits. Cluster analysis could cluster four kinds of medicinal materials into four branches except No.1 medicinal materials. PCA results were superior to cluster analysis. Supervised PLS-DA and OPLS-DA results in multivariate statistical analysis were superior to unsupervised PCA. The classification and identification efficiencies of OPLS-DA were better than those of unsupervised PCA. OPLS-DA aggregated R. dybowskii,its analogues and 2 counterfeits into four groups. Six different protein components were obtained by comprehensive analysis of variable importance in projection (VIP) value, and OPLS-DA Bi load diagram,with relative molecular weights were 51.363,35.838,14.565,17.563,15.358 and 21.696 kDa,respectively. Conclusion:SDS-PAGE maps combined with cluster analysis and multivariate statistical analysis can be used as an effective method to classify and identify R. dybowskii,its analogues and counterfeits. This study provides a reference for the quality evaluation and screening of R. dybowskii.

Specification of Bacteriophage Isolated Against Clinical Methicillin-Resistant Staphylococcus Aureus

OBJECTIVES: The emergence of resistant bacteria is being increasingly reported around the world, potentially threatening millions of lives. Amongst resistant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) is the most challenging to treat. This is due to emergent MRSA strains and less effective traditional antibiotic therapies to Staphylococcal infections. The use of bacteriophages (phages) against MRSA is a new, potential alternate therapy. In this study, morphology, genetic and protein structure of lytic phages against MRSA have been analysed. METHODS: Isolation of livestock and sewage bacteriophages were performed using 0.4 μm membrane filters. Plaque assays were used to determine phage quantification by double layer agar method. Pure plaques were then amplified for further characterization. Sulfate-polyacrylamide gel electrophoresis and random amplification of polymorphic DNA were run for protein evaluation, and genotyping respectively. Transmission electron microscope was also used to detect the structure and taxonomic classification of phage visually. RESULTS: Head and tail morphology of bacteriophages against MRSA were identified by transmission electron microscopy and assigned to the Siphoviridae family and the Caudovirales order. CONCLUSION: Bacteriophages are the most abundant microorganism on Earth and coexist with the bacterial population. They can destroy bacterial cells successfully and effectively. They cannot enter mammalian cells which saves the eukaryotic cells from lytic phage activity. In conclusion, phage therapy may have many potential applications in microbiology and human medicine with no side effect on eukaryotic cells.

Acute phase proteins in serum and cerebrospinal fluid in healthy cattle: possible use for assessment of neurological diseases / Proteínas de fase aguda no soro e no líquido cefalorraquidiano de bovinos hígidos: possíveis usos na avaliação de doenças neurológicas

Use of acute-phase proteins (APPs) for assessment of health and disease in animals has increased greatly within the last decade. The objective was to determine the normal concentration of APPs in the serum and cerebrospinal fluid (CSF) of healthy cattle by polyacrylamide gel electrophoresis. Fifty crossbred animals (350±70kg of BW and 18±1.2 months of age), 25 heifers and 25 steers were used. CSF samples were collected from atlanto-occipital (AO) site and blood samples were obtained from the jugular vein. CSF and serum protein electrophoresis were performed by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Thirty-seven proteins with molecular weights ranging from 7 and 37kDa were identified in CSF of all animals. These eight were nominally identified with immunoglobulin A and G, celuloplasmin, transferrin, albumin, α1-antitripsin, acidic glycoprotein, and haptoglobin. All protein fractions in CSF did not differ between heifers and steers. In sera, 34 proteins with molecular weights between 7 and 244kDa were identified in heifers and steers. Similar proteins were nominally identified in the sera, but only the CSF presented α1-antitripsin. The serum values of acidic glycoprotein and immunoglobulin G were significantly higher in steers compared with heifers. In conclusion, measurement of CSF acute phase protein concentrations can be useful in diagnosing and monitoring the progression of bovine neurological diseases, perhaps even to guide therapeutic procedures. The CSF electrophoretic profile of healthy cattle does not change depending on gender.(AU)

O uso de proteínas de fase aguda (PFAs) para a avaliação da saúde e da doença em animais de produção tem aumentado consideravelmente na última década. O objetivo deste estudo foi determinar a concentração normal de PAFs no soro e no líquido cefalorraquidiano (LCR) de bovinos sadios por meio da eletroforese em gel de poliacrilamida. Foram avaliados cinquenta animais mestiços (350±70kg de PV e 18±1,2 meses de idade), 25 novilhas e 25 novilhos. As amostras de LCR foram colhidas no espaço atlanto-occipital (AO) e as amostras de sangue obtidas da veia jugular. As PAFs do soro e do LCR foram determinadas através da eletroforese em gel poliacrilamida. Trinta e sete proteínas com pesos moleculares que variaram entre 7 e 37kDa foram identificadas no LCR de todos os animais, independente do sexo. Estas oito proteínas foram nominalmente identificadas como imunoglobulina A e G, ceruloplasmina, transferrina, albumina, α1-antitripsina, glicoproteína ácida, e haptoglobina. As frações de proteínas presentes no LCR não diferiram entre novilhas e novilhos. No soro de machos e fêmeas, 34 proteínas com pesos moleculares entre 7 e 244 kDa foram identificadas. As proteínas do soro foram similarmente identificadas, entretanto a α1-antitripsina foi identificada somente no LCR. Os valores séricos de glicoproteína ácida e imunoglobulina G foram significativamente mais elevados nas novilhas em comparação aos novilhos. Em conclusão, a determinação das concentrações de proteínas de fase aguda presentes do LCR pode ser útil no diagnóstico e monitoramento da progressão de doenças neurológicas bovinas, talvez possa ainda direcionar procedimentos terapêuticos. O perfil eletroforético do LCR de bovinos hígidos não se altera em função do sexo.(AU)

Serum levels of proteins and acute-phase proteins in captive emus (Dromaius novaehllandieae) of different ages / Teores séricos de proteínas, inclusive proteínas de fase aguda, em emus (Dromaius novaehollandiae) de diferentes faixas etárias e criados em cativeiro

Protein electrophoresis is a relatively simple technique that allows separating serum protein fractions, and provides important information in the investigation and diagnosis of several diseases. This study determined the levels of acute-phase proteins in the serum of healthy, captive emus (Dromaius novaehollandiae). Animals were divided into two groups (n=11 in each) based on age, with 1-year-old and 4-year-old emus. Acute-phase proteins were separated by SDS-PAGE. Ceruloplasmin, transferrin, albumin, haptoglobin, acidic glycoprotein, IgA, and IgG were detected in the serum of all animals. Protein profiles varied significantly with age (P<0.05). Individuals in the 4-year-old emus group had higher values of ceruloplasmin, transferrin, albumin, haptoglobin, and acidic glycoprotein, compared with the group with 1-year-old animals, showing the role of age in the protein profile of this species. Reference values for acute-phase proteins in healthy emus may be useful in the evaluation of health status and in the diagnosis of diseases affecting the species.(AU)

A eletroforese de proteínas é um método relativamente simples, que permite a separação das proteínas do plasma em frações. Sua interpretação fornece informações importantes para a investigação e o diagnóstico de inúmeras doenças. O objetivo deste estudo foi o de determinar a concentração das proteínas de fase aguda no soro de emus (Dromaius novaehollandiae) hígidos e criados em cativeiro. As aves foram separadas em dois grupos: grupo 1: (n=11), aves com um ano de idade; grupo 2: (n=11), aves com quatro anos de idade. As proteínas de fase aguda foram separadas por eletroforese em gel de poliacrilamida (SDS-PAGE). Identificaram-se as proteínas ceruloplasmina, transferrina, albumina, IgG, haptoglobina, glicoproteína ácida, IgA e IgG no soro de todos os emus. Houve diferença (P<0.05) entre os traçados eletroforéticos em função da faixa etária. As aves do grupo 2 apresentaram valores superiores de ceruloplasmina, transferrina, albumina, haptoglobina e glicoproteína ácida quando comparadas às aves do grupo 1. Conclui-se que o perfil eletroforético de emus sofre alterações conforme a idade analisada. O estabelecimento de valores de referência para as proteínas de fase aguda de emus hígidos poderá auxiliar estudos futuros na avaliação da saúde assim como no diagnóstico de doenças em emus.(AU)

Kinetics of the expressions of radiation-induced plasma proteins of the cardiac territory in electrophoresis / Cinética das expressões de proteínas plasmáticas radioinduzidas do território cardíaco em eletroforese

ABSTRACT Introduction: Radiation induces acute and late alterations in blood proteins. Objective: The present study aims at analyzing in time kinetics the electrophoretic profiles of expression bands of blood protein, in the molecular weight range greater than and equal to that of albumin, modulated by ionizing radiation in the cardiac territory, in animal model. Material and methods: Animals were exposed to a whole-body dose of 5 Gy ionizing radiation (Co-60). Serum samples were collected from isogenic Wistar rats (control and irradiated groups). At a time kinetics of 12, 24, 48, 72, 96 hours and 35 days post-irradiation, thoracolaparotomies were performed with anesthesia, and 0.3 ml of blood was collected between the left ventricle and the pulmonary artery. The samples were held in heparin and their components were separated by centrifugation. Protein bands with similar molecular weight were identified by vertical 10% electrophoresis, silver staining. Results: The findings indicate a systemic acute altered expression of proteins with molecular weight greater than or equal to that of albumin in acrylamide gel, presenting suppression and increased expression due to modulation of preexisting bands, identified in time kinetics. Conclusion: These findings point out to acute alterations of protein expression modulated in time, but also to a late modulation of gene expression.

RESUMO Introdução: A radiação de corpo inteiro induz alterações agudas e tardias no perfil proteico sanguíneo. Objetivo: O presente estudo tem como escopo analisar em cinética de tempo o perfil eletroforético de bandas de expressão das proteínas solúveis do sangue, na faixa de peso molecular superior e igual à albumina, moduladas por radiação ionizante no território cardíaco em modelo animal. Materiais e métodos: Animais foram expostos à radiação de Co-60, em dose de 5 Gy corpo inteiro. Foram coletadas amostras de soro sanguíneo em ratos Wistar isogênicos (no grupo-controle e no irradiado). Em uma cinética de tempo de 12, 24, 48, 72 e 96 horas e 35 dias pós-exposição, foram realizadas toracolaparotomias com anestesia profunda e, posteriormente, foi coletado 0,3 ml de sangue entre ventrículo esquerdo e artéria pulmonar. As amostras foram heparinizadas, sendo, em seguida, separados seus componentes por centrifugação. As bandas proteicas de peso molecular similar foram identificadas por eletroforese vertical a 10%, coradas com prata. Resultados: Os achados indicam alteração aguda sistêmica da expressão das proteínas de peso molecular superior ou igual à albumina em gel de acrilamida, representando supressão e aumento de expressões por meio da modulação de bandas preexistentes identificadas em cinética de tempo. Conclusão: Esses achados apontam tanto para uma alteração aguda modulada no tempo da expressão proteica quanto para uma alteração moderada tardia da expressão gênica.

Expression of Raf kinase inhibitor protein in human hepatoma tissues by two‑dimensional gel electrophoresis and matrix‑assisted laser desorption/ionization time‑of‑flight methods.

PURPOSE: Hepatocellular carcinoma (HCC) is the most common malignant liver tumor. To reduce the mortality and improve the effectiveness of therapy, it is important to search for changes in tumor‑specific biomarkers whose function may involve in disease progression and which may be useful as potential therapeutic targets. MATERIALS AND METHODS: In this study, we use two‑dimensional polyacrylamide gel electrophoresis (2‑DE) and matrix‑assisted laser desorption/ionization time‑of‑flight mass spectrometry to observe proteome alterations of 12 tissue pairs isolated from HCC patients: Normal and tumorous tissue. Comparing the tissue types with each other, 40 protein spots corresponding to fifteen differentially expressed between normal and cancer part of HCC patients. RESULTS: Raf kinase inhibitor protein (RKIP), an inhibitor of Raf‑mediated activation of mitogen‑activated protein kinase/extracellular signal‑regulated kinase, may play an important role in cancer metastasis and cell proliferation and migration of human hepatoma cells. RKIP may be considered as a marker for HCC, because its expression level changes considerably in HCC compared with normal tissue. In addition, we used the methods of Western blotting and real time‑polymerase chain reaction to analysis the protein expression and gene expression of RKIP. The result showed RKIP protein and gene expression in tumor part liver tissues of HCC patient is lower than peritumorous non‑neoplastic liver tissue of the corresponding HCC samples. CONCLUSION: These results strongly suggest that RKIP may be considered to be a marker for HCC and RKIP are down‑regulated in liver cancer cell.

Differential proteomic analysis of transgenic Bt rice and its non-transgenic parental rice / 国际药学研究杂志

Objective: Under the methodology of differential proteomics and bioinformatics, the impact of the exogenous gene on the expression of rice in the proteome is discussed, aiming to explore into the study of genetically modified rice in the proteomics. Methods: The total protein was extracted from genetically modified rice Huahui No.1(HH1) and non-transgenic rice Minghui 63(MH63), the method of two-dimensional gel electrophoresis was applied to generate corresponding proteome two-dimensional polyacrylamide gel(2D-PAGE) electrophoresis spectrum; then, the mass spectrometry and bioinformatics analysis were conducted after the selection of protein spots with significant differences. Results: The comparing and matching of protein spots between transgenic Bt(cry1Ab/1Ac) rice and non-transgenic rice 2D-PAGE profiles identified 28 protein spots with significant differences. With non-transgenic rice as a reference, transgenic Bt rice held 18 relatively high and 10 relatively low expressions; mass spectrometry and bioinformatics retrieval were made on the different protein spots. It was found that the differentiated protein was mainly involved in energy metabolism, protein synthesis, redox stress response and other biological processes. Conclusion: Differences exist between transgenic Bt HH1 and its parental rice MH63 on the expression of proteome; however, there are neither anti-nutritional and allergenic protein, nor new or toxic proteins among these differentiated proteins.

Differential proteomic analysis of transgenic Bt rice and its non-transgenic parental rice / 国际药学研究杂志

Objective Under the methodology of differential proteomics and bioinformatics,the impact of the exogenous gene on the expression of rice in the proteome is discussed,aiming to explore into the study of genetically modified rice in the proteomics. Methods The total protein was extracted from genetically modified rice Huahui No.1(HH1) and non-transgenic rice Minghui 63 (MH63),the method of two-dimensional gel electrophoresis was applied to generate corresponding proteome two-dimensional poly?acrylamide gel(2D-PAGE)electrophoresis spectrum;then,the mass spectrometry and bioinformatics analysis were conducted after the selection of protein spots with significant differences. Results The comparing and matching of protein spots between transgenic Bt (cry1Ab/1Ac)rice and non-transgenic rice 2D-PAGE profiles identified 28 protein spots with significant differences. With non-trans?genic rice as a reference,transgenic Bt rice held 18 relatively high and 10 relatively low expressions;mass spectrometry and bioinfor?matics retrieval were made on the different protein spots. It was found that the differentiated protein was mainly involved in energy me?tabolism,protein synthesis,redox stress response and other biological processes. Conclusion Differences exist between transgenic Bt HH1 and its parental rice MH63 on the expression of proteome;however,there are neither anti-nutritional and allergenic protein, nor new or toxic proteins among these differentiated proteins.

Alterations in Calcium-Binding Properties of Sarcoplasmic Reticulum Membrane Proteins Following Cardiac Injury.

The objective of present work is to investigate metabolic alterations associated with heart failure, particularly one of its manifestations, a sustained hypocalcemia that causes hemodynamic changes contributed to subsequent myocardial injury. Comparative study was carried out using experimental models of pancreatic necrosis (PN) and crush syndrome (CS) accompanied by cardiac damage down to myocardial infarction. Study design: Wistar adult male rats randomly divided into groups (n=12/group). The controls are healthy intact animals. The pancreatic necrosis (PN) and crush syndrome (CS) groups were then randomly subdivided: PN group- into 3, 24 and 72 h groups concerning hemorrhage, early and late pancreatic necrosis respectively; CS group – into 2, 4, 24, and 48 h decompression stages. The rats were sacrificed to analyze spectra and calcium-binding properties of the membrane proteins isolated from the cardiomyocyte sarcoplasmic reticulum (SR). Development of pathological changes in the heart and pancreas were also monitored. Place and Duration of Study: Department of Pathological Biochemistry and Radioisotope Methods, H. Buniatyan Institute of Biochemistry of Natl. Acad. Sci (NAS), Republic of Armenia (RA). Experiments conducted between May 2011 and October 2013. Methodology: To study pathogenesis of hypocalcemia underlying myocardial damage a translocation of radioactive 45CaCI2 into cardiomyocytes and its intracellular distribution was examined. Binding of 45Ca2+ to the SR membrane proteins was measured after proteins separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and radioactivity from the gel plates was counted by a gas-flow meter Berthold–II. Isoelectric focusing of the protein isolated from the SR of cardiomyocyte was performed. Results: Statistically significant changes in mean radio labeled calcium incorporation into a total protein fraction of the cardiomyocyte SR from control (13682±271) were determined by3h of PN (23055±168, P<.001), 24 h of PN (22876±240, P<.01), and by 72 h (3851±271,P<.01), P vs. control. Similarly, these parameters were detected following CS by 2h decompression (24179±225, P<.01), 4-24 hours decompression (21666±124, P<.001) and 48 h decompression (2941±189, P<.001), P vs. control. We demonstrate that drop in the binding calcium level observed was partially due to impaired affinity to calcium of the cardiomyocyte SR calcium-binding proteins during development of both PN and CS despite a simultaneous manifestation of affinity to calcium of the SR 32-kDa protein. Conclusion: In the present study we have clearly shown that both experimental acute pancreatitis and long-term compression injury may cause similar changes,а loss the calcium-binding properties of the cardiomyocyte proteins, particularly those of SR serving as a main calcium depot under physiological circumstances and appear to be involved in common cellular and molecular mechanisms of myocardial injury contributing to hypocalcemia. Simultaneously, both PN and/or CS cause similar manifestations of the new calcium-binding properties of the cardiomyocyte SR 32-kDa membrane protein, and mirrored dynamic changes in its calcium affinity suggested by Scatchard plot analysis indicating a common mechanism that would be a transient attempt of certain heart cells to compensate hypocalcemia, and thus emerge from an otherwise pathological outcome. Thus, the above mentioned changes could be used to identify patients at high risk of cardiovascular disease in different pathologies.

Purification of Catalase Enzyme from Pleurotus Ostreatus.

The oyster mushroom Pleurotus ostreatus is the most commonly cultivated mushroom, and are effective for antitumor, antibacterial, anti viral and hematological agents and in immune modulating treatments. Several compounds from oyster mushrooms, potentially beneficial for human health have been isolated and studied. The aim of this research is to purify an enzyme catalase from Pleurotus ostreatus through Sephadox G-75 column, its molecular weight was determined by polyacrylamide gel electrophoresis and the catalase enzyme stability were observed at various temperature and different pH condition. Under denaturing conditions, polyacrylamide gel electrophoresis revealed dissociation of a major component of molecular weight 62,000 kDa, which constituted 90% of the total protein of the stained gel, suggesting that the native enzyme is tetrameric. The optimum temperature and pH for the purified enzyme catalase from Pleurotus ostreatus enzymatic reaction were 30°C and pH 7.5.

Group A rotavirus and bacterial agents associated with diarrhoea-induced hospitalisations in children below 5 years of age in Jammu.

Out of 210 faecal samples collected from children below 5 years attending different hospitals in Jammu and exhibiting clinical signs of diarrhoea, 41.9% samples were found positive for group A rotavirus by RNA-PAGE. Escherichia coli isolated in the study belonged to nine serogroups, out of which O69 was most frequent, being present in 12.38% samples. E. coli serogroups well recognised as enteropathogens viz. O69, O20 and O153 were present in 27.6% samples. Other bacterial pathogens associated with diarrhoea were present in 8.09% samples, out of which Shigella spp. was found in 4.76% samples followed by Salmonella spp. (2.38%) and Pseudomonas spp. (0.95%).

Partial characterization of superoxide dismutase activity in the Barber pole worm-Haemonchus contortus infecting Capra hircus and abomasal tissue extracts

Objective: To determine the activity of superoxide dismutase (SOD) in the male and female haematophagous caprine worms, Haemonchus contortus infecting Capra hircus, and their E/S products and also to analyse the effect of Haemonchus infection on the level of host SOD. Methods: The SOD activity was analysed by using the pyrogallol autoxidation assay and non-denaturing polyacrylamide gel electrophoresis followed by specific enzyme staining by riboflavin-nitroblue tetrazolium method. Results: The adult females were found to have higher enzyme activity than the male worms. Appreciable amount of SOD activity was also detected in the worm culture medium and female worms secreted more SOD in comparison to the male parasites. The SOD activity was negatively correlated to the worm burden. Statistically significant decrease in SOD activity (P<0.05) was observed in the heavily infected host tissue in comparison to the control non-infected host tissue. SOD profile of the crude extracts of both the sexes revealed polymorphism and a fast migrating activity band being characteristic of E/S products. The SOD activities were found highly sensitive to potassium cyanide indicating the Cu/Zn form of SOD. Conclusions: Haemonchus contortus is a key model parasite for drug and vaccine discovery. The presences of SOD activity in appreciable amount in the parasite as well as its E/S products indicate that it has a well-developed active antioxidant system to protect itself from the host immune attack. SOD could be the target for vaccine development which is the need of the hour as mass drug administration for parasite control has resulted in anthelmintic resistance across the globe and threatens the viability of sheep and goat industry in many regions of the world. The infection with Haemonchus causes a drastic reduction in SOD activity of the host tissue thus effecting its protective potential. One characteristic SOD band was found in the females which was not present in any other preparations and thus could be exploited for further studies on diagnostic/control measures.

Partial characterization of superoxide dismutase activity in the Barber pole worm-Haemonchus contortus infecting Capra hircus and abomasal tissue extracts

Objective: To determine the activity of superoxide dismutase (SOD) in the male and female haematophagous caprine worms, Haemonchus contortus infecting Capra hircus, and their E/S products and also to analyse the effect of Haemonchus infection on the level of host SOD. Methods: The SOD activity was analysed by using the pyrogallol autoxidation assay and non-denaturing polyacrylamide gel electrophoresis followed by specific enzyme staining by riboflavin-nitroblue tetrazolium method. Results: The adult females were found to have higher enzyme activity than the male worms. Appreciable amount of SOD activity was also detected in the worm culture medium and female worms secreted more SOD in comparison to the male parasites. The SOD activity was negatively correlated to the worm burden. Statistically significant decrease in SOD activity (P Conclusions:Haemonchus contortus is a key model parasite for drug and vaccine discovery. The presences of SOD activity in appreciable amount in the parasite as well as its E/S products indicate that it has a well-developed active antioxidant system to protect itself from the host immune attack. SOD could be the target for vaccine development which is the need of the hour as mass drug administration for parasite control has resulted in anthelmintic resistance across the globe and threatens the viability of sheep and goat industry in many regions of the world. The infection with Haemonchus causes a drastic reduction in SOD activity of the host tissue thus effecting its protective potential. One characteristic SOD band was found in the females which was not present in any other preparations and thus could be exploited for further studies on diagnostic/control measures.

Application of improved gel electrophoresis to the detection of genotype polymorphisms / 中华实用儿科临床杂志

Objective To explore the traits of improved polyacrylamide gel electrophoresis (PAGE)-ethidium bromide(EB) staining in the detection of genotype polymorphisms.Methods The methods of PAGE-silver staining,agarose gel electrophoresis (AGE)-EB staining and improved PAGE-EB staining in polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to detect the μ-opioid receptor (OPRM1) A118G genotypes in case group (n =167) infants with intracranial hemorrhage (ICH),and control group(n =163) infants without ICH,to conduct a case study analysis.And the application traits of three methods were compared.Results Genotypes of OPRM1 A118G were GG (169 bp),AG (193 bp,169 bp),AA (193 bp).Both the electrophoresis methods of PAGE-silver staining and PAGE-EB could be used to detect the genotypes of OPRM1 A118G clearly in this study.There was no statistically significant difference between the resolutions of DNA fragments (P =0.884).The first method,which had 13 experiment steps,consuming 4-5 hours,involving 12 kinds of chemical reagents,and the pictures were taken with the camera,was complex,with difficult operation,more time consuming; Compared with the first method,the secondmethod was simple,which had 6 test procedures,consuming 2 hours with 8 reagents,and the pictures were taken by using an automatic gel imager.AGE-EB could not be used to detect genotypes of OPRM1 A118G.Conclusions The method of improved PAGE-EB has the advantages of fast,easy operation,and high resolution,which is worthy of wider application.

Linkage study of DFNB3 responsible for hearing loss in human.

BACKGROUND: Hearing disorders represent a significant health problem worldwide. Recessive inherited cases of the deafness are more prevalent in Pakistan due to consanguineous marriages. Deafness caused by DFNB3 is due to mutation in the gene MYO XVA and its prevalence among Pakistani population is about 5%. MATERIALS AND METHODS: Families with at least two or more individual affected with deafness were selected from different areas of District Okara of Pakistan. Six consanguineous families of different ethnic groups having deaf individuals were studied. All these families had three or more deaf individuals in either two or more sib ships. Family history was taken to minimize the chances of other abnormalities. Pedigrees drawn by using Cyrillic software (version 2.1) showed that all the marriages were consanguineous and the families have recessive mode of inheritance. Three STR markers were selected and amplified on all the samples of six families through PCR. The PCR products were then genotyped on non denaturing polyacrylamide gel electrophoresis (PAGE). Haplotypes were constructed to determine the pattern of inheritance and also to determine whether a family was linked or unlinked with known DFNB3 locus. RESULTS: One out of six families showed linkage to the DFNB3 while rest of the families remained unlinked. Carriers of deafness genes were identified and information was provided to the families on request. CONCLUSION: Knowledge about the genetic causes of deafness provide insight into the variable expression of genes involved in this hereditary problem and may allow the prediction and prevention of associated health problems.

Characterization of salivary protein during ovulatory phase of menstrual cycle through MALDI-TOF/MS.

Context: Predicting ovulation is the basis on which the fertile period is determined. Nowadays there are many methods available to detect the ovulatory period. Unfortunately, these methods are not always effective for accurate detection of ovulation. Hence, an attempt was made to detect ovulation through single dimension sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of protein with the help of saliva ferning. Aims: The aim of this study was to determine the association of protein level with endogenous reproductive hormone level across the menstrual cycle. Settings and Design: Salivary protein and its confirmation were evaluated during menstrual cycle followed by SDS-PAGE and Mass spectrometry. Statistical Method Used: The protein content present in saliva throughout menstrual cycle is trail by SPSS statistical software version. Materials and Methods: Salivary proteins were investigated serially during pre-ovulatory, ovulatory and post-ovulatory periods of normal menstrual cycle in eighteen healthy volunteers. The samples were collected in three consecutive menstrual cycles. Salivary protein was estimated and analyzed by single dimension SDS-PAGE. Results: The results revealed significant variations in protein concentrations during the menstrual cycle. Protein levels were maximum during ovulation and minimum during postovulatory phase. Further, single dimension SDS-PAGE analysis showed seven different fractions of proteins is from 14-90 kilo Dalton (kDa) in the three phases of the menstrual cycle. Conclusions: Among the proteins, 48 kDa protein was more predominantly exhibited during ovulatory phase than pre and post-ovulatory phase. The present study indicates that the protein level and the specific protein band (48 kDa) through MALDI-TOF MS analysis might serve as an indicator for ovulation.