Comparative venomic profiles of three spiders of the genus Phoneutria

Abstract Background: Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genus Phoneutria, mainly from P. nigriventer. In spite of the pharmaceutical potential demonstrated by P. nigriventer toxins, there is limited information on molecules from venoms of the same genus, as their toxins remain poorly characterized. Understanding this diversity and clarifying the differences in the mechanisms of action of spider toxins is of great importance for establishing their true biotechnological potential. This prompted us to compare three different venoms of the Phoneutria genus: P. nigriventer (Pn-V), P. eickstedtae (Pe-V) and P. pertyi (Pp-V). Methods: Biochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp). Results: The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences. Conclusion: Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from Phoneutria genus, exposing their biotechnological potential as a source for searching for new active molecules.

Comparative venomic profiles of three spiders of the genus Phoneutria

Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genus Phoneutria, mainly from P. nigriventer. In spite of the pharmaceutical potential demonstrated by P. nigriventer toxins, there is limited information on molecules from venoms of the same genus, as their toxins remain poorly characterized. Understanding this diversity and clarifying the differences in the mechanisms of action of spider toxins is of great importance for establishing their true biotechnological potential. This prompted us to compare three different venoms of the Phoneutria genus: P. nigriventer (Pn-V), P. eickstedtae (Pe-V) and P. pertyi (Pp-V). Methods: Biochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp). Results: The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences. Conclusion: Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from Phoneutria genus, exposing their biotechnological potential as a source for searching for new active molecules.(AU)

Expresión proteica del fluido folicular asociado a la calidad del oocito de vacas Cebú / Follicular fluid (FF) protein expression associated with oocyte quality in Zebu cattle / Expressão proteica do fluido folicular associado a qualidade do oócito de vacas Cebú

Resumen El objetivo de este estudio fue determinar la expresión de proteínas del fluido folicular (FF) y su relación con la calidad del oocito. Se evaluaron 52 ovarios de planta de faenado de vacas Cebú comercial, mediante la técnica de disección y aspiración folicular se obtuvo FF y oocitos. Las evaluaciones realizadas fueron: calidad del oocito por aspecto citoplasmático y células del cúmulos y perfil de proteínas del FF mediante SDS-PAGE. Se realizó el análisis descriptivo, a través del procedimiento MEANS, análisis de varianza (PROC. ANOVA) y para las diferencias estadísticas significativas se usó la prueba de comparación de Bonferroni con un nivel de significancia del 5%, mediante el paquete estadístico SAS®. El 52% de los oocitos se categorizaron con calidad I-II. El análisis unidimensional de las proteínas del FF evidenció la presencia de 25 bandas de proteína entre 9 y 240 kDa. En folículos <3 mm se expresaron 23 bandas, en folículos de 3 y 6 mm 19 bandas y en folículos >6mm 20 bandas. Las bandas de peso molecular (PM) de 26kDa, 57kDa y 68kDa representan la mayor concentración en el FF; 4 bandas de PM 14 KDa, 34 KDa, 76 y 79 KDa, solo en folículos de <3mm, 2 bandas de PM 9 y 91 KDa solo en folículos de >3 mm. La banda de 32 KDa no se observó en folículos > de 6mm. Las bandas de mayor frecuencia de presentación fueron las de 26, 40, 42, 57, 68, 240 KDa. Las bandas de proteína que se asociaron con la calidad del oocito en forma significativa (p<0,05) fueron las de PM 24, 57, 68 y 164 KDa para FF de folículos <3mm y las bandas de PM 13, 26 y 38 kDa entre 3 y 6mm, y la de 26 kDa a folículos > de 6mm. Los resultados nos indican asociaciones de la calidad del oocito con algunas bandas de proteína.

Abstract This study was aimed at determining follicular fluid (FF) protein expression and its relationship with oocyte quality. FF and oocytes were obtained by dissection and follicular aspiration of the ovaries from fifty-two commercial Zebu from a slaughterhouse. Oocyte quality was measured by cytoplasmic aspect and cumulus cells and FF protein profile by SDS-PAGE. The SAS statistical package's PROC MEANS and analysis of variance (ANOVA) were used for descriptive analysis and the Bonferroni comparison test for assessing significant statistical differences (5% significance level); 52% of the oocytes were categorised as having I-II quality. One-dimensional SDS-PAGE analysis of FF proteins revealed 25 protein bands having 9 kDa to 240 kDa molecular weight (MW); 23 bands were expressed in <3 mm follicles, 19 bands in 3 and 6 mm follicles and 20 bands in >6 mm follicles. The 26kDa, 57kDa and 68kDa bands' MW represented the highest FF concentration whereas only four bands (14 kDa, 34 kDa, 76 and 79 kDa MW) were found in <3mm follicles and only 2 bands (9kDa and 91 kDa MW) in >3 mm follicles. The 32 kDa band was not observed in >6mm follicles. The 26, 40, 42, 57, 68 and 240 KDa bands occurred with the greatest frequency. The protein bands which were significantly associated with oocyte quality (p<0.05) were 24, 57, 68 and 164 kDa MW for <3mm follicles, 13, 26 and 38 kDa MW bands for 3 and 6mm follicles and 26 kDa for >6mm follicles. The results indicated oocyte quality association with some protein bands.

Resumo Este estudo teve como objetivo determinar a expressão de proteínas do fluido folicular (FF) e sua relação com a qualidade do oócito. Avaliou-se 52 ovários de vacas Cebú comercial colhidos em abatedouro e através da técnica de dissecação e aspiração folicular obtivera-se o FF e os oócitos. As avaliações realizadas foram: qualidade do oócito pelo aspecto citoplasmático e células do cúmulos e o perfil de proteínas do FF mediante SDS-PAGE. Realizou-se a análise descritiva, através do procedimento MEANS, análise de variância (PROC. ANOVA) e para identificar as diferenças estatísticas significativas, utilizou-se a prova de comparação de Bonferroni com um nível de significância de 5%, através do pacote estadístico SAS®. O 52% dos oócitos foram classificados como qualidade I e II. As análises unidimensionais das proteínas do FF evidenciaram a presença de 25 bandas de proteína entre 9 y 240 kDa. Em folículos <3 mm expressaram-se 23 bandas, em folículos entre 3 e 6 mm 19 bandas e em folículos >6mm 20 bandas. As bandas de peso molecular (PM) de 26kDa, 57kDa e 68kDa são as mais frequentes no FF; 4 bandas de PM 14 KDa, 34 KDa, 76 y 79 KDa, solo em folículos de <3mm, 2 bandas de PM 9 y 91 KDa solo em folículos de >3 mm. A banda de 32 KDa não foi observada em folículos> de 6mm. As bandas mais frequentes foram 26, 40, 42, 57, 68, 240 KDa. As bandas de proteína que se associaram à qualidade do oócito em forma significativa (p <0,05) foram as de PM 24, 57, 68 e 164 KDa para FF de folículos <3mm e as bandas de PM 13, 26 e 38 kDa entre 3 e 6mm, e a de 26 kDa a folículos >6mm. Os resultados indicam associações entre qualidade do oócito e algumas bandas de proteína.

Study of comparative proteome between normal and inverted karyotypes of human mesenchymal stem cells

Multipotent mesenchymal stem cells have been expanded in vitro for cellular therapy in numerous clinical settings without standardized culture conditions or quality-control schemes. The in vitro expansion is necessary to obtain sufficient cells for clinical applications. However, the expansion may induce genetic and functional abnormalities which may affect the safety and functionality of MSC, especially the chromosomal stability. This study aimed to investigate the protein profile of umbilical cord-derived MSC with normal and inverted karyotypes after expansion in the laboratory. Mass spectrometry analysis was performed and the Bradford method, Scaffold software, String and Cytoscape databases were employed to measure and characterize the protein content of umbilical cord-derived MSC. Networks of protein interactions, hub and bottleneck proteins were identified by proteomics and systems biology approaches. We found that proteins related to cellular stress were super expressed in inverted karyotype cells. Moreover, a high expression of Serpine 1, RHOA, and CTSB was found in these cells, which are proteins related to cancer. The albumin and ubiquitin proteins have been associated with a positive prognosis in cancer and cellular stress, and were up- and down-regulated in normal karyotype cells, respectively. The results suggests that the paracentric inversion inv(3)(p25p13) induced some type of cellular stress and genetic instability in human mesenchymal stem cells. These analyses showed the importance of carrying out studies related to the genetic instability of human mesenchymal stem cells using the protein expression profile as a parameter.

Quality of horse F(ab')2 antitoxins and antirabies immunoglobulins: protein content and anticomplementary activity

Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab′)2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p < 0.05. Results Horse F(ab′)2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates. Conclusions Anti-botulinum AB (bivalent), anti-diphtheric, antitetanic and anti-rabies horse F(ab′)2 immunoglobulins probably contain acceptable levels of aggregates, although other improvements on the preparations must be carried out. Protein profile analysis and in vitro anticomplementary activity of F(ab′)2 immunoglobulin preparations should be included as quality control steps, to ensure acceptable levels of aggregates, contaminants and whole IgG molecules on final products, reducing the chances of adverse reactions in patients.(AU)

Quality of horse F(ab)2 antitoxins and antirabies immunoglobulins: protein content and anticomplementary activity

Background Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab)2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p 0.05. Results Horse F(ab)2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates...

Profile of apoptotic proteins in oral squamous cell carcinoma: A cluster analysis of 171 cases

Oral squamous cell carcinoma (OSCC) is the eighth most prevalent cancer worldwide. In recent large-scale studies, by immunohistochemistry and cluster analysis, several markers were associated with patient survival in various tumors. The aim of this study was to analyze the expression profiles of 23 proteins that have been linked to the inhibition (Bcl-2, Bcl-x, Bcl-xL, Bcl-2-related protein A1, BAG-1, and survivin) and promotion (Bak, Bax, Bim/Bod, Bim-Long, Bad, Bid, PUMA, Apaf-1, caspase-2, caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, Smac/DIABLO, and cytochrome c) of apoptosis in OSCC. METHODS: Two-hundred and twenty nine cases of OSCC, arranged in a tissue microarray, were immunohistochemically analyzed, and the results were quantified on an automated imaging system. The data were analyzed using a random forest clustering method. RESULTS: Overall protein expression patterns defined two chief clusters: an anti-apoptotic cluster (142 cases) and a pro-apoptotic cluster (29 cases). These groups could not be explained by any clinical or pathological characteristic, and overall and disease-free survival did not differ between them. CONCLUSIONS: Although there was no association with survival, the cluster analysis demonstrated specific protein profiles that could be of interest for using targeted therapies: in one of the clusters, the expression of pro-apoptotic proteins was more prominent, demonstrating a pro-apoptotic profile and highlighting the importance of apoptosis during OSCC development.

Impact of folia application of ascorbic acid ad α-tocopherol on antioxiddant activity and some biochemical aspects of flax cultivars under salinity stress / Impacto de las aplicaciones foliares de ácido ascórbico y α- tocoferol en la actividad antioxidante y algunos aspectos bioquímicos de cultivares de lino sometidos a estrés por salinidad.

The interactive effects of saline water (2000, 4000 and 6000 mg/l) and foliar application of 400 mg/l of ascorbic acid (Asc) or α- tocopherol (α-Toco) on three flax cultivars (Sakha 3, Giza 8 and Ariane) were conducted during two successive seasons 2011/2012 and 2012/2013. The results showed that total soluble carbohydrates, free amino acids and proline contents were significantly increased with increasing salinity levels in the all three tested cultivars except free amino acid content of Giza 8 which showed insignificant decrease. While, nucleic acids (DNA and RNA) showed significant decreases compared with the corresponding control. Moreover, applications of vitamins (Asc or α-Toco) as foliar spraying increased all mentioned contents compared to the corresponding salinity levels. On the other hand, lipid peroxidation and activity levels of polyphenol oxidase, peroxidase and catalase enzymes showed significant increases with increasing salinity levels of all tested three cultivars, while the behaviour of superoxide dismutase activity showed an opposite response as compared with the control in Sakha 3 and Giza 8. Treatments with Asc or α-Toco induced significant reduction in lipid peroxidation and activities of polyphenol oxidase, peroxidase of the all three tested cultivars. Meanwhile, superoxide dismutase increased in all three cultivars, and catalase activities increased only in Sakha 3 cultivar under salt stress as compared with reference controls. Some modifications are observed in protein patterns hence some proteins were disappeared, while certain other proteins were selectively increased and synthesised of a new set of proteins were induced, some of these responses were observed under treatments and salinity, while others were induced by either treatments or salinity.

La interacción de los efectos del agua salada (2000, 4000 and 6000 mg/l) y la aplicación foliar de 400 mg/l de ácido ascórbico (Asc) o α-tocopherol (α-Toco) en tres cultivares de lino (Sakha 3, Giza 8 y Ariane) se analizó durante dos estaciones sucesivas (2011- 2012). Los resultados mostraron que los contenidos de carbohidratos solubles totales, aminoácidos libres y prolina aumentaron significativamente con los niveles crecientes de salinidad en los tres cultivares probados, excepto en el cultivar Giza 8, en el cual el contenido de aminoácidos libres se redujo. Al mismo tiempo, los ácidos nucleicos (ADN y ARN) mostraron disminuciones significativas en comparación con los controles correspondientes. Mientras que aplicaciones foliares de vitaminas (Asc o α-Toco) incrementaron los contenidos mencionados en comparación con los niveles de salinidad. De otro lado, la peroxidación de lípidos y la actividad de la polifenol oxidasa, la peroxidasa, y la catalasa mostraron incrementos significativos con los niveles crecientes de salinidad en los tres cultivares analizados. Mientras tanto, la actividad superóxido dismutasa se incrementó en los tres cultivares y la actividad catalasa se incrementó únicamente en el cultivar Sakha 3 bajo estrés salino, en comparación con los controles. Se observaron algunas modificaciones en los patrones de proteínas. Así, algunas proteínas desaparecieron mientras que otras incrementaron e incluso un nuevo set de proteínas fue inducido. Algunas de estas respuestas fueron observadas en los tratamientos y en la salinidad, mientras que otras fueron inducidas por los tratamientos o por la salinidad.

Impact of CO2 Enhancement on Photosynthesis and Protein Profile - Response Studies with a CO2 Responsive Black Gram Genotype.

Black gram (Vigna mungo (L.) Hepper) var. IC-282009 - a highly CO2 responsive genotype for biomass and seed yield was grown in Open top chambers (OTCs) under three levels of CO2 i.e. ambient (390 ppm) and two elevated levels 550ppm and 700ppm to assess photosynthetic acclimation to elevated CO2. Net photosynthetic rate (PN), change in leaf soluble protein profile and leaf carbohydrate constituents such as total soluble sugars, reducing sugars and starch content in leaves was quantified at all three CO2 concentrations. Photosynthetic rate was enhanced by 78% and 30% at flowering stage with 550ppm and 700ppm CO2 as compared with ambient control. It was also observed a higher accumulation of starch, total soluble sugars and reducing sugars in leaves at elevated CO2 levels. However, the leaf protein content recorded a decrease and altered the profile of ploy peptides with enhanced CO2 levels. At elevated CO2 concentrations significant differences were observed in ploy peptide profile at vegetative and flowering stages, the intensity of 260 kDa poly peptide increased at vegetative stage, whereas 72 kDa polypeptide increased at flowering stage, while 52 kDa poly peptide decreased at both stages. Enhanced CO2 concentrations improved the PN though certain polypeptides of leaf protein are down regulated and necessitate further experimentation to confirm their involvement in responsiveness of the selected black gram genotype.

Efeito da administração de propileno glicol e cobalto associado à vitamina B12 sobre o perfil metabólico e a atividade enzimática de ovelhas da raça Santa Inês no periparto / Effect of propylene glycol, cobalt and vitamin B12 on the metabolic profile and enzymatic in Santa Inês ewes in peripartum

(AU)O presente estudo teve por objetivo avaliar a influência da administração de propileno glicol e cobalto associado à vitamina B12 sobre o perfil metabólico e a atividade enzimática de ovelhas da raça Santa Inês no período do periparto. Foram utilizadas 18 ovelhas prenhes, pesando em torno de 40kg. Aproximadamente 30 dias antes da data prevista para o parto foram separadas de maneira aleatória em três grupos e administrados os suplementos conforme a seguir: (G1/n=6) grupo que recebeu propileno glicol (30mL por via oral diariamente); (G2/n=6) grupo que recebeu cobalto (1mg de cloreto de cobalto a 1%, via oral diariamente) associado a vitamina B12 (2mg via intramuscular, semanalmente) e (G3/n=6) grupo controle. As amostras de sangue das ovelhas para avaliação do perfil metabólico e enzimático (glicose, β-hidroxibutirato-BHB, NEFA, proteína total, albumina, uréia, creatinina, AST, GGT, FA e CK) foram colhidas 30 dias antes da data prevista para o parto, uma semana antes (ante-parto), no parto, às 24h, 72h, 5 dias, 15 dias e 30 dias após o parto. Não foi observado cetonúria nos momentos que antecederam ao parto. A administração dos suplementos não influenciou sobre o perfil metabólico, protéico e energético, assim como não houve comprometimento hepático das ovelhas no período do periparto.(AU)

The aim of this study was to evaluate the influence of the administration of propylene glycol and cobalt associated with vitamin B12 on the metabolic profile and enzymatic activity of Santa Inês ewes in the peripartum period. A total of 18 pregnant ewes, weighing around 40kg were used. Approximately 30 days before the expected date of delivery were randomly separated into three groups and administered supplements as follows: (G1/n = 6) group received propylene glycol (30mL orally daily); (G2/n = 6) group receiving cobalt (1mg cobalt chloride 1%, orally daily) associated with vitamin B12 (2mg intramuscular weekly) and (G3/n = 6) control group. Blood samples from ewes to evaluate the enzymatic and metabolic profile (glucose, β-hydroxybutyrate, BHB, NEFA, total protein, albumin, urea, creatinine, AST, GGT, ALP and CK) were taken 30 days before the date set for delivery, one week before (ante partum), delivery at 24h, 72h, 5 days, 15 days and 30 days after delivery. ketonuria was not observed in pre partum. The administration of supplements had no effect on the metabolic profile, protein and energy, and no liver disorders was observed in peripartum.(AU)

A preliminary molecular study on protein profile of vital organs: A new direction for post mortem interval determination.

Estimation of the postmortem interval (PMI) is one of the most important tasks in Forensic Medicine. Six autopsy organ tissues such as brain, lungs, heart, liver, pancreas and kidney were taken at the time of forensic autopsy. All the proteins present in the tissues were extracted and the protein profile was analyzed on the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) starting from 0 day to 10th day after death. The protein profiles showed a consistent degradation pattern which was consistent and reproducible in all the samples with respect to the time interval. In conclusion, the protein profile of the vital body organs appears to be a useful method for estimating the post mortem interval up to 10th day. Advantage of this approach over others is that it can detect the post mortem interval over a long interval (0 - 10 days) with an easily detectable pattern of protein profile.

Acute phase proteins: a potential approach for diagnosing chronic infection by Trypanosoma vivax / Proteínas de fase aguda: uma possível abordagem para diagnóstico de infecção crônica por Trypanosoma vivax

The present study aimed to assess potential changes in acute phase proteins in sheep experimentally infected with Trypanosoma vivax. There were studied eight male sheep, four used as controls and four infected with 10(5) T. vivax trypomastigotes. Blood samples were collected at two points times before infection and then at 5,7, 9, 11, 13, 15, 20, 30, 45, 60, 75, 90, 105 and 120 days post-infection (dpi). Blood samples were centrifuged and allotted, and acute phase proteins were then separated by electrophoresis on acrylamide gel containing sodium dodecyl sulfate. Protein concentrations were determined by computer-assisted densitometry. Total protein was determined by colorimetric biuret method. Trypanosomes were counted daily using a 5 mL aliquot of blood smear on a glass slide under a 22 × 22 mm coverslip. Parasites were counted in 100 microscopic fields (40× magnification), and then multiplied by a correction factor. The results were expressed as parasites per mL of blood. For statistical analyses, we used the Wilcoxon test at 5% significance level. There was found a reduction in several acute phase proteins and increase in antitrypsin and transferrin. This finding can be used for the diagnosis of T. vivax infection, especially in chronic infection.

O objetivo do presente estudo foi verificar possíveis alterações nas proteínas de fase aguda em ovinos infectados experimentalmente com Trypanosoma vivax. Para tanto, foram utilizados oito ovinos machos, sendo quatro usados como controle e quatro infectados com 10(5) tripomastigotas de T. vivax. Colheram-se amostras de sangue em dois tempos antes da infecção e, posteriormente, aos 5, 7, 9, 11, 13, 15, 20, 30, 45, 60, 75, 90, 105 e 120 dias após a infecção (dpi); após centrifugação e aliquotização das amostras. As proteínas de fase aguda foram separadas por eletroforese em gel de acrilamida, contendo dodecil sulfato de sódio, e suas concentrações foram determinadas através de densitometria computadorizada. A dosagem de proteína total foi realizada pelo método colorimétrico do biureto. A contagem dos tripanossomas foi realizada diariamente, utilizando-se uma alíquota de 5 µL de sangue disperso em lâmina de microscopia, sob lamínula de 22 × 22 mm, contando-se os parasitos em 100 campos microscópicos, com objetiva de 40×, multiplicados pelo fator de correção do microscópio, e o resultado expresso em parasitos por mL de sangue. Para a análise estatística, empregou-se o teste de Wilcoxon a 5% de probabilidade. Foi observada a diminuição de diversas proteínas de fase aguda e aumento de antitripsina e transferrina que podem ser utilizadas para auxiliar no diagnóstico da infecção por T. vivax, principalmente na fase crônica da infecção.

Glicemia, proteinograma e perfil de alguns componentes bioquímicos séricos de cabras da raça Bôer no pós-parto / Glycemic, protein profile and serum biochemical profile in Bôer goat in the postpartum period

Objetivou-se testar a hipótese de que ocorre variação de alguns componentes proteicos, bioquímicos e glicêmicos do sangue de cabras no período pós-parto. Utilizaram-se 11 cabras para avaliar as seguintes variáveis séricas: proteína total (PT), albumina, α-globulina, β-globulina, γ-globulina, imunoglobulina G (IgG), aspartatoaminotransferase (AST), fosfatase alcalina (FA), gamaglutamiltransferase (GGT), creatinina,ureia e glicemia. Esses componentes foram determinados nos momentos zero (imediatamente após o parto), dois, sete, 15, 30 e 75 dias pós-parto. A β-globulina, IgG, creatinina e uréia das cabras não apresentaram variações significativas; a glicemia no momento zero foi maior que os valores basais fisiológicos devido ao estímulo da glicogênese determinado pelo aumento de cortisol no parto. Os constituintes sanguíneos das cabras evidenciaram variações no período até 75 dias pós-parto em consequência de causas fisiológicas ligadas ao parto e puerpério.

The objective was to test the hypothesis that there is variation of some protein components, biochemical and glucose from the blood of goats in the postpartum period. ElevenBôer goats were used to evaluate the serum following variables: total protein (TP), albumin, α-globulin, β-globulin, γ-globulin, immunoglobulin G (IgG), aspartate (AST), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), creatinine, urea and glucose. These components were determined in moments zero (immediately after delivery), two, seven, 15, 30 and 75 days postpartum. The β-globulin, IgG, creatinine and urea from the goats did not show significant variations, the blood glucose at time zero was greater than the baseline values due to physiological stimulation of glycogenesis determined by the increase of cortisol in delivery. The blood constituents of goats showed variations in the period to 75 days postpartum as a result physiological causes related to delivery and postpartum.

Glicemia, proteinograma e perfil de alguns componentes bioquímicos séricos de cabritos da raça Bôer / Glycemic, protein profile and serum biochemical profile in Bôer goat kids

Os cabritos recém-nascidos sofrem diversas adaptações à vida extrauterina que afetam diferentes funções orgânicas, pois precisam produzir calor, iniciar a atividade muscular e buscar alimento. Todos estes eventos levam a modificações em diversos constituintes sanguíneos, como proteínas e parâmetros bioquímicos séricos. Outros estudos demonstraram estas variações, mas sem se estender além do período neonatal até a fase de animais jovens. Objetivou-se testar a hipótese de que ocorre variação doproteinograma, dos componentes bioquímicos e da glicemia de cabritos desde o nascimento até 75 dias de vida em função da adaptação à vida extrauterina. Para atingir estes objetivos, foram colhidas amostras de sangue de 25 cabritos nascidos de partos normais, independentemente do sexo. As variáveis séricas proteína total (PT), albumina, α-globulina, β-globulina, γ-globulina, onde se incluem a imunoglobulina G (IgG), aspartatoaminotransferase (AST), fosfatase alcalina (FA), gamaglutamiltransferase (GGT), creatinina, ureia, e glicemia foram determinadas nos momentos zero (logo após o nascimento), dois, sete, 15, 30 e 75 dias pós-parto. Observaram-se diferenças significativas em todas as variáveis entre os momentos, mas somente a concentração de creatinina foi maior que aquelas dos demais momentos, nos tempos zero e de dois dias pós-nascimento, devido, provavelmente, à imaturidade da função renal em animais neonatos. Os constituintes sanguíneos dos cabritos tiveram variações no período avaliado, relacionados a causas fisiológicas e nutricionais.

The newborn goat kids suffer several adjustments to life outside the uterus that affect different body functions that they need to produce heat, muscle activity and start searching for food. All these events lead to changes in several blood constituents such as proteins and serum biochemical parameters. Other studies have shown these variations, but not extend beyond the neonatal period to phase in young animals. The aim was to test the hypothesis that there is variation of the protein profile, and biochemical components of blood glucose in goat kids from birth to 75 days of life in terms of adaptation to extra uterine life. To achieve these objectives have been collected blood samples from 25 goats born by normal delivery, regardless of their sex. The variables serum total protein (TP), albumin, α-globulin, β-globulin, γ-globulin, which includes the immunoglobulin G (IgG), aspartate (AST), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), creatinine and urea, and glucose were determined at times zero (immediately after birth), two, seven, 15, 30 and 75 days old. We observed significant differences in all variables between times, but only the creatinine concentration was higher than those of other moments in time zero and two days old, probably due to immaturity of renal function in newborn animals. The blood constituents of the kids had variations in the study period, related to physiological and nutritional causes.

Caracterização das proteínas de superfície de membrana externa da sorovariedade Hardjo isolada de bovinos em Minas Gerais / Characterization of outer membrane proteins of serovar Hardjo isolated from cattle in Minas Gerais, Brazil

Foram identificadas diferenças no perfil protéico das amostras de Sponselee, Norma e Hardjoprajitno, com bandas protéicas entre 175, 47 kDA e 12,10 kDa. A amostra Sponselee foi a que apresentou maior número de bandas (12), seguida da Norma que apresentou 11 bandas e de Hardjoprajitno que apresentou 9 bandas. Todas as bandas observadas na amostra Sponselee possuíam correspondentes nas outras duas amostras. A amostra Norma não apresentou uma banda em torno de 35,77 kDa e a Hardjoprajitno não apresentou bandas em torno de 89,59 kDa, 35,77 kDa e 12,10 kDa. O reconhecimento dessas proteínas por soros hiperimunes contra cada uma das amostras também mostrou diferenças, sendo que o maior número de proteínas reconhecido em todas as amostras por todos os soros se encontrou entre 35,83 kDa e 29,19 kDa. Os soros contra bovino na amostra Norma só reconheceu proteínas de baixa massa molecular nas amostras Norma (6,80 kDa) e Hardjoprajitno (6,80 kDa e 5,30 kDa). Soro bovino contra a amostra Hardjoprajitno reconheceu uma proteína de 44,33 kDa todas às amostras e proteínas de 4,22 kDa nas amostras Sponselee e Norma e de 10,49 kDa e 6,16 kDa na Hardjoprajitno. As diferentes proteínas identificadas poderiam se constituir em alvos específicos para o desenvolvimento de testes diagnósticos e vacinas contra leptospirose bovina.

Differences in the protein profile of Leptospira sp. strains Sponselee, Norma and Hardjoprajitno were observed, with bands ranging from 175.47 kDa to 12.10 kDa. Strain Sponselee presented a 12-band profile, while strain Norma showed 11 and strain Hardjoprajitno showed 9 bands in the profile. All bands observed in Sponselee strain profile could match bands in the other two strains. Strain Norma lacks a band at 35.77 kDa and strain Hardjoprajitno lacks the bands at 89.59 kDa, 35.77 kDa and 12.10 kDa. The recognition profile from hyperimmune sera was also different for the studied serovar Hadjo strains. The majority of recognized proteins was in the range of 35.83 kDa to 29.19 kDa. Cattle sera against strain Norma only recognized low molecular mass proteins in strains Norma (6.80 kDa) and Hardjoprajitno (6.80 kDa and 5.30 kDa). Bovine sera against strain Hardjoprajitno recognized a 44.33 kDa protein in all studied strains and proteins of 4.22 kDa in strains Sponselee and Norma and of 10.49 kDa and 6.16 kDa in strain Hadjoprajitno. The different identified proteins could become specific targets to the development of diagnostic tests and vaccines against bovine leptospirosis.

Evaluation of IGF-I levels and serum protein profiles of diabetic cats and dogs

In this study, we measured the insulin-like growth factor (IGF)-I levels and evaluated the serum protein profiles of diabetic, insulin-treated, and healthy cats and dogs. The total IGF-I concentrations were 33.74 +/- 3.4 ng/mL for normal, 25.8 +/- 4.5 ng/mL for diabetic, and 180.4 +/- 31.4 ng/mL for insulin-treated cats. IGF-I concentrations were 46.4 +/- 6.6 ng/mL for normal, 25.1 +/- 4.1 ng/mL for diabetic, and 303.0 +/- 61.3 ng/mL for insulin-treated dogs. Total serum protein profiles were analyzed by SDS-PAGE. Fourteen bands ranging from 25 to 240 kDa in size were observed for cats, and 17 bands ranging from 25 to 289 kDa were observed for dogs. The densities of the bands differed among control, diabetic, and insulin-treated animals. In conclusion, we found that serum protein profiles and IGF-I concentrations were altered in both diabetic and insulin-treated animals. When judiciously interpreted in the light of other clinical and laboratory data, the techniques used in our study provide a valuable modality for measuring the severity of diabetes mellitus in dogs and cats.

Perfil proteico e metabolismo oxidativo de cordeiros experimentalmente infectados pelo Haemonchus contortus e suplementados com selênio e vitamina E / Protein Profile and oxidative metabolism of lambs experimentally infected with Haemonchus contortus and supplemented with selenium and vitamin E

Para avaliar a influência da suplementação com selênio e vitamina E sobre o perfil proteico e metabolismo oxidativo de cordeiros infectados experimentalmente pelo Haemonchus contortus, trinta cordeiros fêmeas foram distribuídos em quatro grupos: G1 (n=10): animais infectados; G2 (n=10): infectados e suplementados; G3 (n=5): controle; e G4 (n=5): não infectados e suplementados. Os grupos 1 e 2 receberam 500 larvas de H. contortus (L3), via oral, por um período de 20 dias, com intervalo de dois dias entre as doses. A suplementação nos grupos 2 e 4 foi realizada no dia zero, com 0,1mg kg-1 de Selenito de sódio (1,67 por cento) e com 2.000UI de vitamina E por via intramuscular (IM). Somente a vitamina E foi reaplicada no dia 30. As coletas de sangue para determinação do perfil proteico (proteína total, albumina, alfa, beta e gamaglobulina) e metabolismo oxidativo (espécies reativas ao ácido tiobarbitúrico-TBARS e a enzima glutationa peroxidase (GSPX) foram realizadas nos dias zero, 20, 30, 45, 60 e 80. OPG foi quantificado nos dias 0, 20 ,45 e 80. Em relação aos valores de proteínas totais, albumina, betaglobulina e gamaglobulina, as principais diferenças foram observadas quando os grupos parasitados foram comparados com o grupo somente suplementado; e este manteve valores mais elevados. Conclui-se que não há influência da suplementação com selênio e vitamina E no perfil proteico e metabolismo oxidativo quando os cordeiros se encontram severamente parasitados por H.contortus.

The objective of the present study was to describe the influence of supplementation with selenium and vitamin E on the protein and oxidative profiles of lambs experimentally infected with Haemonchus contortus. Thirty female lambs were divided into four groups as follows: G1 (n=10): infected animals; G2 (n=10): infected and supplemented; G3 (n=5): control; G4 (n=5): non-infected and supplemented. Groups 1 and 2 received 500 H. contortus larvae (L3) orally for a period of 20 days, with 2-day intervals between doses. Supplementation in groups 2 and 4 was performed on day zero by injecting 0.1mg kg-1 of sodium selenite solution (1.67 percent) and 2,000IU vitamin E through the intramuscular (IM) route. Vitamin E alone was injected once again on day thirty. The blood samples to determine the protein profile (total protein, albumin, alpha, beta and gamma) and oxidative metabolism (species reactive to thiobarbituric acid and glutathione peroxidase (GSPX) were performed on days zero, 20, 30, 45, 60 and 80. The OPG was quantified on days 0, 20, 45 and 80. Regarding the values of total proteins, albumin, beta and gamma globulins, the main differences were observed when the parasitized groups were compared to the supplemented, non-infected group, the latter exhibiting higher values. It is concluded that there is no influence of supplementation with vitamin E and selenium in the protein profile and oxidative metabolism when the lambs are severely parasitized with H. contortus.

Efeito da inclusão de fontes lipídicas na dieta de cabras em lactação sobre os parâmetros sanguíneos / Effect of fat source inclusion in the diet of lactating goats

Conduziu-se o estudo do perfil metabólico, com o objetivo de avaliar o estado nutricional ou possíveis distúrbios metabólicos que podem comprometer a saúde e, consequentemente, o desempenho do rebanho. No presente trabalho foi avaliado o perfil metabólico de cabras em lactação, submetidas a dietas com diferentes fontes de lipídios. Foram utilizadas 16 cabras da raça Saanen com peso vivo médio de 35 Kg e produção média diária de 1,2 kg de leite, distribuídas em quadrado latino com três repetições. Os tratamentos experimentais consistiram nas seguintes dietas: sem suplementação lipidica (TC); semente de faveleira (SF); torta de faveleira (TF) e caroço de algodão (CA). O experimento foi desenvolvido em quatro períodos experimentais de 14 dias, sendo 10 dias de adaptação à dieta e quatro dias de coleta de dados. No último dia de cada período foram coletadas amostras de sangue. A inclusão de semente de oleaginosa na dieta de cabras em lactação diminuiu o consumo de matéria seca por unidade metabólica (CMSU) e consumo de proteína bruta por unidade metabólica (CPBU). Dentre os quatro tratamentos não se observou diferença significativa no que se refere à concentração sérica de uréia, creatinina, GGT (gama glutamiltransferase), cálcio, fósforo, magnésio e glicose. Os níveis séricos de albumina dos animais que receberam TF foram maiores (P<0,05) do que os animais do grupo controle. Este trabalho sugere que as fontes de oleaginosas podem ser utilizadas na suplementação lipídica de cabras Saanen em lactação, entretanto animais alimentados com TF apresentaram um perfil metabólico mais saudável em função da suplementação lipídica.

The study of the metabolic profile has as objective to evaluate the nutritional status or possible metabolic disturbances that can damage the health and consequently the herd performance. In this work, the metabolic profile of lactating goats was evaluated, submitted to diets with different fat sources. 12 Saanen goats with BW of 35 kg and daily milk production of 1,2 kg were used, distributed into a Latin square with three repetitions. The diet comprised, without fat supplementation, TC (control) and with fat supplementation: SF (favelone seed); TF (favelone cake) and CA (cotton seed).The experiment was developed in four experimental periods of 14 days, with 10 days of adaptation to diets and four days for samples collection. In the last day of each period were collected blood samples. The inclusion of oleaginous seed into the lactating goats' diet decreased the dry matter consumption for metabolic unit (CMU) and consumption of crude protein for metabolic unit (CPMU). No significant differences in serum urea, creatinine, GGT (gamma glutamyltransferase), calcium, phosphorus, magnesium or glucose values were noticed in any of the four treatments. The serum levels of albumin in the animals that received TF were higher (P<0.05) than the animals of the control group. This work suggests that the oleaginous sources can be used as fat supplementation of lactating Saanen goats; however, animals fed the TF showed a healthier metabolic profile due to the fat supplement.

Protein profile analysis of Malaysian snake venoms by two-dimensional gel electrophoresis

Snake venoms comprise a highly complex mixture of proteins, which requires for their characterization the use of versatile two-dimensional electrophoresis techniques. In the present study, venoms obtained from eight snakes (Ophiophagus hannah, Naja kaouthia, Naja sumatrana, Bungarus fasciatus, Trimeresurus sumatranus, Tropidolaemus wagleri, Enhydrina schistosa and Calloselasma rhodostoma) commonly found in Malaysia were separated based on two independent properties, isoelectric point (pI) and molecular weight (MW). Many differences in snake venoms at the inter-family, inter-subfamily, inter-genus and inter-species levels were revealed. Notably, proteins from individuals of the Viperidae family – Trimeresurus sumatranus, Tropidolaemus wagleri and Calloselasma rhodostoma – were found to be numerous and scattered by the two-dimensional gel electrophoresis (2DE) specifically in regions between 37 and 100 kDa compared to the Elapidae venom proteins. The latter were clustered at the basic and lower molecular mass region (less than 20 kDa). Trains of spots were commonly observed, indicating that these proteins may be derived from post-translational modifications. Ophiophagus hannah (Elapidae) revealed a great amount of protein spots in the higher molecular mass range when compared to Enhydrina schistosa, Naja kaouthia, Naja sumatrana and Bungarus fasciatus. Overall 2DE showed large differences in the venom profile of each species, which might be employed as an ancillary tool to the identification of venomous snake species.

Protein profile analysis of Malaysian snake venoms by two-dimensional gel electrophoresis

Snake venoms comprise a highly complex mixture of proteins, which requires for their characterization the use of versatile two-dimensional electrophoresis techniques. In the present study, venoms obtained from eight snakes (Ophiophagus hannah, Naja kaouthia, Naja sumatrana, Bungarus fasciatus, Trimeresurus sumatranus, Tropidolaemus wagleri, Enhydrina schistosa and Calloselasma rhodostoma) commonly found in Malaysia were separated based on two independent properties, isoelectric point (pI) and molecular weight (MW). Many differences in snake venoms at the inter-family, inter-subfamily, inter-genus and inter-species levels were revealed. Notably, proteins from individuals of the Viperidae family - Trimeresurus sumatranus, Tropidolaemus wagleri and Calloselasma rhodostoma - were found to be numerous and scattered by the two-dimensional gel electrophoresis (2DE) specifically in regions between 37 and 100 kDa compared to the Elapidae venom proteins. The latter were clustered at the basic and lower molecular mass region (less than 20 kDa). Trains of spots were commonly observed, indicating that these proteins may be derived from post-translational modifications. Ophiophagus hannah (Elapidae) revealed a great amount of protein spots in the higher molecular mass range when compared to Enhydrina schistosa, Naja kaouthia, Naja sumatrana and Bungarus fasciatus. Overall 2DE showed large differences in the venom profile of each species, which might be employed as an ancillary tool to the identification of venomous snake species.(AU)