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Bovine coronavirus structural proteins.  

PubMed Central

The tissue culture-adapted strain (Mebus) of bovine coronavirus was grown in the presence of isotopically labeled amino acids, glucosamine, or orthophosphate for the purpose of analyzing the virion structural proteins. Five species of polypeptides were identified when purified virions were solubilized in urea and sodium dodecyl sulfate and resolved by polyacrylamide gel electrophoresis. Four species were glycosylated and had apparent molecular weights of 140,000, 120,000, 100,000, and 26,000. The glycoproteins were susceptible to proteolytic cleavage and enzymatic iodination when intact virions were studied and are thus at least partially external to the virion envelope. The 140,000-molecular-weight glycoprotein is apparently a dimer of 65,000-molecular-weight glycopolypeptides held together by disulfide linkages. Species 5 was phosphorylated and had an apparent molecular weight of 52,000. In the intact virion, it was unaffected by protease and was not enzymatically iodinated. It is therefore apparently an internal protein. Images

King, B; Brian, D A



Application of a mechanistic model of bovine milk protein synthesis to examine the use of isotope labeling methods.  


Two types of models of bovine milk protein synthesis were used to simulate collection and analysis of data from infusion experiments involving isotope-labeled amino acids (AA). Analytical solutions to a system of ordinary differential equations that describe isotope enrichment curves of each AA pool within the mammary gland were derived and are presented. Numerical solutions from a dynamic mechanistic model suggest that normal experimental procedures can affect the shape of enrichment curves and, therefore, results derived from them. Simulation results suggest that standard methods utilizing in vivo isotope kinetics may be of limited value to characterize the metabolism of the bovine mammary gland, especially AA metabolism and milk protein synthesis and secretion. The results clearly demonstrate the flexibility of such models for the testing of many hypotheses and possible experimental protocols. PMID:9785235

Maas, J A; France, J; McBride, B W



Electrohydrodynamic atomization of protein (bovine serum albumin).  


Bovine serum albumin (BSA) was chosen as a model protein. Three solutions of different concentrations of 5, 20 and 50 mg/ml were prepared, characterised and subjected to electrohydrodynamic atomization (EHDA). The 5 and 20 mg/ml solutions were atomized successfully and mode selection (M-S) maps were drawn for both concentrations to find out regions of stable cone-jet mode atomizaton. Droplet relics of these two solutions were investigated by electron microscopy. Samples were investigated by UV spectroscopy and circular dichroism (CD) spectroscopy before and after electrohydrodynamic atomization. We conclude that, particularly at the higher concentration of protein, EHDA does not result in significant structural change of BSA, and therefore is a processing route that can be considered for encapsulating drugs in proteins. PMID:16167100

Pareta, R; Brindley, A; Edirisinghe, M J; Jayasinghe, S N; Luklinska, Z B



Envelope Proteins Containing Single Amino Acid Substitutions Support a Structural Model of the Receptor-Binding Domain of Bovine Leukemia Virus Surface Protein  

PubMed Central

Functional domains of the strikingly conserved envelope (Env) glycoproteins of bovine leukemia virus (BLV) and its close relative, human T-cell leukemia virus type 1 (HTLV-1), are still being defined. We have used BLV Env protein variants to gain insights into the structure and function of this important determinant of viral infectivity. Each of 23 different single amino acid variants found in cDNA clones of env transcripts present after short-term culture of peripheral blood mononuclear cells from BLV-infected sheep was expressed in COS-1 cells and tested for the ability to mediate cell fusion and to be cleaved to surface (SU) and transmembrane (TM) protein subunits. Of 11 Env variants that failed to induce syncytia or did so poorly, 7 contained changes in amino acids identical or chemically conserved in the HTLV-1 Env protein. These seven included the four variants that showed aberrant proteolytic cleavage and poor cell surface expression, underscoring their importance for Env structure. Ten of 12 variants that retained wild-type syncytium-inducing ability clustered in the N-terminal half of BLV SU, which forms the putative receptor-binding domain (RBD). Several variants in the RBD showed evidence of subtle misfolding, as judged by reduced binding to monoclonal antibodies recognizing conformational epitopes F, G, and H formed by the N terminus of SU. We modeled the BLV RBD by aligning putative structural elements with known elements of the ecotropic Friend murine leukemia virus RBD monomer. All the variant RBD residues but one are exposed on the surface of this BLV model. These variants as well as function-altering, antibody-reactive residues defined by other investigators group on one face of the molecular model. They are strikingly absent from the opposite face, implying that it is likely to face inward in Env complexes. This surface might interact with the C-terminal domain of SU or with an adjacent monomer in the Env oligomer. This location suggests an orientation for the monomer of ecotropic Friend murine leukemia virus RBD.

Johnston, Elizabeth R.; Albritton, Lorraine M.; Radke, Kathryn



Bovine plasma proteins increase virulence of Haemophilus somnus in mice.  


The role of bovine serum or plasma proteins in Haemophilus somnus virulence was investigated in a mouse model of septicemia. An increase in virulence was detected when the organism was pre-incubated for 5 min and inoculated with fetal calf serum. When purified bovine serum or plasma proteins were pre-incubated with H. somnus before inoculating into mice, transferrin was found to increase virulence. Bovine lactoferrin was also noted to increase virulence, but to a lesser extent and had a delayed time course when compared with transferrin. Using an ELISA assay, an increased amount of H. somnus whole cells and culture supernatant bound to bovine transferrin when the organism was grown in iron-restricted media. Lactoferrin also bound to H. somnus, but binding was not affected by growth in iron-restricted media and it was eliminated with 2M NaCl, which reversed charge mediated binding. Transferrin, but not lactoferrin, supported growth of H. somnus on iron-depleted agar based media using a disk assay. Therefore, lactoferrin increased virulence by an undetermined mechanism whereas transferrin increased virulence of H. somnus by binding to iron-regulated outer-membrane proteins (IROMPs) and providing iron to the pathogen. PMID:17125964

Geertsema, Roger S; Kimball, Richard A; Corbeil, Lynette B



Fabrication of a surface imprinted hydrogel shell over silica microspheres using bovine serum albumin as a model protein template.  


Surface imprinting is an effective approach to improve the template transfer efficiency in applications of molecularly imprinted polymers as biosensors and separation materials. In this paper, we tried to fabricate a surface imprinted hydrogel over silica microspheres for selective recognition of bovine serum albumin by covalent immobilization of a water-soluble UV sensitive initiator onto the surface of silica beads. The polymerization was initiated by UV radiation with N-[3-(dimethylamino)propyl]methacrylamide and N-isopropylacrylamide as the functional monomer and assistant monomer, respectively, and a thin coat of stimuli-responsive hydrogel yielded over the silica gels. The surface imprinted hydrogels exhibited specific affinity toward the template protein with an association constant (K(a)) of 2.2 x 10(5)L mol(-1) and a maximum binding capacity (Q(max)) of 27.3 mgg(-1) in Tris-HCl buffer (pH 7.0). The rebinding and desorption kinetics of the surface imprinted hydrogels were determined and proven to be extremely fast (about 1 min compared to 3h for the previously prepared bulk imprinted hydrogel). Besides, the hydrogel-silica core-shell particles inherit both the stimuli-responsive property of the hydrogel and the good mechanical strength of the silica beads based on the on-line evaluation with high-performance liquid chromatography. The above comprehensive merits of the obtained surface imprinted hydrogel suggest the presented approach an attractive and broadly applicable way of developing biosensors and high-performance protein separation materials. PMID:19230646

Hua, Zhendong; Zhou, Shuang; Zhao, Meiping



Inulin like lyoprotectant of bovine plasma proteins concentrated by ultrafiltration  

Microsoft Academic Search

The growth of world demand for protein-enriched products generates a great interest in the search of new protein sources of higher nutritional value and, therefore, in the use of cutting edge technologies to achieve that goal. In this work, technologies of ultrafiltration and freeze-drying to process and obtain a protein concentrate from bovine plasma proteins are employed. The effectiveness of

Laura T. Rodríguez Furlán; Antonio Pérez Padilla; Mercedes E. Campderrós



A bovine whey protein extract can induce the generation of regulatory T cells and shows potential to alleviate asthma symptoms in a murine asthma model.  


The number of people with asthma has dramatically increased over the past few decades and the cost of care is more than $11·3 billion per year. The use of steroids is the major treatment to control asthma symptoms, but the side effects are often devastating. Seeking new drugs or new strategies to reduce the dose of steroid taken has always been an important task. A bovine whey protein extract (WPE), which is enriched in transforming growth factor-? (TGF-?), has been demonstrated to have the potential for reducing symptoms associated with mild-to-moderate T-helper cell type 1-mediated psoriasis in human subjects. However, whether WPE also has potential for inhibiting T-helper cell type 2 (Th2)-mediated disease remains unclear. In the present study, using a murine asthma model, we found that sensitised mice fed WPE daily, before they were challenged, resulted in reducing airway inflammation, serum ovalbumin-specific IgE, Th2-related cytokine production and airway hyperresponsiveness. Increase in the regulatory T cell (Treg) population in vitro and in vivo was observed when treated with WPE. According to the results from the TGF-?-blocking antibody study, we suggest that TGF-? is the main component that endows WPE with the potential to reduce the generation of Treg. Thus, the present data suggest that WPE has the potential to alleviate the symptoms of asthma by inducing the generation of Treg. Therefore, regular administration of WPE might be potentially beneficial for patients with asthma. PMID:23068908

Chen, Jiunn-Horng; Huang, Po-Han; Lee, Chen-Chen; Chen, Pin-Yu; Chen, Hui-Chen



Purification of lipopolysaccharide-binding protein from bovine serum.  


Lipopolysaccharide-binding protein (LBP) plays a central role in presentation of bacterial-derived lipopolysaccharide (LPS; endotoxin) to leukocytes such as macrophages and neutrophils. Interaction of LBP with LPS is significant because LBP-LPS complexes promote activation of leukocytes and the immune system, which results in enhanced secretion of a spectrum of proinflammatory cytokines. An improved, simplified method was used to purify bovine LBP from serum. Methodology consisted of ion-exchange chromatography using Bio-Rex 70 resin, followed by gel-filtration chromatography (Sephacryl S-200 resin) of a selected ion-exchange fraction (0.22-0.50 M NaCl). Densitometric scans on silver-stained polyacrylamide gels of chromatographically-derived proteins indicated up to 88.7% purity of the resultant 64kD protein (bovine LBP) in the cleanest fractions. The isoelectric point of bovine LBP was determined to be 6.8. Identity of the protein was substantiated by western-blot analysis, and by N-terminus amino acid sequence analysis with favorable comparison to published sequence data from rabbit, human, and murine LBP Identity was corroborated by use of purified bovine LBP in bioassays which demonstrated enhanced tissue factor expression of LPS (1 ng ml(-1)-stimulated bovine alveolar macrophages. Tissue factor expression was inhibitable in these assays using anti-CD14 monoclonal antibodies, which is also consistent with LBP-mediated activation of cells. When bovine LBP was heated at 56 degrees C for 30 min, the biological activity was reduced by 50% in the macrophage-based bioassays. Biological activity of bovine LBP was completely destroyed by heating at 62 degrees C for 30 min, which compared favorably with data resulting from use of fetal bovine serum. PMID:8792567

Bochsler, P N; Yang, Z; Murphy, C L; Carroll, R C



Bovine and canine acute phase proteins  

Microsoft Academic Search

Acute phase proteins are serum proteins which increase in concentration during the acute phase response to inflammation or infection. The response occurs in all animals, but in different species the response of individual proteins can be significantly different. Of the numerous acute phase proteins which have been identified in humans, a number have been examined in cattle and dogs but

P. D. Eckersall; J. G. Conner



Different Behavior toward Bovine Spongiform Encephalopathy Infection of Bovine Prion Protein Transgenic Mice with One Extra Repeat Octapeptide Insert Mutation  

Microsoft Academic Search

In humans, insert mutations within the repetitive octapeptide region of the prion protein gene (Prnp) are often associated with familial spongiform encephalopathies. In this study, transgenic mice expressing bovine PrP (boTg mice) bearing an additional octapeptide insertion to the wild type (seven octapeptide repeats instead of six) showed an altered course of bovine spongiform encephalopathy (BSE) infection, reflected as reduced

J. Castilla; A. Gutierrez-Adan; A. Brun; B. Pintado; B. Parra; M. A. Ramirez; F. J. Salguero; F. Diaz San Segundo; A. Rabano; M. J. Cano; J. M. Torres



Immunochemical Study of Bovine Serum Proteins.  

National Technical Information Service (NTIS)

With the aid of immunological methods of analysis, this study characterizes certain protein fractions of the blood serum of cattle. Analyses indicate that serum proteins differ not only in their electrophoretic mobility, but also in their antigenic struct...

V. M. Kholod



Preparation of Simulated Human Milk Protein by Low Temperature Microfiltration and Precipitation of Bovine Milk.  

National Technical Information Service (NTIS)

The invention is directed to the modification of bovine milk to simulate human milk protein composition which can be used in infant formulas. This is accomplished by low temperature ultrafiltration or microfiltration of bovine milk has been pretreated at ...

J. H. Woychik



Stability of bovine spongiform encephalopathy prions: absence of prion protein degradation by bovine gut microbiota.  


Bovine spongiform encephalopathy (BSE) is transmitted by the oral route. However, the impacts of anaerobic fermentation processes in cattle on the stability of BSE-associated prion protein (PrP(Sc)) are still unresolved. In this study, experiments were designed to assess the ability of complex ruminal and colonic contents of bovines to degrade BSE-derived PrP(Sc). No significant decrease in PrP(Sc) levels in BSE brain homogenates was detected by Western blotting after up to 66 h of co-incubation with intestinal fluids. These results indicate that BSE-associated PrP(Sc) survive gastrointestinal digestion processes in cattle and might be excreted via faeces. PMID:22353543

Böhnlein, C; Groschup, M H; Maertlbauer, E; Pichner, R; Gareis, M



Identification of highly active flocculant proteins in bovine blood.  


Synthetic polymeric flocculants are used extensively for wastewater remediation, soil stabilization, and reduction in water leakage from unlined canals. Sources of highly active, inexpensive, renewable flocculants are needed to replace synthetic flocculants. High kaolin flocculant activity was documented for bovine blood (BB) and blood plasma with several anticoagulant treatments. BB serum also had high flocculant activity. To address the hypothesis that some blood proteins have strong flocculating activity, the BB proteins were separated by SEC. Then, the major proteins of the flocculant-active fractions were separated by SDS-PAGE. Identity of the major protein components was determined by tryptic digestion and peptide analysis by MALDI TOF MS. The sequence of selected peptides was confirmed using TOF/TOF-MS/MS fragmentation. Hemoglobin dimer (subunits ? and ?) was identified as the major protein component of the active fraction in BB; its high flocculation activity was confirmed by testing a commercial sample of hemoglobin. In the same manner, three proteins from blood plasma (fibrinogen, ?-globulin, ?-2-macroglobulin) were found to be highly active flocculants, but bovine serum albumin, ?-globulin, and ?-globulin were not flocculants. On a mass basis, hemoglobin, ?-globulin, ?-2-macroglobulin were as effective as anionic polyacrylamide (PAM), a widely used synthetic flocculant. The blood proteins acted faster than PAM, and unlike PAM, the blood proteins flocculants did not require calcium salts for their activity. PMID:22194055

Piazza, George J; Nuñez, Alberto; Garcia, Rafael A



Purification of lipopolysaccharide-binding protein from bovine serum  

Microsoft Academic Search

Lipopolysaccharide-binding protein (LBP) plays a central role in presentation of bacterial-derived lipopolysaccharide (LPS; endotoxin) to leukocytes such as macrophages and neutrophils. Interaction of LBP with LPS is significant because LBP-LPS complexes promote activation of leukocytes and the immune system, which results in enhanced secretion of a spectrum of proinflammatory cytokines. An improved, simplified method was used to purify bovine LBP

Philip N. Bochsler; Zhengang Yang; Charles L. Murphy; Roger C. Carroll



Molecular properties of bovine interphotoreceptor retinol-binding protein.  


Interphotoreceptor retinol-binding protein (IRBP) is a large retinol-carrying glycoprotein, located only in the interphotoreceptor (or subretinal) space of vertebrate eyes. It has recently been purified to apparent homogeneity. The present report presents its sedimentation, spectroscopic, and binding properties. The molecular weight of bovine IRBP, determined by sedimentation equilibrium, is 133,000. The sedimentation coefficient is 5.8S. The Stokes radius, 56 A, obtained from gel-filtration chromatography, is much larger than that expected for a globular protein of the same molecular weight. These results indicate that IRBP is asymmetric (it can modeled as a prolate ellipsoid of revolution with axial ratio of about 8:1) and explain the overestimates of molecular weight obtained in previous studies based on size-exclusion methods. The molar absorption coefficients for IRBP (at 280 nm) and for bound retinol are both unaffected by ligand dissociation. Fluorescence of the holoprotein displays neither fine structure nor energy transfer from tryptophan to bound retinol. Circular dichroism suggests a secondary structure containing approximately 15% alpha-helix and approximately 20% beta-structure, unchanged by the presence of ligand. The binding of retinol creates a positive, extrinsic Cotton effect at 330 nm, proportional to the amount of retinol bound. The apparent dissociation constant for all-trans-retinol is 1.3 X 10(-6) M. This relatively loose binding implies that, if required during the visual cycle, IRBP should be able to transfer its ligand to other binding proteins in the neural retina and retinal pigment epithelium. PMID:4039318

Adler, A J; Evans, C D; Stafford, W F



Phosphorylation of bovine leukemia virus Tax protein is required for in vitro transformation but not for transactivation  

Microsoft Academic Search

The Tax proteins of the oncovirinae viruses are phosphorylated transcriptional activators that exhibit oncogenic potential. The role of phosphorylation in their functional activities remains unknown. As a model for the Human T-cell leukemia virus type I (HTLV-I), Bovine Leukemia Virus (BLV) permits the characterization of viral replication and leukemogenesis in vivo. Here, we show that the BLV Tax protein is

Luc Willems; Cathy Grimonpont; Pierre Kerkhofs; Carine Capiau; Dirk Gheysen; Karel Conrath; Roussi Roussef; Robert Mamoun; Daniel Portetelle; Arsène Burny; Emmanuelle Adam; Laurent Lefèbvre; Jean-Claude Twizere; Hubertine Heremans; Richard Kettmann



Experimental anterior spine fusion using bovine bone morphogenetic protein: a study in rabbits  

Microsoft Academic Search

:   We developed an experimental model to study the merit of bovine bone morphogenic protein (bBMP) injection into the intervertebral\\u000a disc to induce anterior interbody fusion. A total of 24 rabbits, divided into three groups of 8 animals each, were used. One\\u000a hundred and fifty ?g of partially purified bBMP was employed in the first group and 10 ?g bBMP

M. Muschik; D. Schlenzka; V. Ritsilä; C. Tennstedt; Kai Uwe Lewandrowski



Biosynthesis and processing of bovine cartilage link proteins  

SciTech Connect

We have examined posttranslational modifications which are responsible for converting an apparently single precursor to the two major forms of link protein in bovine articular cartilage. Resistance to endoglycosidases H and F suggests that Asn-linked oligosaccharides of link protein secreted by bovine chondrocytes in culture are of the complex or hybrid type. There is no evidence for O-linked oligosaccharides. There is no apparent precursor-product relationship between link protein (LP)1 and LP2, since after a short pulse with (3H)leucine two forms are present, consistent with the existence of two glycosylation sites. An immunoprecipitate of LP1 from pulse-labeled chondrocytes was observed to show a decrease in electrophoretic mobility and increased microheterogeneity during transit through the Golgi, whereas LP2 did not change. During processing both LP1 and LP2 become endoglycosidase H resistant. LP1, but not LP2, can be biosynthetically labeled with (35S)sulfate. Incorporation of (35S)sulfate is inhibited by tunicamycin, indicating that the sulfate is associated with Asn-linked carbohydrate. Sulfation may be important for normal processing, secretion, or degradation of link protein and with sialylation may confer considerable charge heterogeneity upon LP1. We conclude that there are considerable biochemical differences between glycoproteins LP1 and LP2 which may provide a basis for functional differences.

Hering, T.M.; Sandell, L.J. (Univ. of Washington, Seattle (USA))



Role for Arachidonic Acid Metabolism and Protein Synthesis in Recombinant Bovine lnterferon-y---lnduced Activation of Bovine Neutrophils  

Microsoft Academic Search

Bovine neutrophils were preincubated with recombinant bovine interferon-y (rbolFN-'y), and the molecular events leading to enhanced antibody-dependent (ADCC) and antibody- independent (AINC) neutrophil-mediated cytotoxicity and reduced random migration un- der agarose were investigated. Addition of ot-amanitin, puromycin, or cycloheximide (RNA and protein synthesis inhibitors) during preincubation and assaying prevented the rbolFN-'y-induced AINC enhancement and migration inhibition, but did not Influence

MarIa J. Steinbeck; Deborah S. A. Webb; James A. Roth


Photo selective protein immobilization using bovine serum albumin  

NASA Astrophysics Data System (ADS)

A simple and selective technique which immobilizes protein onto a solid substrate by using UV illumination has been developed. In protein immobilization, a Bovine serum albumin (BSA) performed bifunctional role as a cross-linker between substrate and proteins and as a blocker inhibiting a nonspecific protein adsorption. A new photo-induced protein immobilization process has been investigated at each step by fluorescence microscopy, ellipsometry, and Fourier transform infrared (FT-IR) spectroscopy. A UV photomask has been used to induce selective protein immobilization on target regions of the surface of the SiO2 substrates under UV illumination with negligible nonspecific binding. The UV illumination also showed improved photostability than the conventional methods which employed bifunctional photo-crosslinker molecules of photo-reactive diazirine. This new UV illumination-based photo-addressable protein immobilization provides a new approach for developing novel protein microarrays for multiplexed sensing as well as other types of bio-immobilization in biomedical devices and biotechnologies.

Kim, Wan-Joong; Kim, Ansoon; Huh, Chul; Park, Chan Woo; Ah, Chil Seong; Kim, Bong Kyu; Yang, Jong-Heon; Chung, Kwang Hyo; Choi, Yo Han; Hong, Jongcheol; Sung, Gun Yong



A bovine papillomavirus E1-related protein binds specifically to bovine papillomavirus DNA.  

PubMed Central

The E1 open reading frame of bovine papillomavirus (BPV) was expressed as a RecA-E1 fusion protein in Escherichia coli. The bacterially expressed RecA-E1 protein exhibited sequence-specific DNA binding activity; strong binding to the region from nucleotides 7819 to 93 on the BPV genome (designated region A) and weak binding to the adjacent region from nucleotides 7457 to 7818 (region B) were observed. The interaction between the BPV-derived RecA-E1 protein and region A appeared to be highly specific for BPV DNA, as no comparable binding was detected with heterologous papillomavirus DNAs. Binding to region A was eliminated by digestion of region A at the unique HpaI site, which suggests that the RecA-E1 binding site(s) was at or near the HpaI recognition sequence. Binding to region B but not region A was observed when nuclear extracts from ID13 cells were used as a source of E1 proteins. The absence of region A binding by ID13 extracts may reflect a negative regulation of E1 DNA binding activity. Images

Wilson, V G; Ludes-Meyers, J



Proteins, isoleucine, lysine and methionine content of bovine, porcine and poultry blood and their fractions  

Microsoft Academic Search

Studies carried out in bovine blood proteins pointed out that they are excellent sources of lysine (Lys), but deficient in isoleucine (Ile) and methionine (Met). The purpose of this investigation was to determine and compare the content of proteins, Lys, Ile and Met in whole blood, red cells and plasma of bovine, porcine and poultry species. Blood from the different

Enrique Márquez; Mariela Bracho; Anangelina Archile; Lisbeth Rangel; Betty Benítez



IGF-1 stimulates protein synthesis by enhanced signaling through mTORC1 in bovine mammary epithelial cells  

Microsoft Academic Search

Using the MAC-T cell line as a model, the effects of insulin-like growth factor (IGF)-1 on the regulation of protein synthesis through the mammalian target of rapamycin complex 1 (mTORC1) signaling in bovine mammary epithelial cells were evaluated. Global rates of protein synthesis increased by 47% within 30min of IGF-1 treatment. The effect of IGF-1 on protein synthesis was associated

S. A. Burgos; J. P. Cant



Control of domain swapping in bovine odorant-binding protein.  

PubMed Central

As revealed by the X-ray structure, bovine odorant-binding protein (OBPb) is a domain swapped dimer [Tegoni, Ramoni, Bignetti, Spinelli and Cambillau (1996) Nat. Struct. Biol. 3, 863-867; Bianchet, Bains, Petosi, Pevsner, Snyder, Monaco and Amzel (1996) Nat. Struct. Biol. 3, 934-939]. This contrasts with all known mammalian OBPs, which are monomers, and in particular with porcine OBP (OBPp), sharing 42.3% identity with OBPb. By the mechanism of domain swapping, monomers are proposed to evolve into dimers and oligomers, as observed in human prion. Comparison of bovine and porcine OBP sequences pointed at OBPp glycine 121, in the hinge linking the beta-barrel to the alpha-helix. The absence of this residue in OBPb might explain why the normal lipocalin beta-turn is not formed. In order to decipher the domain swapping determinants we have produced a mutant of OBPb in which a glycine residue was inserted after position 121, and a mutant of OBPp in which glycine 121 was deleted. The latter mutation did not result in dimerization, while OBPb-121Gly+ became monomeric, suggesting that domain swapping was reversed. Careful structural analysis revealed that besides the presence of a glycine in the hinge, the dimer interface formed by the C-termini and by the presence of the lipocalins conserved disulphide bridge may also control domain swapping.

Ramoni, Roberto; Vincent, Florence; Ashcroft, Alison E; Accornero, Paolo; Grolli, Stefano; Valencia, Christel; Tegoni, Mariella; Cambillau, Christian



Improvement of the antimicrobial and antioxidant activities of camel and bovine whey proteins by limited proteolysis.  


The compositions and structures of bovine and camel milk proteins are different, which define their functional and biological properties. The aim of this study was to investigate the effects of enzymatic hydrolysis of camel and bovine whey proteins (WPs) on their antioxidant and antimicrobial properties. After enzymatic treatment, both the antioxidant and the antimicrobial activities of bovine and camel WPs were improved. The significantly higher antioxidant activity of camel WPs and their hydrolysates as compared with that of bovine WPs and their hydrolysates may result from the differences in amounts and/or in accessibilities of antioxidant amino acid residues present in their primary structures and from the prevalence of alpha-lactalbumin and beta-lactoglobulin as proteolytic substrates in camel and bovine whey, respectively. The results of this study reveal differences in antimicrobial and antioxidant activities between WP hydrolysates of bovine and camel milk and the effects of limited proteolysis on these activities. PMID:20175528

Salami, Maryam; Moosavi-Movahedi, Ali Akbar; Ehsani, Mohammad Reza; Yousefi, Reza; Haertlé, Thomas; Chobert, Jean-Marc; Razavi, Seyed Hadi; Henrich, Robert; Balalaie, Saeed; Ebadi, Seyed Ahmad; Pourtakdoost, Samineh; Niasari-Naslaji, Amir



Dynamics of bovine glial fibrillary acidic protein phosphorylation.  


Recently, the dynamic features of the intermediate filaments (IF) have been revealed. The effect of phosphorylation on the dynamics of bovine glial fibrillary acidic protein (GFAP), the astroglial IF, was studied in vitro with fluorescently labeled GFAP. Soluble GFAP in low ionic strength buffer was rapidly and fully phosphorylated to be used as phosphorylated GFAP. Assembly of GFAP was observed to be inhibited in proportion to the extent of phosphorylation by mixing phosphorylated and non-phosphorylated GFAP at various ratios, and phosphorylated GFAP could not be assembled with non-phosphorylated GFAP into filaments at all. Furthermore, the subunit exchange was suppressed in proportion to the extent of phosphorylation. Phosphorylation affects the dynamic equilibrium of GFAP, and contributes to breaking down GFAP frameworks in mitotic glial cells. PMID:8907324

Nakamura, Y; Takeda, M; Nishimura, T



Phosphorylation of bovine interphotoreceptor retinoid-binding protein (IRBP)  

SciTech Connect

IRBP is the major soluble (glycolipo) protein of the interphotoreceptor matrix (IPM) and a putative intercellular retinoid-transport vehicle. The authors have now examined phosphorylation of proteins in a crude bovine IPM wash using ..gamma..-/sup 32/P-ATP. SDS-polyacrylamide gel electrophoresis (PAGE) of IPM proteins showed several phosphorylated protein bands, one of them migrating in the same position as purified IRBP. When an aliquot of phosphorylated IPM proteins was incubated overnight with /sup 3/H-retinol and subjected to either size-exclusion or ion-exchange HPLC, a peak of /sup 32/P was observed in both cases which coincided with /sup 3/H-retinol binding and had a retention time identical to that of purified IRBP. When phosphorylated IPM was subjected to Con A Sepharose affinity chromatography and the 50mM methyl ..cap alpha..-D-mannoside eluate chromatographed on ion-exchange HPLC, the /sup 32/P-peak was not present although a substantial amount of non-phosphorylated IRBP was recovered as assessed by SDS-PAGE and Western blotting. However, when the Con A Sepharose beads were dissolved in SDS and subjected to SDS-PAGE and Western blotting, a band of phosphorylated IRBP was observed, indicating that the phosphorylated IRBP was more tightly bound to the Con A Sepharose. The authors conclude that a fraction of IRBP can be phosphorylated by a yet to be characterized protein kinase and that the binding characteristics of IRBP are markedly altered by phosphorylation.

Wiggert, B.; Lee, L.; Chader, G.J.



Acute phase protein changes in calves during an outbreak of respiratory disease caused by bovine respiratory syncytial virus  

Microsoft Academic Search

Bovine acute phase proteins (APPs), lipopolysaccharide binding protein (LBP), serum amyloid A (SAA), haptoglobin (Hp) and alpha1-acid glycoprotein (AGP) were evaluated as inflammatory markers during an outbreak of bovine respiratory disease (BRD) caused by bovine respiratory syncytial virus (BRSV). Calves (n=10) presented mild to moderate signs of respiratory disease. Secondary bacterial infections, Pasteurella multocida and Mycoplasma dispar as major species,

Toomas Orro; Tarja Pohjanvirta; Ulla Rikula; Anita Huovilainen; Sakari Alasuutari; Liisa Sihvonen; Sinikka Pelkonen; Timo Soveri




Microsoft Academic Search

Summary The heat-induced gel strength in suspensions of two types of plasma protein isolates and egg albumen were compared to investigate the use of bovine blood plasma in food systems as a replacement for egg albumen and other proteins. A viscosity index, based on a counter-flow back-extrusion model, was used to measure gel strength. The optimum pH for gel formation

D. W. Hickson; C. W. Dill; R. G. Morgan


Differential stability of the bovine prion protein upon urea unfolding  

PubMed Central

Prion diseases, or transmissible spongiform encephalopathies, are a group of infectious neurological diseases associated with the structural conversion of an endogenous protein (PrP) in the central nervous system. There are two major forms of this protein: the native and noninfectious cellular form, PrPC; and the misfolded, infectious, and proteinase K-resistant form, PrPSc. The C-terminal domain of PrPC is mainly ?-helical in structure, whereas PrPSc in known to aggregate into an assembly of ?-sheets, forming amyloid fibrils. To identify the regions of PrPC potentially involved in the initial steps of the conversion to the infectious conformation, we have used high-resolution NMR spectroscopy to characterize the stability and structure of bovine recombinant PrPC (residues 121 to 230) during unfolding with the denaturant urea. Analysis of the 800 MHz 1H NMR spectra reveals region-specific information about the structural changes occurring upon unfolding. Our data suggest that the dissociation of the native ?-sheet of PrPC is a primary step in the urea-induced unfolding process, while strong hydrophobic interactions between helices ?1 and ?3, and between ?2 and ?3, stabilize these regions even at very high concentrations of urea.

Julien, Olivier; Chatterjee, Subhrangsu; Thiessen, Angela; Graether, Steffen P; Sykes, Brian D



Adenosine 3':5'-cyclic monophosphate-binding proteins in bovine and rat tissues.  

PubMed Central

1. At least two classes of high-affinity cyclic AMP-binding proteins have been identified: those derived from cyclic AMP-dependent protein kinases (regulatory subunits) and those that bind a wide range of adenine analogues (adenine analogue-binding proteins). 2. In fresh-tissue extracts, regulatory subunits could be further subdivided into 'type I or 'type II' depending on whether they were derived from 'type I' or 'type II' protein kinase [see Corbin et al. (1975) J. Biol. Chem. 250, 218-225]. 3. The adenine analogue-binding protein was detected in crude tissue supernatant fractions of bovine and rat liver. It differed from the regulatory subunit of cyclic AMP-dependent protein kinase in many of its properties. Under the conditions of assay used, the protein accounted for about 45% of the binding of cyclic AMP to bovine liver supernatants. 4. The adenine analogue-binding protein from bovine liver was partially purified by DEAE-cellulose and Sepharose 6B chromatography. It had mol.wt. 185000 and was trypsin-sensitive. As shown by competition and direct binding experiments, it bound adenosine and AMP in addition to cyclic AMP. At intracellular concentrations of adenine nucleotides, binding of cyclic AMP was essentially completely inhibited in vitro. Adenosine binding was inhibited by only 30% under similar conditions. 5. Rat tissues were examined for the presence of the adenine analogue-binding protein, and, of those examined (adipose tissue, heart, brain, testis, kidney and liver), significant amounts were only found in the liver. The possible physiological role of the adenine analogue-binding protein is discussed. 6. Because the adenine analogue-binding protein or other cyclic AMP-binding proteins in tissues may be products of partial proteolysis of the regulatory subunit of cyclic AMP-dependent protein kinase, the effects of trypsin and aging on partially purified protein kinase and its regulatory subunit from bovine liver were investigated. In all studies, the effects of trypsin and aging were similar. 7. In fresh preparations, the cyclic AMP-dependent protein kinase had mol.wt. 150000. Trypsin treatment converted it into a form of mol.wt 79500. 8. The regulatory subunit of the protein kinase had mol.wt. 87000. It would reassociate with and inhibit the catalytic subunit of the enzyme. Trypsin treatment of the regulatory subunit produced a species of mol.wt. 35500 which bound cyclic AMP but did not reassociate with the catalytic subunit. Trypsin treatment of the protein kinase and dissociation of the product by cyclic AMP produced a regulatory subunit of mol.wt. 46500 which reassociated with the catalytic subunit. 9. These results may be explained by at least two trypsin-sensitive sites on the regulatory subunit. A model for the effects of trypsin is described.

Sugden, P H; Corbin, J D



Identification of highly active flocculant proteins in bovine blood  

Technology Transfer Automated Retrieval System (TEKTRAN)

Bovine blood is an excellent flocculating agent, faster acting and as effective on a mass basis as polyacrylamide, the most widely utilized polymeric flocculant. To determine the molecular basis of flocculation activity, whole bovine blood (BB) and BB plasma were fractionated by size exclusion chro...


Application of recombinant bovine viral diarrhea virus proteins in the diagnosis of bovine viral diarrhea infection in cattle  

Microsoft Academic Search

The National Animal Disease Laboratory (NADL) vaccine strain of bovine viral diarrhea virus (BVDV) genes for gp48 and p80 were expressed in Escherichia coli. The BVDV-NADL gene for gp62 was integrated into a baculovirus genome for expression in Spodoptera frugiperda (Sf-9) insect ovarian cells. The antigenicity of baculovirus expressed BVDV protein was detected by anti-BVDV specific antibodies in an enzyme-linked

J. R. Reddy; J. Kwang; Ogi Okwumabua; S. Kapil; T. M. Loughin; K. F. Lechtenberg; M. M. Chengappa; H. C. Minocha



Kinetic characterization of hydrolysis of camel and bovine milk proteins by pancreatic enzymes  

Microsoft Academic Search

As differences in protein composition between camel and bovine milk may influence their digestibility, hydrolysis of milk proteins of both species using two pancreatic serine proteases (trypsin and chymotrypsin) was studied. Caseins (CNs) were more rapidly hydrolyzed than whey proteins (WPs) because of their greater flexibility and open structures. The extent of hydrolysis by chymotrypsin of CNs in both species

Maryam Salami; Reza Yousefi; Mohammad Reza Ehsani; Michèle Dalgalarrondo; Jean-Marc Chobert; Thomas Haertlé; Seyed Hadi Razavi; Ali Akbar Saboury; Amir Niasari-Naslaji; Ali Akbar Moosavi-Movahedi



Effect of gamma-irradiation on the physicochemical properties of porcine and bovine blood plasma proteins  

Microsoft Academic Search

To elucidate the effect of ?-irradiation on the physicochemical properties of blood plasma proteins, bovine and porcine blood, released from a slaughterhouse, was collected and plasma proteins were prepared. Physicochemical properties of blood plasma protein powders and solutions, such as molecular weight distribution, secondary structure, solubility, and viscosity, were examined after ?-irradiation at 1, 5, 7, and 10 kGy. Oxygen

Seunghyun Lee; Seunghwan Lee; Kyung Bin Song



Comparative study on heat stability and functionality of camel and bovine milk whey proteins.  


Heat stability, emulsifying, and foaming properties of camel whey have been investigated and compared with that of bovine whey. Camel whey is similar to bovine whey in composition, but is deficient in beta-lactoglubulin (beta-LG), a major component of bovine whey. Whether the deficiency in beta-LG will affect stability and functional properties is not yet known. Substantial information on the functional properties of bovine milk whey proteins is available; however, there is little research done on functional properties of camel whey proteins. Therefore, the objective of this study was to investigate the heat stability, emulsifying, and foaming characteristics of camel whey proteins. Calorimetric studies showed no significant difference in heat stability between bovine and camel whey proteins in liquid form. Upon drying, thermograms indicated that the 2 proteins are different in composition and thermal stability. The difference is represented in the absence of beta-LG and the occurrence of protein denaturation peak at a lesser temperature in camel whey. The first marginal thermal transition in bovine whey appeared at 81 degrees C, followed by 2 other transitions at 146 and 198 degrees C. For camel whey, the transitions appeared at 139, 180, and 207 degrees C respectively. The first marginal denaturation peak in bovine whey is due to beta-LG, which is essentially absent in camel whey, while the second peak is due to the mixture of alpha-lactalbumin, serum albumin, and possibly part of the partially stabilized beta-LG structure during the denaturation process. Because camel whey is deficient in beta-LG, the denaturation peak at 139 must be due to the mixture of alpha-lactalbumin and camel serum albumin. In both proteins, the highest thermal transition is due to sugars such as lactose. The solubility study has shown that camel whey is more sensitive to pH than bovine milk whey and that heat stability is lowest near the isoelectric point of the proteins at pH 4.5. The sensitivity to pH resulted in partial denaturation and increased tendency to aggregate, which caused poor and unstable emulsion at pH 5. Both bovine and camel whey proteins have demonstrated good foaming properties; however, the magnitudes of these properties were considerably greater in bovine milk for all of the conditions studied. PMID:19038927

Laleye, L C; Jobe, B; Wasesa, A A H



Diacylglycerol-activated, calcium/phospholipid-dependent protein kinase (protein kinase C) activity in bovine thyroid.  


Bovine thyroid 100,000 X g supernatant contained diacylglycerol-activated, calcium/phospholipid-dependent protein kinase (protein kinase C). The protein kinase C was partially purified using ion-exchange chromatography and characterized. Substrate specificity studies revealed that the enzyme was most active when histone F1 was used as substrate. The thyroid protein kinase C was not stimulated by Ca2+ or phosphatidylserine (PS), but was stimulated by the combination of the two by 570%. Diolein stimulated the kinase by increasing its sensitivity to Ca2+. Other phospholipids could not substitute for PS and were ineffective in stimulating the protein kinase C in the absence of diolein. However, in the presence of diolein some of the other phospholipids were stimulatory albeit not to the extent of PS. Quercitin, a protein kinase C inhibitor in other systems, inhibited the thyroid enzyme in a dose-related manner. Protein kinase C could also be demonstrated using endogenous thyroid proteins as substrate. Separation of these 32P-labelled proteins by electrophoresis and subsequent autoradiography revealed that three proteins were phosphorylated by the protein kinase C of approximate molecular weights 60,000, 45,000, and less than 29,000. These results offer a possible mechanism by which Ca2+ and/or diacylglycerol effects may be mediated in thyroid. PMID:3161511

Friedman, Y; Poleck, T; Henricks, L; Burke, G



Conditioned medium from irradiated bovine pulmonary artery endothelial cells stimulates increased protein synthesis by irradiated bovine lung fibroblasts in vitro  

SciTech Connect

Pulmonary fibrosis, a potentially fatal consequence of radiation exposure, occurs by unknown mechanisms. The hypothesis that endothelial cells, injured by radiation, could alter the biochemical function of lung fibroblasts, was tested by exposing cultures of bovine pulmonary artery endothelial cells to 0 or 5 Gy radiation and then incubating them in fresh medium for 48 h. This endothelial cell conditioned medium (ECCM) was then applied to irradiated or nonirradiated cultures of bovine lung fibroblasts. Forty-eight hours later the fibroblasts were analyzed for their ability to synthesize DNA and protein. The ECCM from injured cells stimulated fibroblast protein synthesis twofold to threefold in irradiated fibroblasts without increasing DNA synthesis. It also stimulated a significant but less marked increase in protein synthesis in nonirradiated fibroblasts. Two-dimensional gel electrophoresis revealed this increased synthesis to be expressed in less than 10% of the 1100 separable fibroblast proteins. This study shows that endothelial cells injured by radiation produce factors that stimulate injured fibroblasts to markedly increase their synthesis of certain intracellular proteins, while not stimulating fibroblast replication.

Flavin, M.P.; Parton, L.A.; Bowman, C.M. (Childrens Hospital of Los Angeles, CA (USA))



Purification and characterization of an isoform of protein kinase C from bovine neutrophils  

SciTech Connect

Protein kinase C (PKC) from bovine neutrophils was purified 1,420-fold. Subcellular fractionation analysis of bovine neutrophil homogenate in the presence of EGTA indicated that more than 95% of the PKC activity was present in the soluble fraction. Whereas bovine brain PKC could be resolved into four isoenzymatic forms by chromatography on a hydroxylapatite column, bovine neutrophil PKC was eluted in a single peak, suggesting that it corresponded to a single isoform. The apparent molecular weight of bovine neutrophil PKC was 82,000, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bovine neutrophil PKC was autophosphorylated in the presence of ({gamma}-{sup 32}P)ATP, provided that the medium was supplemented with Mg{sup 2+}, Ca{sup 2+}, phosphatidylserine, and diacylglycerol; phorbol myristate acetate could substitute for diacylglycerol. Autophosphorylated PKC could be cleaved by trypsin to generate two radiolabeled peptides of M{sub r} 48,000 and 39,000. The labeled amino acids were serine and threonine. During the course of the purification procedure of bovine neutrophil PKC, a protein of M{sub r} 23,000 was found to exhibit a strong propensity to PKC-dependent phosphorylation in the presence of ({gamma}-{sup 32}P)ATP, Mg{sup 2+}, Ca{sup 2+}, phosphatidylserine, and diacylglycerol. This protein was recovered together with PKC in one of the two active peaks eluted from the Mono Q column at the second step of PKC purification. It is suggested that the M{sub r} 23,000 protein might be a natural substrate for bovine neutrophil PKC.

Dianoux, A.C.; Stasia, M.J.; Vignais, P.V. (Centre d'Etudes Nucleaires, Grenoble (France))



Detection of Bovine Spongiform Encephalopathy-Related Prion Protein Gene Promoter Polymorphisms in Local Turkish Cattle  

Microsoft Academic Search

Polymorphisms in open reading frames of the prion protein gene (PRNP) have been shown to be associated with prion disease susceptibility in humans, sheep, and mice. Studies in recent years have\\u000a demonstrated a similar effect of PRNP promoter and intron-1 polymorphisms on bovine spongiform encephalopathy (BSE) susceptibility in cattle. In this study, the\\u000a deletion\\/insertion (indel) polymorphisms of the bovine PRNP

Cemal Ün; Kemal Oztabak; Nehir Özdemir; Dawit Tesfaye; Ahmet Mengi; Karl Schellander



Proteomics-Based Systems Biology Modeling of Bovine Germinal Vesicle Stage Oocyte and Cumulus Cell Interaction  

PubMed Central

Background Oocytes are the female gametes which establish the program of life after fertilization. Interactions between oocyte and the surrounding cumulus cells at germinal vesicle (GV) stage are considered essential for proper maturation or ‘programming’ of oocytes, which is crucial for normal fertilization and embryonic development. However, despite its importance, little is known about the molecular events and pathways involved in this bidirectional communication. Methodology/Principal Findings We used differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT) on bovine GV oocyte and cumulus cells and identified 811 and 1247 proteins in GV oocyte and cumulus cells, respectively; 371 proteins were significantly differentially expressed between each cell type. Systems biology modeling, which included Gene Ontology (GO) and canonical genetic pathway analysis, showed that cumulus cells have higher expression of proteins involved in cell communication, generation of precursor metabolites and energy, as well as transport than GV oocytes. Our data also suggests a hypothesis that oocytes may depend on the presence of cumulus cells to generate specific cellular signals to coordinate their growth and maturation. Conclusions/Significance Systems biology modeling of bovine oocytes and cumulus cells in the context of GO and protein interaction networks identified the signaling pathways associated with the proteins involved in cell-to-cell signaling biological process that may have implications in oocyte competence and maturation. This first comprehensive systems biology modeling of bovine oocytes and cumulus cell proteomes not only provides a foundation for signaling and cell physiology at the GV stage of oocyte development, but are also valuable for comparative studies of other stages of oocyte development at the molecular level.

Peddinti, Divyaswetha; Memili, Erdogan; Burgess, Shane C.



The proliferation-associated nuclear protein Ki-67 in the bovine system: partial characterisation and its application for determination of the proliferation of Theileria-infected bovine cells.  


Theileria annulata-infected bovine cells as well as mitogen-stimulated bovine peripheral blood mononuclear cells (PBMC) express a proliferation-associated nuclear protein equivalent to the human Ki-67 protein. In analogy to the human system, the expression of the bovine Ki-67 protein is restricted to proliferating cells only, since (a) Ki-67 expression paralleled [3H]-thymidine incorporation in concanavalin A (Con A)-stimulated bovine PBMC, (b) Ki-67 was not detectable in quiescent bovine cells, and (c) Ki-67 expression in Theileria-infected cells is related to the presence of the parasites within the cytoplasm of the host cells; upon treatment with the theilericidal drug buparvaquone the parasites are destroyed and the cells cease to proliferate and to express the Ki-67 protein. Western-blot analysis of lysates of proliferating bovine cells revealed that the prototype monoclonal antibody Ki-67 and the new equivalent antibody MIB-1 detected one prominent protein band with an apparent molecular weight of 430 kDa. Two cDNA clones (pUC18.B1.Ki-67 and pUC18.B2.Ki-67) were isolated from a lambdagt11 cDNA library of T. annulata-infected bovine cells by immunoscreening with the monoclonal antibody MIB-1. Comparison of these cDNA sequences with those of the human Ki-67 protein revealed 60-70% identity. Within the "Ki-67 motif", identity proved to be 80% at the amino acid level. The remarkable identity between bovine and human Ki-67 proteins suggests that MIB-1 can be used as a marker for cell proliferation in animal research. In this context we could identify proliferating cells in lymph nodes of Theileria-infected animals and, furthermore, we could distinguish between infected and uninfected proliferating cells using MIB-1 and an antiserum against a recombinant parasite protein designated SA288. PMID:10431723

Shayan, P; Gerlach, C; Hügel, F U; Kay, G; Campbell, J D; Gerdes, J; Ahmed, J S



Bovine mammary explant versus primary cell cultures: Effect of bovine somatotropin and insulinlike growth factor-I on DNA content and protein synthesis  

Microsoft Academic Search

Summary  Cellular DNA, milk protein content, and protein secretion by bovine mammary explants were compared to cultures of confluent\\u000a and growing primary bovine mammary secretory cells over 4 d. Explants were obtained at slaughter from eight Holstein cows\\u000a (120 ± 35 d lactation). Primary cells were grown to confluence, cryopreserved, thawed, and cultured through five passages.\\u000a Explants and cells were cocultured

J. E. Keys; E. Cifrian; A. J. Guidry; H. M. Farrell



THz spectroscopy and molecular modeling of bovine serum albumin under various hydration conditions  

NASA Astrophysics Data System (ADS)

Bovine serum albumin (BSA) is the most abundant protein in bovine plasma; its three dimensional structure is yet unknown. We investigated the structure and dynamics of BSA in lyophilized samples, in 10% w/w and 50% w/w BSA aqueous solutions using THz spectroscopy and molecular modeling. THz spectra were recorded with a spectral resolution of 7.4 GHz. Theoretical spectra were simulated using a structural model of BSA based on the homology with the known structure of human serum albumin (HSA). The agreement between simulated THz spectra and THz spectra recorded experimentally allowed us to validate the BSA model and the solution models. Based on these models we investigated the flexibility of dry BSA and of BSA with one hydration layer. The hydrated structure of BSA is less flexible than the structure free of water molecules, except for residues 54 - 104 that are more mobile in the hydrated structure. We also investigated the fluctuations of the water molecules within the first hydration layer and identified two groups of water molecules: one that exhibits small fluctuations and one of highly mobile water molecules. These molecules are associated to highly mobile regions from the proteins and move in positive correlation with the neighboring protein regions. We also propose a BSA dimerization model in which the molecules strongly interact. The fluctuations of the BSA monomers and of their first hydration layer were investigated. The two molecules display similar fluctuation patterns, but one of them is slightly more flexible.

Mernea, Maria; Calborean, Octavian; Petrescu, Livia; Zatreanu, Diana; Sandu, Oana; Dascalu, Traian; Mihailescu, Dan Florin



Simulation of urea-induced protein unfolding: a lesson from bovine ?-lactoglobulin.  


To investigate the molecular mechanisms involved in the very initial stages of protein unfolding, we carried out one long (1 ?s) simulation of bovine ?-lactoglobulin (BLG) together with three (500 ns) supporting MD runs, in which the unfolding conditions were produced by adding the osmolyte urea to the simulated systems and/or by increasing the thermal energy raising the temperature from 300 to 350 K. BLG was chosen, since it is a well-characterized model protein, for which structural and folding properties have been widely investigated by X-ray and NMR. MD trajectories were analyzed not only in terms of standard progress variables, such as backbone H-bonds, gyration radius width, secondary structure elements, but also through the scrutiny of interactions and dynamical behavior of specific key residues previously pointed out and investigated by NMR and belonging to a well known hydrophobic cluster. MD trajectories simulated in different unfolding conditions suggest that urea destabilizes BLG structure weakening protein::protein hydrophobic interactions and the hydrogen bond network. The early unfolding events, better observed at higher temperature, affect both secondary and tertiary structure of the protein. PMID:21724434

Eberini, Ivano; Emerson, Andrew; Sensi, Cristina; Ragona, Laura; Ricchiuto, Piero; Pedretti, Alessandro; Gianazza, Elisabetta; Tramontano, Anna



Development and Application of Bovine and Porcine Oligonucleotide Arrays with Protein-Based Annotation  

PubMed Central

The design of oligonucleotide sequences for the detection of gene expression in species with disparate volumes of genome and EST sequence information has been broadly studied. However, a congruous strategy has yet to emerge to allow the design of sensitive and specific gene expression detection probes. This study explores the use of a phylogenomic approach to align transcribed sequences to vertebrate protein sequences for the detection of gene families to design genomewide 70-mer oligonucleotide probe sequences for bovine and porcine. The bovine array contains 23,580 probes that target the transcripts of 16,341 genes, about 72% of the total number of bovine genes. The porcine array contains 19,980 probes targeting 15,204 genes, about 76% of the genes in the Ensembl annotation of the pig genome. An initial experiment using the bovine array demonstrates the specificity and sensitivity of the array.

Garbe, John R.; Elsik, Christine G.; Antoniou, Eric; Reecy, James M.; Clark, Karl J.; Venkatraman, Anand; Kim, Jae-Woo; Schnabel, Robert D.; Michael Dickens, C.; Wolfinger, Russell D.; Fahrenkrug, Scott C.; Taylor, Jeremy F.



Comparison of the principal proteins in bovine, caprine, buffalo, equine and camel milk.  


Proteomic analysis of bovine, caprine, buffalo, equine and camel milk highlighted significant interspecies differences. Camel milk was found to be devoid of ?-lactoglobulin, whereas ?-lactoglobulin was the major whey protein in bovine, buffalo, caprine, and equine milk. Five different isoforms of ?-casein were found in camel milk, analogous to the micro-heterogeneity observed for bovine ?-casein. Several spots observed in 2D-electrophoretograms of milk of all species could tentatively be identified as polypeptides arising from the enzymatic hydrolysis of caseins. The understanding gained from the proteomic comparison of these milks may be of relevance both in terms of identifying sources of hypoallergenic alternatives to bovine milk and detection of adulteration of milk samples and products. PMID:22365180

Hinz, Katharina; O'Connor, Paula M; Huppertz, Thom; Ross, R Paul; Kelly, Alan L



Initial stage of cheese production: a molecular modeling study of bovine and camel chymosin complexed with peptides from the chymosin-sensitive region of ?-casein.  


Bovine chymosin has long been the preferred enzyme used to coagulate cow's milk, in the initial stage of cheese production, during which it cleaves a specific bond in the milk protein ?-casein. Recently, camel chymosin has been shown to have a 70% higher clotting activity toward cow's milk and, moreover, to cleave ?-casein more selectively. Bovine chymosin, on the other hand, is a poor clotting agent toward camel's milk. This paper reports a molecular modeling study aimed at understanding this disparity, based on homology modeling and molecular dynamics simulations using peptide fragments of ?-casein from cow and camel in both bovine and camel chymosin. The results show that the complex between bovine chymosin and the fragment of camel ?-casein is indeed less stable in the binding pocket. The results also indicate that this in part may be due to charge repulsion between a lysine residue in bovine chymosin and an arginine residue in the P4 position of camel ?-casein. PMID:21476511

Sørensen, Jesper; Palmer, David S; Qvist, Karsten Bruun; Schiøtt, Birgit



Stabilizing effect of saccharides on bovine plasma protein: a calorimetric study.  


Bovine plasma proteins provide the needed amino acids for the growth and development of an organism. With the purpose of preserving the native structure, related with the protein functional properties, the oligosaccharide inulin was used as protective agent and was compared with glucose and sucrose, during freeze-drying. In the present study, the thermal stability of protein was investigated as a function of type of saccharide in a concentration range of 5-15% (w/v), and at different pHs. The effect of these variables on phase transition, thermal stability and miscibility was assessed by differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). The results of thermal protein properties (denaturation temperature and enthalpy), demonstrated that endothermic transition shifted to higher temperatures, being the stabilizing effect: inulin>glucose>sucrose. The thermal behavior suggests compatibility or interactions between the components of blends. In this way, the micrographs showed a homogeneous distribution of the different phases, corroborating the miscibility in the matrix. The unfolding process was irreversible and could be adequately described by a two-state model. PMID:22445165

Rodriguez Furlán, Laura T; Lecot, Javier; Pérez Padilla, Antonio; Campderrós, Mercedes E; Zaritzky, Noemi



Isolation of Histone-Like Proteins from Mitochondria of Bovine Heart  

Microsoft Academic Search

Two methods for isolating and purifying histone-like proteins from mitochondria of bovine heart are described. In the first, a sonicated extract of the mitochondria was fractionated in three chromatography steps, including affinity chromatography on DNA-cellulose, to purify a protein that resembles very closely the histone-like protein (HM) of yeast mitochondria. In the second method, an acid extract of the heart

E. W. Yamada; H. Dotzlaw; N. J. Huzel



Calmodulinwdependent protein kinase II from bovine cardiac muscle: purification and differential activation by calcium  

Microsoft Academic Search

Calmodulin-dependent protein kinase II was purified to apparent homogeneity with a high yield from the total calmodulin-binding protein fraction of bovine cardiac muscle in a single step by gel filtration column chromatography. This procedure is simple and suitable for adaptation to large scale preparations. The purified calmodulin-dependent protein kinase has a specific enzymic activity of 2.4 ?mol\\/min\\/mg when mixed histone

R. Kakkar; R. V. S Raju; R. K. Sharma



Pulsed Electric Field Induced Aggregation of Food Proteins: Ovalbumin and Bovine Serum Albumin  

Microsoft Academic Search

Ovalbumin (OVA), bovine serum albumin (BSA), and a mixture of the two proteins (OVA?+?BSA) in solution were exposed to pulsed\\u000a electric field (PEF) to investigate the protein interaction and aggregation. The results demonstrated the self-aggregation\\u000a of OVA through disulfide bond due to the exposure of sulfhydryl groups and intermolecular disulfide interactions when PEF\\u000a intensity exceeded 25 kV cm?1. However, no protein self-aggregation

Wei Zhao; Ruijin Yang


Bovine Pericardium Buttress Reinforces Colorectal Anastomoses in a Canine Model  

Microsoft Academic Search

\\u000a Purpose  The consequences of an anastomotic leak or disruption can be devastating, particularly in the colorectal surgery population.\\u000a The purpose of this study was to evaluate and compare colon anastomoses with or without a collagen matrix buttress derived\\u000a from bovine pericardium.\\u000a \\u000a \\u000a \\u000a Methods  A circular stapler was used to create colon-colon anastomoses in a canine model. Twenty animals underwent two anastomoses\\u000a each: one

Gonzalo F. Hagerman; Wolfgang B. Gaertner; George R. Ruth; Michael L. Potter; Richard E. Karulf



Genetic and antigenic analysis of the G attachment protein of bovine respiratory syncytial virus strains  

Microsoft Academic Search

Antigenic and genetic studies of bovine respiratory syncytial virus (BRSV) were made on isolates ob- tained from three continents over 27 years. Anti- genic variation between eight isolates was initially determined using protein G-specific monoclonal antibodies. Four distinct reaction patterns were observed, two of which corresponded to the pre- viously established subgroups A and AB. A third pattern was produced

M. Elvander; C. Baule; A. Ballagi-Porda; S. Bela


S-100 protein subunits in bovine oviduct epithelium: In situ distribution and changes during primary cell culture  

Microsoft Academic Search

Summary  Cultures of bovine oviduct epithelial cells are widely used in co-culture systems to improve the results ofin vitro fertilization. The aim of the present study was to evaluate the suitability of S-100 protein as a differentiation marker\\u000a for bovine oviduct epithelial cellsin vitro. The distribution of S-100? and S-100? was examined immunohistochemically in bovine oviduct epitheliumin situ and in primary

I. Walter; I. Miller



A bovine acellular scaffold for vocal fold reconstruction in a rat model #  

PubMed Central

With a rat model of vocal fold injury, this study examined the in vivo host response to an acellular xenogeneic scaffold derived from the bovine vocal fold lamina propria, and the potential of the scaffold for constructive tissue remodeling. Bilateral wounds were created in the posterior vocal folds of 20 rats, and bovine acellular scaffolds were implanted into the wounds unilaterally, with the contralateral vocal folds as control. The rats were humanely sacrificed after 3 days, 7 days, 1 month, and 3 months, and the coronal sections of their larynges were examined histologically. Expressions of key matrix proteins including collagen I, collagen III, elastin, fibronectin, hyaluronic acid, and glycosaminoglycans were quantified with digital image analysis. Significant infiltration of host inflammatory cells and host fibroblasts in the scaffold implant was observed in the acute stage of wound repair (3 days and 7 days post-surgery). The mean relative densities of collagen I, collagen III, and glycosaminoglycans in the implanted vocal folds were significantly higher than those in the control after 3 days, followed by gradual decreases over 3 months. Histological results showed that the scaffolds were apparently degraded by 3 months, with no fibrotic tissue formation or calcification. These preliminary findings suggested that the bovine acellular scaffold could be a potential xenograft for vocal fold regeneration.

Xu, Chet C.; Chan, Roger W.; Weinberger, Debra G.; Efune, Guy; Pawlowski, Karen S.



A unique property of fetal bovine serum: high levels of protein-glutathione mixed disulfides.  


Fetal bovine serum has been reported to delay or inhibit "spontaneous" neoplastic transformation in vitro as compared with all other sera tested. The present results indicate that fetal bovine serum is also unique in containing high levels of protein-glutathione mixed disulfides (3 to 7 microng glutathione as mixed disulfide per ml serum). The level of mixed disulfide appears to vary in accordance with the period of gestation of the fetal calves used to prepare the serum, decreasing below detectable levels (less than 0.2 microng per ml) with near-term fetal calves. Calf, adult bovine, fetal horse, and swine sera did not contain detectable levels of this type of mixed disulfide. PMID:67079

Bump, E A; Reed, D J



A 23-kDa protein as a substrate for protein kinase C in bovine neutrophils. Purification and partial characterization  

SciTech Connect

In {sup 32}P{sub i}-loaded bovine neutrophils stimulated with phorbol myristate acetate (PMA), radioactivity was preferentially incorporated into a protein of low molecular mass, suggesting a PKC-dependent phosphorylation. This protein, termed 23-kDa protein, was predominantly localized in the cytosol. The apparent molecular mass of the purified protein range between 20 and 23 kDa. In the absence of mercaptoethanol, a dimer accumulated. Homogeneity of the 23-kDa protein was verified by 2D-PAGE analysis. Gel isoelectric focusing (IEF) of the purified 23-kDa protein followed by Coomassie blue staining allowed the visualization of our discrete protein bands with isoelectric points ranging between pH 6.3 and 6.7. Phosphorylation of the 23-kDa protein by ({gamma}-{sup 32}P)ATP in the presence of bovine neutrophil PKC supplemented with Ca{sup 2+}, phosphatidylserine, and diacylglycerol or with PMA occurred on serine and required the presence of mercaptoethanol. IEF of the {sup 32}P-labeled 23-kDa protein followed by autoradiography revealed for discrete bands with distinct isoelectric points similar to those of the bands stained by Coomassie blue after IEF on nonlabeled 23-kDa protein. The bands of the 23-kDa protein resolved by IEF and transfered to nitrocellulose showed ability to bind ({sup 35}S)GTP-{gamma}-S. The immunoreactivity of antibodies raised in rabbits against the bovine neutrophil 23-kDa protein was demonstrated on immunoblots after SDS-PAGE. The 23-kDa protein differed also from several other proteins of similar molecular mass that have been identified in neutrophils, namely, calmodulin, the small subunit of the low-potential cytochrome b, and a low molecular weight protein which is ADP-ribosylated by the botulinum toxin.

Stasia, M.J.; Dianoux, A.C.; Vignais, P.V. (Centre d'Etudes Nucleaires (France))



Sweet-Sensitive Protein from Bovine Taste Buds: Isolation and Assay  

Microsoft Academic Search

Using refractometry and ultraviolet-difference spectroscopy to indicate interaction between proteins and compounds of low molecular weight, we found a protein fraction in bovine tongue extracts that complexes sugars and saccharin. The strengths of the complexes parallel the degrees of sweetness of the compounds, and the effects of pH upon formation of complexes parallel the effects of pH upon sensitivity of

Frank R. Dastoli; Steven Price



Utilization of bovine blood plasma proteins for the production of angiotensin I converting enzyme inhibitory peptides  

Microsoft Academic Search

Hydrolysates of whole bovine plasma and its separated proteins, albumin and globulins, which inhibit the angiotensin I converting enzyme (ACE) were prepared by enzymic hydrolysis with several proteases available for industrial use. Alcalase produced ACE inhibitory peptides from plasma proteins most efficiently and the Alcalase hydrolysate of albumin showed the most high activity (IC50=0.56 mg\\/ml). Sequential ultrafiltration of the hydrolysate

Chang-Kee Hyun; Heuyn-Kil Shin



Responses of Bovine WC1+   T Cells to Protein and Nonprotein Antigens of Mycobacterium bovis  

Microsoft Academic Search

WC1 T cells of Mycobacterium bovis-infected cattle are highly responsive to M. bovis sonic extract (MBSE). In mycobacterial infections of other species, T cells have been shown to respond to protein and nonprotein antigens, but the bovine WC1 T-cell antigenic targets within MBSE require further definition in terms of the dominance of protein versus nonprotein components. The present study sought

Michael D. Welsh; Hilary E. Kennedy; Allister J. Smyth; R. Martyn Girvin; Peter Andersen; John M. Pollock



Multiple, Distinct Forms of Bovine and Human Protein Kinase C Suggest Diversity in Cellular Signaling Pathways  

Microsoft Academic Search

A new family of protein kinase C-related genes has been identified in bovine, human, and rat genomes. The alpha-, beta-, and gamma-type protein kinase sequences are highly homologous, include a kinase domain, and potential calcium-binding sites, and they contain interspersed variable regions. The corresponding genes are located on distinct human chromosomes; the possibility of even greater genetic complexity of this

Lisa Coussens; Peter J. Parker; Lucy Rhee; Teresa L. Yang-Feng; Ellson Chen; Michael D. Waterfield; Uta Francke; Axel Ullrich



Bovine Parvovirus DNA-binding Proteins: Identification by a Combined DNA Hybridization and Immunodetection Assay  

Microsoft Academic Search

SUMMARY We have investigated the interaction between bovine parvovirus (BPV) capsid and non-capsid proteins and restriction fragments of the BPV genome by a combined DNA hybridization and immunodetection assay. 3zp-labelled DNA was bound to nitrocellulose membranes bearing lysates of mock-infected and virus-infected cells whose proteins had been separated by SDS-polyacrylamide gel electrophoresis. The position of bound DNA was determined by




[In vitro fertilization of bovine oocytes in in-vitro protein-free culture system].  


We studied the possibility of fertilization of bovine oocyte-cumulus complexes, matured in vitro in a protein-free medium, in a protein-free culture system without preliminary capacitation of spermatozoa. The development of embryos to the morula-blastocyst and blastocyst stage was considered as a criterion of successful fertilization. It was shown that replacement of bovine serum albumin for polyvinyl alcohol or polyvinylpyrrolidone in Tyrode medium for fertilization did not affect significantly the development to the morula-blastocyst stage and the number of cells in blastocysts. It was also found that replacement bovine serum albumin for polyvinyl alcohol in all used media, Tyrode medium for washing of oocytes, medium for sperm preparation to fertilization, and Tyrode medium for fertilization, did not affect significantly the development to the morula-blastocyst and blastocyst stages, as well as on the number of cells in blastocysts. The results obtained suggest that in vitro fertilization of bovine oocyte-cumulus complexes is possible in a protein-free culture system without significant reduction in the capacity for in vitro development of the obtained embryos and number of cells in blastocysts. PMID:17168379

Smetanina, I G; Tatarinova, L V; Krivokharchenko, A S


Characterization of two proteins of Staphylococcus aureus isolated from bovine clinical mastitis with homology to glyceraldehyde-3-phosphate dehydrogenase  

Microsoft Academic Search

Staphylococcus aureus is the most common causative agent of bovine mastitis and vaccines developed to control this disease showed limited protection due in part to the lack of common antigens among the mastitis isolates. We isolated and identified two genes encoding proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from a S. aureus strain isolated from bovine clinical mastitis. The GapB and

Noriko Goji; Andrew A. Potter; Jose Perez-Casal



Transcytosis of murine-adapted bovine spongiform encephalopathy agents in an in vitro bovine M cell model.  


Transmissible spongiform encephalopathies (TSE), including bovine spongiform encephalopathy (BSE), are fatal neurodegenerative disorders in humans and animals. BSE appears to have spread to cattle through the consumption of feed contaminated with BSE/scrapie agents. In the case of an oral infection, the agents have to cross the gut-epithelial barrier. We recently established a bovine intestinal epithelial cell line (BIE cells) that can differentiate into the M cell type in vitro after lymphocytic stimulation (K. Miyazawa, T. Hondo, T. Kanaya, S. Tanaka, I. Takakura, W. Itani, M. T. Rose, H. Kitazawa, T. Yamaguchi, and H. Aso, Histochem. Cell Biol. 133:125-134, 2010). In this study, we evaluated the role of M cells in the intestinal invasion of the murine-adapted BSE (mBSE) agent using our in vitro bovine intestinal epithelial model. We demonstrate here that M cell-differentiated BIE cells are able to transport the mBSE agent without inactivation at least 30-fold more efficiently than undifferentiated BIE cells in our in vitro model. As M cells in the follicle-associated epithelium are known to have a high ability to transport a variety of macromolecules, viruses, and bacteria from gut lumen to mucosal immune cells, our results indicate the possibility that bovine M cells are able to deliver agents of TSE, not just the mBSE agent. PMID:20861256

Miyazawa, Kohtaro; Kanaya, Takashi; Takakura, Ikuro; Tanaka, Sachi; Hondo, Tetsuya; Watanabe, Hitoshi; Rose, Michael T; Kitazawa, Haruki; Yamaguchi, Takahiro; Katamine, Shigeru; Nishida, Noriyuki; Aso, Hisashi



Transcytosis of Murine-Adapted Bovine Spongiform Encephalopathy Agents in an In Vitro Bovine M Cell Model? † #  

PubMed Central

Transmissible spongiform encephalopathies (TSE), including bovine spongiform encephalopathy (BSE), are fatal neurodegenerative disorders in humans and animals. BSE appears to have spread to cattle through the consumption of feed contaminated with BSE/scrapie agents. In the case of an oral infection, the agents have to cross the gut-epithelial barrier. We recently established a bovine intestinal epithelial cell line (BIE cells) that can differentiate into the M cell type in vitro after lymphocytic stimulation (K. Miyazawa, T. Hondo, T. Kanaya, S. Tanaka, I. Takakura, W. Itani, M. T. Rose, H. Kitazawa, T. Yamaguchi, and H. Aso, Histochem. Cell Biol. 133:125-134, 2010). In this study, we evaluated the role of M cells in the intestinal invasion of the murine-adapted BSE (mBSE) agent using our in vitro bovine intestinal epithelial model. We demonstrate here that M cell-differentiated BIE cells are able to transport the mBSE agent without inactivation at least 30-fold more efficiently than undifferentiated BIE cells in our in vitro model. As M cells in the follicle-associated epithelium are known to have a high ability to transport a variety of macromolecules, viruses, and bacteria from gut lumen to mucosal immune cells, our results indicate the possibility that bovine M cells are able to deliver agents of TSE, not just the mBSE agent.

Miyazawa, Kohtaro; Kanaya, Takashi; Takakura, Ikuro; Tanaka, Sachi; Hondo, Tetsuya; Watanabe, Hitoshi; Rose, Michael T.; Kitazawa, Haruki; Yamaguchi, Takahiro; Katamine, Shigeru; Nishida, Noriyuki; Aso, Hisashi



Central nervous system gene expression changes in a transgenic mouse model for bovine spongiform encephalopathy  

PubMed Central

Gene expression analysis has proven to be a very useful tool to gain knowledge of the factors involved in the pathogenesis of diseases, particularly in the initial or preclinical stages. With the aim of finding new data on the events occurring in the Central Nervous System in animals affected with Bovine Spongiform Encephalopathy, a comprehensive genome wide gene expression study was conducted at different time points of the disease on mice genetically modified to model the bovine species brain in terms of cellular prion protein. An accurate analysis of the information generated by microarray technique was the key point to assess the biological relevance of the data obtained in terms of Transmissible Spongiform Encephalopathy pathogenesis. Validation of the microarray technique was achieved by RT-PCR confirming the RNA change and immunohistochemistry techniques that verified that expression changes were translated into variable levels of protein for selected genes. Our study reveals changes in the expression of genes, some of them not previously associated with prion diseases, at early stages of the disease previous to the detection of the pathological prion protein, that might have a role in neuronal degeneration and several transcriptional changes showing an important imbalance in the Central Nervous System homeostasis in advanced stages of the disease. Genes whose expression is altered at early stages of the disease should be considered as possible therapeutic targets and potential disease markers in preclinical diagnostic tool development. Genes non-previously related to prion diseases should be taken into consideration for further investigations.



Microassay for the quantitation of protein precipitable polyphenols: use of bovine serum albumin–benzidine conjugate as a protein probe  

Microsoft Academic Search

A simple, sensitive and indirect spectrophotometric method for the determination of protein precipitable polyphenols (tannins) has been developed, based on the ability of the polyphenols to precipitate the synthetic, brown coloured azo-protein, bovine serum albumin–benzidine conjugate (BSA–benzidine, mole ratio 1:7), which shows an absorption maxima at 405nm. The amount of unprecipitated BSA–benzidine is measured directly at 405nm, which is inversely

C. V Ratnavathi; R. B Sashidhar



[Detection and isolation of glycoproteins and nuclear proteins in the bovine brain].  


Using ammonium sulphate precipitation, ion exchange chromatography and preparative isoelectrofocusing, 9 organ-specific glycoproteins and 16 specific nuclear proteins were isolated from bovine brain nervous tissue in a homogeneous state. The isolation of proteins was controlled by a solid phase immunoenzymatic analysis. The molecular weight, subunit composition and isoelectric points of the proteins were determined and their ability to interact with immobilized calf thymus DNA and concanavalin A was demonstrated. It was assumed that the multiplicity of specific proteins of brain tissue is a molecular basis which provides for the functional specificity of the nervous tissue at large. PMID:6525367

Mekhtiev, A A; Gruden', M A; Poletaev, A B



Mapping B-cell linear epitopes of NS3 protein of bovine viral diarrhea virus.  


Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus within the family Flaviviridae. NS3 is one of the immunodominance regions of the BVDV viral proteins. To identify the potential B-cell linear antigenic epitopes within BVDV NS3 region, serial overlapping truncations covering the whole region were expressed and purified, and screened by multistep of Western-blot. We found ((1)VCKKITEHERCHVNI(15)), ((20)AFFGVMPRGTTPRAPVR(36)), ((46)RRGLETGWAYTHQGGI(61)), ((281)EGDMATGITYASYGYFC(297)), ((426)YSGEDPANLRVVTSQSPYVVVATNAIESGV(455)) and ((481)FIVTGLKRMAVTVGEQA(497)) can be recognized by the BVDV infected bovine serum. These proteins have been confirmed by indirect enzyme-linked immunosorbent assay (I-ELISA). The results of this study might open new perspectives on the structure and antibody-antigen reaction of the non-structural proteins and may aid in the clinical application as well. PMID:23276751

Li, Yan; Jia, Ying; Wen, Kai; Liu, Hua; Gao, Mingchun; Ma, Bo; Zhang, Wenlong; Wang, Junwei



Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins  

SciTech Connect

Prostaglandin E/sub 2/ (PEG/sub 2/) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet. In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, the authors purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its ..cap alpha.. subunit from two known pertussis toxin substrate G-proteins (G/sub i/ and G/sub 0/) purified from bovine brain. The molecular weight of the ..cap alpha.. subunit was 40,000, which is between those of G/sub i/ and G/sub 0/. The purified protein was also distinguished immunologically from G/sub i/ and G/sub 0/ and was referred to as G/sub am/. Reconstitution of the PGE receptor with pure C/sub am/, G/sub i/, or G/sub 0/ in phospholipid vesicles resulted in a remarkable restoration of (/sup 3/H)PGE/sub 2/ binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. These results indicate that the PGE receptor can couple functionally with G/sub am/, G/sub i/, or G/sub 0/ in phospholipid vesicles and suggest that G/sub am/ may be involved in signal transduction of the PGE receptor in bovine adrenal medulla.

Negishi, M.; Ito, S.; Yokohama, H.; Hayashi, H.; Katada, T.; Ui, M.; Hayaishi, O.



N- and o-glycosylation of a commercial bovine whey protein product.  


Bovine whey protein products are used as a base ingredient in infant formulas to optimize the amino acid pattern to a more human-like composition. Although the protein composition of bovine milk has been studied in detail, glycosylation details of commercial whey protein products are missing. To this end, both the N- and O-glycans of such a protein concentrate were sequentially released, the N-glycans enzymatically and the O-glycans chemically (reducing and nonreducing conditions). For the structural analysis of the N- and O-glycans a combination of MALDI-TOF-MS, one-dimensional (1)H NMR spectroscopy, Wisteria floribunda agglutinin affinity chromatography, HPAEC-PAD profiling, and HPLC-FD profiling (2-aminobenzamide derivatives), together with exoglycosidase treatments, were used. A mixture of over 60 N-glycans and 10 O-glycans was characterized, giving a detailed insight into the glycosylation of a bovine whey protein product, Deminal 90, which is applied as an ingredient for infant formulas. PMID:23194161

van Leeuwen, Sander S; Schoemaker, Ruud J W; Timmer, Christel J A M; Kamerling, Johannis P; Dijkhuizen, Lubbert



Purification of homologous protein carboxyl methyltransferase isozymes from human and bovine erythrocytes  

SciTech Connect

The authors have purified the two major isozymes of the L-isoaspartyl/D-aspartyl protein methyltransferase from both human and bovine erythrocytes. These four enzymes all have polypeptide molecular weights of approximately 26,500 and appear to be monomers in solution. Each of these enzymes cross-reacts with antibodies directed against protein carboxyl methyltransferase I from bovine brain. Their structures also appear to be similar when analyzed by dodecyl sulfate gel electrophoresis for the large fragments produced by digestion with Staphylococcus aureus protease V8 or when analyzed by high-performance liquid chromatography (HPLC) for tryptic peptides. The structural relatedness of these enzymes was confirmed by sequence analysis of a total of 433 residues in 32 tryptic fragments of the human erythrocyte isozymes I and II and of the bovine erythrocyte isozyme II. They found sequence identity or probable identity in 111 out of 112 residues when they compared the human isozymes I and II and identities in 127 out of 134 residues when the human and bovine isozymes II were compared. These results suggest that the erythrocyte isozymes from both organisms may have nearly identical structures and confirm the similarities in the function of these methyltransferases that have been previously demonstrated.

Gilbert, J.M.; Fowler, A.; Bleibaum, J.; Clarke, S.



Bovine Papillomavirus Type 1 Genomes and the E2 Transactivator Protein Are Closely Associated with Mitotic Chromatin  

PubMed Central

The bovine papillomavirus type 1 E2 transactivator protein is required for viral transcriptional regulation and DNA replication and may be important for long-term episomal maintenance of viral genomes within replicating cells (M. Piirsoo, E. Ustav, T. Mandel, A. Stenlund, and M. Ustav, EMBO J. 15:1–11, 1996). We have evidence that, in contrast to most other transcriptional transactivators, the E2 transactivator protein is associated with mitotic chromosomes in dividing cells. The shorter E2-TR and E8/E2 repressor proteins do not bind to mitotic chromatin, and the N-terminal transactivation domain of the E2 protein is necessary for the association. However, the DNA binding function of E2 is not required. We have found that bovine papillomavirus type 1 genomes are also associated with mitotic chromosomes, and we propose a model in which E2-bound viral genomes are transiently associated with cellular chromosomes during mitosis to ensure that viral genomes are segregated to daughter cells in approximately equal numbers.

Skiadopoulos, Mario H.; McBride, Alison A.



Bovine seminal PDC-109 protein: An overview of biochemical and functional properties.  


Although long-term storage of bovine semen is desirable for wider use, successful cryopreservation depends on several factors, including various proteins present in seminal plasma. One such group of proteins, viz. bovine seminal plasma (BSP) proteins represents the major protein fraction in bovine seminal plasma. They constitute three major heparin-binding (HB-) acidic proteins secreted by seminal vesicles, viz. BSP-A1/-A2 (PDC-109), BSP-A3 and BSP-30-kDa. By purification studies it was deduced that PDC-109 is a polypeptide of 109 amino acids and contains two tandem repeating fibronectin type-II (Fn-II) domains, preceded by a 23 residue N-terminal domain. Though BSP-A1 and BSP-A2 are biochemically similar they differ only in glycosylation and their mixture is called PDC-109 or gonadostatins. PDC-109 exists as a polydisperse, multimeric self-associated molecule and possesses multifunctional properties, viz. binding to the surface of plasma membrane of spermatozoa causing conformational change in the sperm surface proteins and enhances motility. Besides binding, PDC-109 protein provokes cholesterol efflux from sperm membrane and promotes sperm reservoir by interacting with oviductal membrane. Interaction of sperm with PDC-109 protein induces sperm capacitation and acrosome reaction. However, prolonged exposure of spermatozoa with free floating PDC-109 protein as during processing for preservation, increases cholesterol efflux from spermatozoa. The efflux of sperm membrane cholesterol and disturbance in cholesterol:phospholipids ratio causes destabilization of plasma membrane thereby inducing cryoinjury to the sperm. In this review, the biochemical, functional properties of PDC-109 protein and its role during semen cryopreservation is summarized. PMID:23489472

Srivastava, N; Jerome, A; Srivastava, S K; Ghosh, S K; Kumar, Amit



Identification of bovine hibernation-specific protein complex and evidence of its regulation in fasting and aging.  


Hibernation-specific protein (HP) is a plasma protein that regulates hibernation in chipmunks. The HP complex (HP20c) consists of three homologous proteins, HP20, HP25 and HP27, all produced by liver and belonging to the C1q family. To date, HP20c has not been identified in any mammalian species except chipmunk and ground squirrel hibernators. Here, we report a bovine HP20 gene isolated from liver tissue and aortic endothelial cells. Total homology between bovine and chipmunk variants was 63% at the amino acid level. Gene expression was highest in the liver. Western blot revealed HP20 protein in foetal, newborn, calf and adult serum, with highest concentrations in the adult. Similar proteins were detected in sera of other ruminants but not in humans, bears, mice or rats. Bovine HP20 protein was found mainly in ovaries, stomach, heart, kidneys, lungs, testes and prostate, but not in the skeletal muscle. Native HP20 was purified from bovine adult serum as a complex containing 25 and 27 kDa proteins. Mass spectrometry revealed that these proteins are orthologues of chipmunk HP25 and HP27, respectively. Interestingly, bovine HP20 was highly expressed in cattle serum after fasting. Native bovine HP20c may be a useful tool for investigating HP function. PMID:23389309

Fujita, Satoshi; Okamoto, Ryuji; Taniguchi, Masaya; Ban-Tokuda, Tomomi; Konishi, Katsuhisa; Goto, Itaru; Yamamoto, Yasunari; Sugimoto, Kazushi; Takamatsu, Nobuhiko; Nakamura, Mashio; Shiraki, Katsuya; Buechler, Christa; Ito, Masaaki



Experimental anterior spine fusion using bovine bone morphogenetic protein: a study in rabbits.  


We developed an experimental model to study the merit of bovine bone morphogenic protein (bBMP) injection into the intervertebral disc to induce anterior interbody fusion. A total of 24 rabbits, divided into three groups of 8 animals each, were used. One hundred and fifty microg of partially purified bBMP was employed in the first group and 10 microg bBMP in the second group. In the control group, a sham operation was performed. The animals were followed radiographically at weekly intervals and animals were killed 3, 6, and 12 weeks postoperatively. After sacrifice, a mechanical and histologic evaluation of fusion was performed. Results of radiographic and histologic evaluation showed bone formation, which had resulted in the bridging of adjacent endplates, in the 150-microg group. In the 10-microg group, new bone formation was less extensive. In the control group, intradiscal bone formation was seen in only 1 animal. Range of motion measurements on flexion/extension films showed significantly decreased motion in segments that were fused with 150-microg of BMP. This study demonstrated the utility of an experimental model which allowed investigation of how anterior spine fusion may be further studied. Intradiscal injection of BMP could ultimately play a role in the development of minimally invasive techniques for anterior spinal fusion. PMID:10982651

Muschik, M; Schlenzka, D; Ritsilä, V; Tennstedt, C; Lewandrowski, K U



Cloning and Expression of Glycolipid Transfer Protein from Bovine and Porcine Brain*  

PubMed Central

Glycolipid transfer protein (GLTP) is a small (23–24 kDa), basic protein (pI ? 9.0) that accelerates the inter-membrane transfer of various glycolipids. Here, we report the first cloning of cDNAs that encode the bovine and porcine GLTPs. The cDNA open reading frame for bovine GLTP was constructed by bridge-overlapping extension polymerase chain reaction (PCR) after obtaining partial coding cDNA clones by hot start, seminested, and rapid amplification of cDNA ends-PCR. The cDNA open reading frame for porcine GLTP was constructed by reverse transcriptase-PCR. The encoded amino acid sequences in the full-length bovine and porcine cDNAs were identical, consisting of 209 amino acid residues, and were nearly the same as the published sequence determined by Edman degradation. The cDNA encoded one additional amino acid at the N terminus (methionine), arginine at positions 10 and 200 instead of lysine, and threonine at position 65 instead of alanine. Expression of GLTP-cDNA in Escherichia coli using pGEX-6P-1 vector resulted in glutathione S-transferase (GST)-GLTP fusion protein. Regulation of growth and induction conditions led to ?50% of expressed fusion protein being soluble and active. Proteolytic cleavage of GST-GLTP fusion protein (bound to GST-Sepharose) and affinity purification resulted in fully active GLTP. Northern blot analyses of bovine tissues showed a single transcript of ?2.2 kilobases and the following hierarchy of mRNA levels: cerebrum > kidney > spleen ? lung ? cerebellum > liver > heart muscle. Reverse transcriptase-PCR analyses of mRNA levels supported the Northern blot results.

Lin, Xin; Mattjus, Peter; Pike, Helen M.; Windebank, Anthony J.; Brown, Rhoderick E.



The effect of protein binding on ivermectin uptake by bovine brain microvessel endothelial cells  

Microsoft Academic Search

The effect of albumin binding on ivermectin uptake and transfer across the endothelial component of the blood-brain barrier (BBB) was determined with anin vitro model comprised of bovine brain microvessel endothelial cell (BMEC) monolayers. Cellular uptake of ivermectin was limited in the absence of albumin and 90% inhibited in the presence of 10% albumin. Cell membrane association of ivermectin, as

K. L. Audus; S. R. Knaub; F. L. Guillot; J. M. Schaeffer



An evaluation of metal bioaccessibility in estuarine sediments using the commercially available protein, bovine serum albumin  

Microsoft Academic Search

The bioaccessibility of metals (Al, Ca, Fe, Mn, Ag, Cd, Co, Cu, Ni, Pb, Sn, Zn) in oxic estuarine sediments has been evaluated using solutions of a commercially available protein (bovine serum albumin; BSA) that mimic the chemical conditions encountered in the gut environment of many deposit-feeding organisms. Over a 20 h incubation period with 5 g L?1 BSA, metal mobilisation was

Judit Kalman; Andrew Turner



Genus specific features of bovine papillomavirus E6, E7, E5 and E8 proteins  

Microsoft Academic Search

Six bovine papillomavirus (BPV) types, BPV-1 to -6, have been classified in genera Delta-papillomavirus (BPV-1 and -2), Epsilon-papillomavirus (BPV-5) and Xi-papillomavirus (BPV-3, -4 and -6). In addition, 16 unclassified putative BPV types have been reported. In the present study, we characterized genus specific features of E6, E7, E5 (formerly E8) and E8 proteins of seven putative BPV types, BAPV-1, -2,

Yoshimi Tomita; Tomoko Ogawa; Zhongri Jin; Hiroshi Shirasawa



Distribution of S-100 protein and its subunits in bovine exocrine glands  

Microsoft Academic Search

The distribution of S-100 protein and its ?- and ?-subunits in bovine exocrine glands was studied by indirect immunohistochemistry.\\u000a The entire spectrum of salivary glands, glands of the respiratory tract, intestinal glands, male and female genital glands,\\u000a and skin glands was examined. S-100 and its ?-subunit were identified in most serous secretory cells of mixed salivary glands,\\u000a although secretory acini

Siegrun Lauboeck; M. Egerbacher



The combination of glycosaminoglycans and fibrous proteins improves cell proliferation and early differentiation of bovine primary skeletal muscle cells.  


Primary muscle cell model systems from farm animals are widely used to acquire knowledge about muscle development, muscle pathologies, overweight issues and tissue regeneration. The morphological properties of a bovine primary muscle cell model system, in addition to cell proliferation and differentiation features, were characterized using immunocytochemistry, western blotting and real-time PCR. We observed a reorganization of the Golgi complex in differentiated cells. The Golgi complex transformed to a highly fragmented network of small stacks of cisternae positioned throughout the myotubes as well as around the nucleus. Different extracellular matrix (ECM) components were used as surface coatings in order to improve cell culture conditions. Our experiments demonstrated improved proliferation and early differentiation for cells grown on surface coatings containing a mixture of both glycosaminoglycans (GAGs) and fibrous proteins. We suggest that GAGs and fibrous proteins mixed together into a composite biomaterial can mimic a natural ECM, and this could improve myogenesis for in vitro cell cultures. PMID:23933398

Rønning, Sissel Beate; Pedersen, Mona Elisabeth; Andersen, Petter Vejle; Hollung, Kristin



Proteomic Analysis of Differentially Expressed Proteins in Bovine Endometrium with Endometritis  

PubMed Central

Endometritis is one of the primary reasons for reproductive failure. In order to investigate endometritis-associated marker proteins, proteomic analysis was performed on bovine endometrium with endometritis. In bovine endometritis, desmin, ?-actin-2, heat-shock protein (HSP) 27, peroxiredoxin-6, luteinizing hormone receptor isoform 1, collectin-43 precursor, deoxyribonuclease-I (DNase-I), and MHC class I heavy chain (MHC-Ih) were up-regulated. In contrast, transferrin, interleukin-2 precursor, hemoglobin ? subunit, and potassium channel tetramerisation domain-containing 11 (KCTD11) were down-regulated in comparison to normal endometrium. The proteomic results were validated by semiquantitative-PCR and immunoblot analysis. The mRNA levels of desmin, transferrin, ?-actin-2, HSP27, KCTD11, and MHC-Ih were up-regulated by over 1.5-fold, and showed a pattern similar to their proteomic profiles. Desmin and ?-actin-2 protein showed positive correlations between proteomic analysis and immunoblot analysis. These results suggest that desmin and ?-actin-2 may play important roles in endometritis-related function, and could be useful markers for the diagnosis of bovine endometritis.

Choe, Changyong; Park, Jeong-Won; Kim, Eun-Suk; Lee, Sung-Gyu; Park, Sun-Young; Lee, Jeong-Soon; Cho, Myung-Je; Kang, Kee Ryeon; Han, Jaehee



In vitro dimerization of the bovine papillomavirus E5 protein transmembrane domain  

PubMed Central

The E5 protein from bovine papillomavirus is a type II membrane protein and the product of the smallest known oncogene. E5 causes tumor formation by binding and activating the platelet derived growth factor beta receptor (PDGF?R). In order to productively interact with the receptor it is thought that E5 binds as a dimer. However, wild-type E5 and various mutants have also been shown to form trimers, tetramers and even higher order oligomers. The residues in E5 that drive and stabilize a dimeric state are also still in question. At present, two different models for the E5 dimer exist in the literature, one symmetric and one asymmetric. There is universal agreement, however, that the transmembrane (TM) domain plays a vital role in stabilizing the functional oligomer, indeed mutation of various TM domain residues can abolish E5 function. In order to better resolve the role of the E5 TM domain in function, we have undertaken the first quantitative in vitro characterization of the E5 TM domain in detergent micelles and liposomes. Circular and linear dichroism analyses verify that the TM domain adopts a stable ?-helical structure and is able to partition efficiently across lipid bilayers. SDS-PAGE and analytical ultracentrifugation confirm for the first time that the TM domain of E5 forms a strong dimer with a standard state free energy of dissociation of 5.0 kcal mol?1. We have used our new results to interpret existing models of E5 dimer formation and provide a direct link between TM helix interactions and E5 function.

Oates, Joanne; Hicks, Matthew; Dafforn, Timothy R.; DiMaio, Daniel; Dixon, Ann M.



Identification and characterization of RHOA-interacting proteins in bovine spermatozoa.  


In somatic cells, RHOA mediates actin dynamics through a GNA13-mediated signaling cascade involving RHO kinase (ROCK), LIM kinase (LIMK), and cofilin. RHOA can be negatively regulated by protein kinase A (PRKA), and it interacts with members of the A-kinase anchoring (AKAP) family via intermediary proteins. In spermatozoa, actin polymerization precedes the acrosome reaction, which is necessary for normal fertility. The present study was undertaken to determine whether the GNA13-mediated RHOA signaling pathway may be involved in acrosome reaction in bovine caudal sperm, and whether AKAPs may be involved in its targeting and regulation. GNA13, RHOA, ROCK2, LIMK2, and cofilin were all detected by Western blot in bovine caudal sperm. Overlay, immunoprecipitation, and subsequent mass spectrometry analysis identified several RHOA-interacting proteins, including proacrosin, angiotensin-converting enzyme, tubulin, aldolase C, and AKAP4. Using overlay and pulldown techniques, we demonstrate that phosphorylation of AKAP3 increases its interaction with the RHOA-interacting proteins PRKAR2 (the type II regulatory subunit of PRKA, formerly RII) and ropporin (ROPN1, a PRKAR2-like protein, or R2D2). Varying calcium concentrations in pulldown assays did not significantly alter binding to R2D2 proteins. These data suggest that the actin-regulating GNA13-mediated RHOA-ROCK-LIMK-cofilin pathway is present in bovine spermatozoa, that RHOA interacts with proteins involved in capacitation and the acrosome reaction, and that RHOA signaling in sperm may be targeted by AKAPs. Finally, AKAP3 binding to PRKAR2 and ROPN1 is regulated by phosphorylation in vitro. PMID:17928627

Fiedler, Sarah E; Bajpai, Malini; Carr, Daniel W



Insulin autoantibodies with high affinity to the bovine milk protein alpha casein.  


Insulin autoantibodies (IAA) can appear in children within months of introducing solid foods to the diet and before clinical type 1 diabetes. The aim of this study was to determine whether infant dietary antigens could be immunizing agents of IAA. To this end, IAA binding to [(125) I]insulin was competed with food preparations and extracts of foods encountered in the infant diet (milk formulas, bovine milk, wheat flour, fowl meal). Bovine milk powder extracts inhibited IAA-positive samples from six of 53 children (age 0·3-14·0 years) participating in German prospective cohorts. Inhibition in these sera ranged from 23 to 100%. Competition was abolished when hydrolyzed milk powder was used. Competition with protein components of bovine milk found that two of the six milk-reactive sera were inhibited strongly by alpha- and beta-casein; none were inhibited by the milk proteins bovine serum albumin or lactoglobulins. The two casein-reactive sera had high affinity to alpha-casein (1·7×10(9) ; 3·1×10(9) ?l/mol), and lesser affinity to beta-casein (4·0×10(8) ; 7·0×10(7) ?l/mol) and insulin (2·6×10(8) ; 1·6×10(8) ?l/mol). No children with milk-reactive IAA developed autoantibodies to other islet autoantigens or diabetes (median follow-up 9·8 years). These results suggest that autoimmunity to insulin can occur infrequently via cross-reactivity to food proteins, but this form of IAA immunization does not appear to be associated with progression to diabetes. PMID:21361910

Adler, K; Mueller, D B; Achenbach, P; Krause, S; Heninger, A-K; Ziegler, A G; Bonifacio, E



Insulin autoantibodies with high affinity to the bovine milk protein alpha casein  

PubMed Central

Insulin autoantibodies (IAA) can appear in children within months of introducing solid foods to the diet and before clinical type 1 diabetes. The aim of this study was to determine whether infant dietary antigens could be immunizing agents of IAA. To this end, IAA binding to [125I]insulin was competed with food preparations and extracts of foods encountered in the infant diet (milk formulas, bovine milk, wheat flour, fowl meal). Bovine milk powder extracts inhibited IAA-positive samples from six of 53 children (age 0·3–14·0 years) participating in German prospective cohorts. Inhibition in these sera ranged from 23 to 100%. Competition was abolished when hydrolyzed milk powder was used. Competition with protein components of bovine milk found that two of the six milk-reactive sera were inhibited strongly by alpha- and beta-casein; none were inhibited by the milk proteins bovine serum albumin or lactoglobulins. The two casein-reactive sera had high affinity to alpha-casein (1·7 × 109; 3·1 × 109 l/mol), and lesser affinity to beta-casein (4·0 × 108; 7·0 × 107 l/mol) and insulin (2·6 × 108; 1·6 × 108 l/mol). No children with milk-reactive IAA developed autoantibodies to other islet autoantigens or diabetes (median follow-up 9·8 years). These results suggest that autoimmunity to insulin can occur infrequently via cross-reactivity to food proteins, but this form of IAA immunization does not appear to be associated with progression to diabetes.

Adler, K; Mueller, D B; Achenbach, P; Krause, S; Heninger, A-K; Ziegler, A G; Bonifacio, E



Identification and characterization of a bovine Iipopolysaccharide-binding protein  

Microsoft Academic Search

Endogenous regulatory mechanisms exist in mammals that enable a rapid response to lipopolysaccha- ride (LPS, endotoxin) stemming from gram-negative bac- terial infections. Serum proteins and cell surface recep- tors exist that bind LPS, and this interaction may either aid in nonpathogenic removal of LPS from the body or potentiate the effects of LPS. We have used a photoreac- tive, thiol-cleavable,

Lajwanti S. Khemlani; Zhengang Yang; Philip N. Bochsler



Measurement of protein synthesis and degradation in C 2 C 22 myoblasts using extracts of muscle from hormone treated bovine  

Microsoft Academic Search

A detailed methodology is described for determination of treatment effects on muscle cell protein synthesis and muscle cell protein degradation in a cell culture system. C2C22 mouse myoblasts were treated with growth media containing muscle extracts from bovine treated with different pharmaceutical agents. Radiolabeled amino acids were added to the growth media to determine treatment effects on protein synthesis and

J. L. Montgomery; W. M. Harper; M. F. Miller; K. J. Morrow; J. R. Blanton



Distinction of different heat-treated bovine milks by native-PAGE fingerprinting of their whey proteins  

Microsoft Academic Search

Native-PAGE (polyacrylamide gel electrophoresis) was used for the simultaneous qualitative and quantitative analysis of whey proteins of raw, commercial and laboratory heat-treated bovine milks. Four whey protein bands, including ?-lactoglobulin variants (?-LG A and B), could be distinctively separated in the gel. The results showed that levels of the major whey proteins were reduced by less than 23% in the

Suju Lin; Jing Sun; Dongdong Cao; Jiankang Cao; Weibo Jiang



Sequence variations of the bovine prion protein gene (PRNP) in native Korean Hanwoo cattle  

PubMed Central

Bovine spongiform encephalopathy (BSE) is one of the fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs) caused by infectious prion proteins. Genetic variations correlated with susceptibility or resistance to TSE in humans and sheep have not been reported for bovine strains including those from Holstein, Jersey, and Japanese Black cattle. Here, we investigated bovine prion protein gene (PRNP) variations in Hanwoo cattle [Bos (B.) taurus coreanae], a native breed in Korea. We identified mutations and polymorphisms in the coding region of PRNP, determined their frequency, and evaluated their significance. We identified four synonymous polymorphisms and two non-synonymous mutations in PRNP, but found no novel polymorphisms. The sequence and number of octapeptide repeats were completely conserved, and the haplotype frequency of the coding region was similar to that of other B. taurus strains. When we examined the 23-bp and 12-bp insertion/deletion (indel) polymorphisms in the non-coding region of PRNP, Hanwoo cattle had a lower deletion allele and 23-bp del/12-bp del haplotype frequency than healthy and BSE-affected animals of other strains. Thus, Hanwoo are seemingly less susceptible to BSE than other strains due to the 23-bp and 12-bp indel polymorphisms.

Choi, Sangho



Antihypertensive Peptides Derived from Bovine Casein and Whey Proteins  

Microsoft Academic Search

Peptides play an important primary role as a supply of essential amino acids and a source of nitrogen. Recent studies have\\u000a reported on another role of peptides: having specific amino acid sequences that can express some biological functions in vivo. For an exhaustive study and supply of biologically active peptides, a large-scale screening of protein sources is necessary.\\u000a Various physiologically

Tadao Saito


The bovine seminal plasma protein PDC109 extracts phosphorylcholine-containing lipids from the outer membrane leaflet  

Microsoft Academic Search

The bovine seminal plasma protein PDC-109 modulates the maturation of bull sperm cells by removing lipids, mainly phosphatidylcholine\\u000a and cholesterol, from their cellular membrane. Here, we have characterized the process of extraction of endogenous phospholipids\\u000a and of their respective analogues. By measuring the PDC-109-mediated release of fluorescent phospholipid analogues from lipid\\u000a vesicles and from biological membranes (human erythrocytes, bovine epididymal

Astrid Tannert; Anke Kurz; Karl-Rudolf Erlemann; Karin Müller; Andreas Herrmann; Jürgen Schiller; Edda Töpfer-Petersen; Puttaswamy Manjunath; Peter Müller



Hemagglutination mediated by the spike protein of cell-adapted bovine torovirus.  


Bovine torovirus (BToV)-Aichi, recently isolated in cultured cells, showed hemagglutination (HA) activity, although the virus has a truncated hemagglutinin-esterase (HE) protein, judging from its gene structure, indicating the existence of another viral protein with HA activity. We examined whether the spike (S) protein possesses HA activity. A BToV antiserum used in this study, reactive to S but not to HE, inhibited HA activity. Furthermore, cells infected with BToV and those expressing S showed hemadsorption (HAD) activity, which was inhibited by the anti-BToV serum; however, HAD activity by expressed HE was not blocked. These data indicate that the S protein of BToV-Aichi is responsible for its HA activity. PMID:23420207

Shimabukuro, Kozue; Ujike, Makoto; Ito, Toshihiro; Tsunemitsu, Hiroshi; Oshitani, Hitoshi; Taguchi, Fumihiro



Identification and characterization of the glucose-transport protein of the bovine blood/brain barrier.  

PubMed Central

The glucose-transport protein from bovine cerebral-cortex microvessels has been identified and characterized by virtue of its ability to bind the ligand [4-3H]cytochalasin B. Microvessel membranes were found to contain a single set of glucose-inhibitable high-affinity cytochalasin B-binding sites [113 +/- 16 (S.E.M.) pmol/mg of membrane protein], with an association constant of 6.8 +/- 1.8 (S.E.M.) micron-1. D-Glucose inhibited the binding to these sites with a Ki of 31 mM. The transport protein was identified by photoaffinity labelling with [4-3H]cytochalasin B and was found to migrate as a broad band of apparent Mr 55,000 on SDS/polyacrylamide gels. Labelling was inhibited by D-glucose, but not by L-glucose. Treatment with endoglycosidase F yielded a sharper band of apparent Mr 46,000, indicating that the transport protein is glycosylated. However, in contrast with the human erythrocyte glucose transporter, digestion with endo-beta-galactosidase had little effect on the electrophoretic mobility of the microvessel protein. Tryptic digestion of the photolabelled protein yielded a radioactive fragment of apparent Mr 18,000, similar to that of the fragment produced by digestion of the labelled human erythrocyte glucose transporter. In addition, a protein of Mr identical with that of the photolabelled transporter was labelled on Western blots of microvessel membranes by antisera raised against the intact erythrocyte transporter and against synthetic peptides corresponding to its N- and C-terminal regions. It is concluded that the glucose-transport protein of bovine cerebral-cortex microvessel endothelial cells shows structural homology with the human erythrocyte glucose transporter. Images Fig. 8.

Kasanicki, M A; Cairns, M T; Davies, A; Gardiner, R M; Baldwin, S A



Sequence variability and protein domain architectures for bovine Toll-like receptors 1, 5, and 10.  


The mammalian Toll-like receptors (TLRs) play an important role in the recognition of invading pathogens and the modulation of innate immune responses. The primary objective of this study was to characterize single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (indels) within bovine TLRs 1, 5, and 10, thereby facilitating future TLR signaling and association studies relevant to bovine innate immunity. Comparative sequence analysis for 10 bovine breeds derived from Bos taurus and Bos indicus revealed 98 polymorphisms (92 SNPs and 6 indels), with at least 14 nonsynonymous SNPs located within predicted TLR domains considered to be of functional significance. Of the 98 polymorphisms detected, 94 are reported here for the first time. Notably, 2 nonsynonymous SNPs were determined to modulate the prediction of a novel leucine-rich repeat (LRR) domain within B. indicusTLR5. Prediction and comparison of TLR protein domain architectures for multiple species revealed seven conserved regions of LRR patterning associated with the three genes investigated. PMID:17719743

Seabury, C M; Cargill, E J; Womack, J E



Translation of bovine leukemia virus virion RNAs in heterologous protein-synthesizing systems.  

PubMed Central

Bovine leukemia virus 60 to 70S RNA was heat denatured, the polyadenylic acid-containing species were separated by velocity sedimentation, and several size classes were translated in a micrococcal nuclease-treated cell-free system from rabbit reticulocytes. The major RNA species sedimented at 38S and migrated as a single component of molecular weight 2.95 x 10(6) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The predominant polypeptides of the in vitro translation of bovine leukemia virus 38S RNA were products with molecular weights of 70,000 and 45,000; minor components with molecular weights of 145,000 and 18,000 were also observed. Two lines of evidence indicate that the 70,000- and 45,000-molecular weight polypeptides represent translation products of the gag gene of the bovine leukemia virus genome (Pr70gag and Pr45gag). First, they are specifically precipitated by a monospecific antiserum to the major internal protein, p24, and second, they are synthesized and correctly processed into virion proteins p24, p15, and p10 in Xenopus laevis oocytes microinjected with bovine leukemia virus 38S RNA. The 145,000-molecular weight polypeptide was immunoprecipitated by the anti-p24 serum and not by an antiserum to the major envelope glycoprotein, gp60. It contained all the tryptic peptides of Pr70gag and additional peptides unique to it, and thus represents in elongation product of Pr70gag in an adjacent gene, presumably the pol gene. The 18,000-molecular weight product was antigenically unrelated to p24 and gp60 and shared no peptides in common with Pr70gag, Pr45gag, or the 145,000-molecular weight polypeptide. It was maximally synthesized on a polyadenylic acid-containing virion 16 to 18S RNA, and we present evidence that this RNA is a 3' end-derived subgenomic fragment of the bovine leukemia virus genome rather than a contaminating cellular RNA. Images

Ghysdael, J; Kettmann, R; Burny, A



Differences in fragmentation between bound and unbound bovine secretory component suggest a model for its interaction with polymeric immunoglobulin.  

PubMed Central

Unbound bovine secretory component was cleaved into two-domain and one-domain fragments by trypsin within 1 h. Bovine secretory component covalently bound to bovine IgA dimer, as in secretory IgA, was much more resistant to fragmentation, which did not proceed beyond the three-domain stage even after 5 h. Bovine secretory component non-covalently bound to bovine IgM or to human IgM or IgA polymer was also relatively resistant to fragmentation, which again was largely arrested at the three-domain stage. A model for the binding of secretory component to polymeric immunoglobulin is proposed.

Beale, D



Enantioselectivity of bovine serum albumin-bonded columns produced with isolated protein fragments  

Microsoft Academic Search

Enantioselectivity of bovine serum albumin (BSA)-bonded columns produced with isolated protein fragments has been investigated. The BSA fragment, BSA-FG75, was isolated by size exclusion chromatography following peptic digest of BSA. The isolated BSA-FG75 was further fractionated to two fractions, BSA-F1 and BSA-F2, by anion-exchange chromatography. BSA-F1 and BSA-F2 had molecular mass of about 35?000 daltons, estimated by matrix-assisted laser desorption

Jun Haginaka; Naoko Kanasugi



Addition of bovine plasma hydrolysates improves the antioxidant properties of soybean and sunflower protein-based films  

Microsoft Academic Search

The effect of adding different amounts of a bovine plasma hydrolysate (BPH) with high antioxidant capacity on the functional properties of protein films based on soybean and sunflower protein was studied. BPH caused a decrease in tensile strength, elastic modulus and glass transition temperature of the films, as well as an increase in their elongation at break and water vapor

Pablo R. Salgado; Graciela B. Fernández; Silvina R. Drago; Adriana N. Mauri



E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity  

SciTech Connect

In addition to members of the Orthomyxoviridae and Paramyxoviridae, several coronaviruses have been shown to possess receptor-destroying activities. Purified bovine coronavirus (BCV) preparations have an esterase activity which inactivates O-acetylsialic acid-containing receptors on erythrocytes. Diisopropyl fluorophosphate (DFP) completely inhibits this receptor-destroying activity of BCV, suggesting that the viral enzyme is a serine esterase. Treatment of purified BCV with (/sup 3/H)DFP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins revealed that the esterase/receptor-destroying activity of BCV is associated with the E3 protein was specifically phosphorylated. This finding suggests that the esterase/receptor-destroying activity of BCV is associated with the E3 protein. Furthermore, treatment of BCV with DFP dramatically reduced its infectivity in a plaque assay. It is assumed that the esterase activity of BCV is required in an early step of virus replication, possible during virus entry or uncoating.

Vlasak, R.; Luytjes, W.; Leider, J.; Spaan, W.; Palese, P.



Relaxation kinetics and the glassiness of proteins: the case of bovine pancreatic trypsin inhibitor.  

PubMed Central

Folded proteins may be regarded as soft active matter under physiological conditions. The densely packed hydrophobic interior, the relatively molten hydrophilic exterior, and the spacer connecting these put together a large number of locally homogeneous regions. For the case of the bovine pancreatic trypsin inhibitor, with the aid of molecular dynamics simulations, we have demonstrated that the kinetics of the relaxation of the internal motions is highly concerted, manifesting the protein's heterogeneity, which may arise from variations in density, local packing, or the local energy landscape. This behavior is characterized in a stretched exponential decay described by an exponent of approximately 0.4 at physiological temperatures. Due to the trapped conformations, configurational entropy becomes smaller, and the associated stretch exponent drops to half of its value below the glass transition range. The temperature dependence of the inverse relaxation time closely follows the Vogel-Tamman-Fulcher expression when the protein is biologically active.

Baysal, Canan; Atilgan, Ali Rana



Optical spectroscopic determination of bovine tropoelastin molecular model  

Microsoft Academic Search

Aqueous solution (CD in water) and solid state (FT-IR on a ZnSe window) spectra of bovine tropoelastin (BTPE) have been recorded and analysed in order to get additional insights into the molecular structure of its elastic elastin polymer. Conformational analyses evidenced high levels of both ordered and unordered secondary structures in the solid and solution states. The structural contents of

L. Debelle; A. J. P. Alix



Properties of dissociatively extracted fetal tooth matrix proteins. I. Principal molecular species in developing bovine enamel.  


A sequential dissociative extraction scheme is described in which tooth matrix proteins are extracted first in 4 M guanidine HCl, pH 7.4, and then in 4 M guanidine HCl, 0.5 M EDTA, pH 7.4, both with protease inhibitors present. The latter step dissolves the mineralized portion of the tissue and extracts noncollagenous proteins closely associated with hydroxyapatite crystallites in the mineralized matrix. In fetal bovine enamel, the initial dissociative extraction step completely removes proline-rich amelogenins from the tissue without dissolving the enamel apatite. The amelogenin proteins consist of several species on polyacrylamide gel electrophoresis with sodium dodecyl sulfate, but display anomalous migration behavior relative to conventional marker proteins in this technique. Subsequent extraction of fetal bovine enamel with guanidine HCl/EDTA removes matrix enamelins, acidic glycoproteins that are tightly bound to the enamel hydroxyapatite. This latter fetal protein type has not been isolated previously. The enamelins are adsorbed strongly by DEAE-cellulose in 7 M urea and totally adsorb to synthetic apatite, even in 4 M guanidine HCl. The enamelins display normal behavior on polyacrylamide gels and stain positively for sialic acid/phosphate and carbohydrate. With advancing tooth maturation, amelogenins disappear while enamelins are conserved. Gel filtration chromatography in 4 M guanidine HCl showed amelogenin components at apparent molecular weights of approximately 25,000, 15,000, 9,500, 7,500, and 6,000, while the enamelins eluted at Mr positions of approximately 72,000, 56,000, 42,000, 30,000, 21,000, 13,000, and 8,000. The gel filtration data showed a clear shift in molecular size population from higher to lower components for both amelogenins and enamelins with progressive enamel maturation. PMID:7430099

Termine, J D; Belcourt, A B; Christner, P J; Conn, K M; Nylen, M U



Morphologic study on experimental allergic neuritis mediated by T cell line specific for bovine P2 protein in Lewis rats.  


Light and electron microscope studies were performed on experimental allergic neuritis (EAN) passively induced in Lewis rats by the intravenous injection of T line cells specific for bovine P2 protein. Histologic changes were almost entirely restricted to the peripheral nervous system, being most severe in the sciatic nerve and lumbosacral nerve roots, whereas the brachial nerve and cervical nerve roots were involved to a lesser extent. The lesions were composed of edema, cellular infiltrates, demyelination, and, subsequently, axonal degeneration. Infiltrated macrophages were observed actively stripping the myelin, and the Schwann cell cytoplasm of affected nerve fibers was pushed to the periphery without distinct evidence of degeneration. The first evidence of pathologic change was severe edema in the sciatic nerve 4 days postinoculation. This edema was demonstrated immunohistochemically by the presence of albumin and fibrinogen in the endoneurial space. Mast cell degranulation was observed in these edematous nerve lesions. The cellular infiltrates which formed perivascular cuffs were composed of not only mononuclear cells but also many granulocytes. In the central nervous system, meningeal cell infiltration was also observed in the spinal cord, and after 7 days postinoculation degeneration of the posterior column was also found. This latter observation is thought to represent degeneration due to axonal damage of lumbosacral posterior roots. These pathologic findings in a T cell-mediated model of EAN were essentially the same as those previously reported in conventionally induced EAN or human Guillain-Barré Syndrome. Thus, T cells specific for bovine P2 protein can induce typical EAN lesions in the Lewis rat. The further investigation of this transfer model of EAN will enable us to clarify the pathogenesis of EAN and Guillain-Barré syndrome. PMID:2410663

Izumo, S; Linington, C; Wekerle, H; Meyermann, R



High-fat diet based on dried bovine brain: an effective animal model of dyslipidemia and insulin resistance  

Microsoft Academic Search

Currently, there are no reports in the literature demonstrating any animal model that ingests one of the fattiest animal food\\u000a source, the bovine brain. We hypothesized that a high-fat diet (HFD), based on dried bovine brain, could be used to develop\\u000a an animal model possessing a spectrum of insulin resistance-related features. The HFD was formulated with 40% dried bovine\\u000a brain

Tiago Gomes Araújo; Ana Catarina Rezende Leite; Caíque Silveira Martins da Fonseca; Bruno Melo Carvalho; Alexandre Ricardo Pereira Schuler; Vera Lúcia de Menezes Lima


In Vitro and In Vivo Oncogenic Potential of Bovine Leukemia Virus G4 Protein  

PubMed Central

In addition to the genes involved in the structure of the viral particle, the bovine leukemia virus (BLV) genome contains a region called X which contains at least four genes. Among them, the tax and rex genes, respectively, are involved in transcriptional and posttranscriptional regulation of viral transcription. Two other genes, R3 and G4, were identified after cloning of the corresponding mRNAs from BLV-infected lymphocytes. Although the function of the two latter genes is still unknown, they appear to have important roles, since deletion of them restricts viral propagation in vivo. In order to assess the oncogenic potential of the R3 and G4 proteins, we first analyzed their ability to immortalize and/or transform primary rat embryo fibroblasts (Refs). In this assay, the G4 but not the R3 protein cooperated with the Ha-ras oncogene to induce tumors in nude mice. It thus appears that G4 exhibited oncogenic potential in vitro. To extend these observations in vivo, the pathology induced by recombinant viruses with mutations in G4 and in R3 and G4 was next evaluated with the sheep animal model. Viral propagation, as measured by semiquantitative PCR, appeared to be reduced when the R3 and G4 genes were deleted. These observations confirm and extend our previous data underlining the biological function of these genes. In addition, we present the results of a clinical survey that involves 39 sheep infected with six different BLV recombinants. Over a period of 40 months, 83% of the sheep infected with a wild-type virus developed leukemias and/or lymphosarcomas. In contrast, none out of 13 sheep infected with viruses with mutations in G4 or in R3 and G4 developed disease. We conclude that in addition to its oncogenic potential in vitro, G4 is required for pathogenesis in vivo. These observations should help us gain insight into the process of leukemogenesis induced by the related human T-cell leukemia virus type 1.

Kerkhofs, Pierre; Heremans, Hubertine; Burny, Arsene; Kettmann, Richard; Willems, Luc



Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein  

SciTech Connect

The benefits of adding bovine serum albumin (BSA) or T4 gene 32 proteins (gp32) to PCR were evaluated with reaction mixtures containing substances that inhibit amplification. Whereas 10- to 1,000-fold more FeCl{sub 3}, hemin, fulvic acids, humic acids, tannic acids, or extracts from feces, freshwater, or marine water were accommodated in PCR when either 400 ng of BSA per {mu}l was included in the reactions, neither BSA nor gp32 relieved interference significantly when minimum inhibitory levels of bile salts, bilirubin, EDTA, NaCl, sodium dodecyl sulfate, or Triton X-100 were present. Use of BSA and gp32 together offered no more relief of inhibition than either alone at its optimal level, and neither protein had any noticeable effect on amplification in the absence of inhibitors. 21 refs., 3 figs.

Kreader, C.A. [Environmental Protection Agency, Cincinnati, OH (United States)



Screening of recombinant proteins as antigens in indirect ELISA for diagnosis of bovine tuberculosis.  


Bovine tuberculosis is an important infectious disease caused by Mycobacterium bovis, which is responsible for considerable economic losses. This disease constitutes a serious public health problem. Control programs in most countries, including Brazil, are based on the identification and slaughter of infected animals, as defined by the skin tuberculin test, which has its constraints. In the present study, the recombinant proteins CFP-10, ESAT-6, Mb0143, MPB83, PE5, PE13, TB10.4, TB15.3 and a chimera of ESAT-6/MPB70/MPB83 (fusion protein) were tested as ELISA antigens for the diagnosis of bovine tuberculosis. The proteins were produced in Escherichia coli, purified and tested in ELISAs with sera from 126 cattle having tested negative in the comparative intradermal tuberculin test (CITT) and 107 sera from cattle having tested positive in the CITT. Also, 236 sera from two BTB-free beef cattle herds were tested. Among the proteins tested, only the ESAT-6/MPB70/MPB83 chimera demonstrated satisfactory agreement with the CITT (kappa index: 0.688), reflecting in 83.2% sensitivity and 86.5% specificity. The ELISA absorbances of the cattle sera from BTB-free herds showed similar levels to those of CITT positive cattle, probably as the result of successive skin tuberculinizations to define the BTB-free status of the herds. However, the ELISA with the ESAT-6/MPB70/MPB83 chimera was useful to discriminate BTB positive and negative cattle in herds prior to the tuberculin skin test. PMID:23419946

Souza, Ingrid If; Melo, Elaine Sp; Ramos, Carlos An; Farias, Thaís A; Osório, Ana Luiza Ar; Jorge, Klaudia Sg; Vidal, Carlos Es; Silva, Altino S; Silva, Márcio R; Pellegrin, Aiesca O; Araújo, Flábio R



Involvement of cortactin and phosphotyrosine proteins in cell–cell contact formation in cultured bovine corneal endothelial cells  

Microsoft Academic Search

Phosphotyrosine proteins involvement, particularly cortactin, was studied in cell–cell contacts of cultured bovine corneal\\u000a endothelial (BCE) cells. These proteins, including ?-catenin, vinculin and cortactin, are localized at cell–cell contacts\\u000a separate from the cortical actin ring. Approximately 50% of cortactin isoforms p80 and p85 were associated with the Triton-insoluble\\u000a fraction while phosphotyrosine proteins were in the soluble fraction. Disruption of cell–cell

Lily Kredy-Farhan; Shlomo Kotev-Emeth; Naphtali Savion



Isolation of a calcium-binding protein of the acrosomal membrane of bovine spermatozoa  

PubMed Central

The mammalian sperm acrosome reaction is a calcium-dependent exocytotic event characterized by extensive fusion between the plasma and the outer acrosomal membrane. The mechanisms by which elevation of cytosolic calcium initiates the membrane fusion process are not understood and the present study was undertaken to identify calcium-binding proteins in the acrosomal membrane (AM) of bovine spermatozoa. Sperm heads, purified from sonicated spermatozoa, were used to isolate an acrosomal membrane-enriched fraction on Percoll density gradients. Using SDS-PAGE and a 45Ca2+-blot overlay assay, calcium-binding proteins of 64, 45, 43, and 39 kDa were identified in the AM enriched fraction. Phase separation analysis with Triton X-114 identified the 64 kDa polypeptide as an integral membrane protein. The 64 kDa polypeptide was purified and utilized to prepare a polyclonal antiserum. Both light and electron microscopic immunocytochemistry demonstrated that the protein was distributed throughout all domains of the acrosomal membrane. These results identify a 64 kDa calcium-binding integral membrane protein of the mammalian acrosome. Its potential function in calcium-dependent membrane fusion events of the acrosome reaction and in fertilization is discussed.

Nagdas, Subir K.; Buchanan, Teresa; McCaskill, Shaina; Mackey, Jared; Alvarez, George E.; Raychoudhury, Samir



Association of the GTP-binding protein Rab3A with bovine adrenal chromaffin granules  

SciTech Connect

The Rab3A protein belongs to a large family of small GTP-binding proteins that are present in eukaryotic cells and that share amino acid identities with the Ras proteins (products of the ras protooncogenes). Rab3A, which is specifically located in nervous and endocrine tissues, is suspected to play a key role in secretion. Its localization was investigated in bovine adrenal gland by using a polyclonal antibody. Rab3A was detected in adrenal medulla but not in adrenal cortex. In cultured adrenal medulla cells, Rab3A was specifically expressed in the catecholamine-secreting chromaffin cells. Subcellular fractionation suggested that Rab3A is about 30% cytosolic and that particulate Rab3A is associated with the membrane of chromaffin granules (the catecholamine storage organelles) and with a second compartment likely to be the plasma membrane. The Rab3A localization on chromaffin granule membranes was confirmed by immunoadsorption with an antibody against dopamine {beta}-hydroxylase. Rab3A was not extracted from this membrane by NaCl or KBr but was partially extracted by urea and totally solubilized by Triton X-100, suggesting either an interaction with an intrinsic protein or a membrane association through fatty acid acylation. This study suggests that Rab3A, which may also be located on other secretory vesicles containing noncharacterized small GTP-binding proteins, is involved in their biogenesis or in the regulated secretion process.

Darchen, F.; Hammel, F.; Monteils, M.P.; Scherman, D. (Centre National de la Recherche Scientifique, Paris (France)); Zahraoui, A.; Tavitian, A. (Institut National de la Sante et de la Recherche Medicale, Paris (France))



Comparative Study on Heat Stability and Functionality of Camel and Bovine Milk Whey Proteins  

Microsoft Academic Search

Heat stability, emulsifying, and foaming properties of camel whey have been investigated and compared with that of bovine whey. Camel whey is similar to bovine whey in composition, but is deficient in ?-lactoglubulin (?-LG), a major component of bovine whey. Whether the deficiency in ?-LG will affect stability and functional properties is not yet known. Substantial information on the functional

L. C. Laleye; B. Jobe; A. A. H. Wasesa



Characterization of cell wall associated proteins of a Staphylococcus aureus isolated from bovine mastitis case by a proteomic approach  

Microsoft Academic Search

Staphylococcus aureus causes different pathologies in humans and animals. In particular, it is involved in intramammary infections in cows, causing economic losses and milk-safety problems. Although it is well-known that surface components (proteins and capsular polysaccharides) and exotoxins are virulence factors involved in the pathogenesis of bovine mastitis, less is known about the precise biochemical identity of such molecules. Therefore,

Francesca Taverna; Armando Negri; Renata Piccinini; Alfonso Zecconi; Simona Nonnis; Severino Ronchi; Gabriella Tedeschi



Effects of Dietary Spray-Dried Bovine Plasma Protein on Broiler Growth Performance and Breast-Meat Yield  

Microsoft Academic Search

SUMMARY Dietary spray-dried plasma protein (SDPP) is effective in improving growth performance of pigs raised in unsanitary conditions. However, little is known about the efficacy of SDPP in improving growth performance and carcass characteristics of broiler chickens. In the present study, graded levels of bovine SDPP (0 to 2% of the diet) were fed to male broiler chickens (Ross 308)

K. Bregendahl; D. U. Ahn; D. W. Trampel; J. M. Campbell



Isothermal Titration Calorimetric Studies on the Interaction of the Major Bovine Seminal Plasma Protein, PDC-109 with Phospholipid Membranes  

PubMed Central

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process.

Anbazhagan, V.; Sankhala, Rajeshwer S.; Singh, Bhanu Pratap; Swamy, Musti J.



A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain.  

PubMed Central

Phosphoprotein phosphatase 2A (PP2A) is one of the four major protein serine/threonine phosphatases found in all eukaryotic cells. We have shown that the 36-kDa catalytic subunit of PP2A is carboxyl methylated in eukaryotic cells, and we have previously identified and purified a novel methyltransferase (MTase) that is responsible for this modification. Here, we describe a novel protein carboxyl methyl-esterase (MEase) from bovine brain that demethylates PP2A. The enzyme has been purified to homogeneity as a monomeric 46-kDa soluble protein. The MEase is highly specific for PP2A. It does not catalyze the demethylation of other protein or peptide methylesters. Moreover, MEase activity is dramatically inhibited by nanomolar concentrations of okadaic acid, a specific inhibitor of PP2A. From these results, we conclude that PP2A methylation is controlled by two specific enzymes, a MTase and a MEase. Since PP2A methylation is highly conserved in eukaryotes ranging from human to yeast, it is likely that this system plays an important role in phosphatase regulation. Images Fig. 2

Lee, J; Chen, Y; Tolstykh, T; Stock, J



Characterization and purification of recombinant bovine viral diarrhea virus particles with epitope-tagged envelope proteins.  


Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus within the family Flaviviridae. The lipid membrane of the virions is supposed to contain the three glycosylated envelope proteins E(rns), E1 and E2, but detailed studies of virus assembly are complicated because no efficient purification method for pestiviruses has been described so far. In this study, we generated infectious BVDV with N-terminally FLAG-tagged E(rns) or E2 proteins, respectively. The expression of the epitope-tagged E(rns) and E2 proteins could be shown by immunofluorescence and Western blot experiments. Furthermore, an affinity tag purification protocol for the isolation and concentration of infectious BVDV was established. In the preparation with a titre of 10(8.75) TCID(50) ml(-1), spherical particles with a diameter of 43-58 nm (mean diameter: 48 nm) could be detected by negative staining electron microscopy, and immunogold labelling located both E(rns) and E2 proteins at the virus membrane. PMID:21346033

Wegelt, Anne; Reimann, Ilona; Granzow, Harald; Beer, Martin



Expression of bovine leukemia virus p24 protein in bacterial cell.  


Bovine leukemia virus (BLV) is a member of the family Retroviridae, genus Deltaretrovirus that has three important gene including gag, pol and env. This virus causes B-cell lymphocytosis and lymphosarcoma in cows. In the first step PCR product of gag gene of BLV isolated in different regions of Iran and BLV-FLK strain were cloned in to a pTZ57R/T vector, then insert were digested by BglII and XhoI restriction enzymes and cloned in to pET-28(a) as an expression vector. For the expression of p24 protein, the pET-28(a) recombinant vector was transformed in BL21(DE3) strain of E. coli competent cell and after induction of the protein having been expressed by IPTG, the presence of gag expressed protein was shown in immunoblotting and SDS-PAGE system. With respect to the remarkable frequency of infection to BLV in Iran and the necessity of controlling it through vaccination with recombinant vaccines of gp51, manufacturing and applying the recombinant p24 protein are vital goals in recognition and distinction between infection and responses caused by vaccine. PMID:19137855

Momtaz, H; Hemmatzadeh, F; Keyvanfar, H



Bovine Viral Diarrhea Virus Core Is an Intrinsically Disordered Protein That Binds RNA?  

PubMed Central

Pestiviruses, including bovine viral diarrhea virus (BVDV), are important animal pathogens and close relatives of hepatitis C virus. Pestivirus particles are composed of an RNA genome, a host-derived lipid envelope, and four virion-encoded structural proteins, core (C), Erns, E1, and E2. Core is a small, highly basic polypeptide that is processed by three enzymatic cleavages before its incorporation into virions. Little is known about its biological properties or its role in virion assembly and structure. We have purified BVDV core protein and characterized it biochemically. We have determined that the processed form of core lacks significant secondary structure and is instead intrinsically disordered. Consistent with its highly basic sequence, we observed that core binds to RNA, although with low affinity and little discernible specificity. We found that BVDV core protein was able to functionally replace the nonspecific RNA binding and condensing region of an unrelated viral capsid protein. Together these results suggest that the in vitro properties of core may reflect its mechanism of action in RNA packaging and virion morphogenesis.

Murray, Catherine L.; Marcotrigiano, Joseph; Rice, Charles M.



Expression in Escherichia coli and purification of soluble forms of the F protein of bovine respiratory syncytial virus.  


Six fragments of the F gene from bovine respiratory syncytial virus (BRSV) were engineered into the pMAL-c2 Escherichia coli expression vector and expressed as C-terminal maltose-binding protein (MBP) fusion products. The resulting polypeptides were partially soluble and single-step purified by affinity chromatography. These fusion proteins were recognized in Western blots by several MAbs directed against human respiratory syncytial virus F protein. In addition, rabbit polyclonal antisera raised against two purified MBP-derived proteins reacted with the BRSV-F protein. PMID:9056494

Naval, J; Piñol, J; Rebordosa, X; Serra-Hartmann, X; Pérez-Pons, J A; Querol, E



Cellular prion protein in the bovine mammary gland is selectively expressed in active lactocytes.  


The cellular prion protein (PrP(c)) is a highly conserved glycoprotein with a still enigmatic physiological function. It is mainly expressed in the central nervous system but accumulating data suggest that PrP(c) is also found in a broad spectrum of non-neuronal tissue. Here we investigated the cell-type-related PrP(c) expression in the bovine mammary gland by using immunohistochemistry (IHC), ELISA, Western blot, and real-time RT-PCR. Specific immunostaining of serial sections revealed that PrP(c) is selectively localized in mammary gland epithelial cells. Particularly strong expression was found at the basolateral surface of those cells showing active secretion. Results obtained by RT-PCR and ELISA complemented IHC findings. No correlation was found between the level of PrP(c) expression and other parameters such as age of the animals under study or stage of lactation. PMID:16864892

Didier, Andrea; Dietrich, Richard; Steffl, Martin; Gareis, Manfred; Groschup, Martin H; Müller-Hellwig, Simone; Märtlbauer, Erwin; Amselgruber, Werner M



Binding of the Inhibitor Protein IF1 to Bovine F1-ATPase  

PubMed Central

In the structure of bovine F1-ATPase inhibited with residues 1–60 of the bovine inhibitor protein IF1, the ?-helical inhibitor interacts with five of the nine subunits of F1-ATPase. In order to understand the contributions of individual amino acid residues to this complex binding mode, N-terminal deletions and point mutations have been introduced, and the binding properties of each mutant inhibitor protein have been examined. The N-terminal region of IF1 destabilizes the interaction of the inhibitor with F1-ATPase and may assist in removing the inhibitor from its binding site when F1Fo-ATPase is making ATP. Binding energy is provided by hydrophobic interactions between residues in the long ?-helix of IF1 and the C-terminal domains of the ?DP-subunit and ?TP-subunit and a salt bridge between residue E30 in the inhibitor and residue R408 in the C-terminal domain of the ?DP-subunit. Several conserved charged amino acids in the long ?-helix of IF1 are also required for establishing inhibitory activity, but in the final inhibited state, they are not in contact with F1-ATPase and occupy aqueous cavities in F1-ATPase. They probably participate in the pathway from the initial interaction of the inhibitor and the enzyme to the final inhibited complex observed in the structure, in which two molecules of ATP are hydrolysed and the rotor of the enzyme turns through two 120° steps. These findings contribute to the fundamental understanding of how the inhibitor functions and to the design of new inhibitors for the systematic analysis of the catalytic cycle of the enzyme.

Bason, John V.; Runswick, Michael J.; Fearnley, Ian M.; Walker, John E.



Acetic Acid Activates the AMP-Activated Protein Kinase Signaling Pathway to Regulate Lipid Metabolism in Bovine Hepatocytes  

PubMed Central

The effect of acetic acid on hepatic lipid metabolism in ruminants differs significantly from that in monogastric animals. Therefore, the aim of this study was to investigate the regulation mechanism of acetic acid on the hepatic lipid metabolism in dairy cows. The AMP-activated protein kinase (AMPK) signaling pathway plays a key role in regulating hepatic lipid metabolism. In vitro, bovine hepatocytes were cultured and treated with different concentrations of sodium acetate (neutralized acetic acid) and BML-275 (an AMPK? inhibitor). Acetic acid consumed a large amount of ATP, resulting in an increase in AMPK? phosphorylation. The increase in AMPK? phosphorylation increased the expression and transcriptional activity of peroxisome proliferator-activated receptor ?, which upregulated the expression of lipid oxidation genes, thereby increasing lipid oxidation in bovine hepatocytes. Furthermore, elevated AMPK? phosphorylation reduced the expression and transcriptional activity of the sterol regulatory element-binding protein 1c and the carbohydrate responsive element-binding protein, which reduced the expression of lipogenic genes, thereby decreasing lipid biosynthesis in bovine hepatocytes. In addition, activated AMPK? inhibited the activity of acetyl-CoA carboxylase. Consequently, the triglyceride content in the acetate-treated hepatocytes was significantly decreased. These results indicate that acetic acid activates the AMPK? signaling pathway to increase lipid oxidation and decrease lipid synthesis in bovine hepatocytes, thereby reducing liver fat accumulation in dairy cows.

Li, Xinwei; Chen, Hui; Guan, Yuan; Li, Xiaobing; Lei, Liancheng; Liu, Juxiong; Yin, Liheng; Liu, Guowen; Wang, Zhe



Analgesic efficacy of sodium salicylate in an amphotericin B-induced bovine synovitis-arthritis model  

Microsoft Academic Search

This study examined the efficacy of sodium salicylate for providing analgesia in an amphotericin B-induced bovine synovitis-arthritis model using 10 male Holstein calves, 4 to 6 mo old and weighing approximately 250 kg. The study used a repeated measures partial crossover design with 2 phases, consisting of 3 treat- ment periods within each phase. Calves were blocked by body weight

JAMIE LEE KOTSCHWAR; J. F. Coetzee; D. E. Anderson; R. Gehring; B. KuKanich; M. D. Apley



Immunosuppressive effect of bovine seminal ribonuclease on a model of corneal transplantation in rabbit  

Microsoft Academic Search

• Background: Bovine seminal ribonuclease (BS RNase) was determined to have a specific suppressive effect on the proliferation of T lymphocytes in vitro. Its immunosuppressive effect was proven in skin grafting in mice as well. • Methods: The immunosuppressive effect of BS RNase was evaluated in tissue cultures and on a model of corneal transplantation in rabbits. The penetration of

Martin Filipec; Zdenka Hašková; Kate?ina Havrlíková; Erik Letko; Vladimír Holá?; Josef Matoušek; Ivan Kalousek



Binding of (/sup 3/H)forskolin to platelet membranes and solubilized proteins from bovine brain  

SciTech Connect

(/sup 3/H)Forskolin ((/sup 3/H)FSK) bound to platelet membranes with a Kd of 20 nM and a Bmax of 125 fmol/mg protein. The Bmax was increased to 400 fmol/mg protein in the presence of GppNHp (or NaF) and MgCl/sub 2/ with no change in Kd. PGE/sub 1/ decreased the EC50 of GppNHp to increase the Bmax for (/sup 3/H)FSK binding from 600 nM to 35 nM. In contrast, PGE/sub 1/ had no effect on the EC50 of NaF to increase (/sup 3/H)FSK binding. (/sup 3/H)FSK binding increased slowly over 60 min when forskolin and GppNHp were added to membranes simultaneously at 20/sup 0/C. Preincubation of membranes with GppNHp at 20/sup 5/C also caused a linear increase in adenylate cyclase specific activity over 60 minutes. (/sup 3/H)FSK bound to solubilized protein from bovine brain membrane with a Kd of 22 nM. GppNHp increased the number of binding sites in solubilized proteins only if membranes were not preincubated with GppNHp prior to solubilization. In conclusion the number of binding sites for (/sup 3/H)FSK is increased by agents that activate adenylate cyclase through the Ns protein. These sites appear to be associated with an activated complex of the Ns protein and adenylate cyclase.

Nelson, C.A.; Seamon, K.B.



Responses of Bovine WC1+ ?? T Cells to Protein and Nonprotein Antigens of Mycobacterium bovis  

PubMed Central

WC1+ ?? T cells of Mycobacterium bovis-infected cattle are highly responsive to M. bovis sonic extract (MBSE). In mycobacterial infections of other species, ?? T cells have been shown to respond to protein and nonprotein antigens, but the bovine WC1+ ?? T-cell antigenic targets within MBSE require further definition in terms of the dominance of protein versus nonprotein components. The present study sought to characterize the WC1+ ?? T-cell antigenic targets, together with the role of interleukin-2 (IL-2), in the context of M. bovis infection. This was achieved by testing crude and defined antigens to assess protein versus nonprotein recognition by WC1+ ?? T cells in comparison with CD4+ ?? T cells. Both cell types proliferated strongly in response to MBSE, with CD4+ T cells being the major producers of gamma interferon (IFN-?). However, enzymatic digestion of the protein in MBSE removed its ability to stimulate CD4+ T-cell responses, whereas some WC1+ ?? T-cell proliferation remained. The most antigenic protein inducing proliferation and IFN-? secretion in WC1+ ?? T-cell cultures was found to be ESAT-6, which is a potential novel diagnostic reagent and vaccine candidate. In addition, WC1+ ?? T-cell proliferation was observed in response to stimulation with prenyl pyrophosphate antigens (isopentenyl pyrophosphate and monomethyl phosphate). High levels of cellular activation (CD25 expression) resulted from MBSE stimulation of WC1+ ?? T cells from infected animals. A similar degree of activation was induced by IL-2 alone, but for WC1+ ?? T-cell division IL-2 was found to act only as a costimulatory signal, enhancing antigen-driven responses. Overall, the data indicate that protein antigens are important stimulators of WC1+ ?? T-cell proliferation and IFN-? secretion in M. bovis infection, with nonprotein antigens inducing significant proliferation. These findings have important implications for diagnostic and vaccine development.

Welsh, Michael D.; Kennedy, Hilary E.; Smyth, Allister J.; Girvin, R. Martyn; Andersen, Peter; Pollock, John M.



Effect of Bovine Papillomavirus E2 Protein-Specific Monoclonal Antibodies on Papillomavirus DNA Replication  

PubMed Central

The bovine papillomavirus type 1 (BPV-1) E2 protein is the master regulator of papillomavirus replication and transcription. We have raised a panel of monoclonal antibodies (MAbs) against the BPV-1 E2 protein and used them to probe the structure and function of the protein. Five MAbs reacted with linear epitopes, and four MAbs recognized conformation-dependent epitopes which mapped within the C-terminal DNA-binding and dimerization domain. MAb 1E2 was able to recognize the replication- and transactivation-defective but not the competent conformation of the transactivation domain of the E2 protein. MAb 5H4 prevented efficiently the formation of E2-DNA as well as E2-dependent E1-E2-origin complexes and also dissociated preformed complexes in a concentration-dependent manner. Cotransfection of several MAbs with the BPV-1 minimal origin plasmid pUCAlu into CHO4.15 cells resulted in a dose-dependent inhibition of replication. Inhibition of replication by MAb 5H4 and the Fab? fragment of 5H4 correlated with their ability to dissociate the E2 protein from the DNA. MAb 3F12 and MAbs 1H10 and 1E4, directed against the hinge region, were also capable of inhibiting BPV-1 origin replication in CHO4.15 cells. However, the Fab? fragments of 1H10 and 3F12 had no effect in the transient replication assay. These data suggest that MAbs directed against the hinge region sterically hinder the inter- or intramolecular interactions required for the replication activity of the E2 protein.

Kurg, Reet; Parik, Juri; Juronen, Erkki; Sedman, Tiina; Abroi, Aare; Liiv, Ingrid; Langel, Ulo; Ustav, Mart



Identification and zinc dependence of the bovine herpesvirus 1 transactivator protein BICP0.  


Bovine herpesvirus 1 (BHV-1) specifies and unspliced early 2.6-kb RNA (ER2.6) which is 3' coterminal with exon 2 of the 2.9-kb immediate-early (IE) RNA. The two transcripts have a common open reading frame (676 codons). The predicted protein, designated BHV-1 infected cell protein 0 (BICP0), contains a zinc finger domain with homology to ICP0 of herpes simplex virus type 1 and protein 61 of varicella-zoster virus, and depending on the promoter, it acts as a strong activator or as a repressor in transient expression assays. In situ immunoadsorbent assays using antisera against synthetic oligopeptides demonstrated that BICP0 accumulates in nuclei of BHV-1-infected cells, as expected for an IE gene product involved in gene regulation. Western blots (immunoblots) revealed a BHV-1-specific 97-kDa protein which was detectable during the IE phase and also at later periods of infection, indicating that the kinetics of BICP0 synthesis is consistent with the switch from IER2.9 to ER2.6. To confirm that ER2.6 encoded the 97-kDa BICP0 protein, a DNA fragment containing BICP0-coding sequences was inserted into the Autographa californica baculovirus genome. A recombinant protein, identified by its reactivity with antipeptide sera, exhibited the same electrophoretic mobility as BICP0 specified by BHV-1. We microinjected Xenopus oocytes with a BICP0 effector plasmid and a promoter-chloramphenicol acetyltransferase plasmid. BICP0-induced stimulation of this promoter was strongly reduced when intracellular zinc was chelated by thionein, indicating that the effect of BICP0 is zinc dependent. PMID:8151780

Fraefel, C; Zeng, J; Choffat, Y; Engels, M; Schwyzer, M; Ackermann, M



Localization of non-conventional protein kinase C isoforms in bovine brain cell nuclei.  

PubMed Central

Using Western blotting and immunofluorescence microscopy we detected the protein kinase C isoforms delta, epsilon and zeta in isolated cell nuclei from bovine cerebral cortex. Both protein kinase C (PKC) delta and PKC epsilon are present in higher concentrations in neuronal than in glial nuclei and are located inside the nucleus and at the nuclear envelope. There they give a punctate staining in immunofluorescence microscopy. PKC zeta is also present both in the nucleoplasm and at the nuclear envelope. PKC eta could not be detected in the cell nuclei and, even in the homogenate of cerebral cortex, this isoform is present only in very low concentrations. The antibody against PKC eta bound strongly to a nucleoplasmic protein with an apparent molecular mass of 99 kDa. The localization of non-conventional PKC isoforms at the cell nucleus strongly indicates that these isoforms are directly involved in the regulation of nuclear processes. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

Rosenberger, U; Shakibaei, M; Buchner, K



Biophysical investigations on the interaction of the major bovine seminal plasma protein, PDC-109, with heparin.  


PDC-109, the major bovine seminal plasma protein, binds to sperm plasma membrane and modulates capacitation in the presence of heparin. In view of this, the PDC-109/heparin interaction has been investigated employing various biophysical approaches. Isothermal titration calorimetric studies yielded the association constant and changes in enthalpy and entropy for the interaction at 25 °C (pH 7.4) as 1.92 (±0.2) × 10(5) M(-1), 18.6 (±1.6) kcal M(-1), and 86.5 (±5.1) cal M(-1) K(-1), respectively, whereas differential scanning calorimetric studies indicated that heparin binding results in a significant increase in the thermal stability of PDC-109. The affinity decreases with increase in pH and ionic strength, consistent with the involvement of electrostatic forces in this interaction. Circular dichroism spectroscopic studies indicated that PDC-109 retains its conformational features even up to 70-75 °C in the presence of heparin, whereas the native protein unfolds at about 55 °C. Atomic force microscopic studies demonstrated that large oligomeric structures are formed upon binding of PDC-109 to heparin, indicating an increase in the local density of the protein, which may be relevant to the ability of heparin to potentiate PDC-109 induced sperm capacitation. PMID:21939260

Sankhala, Rajeshwer S; Damai, Rajani S; Anbazhagan, V; Kumar, C Sudheer; Bulusu, Gopalakrishnan; Swamy, Musti J



[Role of protein kinases in prolactin signaling in bovine oocyte-cumulus complexes].  


Mechanisms of prolactin signal transduction in generative and somatic cells of mammalian ovarian follicles have been studied only to a small extent. In the present work, the involvement oftyrosine kinases and protein kinase C in mediating of the previously revealed modulating effects of prolactin on the nuclear maturation of bovine oocytes and the morphologic-functional state of surrounding cumulus cells was investigated in vitro. Tyrosine kinase inhibitor, genistein, was found to suppress the stimulating action of prolactin on the completion of oocyte nuclear maturation and cumulus expansion, whereas protein kinase C inhibitor, calpostin C, did not affect these hormonal effects. Furthermore, both genistein and calpostin C inhibited the inducing influence of prolactin on the proliferative activity of cumulus cells. At the same time the retarding action ofprolactin on destructive processes in cumulus cells was blocked only in the presence of calpostin C. The results of the study suggest that the stimulatory influence of prolactin on oocyte nuclear maturation and attendant cumulus expansion is achieved with the participation of tyrosine kinases, whereas the modulating action of the hormone on the functional state of cumulus cells depends on activation of not only tyrosine kinases, but also protein kinase C. PMID:21961288

Lebedeva, I Iu; Singina, G N; Tarada?nik, T E; Zinov'eva, N A



Two bovine models of osteogenesis imperfecta exhibit decreased apatite crystal size  

Microsoft Academic Search

Summary  In recent years advances have been made in detailing the changes in both collagen and noncollagenous proteins caused by a\\u000a variety of mutations leading to osteogenesis imperfecta. Much less, however, is known about the mineral phase in the affected\\u000a bone. In this report, we measured the crystallinity of the apatite in bovine OI bone. Line broadening of the 002 reflection

L. W. Fisher; E. D. Eanes; L. J. Denholm; B. R. Heywood; J. D. Termine



The proteomic advantage: label-free quantification of proteins expressed in bovine milk during experimentally induced coliform mastitis.  


Coliform mastitis remains a primary focus of dairy cattle disease research due in part to the lack of efficacious treatment options for the deleterious side effects of exposure to LPS, including profound intra-mammary inflammation. To facilitate new veterinary drug approvals, reliable biomarkers are needed to evaluate the efficacy of adjunctive therapies for the treatment of inflammation associated with coliform mastitis. Most attempts to characterize the host response to LPS, however, have been accomplished using ELISAs. Because a relatively limited number of bovine-specific antibodies are commercially available, reliance on antibodies can be very limiting for biomarker discovery. Conversely, proteomic approaches boast the capability to analyze an unlimited number of protein targets in a single experiment, independent of antibody availability. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), a widely used proteomic strategy for the identification of proteins in complex mixtures, has gained popularity as a means to characterize proteins in various bovine milk fractions, both under normal physiological conditions as well as during clinical mastitis. The biological complexity of bovine milk has, however, precluded the complete annotation of the bovine milk proteome. Conventional approaches to reducing sample complexity, including fractionation and the removal of high abundance proteins, has improved proteome coverage, but the dynamic range of proteins present, and abundance of a relatively small number of proteins, continues to hinder comparative proteomic analyses of bovine milk. Nonetheless, advances in both liquid chromatography and mass spectrometry instrumentation, including nano-flow liquid chromatography (nano-LC), nano-spray ionization, and faster scanning speeds and ionization efficiency of mass spectrometers, have improved analyses of complex samples. In the current paper, we review the proteomic approaches used to conduct comparative analyses of milk from healthy cows and cows with clinical mastitis, as well as proteins related to the host response that have been identified in mastitic milk. Additionally, we present data that suggests the potential utility of LC-MS/MS label-free quantification as an alternative to costly labeling strategies for the relative quantification of individual proteins in complex mixtures. Temporal expression patterns generated using spectral counts, an LC-MS/MS label-free quantification strategy, corresponded well with ELISA data for acute phase proteins with commercially available antibodies. Combined, the capability to identify low abundance proteins, and the potential to generate temporal expression profiles, indicate the advantages of using proteomics as a screening tool in biomarker discovery analyses to assess biologically relevant proteins modulated during disease, including previously uncharacterized targets. PMID:21067814

Boehmer, Jamie L; DeGrasse, Jeffrey A; McFarland, Melinda A; Tall, Elizabeth A; Shefcheck, Kevin J; Ward, Jeffrey L; Bannerman, Douglas D



Multiple protein biomarker assessment for recombinant bovine somatotropin (rbST) abuse in cattle.  


Biomarker profiling, as a rapid screening approach for detection of hormone abuse, requires well selected candidate biomarkers and a thorough in vivo biomarker evaluation as previously done for detection of growth hormone doping in athletes. The bovine equivalent of growth hormone, called recombinant bovine somatotropin (rbST) is (il)legally administered to enhance milk production in dairy cows. In this study, first a generic sample pre-treatment and 4-plex flow cytometric immunoassay (FCIA) were developed for simultaneous measurement of four candidate biomarkers selected from literature: insulin-like growth factor 1 (IGF-1), its binding protein 2 (IGFBP2), osteocalcin and endogenously produced antibodies against rbST. Next, bovine serum samples from two extensive controlled rbST animal treatment studies were used for in vivo validation and biomarker evaluation. Finally, advanced statistic tools were tested for the assessment of biomarker combination quality aiming to correctly identify rbST-treated animals. The statistical prediction tool k-nearest neighbours using a combination of the biomarkers osteocalcin and endogenously produced antibodies against rbST proved to be very reliable and correctly predicted 95% of the treated samples starting from the second rbST injection until the end of the treatment period and even thereafter. With the same biomarker combination, only 12% of untreated animals appeared false-positive. This reliability meets the requirements of Commission Decision 2002/657/EC for screening methods in veterinary control. From the results of this multidisciplinary study, it is concluded that the osteocalcin - anti-rbST-antibodies combination represent fit-for-purpose biomarkers for screening of rbST abuse in dairy cattle and can be reliably measured in both the developed 4-plex FCIA as well as in a cost-effective 2-plex microsphere-based binding assay. This screening method can be incorporated in routine veterinary monitoring programmes: in the European Union for detection of rbST abuse and in the control of rbST-free dairy farms in the United States of America and other countries. PMID:23300820

Ludwig, Susann K J; Smits, Nathalie G E; van der Veer, Grishja; Bremer, Maria G E G; Nielen, Michel W F



Multiple Protein Biomarker Assessment for Recombinant Bovine Somatotropin (rbST) Abuse in Cattle  

PubMed Central

Biomarker profiling, as a rapid screening approach for detection of hormone abuse, requires well selected candidate biomarkers and a thorough in vivo biomarker evaluation as previously done for detection of growth hormone doping in athletes. The bovine equivalent of growth hormone, called recombinant bovine somatotropin (rbST) is (il)legally administered to enhance milk production in dairy cows. In this study, first a generic sample pre-treatment and 4-plex flow cytometric immunoassay (FCIA) were developed for simultaneous measurement of four candidate biomarkers selected from literature: insulin-like growth factor 1 (IGF-1), its binding protein 2 (IGFBP2), osteocalcin and endogenously produced antibodies against rbST. Next, bovine serum samples from two extensive controlled rbST animal treatment studies were used for in vivo validation and biomarker evaluation. Finally, advanced statistic tools were tested for the assessment of biomarker combination quality aiming to correctly identify rbST-treated animals. The statistical prediction tool k-nearest neighbours using a combination of the biomarkers osteocalcin and endogenously produced antibodies against rbST proved to be very reliable and correctly predicted 95% of the treated samples starting from the second rbST injection until the end of the treatment period and even thereafter. With the same biomarker combination, only 12% of untreated animals appeared false-positive. This reliability meets the requirements of Commission Decision 2002/657/EC for screening methods in veterinary control. From the results of this multidisciplinary study, it is concluded that the osteocalcin – anti-rbST-antibodies combination represent fit-for-purpose biomarkers for screening of rbST abuse in dairy cattle and can be reliably measured in both the developed 4-plex FCIA as well as in a cost-effective 2-plex microsphere-based binding assay. This screening method can be incorporated in routine veterinary monitoring programmes: in the European Union for detection of rbST abuse and in the control of rbST-free dairy farms in the United States of America and other countries.

Ludwig, Susann K. J.; Smits, Nathalie G. E.; van der Veer, Grishja; Bremer, Maria G. E. G.; Nielen, Michel W. F.



Structure and Dynamics of the Solvation of Bovine Pancreatic Trypsin Inhibitor in Explicit Water: A Comparative Study of the Effects of Solvent and Protein Polarizability  

SciTech Connect

The research described in this product was performed in part in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. To isolate the effects of the inclusion of polarizability in the force field model on the structure and dynamics of the solvating water in differing electrostatic environments of proteins, we present the results of molecular dynamics simulations of the bovine pancreatic trypsin inhibitor (BPTI) in water with force fields that explicitly include polarization for both the protein and the water. We use three model potentials for water and two model potentials for the protein. Two of the water models and one of the protein models are polarizable. A total of six systems were simulated representing all combinations of these polarizable and nonpolarizable protein and water force fields. We find that all six systems behave in a similar manner in regions of the protein that are weakly electrostatic (either hydrophobic or weakly hydrophilic). However, in the vicinity of regions of the protein with relatively strong electrostatic fields (near positively or negatively charged residues), we observe that the water structure and dynamics are dependent on both the model of the protein and the model of the water. We find that a large part of the dynamical dependence can be described by small changes in the local environments of each region that limit the local density of non-hydrogen-bonded waters, precisely the water molecules that facilitate the dynamical relaxation of the water-water hydrogen bonds. We introduce a simple method for rescaling for this effect. When this is done, we are able to effectively isolate the influence of polarizability on the dynamics. We find that the solvating water’s relaxation is most affected when both the protein and the water models are polarizable. However, when only one model (or neither) is polarizable, the relaxation is similar regardless of the models used.

Kim, Byoung Chan; Young, Tom; Harder, Edward; Friesner, Richard A.; Berne, Bruce J.



Finding biomarkers in non-model species: literature mining of transcription factors involved in bovine embryo development  

PubMed Central

Background Since processes in well-known model organisms have specific features different from those in Bos taurus, the organism under study, a good way to describe gene regulation in ruminant embryos would be a species-specific consideration of closely related species to cattle, sheep and pig. However, as highlighted by a recent report, gene dictionaries in pig are smaller than in cattle, bringing a risk to reduce the gene resources to be mined (and so for sheep dictionaries). Bioinformatics approaches that allow an integration of available information on gene function in model organisms, taking into account their specificity, are thus needed. Besides these closely related and biologically relevant species, there is indeed much more knowledge of (i) trophoblast proliferation and differentiation or (ii) embryogenesis in human and mouse species, which provides opportunities for reconstructing proliferation and/or differentiation processes in other mammalian embryos, including ruminants. The necessary knowledge can be obtained partly from (i) stem cell or cancer research to supply useful information on molecular agents or molecular interactions at work in cell proliferation and (ii) mouse embryogenesis to supply useful information on embryo differentiation. However, the total number of publications for all these topics and species is great and their manual processing would be tedious and time consuming. This is why we used text mining for automated text analysis and automated knowledge extraction. To evaluate the quality of this “mining”, we took advantage of studies that reported gene expression profiles during the elongation of bovine embryos and defined a list of transcription factors (or TF, n?=?64) that we used as biological “gold standard”. When successful, the “mining” approach would identify them all, as well as novel ones. Methods To gain knowledge on molecular-genetic regulations in a non model organism, we offer an approach based on literature-mining and score arrangement of data from model organisms. This approach was applied to identify novel transcription factors during bovine blastocyst elongation, a process that is not observed in rodents and primates. As a result, searching through human and mouse corpuses, we identified numerous bovine homologs, among which 11 to 14% of transcription factors including the gold standard TF as well as novel TF potentially important to gene regulation in ruminant embryo development. The scripts of the workflow are written in Perl and available on demand. They require data input coming from all various databases for any kind of biological issue once the data has been prepared according to keywords for the studied topic and species; we can provide data sample to illustrate the use and functionality of the workflow. Results To do so, we created a workflow that allowed the pipeline processing of literature data and biological data, extracted from Web of Science (WoS) or PubMed but also from Gene Expression Omnibus (GEO), Gene Ontology (GO), Uniprot, HomoloGene, TcoF-DB and TFe (TF encyclopedia). First, the human and mouse homologs of the bovine proteins were selected, filtered by text corpora and arranged by score functions. The score functions were based on the gene name frequencies in corpora. Then, transcription factors were identified using TcoF-DB and double-checked using TFe to characterise TF groups and families. Thus, among a search space of 18,670 bovine homologs, 489 were identified as transcription factors. Among them, 243 were absent from the high-throughput data available at the time of the study. They thus stand so far for putative TF acting during bovine embryo elongation, but might be retrieved from a recent RNA sequencing dataset (Mamo et al. , 2012). Beyond the 246 TF that appeared expressed in bovine elongating tissues, we restricted our interpretation to those occurring within a list of 50 top-ranked genes. Among the transcription factors identified therein, half belonged to the gold standard (ASCL2, c-F



Gz- and not Gi-proteins are coupled to pre-junctional ?-opioid receptors in bovine airways.  


We investigated the signal transmission pathway by which activation of ?-opioid receptors attenuates acetylcholine (ACh) release in bovine trachealis. Electrical stimulation (ES)-induced [(3)H]-ACh release was determined in bovine tracheal smooth muscle strips pre-incubated with either the Gi-protein inhibitor pertussis toxin (PTX, 500ng/ml and 1?g/ml) or the Gz-protein specific inhibitor arachidonic acid (AA, 10(-6)M and 10(-5)M) and then treated with DAMGO (D-Ala(2),N-MePhe(4),Gly-ol(5)-enkephalin) 10(-5)M. Indomethacin 10(-5)M was used to block AA cascade. The inhibitory effect of DAMGO on ES-induced [(3)H]-ACh release was PTX-insensitive, but, by contrast, ablated by AA in a concentration-dependent manner. AA 10(-5)M alone reduced [(3)H]-ACh release, an effect that was prevented by iberiotoxin 10(-7)M, suggesting an involvement of Ca(2+)-activated K(+)-channels. Western blot analysis consistently showed immunoreactive bands against a specific antibody anti-Gz-? subunit at ?40kDa, consistent with the presence of Gz-protein. The present findings suggest that in isolated bovine trachealis, activation of ?-opioid receptors inhibits ACh-release through a signal transmission pathway involving Gz-protein rather than Gi-protein. PMID:23911590

Baroffio, Michele; Brichetto, Lorenzo; Franco, Luisa; Crimi, Emanuele; Rehder, Kai; Brusasco, Vito



Scavenger receptor-mediated recognition of maleyl bovine plasma albumin and the demaleylated protein in human monocyte macrophages  

SciTech Connect

Maleyl bovine plasma albumin competed on an equimolar basis with malondialdehyde low density lipoprotein (LDL) in suppressing the lysosomal hydrolysis of SVI-labeled malondialdehyde LDL mediated by the scavenger receptor of human monocyte macrophages. Maleyl bovine plasma albumin, in which 94% of the amino groups were modified, exhibited an anodic mobility in agarose electrophoresis 1.7 times that of the native protein. Incubation of maleyl bovine plasma albumin at pH 3.5 regenerated the free amino groups and restored the protein to the same electrophoretic mobility as native albumin. Although ligands recognized by the scavenger receptor typically are anionic, the authors propose that addition of new negative charge achieved by maleylation, rather than directly forming the receptor binding site(s), induces conformational changes in albumin as a prerequisite to expression of the recognition domain(s). They conclude that the primary sequence of albumin, rather than addition of new negative charge, provides the recognition determinant(s) essential for interaction of maleyl bovine plasma albumin with the scavenger receptor.

Haberland, M.E.; Fogelman, A.M.



Effective in vitro expansion of CD40-activated human B lymphocytes in a defined bovine protein-free medium.  


CD40-CD154 interaction is used to culture human B lymphocytes, which are now viewed as effectors to potentially promote T lymphocyte response against malignant cells in cell-based therapy. Currently, the media used, based on bovine serum, are raising concerns for patient safety in such therapy. In this study, we have investigated whether human B lymphocytes could be cultured in the absence of bovine serum. Blood CD19(+) B lymphocytes were activated using interaction through CD40 in medium containing defined animals or human proteins and lipids, and were monitored during short-term periods (?15 days). Conventional stem-cell medium and a medium containing human albumin instead of bovine albumin were tested. We observed that the response of B lymphocytes appeared influenced by lot-to-lot variability in low density lipoproteins (LDL). Nevertheless, B lymphocyte proliferation and secretion in serum-free and bovine protein-free media were quite similar to that of cells cultured in medium containing FBS. Our results show that CD40-activated B lymphocytes can be cultured for up to 15 days in a serum-free medium containing human albumin, LDL, ?-tocopherol and chemically-defined lipids. PMID:21723869

Néron, Sonia; Roy, Annie; Dumont, Nellie; Dussault, Nathalie



Identification of a Prion Protein Epitope Modulating Transmission of Bovine Spongiform Encephalopathy Prions to Transgenic Mice  

Microsoft Academic Search

There is considerable concern that bovine prions from cattle with bovine spongiform encephalopathy (BSE) may have been passed to humans (Hu), resulting in a new form of Creutzfeldt-Jakob disease (CJD). We report here the transmission of bovine (Bo) prions to transgenic (Tg) mice expressing BoPrP; one Tg line exhibited incubation times of ≈ 200 days. Like most cattle with BSE,

Michael R. Scott; Jiri Safar; Glenn Telling; Oanh Nguyen; Darlene Groth; Marilyn Torchia; Ruth Koehler; Patrick Tremblay; Dirk Walther; Fred E. Cohen; Stephen J. Dearmond



Phosphorylation of bovine leukemia virus Tax protein is required for in vitro transformation but not for transactivation.  


The Tax proteins of the oncovirinae viruses are phosphorylated transcriptional activators that exhibit oncogenic potential. The role of phosphorylation in their functional activities remains unknown. As a model for the Human T-cell leukemia virus type I (HTLV-I), Bovine Leukemia Virus (BLV) permits the characterization of viral replication and leukemogenesis in vivo. Here, we show that the BLV Tax protein is phosphorylated on serine residues 106 and 293 both in insect and in mammalian cells. These sites can also be efficiently phosphorylated by the cdc2 and MAP kinases in vitro. Mutation of these residues does not affect the capacity of the Tax protein to function as a transactivator. Indeed, the Tax proteins mutated at one or both serines increase LTR-directed viral transcription at levels similar to those obtained with wild-type Tax in cell culture. Moreover, inhibition of Tax phosphorylation by W7, a calmodulin antagonist, does not alter its transactivation activity. Thus, phosphorylation on serines 106 and 293 is not required for transactivation by Tax. However, simultaneous substitution of both serines into alanine residues destroys the capacity of Tax to cooperate with the Ha-ras oncogene to transform primary rat embryo fibroblasts and induce tumors in nude mice. When the serines were replaced with aspartic acid residues, the oncogenic potential of Tax was maintained indicating that the negative charge rather than the phosphate group itself was required for Tax oncogenicity. Finally, to assess the role of the serine residues in vivo, recombinant viruses which express the Tax mutants were constructed and injected into sheep. It appeared that the mutated proviruses replicate at levels similar to the wild-type virus in vivo. We conclude that Tax phosphorylation is dispensable for transactivation and viral replication in vivo but is required for its oncogenic potential in vitro. PMID:9619825

Willems, L; Grimonpont, C; Kerkhofs, P; Capiau, C; Gheysen, D; Conrath, K; Roussef, R; Mamoun, R; Portetelle, D; Burny, A; Adam, E; Lefèbvre, L; Twizere, J C; Heremans, H; Kettmann, R



Subcellular Localization of the Bovine Leukemia Virus R3 and G4 Accessory Proteins  

PubMed Central

Bovine leukemia virus (BLV) is a complex retrovirus that belongs to the Deltaretrovirus genus, which also includes Human T-cell leukemia virus type 1 (HTLV-1). Both viruses contain an X region coding for at least four proteins: Tax and Rex, which are involved in transcriptional and posttranscriptional regulation, respectively, and the accessory proteins R3 and G4 (for BLV) and p12I, p13II, and p30II (for HTLV-1). The present study was aimed at characterizing the subcellular localization of BLV R3 and G4. The results of immunofluorescence experiments on transfected HeLa Tat cells demonstrated that R3 is located in the nucleus and in cellular membranes, as previously reported for HTLV-1 p12I. In contrast, G4, like p13II, is localized both in the nucleus and in mitochondria. In addition, we have shown that G4 harbors a mitochondrial targeting signal consisting of a hydrophobic region and an amphipathic ?-helix. Thus, despite a lack of significant primary sequence homology, R3 and p12I and G4 and p13II exhibit similar targeting properties, suggesting possible overlap in their functional properties.

Lefebvre, Laurent; Ciminale, Vincenzo; Vanderplasschen, Alain; D'Agostino, Donna; Burny, Arsene; Willems, Luc; Kettmann, Richard



Unique quadruplex structure and interaction of an RNA aptamer against bovine prion protein  

PubMed Central

RNA aptamers against bovine prion protein (bPrP) were obtained, most of the obtained aptamers being found to contain the r(GGAGGAGGAGGA) (R12) sequence. Then, it was revealed that R12 binds to both bPrP and its ?-isoform with high affinity. Here, we present the structure of R12. This is the first report on the structure of an RNA aptamer against prion protein. R12 forms an intramolecular parallel quadruplex. The quadruplex contains G:G:G:G tetrad and G(:A):G:G(:A):G hexad planes. Two quadruplexes form a dimer through intermolecular hexad–hexad stacking. Two lysine clusters of bPrP have been identified as binding sites for R12. The electrostatic interaction between the uniquely arranged phosphate groups of R12 and the lysine clusters is suggested to be responsible for the affinity of R12 to bPrP. The stacking interaction between the G:G:G:G tetrad planes and tryptophan residues may also contribute to the affinity. One R12 dimer molecule is supposed to simultaneously bind the two lysine clusters of one bPrP molecule, resulting in even higher affinity. The atomic coordinates of R12 would be useful for the development of R12 as a therapeutic agent against prion diseases and Alzheimer's disease.

Mashima, Tsukasa; Matsugami, Akimasa; Nishikawa, Fumiko; Nishikawa, Satoshi; Katahira, Masato



Characterization of lipid-protein interactions in acetylcholinesterase lipoprotein extracted from bovine erythrocytes.  

PubMed Central

Acetylcholinesterase was released from bovine erythrocytes in hypo-osmotic sodium phosphate buffer. Initially, about 30% of the enzyme was released in a soluble lipoprotein form, and further incubation resulted in the progressive release of the enzyme in a particulate form. Solubilization of the acetylcholinesterase in the particulate fraction with Lubrol WX (2 mg/ml) resulted in the loss of all lipids except a non-exchangeable fraction identified as cardiolipin. Addition of a mixture of erythrocyte phospholipids to the soluble forms and to the Lubrol WX-solubilized enzyme resulted in the formation of particulate forms of the enzyme with increased partial specific volume and Stokes radius, and a break in the Arrhenius plot of the enzyme activity around 20 degrees C. The break in the Arrhenius plot was abolished by treatment of a soluble enzyme preparation with 1.8 M salt (NaCl) in phosphate buffer, conditions that allowed the extraction of cardiolipin from the enzyme by chloroform/methanol. Failure of the high-salt treatment to decrease the Stokes radius made it unlikely that the bound cardiolipin formed a boundary layer or annulus around the protein. It is suggested that cardiolipin is bound to the core of the dimeric protein structure, thereby controlling the acetylcholinesterase activity.

Beauregard, G; Roufogalis, B D



Effects of bovine serum proteins in culture medium on post-warming survival of bovine blastocysts developed in vitro.  


Experiments were conducted to investigate the factors affecting the survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. Zygotes were obtained by in vitro maturation and fertilization of oocytes. Embryos used in this study were developed in vitro at Day 7 and 8 (Day 0 = insemination day) in modified synthetic oviduct fluid medium supplemented with calf serum or BSA. Embryos were cryopreserved in a two-step protocol consisting of exposure to 10% ethylene glycol for 5 min, followed by the original vitrification solution (designated as VS) consisting of 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline for 1 min. After warming, embryos were cultured in modified TCM-199 for an in vitro survival assay. The highest survival rate was obtained from the warmed embryos developed at Day 7 in medium supplemented with BSA (82.6%), and there were significant differences between results with calf scrum and BSA treatment (42.4 and 70.7%, respectively; P < 0.01). However, there were no significant differences in the cell numbers of embryos among the treatments. These results suggest that the survival of embryos developed in medium with BSA is superior to that of embryos developed in medium containing calf serum, although the cell numbers of the embryos developed under both media were similar. PMID:16728072

Ohboshi, S; Etoh, T; Sakamoto, K; Fujihara, N; Yoshida, T; Tomogane, H



A Bovine Model of Respiratory Chlamydia psittaci Infection: Challenge Dose Titration  

PubMed Central

This study aimed to establish and evaluate a bovine respiratory model of experimentally induced acute C. psittaci infection. Calves are natural hosts and pathogenesis may resemble the situation in humans. Intrabronchial inoculation of C. psittaci strain DC15 was performed in calves aged 2–3 months via bronchoscope at four different challenge doses from 106 to 109 inclusion-forming units (ifu) per animal. Control groups received either UV-inactivated C. psittaci or cell culture medium. While 106 ifu/calf resulted in a mild respiratory infection only, the doses of 107 and 108 induced fever, tachypnea, dry cough, and tachycardia that became apparent 2–3 days post inoculation (dpi) and lasted for about one week. In calves exposed to 109 ifu C. psittaci, the respiratory disease was accompanied by severe systemic illness (apathy, tremor, markedly reduced appetite). At the time point of most pronounced clinical signs (3 dpi) the extent of lung lesions was below 10% of pulmonary tissue in calves inoculated with 106 and 107 ifu, about 15% in calves inoculated with 108 and more than 30% in calves inoculated with 109 ifu C. psittaci. Beside clinical signs and pathologic lesions, the bacterial load of lung tissue and markers of pulmonary inflammation (i.e., cell counts, concentration of proteins and eicosanoids in broncho-alveolar lavage fluid) were positively associated with ifu of viable C. psittaci. While any effect of endotoxin has been ruled out, all effects could be attributed to infection by the replicating bacteria. In conclusion, the calf represents a suitable model of respiratory chlamydial infection. Dose titration revealed that both clinically latent and clinically manifest infection can be reproduced experimentally by either 106 or 108 ifu/calf of C. psittaci DC15 while doses above 108 ifu C. psittaci cannot be recommended for further studies for ethical reasons. This defined model of different clinical expressions of chlamydial infection allows studying host-pathogen interactions.

Reinhold, Petra; Ostermann, Carola; Liebler-Tenorio, Elisabeth; Berndt, Angela; Vogel, Anette; Lambertz, Jacqueline; Rothe, Michael; Ruttger, Anke; Schubert, Evelyn; Sachse, Konrad



Human Milk Protein Production in Xenografts of Genetically Engineered Bovine Mammary Epithelial Stem Cells  

Microsoft Academic Search

BackgroundIn the bovine species milk production is well known to correlate with mammary tissue mass. However, most advances in optimizing milk production relied on improvements of breeding and husbandry practices. A better understanding of the cells that generate bovine mammary tissue could facilitate important advances in milk production and have global economic impact. With this possibility in mind, we show

Eugenio Martignani; Peter Eirew; Paolo Accornero; Connie J. Eaves; Mario Baratta; Joseph Najbauer



Nucleotide Sequence of a Bovine Clone Encoding the Angiogenic Protein, Basic Fibroblast Growth Factor  

Microsoft Academic Search

Basic and acidic fibroblast growth factors (FGF's) are potent mitogens for capillary endothelial cells in vitro, stimulate angiogenesis in vivo, and may participate in tissue repair. An oligonucleotide probe for bovine basic FGF was designed from the nucleotide sequence of the amino-terminal exon of bovine acidic FGF, taking into account the 55 percent amino acid sequence homology between the two

Judith A. Abraham; Ayalew Mergia; Jacqueline L. Whang; Annette Tumolo; Jeff Friedman; Kathryn A. Hjerrild; Denis Gospodarowicz; John C. Fiddes



Myristoyl-CoA:Protein N-Myristoyltransferase from Bovine Cardiac Muscle: Molecular Cloning, Kinetic Analysis, and in VitroProteolytic Cleavage by m-Calpain  

Microsoft Academic Search

Myristoyl-CoA:proteinN-myristoyltransferase (NMT) catalyzes the attachment of myristate onto the amino terminal glycine residue of select polypeptides. Cardiac tissue expresses high levels of cAMP-dependent protein kinase whose catalytic subunit is myristoylated; however, cardiac muscle extracts were found to contain low NMT activities. Northern blot analysis of bovine heart poly(A)+RNA probed with bovine spleen NMT cDNA revealed a 1.7-kb mRNA. Western blot

Rajala V. S. Raju; Rakesh Kakkar; Raju S. S. Datla; Jasim Radhi; Rajendra K. Sharma



Distance-constraint approach to protein folding. II. Prediction of three-dimensional structure of bovine pancreatic trypsin inhibitor  

Microsoft Academic Search

The possibility of predicting the three-dimensional structure of bovine pancreatic trypsin inhibitor (BPTI) is examined using a distance-constraint approach. The mean distances between the amino acid residues in globular proteins, calculated in a previous paper, are utilized as distance constraints. In this study, as in previous work of others, root-mean-square deviations of the predicted conformations from the native one of

Hiroshi Wako; Harold A. Scheraga



Binding characteristics of bovine serum albumin encapsulated in sol-gel glasses: An alternative for protein interaction studies  

Microsoft Academic Search

Silica glasses doped with 500–700?g of bovine serum albumin were prepared by the sol-gel method; two pH conditions (pH 5 and 7) were assayed for protein encapsulation. Both biomaterials showed a highly porous structure, with pore sizes in the range 5–28nm. Columns packed with the ground biogels were on-line coupled to a C18 HPLC column for evaluation of the entrapped

Luz E. Vera-Avila; Erika García-Salgado; Martha P. García de Llasera; Araceli Peña-Alvarez



Ultrasensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy  

Microsoft Academic Search

BACKGROUND: The definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) in humans or bovine spongiform encephalopathy (BSE) in cattle currently relies on the post mortem detection of the pathological form of the prion protein (PrPSc) in brain tissue. Infectivity studies indicate that PrPSc may also be present in body fluids, even at presymptomatic stages of the disease, albeit

Lothar Trieschmann; Alexander Navarrete Santos; Katja Kaschig; Sandra Torkler; Elke Maas; Hermann Schätzl; Gerald Böhm



Bone Matrix Proteins: Isolation and Characterization of a Novel Cell-binding Keratan Sulfate Proteoglycan (Osteoadherin) from Bovine Bone  

Microsoft Academic Search

A small cell-binding proteoglycan for which we propose the name osteoadherin was extracted from bovine bone with guanidine hydrochloride-containing EDTA. It was purified to homogeneity using a combi- nation of ion-exchange chromatography, hydroxyapa- tite chromatography, and gel filtration. The M r of the proteoglycan was 85,000 as determined by SDS-PAGE. The protein is rich in aspartic acid, glutamic acid, and

Mikael Wendel; Yngve Sommarin; Dick Heinegård



Bovine hepatic and adipose retinol-binding protein gene expression and relationship with tumor necrosis factor-?.  


Retinol-binding protein (RBP) is the main transport system for retinol in circulation, is a relatively small protein with one binding site for retinol in the all-trans form, and is bound to transthyretin. The objectives of this study were to characterize the temporal pattern of bovine hepatic mRNA expression of RBP during the periparturient period and to determine if a relationship exists between the expression of RBP and that of tumor necrosis factor (TNF)-? in dairy cows. In experiment 1, we assessed hepatic mRNA expression of RBP during the periparturient period. Liver tissues were sampled from periparturient dairy cows (n=9) at -21, -4, +1, +7, and +21 relative to parturition and frozen in liquid N(2). Total RNA was extracted from each tissue sample and cDNA was generated. Gene expressions of RBP and ?-actin (as a housekeeping gene) were measured as relative quantity using reverse transcription-PCR. Data were analyzed using cycle threshold values, adjusted to ?-actin, and significance was determined at P<0.05. Serum samples (-21, -4, +1, +7, and +21 relative to parturition) were analyzed for retinol concentration using a standard HPLC-based method. Cows had variable expression of hepatic RBP and serum retinol over the transition period, with a decline near parturition and a rebound toward prepartum levels later in lactation. In experiment 2, liver and visceral (intestinal) adipose tissues were sampled from dairy cows (n=28) at slaughter. Expression of RBP and TNF-? was detected in all samples and variations among cows were highly significant for both genes. Across tissues, expression of RBP was positively correlated with that of TNF-? (r=0.60). Within adipose tissue, expression of RBP and TNF-? was weakly correlated (r=0.23), whereas in hepatic tissue, expression was strongly correlated (r=0.62). In experiment 3, late-lactation dairy Holstein cows were blocked by parity and feed intake, and randomly assigned to control, recombinant bovine (rb)TNF challenge, or pair-fed control treatment (n=5/treatment). Cows were injected with either rbTNF (subcutaneous injection of 2 µg/kg of body weight in saline) or sterile saline (control and pair-fed control animals) once daily for 7d. Liver biopsy was performed on d 7 and samples were processed for expression of RBP and TNF-?. Although TNF challenge caused an upregulation of hepatic TNF-? expression, as expected, it did not alter hepatic RBP expression. Overall, the temporal pattern of hepatic RBP gene expression during the periparturient period followed, to a great extent, that of plasma retinol. Although a strong positive correlation was previously detected between bovine hepatic RBP and TNF-? transcripts, rbTNF challenge did not cause alter RBP expression. These observations collectively imply that regulation of RBP at the transcription level is influenced by physiological state but may be independent from that of transthyretin, which is altered by proinflammatory stimuli (such as TNF-?) via induction of transcription factor nuclear factor-interleukin 6. PMID:23040032

Rezamand, P; Watts, J S; Hunt, K M; Bradford, B J; Mamedova, L K; Morey, S D



Allostery in a Coarse-Grained Model of Protein Dynamics  

NASA Astrophysics Data System (ADS)

We propose a criterion for optimal parameter selection in coarse-grained models of proteins and develop a refined elastic network model (ENM) of bovine trypsinogen. The unimodal density-of-states distribution of the trypsinogen ENM disagrees with the bimodal distribution obtained from an all-atom model; however, the bimodal distribution is recovered by strengthening interactions between atoms that are backbone neighbors. We use the backbone-enhanced model to analyze allosteric mechanisms of trypsinogen and find relatively strong communication between the regulatory and active sites.

Ming, Dengming; Wall, Michael E.



Responses of bovine WC1(+) gammadelta T cells to protein and nonprotein antigens of Mycobacterium bovis.  


WC1(+) gammadelta T cells of Mycobacterium bovis-infected cattle are highly responsive to M. bovis sonic extract (MBSE). In mycobacterial infections of other species, gammadelta T cells have been shown to respond to protein and nonprotein antigens, but the bovine WC1(+) gammadelta T-cell antigenic targets within MBSE require further definition in terms of the dominance of protein versus nonprotein components. The present study sought to characterize the WC1(+) gammadelta T-cell antigenic targets, together with the role of interleukin-2 (IL-2), in the context of M. bovis infection. This was achieved by testing crude and defined antigens to assess protein versus nonprotein recognition by WC1(+) gammadelta T cells in comparison with CD4(+) alphabeta T cells. Both cell types proliferated strongly in response to MBSE, with CD4(+) T cells being the major producers of gamma interferon (IFN-gamma). However, enzymatic digestion of the protein in MBSE removed its ability to stimulate CD4(+) T-cell responses, whereas some WC1(+) gammadelta T-cell proliferation remained. The most antigenic protein inducing proliferation and IFN-gamma secretion in WC1(+) gammadelta T-cell cultures was found to be ESAT-6, which is a potential novel diagnostic reagent and vaccine candidate. In addition, WC1(+) gammadelta T-cell proliferation was observed in response to stimulation with prenyl pyrophosphate antigens (isopentenyl pyrophosphate and monomethyl phosphate). High levels of cellular activation (CD25 expression) resulted from MBSE stimulation of WC1(+) gammadelta T cells from infected animals. A similar degree of activation was induced by IL-2 alone, but for WC1(+) gammadelta T-cell division IL-2 was found to act only as a costimulatory signal, enhancing antigen-driven responses. Overall, the data indicate that protein antigens are important stimulators of WC1(+) gammadelta T-cell proliferation and IFN-gamma secretion in M. bovis infection, with nonprotein antigens inducing significant proliferation. These findings have important implications for diagnostic and vaccine development. PMID:12379688

Welsh, Michael D; Kennedy, Hilary E; Smyth, Allister J; Girvin, R Martyn; Andersen, Peter; Pollock, John M



A 66-kDa protein of bovine hypophyseal Pars tuberalis induces luteinizing hormone release from rat Pars distalis.  


In this study, evidence for a factor secreted by bovine hypophyseal pars tuberalis that stimulates luteinizing hormone (LH) release from rat pars distalis cells is shown. The secretion products of bovine pars tuberalis cells into the culture medium were assayed on dispersed rat pars distalis cells in 30 min incubations and superfusion experiments. The culture medium from pars tuberalis total cell populations, added at a dose of 6 microg per tube, induced the greater LH release from pars distalis cells, without effect on follicle stimulating hormone (FSH) release. After pars tuberalis cells separation on a discontinuos Percoll gradient, only the culture medium of cells from 50 and 60% strength Percoll were able to release LH from rat pars distalis cells. Therefore, cell fractions from 50 and 60% strenght Percoll were cultured together. To elicit maximal LH release (6 times the basal output), with the addition of 2 microg of pars tuberalis protein was required, suggesting that these cells produce the factor or factors which affect pars distalis gonadotrope cells. After applying the pars tuberalis culture medium on 12% SDS-PAGE, the band with biological activity was that of 66-kDal. Fifty ng protein of its eluate released almost 9 times the basal output of LH from pars distalis cells. Results suggest a modulating effect of a protein from the bovine pars tuberalis on rat cultured gonadotrope cells from the pars distalis. PMID:19181183

Lafarque, Martha; Oliveros, Liliana



Mammalian skeletal muscle C-protein: purification from bovine muscle, binding to titin and the characterization of a full-length human cDNA.  


We report a fast method for the isolation of homogeneous C-protein from bovine skeletal muscle. In electron micrographs C-protein appears as short rods with a relatively uniform length of about 50 nm. Protein sequencing shows a single N-terminal sequence. Radio-labelled C-protein strongly decorates titin II and myosin rods but not myosin heads. Binding to titin II is retained in preparations lacking titin-associated proteins. Antibodies to bovine C-protein were used to screen a lambda gt11 cDNA library constructed from fetal human skeletal muscle. Clone HC38 is 3833 bp long and encodes a protein of 1138 amino acid residues. The start of the predicted sequence fits the N-terminal sequence of the bovine protein. All partial sequences obtained from the bovine protein (348 residues) and the sequence deduced from a partial chicken cDNA (Einheber and Fischman, 1990) can be aligned along the human sequence. The sequences of human and chicken C-proteins share 50% identity and 70% similarity. Along the repeat patterns of the human protein the fibronectin (Fn)-like domains are better conserved than the immunoglobulin (Ig)-like domains. Regions of strong divergence between chicken fast C-protein and human slow C-protein may represent differences in C-protein isoforms. PMID:1429890

Fürst, D O; Vinkemeier, U; Weber, K



Bovine colostrum prevents bacterial translocation in an intestinal ischemia/reperfusion-injured rat model.  


This study evaluated whether or not bovine colostrum (BC) is able to treat or prevent intestinal barrier damage, bacterial translocation, and the related systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS) in an intestinal ischemia/reperfusion (I/R)-injured rat model. Fifty Sprague-Dawley rats were used. The rats' intestinal I/R injuries were induced by clamping the superior mesenteric artery for 30 minutes. After 3 hours of reperfusion and then twice daily reclamping during the experiment, the experimental group was given BC (4 mL/kg/day) perorally, and the other groups received 0.9% saline and low fat milk (LFM) after intestinal I/R injury. Seventy-two hours later we assessed (1) intestinal damage and intestinal permeability, (2) enteric bacterial count and bacterial translocation, (3) serum albumin, protein, and hepatic enzyme levels, (4) pathologic findings of ileum and lung, (5) activity of oxygen-free radical species, and (6) pro-inflammatory cytokines (tumor necrosis factor-alpha and interleukin-1beta). Intestinal damage, intestinal permeability, and bacterial translocation to other organs were significantly reduced in rats fed with BC after I/R when compared to rats fed LFM/saline after I/R (P < .05). In the evaluation of acute lung injury, neutrophils were found only in the lungs of the saline-fed group after I/R, and the wet/dry ratio of the lung tissue was significantly reduced in the BC-fed group after I/R compared to other I/R groups. A marked difference was found between LFM/saline-fed groups and BC-fed groups regarding malondialdehyde (P < .05) and pro-inflammatory cytokines (P < .01). In conclusion, BC may have beneficial effects in treating and preventing intestinal barrier damage, bacterial translocation and the related SIRS and MODS in the intestinal I/R-injured rat model. PMID:19298194

Choi, Han Sung; Jung, Kyung Hee; Lee, Seung Chul; Yim, Sung Vin; Chung, Joo-Ho; Kim, Youn Wha; Jeon, Woo Kyu; Hong, Hoon Pyo; Ko, Young Gwan; Kim, Chul-Ho; Jang, Ki-Hyo; Kang, Soon Ah



A Bovine Model of Respiratory Chlamydia psittaci Infection: Challenge Dose Titration  

Microsoft Academic Search

This study aimed to establish and evaluate a bovine respiratory model of experimentally induced acute C. psittaci infection. Calves are natural hosts and pathogenesis may resemble the situation in humans. Intrabronchial inoculation of C. psittaci strain DC15 was performed in calves aged 2–3 months via bronchoscope at four different challenge doses from 106 to 109 inclusion-forming units (ifu) per animal.

Petra Reinhold; Carola Ostermann; Elisabeth Liebler-Tenorio; Angela Berndt; Anette Vogel; Jacqueline Lambertz; Michael Rothe; Anke Rüttger; Evelyn Schubert; Konrad Sachse



Octanediol treatment of glutaraldehyde fixed bovine pericardium: evidence of anticalcification efficacy in the subcutaneous rat model  

Microsoft Academic Search

Objective: Anticalcification strategies of glutaraldehyde-fixed xenograft tissue aim to extract lipids or to neutralize toxic aldehyde residuals. The purpose of this study was to evaluate the efficacy of octanediol compared to standard treatments of glutaraldehyde-fixed bovine pericardium in the subdermal rat model. Octanediol treatment is an ethanolic solution (40%) containing a long chain aliphatic alcohol (5% 1,2-octanediol) that removes lipids

Elena Pettenazzo; Marialuisa Valente; Gaetano Thiene



Bovine pericardium buttress limits recanalization of the uncut roux-en-Y in a porcine model  

Microsoft Academic Search

In contrast to the traditional Roux-en-Y reconstruction, an uncut Roux-en-Y provides biliopancreatic diversion and may preserve\\u000a myoelectric continuity. Previous iterations of the uncut Roux have been plagued by recanalization of the uncut staple line\\u000a in the afferent small bowel. Our aim was to determine if bovine pericardium buttress prevents recanalization of the stapled\\u000a smallbowel partition in a porcine model. Sixteen

John M. Morton; Tananchai A Lucktong; Scott Trasti; Timothy M. Farrell



Modeling Alkaline Phosphatase Inactivation in Bovine Milk During High-Temperature Short-Time Pasteurization  

Microsoft Academic Search

Alkaline phosphatase (AP) is used as the indicator enzyme for proper pasteurization of bovine milk. Predictive modeling of AP inactivation during high-temperature short-time (HTST) pasteurization would support regulations; thus ensuring the safety of heat treated milk. Activation energy (Ea) of AP in milk was measured experimentally using the capillary tube method, and Ea was found to be 429252 J\\/mol. The

Q. Lu; P. Piyasena; G. S. Mittal



Human Milk Protein Production in Xenografts of Genetically Engineered Bovine Mammary Epithelial Stem Cells  

PubMed Central

Background In the bovine species milk production is well known to correlate with mammary tissue mass. However, most advances in optimizing milk production relied on improvements of breeding and husbandry practices. A better understanding of the cells that generate bovine mammary tissue could facilitate important advances in milk production and have global economic impact. With this possibility in mind, we show that a mammary stem cell population can be functionally identified and isolated from the bovine mammary gland. We also demonstrate that this stem cell population may be a promising target for manipulating the composition of cow's milk using gene transfer. Methods and Findings We show that the in vitro colony-forming cell assay for detecting normal primitive bipotent and lineage-restricted human mammary clonogenic progenitors are applicable to bovine mammary cells. Similarly, the ability of normal human mammary stem cells to regenerate functional bilayered structures in collagen gels placed under the kidney capsule of immunodeficient mice is shared by a subset of bovine mammary cells that lack aldehyde dehydrogenase activity. We also find that this activity is a distinguishing feature of luminal-restricted bovine progenitors. The regenerated structures recapitulate the organization of bovine mammary tissue, and milk could be readily detected in these structures when they were assessed by immunohistochemical analysis. Transplantation of the bovine cells transduced with a lentivirus encoding human ?-CASEIN led to expression of the transgene and secretion of the product by their progeny regenerated in vivo. Conclusions These findings point to a common developmental hierarchy shared by human and bovine mammary glands, providing strong evidence of common mechanisms regulating the maintenance and differentiation of mammary stem cells from both species. These results highlight the potential of novel engineering and transplant strategies for a variety of commercial applications including the production of modified milk components for human consumption.

Martignani, Eugenio; Eirew, Peter; Accornero, Paolo



Photochemistry and stereoselectivity of cellular retinaldehyde-binding protein from bovine retina  

SciTech Connect

11-cis-Retinaldehyde bound to cellular retinaldehyde-binding protein (CRALBP) is unaffected in bovine eyecup preparations by illumination that bleaches approximately 70% of the rhodopsin. Illumination of retinal homogenates to which CRALBP X (/sup 3/H)11-cis-retinaldehyde had been added did not result in a reduction of the specific activity of recovered 11-cis-retinaldehyde, ruling out a bleaching regeneration cycle. The quantum efficiency of photoisomerization for CRALBP X 11-cis-retinaldehyde was determined by comparing the rate of photoisomerization of 11-cis-retinaldehyde bound to purified CRALBP and opsin. The low value obtained (0.07), coupled with a low molar extinction coefficient (15,400 M-1 cm-1), results in a photosensitivity only about 4% that of rhodopsin. CRALBP binds 9-cis- and 11-cis-retinaldehyde, producing complexes with absorption maxima at 405 and 425 nm, respectively. No complexes were detected with 13-cis- and all-trans-retinaldehyde. Following incubation of CRALBP X 11-cis-retinol with an equimolar mixture of 9-, 11-, 13-cis-, and all-trans-retinaldehydes, only 11-cis-retinaldehyde and residual 11-cis-retinol are present on the protein following separation from excess retinoids. A similar result is obtained following incubation of CRALBP X 11-cis-retinol with mixtures of 9- and 11-cis-retinaldehyde ranging in composition from 9:1 to 1:9 (9-cis-:11-cis-,mol/mol). The results indicate that CRALBP X 11-cis-retinol is sufficiently stereoselective in its binding properties to warrant consideration as a component of the mechanism for the generation of 11-cis-retinaldehyde in the dark.

Saari, J.C.; Bredberg, D.L.



Agouti Revisited: Transcript Quantification of the ASIP Gene in Bovine Tissues Related to Protein Expression and Localization  

PubMed Central

Beside its role in melanogenesis, the agouti signaling protein (ASIP) has been related to obesity. The potentially crucial role in adipocyte development makes it a tempting candidate for economic relevant, fat related traits in farm animals. The objective of our study was to characterize the mRNA expression of different ASIP transcripts and of putative targets in different bovine tissues, as well as to study consequences on protein abundance and localization. ASIP mRNA abundance was determined by RT-qPCR in adipose and further tissues of cattle representing different breeds and crosses. ASIP mRNA was up-regulated more than 9-fold in intramuscular fat of Japanese Black cattle compared to Holstein (p<0.001). Further analyses revealed that a transposon-derived transcript was solely responsible for the increased ASIP mRNA abundance. This transcript was observed in single individuals of different breeds indicating a wide spread occurrence of this insertion at the ASIP locus in cattle. The protein was detected in different adipose tissues, skin, lung and liver, but not in skeletal muscle by Western blot with a bovine-specific ASIP antibody. However, the protein abundance was not related to the observed ASIP mRNA over-expression. Immuno-histochemical analyses revealed a putative nuclear localization of ASIP additionally to the expected cytosolic signal in different cell types. The expression of melanocortin receptors (MCR) 1 to 5 as potential targets for ASIP was analyzed by RT-PCR in subcutaneous fat. Only MC1R and MC4R were detected indicating a similar receptor expression like in human adipose tissue. Our results provide evidence for a widespread expression of ASIP in bovine tissues at mRNA and, for the first time, at protein level. ASIP protein is detectable in adipocytes as well as in further cells of adipose tissue. We generated a basis for a more detailed investigation of ASIP function in peripheral tissues of various mammalian species.

Albrecht, Elke; Komolka, Katrin; Kuzinski, Judith; Maak, Steffen



Radiofrequency Ablation (RFA): Development of a Flow Model for Bovine Livers for Extensive Bench Testing  

SciTech Connect

Purpose. To develop a flow model for bovine livers for extensive bench testing of technical improvements or procedure-related developments of radiofrequency ablation excluding animal experiments. Methods. The perfusion of bovine livers directly from the slaughterhouse was simulated in a liver perfusion tank developed for the experimental work. The liver perfusion medium used was a Tyrode solution prepared in accordance with physiologic criteria (as for liver transplants) which was oxygenated by an oxygenator and heated to 36.5 deg. C. Portal vein circulation was regulated via a flow- and pressure-controlled pump and arterial circulation using a dialysis machine. Flow rate and pressure were adjusted as for the physiology of a human liver converted to bovine liver conditions. The fluid discharged from the liver was returned into the perfusion system through the vena cava. Extendable precision swivel arms with the radiofrequency probe attached were mounted on the liver perfusion tank. RFA was conducted with the RF3000 generator and a 2 cm LeVeen needle (Boston Scientific, Ratingen, Germany) in a three-dimensional grid for precise localization of the generated thermolesions. Results. Four bovine livers weighing 8.4 {+-} 0.4 kg each were prepared, connected to the perfusion system, and consecutively perfused for the experiments. Mean arterial flow was 569 {+-} 43 ml/min, arterial pressure 120 mmHg, portovenous flow 1440 {+-} 305 ml/min, and portal pressure 10 mmHg. Macroscopic evaluation after the experiments revealed no thrombi within the hepatic vessels. A total of 136 RF thermolesions were generated with an average number of 34 per liver. Mean RF duration was 2:59 {+-} 2:01 min:sec with an average baseline impedance of 28.2 {+-} 3.4 ohms. The mean diameter of the thermolesions along the puncture channel was 22.98 {+-} 4.34 mm and perpendicular to the channel was 23.27 {+-} 4.82 mm. Conclusion. Extracorporeal perfusion of bovine livers with consecutive standardized RF ablation was feasible. The bovine liver flow model seems to allow extensive, standardized evaluation of technical or procedure-related developments of RF systems.

Lubienski, Andreas [University of Schleswig-Holstein, Institute of Radiology (Germany)], E-mail:; Bitsch, Rudi G. [Ruprecht Karls University Heidelberg, Department of Orthopedics (Germany); Lubienski, Katrin [University of Schleswig-Holstein, Institute of Radiology (Germany); Kauffmann, Guenter [Ruprecht Karls University Heidelberg, Department of Diagnostic Radiology (Germany); Duex, Markus [Hospital Northwest Frankfurt, Department of Radiology (Germany)



Effects of milk protein variants on the protein composition of bovine milk  

Microsoft Academic Search

The effects of ß-lactoglobulin (ß-LG), ß-casein (ß-CN), and -CN variants and ß--CN haplotypes on the relative concentrations of the major milk proteins -lactalbumin (-LA), ß-LG, S1-CN, S2-CN, ß-CN, and -CN and milk production traits were estimated in the milk of 1,912 Dutch Holstein-Friesian cows. We show that in the Dutch Holstein-Friesian population, the allele frequencies have changed in the past

J. M. L. Heck; A. Schennink; Valenberg van H. J. F; H. Bovenhuis; M. H. P. W. Visker; Arendonk van J. A. M; Hooijdonk van A. C. M



Binding of the E1 and E2 Proteins to the Origin of Replication of Bovine Papillomavirus  

Microsoft Academic Search

DNA replication of bovine papillomavirus (BPV) requires two viral proteins encoded from the E1 and E2 openreadingframes.E1andE2aresequence-specificDNAbindingproteinsthatbindtotheircognatebinding sitesintheBPVoriginofreplication(ori).TheE1andE2proteinscaninteractphysicallywitheachother,and this interaction results in cooperative binding when binding sites for both proteins are present. We have analyzed the binding of E1 to the ori in the absence and presence of E2, using DNase I footprint analysis, gel mobility shift assays, and interference




Identification of Babesia bigemina and Babesia bovis merozoite proteins with isolate- and species-common epitopes recognized by antibodies in bovine immune sera.  

PubMed Central

Immunoprecipitation of radiolabeled antigens with bovine antisera indicated that many Babesia bigemina and Babesia bovis merozoite proteins contain isolate-common epitopes, while at least 16 B. bigemina and 8 B. bovis proteins contain species-cross-reactive epitopes. One immunogenic, isolate-common, and species-specific candidate diagnostic protein from each species was identified. Images

McElwain, T F; Palmer, G H; Goff, W L; McGuire, T C



Osmotically unresponsive water fraction on proteins: non-ideal osmotic pressure of bovine serum albumin as a function of pH and salt concentration.  


How much does protein-associated water differ in colligative properties (freezing point, boiling point, vapor pressure and osmotic behavior) from pure bulk water? This question was approached by studying the globular protein bovine serum albumin (BSA), using changes in pH and salt concentration to alter its native structural conformation and state of aggregation. BSA osmotic pressure was investigated experimentally and analyzed using the molecular model of Fullerton et al. [Biochem Cell Biol 1992;70(12):1325]. Analysis yielded both the extent of osmotically unresponsive water (OUW) and the effective molecular weight values of the membrane-impermeable BSA solute. Manipulation of BSA conformation and aggregation by membrane-penetrating cosolutes show that alterations in pH and salt concentration change the amount of bulk water that escapes into BSA from a minimum of 1.4 to a maximum of 11.7 g water per g dry mass BSA. PMID:16376113

Fullerton, Gary D; Kanal, Kalpana M; Cameron, Ivan L



Central nervous system extracellular matrix changes in a transgenic mouse model of bovine spongiform encephalopathy.  


Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy characterised by accumulation of resistant prion protein (PrP(BSE)), neuronal loss, spongiosus and glial cell proliferation. In this study, properties of the extracellular matrix (ECM) were investigated in boTg110 transgenic mice over-expressing the bovine cellular prion protein (PrP(c)) and infected with BSE. Using immunohistochemistry with Wisteria floribunda agglutinin as a specific marker for perineuronal nets (PNNs) and antibodies against aggrecan and hyaluronic acid binding protein, loss of ECM was correlated with PrP(BSE) accumulation and activation of astrocytes and microglia. PrP(BSE) accumulation and glial cell activation were detected from the earliest stages of the disease and increased in the terminal stages. Decreases in PNNs, aggrecan and hyaluronic acid were observed only in the terminal stages and correlated with the distribution of PrP(BSE) and activated glial cells. This study suggests that the loss of PNNs, aggrecan and hyaluronic acid is a consequence of PrP(BSE) accumulation. Degradation of ECM in BSE may be due to secretion of degradative enzymes by activated glial cells. PMID:18789733

Costa, Carme; Tortosa, Raül; Vidal, Enric; Padilla, Danielle; Torres, Juan Maria; Ferrer, Isidre; Pumarola, Martí; Bassols, Anna



Generation and immunity testing of a recombinant adenovirus expressing NcSRS2-NcGRA7 fusion protein of bovine Neospora caninum.  


Neospora caninum is the etiologic agent of bovine neosporosis, which affects the reproductive performance of cattle worldwide. The transmembrane protein, NcSRS2, and dense-granule protein, NcGRA7, were identified as protective antigens based on their ability to induce significant protective immune responses in murine neosporosis models. In the current study, NcSRS2 and NcGRA7 genes were spliced by overlap-extension PCR in a recombinant adenovirus termed Ad5-NcSRS2-NcGRA 7, expressing the NcSRS2-NcGRA7 gene, and the efficacy was evaluated in mice. The results showed that the titer of the recombinant adenovirus was 10(9)TCID50/ml. Three weeks post-boost immunization (w.p.b.i.), the IgG antibody titer in sera was as high as 1:4,096. IFN-? and IL-4 levels were significantly different from the control group (P<0.01). This research established a solid foundation for the development of a recombinant adenovirus vaccine against bovine N. caninum. PMID:23710096

Jia, Li-Jun; Zhang, Shou-Fa; Qian, Nian-Chao; Xuan, Xue-Nan; Yu, Long-Zheng; Zhang, Xue-Mei; Liu, Ming-Ming



Development and statistical validation of a guinea pig model for vaccine potency testing against Infectious Bovine Rhinothracheitis (IBR) virus  

Microsoft Academic Search

Infectious Bovine Rhinothracheitis (IBR) caused by bovine herpesvirus 1 (BoHV-1) infection is distributed worldwide. BoHV-1 either alone or in association with other respiratory cattle pathogens causes significant economic losses to the livestock industry. The aim of this work was to validate a guinea pig model as an alternative method to the current BoHV-1 vaccine potency testing in calves. Guinea pigs

Viviana Parreño; María Virginia López; Daniela Rodriguez; María Marta Vena; Mercedes Izuel; Jorge Filippi; Alejandra Romera; Claudia Faverin; Rodolfo Bellinzoni; Fernando Fernandez; Laura Marangunich



Synthetic peptides from region 65–84 of bovine myelin basic protein: Radioimmunoassays and equilibrium competitive inhibition studies with antibodies prepared against myelin basic protein  

Microsoft Academic Search

Synthetic peptides with various and overlapping sequences represented by the residue region 65–84 of bovine myelin basic protein (MBP-bov) were tested in sodium sulfate radioimmunoassays for their reactivity with 15 rabbit antisera against MBPs from six different animal species and nine pools of syngeneic Lewis rat anti-MBP antisera. Three of the peptides were labeled with125I and studied by direct binding

Eugene D. Day; George A. Hashim; Vincent A. Varitek; Kenneth J. Lazarus; Philip Y. Paterson



Prevalent abnormal prion protein in human appendixes after bovine spongiform encephalopathy epizootic: large scale survey  

PubMed Central

Objectives To carry out a further survey of archived appendix samples to understand better the differences between existing estimates of the prevalence of subclinical infection with prions after the bovine spongiform encephalopathy epizootic and to see whether a broader birth cohort was affected, and to understand better the implications for the management of blood and blood products and for the handling of surgical instruments. Design Irreversibly unlinked and anonymised large scale survey of archived appendix samples. Setting Archived appendix samples from the pathology departments of 41 UK hospitals participating in the earlier survey, and additional hospitals in regions with lower levels of participation in that survey. Sample 32?441 archived appendix samples fixed in formalin and embedded in paraffin and tested for the presence of abnormal prion protein (PrP). Results Of the 32?441 appendix samples 16 were positive for abnormal PrP, indicating an overall prevalence of 493 per million population (95% confidence interval 282 to 801 per million). The prevalence in those born in 1941-60 (733 per million, 269 to 1596 per million) did not differ significantly from those born between 1961 and 1985 (412 per million, 198 to 758 per million) and was similar in both sexes and across the three broad geographical areas sampled. Genetic testing of the positive specimens for the genotype at PRNP codon 129 revealed a high proportion that were valine homozygous compared with the frequency in the normal population, and in stark contrast with confirmed clinical cases of vCJD, all of which were methionine homozygous at PRNP codon 129. Conclusions This study corroborates previous studies and suggests a high prevalence of infection with abnormal PrP, indicating vCJD carrier status in the population compared with the 177 vCJD cases to date. These findings have important implications for the management of blood and blood products and for the handling of surgical instruments.



First principles predictions of the structure and function of g-protein-coupled receptors: validation for bovine rhodopsin.  


G-protein-coupled receptors (GPCRs) are involved in cell communication processes and with mediating such senses as vision, smell, taste, and pain. They constitute a prominent superfamily of drug targets, but an atomic-level structure is available for only one GPCR, bovine rhodopsin, making it difficult to use structure-based methods to design receptor-specific drugs. We have developed the MembStruk first principles computational method for predicting the three-dimensional structure of GPCRs. In this article we validate the MembStruk procedure by comparing its predictions with the high-resolution crystal structure of bovine rhodopsin. The crystal structure of bovine rhodopsin has the second extracellular (EC-II) loop closed over the transmembrane regions by making a disulfide linkage between Cys-110 and Cys-187, but we speculate that opening this loop may play a role in the activation process of the receptor through the cysteine linkage with helix 3. Consequently we predicted two structures for bovine rhodopsin from the primary sequence (with no input from the crystal structure)-one with the EC-II loop closed as in the crystal structure, and the other with the EC-II loop open. The MembStruk-predicted structure of bovine rhodopsin with the closed EC-II loop deviates from the crystal by 2.84 A coordinate root mean-square (CRMS) in the transmembrane region main-chain atoms. The predicted three-dimensional structures for other GPCRs can be validated only by predicting binding sites and energies for various ligands. For such predictions we developed the HierDock first principles computational method. We validate HierDock by predicting the binding site of 11-cis-retinal in the crystal structure of bovine rhodopsin. Scanning the whole protein without using any prior knowledge of the binding site, we find that the best scoring conformation in rhodopsin is 1.1 A CRMS from the crystal structure for the ligand atoms. This predicted conformation has the carbonyl O only 2.82 A from the N of Lys-296. Making this Schiff base bond and minimizing leads to a final conformation only 0.62 A CRMS from the crystal structure. We also used HierDock to predict the binding site of 11-cis-retinal in the MembStruk-predicted structure of bovine rhodopsin (closed loop). Scanning the whole protein structure leads to a structure in which the carbonyl O is only 2.85 A from the N of Lys-296. Making this Schiff base bond and minimizing leads to a final conformation only 2.92 A CRMS from the crystal structure. The good agreement of the ab initio-predicted protein structures and ligand binding site with experiment validates the use of the MembStruk and HierDock first principles' methods. Since these methods are generic and applicable to any GPCR, they should be useful in predicting the structures of other GPCRs and the binding site of ligands to these proteins. PMID:15041637

Trabanino, Rene J; Hall, Spencer E; Vaidehi, Nagarajan; Floriano, Wely B; Kam, Victor W T; Goddard, William A



Isolation, Purification and Characterization of Fatty-Acid-Binding Protein from Milk Fat Globule Membrane: Effect of Bovine Growth Hormone Treatment  

Microsoft Academic Search

Fatty-acid-binding protein (FABP) was purified from bovine milk fat globule membrane (MFGM) by ion-exchange chromatography on DEAE-Sepharose and by gel-filtration on Sephadex G-50. Purified FABP was similar to bovine mammary gland heart (H)-type FABP\\/ mammary derived growth inhibitor (MDGI). It inhibited growth of mammary epithelial cells at nanogram concentrations, had a relative molecular mass of 15 kDa, as determined by



Hydrogen peroxide-and fetal bovine serum-induced DNA synthesis in vascular smooth muscle cells: positive and negative regulation by protein kinase C isoforms  

Microsoft Academic Search

Hydrogen peroxide and fetal bovine serum stimulate DNA synthesis in growth-arrested smooth muscle cells with remarkably similar kinetics and cell density dependence. However, while stimulation with fetal bovine serum results in cell proliferation, that by H202 is followed by cell death. Depletion of conventional and novel protein kinase C isoforms, resulting from a long treatment with phorbol-l2-myristate- 13-acetate, further increases

Mara Fiorani; Orazio Cantoni; Andrea Tasinato; Daniel Boscoboinik; Angelo Azzi



Conservative mutations in the immunosuppressive region of the bovine leukemia virus transmembrane protein affect fusion but not infectivity in vivo.  


Many retroviruses, including bovine leukemia virus (BLV), contain a highly conserved region located about 40 amino acids downstream from the fusion peptide within the sequence of the external domain of the transmembrane (TM) protein. This region is notably thought to be involved in the presentation of the NH2-terminal peptide to allow cell fusion. By using hydrophobic cluster analysis and by analogy with the influenza A hemagglutinin structures, the core of the TM structure including this particular region was predicted to consist, in the BLV and other retroviral envelope proteins, of an alpha-helix followed by a loop region, both docked against a subsequent alpha-helix that forms a triple-stranded coiled coil. The loop region could undergo, as in hemagglutinin, a major refolding into an alpha-helix integrating the coiled coil structure and putting the fusion peptide to one tip of the molecule. Based on this model, we have identified amino acids that may be essential to the BLV TM structure, and a series of mutations were introduced in the BLV env gene of an infectious molecular clone. A first series of mutations was designed to disturb the coiled coil structure (substitutions with proline residues), whereas others would maintain the general TM structure. When expressed by Semliki Forest virus recombinants, all the mutated envelope proteins were stable and efficiently synthesized in baby hamster kidney cells. Both proline-substituted and conservative mutants were strongly affected in their capacity to fuse to CC81 indicator cells. In addition, it appeared that the integrity of the TM coiled coil structure is essential for envelope protein multimerization, as analyzed by metrizamide gradient centrifugation. Finally, to gain insight into the role of this coiled coil in the infectious potential of BLV in vivo, the mutated TM genes were introduced in an infectious and pathogenic molecular clone and injected into sheep. It appeared that only the conservative mutations (A60V and A64S) allowed maintenance of viral infectivity in vivo. Since these mutations destroyed the ability to induce syncytia, we conclude that efficient fusion capacity of the recombinant envelopes is not a prerequisite for the infectious potential of BLV in vivo. Viral propagation of these mutants was strongly affected in some of the infected sheep. However, the proviral loads within half of the infected animals (2 out of 2 for A60V and 1 out of 4 for A64S) were close to the wild-type levels. In these sheep, it thus appears that the A60V and A64S mutants propagate efficiently despite being unable to induce syncytia in cell culture. PMID:9582317

Gatot, J S; Callebaut, I; Mornon, J P; Portetelle, D; Burny, A; Kerkhofs, P; Kettmann, R; Willems, L



Involvement of the cyclic AMP-responsive element binding protein in bovine leukemia virus expression in vivo.  

PubMed Central

The TAR element (Tax-responsive element; also called TxRE) is a major determinant of the regulation of bovine leukemia virus (BLV) expression. In order to gain insight into the mechanisms of viral expression, complexes formed between proteins and the TAR enhancer DNA were analyzed by gel retardation assays. We report here that nuclear lysates from ex vivo-isolated B lymphocytes contain proteins that specifically bind to TAR. An antibody directed toward the cyclic AMP-responsive element binding (CREB) protein supershifted a complex (C1) present only in BLV-infected B lymphocytes. The CREB protein thus appears to be a major transcription factor involved in BLV expression in vivo. Images

Adam, E; Kerkhofs, P; Mammerickx, M; Kettmann, R; Burny, A; Droogmans, L; Willems, L



Modeling Protein Self Assembly  

ERIC Educational Resources Information Center

|Understanding the structure and function of proteins is an important part of the standards-based science curriculum. Proteins serve vital roles within the cell and malfunctions in protein self assembly are implicated in degenerative diseases. Experience indicates that this topic is a difficult one for many students. We have found that the concept…

Baker, William P.; Jones, Carleton Buck; Hull, Elizabeth



Modeling Protein Domain Function  

ERIC Educational Resources Information Center

|This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth



Structural equation models to estimate risk of infection and tolerance to bovine mastitis  

PubMed Central

Background One method to improve durably animal welfare is to select, as reproducers, animals with the highest ability to resist or tolerate infection. To do so, it is necessary to distinguish direct and indirect mechanisms of resistance and tolerance because selection on these traits is believed to have different epidemiological and evolutionary consequences. Methods We propose structural equation models with latent variables (1) to quantify the latent risk of infection and to identify, among the many potential mediators of infection, the few ones that influence it significantly and (2) to estimate direct and indirect levels of tolerance of animals infected naturally with pathogens. We applied the method to two surveys of bovine mastitis in the Walloon region of Belgium, in which we recorded herd management practices, mastitis frequency, and results of bacteriological analyses of milk samples. Results and discussion Structural equation models suggested that, among more than 35 surveyed herd characteristics, only nine (age, addition of urea in the rations, treatment of subclinical mastitis, presence of dirty liner, cows with hyperkeratotic teats, machine stripping, pre- and post-milking teat disinfection, and housing of milking cows in cubicles) were directly and significantly related to a latent measure of bovine mastitis, and that treatment of subclinical mastitis was involved in the pathway between post-milking teat disinfection and latent mastitis. These models also allowed the separation of direct and indirect effects of bacterial infection on milk productivity. Results suggested that infected cows were tolerant but not resistant to mastitis pathogens. Conclusions We revealed the advantages of structural equation models, compared to classical models, for dissecting measurements of resistance and tolerance to infectious diseases, here bovine mastitis. Using our method, we identified nine major risk factors that were directly associated with an increased risk of mastitis and suggested that cows were tolerant but not resistant to mastitis. Selection should aim at improved resistance to infection by mastitis pathogens, although further investigations are needed due to the limitations of the data used in this study.



A bovine model of respiratory Chlamydia psittaci infection: challenge dose titration.  


This study aimed to establish and evaluate a bovine respiratory model of experimentally induced acute C. psittaci infection. Calves are natural hosts and pathogenesis may resemble the situation in humans. Intrabronchial inoculation of C. psittaci strain DC15 was performed in calves aged 2-3 months via bronchoscope at four different challenge doses from 10(6) to 10(9) inclusion-forming units (ifu) per animal. Control groups received either UV-inactivated C. psittaci or cell culture medium. While 10(6) ifu/calf resulted in a mild respiratory infection only, the doses of 10(7) and 10(8) induced fever, tachypnea, dry cough, and tachycardia that became apparent 2-3 days post inoculation (dpi) and lasted for about one week. In calves exposed to 10(9) ifu C. psittaci, the respiratory disease was accompanied by severe systemic illness (apathy, tremor, markedly reduced appetite). At the time point of most pronounced clinical signs (3 dpi) the extent of lung lesions was below 10% of pulmonary tissue in calves inoculated with 10(6) and 10(7) ifu, about 15% in calves inoculated with 10(8) and more than 30% in calves inoculated with 10(9) ifu C. psittaci. Beside clinical signs and pathologic lesions, the bacterial load of lung tissue and markers of pulmonary inflammation (i.e., cell counts, concentration of proteins and eicosanoids in broncho-alveolar lavage fluid) were positively associated with ifu of viable C. psittaci. While any effect of endotoxin has been ruled out, all effects could be attributed to infection by the replicating bacteria. In conclusion, the calf represents a suitable model of respiratory chlamydial infection. Dose titration revealed that both clinically latent and clinically manifest infection can be reproduced experimentally by either 10(6) or 10(8) ifu/calf of C. psittaci DC15 while doses above 10(8) ifu C. psittaci cannot be recommended for further studies for ethical reasons. This defined model of different clinical expressions of chlamydial infection allows studying host-pathogen interactions. PMID:22299031

Reinhold, Petra; Ostermann, Carola; Liebler-Tenorio, Elisabeth; Berndt, Angela; Vogel, Anette; Lambertz, Jacqueline; Rothe, Michael; Rüttger, Anke; Schubert, Evelyn; Sachse, Konrad



Perfusion-Cultured Bovine Anterior Segments as an Ex Vivo Model for Studying Glucocorticoid-Induced Ocular Hypertension and Glaucoma  

PubMed Central

Purpose. To determine whether perfusion-cultured bovine anterior segments would be a suitable model for glaucoma research. Methods. Fresh bovine eyes were dissected and sealed on a custom-made acrylic dish with an O-ring. Perfusion medium was infused by a syringe pump at a constant infusion rate of 5 ?L/min. After intraocular pressure (IOP) was stable, bovine eyes were perfused with medium containing either a vehicle control (0.1% ethanol [ETH]) or dexamethasone (DEX) for up to 7 days. IOP was recorded by a pressure transducer and a computerized system. Perfusion medium was collected for Western immunoblot analysis of myocilin (MYOC). Results. The morphology of the bovine trabecular meshwork after perfusion culture was similar to that of freshly dissected, nonperfused bovine eyes. Treatment with DEX elevated IOP in some bovine eyes, whereas others showed little change. The authors analyzed the data from 18 ETH-treated control eyes and defined 2.82 mm Hg as the threshold of ocular hypertension (OHT), which equals mean pressure change + 2× SD. Approximately 40% (12/29) of the bovine eyes were DEX responders, which is very close to the DEX-responsive rates observed in human and monkey eyes. Western blot data showed that DEX treatment induced the expression of the DEX-inducible gene MYOC only in the perfusion-cultured anterior segments with DEX-induced OHT. Conclusions. OHT can be induced by DEX in perfusion-cultured bovine anterior segments. This is a fast, convenient, affordable, and reliable model for studying DEX-induced OHT and the mechanisms of trabecular outflow.

Tovar-Vidales, Tara; Yorio, Thomas; Wordinger, Robert J.; Clark, Abbot F.



Mutations in the bovine leukemia virus Tax protein can abrogate the long terminal repeat-directed transactivating activity without concomitant loss of transforming potential.  


The bovine leukemia virus Tax protein transactivates gene expression directed by the viral long terminal repeat (LTR) and contributes to immortalization of primary cells. Theoretical analysis of the protein sequence revealed the presence of a putative zinc finger structure at its amino end. Selected mutations in that region completely abolished transactivation, demonstrating its importance for LTR-directed gene regulation. However, these mutations did not interfere with the ability of tax to bind zinc or to contribute to immortalization of primary cells. Thus, transactivation of bovine leukemia virus LTR and target cell transformation are independent functions of Tax and involve different functional domains of the protein. PMID:1315045

Willems, L; Grimonpont, C; Heremans, H; Rebeyrotte, N; Chen, G; Portetelle, D; Burny, A; Kettmann, R



A novel in vitro bovine cartilage punch model for assessing the regeneration of focal cartilage defects with biocompatible bacterial nanocellulose.  


INTRODUCTION: Current therapies for articular cartilage defects fail to achieve qualitatively sufficient tissue regeneration, possibly because of a mismatch between the speed of cartilage rebuilding and the resorption of degradable implant polymers. The present study focused on the self-healing capacity of resident cartilage cells in conjunction with cell-free and biocompatible (but non-resorbable) bacterial nanocellulose (BNC). This was tested in a novel in vitro bovine cartilage punch model. METHODS: Standardized bovine cartilage discs with a central defect filled with BNC were cultured for up to 8 weeks with/without stimulation with TGF-beta1. Cartilage formation and integrity were analyzed by histology, immunohistochemistry, and electron microscopy. Content, release, and neosynthesis of the matrix molecules proteoglycan/aggrecan, collagen II, and collagen I were also quantified. Finally, gene expression of these molecules was profiled in resident chondrocytes and chondrocytes migrated onto the cartilage surface or the implant material. RESULTS: Non-stimulated and especially TGF-beta1-stimulated cartilage discs displayed a preserved structural and functional integrity of the chondrocytes and surrounding matrix, remained vital in long-term culture (8 weeks) without signs of degeneration, and showed substantial synthesis of cartilage-specific molecules at the protein and mRNA level. Whereas mobilization of chondrocytes from the matrix onto the surface of cartilage and implant was pivotal for successful seeding of cell-free BNC, chondrocytes did not immigrate into the central BNC area, possibly due to the relatively small diameter of its pores (2-5 um). Chondrocytes on the BNC surface showed signs of successful redifferentiation over time, including increase of aggrecan/collagen type II mRNA, decrease of collagen type I mRNA, and initial deposition of proteoglycan and collagen type II in long-term high-density pellet cultures. Although TGF-beta1 stimulation showed protective effects on matrix integrity, effects on other parameters were limited. CONCLUSIONS: The present bovine cartilage punch model represents a robust, reproducible, and highly suitable tool for the long-term culture of cartilage, maintaining matrix integrity and homoeostasis. As an alternative to animal studies, this model may closely reflect early stages of cartilage regeneration, allowing the evaluation of promising biomaterials with/without chondrogenic factors. PMID:23673274

Pretzel, David; Linss, Stefanie; Ahrem, Hannes; Endres, Michaela; Kaps, Christian; Klemm, Dieter; Kinne, Raimund W



A pregnant mouse model for bovine Tritrichomonas foetus infection.  


The economically important effects of Tritrichomonas foetus infection in cattle are abortion and infertility, yet there has not been an animal model to examine the parasite-host interactions during gestation. In this study, 5- and 7- to 8-week-old BALB/cAnNCr, BALB/cJ, and SCID/NCr mice on a BALB/c background were intravaginally infected with T. foetus. All BALB/cAnNCr and BALB/cJ mice, and 89% of SCID/NCr mice sustained infections for 13 weeks, if inoculated before 5 weeks of age. Infection rates were lower in all mouse strains inoculated at 7 weeks of age, although BALB/cAnNCr mice were significantly more susceptible than BALB/cJ or SCID/NCr mice. Vaginal bacterial flora did not account for the variation in mouse-strain susceptibility, although coagulase-negative staphylococci in vaginal flora were associated with failure of T. foetus to infect. As with infected cattle, T. foetus-specific vaginal immunoglobulin (Ig) G and IgA antibodies were elevated after infection. The number and viability of day-10 fetuses were reduced in mice infected at 5 weeks of age and bred 12 weeks after infection. Lesions in pregnant and nonpregnant infected mice, including suppurative and eosinophilic vaginitis; cervicitis; endometritis with distension of the uterine lumen; endometrial ulceration; and glandular ectasia, with neutrophils in the glandular lumen and loss of gland epithelium, were similar to those in cattle. The decidua and placenta were multifocally necrotic. Immunohistochemistry demonstrated trichomonads in vaginal folds and uterine glands, and adjacent to fetal tissues. In summary, experimentally infected BALB/cAnNCr mice showed many pathologic similarities to cattle and may serve as a model to study host-trichomonad interactions. PMID:18984788

Agnew, D W; Corbeil, L B; Munson, L; Byrne, B A; BonDurant, R H



Postfabrication encapsulation of model protein drugs in a negatively thermosensitive hydrogel  

Microsoft Academic Search

A postfabrication encapsulation technique was developed for loading model protein drugs into an intelligent and biodegradable hydrogel film, which exhi- bits negative thermosensitivity with a desirable phase transition temperature between refrigerator temperature and body temperature. The hydrogel comprises mainly poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymer, and oligo(lactide). The model proteins Hemoglobin and Bovine Serum Albumin were loaded intothehydrogelfilmsbysoakingthegelsat48C,atwhichthehydrogelfilmwasswollen. The loaded

Ying Zhang; Wen Zhu; Biaobing Wang; Lin Yu; Jiandong Ding



In Vitro Digestion of Proteins and Growth Factors in a Bovine Whey Protein Extract as Determined Using a Computer-Controlled Dynamic Gastrointestinal System (TIM1)  

Microsoft Academic Search

The digestion of major whey proteins\\/peptides, transforming growth factor-beta2 (TGF-?2) and insulin-like growth factor-I\\u000a (IGF-I) in a bovine whey protein extract (BWPE) was investigated in vitro using a dynamic gastrointestinal digestion system\\u000a (TIM-1). ?-lactoglobulin and glycomacropeptide were the whey components most resistant to 3 h of gastric digestion (pepsin)\\u000a and were also the last to be cleared from the gastric compartment.

Samira Nabil; Sylvie F. Gauthier; Réjean Drouin; Patrice E. Poubelle; Yves Pouliot


Sclera-Choroid-RPE Transport of Eight ?-Blockers in Human, Bovine, Porcine, Rabbit, and Rat Models  

PubMed Central

Purpose. To determine the influence of drug lipophilicity, ocular pigmentation, and species differences on transscleral solute transport. Methods. The transport of eight ?-blockers across excised sclera/sclera-choroid-RPE (SCRPE) of albino rabbit, pigmented rabbit, human, porcine, and bovine eyes was determined over 6 hours. The ex vivo transscleral ?-blocker transport to the vitreous at the end of 6 hours was determined in euthanatized, pigmented Brown Norway rats. The thicknesses of the sclera and SCRPE and the melanin content in choroid-RPE (CRPE) were measured to determine whether species differences in drug transport can be explained on this basis. Results. Solute lipophilicity inversely correlated with the SCRPE cumulative percentage of transport in all species (R2 ? 0.80). The CRPE impeded the SCRPE transport of all ?-blockers (51%–64% resistance in the rabbits; 84%–99.8% in the bovine and porcine eyes) more than the sclera, with the impedance increasing with lipophilicity. SCRPE transport followed the trend albino rabbit > pigmented rabbit > human > porcine > bovine, and a cross-species comparison showed good Spearman's rho correlation (R2 ? 0.85). Bovine (R2 = 0.84), porcine (R2 = 0.84), and human (R2 = 0.71) SCRPE transport was more predictive than that in the rabbit models (R2 = 0.60–0.61) of transscleral solute transport to the vitreous in rats. The CRPE concentrations were higher in pigmented rabbits than in albino rabbits. The melanin content of the CRPE exhibited the trend albino rabbit ? pigmented rabbit < porcine ? bovine < rat. Normalization to scleral thickness abolished the species differences in scleral transport. Normalization to SCRPE thickness and melanin content significantly reduced species differences in SCRPE transport. Conclusions. Owing to the presence of pigment and drug binding, choroid-RPE is the principal barrier to transscleral ?-blocker transport, with the barrier being more significant for lipophilic ?-blockers. Although different in magnitude between species, sclera/SCRPE transport can be correlated between species. Tissue thickness accounts for the species differences in scleral transport. Differences in tissue thickness and melanin content largely account for the species differences in SCRPE transport.

Kadam, Rajendra S.; Cheruvu, Narayan P. S.; Edelhauser, Henry F.



Mutations in the Bovine Leukemia Virus Tax Protein Can Abrogate the Long Terminal Repeat-Directed Transactivating Activity Without Concomitant Loss of Transforming Potential  

Microsoft Academic Search

The bovine leukemia virus Tax protein trans-activates gene expression directed by the viral long terminal repeat (LTR) and contributes to immortalization of primary cells. Theoretical analysis of the protein sequence revealed the presence of a putative zinc finger structure at its amino end. Selected mutations in that region completely abolished transactivation, demonstrating its importance for LTR-directed gene regulation. However, these

Luc Willems; Catherine Grimonpont; Hubertine Heremans; Nicole Rebeyrotte; Gao Chen; Daniel Portetelle; Arsene Burny; Richard Kettmann



Osmotically unresponsive water fraction on proteins: Non-ideal osmotic pressure of bovine serum albumin as a function of pH and salt concentration  

Microsoft Academic Search

How much does protein-associated water differ in colligative properties (freezing point, boiling point, vapor pressure and osmotic behavior) from pure bulk water? This question was approached by studying the globular protein bovine serum albumin (BSA), using changes in pH and salt concentration to alter its native structural conformation and state of aggregation. BSA osmotic pressure was investigated experimentally and analyzed

Gary D. Fullerton; Kalpana M. Kanal; Ivan L. Cameron



Polymer-Induced Heteronucleation for Protein Single Crystal Growth: Structural Elucidation of Bovine Liver Catalase and Concanavalin A Forms  

SciTech Connect

Obtaining single crystals for X-ray diffraction remains a major bottleneck in structural biology; when existing crystal growth methods fail to yield suitable crystals, often the target rather than the crystallization approach is reconsidered. Here we demonstrate that polymer-induced heteronucleation, a powerful technique that has been used for small molecule crystallization form discovery, can be applied to protein crystallization by optimizing the heteronucleant composition and crystallization formats for crystallizing a wide range of protein targets. Applying these advances to two benchmark proteins resulted in dramatically increased crystal size, enabling structure determination, for a half century old form of bovine liver catalase (BLC) that had previously only been characterized by electron microscopy, and the discovery of two new forms of concanavalin A (conA) from the Jack bean and accompanying structural elucidation of one of these forms.

Foroughi, Leila M.; Kang, You-Na; Matzger, Adam J. (Michigan)



Dynamics of mitotic activity and expression of viral proteins gp51 and p24 of bovine leukemia virus producing cells.  


The dynamics of expression of viral proteins gp51 and p24, virus particle production and mitotic activity of a highly productive bovine leukemia virus clone of the fetal lamb kidney (FLK) cell line were studied. The period of the highest protein production (20-44 h after incubation) was established by the indirect immunofluorescence method using specific monoclonal antibodies. It was followed by a complete formation of the cell monolayer and the most intensive period of the mitotic activity determined by the 3H-thymidine labeling method. After the active synthesis of viral protein, the formation of mature viral particles and their shedding in the intercellular spaces was established electron-microscopically and by the syncytia induction test. A similar comparative study was carried out with a BLV producing short-term lymphocyte culture (STLC). PMID:8157133

Roussev, R; Polianova, M; Portetelle, D; Ivanovna, O



Microwave Ablation Compared with Radiofrequency Ablation for Breast Tissue in an Ex Vivo Bovine Udder Model  

Microsoft Academic Search

Purpose  To compare the effectiveness of microwave (MW) ablation with radiofrequency (RF) ablation for treating breast tissue in a\\u000a nonperfused ex vivo model of healthy bovine udder tissue.\\u000a \\u000a \\u000a \\u000a \\u000a Materials and Methods  MW ablations were performed at power outputs of 25W, 35W, and 45W using a 915-MHz frequency generator and a 2-cm active tip\\u000a antenna. RF ablations were performed with a bipolar RF

Toshihiro TanakaSaskia; Saskia Westphal; Peter Isfort; Till Braunschweig; Tobias Penzkofer; Philipp Bruners; Kimihiko Kichikawa; Thomas Schmitz-Rode; Andreas H. Mahnken


A new aspect to chaperone-like activity of bovine ?-casein by protein-protein interactions study.  


In the present work, chaperone-like activity of bovine ?-casein (?-CN) against thermal denaturation and aggregation processes of bovine carbonic anhydrase (BCA) was investigated. We used different instrument and new developed methods for surveillance the structural alteration of BCA and its functional properties upon interaction with bovine ?-CN. The thermodynamic data obtained by DSC showed that interaction of ?-CN can enhance the thermal stability of enzyme but due to decrease of activation energy of aggregation, the kinetic of process as driven force accelerated the thermal aggregation. In the presence of ?-CN due to enhancement of hydrophobicity and favoring the formation of first intermediate of CA, aggregation conveniently occurred with a higher rate at a low temperature. PMID:22903015

Sharifizadeh, Ahmad; Saboury, Ali Akbar; Moosavi-Movahedi, Ali Akbar; Salami, Maryam; Yousefi, Reza



Increased Levels of LPS-Binding Protein in Bovine Blood and Milk Following Bacterial Lipopolysaccharide Challenge  

Microsoft Academic Search

Several species of gram-negative bacteria, including Escherichia coli, Klebsiella pneumoniae, and various species of Enterobacter, are common mastitis patho- gens. Allof thesebacteria arecharacterized bythe pres- ence of endotoxin or lipopolysaccharide (LPS) in their outer membrane. The bovine mammary gland is highly sensitive to LPS, and LPS has been implicated, in part, in the pathogenesis of gram-negative mastitis. Recogni- tion of

Douglas D. Bannerman; Max J. Paape; William R. Hare; Eun Jung Sohn



Tau Protein Expression in Adult Bovine Oligodendrocytes: Functional and Pathological Significance  

Microsoft Academic Search

In tauopathies, overexpression of tau exon 10 is linked to degeneration and abnormal tau deposition in neurons and oligodendroglia (OLGs). To compare exon 10 expression in normal neurons and OLGs, adult bovine brain was examined for the expression of tau in gray matter and cultured OLGs isolated from white matter. Using exon-specific antibodies, we found that both types of tissues

Hanna Ksiezak-Reding; Muhammad Farooq; Liang-sheng Yang; Dennis W. Dickson; Patrizia LoPresti



Identification of a prion protein epitope modulating transmission of bovine spongiform encephalopathy prions to transgenic mice  

PubMed Central

There is considerable concern that bovine prions from cattle with bovine spongiform encephalopathy (BSE) may have been passed to humans (Hu), resulting in a new form of Creutzfeldt–Jakob disease (CJD). We report here the transmission of bovine (Bo) prions to transgenic (Tg) mice expressing BoPrP; one Tg line exhibited incubation times of ?200 days. Like most cattle with BSE, vacuolation and astrocytic gliosis were confined in the brainstems of these Tg mice. Unexpectedly, mice expressing a chimeric Bo/Mo PrP transgene were resistant to BSE prions whereas mice expressing Hu or Hu/Mo PrP transgenes were susceptible to Hu prions. A comparison of differences in Mo, Bo, and Hu residues within the C terminus of PrP defines an epitope that modulates conversion of PrPC into PrPSc and, as such, controls prion transmission across species. Development of susceptible Tg(BoPrP) mice provides a means of measuring bovine prions that may prove critical in minimizing future human exposure.

Scott, Michael R.; Safar, Jiri; Telling, Glenn; Nguyen, Oanh; Groth, Darlene; Torchia, Marilyn; Koehler, Ruth; Tremblay, Patrick; Walther, Dirk; Cohen, Fred E.; DeArmond, Stephen J.; Prusiner, Stanley B.



Relationships among bovine heat shock protein 70 genotype, forage type, and plasma concentrations of HSP-70.  

Technology Transfer Automated Retrieval System (TEKTRAN)

The bovine HSP-70 gene is polymorphic, shows breed bias, and is related to milk production. Our objective was to determine if composite HSP-70 genotypes were related to plasma concentration of HSP-70. Genomic DNA and plasma samples were collected from 71 cows. The cows were Angus (n = 18; Bos taurus...


The Mouse Homolog of the Bovine Leukemia Virus Receptor Is Closely Related to the d Subunit of Adaptor-Related Protein Complex AP3, Not Associated with the Cell Surface  

Microsoft Academic Search

A mouse cDNA (mBLVR1) which was highly homologous to the bovine cDNA of the bovine leukemia virus receptor (BLVR) gene was cloned. The mBLVR1 cDNA, of 4,730 bp, covered nearly the full length of the mRNA (about 5 kb) and included an open reading frame (ORF) encoding a protein of 1,199 amino acids. While the bovine BLVR protein was thought




Agouti revisited: transcript quantification of the ASIP gene in bovine tissues related to protein expression and localization.  


Beside its role in melanogenesis, the agouti signaling protein (ASIP) has been related to obesity. The potentially crucial role in adipocyte development makes it a tempting candidate for economic relevant, fat related traits in farm animals. The objective of our study was to characterize the mRNA expression of different ASIP transcripts and of putative targets in different bovine tissues, as well as to study consequences on protein abundance and localization. ASIP mRNA abundance was determined by RT-qPCR in adipose and further tissues of cattle representing different breeds and crosses. ASIP mRNA was up-regulated more than 9-fold in intramuscular fat of Japanese Black cattle compared to Holstein (p<0.001). Further analyses revealed that a transposon-derived transcript was solely responsible for the increased ASIP mRNA abundance. This transcript was observed in single individuals of different breeds indicating a wide spread occurrence of this insertion at the ASIP locus in cattle. The protein was detected in different adipose tissues, skin, lung and liver, but not in skeletal muscle by Western blot with a bovine-specific ASIP antibody. However, the protein abundance was not related to the observed ASIP mRNA over-expression. Immuno-histochemical analyses revealed a putative nuclear localization of ASIP additionally to the expected cytosolic signal in different cell types. The expression of melanocortin receptors (MCR) 1 to 5 as potential targets for ASIP was analyzed by RT-PCR in subcutaneous fat. Only MC1R and MC4R were detected indicating a similar receptor expression like in human adipose tissue. Our results provide evidence for a widespread expression of ASIP in bovine tissues at mRNA and, for the first time, at protein level. ASIP protein is detectable in adipocytes as well as in further cells of adipose tissue. We generated a basis for a more detailed investigation of ASIP function in peripheral tissues of various mammalian species. PMID:22530003

Albrecht, Elke; Komolka, Katrin; Kuzinski, Judith; Maak, Steffen



Purified bovine plasma blocking factor decreases Bovine leukemia virus p24 expression while increasing protein synthesis and transcriptional activity of peripheral blood mononuclear cells in short-term culture  

PubMed Central

Abstract Bovine leukemia virus (BLV) induces a persistent infection in the B-cells causing polyclonal expansion of B-cells in one-third of infected cattle and lymphosarcoma in less than 5% of infected cattle. While BLV is difficult to detect in vivo, it is readily produced by cultured lymphocytes and is diminished when supplemented by bovine plasma. This phenomenon is attributed to a poorly characterized plasma blocking factor (PBF). We assessed the effects of bovine plasma on cell viability and BLV p24 expression, and the effects of purified PBF on protein synthesis and gene expression of short-term cultures of bovine lymphocytes. The addition of 25% plasma or semi-purified PBF to cultures had no significant effect on cell viability but caused significant decreases in BLV p24 production and significantly increased de novo protein synthesis. Utilizing a human microarray, the RNA messages of 83 genes involved in cell division, cell metabolism, and gene regulation were up-regulated.



The S protein of bovine coronavirus is a hemagglutinin recognizing 9-O-acetylated sialic acid as a receptor determinant.  

PubMed Central

The S protein of bovine coronavirus (BCV) has been isolated from the viral membrane and purified by gradient centrifugation. Purified S protein was identified as a viral hemagglutinin. Inactivation of the cellular receptors by sialate 9-O-acetylesterase and generation of receptors by sialylation of erythrocytes with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicate that S protein recognizes 9-O-acetylated sialic acid as a receptor determinant as has been shown previously for intact virions. The second glycoprotein of BCV, HE, which has been thought previously to be responsible for the hemagglutinating activity of BCV, is a less efficient hemagglutinin; it agglutinates mouse and rat erythrocytes, but in contrast to S protein, it is unable to agglutinate chicken erythrocytes, which contain a lower level of Neu5,9Ac2 on their surface. S protein is proposed to be responsible for the primary attachment of virus to cell surface. S protein is proposed to be responsible for the primary attachement of virus to cell surface receptors. The potential of S protein as a probe for the detection of Neu5,9Ac2-containing glycoconjugates is demonstrated. Images

Schultze, B; Gross, H J; Brossmer, R; Herrler, G



Brain-specific hyaluronate-binding protein: an immunohistological study with monoclonal antibodies of human and bovine central nervous system.  

PubMed Central

Hyaluronectin is a protein isolated from acid extracts of human brain by affinity chromatography on immobilized hyaluronate. With polyclonal antibodies, it was immunohistologically localized in the rat at the nodes of Ranvier of central and peripheral myelinated fibers and in mesenchymal tissues. Compared to adult rat, hyaluronectin-immunoreactive material was more abundant in embryonal rat brain and mesenchyma. We report a different localization in human and bovine tissues with monoclonal antibodies reacting with human hyaluronectin by NaDodSO4/PAGE and immunoblotting but not staining rat tissues by immunohistology. In human and calf the antigen reacting with hyaluronectin monoclonal antibodies was brain specific, while several peripheral tissues were stained by the polyclonal antibodies. In human and bovine central nervous system monoclonal antibodies stained white matter and tissues formed predominantly by glial fibers (e.g., subependymal glia). In white matter hyaluronectin-immunoreactive material formed a delicate mesh surrounding individual myelinated fibers, a pattern compatible with the distribution of fine astroglial processes in this location. Gray matter did not stain with monoclonal antibodies, the granular layer of the cerebellum excepted. The findings suggest that human hyaluronectin is heterogeneous and comprises at least two fractions. The main fraction is a brain-specific protein, probably produced by white matter astrocytes. Another fraction cross-reacting with rat is more abundant in embryonal tissues, including mesenchyma and brain. Images

Bignami, A; Dahl, D



Cell Arrest and Cell Death in Mammalian Preimplantation Development: Lessons from the Bovine Model  

PubMed Central

Background The causes, modes, biological role and prospective significance of cell death in preimplantation development in humans and other mammals are still poorly understood. Early bovine embryos represent a very attractive experimental model for the investigation of this fundamental and important issue. Methods and Findings To obtain reference data on the temporal and spatial occurrence of cell death in early bovine embryogenesis, three-dimensionally preserved embryos of different ages and stages of development up to hatched blastocysts were examined in toto by confocal laser scanning microscopy. In parallel, transcript abundance profiles for selected apoptosis-related genes were analyzed by real-time reverse transcriptase-polymerase chain reaction. Our study documents that in vitro as well as in vivo, the first four cleavage cycles are prone to a high failure rate including different types of permanent cell cycle arrest and subsequent non-apoptotic blastomere death. In vitro produced and in vivo derived blastocysts showed a significant incidence of cell death in the inner cell mass (ICM), but only in part with morphological features of apoptosis. Importantly, transcripts for CASP3, CASP9, CASP8 and FAS/FASLG were not detectable or found at very low abundances. Conclusions In vitro and in vivo, errors and failures of the first and the next three cleavage divisions frequently cause immediate embryo death or lead to aberrant subsequent development, and are the main source of developmental heterogeneity. A substantial occurrence of cell death in the ICM even in fast developing blastocysts strongly suggests a regular developmentally controlled elimination of cells, while the nature and mechanisms of ICM cell death are unclear. Morphological findings as well as transcript levels measured for important apoptosis-related genes are in conflict with the view that classical caspase-mediated apoptosis is the major cause of cell death in early bovine development.

Leidenfrost, Sandra; Boelhauve, Marc; Reichenbach, Myriam; Gungor, Tuna; Reichenbach, Horst-Dieter; Sinowatz, Fred; Wolf, Eckhard; Habermann, Felix A.



Short communication: Identification of the bovine sterol regulatory element binding protein-1c promoter and its activation by liver X receptor.  


Sterol regulatory element binding proteins (SREBP) are a family of transcription factors that regulate cholesterogenesis and lipogenesis. Sterol regulatory element binding proteins-1a and -1c are transcribed from the same gene by the use of alternate promoters, and only differ at their first exon. Sterol regulatory element binding protein-1c is hypothesized to be an important regulator of genes involved in milk fat synthesis in the lactating dairy cow. However, the bovine SREBP-1c promoter has not been previously characterized, and studies to date that have investigated the role of SREBP-1 in the bovine mammary gland have not distinguished between isoforms 1a and 1c. The purpose of this study was to characterize the bovine SREBP-1c promoter and to investigate the DNA elements involved in the regulation of SREBP-1c expression by the liver X receptor agonist T0901317 in 2 different bovine mammary epithelial cell lines. Luciferase reporter constructs containing the wild-type SREBP-1c promoter or constructs with mutated liver X receptor response elements or sterol response element were transfected into MacT cells and bovine mammary epithelial (BME-UV) cells. We have demonstrated that the liver X receptor response elements sites in the SREBP-1c promoter are necessary for mediating the T0901317 response, and that stimulation through the sterol response element site plays only a minor role in this pathway. This report describes the bovine SREBP-1c promoter and its regulation by liver X receptor in bovine mammary epithelial cells. PMID:21094755

Lengi, A J; Corl, B A



Oncoviral Bovine Leukemia Virus G4 and Human T-Cell Leukemia Virus Type 1 p13II Accessory Proteins Interact with Farnesyl Pyrophosphate Synthetase  

PubMed Central

G4 and p13II are accessory proteins encoded by the X region of bovine leukemia virus and human T-cell leukemia virus type 1 (HTLV-1), respectively. Disruption of the G4 and p13II open reading frames interferes with viral spread in animal model systems, indicating that the corresponding proteins play a key role in viral replication. In addition, G4 is oncogenic in primary cell cultures and is absolutely required for efficient onset of leukemogenesis in sheep. To gain insight into the function of these proteins, we utilized the yeast two-hybrid system to identify protein partners of G4. Results revealed that G4 interacts with farnesyl pyrophosphate synthetase (FPPS), a protein involved in the mevalonate/squalene pathway and in synthesis of FPP, a substrate required for prenylation of Ras. The specificity of the interaction was verified by glutathione S-transferase (GST) pull-down assays and by coimmunoprecipitation experiments. Furthermore, confocal microscopy showed that the subcellular localization of G4 was profoundly affected by FPPS. The G4 protein itself was not prenylated, at least in rabbit reticulocyte lysate-based assays. The domain of G4 required for binding to FPPS was restricted to an amphipathic ?-helix rich in arginine residues. Subtle mutation of this ?-helix abrogated G4 oncogenic potential in vitro, providing a biological relevance for FPPS-G4 complex formation in cells. Finally, HTLV-1 p13II was also found to specifically interact with FPPS (in yeast as well as in GST pull-down assays) and to colocalize with G4 in mitochondria, suggesting a functional analogy between these oncoviral accessory proteins. Identification of FPPS as a molecular partner for p13II and G4 accessory proteins opens new prospects for treatment of retrovirus-induced leukemia.

Lefebvre, Laurent; Vanderplasschen, Alain; Ciminale, Vincenzo; Heremans, Hubertine; Dangoisse, Olivier; Jauniaux, Jean-Claude; Toussaint, Jean-Francois; Zelnik, Vlado; Burny, Arsene; Kettmann, Richard; Willems, Luc



Modelling of proteins in membranes.  


This review describes some recent theories and simulations of mesoscopic and microscopic models of lipid membranes with embedded or attached proteins. We summarize results supporting our understanding of phenomena for which the activities of proteins in membranes are expected to be significantly affected by the lipid environment. Theoretical predictions are pointed out, and compared to experimental findings, if available. Among others, the following phenomena are discussed: interactions of interfacially adsorbed peptides, pore-forming amphipathic peptides, adsorption of charged proteins onto oppositely charged lipid membranes, lipid-induced tilting of proteins embedded in lipid bilayers, protein-induced bilayer deformations, protein insertion and assembly, and lipid-controlled functioning of membrane proteins. PMID:16620797

Sperotto, Maria Maddalena; May, Sylvio; Baumgaertner, Artur



Intragastric Administration of Lactobacillus casei Expressing Bovine Rotavirus VP7 Protein Induced Specific Antibody Production  

Microsoft Academic Search

The aim of this study is to explore the mucosal and systemic antibody immune responses induced by recombinant L.casei expressing the major immunoprotective antigen VP7 of bovine rotavirus (BRV). Two inducible expression vectors containing the antigenic dominant region of VP7 gene, pPG-1-VP7 (cell-surface expression vector) and pPG-2-VP7 (secretory vector), were constructed and electrotransformed into L. casei 393 to obtain positive

Songmei Liu; Guanqun Zhang; Didi Liu; Yijing Li



MATER protein expression and intracellular localization throughout folliculogenesis and preimplantation embryo development in the bovine  

Microsoft Academic Search

BACKGROUND: Mater (Maternal Antigen that Embryos Require), also known as Nalp5 (NACHT, leucine rich repeat and PYD containing 5), is an oocyte-specific maternal effect gene required for early embryonic development beyond the two-cell stage in mouse. We previously characterized the bovine orthologue MATER as an oocyte marker gene in cattle, and this gene was recently assigned to a QTL region

Sophie Pennetier; Christine Perreau; Svetlana Uzbekova; Aurore Thélie; Bernadette Delaleu; Pascal Mermillod; Rozenn Dalbiès-Tran



Modelling of proteins in membranes  

Microsoft Academic Search

This review describes some recent theories and simulations of mesoscopic and microscopic models of lipid membranes with embedded or attached proteins. We summarize results supporting our understanding of phenomena for which the activities of proteins in membranes are expected to be significantly affected by the lipid environment. Theoretical predictions are pointed out, and compared to experimental findings, if available. Among

Maria Maddalena Sperotto; Sylvio May; Artur Baumgaertner



An Immunoglobulin Binding Protein (Antigen 5) of the Stable Fly (Diptera: Muscidae) Salivary Gland Stimulates Bovine Immune Responses  

PubMed Central

The stable fly, Stomoxys calcitrans, is an economically important pest of livestock. Prior studies demonstrated lymphocyte suppression by crude salivary gland extract (SGE) of the stable fly. A dominant 27 kDa protein identified in the SGE was reported to stimulate immunodominant antibody responses in exposed cattle. The purpose of this study was to determine if this protein, now identified as a homolog of insect proteins named antigen 5 (Ag5), was responsible for the lymphocyte suppression and if naïve calves can mount an immune response to it. Calves raised in the winter months were immunized with recombinant Ag5 (rAg5) expressed in Drosophila S2 cells or with “natural” Ag5 protein isolated by preparative gel electrophoresis of SGE. Control calves were immunized with adjuvant alone. Rising antibody concentrations to rAg5 were detected in two of three calves immunized with rAg5 and one of three calves immunized with natural Ag5. Recall lymphocyte responses to rAg5 were detected at 21 and 28 DPI in calves immunized with rAg5 but not in calves immunized with the natural Ag5 or those exposed to adjuvant alone. Mitogen-stimulated bovine lymphocyte responses were not suppressed by rAg5. Further investigation using immunoblotting revealed that rAg5 binds to the Fc and F(ab’)2 portions of bovine IgG, but not to an Fab fragment. These findings suggest that Ag5 of the stable fly salivary gland is not immunosuppressive, but has immunoglobulin binding properties and can invoke specific antibody and memory lymphocyte responses in immunized calves.




An immunoglobulin binding protein (antigen 5) of the stable fly (Diptera: Muscidae) salivary gland stimulates bovine immune responses.  


The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is an economically important pest of livestock. Previous studies demonstrated lymphocyte suppression by crude salivary gland extract (SGE) of the stable fly. A dominant 27-kDa protein identified in the SGE was reported to stimulate immunodominant antibody responses in exposed cattle. The purpose of this study was to determine whether this protein, now identified as ahomolog of insect proteins named antigen 5 (Ag5), was responsible for the lymphocyte suppression and whether naive calves can mount an immune response to it. Calves raised in the winter were immunized with recombinant Ag5 (rAg5) expressed in Drosophila S2 cells or with "natural" Ag5 protein isolated by preparative gel electrophoresis of SGE. Control calves were immunized with adjuvant alone. Rising antibody concentrations to rAg5 were detected in two of three calves immunized with rAg5 and one of three calves immunized with natural Ag5. Recall lymphocyte responses to rAg5 were detected at 21 and 28 d postimmunization in calves immunized with rAg5 but not in calves immunized with the natural Ag5 or those exposed to adjuvant alone. Mitogen-stimulated bovine lymphocyte responses were not suppressed by rAg5. Further investigation using immunoblotting revealed that rAg5 binds to the Fc and F (ab')2 portions of bovine IgG, but not to an Fab fragment. These findings suggest that Ag5 of the stable fly salivary gland is not immunosuppressive but that it has immunoglobulin binding properties and can invoke specific antibody and memory lymphocyte responses in immunized calves. PMID:18283948

Ameri, M; Wang, X; Wilkerson, M J; Kanost, M R; Broce, A B



Bovine proteins containing poly-glutamine repeats are often polymorphic and enriched for components of transcriptional regulatory complexes  

PubMed Central

Background About forty human diseases are caused by repeat instability mutations. A distinct subset of these diseases is the result of extreme expansions of polymorphic trinucleotide repeats; typically CAG repeats encoding poly-glutamine (poly-Q) tracts in proteins. Polymorphic repeat length variation is also apparent in human poly-Q encoding genes from normal individuals. As these coding sequence repeats are subject to selection in mammals, it has been suggested that normal variations in some of these typically highly conserved genes are implicated in morphological differences between species and phenotypic variations within species. At present, poly-Q encoding genes in non-human mammalian species are poorly documented, as are their functions and propensities for polymorphic variation. Results The current investigation identified 178 bovine poly-Q encoding genes (Q ? 5) and within this group, 26 genes with orthologs in both human and mouse that did not contain poly-Q repeats. The bovine poly-Q encoding genes typically had ubiquitous expression patterns although there was bias towards expression in epithelia, brain and testes. They were also characterised by unusually large sizes. Analysis of gene ontology terms revealed that the encoded proteins were strongly enriched for functions associated with transcriptional regulation and many contributed to physical interaction networks in the nucleus where they presumably act cooperatively in transcriptional regulatory complexes. In addition, the coding sequence CAG repeats in some bovine genes impacted mRNA splicing thereby generating unusual transcriptional diversity, which in at least one instance was tissue-specific. The poly-Q encoding genes were prioritised using multiple criteria for their likelihood of being polymorphic and then the highest ranking group was experimentally tested for polymorphic variation within a cattle diversity panel. Extensive and meiotically stable variation was identified. Conclusions Transcriptional diversity can potentially be generated in poly-Q encoding genes by the impact of CAG repeat tracts on mRNA alternative splicing. This effect, combined with the physical interactions of the encoded proteins in large transcriptional regulatory complexes suggests that polymorphic variations of proteins in these complexes have strong potential to affect phenotype.



sup 1 H NMR study of the influence of hydrophobic contacts on protein-prosthetic group recognition in bovine and rat ferricytochrome b sub 5  

SciTech Connect

The proton nuclear magnetic resonance spectra of the soluble fragment of native bovine and genetically engineered wild-type rat ferricytochrome b{sub 5} reconstituted with a wide variety of hemes chemically modified at 2- and/or 4-positions have been recorded and analyzed. While all but one nonsymmetric heme yielded comparable amounts of the two heme orientations immediately after reconstitution, the relative proportion of the two orientations at equilibrium varied widely. The unpaired spin density distribution in the heme {pi} system leads to substituent hyperfine shift patterns in these paramagnetic complexes that are completely diagnostic of the heme orientation in the protein matrix. An empirical assignment strategy is outlined and applied which allows unequivocal assignment of the absolute orientation of a derivatized heme within the protein matrix. Using a series of hemes lacking 2-fold symmetry solely due to a single substitution, the preferences for localized site occupation of vinyls, methyls, and hydrogens are developed. The differences in this heme orientational preference among bovine, rat, and chicken ferricytochromes b{sub 5} could be correlated with the relative steric bulk of the residues at positions 23 and 25. Detailed thermodynamic analysis of the orientational preferences of native protoheme reveals that, while the same orientation as found in X-ray crystal structures of bovine cytochrome b{sub 5} predominate at 25{degree}C in both proteins, the preference in the bovine protein is primarily for enthalpic reasons while in the rat protein the preference is due to entropic factors.

Lee, K-B.; La Mar, G.N.; Kehres, L.A.; Fujinari, E.M.; Smith, K.M. (Univ. of California, Davis (USA)); Pochapsky, T.C.; Sligar, S.G. (Univ. of Illinois, Urbana (USA))



Proteins other than the locus of enterocyte effacement-encoded proteins contribute to Escherichia coli O157:H7 adherence to bovine rectoanal junction stratified squamous epithelial cells  

PubMed Central

Background In this study, we present evidence that proteins encoded by the Locus of Enterocyte Effacement (LEE), considered critical for Escherichia coli O157 (O157) adherence to follicle-associated epithelial (FAE) cells at the bovine recto-anal junction (RAJ), do not appear to contribute to O157 adherence to squamous epithelial (RSE) cells also constituting this primary site of O157 colonization in cattle. Results Antisera targeting intimin-?, the primary O157 adhesin, and other essential LEE proteins failed to block O157 adherence to RSE cells, when this pathogen was grown in DMEM, a culture medium that enhances expression of LEE proteins. In addition, RSE adherence of a DMEM-grown-O157 mutant lacking the intimin protein was comparable to that seen with its wild-type parent O157 strain grown in the same media. These adherence patterns were in complete contrast to that observed with HEp-2 cells (the adherence to which is mediated by intimin-?), assayed under same conditions. This suggested that proteins other than intimin-? that contribute to adherence to RSE cells are expressed by this pathogen during growth in DMEM. To identify such proteins, we defined the proteome of DMEM-grown-O157 (DMEM-proteome). GeLC-MS/MS revealed that the O157 DMEM-proteome comprised 684 proteins including several components of the cattle and human O157 immunome, orthologs of adhesins, hypothetical secreted and outer membrane proteins, in addition to the known virulence and LEE proteins. Bioinformatics-based analysis of the components of the O157 DMEM proteome revealed several new O157-specific proteins with adhesin potential. Conclusion Proteins other than LEE and intimin-? proteins are involved in O157 adherence to RSE cells at the bovine RAJ. Such proteins, with adhesin potential, are expressed by this human pathogen during growth in DMEM. Ongoing experiments to evaluate their role in RSE adherence should provide both valuable insights into the O157-RSE interactions and new targets for more efficacious anti-adhesion O157 vaccines.



Antibody responses against the G and F proteins of bovine respiratory syncytial virus after experimental and natural infections.  

PubMed Central

Antibodies against the two major surface glycoproteins of bovine respiratory syncytial virus (BRSV), G and F, play a role in protection against BRSV-associated disease, but only the antibody response against the F protein has been well described. Therefore, we used a novel peptide-based enzyme-linked immunosorbent assay (G peptide-ELISA) to compare immunoglobulin G (IgG) and IgG subclass antibody responses against the G protein with the antibody response against the F protein, as measured by a conventional BRSV ELISA (F-ELISA). Experimental infection of cattle induced significantly lower antibody titers than did natural infection. After natural primary infection, G peptide-specific antibodies declined more rapidly and to lower levels than the F protein-specific antibodies. As a consequence, the G peptide-ELISA detected more reinfections than did the F-ELISA. Ratios of G- and F-specific IgG1/IgG2 antibody titers did not differ markedly after infection or vaccination. Interestingly, after natural infection calves did not develop an IgG2 response to the complete G protein. In contrast, adult cattle had high IgG2 titers against this protein. Vaccination with a live vaccine induced low antibody titers, similar to the titers after experimental infection, whereas vaccination with an inactivated vaccine induced high titers. The results indicate that the kinetics of the G- and F-specific antibody responses differ. Furthermore, the IgG subclass response against the unglycosylated central region of the G protein is similar to the IgG subclass response to the F protein, but the IgG subclass response differs from the response to the complete G protein.

Schrijver, R S; Langedijk, J P; van der Poel, W H; Middel, W G; Kramps, J A; van Oirschot, J T



Upregulation of equine matrix metalloproteinase 1 by bovine papillomavirus type 1 is through the transcription factor activator protein-1.  


Equine sarcoids represent the most common skin tumours in equids worldwide, characterized by extensive invasion and infiltration of lymphatics, rare regression and high recurrence after surgical intervention. Bovine papillomavirus type 1 (BPV-1) activity is necessary for the transformation phenotype of equine fibroblasts. Among the many changes induced by BPV-1, matrix metalloproteinase 1 (MMP-1) upregulation contributes to the invasiveness of equine fibroblasts. However, it is not yet known how BPV-1 proteins regulate equine MMP-1 expression. To elucidate this mechanism, the equine MMP-1 promoter was cloned and analysed. A putative activator protein-1 (AP-1)-binding site was demonstrated to be crucial for upregulated MMP-1 promoter activity by BPV-1. BPV-1 E6 and E7 proteins increased MMP-1 promoter activity, and inhibition of BPV-1 gene expression by small interfering RNA significantly reduced the promoter activity. c-Jun and Fra-1, two components of the AP-1 transcription factor complex, were overexpressed and activated by BPV-1 in equine fibroblasts. Finally, BPV-1 E5, E6 and E7 proteins increased MMP-1 mRNA and protein expression. In conclusion, the expression of MMP-1 can be enhanced by BPV-1 oncoproteins E6 and E7 through the AP-1 transcription factor and by E5 via an indirect mechanism. These findings shed light on the mechanism of BPV-1-mediated equine fibroblast infiltration and indicate that both BPV-1 oncoproteins and AP-1 could be potential targets for equine sarcoid therapy. PMID:21775582

Yuan, ZhengQiang; Gault, Elizabeth A; Campo, M Saveria; Nasir, Lubna



Production, Characterization, and Use of Monoclonal Antibodies Against gp51 Protein to Diagnose Bovine Leukemia Virus Infection  

PubMed Central

Abstract Enzootic bovine leukosis (EBL) is a retroviral infection that causes persistent lymphocytosis and lymphosarcoma in cattle. The economic importance of infection by bovine leukemia virus (BLV) is due to several factors, including losses in exportation, treatment of secondary infection, and reduction in dairy production. To facilitate the development of a national test that is sensitive, simple, and applicable on a large scale, this work aimed to produce and characterize monoclonal antibodies (mAbs) against gp51 protein from BLV for use in an enzyme-linked immunosorbent assay (ELISA) test. Two hundred seventy-four hybridomas were generated, from which 37 were mAbs secretory clones screened by indirect ELISA. The specificity of the mAbs generated against gp51 was verified by Western blot analysis, and the isotypes were characterized for isotyping in IgG1 and IgM. To evaluate the test, 250 sera were tested by agar gel immunodiffusion and mAb-ELISA. The values obtained for the mAb-ELISA test were 95% sensitivity and 90% specificity.

Troiano, Ludmilla D.C.; Agottani, Jorge V.B.; Brodzinski, Josiane; Penha, Tania R.; Ozaki, Silvia C.



Acrosin activity regulation by protein kinase C and tyrosine kinase in bovine sperm acrosome exocytosis induced by lysophosphatidylcholine.  


Acrosin is an important proteolytic enzyme that is capable of hydrolysing the zona pellucida in bovine oocyte. Lysophosphatydic acid (LPA) derivated from lysophosphatidylcholine (LPC) is known to trigger the acrosome exocytosis. The present study was aimed at examining the acrosin activity variations in LPC-induced acrosome exocytosis and its regulation by tyrosine kinase, protein kinase C (PKC) and voltage-dependent calcium channels (VDCC) in spermatozoa previously capacitated with heparin or quercetin. The enzyme activities were spectrophotometrically measured using N-?-benzoyl-DL-arginine p-nitroanilide as an acrosin-specific substrate. The capacitation and acrosomal reaction were evaluated by chlorotetracycline assay, and the viability and acrosome integrity were evaluated by the trypan blue stain/differential interference contrast. It was observed that LPC induced acrosome exocytosis and increased the activity of acrosin in spermatozoa previously capacitated with heparin. In heparin/LPC-treated samples, it was observed that the inhibition of tyrosine kinase and PKC blocked the acrosome exocytosis and the acrosin activity (p < 0.05). Under these conditions, in heparin-capacitated spermatozoa, the LPC provokes an acrosin activity increase that is independent of calcium influx through VDCC Type L. In cryopreserved bovine spermatozoa, LPC might require modulation, mainly tyrosine kinase participation with respect to PKC activity to induce acrosome exocytosis and increase acrosin activity. PMID:22335484

Pérez Aguirreburualde, M S; Fernández, S; Córdoba, M



CHAPS solubilization of a G-protein sensitive 5-HT sub 1 A receptor from bovine hippocampus  

SciTech Connect

The binding of ({sup 3}H) 8-OH-DPAT to membrane-bound 5-HT{sub 1A} receptors from bovine hippocampus was saturable and corresponded to a single high-affinity state. Solubilization of the bovine hippocampal membranes with 10 mM CHAPS containing 200 mM NaCl, renders a preparation which binds ({sup 3}H) 8-OH-DPAT with high affinity and is guanine nucleotide sensitive and ketanserin insensitive. 50% of ({sup 3}H) 8-OH-DPAT binding activity is solubilized. The presence of GMP-P(NH)P promotes a low-affinity state which is characteristic of receptors coupled to G-proteins. GMP-P(NH)P markedly accelerates the dissociation ({sup 3}H) 8-OH-DPAT from solubilized membranes while having negligible effects on association. Thus, the agonist can activate the ternary complex rather than to promote its formation. 8-OH-DPAT, WB 4101 and 5-carboxamidotryptamine dose responsively inhibit soluble ({sup 3}H) 8-OH-DPAT binding with IC{sub 50} values of 16.1, 15.6 and 1.3 nM, respectively. The CHAPS solubilized membrane preparation retains many of the ({sup 3}H) 8-OH-DPAT binding characteristics of the membrane bound form.

Kline, T. (Mt. Sinai School of Medicine, New York, NY (USA)); Park, H.; Meyerson, L.R. (American Cyanamid Co., Mahwah, NJ (USA))



Virulence of human and bovine isolates of group B streptococci (types Ia and III) in experimental pregnant mouse models.  

PubMed Central

Two experimental mouse models were tested for their suitability in measuring virulence of two human and two bovine isolates (types Ia and III) of group B streptococci. In the first model, the kinetics of the number of bacteria in the spleen, liver, and placenta of mice inoculated intravenously on day 16 of pregnancy were monitored for 48 h after infection. In the second model, lethality and abortion were recorded for mice inoculated on day 13 of pregnancy. Levels of colonization in spleens or livers and lethality were significantly greater (P less than 0.001) for human isolates than for bovine isolates. In contrast, no statistically significant differences in the ability to colonize placentas and to induce abortions were noted between human and bovine isolates. The results showed that pregnant mice were more sensitive than nonpregnant mice to a challenge with group B streptococci. The results also suggest that placental colonization and abortion could be a suitable mouse model in evaluating the virulence of human and bovine isolates of group B streptococci.

Poutrel, B; Dore, J



Effects of embryo size at transfer (whole versus demi) and early pregnancy progesterone supplementation on embryo growth and pregnancy-specific protein bovine concentrations in recipient dairy heifers  

Microsoft Academic Search

The objectives of this study were to evaluate embryonic size and survival, plasma progesterone (P4) and pregnancy-specific protein bovine (PSPB) concentrations in early pregnancies (n = 99) following the transfer of one whole (n = 66) or one demi (n = 33) embryo to recipient virgin dairy heifers. The experiment was designed to evaluate the fixed effects of embryo size

L. Lopes-da-Costa; J. Chagas e Silva; M. C. Deloche; N. Jeanguyot; P. Humblot; A. E. M. Horta



Antibody responses against non-structural protein 3 of bovine viral diarrhoea virus in milk and serum samples from animals immunised with an inactivated vaccine  

Microsoft Academic Search

Antibodies against non-structural protein 3 (NS3, p80) of bovine viral diarrhoea virus (BVDV) were determined in milk from cows vaccinated with an inactivated BVDV vaccine and compared to serum antibody levels. Animals in one herd were vaccinated with an inactivated BVDV vaccine according to the standard protocol and animals from a second herd with an intensive schedule. Serum and milk

Marcelino Álvarez; Jorge Donate; Birgit Makoschey


Bovine Leukemia Virus SU Protein Interacts with Zinc, and Mutations within Two Interacting Regions Differently Affect Viral Fusion and Infectivity In Vivo  

Microsoft Academic Search

Bovine leukemia virus (BLV) and human T-cell lymphotropic virus type 1 (HTLV-1) belong to the genus of deltaretroviruses. Their entry into the host cell is supposed to be mediated by interactions of the extracellular (SU) envelope glycoproteins with cellular receptors. To gain insight into the mechanisms governing this process, we investigated the ability of SU proteins to interact with specific

Jean-Stephane Gatot; Isabelle Callebaut; Carine Van Lint; Dominique Demonte; Pierre Kerkhofs; Daniel Portetelle; Arsene Burny; Luc Willems; Richard Kettmann



Composite implant of native bovine bone morphogenetic protein (BMP), collagen carrier and biocoral in the treatment of resistant ulnar nonunions: report of five preliminary cases  

Microsoft Academic Search

Introduction Bone morphogenetic protein (BMP) has been shown to induce bone formation and union in long bone defects and nonunions. There are, however, no previous reports of BMP being used for ulnar nonunions. We report on five cases of resistant ulnar nonunions treated with a composite implant consisting of a biocoral frame, collagen carrier, and bovine BMP. Materials and methods

Sauli Kujala; Timo Raatikainen; Jorma Ryhänen; Outi Kaarela; Pekka Jalovaara



Metal analysis by energy dispersive x-ray fluorescence of bovine brain tubulin and microtubule-associated proteins prepared by phosphocellulose chromatography.  


It has been shown by trace metal analysis that tubulin isolated from bovine brain does not contain strongly bound transition metal ions. The traces of zinc and iron found in the fraction of microtubule-associated proteins might originate from previously reported phosphatase activity (Larsson, H., Wallin, M. and Edström, A. (1979) J. Neurochem. 32, 155--161). PMID:7397220

Wallin, M; Deinum, J; Rindby, A; Lagercrantz, C



Expression of the bovine viral diarrhoea virus Osloss p80 protein: its use as ELISA antigen for cattle serum antibody detection  

Microsoft Academic Search

The putative gene encoding the cytopathic bovine viral diarrhoea virus (BVDV) Osloss strain p80 protein was amplified by PCR and inserted into a T7 promoter- based vector for expression in Escherichia coli. Bacterial expression led to cytoplasmic insoluble inclusion bodies which were denatured by urea treatment and renatured by dialysis. Rabbit antisera were raised against this p80 recombinant antigen and

Nathalie Vanderheijden; L. De Moerlooze; D. Vandenbergh; G. Chappuis; A. Renard; C. Lecomte



Isolation and characterization of two novel peptide amides originating from myelin basic protein in bovine brain  

Microsoft Academic Search

During a systematic search for peptides that possess the C-terminal amide structure, two novel peptide amides, one with a tyrosine amide and the other with an alanine amide were isolated from bovine brain by acid extraction and sequential steps of reversed phase HPLC. Microsequence, amino acid and mass spectral analyses revealed the structures: Ac-Ala-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-amide and Ac-Ala-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu-Ala-Ser-Ala-amide. These 12 and 16

Ken Takamatsu; Kazuhiko Tatemoto I



Expression of the bovine papillomavirus type 1 E5B gene reveals a protein-protein interaction of the E5A and E5B gene products.  


The bovine papillomavirus type 1 (BPV-1) genome has been shown to contain a small open-reading frame designated E5B (nucleotides 4013-4167) which is predicted to encode a hydrophobic, 52 amino acid protein. In order to detect and characterize the E5B protein, an 18 nucleotide sequence encoding a 6 amino acid epitope was added to the 3' end of the E5B open-reading frame which was then expressed in COS-1 cells using a SV40 vector. Immunoprecipitation, immunofluorescence, and cell fractionation studies identified the E5B protein as a 4-kDa protein and localized it primarily to membranes of the endoplasmic reticulum and nucleus. Unlike the E5A protein of BPV-1, E5B did not form dimers (despite containing a cysteine residue) or form complexes with growth factor receptors such as the PDGF receptor or erb B-2 receptor. Interestingly, the E5B protein formed physical complexes with the hydrophobic E5A oncoprotein, apparently via transmembrane interactions. Additionally, expression of E5B inhibited the transforming capability of BPV-1 E5A. These observations suggest that the expression of this viral protein may play a significant role in BPV/host cell interactions. PMID:12667807

Wlazlo, A P; Sparkowski, J J; Jenson, A B; Schlegel, R



Bovine oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP).  


Despite the long-held assumption that reprogramming factors are present in mammalian oocytes at the second metaphase stage, the molecular nature of these factors is not known. Here, we demonstrated that oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP). Injection of TCTP double-stranded RNA into germinal vesicle oocytes decreased the potential of nuclear-transferred (NT) oocytes, but not in vitro fertilized oocytes, to develop into blastocysts. Phosphorylated TCTP is considered to facilitate the first step of somatic cell reprogramming. After transfer of blastocysts that developed from NT oocytes fused with cumulus cells in which phosphorylated TCTP peptide was previously incorporated, the recipient pregnancy rate (47%) increased and the abortion rate (13%) decreased. Moreover, all seven cloned calves survived for at least 1 month after parturition, and had no morphologic abnormalities. The present study demonstrated that pretreatment of donor cells with phosphorylated TCTP peptide has a beneficial effect on the potential of bovine somatic cell nuclei to develop into normal cloned calves. Before widespread application of TCTP for bovine cloning, however, a large-scale embryo transfer study using different donor cell lines of various origins is necessary. PMID:17579559

Tani, Tetsuya; Shimada, Hiroaki; Kato, Yoko; Tsunoda, Yukio



Porous acellular bovine pericardia seeded with mesenchymal stem cells as a patch to repair a myocardial defect in a syngeneic rat model  

Microsoft Academic Search

A patch is often mandatory to repair myocardial defects; however, currently available patches lack the possibility of regeneration. To overcome this limitation, a porous acellular bovine pericardium seeded with BrdU-labeled mesenchymal stem cells (MSCs) was prepared (the MSC patch) to repair a surgically created myocardial defect in the right ventricle of a syngeneic rat model. The bovine pericardium before cell

Hao-Ji Wei; Sung-Ching Chen; Yen Chang; Shiaw-Min Hwang; Wei-Wen Lin; Po-Hong Lai; Huihua Kenny Chiang; Lee-Feng Hsu; Hang-Hsing Yang; Hsing-Wen Sung



Protein crystal growth in microgravity review of large scale temperature induction method: Bovine insulin, human insulin and human ?-interferon  

NASA Astrophysics Data System (ADS)

The Protein Crystal Growth Facility (PCF) is space-flight hardware that accommodates large scale protein crystal growth experiments using temperature change as the inductive step. Recent modifications include specialized instrumentation for monitoring crystal nucleation with laser light scattering. This paper reviews results from its first seven flights on the Space Shuttle, the last with laser light scattering instrumentation in place. The PCF's objective is twofold: (1) the production of high quality protein crystals for x-ray analysis and subsequent structure-based drug design and (2) preparation of a large quantity of relatively contaminant free crystals for use as time-release protein pharmaceuticals. The first three Shuttle flights with bovine insulin constituted the PCF's proof of concept, demonstrating that the space-grown crystals were larger and diffracted to higher resolution than their earth-grown counterparts. The later four PCF missions were used to grow recombinant human insulin crystals for x-ray analysis and continue productions trials aimed at the development of a processing facility for crystalline recombinant a-interferon.

Long, Marianna M.; Bishop, John Bradford; Delucas, Lawrence J.; Nagabhushan, Tattanhalli L.; Reichert, Paul; Smith, G. David



High-resolution X-ray crystal structure of bovine H-protein using the high-pressure cryocooling method  

PubMed Central

Recently, many technical improvements in macromolecular X-ray crystallography have increased the number of structures deposited in the Protein Data Bank and improved the resolution limit of protein structures. Almost all high-resolution structures have been determined using a synchrotron radiation source in conjunction with cryocooling techniques, which are required in order to minimize radiation damage. However, optimization of cryoprotectant conditions is a time-consuming and difficult step. To overcome this problem, the high-pressure cryocooling method was developed (Kim et al., 2005 ?) and successfully applied to many protein-structure analyses. In this report, using the high-pressure cryocooling method, the X-ray crystal structure of bovine H-protein was determined at 0.86?Å resolution. Structural comparisons between high- and ambient-pressure cryocooled crystals at ultra-high resolution illustrate the versatility of this technique. This is the first ultra-high-resolution X-ray structure obtained using the high-pressure cryocooling method.

Higashiura, Akifumi; Ohta, Kazunori; Masaki, Mika; Sato, Masaru; Inaka, Koji; Tanaka, Hiroaki; Nakagawa, Atsushi



High-resolution X-ray crystal structure of bovine H-protein using the high-pressure cryocooling method.  


Recently, many technical improvements in macromolecular X-ray crystallography have increased the number of structures deposited in the Protein Data Bank and improved the resolution limit of protein structures. Almost all high-resolution structures have been determined using a synchrotron radiation source in conjunction with cryocooling techniques, which are required in order to minimize radiation damage. However, optimization of cryoprotectant conditions is a time-consuming and difficult step. To overcome this problem, the high-pressure cryocooling method was developed (Kim et al., 2005) and successfully applied to many protein-structure analyses. In this report, using the high-pressure cryocooling method, the X-ray crystal structure of bovine H-protein was determined at 0.86?Å resolution. Structural comparisons between high- and ambient-pressure cryocooled crystals at ultra-high resolution illustrate the versatility of this technique. This is the first ultra-high-resolution X-ray structure obtained using the high-pressure cryocooling method. PMID:24121354

Higashiura, Akifumi; Ohta, Kazunori; Masaki, Mika; Sato, Masaru; Inaka, Koji; Tanaka, Hiroaki; Nakagawa, Atsushi



Evaluation of Multiplexed Foot-and-Mouth Disease Nonstructural Protein Antibody Assay Against Standardized Bovine Serum Panel  

SciTech Connect

Liquid array technology has previously been used to show proof-of-principle of a multiplexed non structural protein serological assay to differentiate foot-and-mouth infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B and 3D and recombinant protein signature 3ABC in combination with four controls. To determine diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright, UK. This sera panel has been used to assess the performance of other singleplex ELISA-based non-structural protein antibody assays. The 3ABC signature in the multiplexed assay showed comparative performance to a commercially available non-structural protein 3ABC ELISA (Cedi test{reg_sign}) and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex is acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promotes further assay development and optimization to generate an assay for routine use in foot-and-mouth disease surveillance.

Perkins, J; Parida, S; Clavijo, A



Endothelin stimulates a sustained 1,2-diacylglycerol increase and protein kinase C activation in bovine aortic smooth muscle cells  

SciTech Connect

Endothelin is a long-lasting potent vasoconstrictor peptide. We report here that in bovine aortic smooth muscle cells, endothelin biphasically increased total cellular diacylglycerol (DAG) content. When cellular DAG was labeled with (/sup 14/C) glycerol for 48h, endothelin stimulated (/sup 14/C)DAG formation in a biphasic pattern. Only one prolonged phase of DAG accumulation was observed when cells were labeled with (/sup 3/H)glycerol for 2 h. Endothelin induced an increase in the membranous protein kinase C (PKC) activities, which lasted for more than 20 min. These data suggest that (i) endothelin stimulates a sustained generation of DAG, (ii) this accumulation of DAG results in a sustained translocation of cytosolic PKC activities to the membrane.

Lee, T.S.; Chao, T.; Hu, K.Q.; King, G.L.



Cyst(e)ine residues of bovine white-matter proteolipid proteins. Role of disulphides in proteolipid conformation.  

PubMed Central

Cyst(e)ine residues of bovine white-matter proteolipid proteins were characterized in a highly purified preparation. From a total of 10.6 cyst(e)ine residues/molecule of protein, as determined by performic acid oxidation, 2.5-3 thiol groups were freely accessible to iodoacetamide, iodoacetic acid and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), when the proteins were solubilized in chloroform/methanol (C/M) (2:1, v/v). The presence of lipids had no effect on thiol-group exposure. One thiol group available to DTNB in C/M could not be detected when proteolipids were solubilized in the more polar solvent n-butanol. In a C/M solution of purified proteolipid proteins, SDS did not increase the number of reactive thiol groups, but the cleavage of one disulphide bridge made it possible to alkylate six more groups. C.d. and fluorescence studies showed that rupture of this disulphide bond changed the protein conformation, which was reflected in partial loss of helical structure and in a greater exposure to the solvent of at least one tryptophan residue. Cyst(e)ine residues were also characterized in the different components [PLP (principal proteolipid protein), DM20 and LMW (low-Mr proteins)] of the proteolipid preparation. Although the numbers of cyst(e)ine residues in PLP and DM20 were similar, in LMW fewer residues were alkylated under four different experimental conditions. The differences, however, are not simply related to differences in Mr.

Oteiza, P I; Adamo, A M; Aloise, P A; Paladini, A C; Paladini, A A; Soto, E F



The use of aqueous two-phase systems to concentrate and purify bovine leukemia virus outer envelope protein gp51.  


Enzootic bovine leucosis is a chronic lymphoproliferative disease of cattle. The causative agent, bovine leukemia virus (BLV), is related to the human retroviruses HTLV-I and -II. The external env-protein of BLV, a glycoprotein of 51 kDa, carries neutralizing epitopes and should be an essential component in a vaccine against the virus. Problems have been encountered with the concentration and purification of intact virions of BLV and other retroviruses. During centrifugation procedures the external env-proteins are to a great extent detached and consequently poorly recovered with the virion particles. Therefore, other methods are sought to obtain a high yield of the external glycoproteins. The use of two-phase systems based on water soluble polymers is described for the extraction of BLV-gp51 from culture medium. Several polymer systems were tested and the results showed that some were attractive for large scale application. The classical combination dextran-polyethylene glycol gave promising results; a partition coefficient of about 0.02 was obtained for the distribution of the gp51 between the top and combined inter- and bottom phases. In a single extraction step it was possible to obtain 45% of the glycoprotein in a small volume bottom phase and at the same time about 15-fold purified. That should be compared with a recovery of less than 20% with the conventional centrifugation procedures. It is concluded that extraction in phase systems based on water soluble polymers is a methodology well suited for the concentration and purification of BLV-gp51. PMID:2474306

Hammar, L; Merza, M; Malm, K; Eriksson, S; Morein, B



The bovine insulin-like growth factor (IGF) binding protein purified from conditioned medium requires the N-terminal tripeptide in IGF-1 for binding.  


The insulin-like growth factor binding protein (BP) secreted by bovine kidney (MDBK) cells has been purified by affinity chromatography on a rat IGF-2 Sepharose column. Purified BP migrated as a single band of Mr 40,000 upon SDS polyacrylamide gel electrophoresis. An N-terminal sequence of 53 residues was obtained which was very similar up to residue 21 to the corresponding rat BRL-3A BP sequence. In competitive binding experiments with bovine IGF-1 and IGF-2, and recombinant human IGF-1, BP had a similar affinity for these ligand when IGF-1 tracer was used, but a higher affinity for IGF-2 with IGF-2 as radioligand. The N-terminal destripeptide truncated form of bovine IGF-1, which has enhanced biological activity, was found to have a markedly reduced affinity for BP compared to intact IGF-1. The increased bioactivity of destripeptide IGF-1 can be explained by this reduced affinity for BP. PMID:2450535

Szabo, L; Mottershead, D G; Ballard, F J; Wallace, J C



Investigation on the interaction of tetrachloride fluorescein–bovine serum albumin-?-cyclodextrin and the determination of protein by flow injection analysis  

Microsoft Academic Search

In this paper, a simple and sensitive flow injection analysis (FIA) for the determination of protein with spectroscopic probe was developed. This method was based on the investigation of the interaction of tetrachloride fluorescein (2,4,5,7-tetrachloro-3,6-fluorandiol)–bovine serum albumin (BSA), the coupling reaction of protein with tetrachloride fluorescein (TCFS) which was used as a spectroscopic probe in the presence of ?-cyclodextrin (?-CD).

Xiashi Zhu; Yanyan Hu; Aiqin Gong



Use of recombinant proteins MPB70 or MPB83 as capture antigens in ELISAs to confirm bovine tuberculosis infections in Brazil  

Microsoft Academic Search

The objective was to evaluate the use of two indirect IgG-ELISA tests (with recombinant proteins MPB70 or MPB83, respectively, as capture antigens) as confirmatory tests for diagnosis of bovine tuberculosis in a herd of naturally infected dairy cows. Results for ELISA-MPB70 and ELISA-MPB83 were similar (kappa statistic=0.92) on Days 0 (day of intradermal injection with purified protein derivatives, PPD), 7,

C. D. Marassi; L. Medeiros; J. McNair; W. Lilenbaum



Endotoxin-free purification for the isolation of Bovine Viral Diarrhoea Virus E2 protein from insoluble inclusion body aggregates  

PubMed Central

Background Protein expression in Escherichia coli may result in the recombinant protein being expressed as insoluble inclusion bodies. In addition, proteins purified from E. coli contain endotoxins which need to be removed for in vivo applications. The structural protein, E2, from Bovine Viral Diarrhoea Virus (BVDV) is a major immunogenic determinant, and is an ideal candidate as a subunit vaccine. The E2 protein contains 17 cysteine residues creating difficulties in E. coli expression. In this report we outline a procedure for successfully producing soluble and endotoxin-free BVDV E2 protein from inclusion bodies (IB). Results The expression of a truncated form of BVDV-E2 protein (E2-T1) in E. coli resulted in predominantly aggregated insoluble IB. Solubilisation of E2-T1 with high purity and stability from IB aggregates was achieved using a strong reducing buffer containing 100 mM Dithiothreitol. Refolding by dialysis into 50 mM Tris (pH 7.0) containing 0.2% Igepal CA630 resulted in a soluble but aggregated protein solution. The novel application of a two-phase extraction of inclusion body preparations with Triton X-114 reduced endotoxin in solubilised E2-T1 to levels suitable for in vivo use without affecting protein yields. Dynamic light scattering analyses showed 37.5% of the protein was monomeric, the remaining comprised of soluble aggregates. Mice immunised with E2-T1 developed a high titre antibody response by ELISA. Western hybridisation analysis showed E2-T1 was recognised by sera from immunised mice and also by several BVDV-E2 polyclonal and monoclonal antibodies. Conclusion We have developed a procedure using E. coli to produce soluble E2-T1 protein from IB, and due to their insoluble nature we utilised a novel approach using Triton X-114 to efficiently remove endotoxin. The resultant protein is immunogenic and detectable by BVDV-E2 specific antibodies indicating its usefulness for diagnostic applications and as a subunit vaccine. The optimised E. coli expression system for E2-T1 combined with methodologies for solubilisation, refolding and integrated endotoxin removal presented in this study should prove useful for other vaccine applications.



Diffusion models of protein folding  

PubMed Central

In theory and in the analysis of experiments, protein folding is often described as diffusion along a single coordinate. We explore here the application of a one-dimensional diffusion model to interpret simulations of protein folding, where the parameters of a model that “best” describes the simulation trajectories are determined using a Bayesian analysis. We discuss the requirements for such a model to be a good approximation to the global dynamics, and several methods for testing its accuracy. For example, one test considers the effect of an added bias potential on the fitted free energies and diffusion coefficients. Such a bias may also be used to extend our approach to determining parameters for the model to systems which would not normally explore the full coordinate range on accessible time scales. Alternatively, the propagators predicted from the model at different “lag” times may be compared with observations from simulation. We then present some applications of the model to protein folding, including Kramers-like turnover in folding rates of coarse-grained models, the effect of non-native interactions on folding, and the effect of the chosen coordinate on the observed position-dependence of the diffusion coefficients. Lastly, we consider how our results are useful for the interpretation of experiments, and how this type of Bayesian analysis may eventually be applied directly to analyse experimental data.



[Identification of extrinsic proteins in boneless cooked ham by SDS-PAGE: detection level in model systems].  


Protein extraction and separation in polyacrylamide slab gel electrophoresis with Laemmli system (SDS-PAGE) were used to establish the detection level of protein raw materials in mixtures with porcine meat in boneless cooked ham. Model systems of boneless cooked ham with soy protein isolates, caseinate, skim powdered milk, bovine plasma, porcine plasma and whey proteins were studied. The quantification level of this method was 0.5% for soy protein isolates, caseinate and bovine plasma and 1.0% for porcine plasma, milk powder and whey proteins in boneless cooked ham. The electrophoretic method proved to be useful to identify some proteinic raw materials in porcine meat products and verify compliance with Argentine legislation. It may be used as a control methodology. PMID:17249490

López, Laura Beatríz; Greco, Carola Beatriz; Ronayne de Ferrer, Patricia; Valencia, Mirta Eva



Primary structure of bovine pituitary secretory protein I (chromogranin A) deduced from the cDNA sequence  

SciTech Connect

Secretory protein I (SP-I), also referred to as chromogranin A, is an acidic glycoprotein that has been found in every tissue of endocrine and neuroendocrine origin examined but never in exocrine or epithelial cells. Its co-storage and co-secretion with peptide hormones and neurotransmitters suggest that it has an important endocrine or secretory function. The authors have isolated cDNA clones from a bovine pituitary lambdagt11 expression library using an antiserum to parathyroid SP-I. The largest clone (SP4B) hybridized to a transcript of 2.1 kilobases in RNA from parathyroid, pituitary, and adrenal medulla. Immunoblots of bacterial lysates derived from SP4B lysognes demonstrated specific antibody binding to an SP4B/..beta..-galactosidase fusion protein (160 kDa) with a cDNA-derived component of 46 kDa. Radioimmunoassay of the bacterial lystates with SP-I antiserum yielded parallel displacement curves of /sup 125/I-labeled SP-I by the SP4B lysate and authentic SP-I. SP4B contains a cDNA of 1614 nucleotides that encodes a 449-amino acid protein (calculated mass, 50 kDa). The nucleotide sequences of the pituitary SP-I cDNA and adrenal medullary SP-I cDNAs are nearly identical. Analysis of genomic DNA suggests that pituitary, adrenal, and parathyroid SP-I are products of the same gene.

Ahn, T.G.; Cohn, D.V.; Gorr, S.U.; Ornstein, D.L.; Kashdan, M.A.; Levine, M.A.



A model of the spread of the bovine viral-diarrhoea virus within a dairy herd.  


Wet BVDSim (a stochastic simulation model) was developed to study the dynamics of the spread of the bovine viral-diarrhoea virus (BVDV) within a dairy herd. This model took into account herd-management factors (common in several countries), which influence BVDV spread. BVDSim was designed as a discrete-entity and discrete-event simulation model. It relied on two processes defined at the individual-animal level, with interactions. The first process was a semi-Markov process and modelled the herd structure and dynamics (demography, herd management). The second process was a Markov process and modelled horizontal and vertical virus transmission. Because the horizontal transmission occurs by contacts (nose-to-nose) and indirectly, transmission varied with the separation of animals into subgroups. Vertical transmission resulted in birth of persistently infected (PI) calves. Other possible consequences of a BVDV infection during the pregnancy period were considered (pregnancy loss, immunity of calves). The outcomes of infection were modelled according to the stage of pregnancy at time of infection. BVDV pregnancy loss was followed either by culling or by a new artificial insemination depending on the modelled farmer's decision. Consistency of the herd dynamics in the absence of any BVDV infection was verified. To explore the model behaviour, the virus spread was simulated over 10 years after the introduction of a near-calving PI heifer into a susceptible 38 cow herd. Different dynamics of the virus spread were simulated, from early clearance to persistence of the virus 10 years after its introduction. Sensitivity of the model to the uncertainty on transmission coefficient was analysed. Qualitative validation consisted in comparing the bulk-milk ELISA results over time in a sample of herds detected with a new infection with the ones derived from simulations. PMID:15158572

Viet, Anne-France; Fourichon, Christine; Seegers, Henri; Jacob, Christine; Guihenneuc-Jouyaux, Chantal



The major tyrosine-sulfated protein of the bovine anterior pituitary is a secretory protein present in gonadotrophs, thyrotrophs, mammotrophs, and corticotrophs  

PubMed Central

The anterior pituitary is a complex secretory tissue known to contain several sulfated macromolecules. In the present study, we identified the major tyrosine-sulfated protein of the bovine anterior pituitary and investigated its cellular and subcellular localization. This protein consisted of two tyrosine-sulfated polypeptides of molecular weight 86,000 and 84,000 that were highly homologous to each other. In agreement with previous biochemical studies, the tyrosine-sulfated protein of Mr 86,000/84,000 was found to be secretory, as it was observed in the matrix of secretory granules by immunoelectron microscopy. Immunofluorescence studies indicated that the tyrosine- sulfated, secretory protein of Mr 86,000/84,000, referred to as TSP 86/84, was present in all endocrine cells except for some somatotrophic cells. Higher levels of immunoreactivity for TSP 86/84 were observed in gonadotrophic and thyrotrophic than in mammotrophic and corticotrophic cells. This appeared to result from the occurrence of TSP 86/84 in all secretory granules of the former cells and in only some secretory granules of the latter cells. We discuss the possibility that TSP 86/84 may have a role in the packaging of several distinct peptides hormones into secretory granules. One, though not the only, possible function of tyrosine sulfation may concern the sorting of this protein in the Golgi complex.



Optimization and characterization of an in vitro bovine mammary cell culture system to study regulation of milk protein synthesis and mammary differentiation  

SciTech Connect

A long term bovine mammary cell culture system that maintains normal mammary cell function was established and optimized to study milk protein synthesis and secretion and mammary differentiation. This culture system used bovine mammary acini isolated from developing or lactating mammary gland by enzymatic dissociation, and cryopreserved until thawed and plated for growth in vitro for these studies. Cells in M199 with lactogenic hormones {plus minus} fetal calf serum (FCS) were cultured on plastic, 100ul and 500ul type I collagen, and Matrigel, or embedded within type I collagen. Cell morphology, cell number, and total TCA-precipitable {sup 35}S-labelled proteins were monitored. Milk protein ({alpha}{sub s,1}-casein, lactoferrin (LF), {alpha}-lactalbumin, and {beta}-lactoglobulin) secretion and intracellular levels were determined by an ELISA assay.

Talhouk, R.S.



Bovine Adipose Triglyceride Lipase is Not Altered and Adipocyte Fatty Acid-Binding Protein is Increased by Dietary Flaxseed  

Microsoft Academic Search

In this paper, we report the full-length coding sequence of bovine ATGL cDNA and analyze its expression in bovine tissues.\\u000a Similar to human, mouse, and pig ATGL sequences, bovine ATGL has a highly conserved patatin domain that is necessary for lipolytic\\u000a function in mice and humans. This suggests that ATGL is functionally intact as a triglyceride lipase in cattle. Tissue

Jeffrey Deiuliis; Jonghyun Shin; Eric Murphy; Scott L. Kronberg; Maurice L. Eastridge; Yeunsu Suh; Jong-Taek Yoon; Kichoon Lee



Interactions of tubulin and microtubule-associated proteins. Conformation and stability of the oligomeric species from glycerol-cycled microtubule protein of bovine brain.  


1. The conformation of bovine microtubule protein prepared by cycles of assembly and disassembly in the presence of glycerol has been studied by near-u.v. circular dichroism (c.d.) over a range of protein concentrations. The effects on the conformational properties of ionic strength and of a pH range from 6 to 7.5 have been correlated with the known oligomeric composition of microtubule protein preparations, as determined by the sedimentation behaviour of this preparation [Bayley, Charlwood, Clark & Martin (1982) Eur. J. Biochem.121, 579-585]. 2. The formation of 30S oligomeric ring species, either by decreasing ionic strength at pH6.5 or by changing pH in the presence of 0.1m-NaCl, correlates with a significant change in tubulin c.d. Formation of 18S oligomer by changing pH at ionic strength 0.2 produced no comparable effect. The c.d. of tubulin dimer itself is not affected by ionic strength and pH over the same range. 3. The results are interpreted as a small conformational adjustment between tubulin and specific microtubule-associated proteins on forming 30S oligomeric species, due to interaction with the high-molecular-weight-group proteins. The possible significance of this is discussed with respect to microtubule assembly in vitro. 4. By using this conformational parameter, together with equilibrium and kinetic light-scattering studies, the sensitivity of glycerol-cycled microtubule protein to dilution is shown to be strongly pH-dependent, the oligomers being much more stable at pH6.4 than at pH6.9. 5. Oligomeric complexes of tubulin with microtubule-associated proteins show marked stability under conditions similar to those for efficient microtubule assembly in vitro. Oligomeric material therefore must be incorporated directly during assembly in vitro from microtubule protein. PMID:7115306

Martin, S R; Clark, D C; Mayley, P M



Proteomic Analysis of Bovine Sperm YWHA Binding Partners Identify Proteins Involved in Signaling and Metabolism1  

PubMed Central

Posttranslational modification of proteins by phosphorylation is involved in regulation of sperm function. Protein phosphatase 1 gamma isoform 2 (PPP1CC_v2) and protein YWHA (also known as 14-3-3) are likely to be key molecules in pathways involving sperm protein phosphorylation. We have shown that phosphorylated PPP1CC_v2 is bound to protein YWHAZ in spermatozoa. In somatic cells, protein YWHA is known to bind a number of phosphoproteins involved in signaling and energy metabolism. Thus, in addition to PPP1CC_v2, it is likely that sperm contain other YWHA-binding proteins. A goal of the present study was to identify these sperm YWHA-binding proteins. The binding proteins were isolated by affinity chromatography with GST-YWHAZ followed by elution with a peptide, R-11, which is known to disrupt YWHA complexes. The YWHA-binding proteins in sperm can be classified as those involved in fertilization, acrosome reaction, energy metabolism, protein folding, and ubiquitin-mediated proteolysis. A subset of these putative YWHA-binding proteins contain known amino acid consensus motifs, not only for YWHA binding but also for PPP1C binding. Identification of sperm PPP1CC_v2-binding proteins by microcystin-agarose chromatography confirmed that PPP1CC_v2 and YWHA interactomes contain several common proteins. These are metabolic enzymes phosphoglycerate kinase 2, hexokinase 1, and glucose phosphate isomerase; proteins involved in sperm-egg fusion; angiotensin-converting enzyme, sperm adhesion molecule, and chaperones; heat shock 70-kDa protein 5 (glucose-regulated protein 78 kDa; and heat shock 70-kDa protein 1-like. These proteins are likely to be phosphoproteins and potential PPP1CC_v2 substrates. Our data suggest that in addition to potential regulation of a number of important sperm functions, YWHA may act as an adaptor molecule for a subset of PPP1CC_v2 substrates.

Puri, Pawan; Myers, Kimberley; Kline, Douglas; Vijayaraghavan, Srinivasan



An isotope dilution model for partitioning leucine uptake by the bovine mammary gland  

Microsoft Academic Search

A model of leucine uptake and partition by the mammary gland (i.e. the udder) of the lactating dairy cow is constructed and solved in the steady state. Model solution permits—if assumptions are made—calculation of leucine uptake from the arterial blood supply, leucine export into the venous drainage, leucine oxidation, and synthesis and degradation of milk protein and constitutive tissue protein

B. J Bequette; G. E Lobley; J. A Metcalf; D Wray-Cahen; M. S Dhanoa; F. R. C Backwell; M. D Hanigan; J. C Macrae; D. E Beever



Proteomic analysis of bovine nucleolus.  


Nucleolus is the most prominent subnuclear structure, which performs a wide variety of functions in the eukaryotic cellular processes. In order to understand the structural and functional role of the nucleoli in bovine cells, we analyzed the proteomic composition of the bovine nucleoli. The nucleoli were isolated from Madin Darby bovine kidney cells and subjected to proteomic analysis by LC-MS/MS after fractionation by SDS-PAGE and strong cation exchange chromatography. Analysis of the data using the Mascot database search and the GPM database search identified 311 proteins in the bovine nucleoli, which contained 22 proteins previously not identified in the proteomic analysis of human nucleoli. Analysis of the identified proteins using the GoMiner software suggested that the bovine nucleoli contained proteins involved in ribosomal biogenesis, cell cycle control, transcriptional, translational and post-translational regulation, transport, and structural organization. PMID:20970743

Patel, Amrutlal K; Olson, Doug; Tikoo, Suresh K



Regulation of Innate Immune Responses by Bovine Herpesvirus 1 and Infected Cell Protein 0 (bICP0)  

PubMed Central

Bovine herpesvirus 1 (BoHV-1) infected cell protein 0 (bICP0) is an important transcriptional regulatory protein that stimulates productive infection. In transient transfection assays, bICP0 also inhibits interferon dependent transcription. bICP0 can induce degradation of interferon stimulatory factor 3 (IRF3), a cellular transcription factor that is crucial for activating beta interferon (IFN-?) promoter activity. Recent studies also concluded that interactions between bICP0 and IRF7 inhibit trans-activation of IFN-? promoter activity. The C3HC4 zinc RING (really important new gene) finger located near the amino terminus of bICP0 is important for all known functions of bICP0. A recombinant virus that contains a single amino acid change in a well conserved cysteine residue of the C3HC4 zinc RING finger of bICP0 grows poorly in cultured cells, and does not reactivate from latency in cattle confirming that the C3HC4 zinc RING finger is crucial for viral growth and pathogenesis. A bICP0 deletion mutant does not induce plaques in permissive cells, but induces autophagy in a cell type dependent manner. In summary, the ability of bICP0 to stimulate productive infection, and repress IFN dependent transcription plays a crucial role in the BoHV-1 infection cycle.

Jones, Clinton



Inositol phosphates compete with nucleic acids for binding to bovine leukemia virus matrix protein: implications for deltaretroviral assembly.  


The matrix (MA) domain of retroviral Gag proteins plays a crucial role in virion assembly. In human immunodeficiency virus type 1 (HIV-1), a lentivirus, the presence of phosphatidylinositol-(4,5)-bisphosphate triggers a conformational change allowing the MA domain to bind the plasma membrane (PM). In this study, the MA protein from bovine leukemia virus (BLV) was used to investigate the mechanism of viral Gag binding to the membrane during replication of a deltaretrovirus. Fluorescence spectroscopy was used to measure the binding affinity of MA for two RNA constructs derived from the BLV genome as well as for single-stranded DNA (ssDNA). The importance of electrostatic interactions and the ability of inositol hexakisphosphate (IP6) to compete with nucleic acids for binding to MA were also investigated. Our data show that IP6 effectively competes with RNA and DNA for BLV MA binding, while [NaCl] of greater than 100 mM is required to produce any observable effect on DNA-MA binding. These results suggest that BLV assembly may be highly dependent on the specific interaction of the MA domain with components of the PM, as observed previously with HIV-1. The mode of MA binding to nucleic acids and the implications for BLV assembly are discussed. PMID:23504872

Qualley, Dominic F; Lackey, Crystal M; Paterson, Justin P



Distinctions between bovine herpesvirus 1 and herpes simplex virus type 1 VP22 tegument protein subcellular associations.  


The alphaherpesvirus tegument protein VP22 has been characterized with multiple traits including microtubule reorganization, nuclear localization, and nonclassical intercellular trafficking. However, all these data were derived from studies using herpes simplex virus type 1 (HSV-1) and may not apply to VP22 homologs of other alphaherpesviruses. We compared subcellular attributes of HSV-1 VP22 (HVP22) with bovine herpesvirus 1 (BHV-1) VP22 (BVP22) using green fluorescent protein (GFP)-fused VP22 expression vectors. Fluorescence microscopy of cell lines transfected with these constructs revealed differences as well as similarities between the two VP22 homologs. Compared to that of HVP22, the BVP22 microtubule interaction was much less pronounced. The VP22 nuclear interaction varied, with a marbled or halo appearance for BVP22 and a speckled or nucleolus-bound appearance for HVP22. Both VP22 homologs associated with chromatin at various stages of mitosis and could traffic from expressing cells to the nuclei of nonexpressing cells. However, distinct qualitative differences in microtubule, nuclear, and chromatin association as well as trafficking were observed. The differences in VP22 homolog characteristics revealed in this study will help define VP22 function within HSV-1 and BHV-1 infection. PMID:10708447

Harms, J S; Ren, X; Oliveira, S C; Splitter, G A



Concentrated bovine milk whey active proteins facilitate osteogenesis through activation of the JNK-ATF4 pathway.  


Concentrated fractions of low molecular weight whey proteins (1-30 kDa), that is concentrated bovine milk whey active proteins (CBP), have been found to enhance bone formation in both in vivo and clinical studies, but the underlying mechanisms are poorly understood. In this study, we found that CBP promoted osteoblastic differentiation in normal human osteoblasts, and determined the involvement of the c-jun NH2-terminal kinase (JNK)-activating transcription factor 4 (ATF4) pathway. We observed that alkaline phosphatase activity and mineralization were significantly induced by CBP treatment. In addition, mRNA expression of ATF4 was intensely elevated in CBP-treated osteoblasts, indicating that the late-phase events of differentiation were promoted. We found that CBP activated the phosphorylation of JNK and extracellular signal-regulated kinase (ERK). Furthermore, pathway analyses using the various signaling pathway-specific inhibitors revealed that JNK activation, but not ERK activation, is essential for CBP-induced mineralization and ATF4 expression. Our results indicate that the JNK-mediated ATF4 pathway is required for CBP-promotive osteogenesis. PMID:22790938

Tsuji-Naito, Kentaro; Jack, Ralph W



Bovine Spongiform Encephalopathy  

Technology Transfer Automated Retrieval System (TEKTRAN)

Bovine spongiform encephalopathy (BSE) is caused by a novel contagion, known to as a prion. Prions are proteins capable of converting a normal cellular protein into a prion, thereby propagating an infection. BSE is the first known prion zoonotic. As such it has attracted broad scientific and, to a r...


Association of bovine respiratory disease with clinical status and acute phase proteins in calves  

Microsoft Academic Search

Eighty-four calves with respiratory disease from 18 herds in different parts of Finland were chosen for a study evaluating the capacity of different respiratory pathogens to cause changes in different acute phase protein concentrations, white blood cell (WBC) count and clinical signs. The selected acute phase proteins were fibrinogen, haptoglobin, serum amyloid-A, lipopolysaccharide binding protein and ?1-acid glycoprotein. From each

S. Nikunen; H. Härtel; T. Orro; E. Neuvonen; R. Tanskanen; S.-L. Kivelä; S. Sankari; P. Aho; S. Pyörälä; H. Saloniemi; T. Soveri



Characterization and isolation of a high-density-lipoprotein-binding protein from bovine corpus luteum plasma membrane.  

PubMed Central

The ovary uses the cholesterol from high-density lipoproteins (HDL) as a substrate source for steroid hormone production. It is not clear, however, how ovarian cells acquire the lipoprotein cholesterol. This study describes the characterization and isolation of a high-affinity-binding protein for apolipoprotein E-free HDL from the plasma-membrane fraction of bovine corpora lutea. Plasma membranes were prepared by differential centrifugation with 5-6-fold enrichment of 5'-nucleotidase activity. The binding of 125I-HDL to the plasma membranes was time-dependent, and there appeared to be a single high-affinity site with a Kd of 6.7 micrograms of HDL/ml of assay buffer. The binding was not affected by high concentrations of low-density lipoproteins or the Ca2+ chelator EDTA, nor by changes in pH in the range 6.5-9.0. The binding was affected by the salt concentration in the buffer, with a dose-dependent increase that reached a maximum at 150-250 mM-NaCl. Binding was increased in the presence of high concentrations of KCl and KBr, and most significantly increased by high concentrations of bivalent metal ions. Ligand-blot analysis under reducing conditions revealed that the binding protein was a single polypeptide of about 108 kDa that was associated with the plasma-membrane fraction. This HDL-binding protein was purified to homogeneity by solubilization with Triton X-100, poly(ethylene glycol) precipitation, DEAE-Sephadex chromatography, and preparative SDS/PAGE. The purified binding protein is a single polypeptide of 108 kDa that retains high affinity and specificity for HDL as assayed by ligand blotting. Images Fig. 6. Fig. 8. Fig. 9. Fig. 10.

Ferreri, K; Menon, K M



Mutations in domain a? of protein disulfide isomerase affect the folding pathway of bovine pancreatic ribonuclease A  

PubMed Central

Protein disulfide isomerase (PDI, EC, an enzyme and chaperone, catalyses disulfide bond formation and rearrangements in protein folding. It is also a subunit in two proteins, the enzyme collagen prolyl 4-hydroxylase and the microsomal triglyceride transfer protein. It consists of two catalytically active domains, a and a?, and two inactive ones, b and b?, all four domains having the thioredoxin fold. Domain b? contains the primary peptide binding site, but a? is also critical for several of the major PDI functions. Mass spectrometry was used here to follow the folding pathway of bovine pancreatic ribonuclease A (RNase A) in the presence of three PDI mutants, F449R, ?455–457, and abb?, and the individual domains a and a?. The first two mutants contained alterations in the last ? helix of domain a?, while the third lacked the entire domain a?. All mutants produced genuine, correctly folded RNase A, but the appearance rate of 50% of the product, as compared to wild-type PDI, was reduced 2.5-fold in the case of PDI ?455–457, 7.5-fold to eightfold in the cases of PDI F449R and PDI abb?, and over 15-fold in the cases of the individual domains a and a?. In addition, PDI F449R and PDI abb? affected the distribution of folding intermediates. Domains a and a? catalyzed the early steps in the folding but no disulfide rearrangements, and therefore the rate observed in the presence of these individual domains was similar to that of the spontaneous process.

Ruoppolo, Margherita; Orru, Stefania; Talamo, Fabio; Ljung, Johanna; Pirneskoski, Annamari; Kivirikko, Kari I.; Marino, Gennaro; Koivunen, Peppi



Determination of soluble immunoglobulin G in bovine colostrum products by Protein G affinity chromatography-turbidity correction and method validation.  


Immunoglobulin-containing food products and nutraceuticals such as bovine colostrum are of interest to consumers as they may provide health benefits. Commercial scale colostrum products are valued for their immunoglobulin G (IgG) content and therefore require accurate analysis. One of the most commonly used methods for determining total soluble IgG in colostrum products is based on affinity chromatography using a Protein G column and UV detection. This paper documents improvements to the accuracy of the Protein G analysis of IgG in colostrum products, especially those containing aggregated forms of IgG. Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis confirmed that aggregated IgG measured by Protein G does not contain significant amounts of casein or other milk proteins. Size exclusion chromatography identified the content of soluble IgG as mainly monomeric IgG and aggregated material MW > 450 kDa with small amounts of dimer and trimer. The turbidity of the eluting IgG, mainly associated with aggregated IgG, had a significant effect on the quantitative results. Practical techniques were developed to correct affinity LC data for turbidity on an accurate, consistent, and efficient basis. The method was validated in two laboratories using a variety of colostrum powders. Precision for IgG was 2-3% (RSD(r)) and 3-12% (RSD(R)). Recovery was 100.2 ± 2.4% (mean ± RSD, n = 10). Greater amounts of aggregated IgG were solubilized by a higher solution:sample ratio and extended times of mixing or sonication, especially for freeze-dried material. It is concluded that the method without acid precipitation and with turbidity correction provides accurate, precise, and robust data for total soluble IgG and is suitable for product specification and quality control of colostrum products. PMID:21524111

Holland, Patrick T; Cargill, Anne; Selwood, Andrew I; Arnold, Kate; Krammer, Jacqueline L; Pearce, Kevin N



Protein Self-Association in Solution: The Bovine Pancreatic Trypsin Inhibitor Decamer  

PubMed Central

We have used magnetic relaxation dispersion to study bovine pancreatic trypsin inhibitor (BPTI) self-association as a function of pH, salt type and concentration, and temperature. The magnetic relaxation dispersion method sensitively detects stable oligomers without being affected by other interactions. We find that BPTI decamers form cooperatively under a wide range of solution conditions with no sign of dimers or other small oligomers. Decamer formation is opposed by electrostatic repulsion among numerous cationic residues confined within a narrow channel. Accordingly, the decamer population increases with increasing pH, as cationic residues are deprotonated, and with increasing salt concentration. The salt effect cannot be described in terms of Debye screening, but involves the ion-specific sequestering of anions within the narrow channel. The lifetime of the BPTI decamer is 101 ± 4 min at 27°C. We propose that the BPTI decamer, with a heparin chain threading the decamer channel, plays a functional role in the mast cell. We also detect a higher oligomer that appears to be a subcritical nucleation cluster of 3–5 decamers. We argue that monomeric crystals form at high pH despite a high decamer population in solution, because the ion pairs that provide the critical decamer-decamer contacts are disrupted at high pH.

Gottschalk, Michael; Venu, Kandadai; Halle, Bertil



Conformational changes of the chaperone SecB upon binding to a model substrate--bovine pancreatic trypsin inhibitor (BPTI).  


SecB is a homotetrameric cytosolic chaperone that forms part of the protein translocation machinery in E. coli. Due to SecB, nascent polypeptides are maintained in an unfolded translocation-competent state devoid of tertiary structure and thus are guided to the translocon. In vitro SecB rapidly binds to a variety of ligands in a non-native state. We have previously investigated the bound state conformation of the model substrate bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin labeling and pyrene fluorescence methods. It was shown that SecB undergoes a conformational change during the process of substrate binding. Here, we generated SecB mutants containing but a single cysteine per subunit or an exposed highly reactive new cysteine after removal of the nearby intrinsic cysteines. Quantitative spin labeling was achieved with the methanethiosulfonate spin label (MTS) at positions C97 or E90C, respectively. Highfield (W-band) electron paramagnetic resonance (EPR) measurements revealed that with BPTI present the spin labels are exposed to a more polar/hydrophilic environment. Nanoscale distance measurements with double electron-electron resonance (DEER) were in excellent agreement with distances obtained by molecular modeling. Binding of BPTI also led to a slight change in distances between labels at C97 but not at E90C. While the shorter distance in the tetramer increased, the larger diagonal distance decreased. These findings can be explained by a widening of the tetrameric structure upon substrate binding much like the opening of two pairs of scissors. PMID:21848506

Haimann, Michaela M; Akdogan, Yasar; Philipp, Reinhard; Varadarajan, Raghavan; Hinderberger, Dariush; Trommer, Wolfgang E



Fatty acid-binding protein activities in bovine muscle, liver and adipose tissue  

SciTech Connect

Subcutaneous adipose tissue, sternomandibularis muscle and liver were obtained from steers immediately postmortem. Muscle strips and adipose tissue snips were incubated with 0.75 mM (1- UC)palmitate and 5 mM glucose. Muscle strips esterified palmitate at the rate of 2.5 nmol/min per gram tissue, which was 30% of the rate observed for adipose tissue. Fatty acid-binding protein activity was measured in 104,000 x g supernatant fractions of liver, muscle and adipose tissue homogenates. Muscle and adipose tissue fractions bound 840 and 140 pmol (1- UC)palmitoyl-CoA per gram tissue, respectively. Fatty acid-binding protein activity was greater in adipose tissue than in muscle when data were expressed per milligram protein. Fatty acid binding-protein activity was correlated with the rate of palmitate esterification within each tissue. Liver contained the highest fatty acid-binding protein activity.

Smith, S.B.; Ekeren, P.A.; Sanders, J.O.



Experimental reactivation of bovine herpesvirus 1 (BHV-1) by means of corticosteroids in an intranasal rabbit model  

Microsoft Academic Search

Summary Intranasal inoculation of the rabbit was shown to be a viable alternative to eye inoculation as a model to study latency and reactivation of bovine herpesvirus 1 (BHV-1). In four different experiments, separate groups of rabbits were intranasally inoculated with BHV-1. In two experiments some rabbits were inoculated instead with a TK-defective (TK-) mutant strain of BHV-1. The development

G. A. Brown; H. J. Field



Immunocytochemical Characterization of Delta-Opioid and Mu-Opioid Receptor Protein in the Bovine Pineal Gland  

Microsoft Academic Search

Opioidergic innervation has been identified in the mammalian pineal gland. Recently, opioid receptors in bovine pineal glands have been characterized; the activation of these receptors leads to the stimulation of melatonin synthesis. In this study, the precise localization of opioid receptors in bovine pineal glands was determined by an immunohistochemical technique using antibodies raised against delta-opioid and mu-opioid receptors. Immunoreactivity

Pansiri Phansuwan-Pujito; Manuchair Ebadi; Piyarat Govitrapong



Phylogenetic mixture models for proteins.  


Standard protein substitution models use a single amino acid replacement rate matrix that summarizes the biological, chemical and physical properties of amino acids. However, site evolution is highly heterogeneous and depends on many factors: genetic code; solvent exposure; secondary and tertiary structure; protein function; etc. These impact the substitution pattern and, in most cases, a single replacement matrix is not enough to represent all the complexity of the evolutionary processes. This paper explores in maximum-likelihood framework phylogenetic mixture models that combine several amino acid replacement matrices to better fit protein evolution.We learn these mixture models from a large alignment database extracted from HSSP, and test the performance using independent alignments from TREEBASE.We compare unsupervised learning approaches, where the site categories are unknown, to supervised ones, where in estimations we use the known category of each site, based on its exposure or its secondary structure. All our models are combined with gamma-distributed rates across sites. Results show that highly significant likelihood gains are obtained when using mixture models compared with the best available single replacement matrices. Mixtures of matrices also improve over mixtures of profiles in the manner of the CAT model. The unsupervised approach tends to be better than the supervised one, but it appears difficult to implement and highly sensitive to the starting values of the parameters, meaning that the supervised approach is still of interest for initialization and model comparison. Using an unsupervised model involving three matrices, the average AIC gain per site with TREEBASE test alignments is 0.31, 0.49 and 0.61 compared with LG (named after Le & Gascuel 2008 Mol. Biol. Evol. 25, 1307-1320), WAG and JTT, respectively. This three-matrix model is significantly better than LG for 34 alignments (among 57), and significantly worse for 1 alignment only. Moreover, tree topologies inferred with our mixture models frequently differ from those obtained with single matrices, indicating that using these mixtures impacts not only the likelihood value but also the output tree. All our models and a PhyML implementation are available from PMID:18852096

Le, Si Quang; Lartillot, Nicolas; Gascuel, Olivier



Proteins other than the Locus of Enterocyte Effacement-encoded proteins may contribute to Escherichia coli O157:H7 adherence to bovine rectoanal junction stratified squamous epithelial cells  

Technology Transfer Automated Retrieval System (TEKTRAN)

In this study, the Type III Secretion System (TTSS) proteins considered critical for Escherichia coli O157 (O157) adherence to the follicle-associated epithelial (FAE) cells at the bovine recto-anal junction (RAJ), did not appear to contribute to O157 adherence to the RAJ squamous epithelial (RSE) ...


Variable-Target-Function and Build-up Procedures for the Calculation of Protein Conformation. Application to Bovine Pancreatic Trypsin Inhibitor Using Limited Simulated Nuclear Magnetic Resonance Data  

Microsoft Academic Search

An implementation of the variable-target-function procedure, first introduced by Braun and G? [W. Braun and N. G?, J. Mol. Biol. 186, 611–626 (1985)], has been used to generate conformations of the small protein bovine pancreatic trypsin inhibitor (BPTI), given a limited set of simulated data that could be obtained by nuclear magnetic resonance (NMR) techniques. A hybrid strategy was also

Maximiliano Vásquez; Harold A. Scheraga



Lactobacillus bulgaricus Proteinase Expressed in Lactococcus lactis Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine  Lactoglobulin Fusion Proteins  

Microsoft Academic Search

Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine -lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recom- binant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as

Eric Bernasconi; Jacques-Edouard Germond; Michèle Delley; R. Fritsche; B. Corthesy



Copper deficiency in the young bovine results in dramatic decreases in brain copper concentration but does not alter brain prion protein biology  

Microsoft Academic Search

An Mn for Cu substitution on cellular prion proteins (PrPc) in the brain that results in bio- chemical changes to PrPc has been implicated in the pathogenesis of transmissible spongiform encephal- opathies. Recent research in the mature bovine does not support this theory. The present study tested this hypothesis by using progeny from gestating cows re- ceiving Cu-deficient diets or

L. R. Legleiter; J. W. Spears; H. C. Liu



Use of topical bovine thrombin in an anti-coagulated rat model of hepatic injury.  


The need for surgical hemostasis in patients treated with anticoagulant medications is a concern. This study assessed a bovine-derived topical hemostat (FastAct, FA) using a partial hepatectomy hemorrhage model in anticoagulated rats. Ninety rats were randomly assigned to receive preoperative heparin, warfarin, or nothing (n=30/treatment). Within each treatment group, FA, saline, direct pressure (DP), electrocautery, or nothing (n=6/group) was applied to the hepatectomy site. Eight additional rats were used for assessment of the preoperative anticoagulant regimen. Rats that were not anticoagulated and received FA had faster clot times and less hemorrhage than those receiving DP (P<0.05). In warfarin-pretreated rats, FA resulted in faster coagulation times than saline or DP and less hemorrhage than saline (P<0.05). No differences were detected in heparinized rats. Across all groups, rats receiving FA lost less blood and formed clots more frequently than saline (P<0.05). FA may be useful to treat hemorrhage from hepatic lacerations in anticoagulated patients. PMID:22633173

Schmiedt, Chad W; Köhler, Rickard; Brainard, Benjamin M



Effects of myocardial contractility on microemboli production by mechanical heart valves in a bovine model.  


Microemboli caused by mechanical heart valves have the potential to cause cerebrovascular events. We investigated the effects of myocardial contractility and heart rate on microemboli production in association with conventional and experimental mechanical heart valves implanted in the mitral position in a bovine model. In 10 calves, the mitral valves were replaced with mechanical valves. Doppler recordings were analyzed for high-intensity transient signals, which are ultrasound reflections from circulating microemboli. The animals were studied at rest, during pacing at 160 bpm, after dobutamine infusion, and after esmolol infusion. The incidence of high intensity transient signals was expressed as signal frequency (signals per hour) and as signal rate (signals per 100 heart cycles). With a 68% increase in the heart rate, signal frequency increased by 135%, but signal rate increased by only 41 %. With a 144% increase in myocardial contractility, signal rate increased by 264 %. With a 31 % decrease in contractility, signal rate decreased by 62 %. We conclude that microemboli production by mechanical heart valves varies with myocardial contractility and heart rate. The fact that contractility affects the incidence of high-intensity transient signals suggests that the microemboli are gaseous in nature, that their production is pressure driven, and that cavitation is a possible cause. It is likely that mechanical heart valve design is responsible for the quantity of microemboli production. PMID:11093405

Deklunder, G; Lecroart, J L; Conger, J L; Lapeyre, D; Gregoric, I; Rose, H; Tamez, D; Frazier, O H



Isolation of calcium-binding proteins on selective adsorbents. Application to purification of bovine calmodulin.  


We report the fractionation of calcium-binding proteins using immobilized metal ion affinity chromatography (IMAC) with hard metal ions. Various hard metal ions (Mn2+, La3+, Nd3+, Eu(3 were immobilized on cross-linked agarose substituted with Tris(carboxymethyl)ethylenediamine (TED) and used as an adsorbent. After systematic studies, europium was selected for further work on the fractionation of calcium-binding proteins. It was found that the presence of Ca2+ in the sample and the solvent strongly promoted the adsorption and selectivity. Selective elution was accomplished in stepwise mode by the addition of calcium chelators such as malonate, citrate and phosphate. Calmodulin of high purity was isolated from a crude extract. Similar behavior of other calcium-binding proteins indicates that the reported chromatographic procedure can be generally applied to such proteins. PMID:8653201

Chaga, G S; Ersson, B; Porath, J O



Determination of protein-ligand binding affinity by NMR: observations from serum albumin model systems.  


The usefulness of bovine serum albumin (BSA) as a model protein for testing NMR methods for the study of protein-ligand interactions is discussed. Isothermal titration calorimetry established the binding affinity and stoichiometry of the specific binding site for L-tryptophan, D-tryptophan, naproxen, ibuprofen, salicylic acid and warfarin. The binding affinities of the same ligands determined by NMR methods are universally weaker (larger KD). This is because the NMR methods are susceptible to interference from additional non-specific binding. The L-tryptophan-BSA and naproxen-BSA systems were the best behaved model systems. PMID:15816062

Fielding, Lee; Rutherford, Samantha; Fletcher, Dan



Bovine leukemia virus structural gene vectors are immunogenic and lack pathogenicity in a rabbit model.  


Infection with a replication-competent bovine leukemia virus structural gene vector (BLV SGV) is an innovative vaccination approach to prevent disease by complex retroviruses. Previously we developed BLV SGV that constitutively expresses BLV gag, pol, and env and related cis-acting sequences but lacks tax, rex, RIII, and GIV and most of the BLV long terminal repeat sequences, including the cis-acting Tax and Rex response elements. The novel SGV virus is replication competent and replicates a selectable vector to a titer similar to that of the parental BLV in cell culture. The overall goal of this study was to test the hypothesis that infection with BLV SGV is nonpathogenic in rabbits. BLV infection of rabbits by inoculation of cell-free BLV or cell-associated BLV typically causes an immunodeficiency-like syndrome and death by 1 year postinfection. We sought to evaluate whether in vivo transfection of BLV provirus recapitulates pathogenic BLV infection and to compare BLV and BLV SGV with respect to infection, immunogenicity, and clinical outcome. Three groups of rabbits were subjected to in vivo transfection with BLV, BLV SGV, or negative control DNA. The results of our 20-month study indicate that in vivo transfection of rabbits with BLV recapitulates the fatal BLV infection produced by cell-free or cell-associated BLV. The BLV-infected rabbits exhibited sudden onset of clinical decline and immunodeficiency-like symptoms that culminated in death. BLV and BLV SGV infected peripheral blood mononuclear cells and induced similar levels of seroconversion to BLV structural proteins. However, BLV SGV exhibited a reduced proviral load and did not trigger the immunodeficiency-like syndrome. These results are consistent with the hypothesis that BLV SGV is infectious and immunogenic and lacks BLV pathogenicity in rabbits, and they support the use of this modified proviral vector delivery system for vaccines against complex retroviruses like BLV. PMID:10482566

Kucerova, L; Altanerova, V; Altaner, C; Boris-Lawrie, K



Expression, localization, and functional model of cholesterol transporters in lactating and nonlactating mammary tissues of murine, bovine, and human origin.  


Members of the ATP-binding cassette (ABC) transporters play a pivotal role in cellular lipid efflux. To identify candidate cholesterol transporters implicated in lipid homeostasis and mammary gland (MG) physiology, we compared expression and localization of ABCA1, ABCG1, and ABCA7 and their regulatory genes in mammary tissues of different species during the pregnancy-lactation cycle. Murine and bovine mammary glands (MGs) were investigated during different functional stages. The abundance of mRNAs was determined by quantitative RT-PCR. Furthermore, transporter proteins were localized in murine, bovine, and human MGs by immunohistochemistry. In the murine MG, ABCA1 mRNA abundance was elevated during nonlactating compared with lactating stages, whereas ABCA7 and ABCA1 mRNA profiles were not altered. In the bovine MG, ABCA1, ABCG1, and ABCA7 mRNAs abundances were increased during nonlactating stages compared with lactation. Furthermore, associations between mRNA levels of transporters and their regulatory genes LXRalpha, PPARgamma, and SREBPs were found. ABCA1, ABCG1, and ABCA7 proteins were localized in glandular MG epithelial cells (MEC) during lactation, whereas during nonlactating stages, depending on species, the proteins showed distinct localization patterns in MEC and adipocytes. Our results demonstrate that ABCA1, ABCG1, and ABCA7 are differentially expressed between lactation and nonlactating stages and in association with regulatory genes. Combined expression and localization data suggest that the selected cholesterol transporters are universal MG transporters involved in transport and storage of cholesterol and in lipid homeostasis of MEC. Because of the species-specific expression patterns of transporters in mammary tissue, mechanisms of cholesterol homeostasis seem to be differentially regulated between species. PMID:20445153

Mani, Orlando; Körner, Meike; Sorensen, Martin T; Sejrsen, Kristen; Wotzkow, Carlos; Ontsouka, Corneille E; Friis, Robert R; Bruckmaier, Rupert M; Albrecht, Christiane



Bovine Colostrum Increases Pore-Forming Claudin-2 Protein Expression but Paradoxically Not Ion Permeability Possibly by a Change of the Intestinal Cytokine Milieu  

PubMed Central

An impaired intestinal barrier function is involved in the pathogenesis of inflammatory bowel disease (IBD). Several nutritional factors are supposed to be effective in IBD treatment but scientific data about the effects on the intestinal integrity remain scarce. Bovine colostrum was shown to exert beneficial effects in DSS-induced murine colitis, and the present study was undertaken to explore the underlying molecular mechanisms. Western blot revealed increased claudin-2 expression in the distal ileum of healthy mice after feeding with colostrum for 14 days, whereas other tight junction proteins (claudin-3, 4, 10, 15) remained unchanged. The colostrum-induced claudin-2 induction was confirmed in differentiated Caco-2 cells after culture with colostrum for 48 h. Paradoxically, the elevation of claudin-2, which forms a cation-selective pore, was neither accompanied by increased ion permeability nor impaired barrier function. In an in situ perfusion model, 1 h exposure of the colonic mucosa to colostrum induced significantly increased mRNA levels of barrier-strengthening cytokine transforming growth factor-?, while interleukine-2, interleukine-6, interleukine-10, interleukine-13, and tumor-necrosis factor-? remained unchanged. Thus, modulation of the intestinal transforming growth factor-? expression might have compensated the claudin-2 increase and contributed to the observed barrier strengthening effects of colostrum in vivo and in vitro.

Maletzki, Claudia; Lamprecht, Georg



Binding of a substrate analog to a domain swapping protein: X-ray structure of the complex of bovine seminal ribonuclease with uridylyl(2',5')adenosine.  

PubMed Central

Bovine seminal ribonuclease (BS-RNase) is a unique member of the pancreatic-like ribonuclease superfamily. The native enzyme is a mixture of two dimeric forms with distinct structural features. The most abundant form is characterized by the swapping of N-terminal fragments. In this paper, the crystal structure of the complex between the swapping dimer and uridylyl(2',5')adenosine is reported at 2.06 A resolution. The refined model has a crystallographic R-factor of 0.184 and good stereochemistry. The quality of the electron density maps enables the structure of both the inhibitor and active site residues to be unambiguously determined. The overall architecture of the active site is similar to that of RNase A. The dinucleotide adopts an extended conformation with the pyrimidine and purine base interacting with Thr45 and Asn71, respectively. Several residues (Gln11, His12, Lys41, His119, and Phe120) bind the oxygens of the phosphate group. The structural similarity of the active sites of BS-RNase and RNase A includes some specific water molecules believed to be relevant to catalytic activity. Upon binding of the dinucleotide, small but significant modifications of the tertiary and quaternary structure of the protein are observed. The ensuing correlation of these modifications with the catalytic activity of the enzyme is discussed.

Vitagliano, L.; Adinolfi, S.; Riccio, A.; Sica, F.; Zagari, A.; Mazzarella, L.



DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gs?)-encoding (GNAS) genomic imprinting domain are associated with performance traits  

PubMed Central

Background Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs) spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS) domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486) were located upstream of the GNAS gene, while one SNP (rs41694646) was located in the second intron of the GNAS gene. The final SNP (rs41694656) was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192. Results SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646) is associated (P ? 0.05) with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf) and gestation length. Association (P ? 0.01) with direct calving difficulty (i.e. due to calf size) and maternal calving difficulty (i.e. due to the maternal pelvic width size) was also observed at the rs43101491 SNP. Following adjustment for multiple-testing, significant association (q ? 0.05) remained between the rs41694646 SNP and four traits (animal stature, body depth, direct calving difficulty and milk yield) only. Notably, the single SNP in the bovine NESP55 gene (rs41694656) was associated (P ? 0.01) with somatic cell count--an often-cited indicator of resistance to mastitis and overall health status of the mammary system--and previous studies have demonstrated that the chromosomal region to where the GNAS domain maps underlies an important quantitative trait locus for this trait. This association, however, was not significant after adjustment for multiple testing. The three remaining SNPs assayed were not associated with any of the performance traits analysed in this study. Analysis of all pairwise linkage disequilibrium (r2) values suggests that most allele substitution effects for the assayed SNPs observed are independent. Finally, the polymorphic coding SNP in the putative bovine NESP55 gene was used to test the imprinting status of this gene across a range of foetal bovine tissues. Conclusions Previous studies in other mammalian species have shown that DNA sequence variation within the imprinted GNAS gene cluster contributes to several physiological and metabolic disorders, including obesity in humans and mice. Similarly, the results presented here indicate an important role for the imprinted GNAS cluster in underlying complex performance traits in cattle such as animal growth, calving, fertility and health. These findings suggest that GNAS domain-associated polymorphisms may serve as important genetic markers for future livestock breeding programs and support previous studies that candidate imprinted loci may act as molecular targets for the genetic improvement of agricultural populations. In addition, we present new evidence that the bovine NESP55 gene is epigenetically regulated as a maternally expressed imprinted gene in placental and intestinal tissues from 8-10 week old bovine foetuses.



Prediction of Protein-protein Interactions Using Alpha Shape Modeling  

NASA Astrophysics Data System (ADS)

Protein-protein interactions play important roles in a lot of biological progress. Previous studies about protein-protein interactions were mainly based on sequence analysis. As more 3D structural information can be obtained from protein-protein complexes, structural analysis becomes feasible and useful. In this study, we used structural alignment to predict the protein-binding site and apply 3D alpha shape modeling to analyze the interface characteristics. We have developed a method for protein-protein interaction prediction. The result indicates good performance of our method in discriminating protein-binding structures from non-protein binding structures. Our method outperforms the previous methods based on the Matthews correlation coefficient.

Zhou, Weiqiang; Yan, Hong; Fan, Xiaodan; Hao, Quan



Preparation and characterization of bovine serum albumin surface-imprinted thermosensitive magnetic polymer microsphere and its application for protein recognition.  


A novel bovine serum albumin surface-imprinted thermosensitive magnetic composite microsphere was successfully prepared by the surface grafting copolymerization method in the presence of temperature-sensitive monomer N-isopropylacrylamide (NIPAM), functional monomer methacrylic acid (MAA) and cross-linking agent N,N'-methylenebisacrylamide (MBA). The structure and component of the thermosensitive magnetic molecularly imprinted microsphere were investigated by transmission electron microscopy (TEM), Fourier transform infrared (FT-IR), vibrating sample magnetometer (VSM) and thermogravimetric analysis (TGA). The results of thermosensitivity, adsorption capacity, selectivity and reusability showed the formation of a thermosensitivity grafting polymer layer P(NIPAM-MAA-MBA) on the surface of Fe3O4@SiO2 and the good adsorption capacity and specific recognition for template protein. When the adsorption temperature was higher than the lower critical solution temperature (LCST) of poly(N-isopropylacrylamide) (PNIPAM), shape memory effect of imprinted cavities would be more effective. In other words, it was more conducive to capture template molecules under this condition and the imprinting factor would be higher. On the other hand, when the desorption temperature was lower than LCST of PNIPAM, the decrease of shape memory effect between imprinted cavities and template molecules would facilitate the release of template molecules from the imprinted cavities. Based on this property, the adsorption and desorption of template molecules could be regulated by system temperature indirectly which benefited from the existence of thermosensitivity imprinting layer. PMID:23973936

Li, Xiangjie; Zhang, Baoliang; Li, Wei; Lei, Xingfeng; Fan, Xinlong; Tian, Lei; Zhang, Hepeng; Zhang, Qiuyu



Experimental H-type bovine spongiform encephalopathy characterized by plaques and glial- and stellate-type prion protein deposits  

PubMed Central

Atypical bovine spongiform encephalopathy (BSE) has recently been identified in Europe, North America, and Japan. It is classified as H-type and L-type BSE according to the molecular mass of the disease-associated prion protein (PrPSc). To investigate the topographical distribution and deposition patterns of immunolabeled PrPSc, H-type BSE isolate was inoculated intracerebrally into cattle. H-type BSE was successfully transmitted to 3 calves, with incubation periods between 500 and 600 days. Moderate to severe spongiform changes were detected in the cerebral and cerebellar cortices, basal ganglia, thalamus, and brainstem. H-type BSE was characterized by the presence of PrP-immunopositive amyloid plaques in the white matter of the cerebrum, basal ganglia, and thalamus. Moreover, intraglial-type immunolabeled PrPSc was prominent throughout the brain. Stellate-type immunolabeled PrPSc was conspicuous in the gray matter of the cerebral cortex, basal ganglia, and thalamus, but not in the brainstem. In addition, PrPSc accumulation was detected in the peripheral nervous tissues, such as trigeminal ganglia, dorsal root ganglia, optic nerve, retina, and neurohypophysis. Cattle are susceptible to H-type BSE with a shorter incubation period, showing distinct and distinguishable phenotypes of PrPSc accumulation.



Glucocorticoid effects on extracellular matrix proteins and integrins in bovine trabecular meshwork cells in relation to glaucoma.  


The trabecular meshwork (TM) is a specialized eye tissue essential for regulation of the aqueous humor outflow and control of the intraocular pressure. Disturbances of TM cells may lead to elevated intraocular pressure and glaucoma. This study assessed the dexamethasone effects on levels of extracellular matrix proteins and their integrin receptors in bovine TM cells. Instillation of glucocorticoids such as dexamethasone is known to result in ocular hypertension. The histologic changes induced resemble those seen in glaucoma. Examination of the effects of glucocorticoid therefore may provide insights into the pathogenesis of glaucoma. TM cells in either tissue culture or organ cultures were treated with 0 (control), 0.1, or 1 microM of dexamethasone for 72 h. Immunostaining, Western, Northern and dot blot analyses showed that dexamethasone caused an increase in levels of fibronectin and collagen type IV in tissue-cultured TM cells. Increased focal contacts were also observed but the levels of laminin and collagen type I were unaffected. The dexamethasone effect was similarly demonstrated in organ cultures, with the exception that collagen type I also was enhanced. These results suggest that dexamethasone modulates extracellular matrices in the TM. Glucocorticoid may exert its effect through such a modulation in the development of steroid glaucoma. PMID:9852235

Zhou, L; Li, Y; Yue, B Y



p38 mitogen-activated protein kinase is crucial for bovine papillomavirus type-1 transformation of equine fibroblasts.  


Equine sarcoids represent the most common skin tumours in equids worldwide, characterized by extensive invasion and infiltration of lymphatics, rare regression and high recurrence after surgical intervention. Bovine papillomavirus type-1 (BPV-1) and less commonly BPV-2 are the causative agents of the diseases. It has been demonstrated that BPV-1 viral gene expression is necessary for maintaining the transformation phenotype. However, the underlying mechanism for BPV-1 transformation remains largely unknown, and the cellular factors involved in transformation are not fully understood. Previously mitogen-activated protein kinase (MAPK) signalling pathway has been shown to be important for cellular transformation. This study investigated the role of p38 MAPK (p38) in the transformation of equine fibroblasts by BPV-1. Elevated expression of phosphorylated p38 was observed in BPV-1 expressing fibroblasts due to the expression of BPV-1 E5 and E6. The phosphorylation of the MK2 kinase, a substrate of p38, was also enhanced. Inhibition of p38 activity by its selective inhibitor SB203580 changed cell morphology, reduced the proliferation of sarcoid fibroblasts and inhibited cellular invasiveness, indicating the indispensable role of p38 in BPV-1 transformation of equine fibroblasts. These findings provide new insights into the pathogenesis of equine sarcoids and suggest that p38 could be a potential target for equine sarcoid therapy. PMID:21471309

Yuan, ZhengQiang; Gault, Elizabeth A; Campo, M Saveria; Nasir, Lubna



Membrane insertion and lipid-protein interactions of bovine seminal plasma protein PDC-109 investigated by spin-label electron spin resonance spectroscopy.  

PubMed Central

The interaction of the major acidic bovine seminal plasma protein, PDC-109, with dimyristoylphosphatidylcholine (DMPC) membranes has been investigated by spin-label electron spin resonance spectroscopy. Studies employing phosphatidylcholine spin labels, bearing the spin labels at different positions along the sn-2 acyl chain indicate that the protein penetrates into the hydrophobic interior of the membrane and interacts with the lipid acyl chains up to the 14th C atom. Binding of PDC-109 at high protein/lipid ratios (PDC-109:DMPC = 1:2, w/w) results in a considerable decrease in the chain segmental mobility of the lipid as seen by spin-label electron spin resonance spectroscopy. A further interesting new observation is that, at high concentrations, PDC-109 is capable of (partially) solubilizing DMPC bilayers. The selectivity of PDC-109 in its interaction with membrane lipids was investigated by using different spin-labeled phospholipid and steroid probes in the DMPC host membrane. These studies indicate that the protein exhibits highest selectivity for the choline phospholipids phosphatidylcholine and sphingomyelin under physiological conditions of pH and ionic strength. The selectivity for different lipids is in the following order: phosphatidylcholine approximately sphingomyelin > or = phosphatidic acid (pH 6.0) > phosphatidylglycerol approximately phosphatidylserine approximately and rostanol > phosphatidylethanolamine > or = N-acyl phosphatidylethanolamine >> cholestane. Thus, the lipids bearing the phosphocholine moiety in the headgroup are clearly the lipids most strongly recognized by PDC-109. However, these studies demonstrate that this protein also recognizes other lipids such as phosphatidylglycerol and the sterol androstanol, albeit with somewhat reduced affinity.

Ramakrishnan, M; Anbazhagan, V; Pratap, T V; Marsh, D; Swamy, M J



Enterohemorrhagic Escherichia coli induce attaching and effacing lesions and hemorrhagic colitis in human and bovine intestinal xenograft models.  


Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important cause of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans worldwide. The two major virulence determinants of EHEC are the Shiga toxins (Stx) and the type III secretion system (T3SS), including the injected effectors. Lack of a good model system hinders the study of EHEC virulence. Here, we investigated whether bovine and human intestinal xenografts in SCID mice can be useful for studying EHEC and host tissue interactions. Fully developed, germ-free human and bovine small intestine and colon were established by subcutaneous transplantation of human and bovine fetal gut into SCID mice. Xenografts were allowed to develop for 3-4 months and thereafter were infected by direct intraluminal inoculation of Stx-negative derivatives of EHEC O157:H7, strain EDL933. The small intestine and colon xenografts closely mimicked the respective native tissues. Upon infection, EHEC induced formation of typical attaching and effacing lesions and tissue damage that resembled hemorrhagic colitis in colon xenografts. By contrast, xenografts infected with an EHEC mutant deficient in T3SS remained undamaged. Furthermore, EHEC did not attach to or damage the epithelium of small intestinal tissue, and these xenografts remained intact. EHEC damaged the colon in a T3SS-dependent manner, and this model is therefore useful for studying the molecular details of EHEC interactions with live human and bovine intestinal tissue. Furthermore, we demonstrate that Stx and gut microflora are not essential for EHEC virulence in the human gut. PMID:20959635

Golan, Lilach; Gonen, Erez; Yagel, Simcha; Rosenshine, Ilan; Shpigel, Nahum Y



Enterohemorrhagic Escherichia coli induce attaching and effacing lesions and hemorrhagic colitis in human and bovine intestinal xenograft models  

PubMed Central

SUMMARY Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important cause of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans worldwide. The two major virulence determinants of EHEC are the Shiga toxins (Stx) and the type III secretion system (T3SS), including the injected effectors. Lack of a good model system hinders the study of EHEC virulence. Here, we investigated whether bovine and human intestinal xenografts in SCID mice can be useful for studying EHEC and host tissue interactions. Fully developed, germ-free human and bovine small intestine and colon were established by subcutaneous transplantation of human and bovine fetal gut into SCID mice. Xenografts were allowed to develop for 3–4 months and thereafter were infected by direct intraluminal inoculation of Stx-negative derivatives of EHEC O157:H7, strain EDL933. The small intestine and colon xenografts closely mimicked the respective native tissues. Upon infection, EHEC induced formation of typical attaching and effacing lesions and tissue damage that resembled hemorrhagic colitis in colon xenografts. By contrast, xenografts infected with an EHEC mutant deficient in T3SS remained undamaged. Furthermore, EHEC did not attach to or damage the epithelium of small intestinal tissue, and these xenografts remained intact. EHEC damaged the colon in a T3SS-dependent manner, and this model is therefore useful for studying the molecular details of EHEC interactions with live human and bovine intestinal tissue. Furthermore, we demonstrate that Stx and gut microflora are not essential for EHEC virulence in the human gut.

Golan, Lilach; Gonen, Erez; Yagel, Simcha; Rosenshine, Ilan; Shpigel, Nahum Y.



Bovine herpesvirus 1 UL49.5 homolog gene encodes a novel viral envelope protein that forms a disulfide-linked complex with a second virion structural protein.  

PubMed Central

We previously reported that the genome of bovine herpesvirus 1 (BHV-1) contains an open reading frame (ORF) homologous to the herpes simplex virus UL49.5 ORF, and as with the herpes simplex virus UL49.5 ORF, the deduced amino acid sequence of the BHV-1 UL49.5 homolog (UL49.5h) contains features characteristic of an integral membrane protein, implying that it may constitute a functional gene encoding a novel viral envelope protein. This communication reports on the identification of the BHV-1 UL49.5h gene product. By employing an antibody against a synthetic BHV-1 UL49.5h peptide and an UL49.5h gene deletion mutant, the primary product of BHV-UL49.5h gene was identified as a polypeptide with a size of approximately 9 kDa; in both infected cells and isolated virions, the UL49.5h products were found to exist in three forms; monomer, disulfide-linked homodimer, and disulfide-linked heterodimer containing a second viral protein with a size of about 39 kDa. O-Glycosidase digestion and [3H]glucosamine labelling experiments showed that the UL49.5h protein is not glycosylated. Although the deduced amino acid sequence contains putative sites for myristylation and phosphorylation, we were unable to detect either modification. Surface labelling and trypsin digestion protection experiments showed that the BHV-1 UL49.5h protein was present on the surface of infected cells and on the surface of mature virions. Nonionic detergent partition of isolated virions revealed that the UL49.5h protein is more tightly associated with the virion tegument-nucleocapsid structure than envelope protein gD. The results from this study demonstrate that the BHV-1 UL49.5h gene encodes a nonglycosylated virion envelope protein which may associate with virion internal structures by forming a complex with the 39-kDa virion structural protein.

Liang, X; Chow, B; Raggo, C; Babiuk, L A



Modeling of bovine spongiform encephalopathy in a two-species feedback loop.  


Bovine spongiform encephalopathy, otherwise known as mad cow disease, can spread when an individual cow consumes feed containing the infected tissues of another individual, forming a one-species feedback loop. Such feedback is the primary means of transmission for BSE during epidemic conditions. Following outbreaks in the European Union and elsewhere, many governments enacted legislation designed to limit the spread of such diseases via elimination or reduction of one-species feedback loops in agricultural systems. However, two-species feedback loops-those in which infectious material from one-species is consumed by a secondary species whose tissue is then consumed by the first species-were not universally prohibited and have not been studied before. Here we present a basic ecological disease model which examines the rôle feedback loops may play in the spread of BSE and related diseases. Our model shows that there are critical thresholds between the infection's expansion and decrease related to the lifespan of the hosts, the growth rate of the prions, and the amount of prions circulating between hosts. The ecological disease dynamics can be intrinsically oscillatory, having outbreaks as well as refractory periods which can make it appear that the disease is under control while it is still increasing. We show that non-susceptible species that have been intentionally inserted into a feedback loop to stop the spread of disease do not, strictly by themselves, guarantee its control, though they may give that appearance by increasing the refractory period of an epidemic's oscillations. We suggest ways in which age-related dynamics and cross-species coupling should be considered in continuing evaluations aimed at maintaining a safe food supply. PMID:23746801

Barnes, Richard; Lehman, Clarence



Modeling Enzymatic Reactions in Proteins.  

NASA Astrophysics Data System (ADS)

We will discuss application of our density functional (DFT)-based QM/MM methodology to modeling a variety of protein active sites, including methane monooxygenase, myoglobin, and cytochrome P450. In addition to the calculation of intermediates, transition states, and rate constants, we will discuss modeling of reactions requiring protein conformational changes. Our methodology reliably achieves small errors as a result of imposition of the QM/MM boundary. However, the accuracy of DFT methods can vary significantly with the type of system under study. We will discuss a novel approach to the reduction of errors in gradient corrected and hybrid DFT functionals, using empirical localized orbital corrections (DFT-LOC), which addresses this problem effectively. For example, the mean unsigned error in atomization energies for the G3 data set using the B3LYP-LOC model is 0.8 kcal/mole, as compared with 4.8 kcal/mole for B3LYP and 1.0 kcal/mole for G3 theory.

Friesner, Richard



Time-of-flight neutron diffraction study of bovine ?-chymotrypsin at the Protein Crystallography Station.  


The overarching goal of this research project is to determine, for a subset of proteins, exact hydrogen positions using neutron diffraction, thereby improving H-atom placement in proteins so that they may be better used in various computational methods that are critically dependent upon said placement. In order to be considered applicable for neutron diffraction studies, the protein of choice must be amenable to ultrahigh-resolution X-ray crystallography, be able to form large crystals (1 mm(3) or greater) and have a modestly sized unit cell (no dimension longer than 100 Å). As such, ?-chymotrypsin is a perfect candidate for neutron diffraction. To understand and probe the role of specific active-site residues and hydrogen-bonding patterns in ?-chymotrypsin, neutron diffraction studies were initiated at the Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center (LANSCE). A large single crystal was subjected to H/D exchange prior to data collection. Time-of-flight neutron diffraction data were collected to 2.0 Å resolution at the PCS with ~85% completeness. Here, the first time-of-flight neutron data collection from ?-chymotrypsin is reported. PMID:21543868

Lazar, Louis M; Fisher, S Zoe; Moulin, Aaron G; Kovalevsky, Andrey; Novak, Walter R P; Langan, Paul; Petsko, Gregory A; Ringe, Dagmar



Time-of-flight neutron diffraction study of bovine ?--chymotrypsin at the Protein Crystallography Station  

PubMed Central

The overarching goal of this research project is to determine, for a subset of proteins, exact hydrogen positions using neutron diffraction, thereby improving H-atom placement in proteins so that they may be better used in various computational methods that are critically dependent upon said placement. In order to be considered applicable for neutron diffraction studies, the protein of choice must be amenable to ultrahigh-resolution X-ray crystallography, be able to form large crystals (1?mm3 or greater) and have a modestly sized unit cell (no dimension longer than 100?Å). As such, ?-chymotrypsin is a perfect candidate for neutron diffraction. To understand and probe the role of specific active-site residues and hydrogen-bonding patterns in ?-chymotrypsin, neutron diffraction studies were initiated at the Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center (LANSCE). A large single crystal was subjected to H/D exchange prior to data collection. Time-of-flight neutron diffraction data were collected to 2.0?Å resolution at the PCS with ?85% completeness. Here, the first time-of-flight neutron data collection from ?-­chymotrypsin is reported.

Lazar, Louis M.; Fisher, S. Zoe; Moulin, Aaron G.; Kovalevsky, Andrey; Novak, Walter R. P.; Langan, Paul; Petsko, Gregory A.; Ringe, Dagmar




Technology Transfer Automated Retrieval System (TEKTRAN)

FLICE-like inhibitory protein (FLIP) inhibits apoptosis and NF-' B activation induced by pro-inflammatory mediators in humans and mice. These processes are critical events in the pathogenesis of a variety of diseases in cattle, including mastitis. Because FLIP is known to moderate these events in ot...



Microsoft Academic Search

Recipient beef heifers, pregnant with single demi-embryos, were paired according to identical twin or full-sib embryo. Within pair, recipient heifers were assigned to one of two isocaloric diets containing a control or restricted level of protein (91 vs 55% of National Research Council recom- mendations) on d 190 of gestation. Following parturition, calves were weighed, fed 1 liter of colo-

G. E. Carstens; D. E. Johnson; M. D. Holland; K. G. Odde


Clinical and Pathologic Features of H-Type Bovine Spongiform Encephalopathy Associated with E211K Prion Protein Polymorphism  

PubMed Central

The majority of bovine spongiform encephalopathy (BSE) cases have been ascribed to the classical form of the disease. H-type and L-type BSE cases have atypical molecular profiles compared to classical BSE and are thought to arise spontaneously. However, one case of H-type BSE was associated with a heritable E211K mutation in the prion protein gene. The purpose of this study was to describe transmission of this unique isolate of H-type BSE when inoculated into a calf of the same genotype by the intracranial route. Electroretinograms were used to demonstrate preclinical deficits in retinal function, and optical coherence tomography was used to demonstrate an antemortem decrease in retinal thickness. The calf rapidly progressed to clinical disease (9.4 months) and was necropsied. Widespread distribution of abnormal prion protein was demonstrated within neural tissues by western blot and immunohistochemistry. While this isolate is categorized as BSE-H due to a higher molecular mass of the unglycosylated PrPSc isoform, a strong labeling of all 3 PrPSc bands with monoclonal antibodies 6H4 and P4, and a second unglycosylated band at approximately 14 kDa when developed with antibodies that bind in the C-terminal region, it is unique from other described cases of BSE-H because of an additional band 23 kDa demonstrated on western blots of the cerebellum. This work demonstrates that this isolate is transmissible, has a BSE-H phenotype when transmitted to cattle with the K211 polymorphism, and has molecular features that distinguish it from other cases of BSE-H described in the literature.

Greenlee, Justin J.; Smith, Jodi D.; West Greenlee, M. Heather; Nicholson, Eric M.



Epididymosomes transfer epididymal sperm binding protein 1 (ELSPBP1) to dead spermatozoa during epididymal transit in bovine.  


Previously, we showed that epididymal sperm binding protein 1 (ELSPBP1) characterizes spermatozoa already dead before ejaculation in bovine. In this study, we investigated the presence of ELSPBP1 in bull genital tract as well as its acquisition by spermatozoa during epididymal transit. As assessed by real-time RT-PCR, ELSPBP1 was highly expressed in the caput and the corpus epididymis but was present in lower expression levels in the testis and the cauda epididymis. Immunohistochemistry revealed the same expression pattern. However, Western blot on tissue homogenates showed some discrepancies, as ELSPBP1 was found in a comparable concentration all along the epididymis. This difference was due to the presence of ELSPBP1 in the epididymal fluid. In both caput and cauda epididymal fluid, ELSPBP1 was associated with the epididymosomes, small membranous vesicles secreted by epithelial cells of the epididymis and implicated in the transfer of proteins to spermatozoa. As assessed by immunocytometry, ELSPBP1 was found on a subset of dead spermatozoa in caput epididymis but was found on all dead spermatozoa in cauda epididymis. To assess ELSPBP1 acquisition by spermatozoa, caput epididymal spermatozoa were incubated with cauda epididymosomes under various conditions. ELSPBP1 detection by immunocytometry assay revealed that only spermatozoa already dead before incubation were receptive to ELSPBP1 transfer by epididymosomes. This receptivity was enhanced by the presence of zinc in the incubation medium. This specificity for a sperm subpopulation suggests that an underlying mechanism is involved and that ELSPBP1 could be a tag for the recognition of dead spermatozoa during epididymal transit. PMID:22875906

D'Amours, Olivier; Frenette, Gilles; Bordeleau, Louis-Jean; Allard, Nancy; Leclerc, Pierre; Blondin, Patrick; Sullivan, Robert



Transmission of bovine leukaemia virus within dairy herds by simulation modelling.  


In Argentina, bovine leukaemia virus (BLV) infection is common in dairy herds. The country currently has a National Voluntary Control Programme but relatively few farms have enrolled. However, there is increased interest from authorities and farmers to implement regional compulsory programmes but there is scarce quantitative information of the transmission of BLV in cattle herds. This information is a prerequisite to develop effective BLV control strategies. Mathematical modelling offers ways of integrating population-level knowledge and epidemiological data to predict the outcomes of intervention scenarios. The purpose of the current paper is to gain understanding about the dynamics of the transmission of BLV in dairy herds from Argentina by simulation and to compare various BLV transmission models and select the one that is most appropriate. The hypothetical herd is conceptually described in terms of BLV status as a population of individuals that are protected by maternal antibodies (M), that are susceptible (S), that are in the latent period (E) or that are infectious (I). BLV is spread by horizontal and vertical transmission. We used an age-structured population model and within-herd transmission was simulated by Monte Carlo techniques. The next-generation approach has been used for the systematic computation of the basic reproduction ratio (R0). Parameter values for disease transmission were derived from previously published data; rates of entry, exit or transition between age groups were calculated based on our previous study, observational data, expert opinions and literature. With these parameter values the probability of a minor outbreak was estimated to be 10%, the probability of extinction was estimated as <0.001% and the expected time to extinction as more than 80 years. The probability of a minor outbreak and changes in prevalence were different when the index case was an adult cow compared to introduction by a heifer. Prediction of prevalences from MSI models fit the data satisfactorily. R0 was estimated as 9.5. The sensitivity analysis on R0 showed that all measures directed to reduce the transmission rate are potentially effective given operational control measures. An important prediction of these models is that, even in a relatively small, closed dairy herd, the time-scale for a BLV outbreak may be as long as several years and within-herd control of BLV requires intensive efforts. PMID:17076940

Monti, G E; Frankena, K; De Jong, M C M



Cationic peptides obtained by reversible disaggregation of antibacterial proteins of bovine seminal plasma  

Microsoft Academic Search

A series of experiments was conducted to investigate the aggregating and disaggregating properties of anti-bacterial proteins. in seminal plasma. Little of the antibactenal activity of bovme seminal plasma diffused through dialysis membrane of retentivity 10 kDa at pH 7, but at pH 3 or with 0.1 mol\\/litre citrate at pH 7 most of the activity diffused through. Some of this

P. Shannon; B. Curson; P. C. Molan



Immobilisation of bovine enterokinase and application of the immobilised enzyme in fusion protein cleavage  

Microsoft Academic Search

Two immobilisation methods for enterokinase were developed, which yielded high remaining activities for the cleavage of the\\u000a fusion protein MUC1-IgG Fc. Different carrier materials were compared regarding remaining enzyme activity and storage stability.\\u000a Immobilisation procedures involving support material activation using glutardialdehyde were found to result in low remaining\\u000a activities. Applying less aggressive activation procedures, remaining activities of ?60% were received

Tina Kubitzki; Thomas Noll; Stephan Lütz



Bovine Rotavirus Nonstructural Protein 4 Produced by Lactococcus lactis Is Antigenic and Immunogenic  

PubMed Central

Rotavirus nonstructural protein 4 (NSP4) can induce diarrhea in mice. To get insight into the biological effects of NSP4, production of large quantities of this protein is necessary. We first tried to produce the protein in Escherichia coli, but the nsp4 gene proved to be unstable. The capacity of the generally regarded as safe organism Lactococcus lactis to produce NSP4 either intra- or extracellularly was then investigated by using the nisin-controlled expression system. Production of recombinant NSP4 (rNSP4) was observed in L. lactis for both locations. In spite of a very low secretion efficiency, the highest level of production was obtained with the fusion between a lactococcal signal peptide and rNSP4. Cultures of the rNSP4-secreting strain were injected into rabbits, and a specific immune response was elicited. The anti-rNSP4 antibodies produced in these rabbits recognized NSP4 in MA104 cells infected by rotavirus. We showed that L. lactis is able to produce antigenic and immunogenic rNSP4 and thus is a good organism for producing viral antigens.

Enouf, Vincent; Langella, Philippe; Commissaire, Jacqueline; Cohen, Jean; Corthier, Gerard



Infectivity of scrapie prion protein (PrPSc) following in vitro digestion with bovine gastrointestinal microbiota.  


The influence of a complex microflora residing in the gastrointestinal tract of cattle on the prion protein plays a crucial role with respect to early pathogenesis and the potential infectivity of faeces resulting in contamination of the environment. It is unknown whether infectious prion proteins, considered to be very stable, are inactivated by microbial processes in the gastrointestinal tract of animals during digestion. In our previous study it was shown that the scrapie-associated prion protein was degraded by ruminal and colonic microbiota of cattle, as indicated by a loss of anti-prion antibody 3F4 immunoreactivity in Western blot. Subsequently, in this study hamster bioassays with the pre-treated samples were performed. Although the PrP(Sc) signal was reduced up to immunochemically undetectable levels within 40 h of pre-treatment, significant residual prion infectivity was retained after degradation of infected hamster brain through the gastrointestinal microflora of cattle. The data presented here show that the loss of anti-prion antibody 3F4 immunoreactivity is obviously not correlated with a biological inactivation of PrP(Sc). These results highlight the deficiency of using Western blot in transmissible spongiform encephalopathies inactivation assessment studies and, additionally, point to the possibility of environmental contamination with faeces containing PrP(Sc) following an oral ingestion of prions. PMID:17542960

Scherbel, C; Pichner, R; Groschup, M H; Mueller-Hellwig, S; Scherer, S; Dietrich, R; Maertlbauer, E; Gareis, M



Identification and analysis of a novel protein-tyrosine kinase from bovine thymus  

SciTech Connect

A cytosolic protein-tyrosine kinase has been identified and purified to near homogeneity from calf thymus by using the phosphorylation of the tyrosine-containing peptide angiotensin I as an assay. Specific peptide phosphorylating activity was enhanced by carrying out the assay at high ionic strength (2M NaCl). The inclusion of NaCl at this concentration acts to stimulate endogenous protein-tyrosine kinase activity while simultaneously inhibiting other endogenous kinases. The purification procedure involved extraction of the enzyme from calf-thymus and sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-sepharose, butylagarose, and Sephadex G-75. Analysis of the most highly purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single Coomassie blue-stained band of 41 KDa. This molecular weight was consistent with results obtained from gel filtration, indicating that the enzyme exists as a monomer. The enzyme has also been found to catalyze an autophosphorylation reaction. Incubation of the enzyme with Mn/sup 2 +/ and (..gamma..-/sup 32/P)ATP led to its modification on a tyrosine residue. Phosphopeptide mapping experiments indicated that the 41 KDa kinase was distinct from p56, the major membrane-associated protein-tyrosine kinase in T lymphocytes.

Zioncheck, T.F.; Harrison, M.L.; Geahlen, R.L.



A potent, heat-stable protein inhibitor of [branched-chain alpha-keto acid dehydrogenase]-phosphatase from bovine kidney mitochondria.  

PubMed Central

A heat- and acid-stable protein inhibitor of the [branched-chain alpha-keto acid dehydrogenase]-phosphatase was purified over 100,000-fold from extracts of bovine kidney mitochondria. The nearly homogeneous protein was recovered with a yield of 4-8%. The apparent molecular weight of the inhibitor is about 36,000. This protein is a noncompetitive inhibitor of the phosphatase, and the inhibitor constant (Ki) is about 0.13 nM. The inhibition was reversed 50% by about 1.3 mM Mg2+ and about 0.1 mM spermine. This protein inhibitor is different from the cytosolic protein phosphatase inhibitors 1 and 2. Images

Damuni, Z; Humphreys, J S; Reed, L J



The SU and TM Envelope Protein Subunits of Bovine Leukemia Virus Are Linked by Disulfide Bonds, both in Cells and in Virions  

PubMed Central

After the polyprotein precursor of retroviral envelope proteins is proteolytically cleaved, the surface (SU) and transmembrane (TM) subunits remain associated with each other by noncovalent interactions or by disulfide bonds. Disulfide linkages confer a relatively stable association between the SU and TM envelope protein subunits of Rous sarcoma virus and murine leukemia virus. In contrast, the noncovalent association between SU and TM of human immunodeficiency virus leads to significant shedding of SU from the surface of infected cells. The SU and TM proteins of bovine leukemia virus (BLV) initially were reported to be disulfide linked but later were concluded not to be, since TM is often lost during purification of SU protein. Here, we show that SU and TM of BLV do, indeed, associate through disulfide bonds, whether the envelope proteins are overexpressed in transfected cells, are produced in virus-infected cells, or are present in newly produced virions.

Johnston, Elizabeth R.; Radke, Kathryn



Limited proteolysis of bovine alpha-lactalbumin: isolation and characterization of protein domains.  

PubMed Central

The partly folded states of alpha-lactalbumin (alpha-LA) exposed to acid solution at pH 2.0 (A-state) or at neutral pH upon EDTA-mediated removal of the single protein-bound calcium ion (apo form) have been probed by limited proteolysis experiments. These states are nowadays commonly considered to be molten globules and thus protein-folding intermediates. Pepsin was used for proteolysis at acid pH, while proteinase K and chymotrypsin at neutral pH. The expectations were that these proteolytic probes would detect sites and/or chain regions in the partly folded states of alpha-LA sufficiently dynamic, or even unfolded, capable of binding and adaptation to the specific stereochemistry of the protease's active site. A time-course analysis of the proteolytic events revealed that the fast, initial proteolytic cuts of the 123-residue chain of alpha-LA in its A-state or apo form by the three proteases occur at the same chain region 39-54, the actual site(s) of cleavage depending upon the protease employed. This region in native alpha-LA encompasses the beta-sheets of the protein. Subsequent cleavages occur mostly at chain regions 31-35 and 95-105. Four fragment species of alpha-LA have been isolated by reverse-phase high-performance liquid chromatography, and their conformational properties examined by circular dichroism and fluorescence emission spectroscopy. The single chain fragment 53-103, containing all the binding sites for calcium in native alpha-LA and cross-linked by two disulfide bridges, maintains in aqueous buffer and in the presence of calcium ions a folded structure characterized by the same content of alpha-helix of the corresponding chain segment in native alpha-LA. Evidence for some structure was also obtained for the two-chain species 1-40 and 104-123, as well as 1-31 and 105-123, both systems being covalently linked by two disulfide bonds. In contrast, the protein species given by fragment 1-34 connected to fragment 54-123 or 57-123 via four disulfide bridges adopts in solution a folded structure with the helical content expected for a native-like conformation. Of interest, the proteolytic fragment species herewith isolated correspond to the structural domains and subdomains of alpha-LA that can be identified by computational analysis of the three-dimensional structure of native alpha-LA (Siddiqui AS, Barton GI, 1995, Protein Sci 4:872-884). The fast, initial cleavages at the level of the beta-sheet region of native alpha-LA indicate that this region is highly mobile or even unfolded in the alpha-LA molten globule(s), while the rest of the protein chain maintains sufficient structure and rigidity to prevent extensive proteolysis. The subsequent cleavages at chain segment 95-105 indicate that also this region is somewhat mobile in the A-state or apo form of the protein. It is concluded that the overall domain topology of native alpha-LA is maintained in acid or at neutral pH upon calcium depletion. Moreover, the molecular properties of the partly folded states of alpha-LA deduced here from proteolysis experiments do correlate with those derived from previous NMR and other physicochemical measurements.

Polverino de Laureto, P.; Scaramella, E.; Frigo, M.; Wondrich, F. G.; De Filippis, V.; Zambonin, M.; Fontana, A.



Vitamin D Signaling in the Bovine Immune System: A Model for Understanding Human Vitamin D Requirements  

PubMed Central

The endocrine physiology of vitamin D in cattle has been rigorously investigated and has yielded information on vitamin D requirements, endocrine function in health and disease, general metabolism, and maintenance of calcium homeostasis in cattle. These results are relevant to human vitamin D endocrinology. The current debate regarding vitamin D requirements is centered on the requirements for proper intracrine and paracrine vitamin D signaling. Studies in adult and young cattle can provide valuable insight for understanding vitamin D requirements as they relate to innate and adaptive immune responses during infectious disease. In cattle, toll-like receptor recognition activates intracrine and paracrine vitamin D signaling mechanism in the immune system that regulates innate and adaptive immune responses in the presence of adequate 25-hydroxyvitamin D. Furthermore, experiments with mastitis in dairy cattle have provided in vivo evidence for the intracrine vitamin D signaling mechanism in macrophages as well as vitamin D mediated suppression of infection. Epidemiological evidence indicates that circulating concentrations above 32 ng/mL of 25-hydroxyvitamin D are necessary for optimal vitamin D signaling in the immune system, but experimental evidence is lacking for that value. Experiments in cattle can provide that evidence as circulating 25-hydroxyvitamin D concentrations can be experimentally manipulated within ranges that are normal for humans and cattle. Additionally, young and adult cattle can be experimentally infected with bacteria and viruses associated with significant diseases in both cattle and humans. Utilizing the bovine model to further delineate the immunomodulatory role of vitamin D will provide potentially valuable insights into the vitamin D requirements of both humans and cattle, especially as they relate to immune response capacity and infectious disease resistance.

Nelson, Corwin D.; Reinhardt, Timothy A.; Lippolis, John D.; Sacco, Randy E.; Nonnecke, Brian J.



Regulation of a swelling-activated chloride current in bovine endothelium by protein tyrosine phosphorylation and G proteins  

PubMed Central

The role of protein tyrosine phosphorylation and of G proteins in the activation of a swelling-activated Cl? current (ICl,swell) in calf pulmonary artery endothelial (CPAE) cells was studied using the whole-cell patch clamp technique. ICl,swell was activated by reducing the extracellular osmolality by either 12.5% (mild hypotonicity) or 25% (strong hypotonicity).The protein tyrosine kinase (PTK) inhibitors tyrphostin B46, tyrphostin A25 and genistein inhibited ICl,swell with IC50 values of, respectively, 9.2 ± 0.2, 61.4 ± 1.7 and 62.9 ± 1.3?M. Tyrphostin A1, a tyrphostin analogue with little effect on PTK activity, and daidzein, an inactive genistein analogue, were without effect on ICl,swell.The protein tyrosine phosphatase (PTP) inhibitors Na3VO4 (200 ?M) and dephostatin (20 ?M) potentiated ICl,swell activated by mild hypotonicity by 47 ± 9 and 69 ± 15%, respectively.Intracellular perfusion with GTP?S (100 ?M) transiently activated a Cl? current with an identical biophysical and pharmacological profile to ICl,swell. This current was inhibited by the tested PTK inhibitors and potentiated by the PTP inhibitors. Hypertonicity-induced cell shrinkage completely inhibited the GTP?S-activated Cl? current.Intracellular perfusion with GDP?S (1 mM) caused a time-dependent inhibition of ICl,swell, which was more pronounced when the current was activated by mild hypotonicity.Our results demonstrate that the activity of endothelial swelling-activated Cl? channels is dependent on tyrosine phosphorylation and suggest that G proteins regulate the sensitivity to cell swelling.

Voets, Thomas; Manolopoulos, Vangelis; Eggermont, Jan; Ellory, Clive; Droogmans, Guy; Nilius, Bernd



Herpes simplex (HSV-1) infection of bovine aorta smooth muscle cells (SMC) inhibits matrix protein synthesis  

SciTech Connect

Studies from this laboratory have shown that HSV-1 infection suppresses matrix protein synthesis by endothelial cells in vitro. In this study the authors have investigated the effects of HSV-1 infection on SMC. Monolayers of SMC were infected with HSV-1 at a multiplicity of infection (MOI) ranging from 0.1 to 20. Viral replication and release to the medium was measured by plaque assay in Vero cells. At an MOI of 0.1, 10 or 20, viral replication occurred and maximum virus titers were achieved by 24 hrs. post-infection. Virus release in the medium began during the first 12 hrs. post-infection and reached maximum at 24 hrs. Infected and uninfected cultures of SMC were pulse labeled with either (/sup 14/C)proline or (/sup 35/S)-methionine at different hrs. post-infection. Incorporation of radioactivity into non-dialyzable protein was determined in fluorograms following SDS-PAGE of the cell-matrix or medium fractions. The synthesis of fibronectin and collagen Types I and III was suppressed and the degree of suppression was dependent on the duration of infection and on the virus dose. These data suggest that SMC can support HSV-1 replication in vitro and that such infection can lead to altered extracellular matrix synthesis.

Lashgari, M.S.; Friedman, H.M.; Kefalides, N.A.



Enantioselectivity of bovine serum albumin-bonded columns produced with isolated protein fragments. II. Characterization of protein fragments and chiral binding sites.  


Enantioselectivity of bovine serum albumin (BSA)-bonded columns produced with isolated protein fragments has been investigated. The BSA fragment, BSA-FG75, was isolated by size exclusion chromatography following peptic digest of BSA. The isolated BSA-FG75 was further fractionated to two fractions, BSA-F1 and BSA-F2, by anion-exchange chromatography. BSA-F1 and BSA-F2 had molecular mass of about 35000 daltons, estimated by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, BSA-F1 has amino acid residues 1-307 estimated by electrospray ionization (ESI) mass spectrometry, while BSA-F2 is an N-terminal half BSA fragment. The BSA, BSA-FG75, BSA-F1 and BSA-F2 proteins were bound to aminopropyl-silica gels activated by N,N'-disuccinimidyl carbonate. The bound amounts of the BSA fragments were 2.2-2.7 times more than that of the intact BSA. Chiral recognition of 2-arylpropionic acid derivatives, benzodiazepines, warfarin and benzoin was obtained with the BSA fragment-bonded columns. The non-enantioselective interactions of benzoin and benzodiazepines except for clorazepate with BSA fragments were increased with protein surface coverages, while those of 2-arylpropionic acid, clorazepate and warfarin were decreased. The BSA fragment columns gave higher enantioselectivity for lorazepam and benzoin, and lower enantioselectivity for other compounds tested, compared with the BSA column. These results might be due to changes in the globular structure of the BSA fragment and/or changes in the local environment around the binding sites. PMID:9188181

Haginaka, J; Kanasugi, N



Validation of excised bovine nasal mucosa as in vitro model to study drug transport and metabolic pathways in nasal epithelium.  


The present work aims at the validation of excised bovine nasal mucosa as an in vitro model to address transport and metabolism pathways relative to the nasal mucosal uptake of therapeutic peptides. Preservation of the viability of the excised tissue in the course of in vitro studies of up to 3 h was demonstrated by (i) positive viability staining, (ii) constant transepithelial electrical resistance (42 +/- 12 Omega cm(2)), (iii) constant rates of metabolic turnover, and (iv) linear permeation profiles of therapeutic peptides and (3)H-mannitol. Using 1-leucine-4-methoxy-2-naphthylamide as a model substrate, we observed no difference between bovine and human nasal aminopeptidase activity. By a series of therapeutic peptides, no direct correlation was found between their effective permeability coefficients (from 0. 1 x 10(-5) to 5 x 10(-5) cm s(-1)) and their respective molecular masses (from 417 to 3,432 Da), indicating that other factors dominate nasal permeability. For instance, the permeabilities of metabolically labile peptides were concentration dependent and saturable, as demonstrated for two short thymopoietin fragments, Arg-Lys-Asp (TP3) and Arg-Lys-Asp-Val (TP4). By permeation studies using gonadorelin and two gonadorelin derivatives, buserelin and Hoe 013, without and in the presence of the chemical enhancer bacitracin, we also verified the ability of the model to assess chemical enhancer effects and their reversibility. In conclusion, our work demonstrates the potential of the investigated in vitro model, excised bovine nasal mucosa, to explore mechanistic aspects of nasal transport and metabolism of therapeutic peptides. PMID:10707019

Schmidt, M C; Simmen, D; Hilbe, M; Boderke, P; Ditzinger, G; Sandow, J; Lang, S; Rubas, W; Merkle, H P



Interfacial behaviour of bovine testis hyaluronidase  

PubMed Central

The interfacial properties of bovine testicular hyaluronidase were investigated by demonstrating the association of hyaluronidase activity with membranes prepared from bovine testis. Protein adsorption to the air/water interface was investigated using surface pressure-area isotherms. In whichever way the interfacial films were obtained (protein injection or deposition), the hyaluronidase exhibited a significant affinity for the air/water interface. The isotherm obtained 180 min after protein injection into a pH 5.3 subphase was similar to the isotherm obtained after spreading the same amount of protein onto the same subphase, indicating that bovine testicular hyaluronidase molecules adopted a similar arrangement and/or conformation at the interface. Increasing the subphase pH from 5.3 to 8 resulted in changes of the protein isotherms. These modifications, which could correspond to the small pH-induced conformational changes observed by Fourier-transform IR spectroscopy, were discussed in relation to the pH influence on the hyaluronidase activity. Adding hyaluronic acid, the enzyme substrate, to the subphase tested the stability of the interfacial properties of hyaluronidase. The presence of hyaluronic acid in the subphase did not modify the protein adsorption and allowed substrate binding to a preformed film of hyaluronidase at pH 5.3, the optimal pH for the enzyme activity. Such effects of hyaluronic acid were not observed when the subphase was constituted of pure water, a medium where the enzyme activity was negligible. These influences of hyaluronic acid were discussed in relation to the modelled structure of bovine testis hyaluronidase where a hydrophobic region was proposed to be opposite of the catalytic site.

Belem-Goncalves, Silvia; Tsan, Pascale; Lancelin, Jean-Marc; Alves, Tito L. M.; Salim, Vera M.; Besson, Francoise



Application of systems analysis in modelling the risk of bovine spongiform encephalopathy (BSE)  

Microsoft Academic Search

Bovine spongiform encephalopathy (BSE), widely known as “mad cow disease”, has virtually crippled the British livestock industry. Even though, no cases of BSE have been reported in the United States (US), a similar epidemic in the US would be catastrophic. The added concern for the risk of introduction of the human disease called variant Creutzfeldt-Jacob disease that has been linked

T. Habtemariam; B. Tameru; D. Nganwa; L. Ayanwale; A. Ahmed; D. Oryang; H. AbdelRahman; G. Gray; J. Cohen; S. Kreindel



Modeling Mitochondrial Protein Evolution Using Structural Information  

Microsoft Academic Search

.   We present two new models of protein sequence evolution based on structural properties of mitochondrial proteins. We compare\\u000a these models with others currently used in phylogenetic analyses, investigating their performance over both short and long\\u000a evolutionary distances. We find that our models that incorporate secondary structure information from mitochondrial proteins\\u000a are statistically comparable with existing models when studying 13

Pietro Liò; Nick Goldman



Effect of pregnancy and progesterone concentration on expression of genes encoding for transporters or secreted proteins in the bovine endometrium.  


The objective of this study was to determine the temporal and spatial expression patterns of genes encoding transporters, as well as selected secreted proteins that may be regulated by progesterone (P4) and/or the presence of the conceptus in the bovine endometrium. Estrus-synchronized beef heifers were randomly assigned to either: 1) pregnant, high P4; 2) pregnant, normal P4; 3) cyclic, high P4; or 4) cyclic, normal P4. Uteri were collected on days 5, 7, 13, and 16 of the estrous cycle or pregnancy. Localization of mRNAs for ANPEP, CTGF, LPL, LTF, and SLC5A1 in the uteri was determined by radioactive in situ hybridization, and expression quantified in the endometria by quantitative real-time PCR. ANPEP localized to luminal (LE) and superficial glandular (sGE) epithelia of all heifers on days 5 and 7 only. SLC5A1 mRNA was detected in the LE and sGE on days 13 and 16 in all heifers, and expression increased on day 16 in pregnant groups. CTGF localized weakly to the LE and GE on days 5 and 7 but increased on days 13 and 16 with an increase (P < 0.05) in CTGF expression in high P4 (day 7) and pregnant heifers (day 16). Both LPL and LTF localized to the GE only on days 5 and 7. In conclusion we have characterized the temporal expression pattern of these genes and modulation of their transcript abundance by P4 (CTGF, LPL) and/or the conceptus (CTGF, SLC5A1) likely modifies the uterine microenvironment, enhancing histotroph composition and contributing to advanced conceptus elongation. PMID:19996158

Forde, N; Spencer, T E; Bazer, F W; Song, G; Roche, J F; Lonergan, P



Discrimination between Scrapie and Bovine Spongiform Encephalopathy in Sheep by Molecular Size, Immunoreactivity, and Glycoprofile of Prion Protein  

PubMed Central

A procedure for discrimination between scrapie and bovine spongiform encephalopathy (BSE) in sheep is of importance for establishing whether BSE has entered the sheep population. Since BSE has not yet been found in sheep at the farm level, such discrimination procedures can be developed only with experimental sheep BSE. Two distinctive molecular features of the prion protein (PrP)—molecular size and glycosylation profile—in proteinase K digests of brain stem tissue from sheep were used here; upon Western blotting, these features led to an unequivocal discrimination among natural scrapie, experimental scrapie, and experimental BSE. The higher electrophoretic mobility of PrP in sheep BSE could be best observed after deglycosylation treatment with N-glycosidase F. A simpler method for confirmation of this size difference involved comparison of the ratios for the binding of two monoclonal antibodies: P4 and 66.94b4. Based on epitope mapping studies with P4 and peptides, it appeared that N-terminal amino acid sequence WGQGGSH was intact only in sheep scrapie digests. Another feature typical for PrP in sheep BSE was the large fraction of diglycosylated PrP (70% or more). These data were obtained for a large group of positive sheep, consisting of 7 sheep with experimental BSE infection (genotypes: six ARQ/ARQ and one AHQ/AHQ), 48 sheep naturally infected with scrapie (six different genotypes), and 3 sheep with primary experimental scrapie infection. Routine tests of slaughter material serve well for the initial detection of both BSE and scrapie. With Western blotting as a rapid follow-up test, a 66.94b4/P4 antibody binding ratio above 1.5 is a practical indicator for serious suspicion of BSE infection in sheep.

Thuring, C. M. A.; Erkens, J. H. F.; Jacobs, J. G.; Bossers, A.; Van Keulen, L. J. M.; Garssen, G. J.; Van Zijderveld, F. G.; Ryder, S. J.; Groschup, M. H.; Sweeney, T.; Langeveld, J. P. M.



Subunit dissociation and protein unfolding in the bovine heart cytochrome oxidase complex induced by guanidine hydrochloride.  


The response of cytochrome oxidase to the denaturant guanidine hydrochloride (Gdn.HCl) occurs in two stages. The first stage is a sharp transition centered at 1 M Gdn.HCl, whereas the second stage occurs from 3 to 7 M Gdn.HCl. In the first phase, changes occur in several spectroscopic properties: (1) the tryptophan fluorescence increases from 37% of that of N-acetyltryptophanamide to 85%; (2) the emission maximum shifts from 328 to 333 nm; (3) the circular dichroism (CD) signal at 222 nm diminishes by 30%; and (4) the Soret CD signal at 426 nm is completely abolished. These spectroscopic changes are accompanied by complete loss of the oxidase's steady-state electron-transfer activity. Of the 13 available sulfhydryl residues, 2 are reactive in the isolated enzyme, but this number increases to almost 10 in the first stage of denaturation. Subunits III, VIb, VIc, and VII dissociate from the protein complex at 0.5 M Gdn.HCl, but only subunit VII can be recovered after gel filtration chromatography [nomenclature according to Buse et al. (1985)]. In 2.5 M Gdn.HCl, the heme groups are found with a complex consisting predominantly of subunits I, II, and IV. In the second phase of denaturation, there is further disruption in the structure of the oxidase as indicated by continued decline in the ultraviolet CD signal and shift to longer wavelength of the tryptophan emission spectrum. However, the fluorescence quantum yield and number of reactive sulfhydryl groups decrease as the denaturant level is raised. Gel filtration chromatography reveals that protein and heme form a high molecular weight aggregate at 5 M Gdn.HCl.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2844238

Hill, B C; Cook, K; Robinson, N C



Jumonji domain-containing protein 3 regulates histone 3 lysine 27 methylation during bovine preimplantation development.  


Understanding the mechanisms of epigenetic remodeling that follow fertilization is a fundamental step toward understanding the bases of early embryonic development and pluripotency. Extensive and dynamic chromatin remodeling is observed after fertilization, including DNA methylation and histone modifications. These changes underlie the transition from gametic to embryonic chromatin and are thought to facilitate embryonic genome activation. In particular, trimethylation of histone 3 lysine 27 (H3K27me3) is associated with gene-specific transcription repression. Global levels of this epigenetic mark are high in oocyte chromatin and decrease to minimal levels at the time of embryonic genome activation. We provide evidence that the decrease in H3K27me3 observed during early development is cell-cycle independent, suggesting an active mechanism for removal of this epigenetic mark. Among H3K27me3-specific demethylases, Jumonji domain-containing protein 3 (JMJD3), but not ubiquitously transcribed tetratricopeptide repeat X (UTX), present high transcript levels in oocytes. Soon after fertilization JMJD3 protein levels increase, concurrent with a decrease in mRNA levels. This pattern of expression suggests maternal inheritance of JMJD3. Knockdown of JMJD3 by siRNA injection in parthenogenetically activated metaphase II oocytes resulted in inhibition of the H3K27me3 decrease normally observed in preimplantation embryos. Moreover, knockdown of JMJD3 in oocytes reduced the rate of blastocyst development. Overall, these results indicate that JMJD3 is involved in active demethylation of H3K27me3 during early embryo development and that this mark plays an important role during the progression of embryos to blastocysts. PMID:22308433

Canovas, Sebastian; Cibelli, Jose B; Ross, Pablo J



Expression of SULT1E1 protein in bovine placentomes: evidence for localization in uninucleated trophoblast cells.  


The bovine placenta produces considerable amounts of pregnancy-associated estrogens, predominantly estrone sulfate, which does not interact with classical nuclear estrogen receptors. Thus, bovine placental estrogens may rather act as local regulatory factors than as hormones in a classical sense. To obtain information on the local availability of free estrogens in bovine placentomes, the expression pattern of the estrogen specific sulfotransferase SULT1E1 was characterized using antisera against the bovine and human enzyme, respectively. In western blot both antisera detected a band of the expected molecular weight (approx. 33 kDa) in placentomes and fetal liver. In immunohistochemistry the two antisera yielded virtually identical results. In placentomes distinct staining was restricted to the cytosol of uninucleated trophoblast cells (UTC). The staining pattern in UTC, immature and mature trophoblast giant cells (TGC) is consistent with a down-regulation of SULT1E1 during TGC differentiation. During early and midgestation staining intensity in UTC was higher in the trophoblast covering the chorionic plate and basal parts of stem villi compared to other regions of the villous tree, whereas during late gestation and at parturition an almost ubiquitous distinct staining of UTC was found. Correspondingly, relative SULT1E1-mRNA levels as measured by quantitative real-time RT-PCR increased significantly during late gestation (p = 0.0043). Comparative measurements showed that SULT1E1-mRNA levels in adult bovine organs were considerably lower compared to placentomes and fetal liver. The results suggest that the activities of free estrogens produced in bovine TGC are curtailed by SULT1E1 expressed in UTC and in fetal liver. Bovine pregnancy-associated estrogens produced in trophoblast giant cells are predominantly sulfonated in uninucleated trophoblast cells. PMID:21474178

Khatri, P; Frenette, G; Sullivan, R; Hoffmann, B; Schuler, G



The Region between Amino Acids 245 and 265 of the Bovine Leukemia Virus (BLV) Tax Protein Restricts Transactivation Not Only via the BLV Enhancer but Also via Other Retrovirus Enhancers  

Microsoft Academic Search

Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis and is closely related to human T-cell leukemia virus type 1 (HTLV-1). The Tax protein of BLV acts through the 5* long terminal repeat (LTR) of BLV and activates the transcription of BLV. In this study, we amplified tax genes from BLV-infected cattle using PCR. We cloned the genes and




Bovine in vitro oocyte maturation as a model for manipulation of the gamma-glutamyl cycle and intraoocyte glutathione.  


Glutathione (GSH) is the main non-enzymatic defence against oxidative stress and is a critical intracellular component required for oocyte maturation. In the present study, several modulators of intracellular GSH were assessed for their effect on the in vitro maturation (IVM) and intracellular GSH content of bovine metaphase (MII) oocytes. Of the five GSH modulators tested, only the cell-permeable GSH donor glutathione ethyl ester (GSH-OEt) significantly increased the GSH content of IVM MII oocytes in a concentration-dependent manner without adversely affecting oocyte maturation rate. The GSH level in IVM MII oocytes was greatly influenced by the presence or absence of cumulus cells and severely restricted when oocytes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine MII oocytes. Supplementation of the maturation medium with bovine serum albumin (BSA) or fetal calf serum (FCS) affected the GSH content of IVM MII oocytes, with greater levels attained under BSA culture conditions. The addition of GSH-OEt to the maturation medium increased the GSH content of IVM MII oocytes, irrespective of protein source. Spindle morphology, as assessed by immunocytochemistry and confocal microscopy, displayed distinct alterations in response to changes in oocyte GSH levels. GSH depletion caused by BSO treatment tended to widen spindle poles and significantly increased spindle area. Supplementation of the IVM medium with GSH-OEt increased spindle length, but did not significantly alter spindle area or spindle morphology. GSH-OEt represents a novel oocyte-permeable and cumulus cell-independent approach for effective elevation of mammalian oocyte GSH levels. PMID:18577355

Curnow, E C; Ryan, J; Saunders, D; Hayes, E S



Establishment and characterization of a lactating bovine mammary epithelial cell model for the study of milk synthesis.  


This study sought to establish an in vitro lactating BMEC (bovine mammary epithelial cell) model, which may maintain the native function for a period of time. Mammary tissues of midlactation Holstein dairy cows were dispersed and cultured in a medium containing insulin, prolactin, hydrocortisone, transferrin, epidermal growth factor and fetal calf serum. After the cells migrating from the tissue reached approximately 80% of confluency, the tissues were removed, and secretory epithelial cells were enriched by digesting with 0.25% trypsin repeatedly to remove fibroblasts. The BMEC cells plated on plastic dishes displayed a monolayer, cobblestone, epithelial-like morphology and formed alveoli-like structures and island monolayer aggregates which are the typical characteristics of the mammary epithelial cells. The isolated cells were identified as of epithelial origin by staining with antibody against cytokeratin 18. A one-half logarithmically growth curve and abundant microvilli and cytoplasmic lipid droplets were observed in these cells. The transcription of the alphas1 casein gene and synthesis of alphas caseins were also detected in the model. Thus, our lactating BMEC model can be an effective model in vitro for studies of milk synthesis in the bovine mammary gland. PMID:20214659

Zhao, Ke; Liu, Hong-Yun; Zhou, Miao-Miao; Liu, Jian-Xin



Use of ESAT-6-CFP-10 fusion protein in the bovine interferon-gamma ELISPOT assay for diagnosis of Mycobacterium bovis infection in cattle.  


Bovine tuberculosis (BTB) is a chronic bacterial disease, and a major animal health problem with zoonotic implications. Screening of mycobacterial infections in bovines is traditionally done using the single intradermal tuberculin test. Though the test is widely used, it has its own disadvantages and they include its inability to distinguish between pathogenic and non-pathogenic mycobacterial infections owing to its low specificity. Furthermore, the associated operative difficulties of this test have driven the quest for discovery of new antigens and diagnostic assays leading to the development of the interferon (IFN)- test. Presently, combinatorial testing using the skin test and the interferon gamma assays are being used in the diagnosis of BTB in various control and surveillance programs. In this study, we report the cloning, expression and purification of ESAT-6-CFP-10 fusion protein and its further use in the development of the IFN- gamma ELISPOT assay for accurate diagnosis of BTB in cattle. The BTB diagnosis employing the ELISPOT assay was evaluated using peripheral blood mononuclear cells from culture positive and culture negative cattle. The ELISPOT assay showed higher specificity and sensitivity in detecting BTB when a recombinant ESAT-6-CFP-10 fusion protein was used. The present study indicated that the usefulness of the fusion protein can replace the ESAT-6, CFP-10 or combination of both proteins for detecting BTB in IFN-gamma ELISPOT assay. PMID:22691409

Parthasarathy, Sugumar; Veerasami, Maroudam; Appana, Gangadharrao; Chandran, Dev; Das, Dipankar; Srinivasan, Villuppanoor Alwar



Biophysical modeling of mismatch repair proteins  

NASA Astrophysics Data System (ADS)

Mismatch repair proteins play a vital role in the bology of cancer due to their dual functions as repair proteins and as sensors of DNA damage. Computational modeling of mismatch repair proteins in conjunction with biological experimentation has demonstrated the role of long-range communication in the functions of these proteins. Furthermore, different conformations have been shown to be associated with different cellular functions, and these differences are being exploited in drug discovery. The latest results in this modeling will be presented.

Salsbury, Freddie



Modelling protein-protein interaction networks via a stickiness index  

PubMed Central

What type of connectivity structure are we seeing in protein–protein interaction networks? A number of random graph models have been mooted. After fitting model parameters to real data, the models can be judged by their success in reproducing key network properties. Here, we propose a very simple random graph model that inserts a connection according to the degree, or ‘stickiness’, of the two proteins involved. This model can be regarded as a testable distillation of more sophisticated versions that attempt to account for the presence of interaction surfaces or binding domains. By computing a range of network similarity measures, including relative graphlet frequency distance, we find that our model outperforms other random graph classes. In particular, we show that given the underlying degree information, fitting a stickiness model produces better results than simply choosing a degree-matching graph uniformly at random. Therefore, the results lend support to the basic modelling methodology.

Przulj, Natasa; Higham, Desmond J



The Importance of Protein-Protein Interactions on the pH-Induced Conformational Changes of Bovine Serum Albumin: A Small-Angle X-Ray Scattering Study  

PubMed Central

Abstract The combined effects of concentration and pH on the conformational states of bovine serum albumin (BSA) are investigated by small-angle x-ray scattering. Serum albumins, at physiological conditions, are found at concentrations of ?35–45 mg/mL (42 mg/mL in the case of humans). In this work, BSA at three different concentrations (10, 25, and 50 mg/mL) and pH values (2.0–9.0) have been studied. Data were analyzed by means of the Global Fitting procedure, with the protein form factor calculated from human serum albumin (HSA) crystallographic structure and the interference function described, considering repulsive and attractive interaction potentials within a random phase approximation. Small-angle x-ray scattering data show that BSA maintains its native state from pH 4.0 up to 9.0 at all investigated concentrations. A pH-dependence of the absolute net protein charge is shown and the charge number per BSA is quantified to 10(2), 8(1), 13(2), 20(2), and 26(2) for pH values 4.0, 5.4, 7.0, 8.0, and 9.0, respectively. The attractive potential diminishes as BSA concentration increases. The coexistence of monomers and dimers is observed at 50 mg/mL and pH 5.4, near the BSA isoelectric point. Samples at pH 2.0 show a different behavior, because BSA overall shape changes as a function of concentration. At 10 mg/mL, BSA is partially unfolded and a strong repulsive protein-protein interaction occurs due to the high amount of exposed charge. At 25 and 50 mg/mL, BSA undergoes some re-folding, which likely results in a molten-globule state. This work concludes by confirming that the protein concentration plays an important role on the pH-unfolded BSA state, due to a delicate compromise between interaction forces and crowding effects.

Barbosa, Leandro R.S.; Ortore, Maria Grazia; Spinozzi, Francesco; Mariani, Paolo; Bernstorff, Sigrid; Itri, Rosangela



Small interfering RNAs targeting viral structural envelope protein genes and the 5?-UTR inhibit replication of bovine viral diarrhea virus in MDBK cells.  


Bovine viral diarrhea viruses (BVDVs) are important pathogens of cattle that occur worldwide, and for which no antiviral therapy is available. In the present study, the inhibitory effect of small interfering (si) RNAs on bovine viral diarrhea virus 1 (BVDV-1) replication in cultured bovine cells was explored. Four synthetic siRNAs were designed to target structural envelope region genes (Erns, E1, and E2) and one cocktail of siRNA was generated to target the 5?-UTR of the BVDV-1 genome. The inhibitory effects of siRNAs were assessed by determination of infectious viral titer, viral antigen and viral RNA. The siRNA cocktail and three of the synthetic siRNAs produced moderate anti-BVDV-1 effect in vitro as shown by 25%-40% reduction in BVDV-1 antigen production, 7.9-19.9-fold reduction in viral titer and 21-48-fold reduction in BVDV-1 RNA copy number. Our findings suggest that siRNA cocktail targeted at the 5?-UTR is a stronger inhibitor of BVDV-1 replication and the targets for siRNA inhibition can be extended to BVDV-1 structural envelope protein genes. PMID:21978163

Mishra, N; Rajukumar, K; Kalaiyarasu, S; Behera, S P; Nema, R K; Dubey, S C



Spatio-temporal expression patterns of aurora kinases a, B, and C and cytoplasmic polyadenylation-element-binding protein in bovine oocytes during meiotic maturation.  


Maturation of immature bovine oocytes requires cytoplasmic polyadenylation and synthesis of a number of proteins involved in meiotic progression and metaphase-II arrest. Aurora serine-threonine kinases--localized in centrosomes, chromosomes, and midbody--regulate chromosome segregation and cytokinesis in somatic cells. In frog and mouse oocytes, Aurora A regulates polyadenylation-dependent translation of several mRNAs such as MOS and CCNB1, presumably by phosphorylating CPEB, and Aurora B phosphorylates histone H3 during meiosis. We analyzed the expression of three Aurora kinase genes--AURKA, AURKB, and AURKC--in bovine oocytes during meiosis by reverse transcription followed by quantitative real-time PCR and immunodetection. Aurora A was the most abundant form in oocytes, both at mRNA and protein levels. AURKA protein progressively accumulated in the oocyte cytoplasm during antral follicle growth and in vitro maturation. AURKB associated with metaphase chromosomes. AURKB, AURKC, and Thr-phosphorylated AURKA were detected at a contractile ring/midbody during the first polar body extrusion. CPEB, localized in oocyte cytoplasm, was hyperphosphorylated during prophase/metaphase-I transition. Most CPEB degraded in metaphase-II oocytes and remnants remained localized in a contractile ring. Roscovitine, U0126, and metformin inhibited meiotic divisions; they all induced a decrease of CCNB1 and phospho-MAPK3/1 levels and prevented CPEB degradation. However, only metformin depleted AURKA. The Aurora kinase inhibitor VX680 at 100 nmol/L did not inhibit meiosis but led to multinuclear oocytes due to the failure of the polar body extrusion. Thus, in bovine oocyte meiosis, massive destruction of CPEB accompanies metaphase-I/II transition, and Aurora kinases participate in regulating segregation of the chromosomes, maintenance of metaphase-II, and formation of the first polar body. PMID:17687118

Uzbekova, Svetlana; Arlot-Bonnemains, Yannick; Dupont, Joëlle; Dalbiès-Tran, Rozenn; Papillier, Pascal; Pennetier, Sophie; Thélie, Aurore; Perreau, Christine; Mermillod, Pascal; Prigent, Claude; Uzbekov, Rustem



Evaluation of the Recombinant 10-Kilodalton Immunodominant Region of the BP26 Protein of Brucella abortus for Specific Diagnosis of Bovine Brucellosis ?  

PubMed Central

Brucellosis is a disease with worldwide distribution affecting animals and human beings. Brucella abortus is the causative agent of bovine brucellosis. The cross-reactions of currently available diagnostic procedures for B. abortus infection result in false-positive reactions, which make the procedures unreliable. These tests are also unable to differentiate Brucella-infected and -vaccinated animals. The present work is focused on the use of a nonlipopolysaccharide (LPS) diagnostic antigen, a recombinant 10-kDa (r10-kDa) protein of B. abortus, for specific diagnosis of brucellosis. The purified recombinant protein was used as a diagnostic antigen in plate enzyme-linked immunosorbent assay (p-ELISA) format to screen 408 bovine serum samples (70 presumptively negative, 308 random, and 30 vaccinated), and the results were compared with those of the Rose Bengal plate agglutination test (RBPT) and the standard tube agglutination test (STAT). Statistical analysis in presumptive negative samples revealed 100 and 98.41% specificity of p-ELISA with RBPT and STAT, and an agreement of 91.43% with the tests using Cohen's kappa statistics. In random samples, the agreement of p-ELISA was 77.92% and 80.52% with RBPT and STAT, respectively. p-ELISA investigation of vaccinated samples reported no false-positive results, whereas RBPT and STAT reported 30% and 96.6% false-positive results, respectively. The data suggest that p-ELISA with r10-kDa protein may be a useful method for diagnosis of bovine brucellosis. Furthermore, p-ELISA may also be used as a tool for differentiating Brucella-vaccinated and naturally infected animals.

Tiwari, Arvind Kumar; Kumar, Subodh; Pal, Vijai; Bhardwaj, Bhupendra; Rai, Ganga Prasad



Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model  

Microsoft Academic Search

Infection with a replication-competent bovine leukemia virus structural gene vector (BLV SGV) is an innovative vaccination approach to prevent disease by complex retroviruses. Previously we developed BLV SGV that constitutively expresses BLV gag, pol, and env and related cis-acting sequences but lacks tax, rex, RIII, and GIV and most of the BLV long terminal repeat sequences, including the cis-acting Tax




Perifusion model system to culture bovine hypothalamic slices in series with dispersed anterior pituitary cells  

Microsoft Academic Search

Summary  Dispersed bovine anterior pituitary cells were incubated either in static or perifusion cultures to assess basal growth hormone\\u000a release as well as stimulatory and inhibitory effects of growth hormone-releasing hormone and somatostatin, respectively,\\u000a on growth hormone release. Total concentrations of growth hormones over a 12-hour incubation period were fivefold greater\\u000a in perifused than in static cultures (2034 ± 160 vs.

H. A. Hassan; R. A. Merkel



Metabolism of soluble rapeseed meal (Brassica rapa L.) protein during incubations with buffered bovine rumen contents in vitro.  


Accurate quantitative information on the fate of dietary protein in the rumen is central to modern metabolizable protein systems developed to improve the efficiency of nitrogen utilization in ruminants. An in vitro method was developed to estimate the rate of soluble rapeseed meal (Brassica rapa L.) protein (SRMP) degradation. Unlabeled and (15)N-labeled solvent-extracted rapeseed meal were incubated alone or as an equal mixture (125 mg of N/L) with buffered rumen contents and a mixture of carbohydrates formulated to provide a constant source of fermentable energy during the course of all incubations. Incubations were made over 0.33, 0.67, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, and 10.0 h. Enrichment of (14)N and (15)N isotopes in total N of ammonia (AN), soluble nonammonia (SNAN), and insoluble (ISN) fractions liberated during incubations with test proteins was determined. A model with 4 pools that accounted for both intracellular and extracellular N transformations was used to estimate the rate of SRMP degradation. Parameter values used in the model were adjusted based on the size of A(14)N, A(15)N, SNA(14)N, SNA(15)N, IS(14)N, and IS(15)N pools, measured at different time points during incubations with buffered rumen fluid. The mean rate of N degradation for SRMP was estimated at 0.126 (SD 0.0499) h(-1). No substantive difference in the rate of protein degradation or microbial protein synthesis was observed during incubations of labeled and unlabeled substrates with rumen fluid. In conclusion, combined use of data from incubations of unlabeled and (15)N-labeled rapeseed protein with buffered rumen inoculum provided sufficient information to allow for estimation of parameter values in a complex dynamic model of soluble protein degradation. Results indicate the potential of the technique to evaluate the degradability of SNAN of other dietary protein sources and implicate ruminal escape of soluble rapeseed protein as an important source of amino acids in ruminants. PMID:23127902

Stefa?ski, T; Ahvenjärvi, S; Huhtanen, P; Shingfield, K J



The use of bovine screws to promote bone formation using a tibia model in dogs.  


The objective of this study was to evaluate the use of a unique resorbable bovine bone screw to stimulate bone formation. Bovine bone screws were inserted in the tibia of beagle dogs. Each animal received 8 screws, divided into groups A (screws + no membranes), B (screws + titanium reinforced membranes), and C (bone defects treated with autogenous bone grafts). Animals were killed at 2, 4, and 6 months. New bone was measured with a periodontal probe and reported an average of 7.4 mm in vertical bone gain for group B, 3.6 mm for group A, and 1.7 mm for group C. Submission to Kruskal-Wallis test showed statistical differences among groups (P < .05). Histologic examination revealed an intimate contact between the newly formed bone and the resorbing bone screws. We conclude that bovine bone screws provide an environment for new bone formation and thus may provide an alternative therapy for enhancing bone formation vertically, including for regenerative procedures as well as before implant therapy. PMID:23058228

Bianchini, Marco Aurélio; Pontual, Marco Antônio B; Bez, Leonardo; Benfatti, César Augusto M; Boabaid, Fernanda; Somerman, Martha J; Magini, Ricardo S



Expression of the bovine viral diarrhoea virus Osloss p80 protein: its use as ELISA antigen for cattle serum antibody detection.  


The putative gene encoding the cytopathic bovine viral diarrhoea virus (BVDV) Osloss strain p80 protein was amplified by PCR and inserted into a T7 promoter-based vector for expression in Escherichia coli. Bacterial expression led to cytoplasmic insoluble inclusion bodies which were denatured by urea treatment and renatured by dialysis. Rabbit antisera were raised against this p80 recombinant antigen and assayed for the immunoprecipitation of either p120 or p80 protein from cytopathic or non-cytopathic BVDV biotype-infected bovine cells. The p80 gene sequence was also integrated into a baculovirus genome for its expression in Spodoptera frugiperda insect cells. The recombinant proteins isolated from bacteria or insect cells showed distinct antigenic properties when analysed by ELISA. Their ability to detect anti-BVDV specific antibodies was examined in a monoclonal antibody-based competitive ELISA performed on a series of field cattle sera. This comparative assay revealed the superiority of the insect cell-mediated expression to mimic the natural BVDV antigen produced by cell culture. The baculovirus/insect cell recombinant antigen gave the highest correlation between the ELISA-detected antibodies and the corresponding virus neutralization data. PMID:8393083

Vanderheijden, N; De Moerlooze, L; Vandenbergh, D; Chappuis, G; Renard, A; Lecomte, C



Transcriptional profiling of immune genes in bovine monocyte-derived macrophages exposed to bacterial antigens  

Microsoft Academic Search

The involvement of Toll-like receptors (TLRs) and other immune signalling genes during challenge of bovine macrophages with bacterial products derived from disease-causing bacteria in cattle was investigated. An in vitro cell culture model of bovine monocyte derived macrophages (MDM) was established and these cells were exposed to purified protein derivative (PPD-b) derived from Mycobacterium bovis and to lipopolysachharide (LPS) derived

Maria Taraktsoglou; Urszula Szalabska; David A. Magee; John A. Browne; Torres Sweeney; Eamonn Gormley; David E. MacHugh



Immunoaffinity purification of two major proteins of bovine leukemia virus (gp51 and p24) and their use for discrimination between vaccinated and infected animals.  


The outer envelope glycoprotein gp51 and the core protein p24 of bovine leukemia virus (BLV), were purified from culture media of FLK-BLV cells by a single-step procedure, using immunoaffinity chromatography based on monoclonal antibodies to the respective proteins. About 90% of the envelope glycoprotein in the culture medium was recovered as a highly purified product. Both purified protein (gp51 and p24) preparations, were found to be highly specific antigens by ELISA, and did not cross-react with sera raised against the other antigen. The conformational epitopes on the purified gp51 were preserved as judged by their reactions with the corresponding monoclonal antibodies. The p24 ELISA reacted only with sera from naturally infected animals and not with sera from animals immunized with an experimental gp51-iscom vaccine. The p24 antigen is therefore useful for discriminating between BLV-infected animals and those immunized with a gp51 subunit vaccine. PMID:1723735

Merza, M; Sundquist, B; Söber, J; Morein, B



Involvement of different protein kinases and phospholipases A2 in phorbol ester (TPA)-induced arachidonic acid liberation in bovine platelets.  

PubMed Central

The effect of various phospholipase A2 and protein kinase inhibitors on the arachidonic acid liberation in bovine platelets induced by the protein kinase activator 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied. TPA stimulates arachidonic acid release mainly by activating group IV cytosolic PLA2 (cPLA2), since inhibitors of this enzyme markedly inhibited arachidonic acid formation. However, group VI Ca2+-independent PLA2 (iPLA2) seems to contribute to the arachidonic acid liberation too, since the relatively specific iPLA2 inhibitor bromoenol lactone (BEL) decreased arachidonic acid generation in part. The pronounced inhibition of the TPA-induced arachidonic acid release by the protein kinase C (PKC) inhibitors GF 109203X and Ro 31-82220, respectively, and by the p38 MAP kinase inhibitor SB 202190 suggests that the activation of the PLA2s by TPA is mediated via PKC and p38 MAP kinase.

Lehr, M; Griessbach, K



Comparison of amino acid sequence of bovine coagulation Factor IX (Christmas Factor) with that of other vitamin K-dependent plasma proteins.  

PubMed Central

The amino acid sequence of bovine blood coagulation Factor IX (Christmas Factor) is presented and compared with the sequences of other vitamin K-dependent plasma proteins and pancreatic trypsinogen. The 416-residue sequence of Factor IX was determined largely by automated Edman degradation of two large segments, containing 181 and 235 residues, isolated after activating Factor IX with a protease from Russell's viper venom. Subfragments of the two segments were produced by enzymatic digestion and by chemical cleavage of methionyl, tryptophyl, and asparaginyl-glycyl bonds. Comparison of the amino acid sequences of Factor IX, Factor X, and Protein C demonstrates that they are homologous throughout. Their homology with prothrombin, however, is restricted to the amino-terminal region, which is rich in gamma-carboxyglutamic acid, and the carboxyl-terminal region, which represents the catalytic domain of these proteins and corresponds to that of pancreatic serine proteases.

Katayama, K; Ericsson, L H; Enfield, D L; Walsh, K A; Neurath, H; Davie, E W; Titani, K



The effect of the number of observations used for Fourier transform infrared model calibration for bovine milk fat composition on the estimated genetic parameters of the predicted data  

Microsoft Academic Search

Fourier transform infrared spectroscopy is a suitable method to determine bovine milk fat composition. However, the determination of fat composition by gas chromatography, required for calibration of the infrared prediction model, is expensive and labor intensive. It has recently been shown that the number of calibration samples is strongly related to the model's validation r2 (i.e., accuracy of prediction). However,

M. J. M. Rutten; H. Bovenhuis; Arendonk van J. A. M



Hidden Markov Models in Computational Biology Applications to Protein Modeling  

Microsoft Academic Search

Ridden .Markov Models (HMMs) are applied t.0 the problems of statistical modeling, database searching and multiple sequence alignment of protein families and protein domains. These methods are demonstrated on the globin family, the protein kinase catalytic domain, and the EF-hand calcium binding motif. In each case the parameters of an HMM are estimated from a training set of unaligned sequences.

Anders Krogh; Michael Brown; I. Saira



Outer Membrane Protein A of Bovine and Ovine Isolates of Mannheimia haemolytica Is Surface Exposed and Contains Host Species-Specific Epitopes ?  

PubMed Central

Mannheimia haemolytica is the etiological agent of pneumonic pasteurellosis of cattle and sheep; two different OmpA subclasses, OmpA1 and OmpA2, are associated with bovine and ovine isolates, respectively. These proteins differ at the distal ends of four external loops, are involved in adherence, and are likely to play important roles in host adaptation. M. haemolytica is surrounded by a polysaccharide capsule, and the degree of OmpA surface exposure is unknown. To investigate surface exposure and immune specificity of OmpA among bovine and ovine M. haemolytica isolates, recombinant proteins representing the transmembrane domain of OmpA from a bovine serotype A1 isolate (rOmpA1) and an ovine serotype A2 isolate (rOmpA2) were overexpressed, purified, and used to generate anti-rOmpA1 and anti-rOmpA2 antibodies, respectively. Immunogold electron microscopy and immunofluorescence techniques demonstrated that OmpA1 and OmpA2 are surface exposed, and are not masked by the polysaccharide capsule, in a selection of M. haemolytica isolates of various serotypes and grown under different growth conditions. To explore epitope specificity, anti-rOmpA1 and anti-rOmpA2 antibodies were cross-absorbed with the heterologous isolate to remove cross-reacting antibodies. These cross-absorbed antibodies were highly specific and recognized only the OmpA protein of the homologous isolate in Western blot assays. A wider examination of the binding specificities of these antibodies for M. haemolytica isolates representing different OmpA subclasses revealed that cross-absorbed anti-rOmpA1 antibodies recognized OmpA1-type proteins but not OmpA2-type proteins; conversely, cross-absorbed anti-rOmpA2 antibodies recognized OmpA2-type proteins but not OmpA1-type proteins. Our results demonstrate that OmpA1 and OmpA2 are surface exposed and could potentially bind to different receptors in cattle and sheep.

Hounsome, Jonathan D. A.; Baillie, Susan; Noofeli, Mojtaba; Riboldi-Tunnicliffe, Alan; Burchmore, Richard J. S.; Isaacs, Neil W.; Davies, Robert L.



Systematic identification of yeast proteins extracted into model wine during aging on the yeast lees.  


Total protein and protein-associated mannan concentrations were measured, and individual proteins were identified during extraction into model wines over 9 months of aging on the yeast lees following completion of fermentations by seven wine strains of Saccharomyces cerevisiae. In aged wines, protein-associated mannan increased about 6-fold (+/-66%), while total protein only increased 2-fold (+/-20%), which resulted in a significantly greater protein-associated mannan/total protein ratio for three strains. A total of 219 proteins were identified among all wine samples taken over the entire time course. Of the 17 "long-lived" proteins detected in all 9 month samples, 13 were cell wall mannoproteins, and four were glycolytic enzymes. Most cytosolic proteins were not detected after 6 months. Native mannosylated yeast invertase was assayed for binding to wine tannin and was found to have a 10-fold lower affinity than nonglycosylated bovine serum albumin. Enrichment of mannoproteins in the aged model wines implies greater solution stability than other yeast proteins and the possibility that their contributions to wine quality may persist long after bottling. PMID:20108898

Rowe, Jeffrey D; Harbertson, James F; Osborne, James P; Freitag, Michael; Lim, Juyun; Bakalinsky, Alan T



Insulin-like activity and immunoreactive insulin content of gel-filtered protein fractions of bovine serum  

Microsoft Academic Search

Summary  Serum globulin fractions isolated by gel filtration from fasting bovine sera with simultaneous exclusion of low molecular endogenous immunoreactive insulin (IRI), were found to contain immunoreactive insulin-like material in quantities which equal or surpass the directly assayable IRI content of native fasting sera. In one additional and clearly aberrant serum, independently isolated globulin fractions were found to contain 306 U

W. Konijnendijk; P. R. Bouman



Substrate phage display for protease substrate sequence characterization: bovine factor xa as a model system.  


Regulatory proteases modulate proteomic dynamics with a spectrum of specificities against substrate proteins. Substrate phage display is one of the key methodologies in producing substrate sequence information in vitro. Factor Xa, a key regulatory protease in the blood coagulation system, is used as a model system to demonstrate a high-throughput procedure to quantitatively characterize substrate sequences and their susceptibilities for enzymatic cleavage. This methodology can be generalized to proteases for which the active forms (not necessarily purified forms) are available for the in vitro experiments. PMID:24146400

Hsu, Hung-Ju; Yang, An-Suei



Chemical Modification of Lysozyme, Glucose 6-Phosphate Dehydrogenase, and Bovine Eye Lens Proteins Induced by Peroxyl Radicals: Role of Oxidizable Amino Acid Residues.  


Chemical and structural alterations to lysozyme (LYSO), glucose 6-phosphate dehydrogenase (G6PD), and bovine eye lens proteins (BLP) promoted by peroxyl radicals generated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) under aerobic conditions were investigated. SDS-PAGE analysis of the AAPH-treated proteins revealed the occurrence of protein aggregation, cross-linking, and fragmentation; BLP, which are naturally organized in globular assemblies, were the most affected proteins. Transmission electron microscopy (TEM) analysis of BLP shows the formation of complex protein aggregates after treatment with AAPH. These structural modifications were accompanied by the formation of protein carbonyl groups and protein hydroperoxides. The yield of carbonyls was lower than that for protein hydroperoxide generation and was unrelated to protein fragmentation. The oxidized proteins were also characterized by significant oxidation of Met, Trp, and Tyr (but not other) residues, and low levels of dityrosine. As the dityrosine yield is too low to account for the observed cross-linking, we propose that aggregation is associated with tryptophan oxidation and Trp-derived cross-links. It is also proposed that Trp oxidation products play a fundamental role in nonrandom fragmentation and carbonyl group formation particularly for LYSO and G6PD. These data point to a complex mechanism of peroxyl-radical mediated modification of proteins with monomeric (LYSO), dimeric (G6PD), and multimeric (BLP) structural organization, which not only results in oxidation of protein side chains but also gives rise to radical-mediated protein cross-links and fragmentation, with Trp species being critical intermediates. PMID:23252580

Arenas, Andrea; López-Alarcón, Camilo; Kogan, Marcelo; Lissi, Eduardo; Davies, Michael J; Silva, Eduardo



Isolation of cDNAs encoding desmosomal plaque proteins: evidence that bovine desmoplakins I and II are derived from two mRNAs and a single gene.  

PubMed Central

Desmoplakins (DPs) I and II (approximately equal to 240 and approximately equal to 210 kDa) are major components of the internal portion of the desmosomal cytoplasmic plaque. Desmosomes play a crucial role in cell-cell adhesion and serve as specific attachment sites for cytoplasmic intermediate filaments. Although DP-I and -II are closely related molecules, their structure (i.e., amino acid or DNA sequence) has not been determined. In addition, it is not known whether these proteins are derived from one or more genes or whether they result from posttranscriptional or posttranslational events. This paper describes the isolation and characterization of eight DP cDNA clones from a bovine lambda gt11 expression library. Fusion proteins from six of these clones selected antibodies that reacted with DP-I and -II and two selected antibodies that reacted with DP-I alone. Antibodies made against fusion protein produced by the DP1A clone reacted specifically with DP-I and -II on immunoblots. When used for indirect immunofluorescence on bovine tongue cryostat sections and cultured mouse keratinocytes, these antibodies produced a typical desmosomal staining pattern. RNA blot analysis demonstrated hybridization of three DP-I/II cDNA probes with two messages of approximately equal to 7.5 and approximately equal to 9.5 kilobases in bovine tongue RNA. In contrast, a cDNA clone that affinity-purified antibodies reacting with DP-I only hybridized exclusively with the 9.5-kilobase band. Southern blots of genomic DNA digested with a panel of restriction enzymes were hybridized with one probe derived from a DP-I/II clone and with one from a DP-I clone. Both probes hybridized with single bands of the same size in each digested sample of DNA. Together, these data suggest that DP-I and DP-II are translated from two separate messages in bovine tongue and that these messages may be derived from a single gene. Images

Green, K J; Goldman, R D; Chisholm, R L



An economic model to evaluate the mitigation programme for bovine viral diarrhoea in Switzerland.  


Economic analyses are indispensable as sources of information to help policy makers make decisions about mitigation resource use. The aim of this study was to conduct an economic evaluation of the Swiss national mitigation programme for bovine viral diarrhoea virus (BVDV), which was implemented in 2008 and concludes in 2017. The eradication phase of the mitigation programme comprised testing and slaughtering of all persistently infected (PI) animals found. First, the whole population was antigen tested and all PI cattle removed. Since October 2008, all newborn calves have been subject to antigen testing to identify and slaughter PI calves. All mothers of PI calves were retested and slaughtered if the test was positive. Antigen testing in calves and elimination of virus-carriers was envisaged to be conducted until the end of 2011. Subsequently, a surveillance programme will document disease freedom or detect disease if it recurs. Four alternative surveillance strategies based on antibody testing in blood from newborn calves and/or milk from primiparous cows were proposed by Federal Veterinary Office servants in charge of the BVDV mitigation programme. A simple economic spreadsheet model was developed to estimate and compare the costs and benefits of the BVDV mitigation programme. In an independent project, the impact of the mitigation programme on the disease dynamics in the population was simulated using a stochastic compartment model. Mitigation costs accrued from materials, labour, and processes such as handling and testing samples, and recording results. Benefits were disease costs avoided by having the mitigation programme in place compared to a baseline of endemic disease equilibrium. Cumulative eradication costs and benefits were estimated to determine the break-even point for the eradication component of the programme. The margin over eradication cost therefore equalled the maximum expenditure potentially available for surveillance without the net benefit from the mitigation programme overall becoming zero. Costs of the four surveillance strategies and the net benefit of the mitigation programme were estimated. Simulations were run for the years 2008-2017 with 20,000 iterations in @Risk for Excel. The mean baseline disease costs were estimated to be 16.04 m CHF (1 Swiss Franc, CHF=0.73 € at the time of analysis) (90% central range, CR: 14.71-17.39 m CHF) in 2008 and 14.89 m CHF (90% CR: 13.72-16.08 m CHF) in 2009. The break-even point was estimated to be reached in 2012 and the margin over eradication cost 63.15m CHF (90% CR: 53.72-72.82 m CHF). The discounted cost for each surveillance strategy was found to be smaller than the margin, so the mitigation programme overall is expected to have a positive net economic benefit irrespective of the strategy adopted. For economic efficiency, the least cost surveillance alternative must be selected. PMID:22402180

Häsler, B; Howe, K S; Presi, P; Stärk, K D C



The use of embryonic stem cell derived bioactive material as a new protein supplement for the in vitro culture of bovine embryos.  


Embryonic stem (ES) cells are expanded versions of the inner cell mass cells that compose the early mammalian blastocyst. Components derived from ES cells may contain various bioactive materials (BM) helpful for early preimplantation embryo growth. In this study, we examined the effect of human ES cell derived BM (hES-BM) on in vitro culture of bovine embryos. When bovine parthenogenetic day 2 embryos were cultured in 10% hES-BM, a significantly higher embryo development rate (44.3%) and increased cell numbers were observed relative to control medium containing 3 mg/ml BSA (19.5%; P<0.01). Among the various concentrations (5, 10 and 15%) and days of treatment (2 or 4 days) tested, 10% hES-BM treatment for 4 days provided the best culture environment to support the growth of bovine embryos in vitro (P<0.05). Little difference was observed between 10% hES-BM and 10% FBS treatment in the examined parthenogenetic or in vitro fertilized embryos, although the hES-BM group developed at a slightly better rate. However, the ICM cell numbers were significantly higher in the hES-BM group in irrespective of embryo origin (P<0.05). In addition, the relative levels of pluripotency (Oct4, × 1.8 fold; Nanog. × 3.3 fold), embryogenesis (Stat3, × 2.8 fold; FGF4, × 18.8 fold; E-cad, × 2.0 fold) and growth (Glut5, × 2.6 fold) genes were significantly higher in the 10% hES-BM group than in the 10% FBS group (P<0.05), while the levels of other genes (Bax, Bcl2, MnSOD and Connexin43) were not different. This is the first report examining the positive effects of hES-BM on bovine embryo development in vitro. Based on our results, we conclude that hES-BM can be used as a new protein supplement for bovine preimplantation embryo development. PMID:21289468

Kim, Eun Young; Lee, Jun Beom; Park, Hyo Young; Jeong, Chang Jin; Riu, Key Zung; Park, Se Pill



Assessment of a Protein Cocktail-Based Skin Test for Bovine Tuberculosis in a Double-Blind Field Test in Cattle  

PubMed Central

Bovine tuberculosis (bTB) is a worldwide zoonosis caused mainly by Mycobacterium bovis. The traditional diagnostic method used often is the tuberculin skin test, which uses bovine purified protein derivatives (PPD-B). However, it is difficult to maintain uniformity of PPD-B from batch to batch, and it shares common antigens with nonpathogenic environmental mycobacteria. To overcome these problems, M. bovis-specific antigens that showed good T cell stimulation, such as CFP-10, ESAT-6, Rv3615c, etc., have been used in the skin test, but there have been no large-scale clinical studies on these antigens. In this study, two combinations (CFP-10/ESAT-6/TB10.4 protein cocktail and CFP-10/ESAT-6/Rv3872/MPT63 protein cocktail) were developed and used as stimuli in the skin test. Cattle were double-blind tested to assess the efficiency of the protein cocktail-based skin tests. The results showed that the CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test can differentiate TB-infected cattle from Mycobacterium avium-infected ones and that it shows a high degree of agreement with the traditional tuberculin skin test (? = 0.8536) and gamma interferon (IFN-?) release assay (? = 0.8154). Compared to the tuberculin skin test, the relative sensitivity and relative specificity of the CFP-10/ESAT-6/TB10.4-based skin test were 87% and 97%, respectively., The relative sensitivity and relative specificity of the CFP-10/ESAT-6/TB10.4-based skin test were 93% and 92%, respectively, on comparison with the IFN-? release assay. The correlation between the increases in skin thickness observed after the inoculation of stimuli was high (PPD-B versus CFP-10/ESAT-6/TB10.4, Spearman r of 0.8435). The correlation between the optical density at 450 nm (OD450) obtained after blood stimulation with PPD-B and the increase in skin thickness observed after inoculation of the CFP-10/ESAT-6/TB10.4 protein cocktail was high (Spearman r = 0.7335). Therefore, the CFP-10/ESAT-6/TB10.4-based skin test responses correlate to traditional measures of bovine TB evaluation, including skin test and gamma interferon release assay.

Xin, Ting; Jia, Hong; Ding, Jiabo; Li, Pingjun; Yang, Hongjun; Hou, Shaohua; Yuan, Weifeng; Guo, Xiaoyu; Wang, Haichun; Liang, Qianqian; Li, Ming



Purification and characterization from bovine brain cytosol of a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A, a ras p21-like GTP-binding protein.  


A novel regulatory protein for smg p25A, a ras p21-like GTP-binding protein, was purified to near homogeneity from bovine brain cytosol. This regulatory protein, designated here as smg p25A GDP dissociation inhibitor (GDI), inhibited the dissociation of GDP, but not of guanosine 5'-(3-O-thio)triphosphate (GTPgamma S), from smg p25A. smg p25A GDI also inhibited the binding of GTPgamma S to the GDP-bound form of smg p25A but not of that to the guanine nucleotide-free form. GDI did not stimulate the GTPase activity of smg p25A and by itself showed neither GTPgammaS-binding nor GTPase activity. GDI was inactive for other ras p21/ras p21-like GTP-binding proteins including c-Ha-ras p21, rhoB p20, and smg p21. The Mr value of GDI was estimated to be about 54,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, about 65,000 from the S value (4.5 S), and about 82,000 by gel filtration. The isoelectric point of GDI was about pH 5.6. The activities of GDI were killed by tryptic digestion or heat boiling. These results indicate that bovine brain cytosol contains a regulatory protein for smg p25A that inhibits the dissociation of GDP from and thereby the subsequent binding of GTP to this protein. PMID:2105320

Sasaki, T; Kikuchi, A; Araki, S; Hata, Y; Isomura, M; Kuroda, S; Takai, Y



Bovine Genome Database: integrated tools for genome annotation and discovery  

PubMed Central

The Bovine Genome Database (BGD; strives to improve annotation of the bovine genome and to integrate the genome sequence with other genomics data. BGD includes GBrowse genome browsers, the Apollo Annotation Editor, a quantitative trait loci (QTL) viewer, BLAST databases and gene pages. Genome browsers, available for both scaffold and chromosome coordinate systems, display the bovine Official Gene Set (OGS), RefSeq and Ensembl gene models, non-coding RNA, repeats, pseudogenes, single-nucleotide polymorphism, markers, QTL and alignments to complementary DNAs, ESTs and protein homologs. The Bovine QTL viewer is connected to the BGD Chromosome GBrowse, allowing for the identification of candidate genes underlying QTL. The Apollo Annotation Editor connects directly to the BGD Chado database to provide researchers with remote access to gene evidence in a graphical interface that allows editing and creating new gene models. Researchers may upload their annotations to the BGD server for review and integration into the subsequent release of the OGS. Gene pages display information for individual OGS gene models, including gene structure, transcript variants, functional descriptions, gene symbols, Gene Ontology terms, annotator comments and links to National Center for Biotechnology Information and Ensembl. Each gene page is linked to a wiki page to allow input from the research community.

Childers, Christopher P.; Reese, Justin T.; Sundaram, Jaideep P.; Vile, Donald C.; Dickens, C. Michael; Childs, Kevin L.; Salih, Hanni; Bennett, Anna K.; Hagen, Darren E.; Adelson, David L.; Elsik, Christine G.



PreImplantation Factor (PIF) correlates with early mammalian embryo development-bovine and murine models  

PubMed Central

Background PreImplantation Factor (PIF), a novel peptide secreted by viable embryos is essential for pregnancy: PIF modulates local immunity, promotes decidual pro-adhesion molecules and enhances trophoblast invasion. To determine the role of PIF in post-fertilization embryo development, we measured the peptide's concentration in the culture medium and tested endogenous PIF's potential trophic effects and direct interaction with the embryo. Methods Determine PIF levels in culture medium of multiple mouse and single bovine embryos cultured up to the blastocyst stage using PIF-ELISA. Examine the inhibitory effects of anti-PIF-monoclonal antibody (mAb) added to medium on cultured mouse embryos development. Test FITC-PIF uptake by cultured bovine blastocysts using fluorescent microscopy. Results PIF levels in mouse embryo culture medium significantly increased from the morula to the blastocyst stage (ANOVA, P = 0.01). In contrast, atretic embryos medium was similar to the medium only control. Detectable - though low - PIF levels were secreted already by 2-cell stage mouse embryos. In single bovine IVF-derived embryos, PIF levels in medium at day 3 of culture were higher than non-cleaving embryos (control) (P = 0.01) and at day 7 were higher than day 3 (P = 0.03). In non-cleaving embryos culture medium was similar to medium alone (control). Anti-PIF-mAb added to mouse embryo cultures lowered blastocyst formation rate 3-fold in a dose-dependent manner (2-way contingency table, multiple groups, X2; P = 0.01) as compared with non-specific mouse mAb, and medium alone, control. FITC-PIF was taken-up by cultured bovine blastocysts, but not by scrambled FITC-PIF (control). Conclusions PIF is an early embryo viability marker that has a direct supportive role on embryo development in culture. PIF-ELISA use to assess IVF embryo quality prior to transfer is warranted. Overall, our data supports PIF's endogenous self sustaining role in embryo development and the utility of PIF- ELISA to detect viable embryos in a non-invasive manner.



Biophysical studies on the interactions of jatrorrhizine with bovine serum albumin by spectroscopic and molecular modeling methods.  


The interaction between jatrorrhizine (JAT) and bovine serum albumin (BSA) has been studied. The studies were carried out in a buffer medium at pH 7.4 using fluorescence spectroscopy, UV-vis spectroscopy, and molecular modeling methods. The results of fluorescence quenching and UV-vis absorption spectra experiments indicated the formation of the complex of BSA-JAT. Binding parameters were determined using the Stern-Volmer equation and Scatchard equation. The results of thermodynamic parameters ?G, ?H and ?S at different temperatures indicate that the electrostatic interactions and hydrogen bonds play a major role for JAT-BSA association. Site marker competitive displacement experiments and molecular modeling calculation demonstrating that JAT is mainly located within the hydrophobic pocket of the subdomain IIIA of BSA. Furthermore, The distance between donor (BSA) and acceptor (JAT) was estimated according to fluorescence resonance energy transfer. PMID:23645029

Mi, Ran; Li, Pei-Qi; Hu, Yan-Jun; Fan, Xiao-Yang; Li, Hai-Ying; Yu, Xue-Cheng; Ouyang, Yu



A soft and transparent handleable protein model  

NASA Astrophysics Data System (ADS)

The field of structural biology currently relies on computer-generated graphical representations of three-dimensional (3D) structures to conceptualize biomolecules. As the size and complexity of the molecular structure increases, model generation and peer discussions become more difficult. It is even more problematic when discussing protein-protein interactions wherein large surface area contact is considered. This report demonstrates the viability of a new handleable protein molecular model with a soft and transparent silicone body similar to the molecule's surface. A full-color printed main chain structure embedded in the silicone body enables users to simultaneously feel the molecular surface, view through the main chain structure, and manually simulate molecular docking. The interactive, hands-on experience deepens the user's intuitive understanding of the complicated 3D protein structure and elucidates ligand binding and protein-protein interactions. This model would be an effective discussion tool for the classroom or laboratory that stimulates inspired learning in this study field.

Kawakami, Masaru



Adipogenesis of bovine perimuscular preadipocytes  

SciTech Connect

In this study, non-transformed progeny adipofibroblasts, derived from mature adipocyte dedifferentiation, was used as a novel in vitro model to study adipogenic gene expression in cattle. Adipofibroblasts from dedifferentiated mature perimuscular fat (PMF) tissue were cultured with differentiation stimulants until the cells exhibited morphological differentiation. Treated cells were harvested from day 2 to 16 for RNA extraction, whereas control cells were cultured without addition of stimulants. Results from time course gene expression assays by quantitative real-time PCR revealed that peroxisome proliferator-activated receptor gamma (PPAR-{gamma}), sterol regulatory element binding protein 1 (SREBP-1) and their six down-stream genes were co-expressed at day 2 post-differentiation induction. When compared to other adipogenesis culture systems, the adipogenic gene expression of bovine PMF adipofibroblasts culture was different, especially to the rodent model. Collectively, these results demonstrated PPAR-{gamma} and SREBP-1 cooperatively play a key role to regulate the re-differentiation of bovine adipofibroblasts, during early conversion stages in vitro.

Taniguchi, Masaaki; Le Luo Guan; Zhang Bing [Department of Agricultural, Food and Nutritional Science, University of Alberta, 410 Ag-For Centre, Edmonton, Alta., T6G 2P5 (Canada); Dodson, Michael V. [Department of Animal Sciences, Washington State University, P.O. Box 646310, Pullman, WA 99164 (United States); Okine, Erasmus [Department of Agricultural, Food and Nutritional Science, University of Alberta, 410 Ag-For Centre, Edmonton, Alta., T6G 2P5 (Canada); Moore, Stephen S. [Department of Agricultural, Food and Nutritional Science, University of Alberta, 410 Ag-For Centre, Edmonton, Alta., T6G 2P5 (Canada)], E-mail:



A network model to correlate conformational change and the impedance spectrum of single proteins  

Microsoft Academic Search

Integrated nanodevices based on proteins or biomolecules are attracting increasing interest in today's research. In fact, it has been shown that proteins such as azurin and bacteriorhodopsin manifest some electrical properties that are promising for the development of active components of molecular electronic devices. Here we focus on two relevant kinds of protein: bovine rhodopsin, prototype of G-protein-coupled-receptor (GPCR) proteins,

Eleonora Alfinito; Cecilia Pennetta; Lino Reggiani



Bovine DNase I: gene organization, mRNA expression, and changes in the topological distribution of the protein during apoptosis in lens epithelial cells.  


Genomic DNA sequencing and alignment with the known DNase I mRNA showed that the bovine gene consists of 9 exons and that only the last 8 encode the protein, since initial ATG was found at exon II. RT-PCR was used to identify DNase I mRNA in lens epithelium in vivo and in cultured epithelial cells. We found DNase I transcripts having the same nucleotide sequence as the pancreas form and others lacking almost all exon V. The lens protein presented a slightly higher relative molecular weight than the pancreatic enzyme. Lens DNase I was located in secretory pathway organelles and excluded from the nucleus. Nevertheless, in apoptotic lens epithelial cells in vitro, DNase I translocated to the nucleus and co-localized with TUNEL positive chromatin aggregates. These results indicate that cells in the lens epithelium constitutively express DNase I, and suggest a direct involvement of this nuclease in the final phases of chromatin degradation. PMID:14680812

De María, Alicia; Arruti, Cristina



The YXXL signalling motifs of the bovine leukemia virus transmembrane protein are required for in vivo infection and maintenance of high viral loads.  


The bovine leukemia virus (BLV) transmembrane protein (gp30) contains three YXXL motifs at its carboxyterminal end. Two of these motifs have been implicated in vitro in signal transduction pathways from the external to the intracellular compartment. In order to analyze the biological relevance of these motifs in vivo, recombinant BLV proviruses were constructed. A mutation of the tyrosine residue of the second YXXL motif completely destroyed the infectious potential of the virus in sheep. In contrast, the tyrosine of the first motif appeared to be dispensable for infectivity. However, the propagation of the recombinant virus within the animal was greatly impaired (as demonstrated by PCR and enzyme-linked immunosorbent assay). These recombinant BLVs thus exhibit an attenuated phenotype. Altogether, our data demonstrate the importance of the YXXL motifs of the BLV transmembrane protein for in vivo infection and viral propagation. PMID:7769672

Willems, L; Gatot, J S; Mammerickx, M; Portetelle, D; Burny, A; Kerkhofs, P; Kettmann, R



The YXXL signalling motifs of the bovine leukemia virus transmembrane protein are required for in vivo infection and maintenance of high viral loads.  

PubMed Central

The bovine leukemia virus (BLV) transmembrane protein (gp30) contains three YXXL motifs at its carboxyterminal end. Two of these motifs have been implicated in vitro in signal transduction pathways from the external to the intracellular compartment. In order to analyze the biological relevance of these motifs in vivo, recombinant BLV proviruses were constructed. A mutation of the tyrosine residue of the second YXXL motif completely destroyed the infectious potential of the virus in sheep. In contrast, the tyrosine of the first motif appeared to be dispensable for infectivity. However, the propagation of the recombinant virus within the animal was greatly impaired (as demonstrated by PCR and enzyme-linked immunosorbent assay). These recombinant BLVs thus exhibit an attenuated phenotype. Altogether, our data demonstrate the importance of the YXXL motifs of the BLV transmembrane protein for in vivo infection and viral propagation.

Willems, L; Gatot, J S; Mammerickx, M; Portetelle, D; Burny, A; Kerkhofs, P; Kettmann, R



Effects of insulin, recombinant bovine somatotropin, and their interaction on insulin-like growth factor-I secretion and milk protein production in dairy cows.  


This trial was designed to test the effects of insulin, recombinant bovine somatotropin (rbST), and their interaction on milk protein and selected blood parameters in dairy cows. Eight Holstein cows (86 +/- 10 d in milk) were divided in two groups and used in two replicates of a Latin square design with four animals, four periods, and four treatments: 1) intravenous infusion of saline, 2) infusion of saline and subcutaneous administration of 40 mg of rbST per day, 3) intravenous infusion of 12 mg of insulin per day coupled with glucose infusion, and 4) rbST administration combined with insulin and glucose infusion. The glucose infusion rate was adjusted to maintain euglycemia. Each experimental period lasted 14 d: treatments were administered during the first 6 d, and no treatment was administered during the following 8-d resting phase. The average daily amount of glucose infusion needed to avoid hypoglycemia was 2.8 kg/cow when only insulin was infused as opposed to 2.2 kg/cow when both insulin and rbST were administered, indicating that either rbST causes a peripheral resistance to insulin or rbST increased liver gluconeogenesis or both. Data from the last 3 d of infusion were analyzed by using the SAS system for mixed models. Percent protein of milk tended to be lower (2.84 vs. 2.79%) and milk urea content was lower (16.6 vs. 14.8 mg/dl) during rbST administration, regardless of insulin infusion. Insulin infusion increased percent protein (2.78 vs. 2.85%) and percent casein (2.36 vs. 2.46%) and decreased milk urea content (17.1 vs. 14.3 mg/dl) regardless of rbST administration. For milk yield, protein yield, casein yield, lactose percent, and lactose yield, there were significant interactions between insulin and rbST administration. For example, casein yield averaged 1.17, 1.12, 1.20, and 1.28 kg/d for saline, insulin, rbST, and insulin combined with rbST, respectively. Similarly, there was a significant interaction between insulin and rbST on IGF-I levels, which were 122.5, 181.3, 342.3, and 492.2 ng/ml for saline, insulin, rbST, and insulin combined with rbST, respectively. In conclusion, these results clearly demonstrated that insulin interacts with bST in early lactation to improve milk protein synthesis and yield in dairy cows. These effects are probably mediated through a combination of bST nutrient mobilization, bST-induced gluconeogenesis, bST-induced insulin peripheral resistance, and bST/insulin synergism on insulin-like growth factor-I secretion and on mammary epithelial tissue. PMID:12018418

Molento, C F M; Block, E; Cue, R I; Petitclerc, D



Rho GTPase activating protein 15 (arhGAP15) siRNA effect apoptosis-induced by ethanol in bovine fibroblast cells.  


The Rho GTPases are the sub-group of Ras super family and identified in all eukaryotes. The Rho GTPases effect different cellular signaling pathways involved in a number of diseases such as cancer, neurological and cardiovascular disorders. Members of Rho GTPases including RhoA, RhoC and Rac1 play a major role in regulation of apoptosis in different kind of stress conditions. Here we investigated the Rho GTPase activating protein 15 (ArhGAP15) gene knock-down effect on apoptosis induced by ethanol in bovine fibroblast cells. The bovine Fibroblast cells were treated and transfected with two different concentrations (50 and 100 nM) of ArhGAP15 siRNA for 48 h respectively. Both concentrations of siRNA were effective and the results of RT-PCR revealed an efficient knock-down of ArhGAP15 mRNA in fibroblast cells. Further, the normal cells exposed to a 100 mM ethanol concentration showed a reduction in cell viability and induced the ratio of apoptosis related Bax/Bcl-2 proteins compared with ArhGAP15 siRNA transfected ethanol treated cells. Ethanol also increased caspase-3 expression in normal fibroblast cells compared with transfected cells. The ArhGAP15 knock-down cells treated with ethanol decreased Bax/Bcl-2 ratio and lower caspase-3 protein levels in ArhGAP15 knocked-down cells. Our results suggest that apoptosis induced by ethanol involves the activation of Rho GTPase activating protein 15 and silencing of the said gene protects apoptosis. PMID:23625437

Ullah, Ikram; Lee, Hae Young; Kim, Min Jung; Shah, Shahid Ali; Badshah, Haroon; Kim, Tae Hyun; Chung, Hak-Jae; Yang, Byoung Chul; Kim, Myeong Ok



Models of globular proteins in aqueous solutions  

NASA Astrophysics Data System (ADS)

Protein crystallization is a continuing area of research. Currently, there is no universal theory for the conditions required to crystallize proteins. A better understanding of protein crystallization will be helpful in determining protein structure and preventing and treating certain diseases. In this thesis, we will extend the understanding of globular proteins in aqueous solutions by analyzing various models for protein interactions. Experiments have shown that the liquid-liquid phase separation curves for lysozyme in solution with salt depend on salt type and salt concentration. We analyze a simple square well model for this system whose well depth depends on salt type and salt concentration, to determine the phase coexistence surfaces from experimental data. The surfaces, calculated from a single Monte Carlo simulation and a simple scaling argument, are shown as a function of temperature, salt concentration and protein concentration for two typical salts. Urate Oxidase from Asperigillus flavus is a protein used for studying the effects of polymers on the crystallization of large proteins. Experiments have determined some aspects of the phase diagram. We use Monte Carlo techniques and perturbation theory to predict the phase diagram for a model of urate oxidase in solution with PEG. The model used includes an electrostatic interaction, van der Waals attraction, and a polymerinduced depletion interaction. The results agree quantitatively with experiments. Anisotropy plays a role in globular protein interactions, including the formation of hemoglobin fibers in sickle cell disease. Also, the solvent conditions have been shown to play a strong role in the phase behavior of some aqueous protein solutions. Each has previously been treated separately in theoretical studies. Here we propose and analyze a simple, combined model that treats both anisotropy and solvent effects. We find that this model qualitatively explains some phase behavior, including the existence of a lower critical point under certain conditions.

Wentzel, Nathaniel James


Cooperation of hTERT, SV40 T Antigen and Oncogenic Ras in Tumorigenesis: A Cell Transplantation Model Using Bovine Adrenocortical Cells1  

Microsoft Academic Search

Expression of TERT, the reverse transcriptase compo- nent of telomerase, is necessary to convert normal human cells to cancer cells. Despite this, ''telomeriza- tion'' by hTERT does not appear to alter the normal properties of cells. In a cell transplantation model in which bovine adrenocortical cells form vascularized tissue structures beneath the kidney capsule in scid mice, telomerization does not

Michael Thomas; Tetsuya Suwa; Lianqing Yang; Lifang Zhao; Christina L. Hawks; Peter J. Hornsby



Microscopic model of protein crystal growth.  


A microscopic, reversible model to study protein crystal nucleation and growth is presented. The probability of monomer attachment to the growing crystal was assumed to be proportional to the protein volume fraction and the orientational factor representing the anisotropy of protein molecules. The rate of detachment depended on the free energy of association of the given monomer in the lattice, as calculated from the buried surface area. The proposed algorithm allowed the simulation of the process of crystal growth from free monomers to complexes having 10(5) molecules, i.e. microcrystals with already formed faces. These simulations correctly reproduced the crystal morphology of the chosen model system--the tetragonal lysozyme crystal. We predicted the critical size, after which the growth rate rapidly increased to approximately 50 protein monomers. The major factors determining the protein crystallisation kinetics were the geometry of the protein molecules and the resulting number of kinetics traps on the growth pathway. PMID:11036969

Kierzek, A M; Pokarowski, P; Zielenkiewicz, P



Model study of protein unfolding by interfaces  

NASA Astrophysics Data System (ADS)

We study interface-induced protein unfolding on hydrophobic and polar interfaces by means of a two-dimensional lattice model and an exhaustive enumeration ground-state structure search, for a set of model proteins of length 20 residues. We compare the effects of the two types of interfaces, and search for criteria that influence the retention of a protein’s native-state structure upon adsorption. We find that the unfolding proceeds by a large, sudden loss of native contacts. The unfolding at polar interfaces exhibits similar behavior to that at hydrophobic interfaces but with a much weaker interface coupling strength. Further, we find that the resistance of proteins to unfolding in our model is positively correlated with the magnitude of the folding energy in the native-state structure, the thermal stability (or energy gap) for that structure, and the interface energy for native-state adsorption. We find these factors to be of roughly equal importance.

Chakarova, S. D.; Carlsson, A. E.



Analysis of the effect of the bovine adenosine triphosphate-binding cassette transporter G2 single nucleotide polymorphism Y581S on transcellular transport of veterinary drugs using new cell culture models.  


In commercial dairy production, the risk of drug residues and environmental pollutants in milk from ruminants has become an outstanding problem. One of the main determinants of active drug secretion into milk is the ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). It is located in several organs associated with drug absorption, metabolism, and excretion, and its expression is highly induced during lactation in the mammary gland of ruminants, mice, and humans. As a consequence, potential contamination of milk could expose suckling infants to xenotoxins. In cows, a SNP for this protein affecting quality and quantity of milk production has been described previously (Y581S). In this study, our main purpose was to determine whether this polymorphism has an effect on transcellular transport of veterinary drugs because this could alter substrate pharmacokinetics and milk residues. We stably expressed the wild-type bovine ABCG2 and the Y581S variant in Madin-Darby canine kidney epithelial cells (MDCKII) and MEF3.8 cell lines generating cell models in which the functionality of the bovine transporter could be addressed. Functional studies confirmed the greater functional activity in mitoxantrone accumulation assays for the Y581S variant with a greater relative V(MAX) value (P = 0.040) and showed for the first time that the Y581S variant presents greater transcellular transport of the model ABCG2 substrate nitrofurantoin (P = 0.024) and of 3 veterinary antibiotics, the fluoroquinolone agents enrofloxacin (P = 0.035), danofloxacin (P = 0.001), and difloxacin (P = 0.008), identified as new substrates of the bovine ABCG2. In addition, the inhibitory effect of the macrocyclic lactone ivermectin on the activity of wild-type bovine ABCG2 and the Y581S variant was also confirmed, showing a greater inhibitory potency on the wild-type protein at all the concentrations tested (5 ?M, P = 0.017; 10 ?M, P = 0.001; 25 ?M, P = 0.008; and 50 ?M, P = 0.003). Differential transport activity depending on the genotype together with the differential inhibition pattern might have clinical consequences, including changes in substrate pharmacokinetics (and subsequently pharmacodynamics) and more specifically, changes in secretion of ABCG2 substrates into milk, potentially implying important consequences to veterinary therapeutics. PMID:21821808

Real, R; González-Lobato, L; Baro, M F; Valbuena, S; de la Fuente, A; Prieto, J G; Alvarez, A I; Marques, M M; Merino, G



An augmented ribbon model of protein structure  

Microsoft Academic Search

In this paper, work on a new model,an augmented ribbon model, for describing a number of features of primary, secondary, and super-secondary protein structure in a qualitative but mathematically rigorous way is discussed. The structural features that can be treated by the model include connectivity, directionality, chirality, orientation, and proximity which, in many cases, are difficult to deal with using

Victor A. Nicholson; Gerald M. Maggiora



Computational model for protein unfolding simulation  

NASA Astrophysics Data System (ADS)

The protein folding problem is one of the fundamental and important questions in molecular biology. However, the all-atom molecular dynamics studies of protein folding and unfolding are still computationally expensive and severely limited by the time scale of simulation. In this paper, a simple and fast protein unfolding method is proposed based on the conformational stability analyses and structure modeling. In this method, two structure-based conditions are considered to identify the unstable regions of proteins during the unfolding processes. The protein unfolding trajectories are mimicked through iterative structure modeling according to conformational stability analyses. Two proteins, chymotrypsin inhibitor 2 (CI2) and ? -spectrin SH3 domain (SH3) were simulated by this method. Their unfolding pathways are consistent with the previous molecular dynamics simulations. Furthermore, the transition states of the two proteins were identified in unfolding processes and the theoretical ? values of these transition states showed significant correlations with the experimental data (the correlation coefficients are >0.8). The results indicate that this method is effective in studying protein unfolding. Moreover, we analyzed and discussed the influence of parameters on the unfolding simulation. This simple coarse-grained model may provide a general and fast approach for the mechanism studies of protein folding.

Tian, Xu-Hong; Zheng, Ye-Han; Jiao, Xiong; Liu, Cai-Xing; Chang, Shan



Modelling bovine babesiosis: a tool to simulate scenarios for pathogen spread and to test control measures for the disease.  


Tick-borne diseases are of increasing concern in many countries, particularly as a consequence of changes in land use and climate. Ticks are vectors of numerous pathogens (viruses, bacteria, protozoa) that can be harmful to humans and animals. In the context of animal health, bovine babesiosis poses a recurrent threat to cattle herds. In this study, we use a modeling approach to investigate the spread of babesiosis and evaluate control measures. A previously developed tick population dynamics model (here, Ixodes ricinus) is coupled with a pathogen spread model (here, the protozoan Babesia divergens), which describes pathogen spread in a dairy herd through the following processes: transmission, acquisition, transovarial transmission, transstadial persistence, and clearance of the pathogen. An assessment of the simulated B. divergens prevalence levels in ticks and cattle in the context of existing knowledge and data suggested that the model provides a realistic representation of pathogen spread. The model was then used to evaluate the influence of host density and the effect of acaricides on B. divergens prevalence in cattle. Increasing deer density results in an increase in prevalence in cattle whereas increasing cattle stocking rate results in a slight decrease. A potential increase in deer density would thus have an amplification effect on disease spread due to the increase in the number of infected ticks. Regular use of acaricides produces a reduction in pathogen prevalence in cattle. This model could be adapted to other tick-borne diseases. PMID:22341037

Hoch, Thierry; Goebel, Julien; Agoulon, Albert; Malandrin, Laurence



Stretching lattice models of protein folding  

Microsoft Academic Search

A new class of experiments that probe folding of individual protein domains uses mechanical stretching to cause the transition. We show how stretching forces can be incorporated in lattice models of folding. For fast folding proteins, the analysis suggests a complex relation between the force dependence and the reaction coordinate for folding. Several experimental groups recently have succeeded in moni-




Modelling protein crystallisation using morphological population balance models  

Microsoft Academic Search

Protein crystallisation is known to be affected by many factors and inherently difficult to control. Being able to model the crystal growth behaviour, especially at process scale for the population of particles in a crystalliser will no doubt greatly help the formulation and controlled manufacture of protein crystals. In this paper, a morphological population balance model for crystallisation of tetragonal

Jing J. Liu; Cai Y. Ma; Yang D. Hu; Xue Z. Wang



A cyclic AMP-responsive DNA-binding protein (CREB2) is a cellular transactivator of the bovine leukemia virus long terminal repeat.  


To gain insight into the cellular regulation of bovine leukemia virus (BLV) trans activation, a lambda-gt11 cDNA library was constructed with mRNA isolated from a BLV-induced tumor and the recombinant proteins were screened with an oligonucleotide corresponding to the tax activation-responsive element (TAR). Two clones (called TAR-binding protein) were isolated from 750,000 lambda-gt11 plaques. The binding specificity was confirmed by Southwestern (DNA-protein) and gel retardation assays. Nucleotide sequence analysis revealed that TAR-binding protein is very similar to the CREB2 protein. It contains a leucine zipper structure required for dimerization, a basic amino acid domain, and multiple potential phosphorylation sites. A vector expressing CREB2 was transfected into D17 osteosarcoma cells. In the absence of the tax transactivator, the CREB2 protein and the cyclic AMP-dependent protein kinase A activate the BLV long terminal repeat at a basal expression level: trans activation reached 10% of the values obtained in the presence of tax alone. These data demonstrate that CREB2 is a cellular factor able to induce BLV long terminal repeat expression in the absence of tax protein and could thus be involved in the early stages of viral infection. In addition, we observed that in vitro tax-induced trans activation can be activated or inhibited by CREB2 depending on the presence or absence of protein kinase A. These data suggest that the cyclic AMP pathway plays a role in the regulation of viral expression in BLV-infected animals. PMID:1309910

Willems, L; Kettmann, R; Chen, G; Portetelle, D; Burny, A; Derse, D



Comparative analysis of vector biodistribution, persistence and gene expression following intravenous delivery of bovine, porcine and human adenoviral vectors in a mouse model.  


Nonhuman adenoviruses including bovine adenovirus serotype 3 (BAd3) and porcine adenovirus serotype 3 (PAd3) can circumvent pre-existing immunity against human adenovirus serotype 5 (HAd5) and are being developed as alternative vectors for gene delivery. To assess the usefulness of these vectors for in vivo gene delivery, we compared biodistribution, persistence, state of vector genome, and transgene and vector gene expression by replication-defective BAd3 and PAd3 vectors with those of HAd5 vector in a FVB/n mouse model following intravenous inoculation. BAd3 vector efficiently transduced the heart, kidney and lung in addition to the liver and spleen and persisted for a longer duration compared to PAd3 or HAd5 vectors. Biodistribution of PAd3 vector was comparable to that of HAd5 vector but showed more rapid vector clearance. Only linear episomal forms of BAd3, PAd3, and HAd5 vector genomes were detected. All three vectors efficiently expressed the green fluorescent protein (GFP) transgene proportionate to the vector genome copy number in various tissues. Furthermore, leaky expression of vector genes, both the early (E4) and the late (hexon) was observed in all three vectors and gradually declined with time. These results suggest that BAd3 and PAd3 vectors could serve as an alternative or supplement to HAd5 for gene delivery applications. PMID:19211122

Sharma, Anurag; Bangari, Dinesh S; Tandon, Manish; Pandey, Aseem; HogenEsch, Harm; Mittal, Suresh K