Proteomic analysis of dog tears for potential cancer markers

The first reference map of the proteome of pooled normal dog tears was created using 2-dimensional polyacrylamide gel electrophoresis and the identity of a number of the major species determined using matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF) and peptide mass fingerprint matching on protein sequence databases. In order to understand the changes in protein expression in the tear film of dogs with cancer, tears from such animals were similarly examined. A number of differences were found between the tears of healthy dogs and the dogs with cancer. Differences were found in levels of actin and albumin and in an unidentified protein which may be analogous to human lacryglobulin. These findings suggest that it may be possible to develop tear film analysis to provide a simple non-invasive test for the diagnosis and/or management of canine cancers. (C) 2007 Elsevier Ltd. All rights reserved.

Proteínas séricas de potros da raça Puro Sangue Árabe recém-desmamados ou com mais de trinta dias de desmame

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Serum protein concentrations, including acute phase proteins, in calves experimentally infected with Salmonella Dublin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification of a bisegmented double-stranded RNA virus (picobirnavirus) in calf faeces

To determine the incidence of rotavirus infection among dairy herds in the State of Sdo Paulo, Brazil, 576 faecal samples obtained from calves aged 1-45 days with and without diarrhoea, reared on 63 dairy cattle farms, were analyzed. Polyacrylamide gel electrophoresis (PAGE) identified 28 samples positive for group A rotavirus, while four samples, two diarrhoeic and two non-diarrhoeic, showed a bisegmented genome with a typical picobirnavirus pattern. Electron microscopy revealed spherical virus particles with a diameter of 37 nm and without a defined surface structure. The present study is the first report of a bisegmented virus identified in cattle in Brazil. (C) 2003 Elsevier B.V. Ltd. All rights reserved.

Rotavírus bovino: fatores de risco, prevalência e caracterização antigênica de amostras em rebanhos leiteiros no estado de São Paulo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Prevalência de rotavírus do grupo A em fezes diarréicas de bezerros de corte em sistema semi-intensivo de produção

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular characterization of group A bovine rotavirus in southeastern and central-western Brazil, 2009-2010

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Toxoplasma gondii: isolation of tachyzoites rhoptries and incorporation into Iscom

Rhoptries have been isolated from Toxoplasma gondii tachyzoites by subcellular fractionation in isopynic density sucrose gradient. Five bands were observed, and transmission electron microscopy of these indicated that rhoptries were in band 3. This band had a density of 1.17g/cm(3). Fraction 1 had membrane structures of the parasite. Fraction 2 contained membranes and mitochondria (Fig. 1 B). Fraction 4 had mostly conoid structure (Fig. 2B) and fraction 5 showed ghosts. The electrophoretic and Western blotting analysis of the fractions indicated the presence of a number of proteins. Iscoms were constructed from band 3, which contained the rhoptry structures. Iscom showed a only protein incorporated of 55 kDa. Isolation of the parasite organelles has got in this work is necessary to identification, characterization, and function elucidation of the organelle proteins. (C) 2004 Elsevier B.V. All rights reserved.

Inorganic pyrophosphate-phosphohydrolytic activity associated with rat osseous plate alkaline phosphatase

Purified membrane-bound alkaline phosphatase from rat osseous plate hydrolyzed pyrophosphate in the presence of magnesium ions, with a specific activity of 92.7 U/mg. Optimal apparent pH for pyrophosphatase activity was 8.0 and it remained unchanged on increasing the pyrophosphate concentration. In the absence of magnesium ions the enzyme had a K-m = 88 mu M and V = 36.7 U/mg for pyrophosphate and no inhibition by excess substrate was observed. Pyrophosphatase activity was rapidly destroyed at temperatures above 40 degrees C, but magnesium ions apparently protected the enzyme against danaturation. Sodium metavanadate (Ki = 1.0 mM) was a competitive inhibitor of pyrophosphatase activity, while levamisole (Ki = 8.2 mM) and theophylline (Ki = 7.4 mM) were uncompetitive inhibitors. Magnesium ions (K-0.5 = 1.7 mu M) stimulated pyrophosphatase activity, while cobalt (Ki = 48.5 mu M) and zinc (Ki = 22.0 mu M) ions were non-competitive inhibitors. Manganese and calcium ions had no effect on pyrophosphatase activity. The M-w of the pyrophosphatase: protein was 130 kDa by gel filtration, but a value of 65 kDa was obtained by dissociative gel electrophoresis, suggesting that it was a dimer of apparently identical subunits. These results suggested that pyrophosphatase activity stems from the membrane-bound osseous plate alkaline phosphatase and not from a different protein.

Molecular Identification and Characterization of Ileal and Cecal Fungus Communities in Broilers Given Probiotics, Specific Essential Oil Blends, and Under Mixed Eimeria Infection

Broiler digestive tract fungal communities have gained far less scrutiny than that given corresponding bacterial communities. Attention given poultry-associated fungi have focused primarily on feed-associated toxin-producers, yeast, and yeast products. The current project focused on the use of pyrosequencing and denaturing gradient gel electrophoresis (DGGE) to identify and monitor broiler digestive fungal communities. Eight different treatments were included. Four controls were an Uninfected-Unmedicated Control, an Unmedicated-Infected Control, the antibiotic bacitracin methylene disalicylate plus the ionophore monensin as Positive Control, and the ionophore monensin alone as a Negative Control. Four treatments were two probiotics (BC-30 and Calsporin) and two specific essential oil blends (Crina Poultry Plus and Crina Poultry AF). All chickens except the Unmedicated-Uninfected Control were given, at 15 days of age, a standard oral Eimeria inoculum of sporulated oocysts. Ileal and cecal digesta were collected at pre-Eimeria infection at 14 days of age and at 7 days post-Eimeria infection at 22 days of age. Extracted cecal DNA was analyzed by pyrosequencing to examine the impact of diet supplements and Eimeria infection on individual constituents in the fungal community, while DGGE was used to compare more qualitative changes in ileal and cecal communities. Pyrosequencing identified three phyla, seven classes, eight orders, 13 families, 17 genera, and 23 fungal species. Ileal and cecal DGGE patterns showed fungal communities were clustered mainly into pre- and post-infection patterns. Post-infection Unmedicated-Uninfected patterns were clustered with pre-infection groups demonstrating a strong effect of Eimeria infection on digestive fungal populations. These combined techniques offered added versatility towards unraveling the effects of enteropathogen infection and performance enhancing feed additives on broiler digestive microflora.

POLYPHENOLOXIDASE FROM ATEMOYA FRUIT (ANNONA CHERIMOLA MILL. ANNONA SQUAMOSA L.)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Enzymatic Hydrolysis of Sweet Lupin, Chickpea, and Lentil 11S Globulins Decreases their Antigenic Activity

We investigated the effects of treatments with the enzymes pepsin and trypsin on the in vitro immunological reactivity of the major globulins found in the seeds of sweet lupin, chickpea, and lentil. Polyclonal major globulin-specific antiserum was obtained by immunization of rabbits with a solution of the 11 S globulin of each legume. The globulins were hydrolyzed with pepsin and trypsin for 1, 5, 15, and 30 min. The native globulins and their hydrolysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to identify the polypeptide bands with antigenic activity, and the hypoantigenicity of the hydrolysates was analyzed by enzyme-linked immunosorbent assay. Our results show that enzymatic treatment of the major storage protein (11 S globulin) of sweet lupin, chickpea, and lentil with pepsin or trypsin lead to the formation of large amounts of short peptides and free amino acids that do not allow antibody binding, resulting in a weakened immunoreactivity.

Protein Hydrolysis by Immobilized and Stabilized Trypsin

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Isolation, solubility and in vitro hydrolysis of chickpea vicilin-like protein

The chickpea vicilin-like globulin was isolated and chromatographed on Sepharose CL-6B and Sephacryl S-300. The native globulin with a molecular weight of 140 kDa was resolved in Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in seven polypeptide bands in the range of 12.4-67 kDa. The solubility profile of the protein in water and NaCl solutions was typical of a legume globulin. The purified vicilin-like globulin, native and heated, was hydrolyzed by pepsin, trypsin and chymotrypsin. The hydrolysis patterns indicated that the native vicilin-like protein was only partially degraded by the enzymes in comparison with casein. Heating increased its susceptibility to hydrolysis relative to the native form, for all the enzymes. However, the results obtained by the pH-drop method revealed that the in vitro digestibility of the vicilin-like protein was not altered by heating, while 11 S-like and total globulins suffered a small increase, indicating that the structural characteristics of storage globulins may be important factors limiting the protein digestion. (c) 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus

The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose and glucose as carbon source, This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6 HR 10/30 columns. The purified enzyme showed a relative molecular weight of 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 77 kDa by gel filtration in Superose 6 HR 10/30, Analysis by SDS-PAGE showed a unique polypeptide band with Coomassie Blue stain and nondenaturing PAGE of the purified enzyme obtained from media with different carbon sources showed the band, too, when stained for glucose oxidase activity, the optimal hydrolysis temperature for sucrose, raffinose and inulin was 55 degrees C and the optimal pH for sucrose was 4.75, the apparent K-m values for sucrose, raffinose and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively, Thin layer chromatography showed that inulinase from K. marxianus var. bulgaricus was capable of hydrolyzing different substrates (sucrose, raffinose and inulin), releasing monosaccharides and oligosaccharides, the results obtained suggest the hypothesis that enzyme production was constitutive.