Science.gov

Sample records for model protein bovine

  1. Protein Crystal Bovine Insulin

    NASA Technical Reports Server (NTRS)

    1991-01-01

    The comparison of protein crystal, Bovine Insulin space-grown (left) and earth-grown (right). Facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  2. In vitro digestibility and immunoreactivity of bovine milk proteins.

    PubMed

    Do, Andrew B; Williams, Kristina; Toomer, Ondulla T

    2016-01-01

    Current models of digestibility solely utilize pepsin stability to assess the safety of allergenic food proteins. However, in vivo complete protein digestion requires acid denaturation and pepsin, trypsin, and/or chymotrypsin cleavage. This study aimed to identify the immunoreactivity and allergenicity of stable bovine milk proteins, using an improved digestibility model to simulate physiological gastric and intestinal conditions in vitro. Gel electrophoresis and immunoblot analysis were used to determine protein stability and immunoreactivity, respectively. Immunoreactivity of bovine milk proteins, β-lactoglobulin (β-LG) and casein (CN) was greatly diminished with gastric simulation (0-60 min), but some proteins were stable and immunoreactive with simulated intestinal digestive conditions (0-60 min). This study demonstrates the need for improved digestibility models for more accurate assessment of the behavior of food allergens in vivo. PMID:26213013

  3. Bovine model of respiratory syncytial virus infection.

    PubMed

    Taylor, Geraldine

    2013-01-01

    Bovine respiratory syncytial virus (BRSV), which is an important cause of respiratory disease in young calves, is genetically and antigenically closely related to human (H)RSV. The epidemiology and pathogenesis of infection with these viruses are similar. The viruses are host-specific and infection produces a spectrum of disease ranging from subclinical to severe bronchiolitis and pneumonia, with the peak incidence of severe disease in individuals less than 6 months of age. BRSV infection in calves reproduces many of the clinical signs associated with HRSV in infants, including fever, rhinorrhoea, coughing, harsh breath sounds and rapid breathing. Although BRSV vaccines have been commercially available for decades, there is a need for greater efficacy. The development of effective BRSV and HRSV vaccines face similar challenges, such as the need to vaccinate at an early age in the presence of maternal antibodies, the failure of natural infection to prevent reinfection, and a history of vaccine-augmented disease. Neutralising monoclonal antibodies (mAbs) to the fusion (F) protein of HRSV, which can protect infants from severe HRSV disease, recognise the F protein of BRSV, and vice versa. Furthermore, bovine and human CD8(+) T-cells, which are known to be important in recovery from RSV infection, recognise similar proteins that are conserved between HRSV and BRSV. Therefore, not only can the bovine model of RSV be used to evaluate vaccine concepts, it can also be used as part of the preclinical assessment of certain HRSV candidate vaccines. PMID:24362697

  4. Models of bovine babesiosis including juvenile cattle.

    PubMed

    Saad-Roy, C M; Shuai, Zhisheng; van den Driessche, P

    2015-03-01

    Bovine Babesiosis in cattle is caused by the transmission of protozoa of Babesia spp. by ticks as vectors. Juvenile cattle (<9 months of age) have resistance to Bovine Babesiosis, rarely show symptoms, and acquire immunity upon recovery. Susceptibility to the disease varies between breeds of cattle. Models of the dynamics of Bovine Babesiosis transmitted by the cattle tick that include these factors are formulated as systems of ordinary differential equations. Basic reproduction numbers are calculated, and it is proved that if these numbers are below the threshold value of one, then Bovine Babesiosis dies out. However, above the threshold number of one, the disease may approach an endemic state. In this case, control measures are suggested by determining target reproduction numbers. The percentage of a particular population (for example, the adult bovine population) needed to be controlled to eradicate the disease is evaluated numerically using Columbia data from the literature. PMID:25715822

  5. Sequence and structural implications of a bovine corneal keratan sulfate proteoglycan core protein. Protein 37B represents bovine lumican and proteins 37A and 25 are unique

    NASA Technical Reports Server (NTRS)

    Funderburgh, J. L.; Funderburgh, M. L.; Brown, S. J.; Vergnes, J. P.; Hassell, J. R.; Mann, M. M.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Amino acid sequence from tryptic peptides of three different bovine corneal keratan sulfate proteoglycan (KSPG) core proteins (designated 37A, 37B, and 25) showed similarities to the sequence of a chicken KSPG core protein lumican. Bovine lumican cDNA was isolated from a bovine corneal expression library by screening with chicken lumican cDNA. The bovine cDNA codes for a 342-amino acid protein, M(r) 38,712, containing amino acid sequences identified in the 37B KSPG core protein. The bovine lumican is 68% identical to chicken lumican, with an 83% identity excluding the N-terminal 40 amino acids. Location of 6 cysteine and 4 consensus N-glycosylation sites in the bovine sequence were identical to those in chicken lumican. Bovine lumican had about 50% identity to bovine fibromodulin and 20% identity to bovine decorin and biglycan. About two-thirds of the lumican protein consists of a series of 10 amino acid leucine-rich repeats that occur in regions of calculated high beta-hydrophobic moment, suggesting that the leucine-rich repeats contribute to beta-sheet formation in these proteins. Sequences obtained from 37A and 25 core proteins were absent in bovine lumican, thus predicting a unique primary structure and separate mRNA for each of the three bovine KSPG core proteins.

  6. Electrostatic Properties of Phase-Separating Bovine Lens Proteins.

    NASA Astrophysics Data System (ADS)

    Kovach, Ian Shand

    1992-01-01

    Acid-base titration experiments were conducted to determine the net charge on four bovine lens proteins: gamma_{rm II}, gamma_{rm IIIa}, gamma_{rm IIIb}, gamma_{rm IV}. In addition to the dependence of protein charge on pH, the effects of ionic strength and identity on protein charge were investigated. Titration curves were obtained in 0.1 M KCl, 0.1 M NaCl, 0.1 M NaBr, and 0.01 M KCl. Three theoretical methods, the Linderstrom-Lang model, a modification of the Linderstrom-Lang model which does not linearize the Poisson-Boltzmann equation, and the Kirkwood-Tanford model were used to correct intrinsic proton binding energies in an effort to predict protein charge as a function of solution conditions. The electrostatic interaction energy between protein and solvent was determined using the Linderstrom-Lang model. The acid-base titration curves of the four proteins were found to be very similar, with the exception of basic range titration of gamma_{rm IIIb} which exhibited slightly greater negative charge near pH 10. No evidence of protein charge dependence on the identity of electrolyte was observed. A dependence of protein charge on ionic strength was observed. Decreasing ionic strength was found to correlate with decreasing magnitude of net protein charge at a given pH. The theoretical Linderstrom-Lang titration curves were found to be in fair agreement with the experimental results over a pH range of 5 to 11, and in poor agreement in the pH extremes. The nonlinearized variation of the Linderstrom-Lang model was found to be in good agreement with the titration curves from pH 2 to pH 11. The theoretical Kirkwood-Tanford titration curves were determined for gamma_{rm II} and also found to be in excellent agreement with experimental results. The precise location of protein charge on gamma_{rm II}, as a function of pH, was determined using the Kirkwood-Tanford model. (Copies available exclusively from MIT Libraries, Rm. 14-0551, Cambridge, MA 02139-4307. Ph. 617-253-5668; Fax 617-253-1690.).

  7. Optimization of hydrolysis conditions for bovine plasma protein using response surface methodology.

    PubMed

    Seo, Hyun-Woo; Jung, Eun-Young; Go, Gwang-Woong; Kim, Gap-Don; Joo, Seon-Tea; Yang, Han-Sul

    2015-10-15

    The purpose of this study was to establish optimal conditions for the hydrolysis of bovine plasma protein. Response surface methodology was used to model and optimize responses [degree of hydrolysis (DH), 2,2-diphenyl-1-picrydrazyl (DPPH) radical-scavenging activity and Fe(2+)-chelating activity]. Hydrolysis conditions, such as hydrolysis temperature (46.6-63.4 °C), hydrolysis time (98-502 min), and hydrolysis pH (6.32-9.68) were selected as the main processing conditions in the hydrolysis of bovine plasma protein. Optimal conditions for maximum DH (%), DPPH radical-scavenging activity (%) and Fe(2+)-chelating activity (%) of the hydrolyzed bovine plasma protein, were respectively established. We discovered the following three conditions for optimal hydrolysis of bovine plasma: pH of 7.82-8.32, temperature of 54.1 °C, and time of 338.4-398.4 min. We consequently succeeded in hydrolyzing bovine plasma protein under these conditions and confirmed the various desirable properties of optimal hydrolysis. PMID:25952847

  8. Bovine Immunoglobulin/Protein Isolate Binds Pro-Inflammatory Bacterial Compounds and Prevents Immune Activation in an Intestinal Co-Culture Model

    PubMed Central

    Detzel, Christopher J.; Horgan, Alan; Henderson, Abigail L.; Petschow, Bryon W.; Warner, Christopher D.; Maas, Kenneth J.; Weaver, Eric M.

    2015-01-01

    Intestinal barrier dysfunction is associated with chronic gastrointestinal tract inflammation and diseases such as IBD and IBS. Serum-derived bovine immunoglobulin/protein isolate (SBI) is a specially formulated protein preparation (>90%) for oral administration. The composition of SBI is greater than 60% immunoglobulin including contributions from IgG, IgA, and IgM. Immunoglobulin within the lumen of the gut has been recognized to have anti-inflammatory properties and is involved in maintaining gut homeostasis. The binding of common intestinal antigens (LPS and Lipid A) and the ligand Pam3CSK4, by IgG, IgA, and IgM in SBI was shown using a modified ELISA technique. Each of these antigens stimulated IL-8 and TNF-? cytokine production by THP-1 monocytes. Immune exclusion occurred as SBI (?50 mg/mL) bound free antigen in a dose dependent manner that inhibited cytokine production by THP-1 monocytes in response to 10 ng/mL LPS or 200 ng/mL Lipid A. Conversely, Pam3CSK4 stimulation of THP-1 monocytes was unaffected by SBI/antigen binding. A co-culture model of the intestinal epithelium consisted of a C2BBe1 monolayer separating an apical compartment from a basal compartment containing THP-1 monocytes. The C2BBe1 monolayer was permeabilized with dimethyl palmitoyl ammonio propanesulfonate (PPS) to simulate a damaged epithelial barrier. Results indicate that Pam3CSK4 was able to translocate across the PPS-damaged C2BBe1 monolayer. However, binding of Pam3CSK4 by immunoglobulins in SBI prevented Pam3CSK4 translocation across the damaged C2BBe1 barrier. These results demonstrated steric exclusion of antigen by SBI which prevented apical to basal translocation of antigen due to changes in the physical properties of Pam3CSK4, most likely as a result of immunoglobulin binding. This study demonstrates that immunoglobulins in SBI can reduce antigen-associated inflammation through immune and steric exclusion mechanisms and furthers the mechanistic understanding of how SBI might improve immune status and reduce inflammation in various intestinal disease states. PMID:25830826

  9. Bovine immunoglobulin protein isolates for the nutritional management of enteropathy

    PubMed Central

    Petschow, Bryon W; Blikslager, Anthony T; Weaver, Eric M; Campbell, Joy M; Polo, Javier; Shaw, Audrey L; Burnett, Bruce P; Klein, Gerald L; Rhoads, J Marc

    2014-01-01

    The gastrointestinal tract is responsible for a multitude of digestive and immune functions which depend upon the balanced interaction of the intestinal microbiota, diet, gut barrier function, and mucosal immune response. Disruptions in one or more of these factors can lead to intestinal disorders or enteropathies which are characterized by intestinal inflammation, increased gut permeability, and reduced capacity to absorb nutrients. Enteropathy is frequently associated with human immunodeficiency virus (HIV) infection, inflammatory bowel disease, autoimmune enteropathy, radiation enteritis, and irritable bowel syndrome (IBS), where pathologic changes in the intestinal tract lead to abdominal discomfort, bloating, abnormal bowel function (e.g., diarrhea, urgency, constipation and malabsorption). Unfortunately, effective therapies for the management of enteropathy and restoring intestinal health are still not available. An accumulating body of preclinical studies has demonstrated that oral administration of plasma- or serum-derived protein concentrates containing high levels of immunoglobulins can improve weight, normalize gut barrier function, and reduce the severity of enteropathy in animal models. Recent studies in humans, using serum-derived bovine immunoglobulin/protein isolate, demonstrate that such protein preparations are safe and improve symptoms, nutritional status, and various biomarkers associated with enteropathy. Benefits have been shown in patients with HIV infection or diarrhea-predominant IBS. This review summarizes preclinical and clinical studies with plasma/serum protein concentrates and describes the effects on host nutrition, intestinal function, and markers of intestinal inflammation. It supports the concept that immunoglobulin-containing protein preparations may offer a new strategy for restoring functional homeostasis in the intestinal tract of patients with enteropathy. PMID:25206275

  10. Bovine immunoglobulin protein isolates for the nutritional management of enteropathy.

    PubMed

    Petschow, Bryon W; Blikslager, Anthony T; Weaver, Eric M; Campbell, Joy M; Polo, Javier; Shaw, Audrey L; Burnett, Bruce P; Klein, Gerald L; Rhoads, J Marc

    2014-09-01

    The gastrointestinal tract is responsible for a multitude of digestive and immune functions which depend upon the balanced interaction of the intestinal microbiota, diet, gut barrier function, and mucosal immune response. Disruptions in one or more of these factors can lead to intestinal disorders or enteropathies which are characterized by intestinal inflammation, increased gut permeability, and reduced capacity to absorb nutrients. Enteropathy is frequently associated with human immunodeficiency virus (HIV) infection, inflammatory bowel disease, autoimmune enteropathy, radiation enteritis, and irritable bowel syndrome (IBS), where pathologic changes in the intestinal tract lead to abdominal discomfort, bloating, abnormal bowel function (e.g., diarrhea, urgency, constipation and malabsorption). Unfortunately, effective therapies for the management of enteropathy and restoring intestinal health are still not available. An accumulating body of preclinical studies has demonstrated that oral administration of plasma- or serum-derived protein concentrates containing high levels of immunoglobulins can improve weight, normalize gut barrier function, and reduce the severity of enteropathy in animal models. Recent studies in humans, using serum-derived bovine immunoglobulin/protein isolate, demonstrate that such protein preparations are safe and improve symptoms, nutritional status, and various biomarkers associated with enteropathy. Benefits have been shown in patients with HIV infection or diarrhea-predominant IBS. This review summarizes preclinical and clinical studies with plasma/serum protein concentrates and describes the effects on host nutrition, intestinal function, and markers of intestinal inflammation. It supports the concept that immunoglobulin-containing protein preparations may offer a new strategy for restoring functional homeostasis in the intestinal tract of patients with enteropathy. PMID:25206275

  11. Cellular Requirements for Bovine Immunodeficiency Virus Vif-Mediated Inactivation of Bovine APOBEC3 Proteins

    PubMed Central

    Zhang, Wenyan; Wang, Hong; Li, Zhaolong; Liu, Xin; Liu, Guanchen; Harris, Reuben S.

    2014-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) viral infectivity factor (Vif) form a CRL5 E3 ubiquitin ligase complex to suppress virus restriction by host APOBEC3 (A3) proteins. The primate lentiviral Vif complex is composed of the unique cofactor core binding factor ? (CBF-?) and canonical ligase components Cullin 5 (CUL5), Elongin B/C (ELOB/C), and RBX2. However, the mechanism by which the Vif protein of the related lentivirus bovine immunodeficiency virus (BIV) overcomes its host A3 proteins is less clear. In this study, we show that BIV Vif interacts with Cullin 2 (CUL2), ELOB/C, and RBX1, but not with CBF-? or CUL5, to form a CRL2 E3 ubiquitin ligase and degrade the restrictive bovine A3 proteins (A3Z2Z3 and A3Z3). RNA interference-mediated knockdown of ELOB or CUL2 inhibited BIV Vif-mediated degradation of these A3 proteins, whereas knockdown of CUL5 or CBF-? did not. BIV Vif with mutations in the BC box (Vif SLQ-AAA) or putative VHL box (Vif YI-AA), which cannot interact with ELOB/C or CUL2, respectively, lost the ability to counteract bovine A3 proteins. Moreover, CUL2 and UBE2M dominant negative mutants competitively inhibited the BIV Vif-mediated degradation mechanism. Thus, although the general strategy for inhibiting A3 proteins is conserved between HIV-1/SIV and BIV, the precise mechanisms can differ substantially, with only the HIV-1/SIV Vif proteins requiring CBF-? as a cofactor, HIV-1/SIV Vif using CUL5-RBX2, and BIV Vif using CUL2-RBX1. IMPORTANCE Primate lentivirus HIV-1 and SIV Vif proteins form a ubiquitin ligase complex to target host antiviral APOBEC3 proteins for degradation. However, the mechanism by which the nonprimate lentivirus BIV Vif inhibits bovine APOBEC3 proteins is unclear. In the present study, we determined the mechanism for BIV Vif-mediated degradation of bovine APOBEC3 proteins and found that it differs from the mechanism of HIV-1/SIV Vif by being CBF-? independent and requiring different ubiquitin ligase scaffolding proteins (CUL2-RBX1 instead of CUL5-RBX2). BIV Vif is the only known retroviral protein that can interact with CUL2. This information broadens our understanding of the distinct mechanisms by which the Vif proteins of different lentiviruses facilitate viral infection. This novel mechanism for assembly of the BIV Vif-APOBEC3 ubiquitin ligase complex advances our understanding of viral hijacking of host E3 ubiquitin ligases and illustrates the evolutionary flexibility of lentiviruses. PMID:25142583

  12. Antigen-free bovine cancellous bone loaded with recombinant human bone morphogenetic protein-2 for the repair of tibial bone defects in goat model.

    PubMed

    Li, Donghai; Deng, Liqing; Yang, Zhouyuan; Xie, Xiaowei; Kang, Pengde; Tan, Zhen

    2016-04-01

    Antigen-free bovine cancellous bone has good performances of porous network structures and mechanics with antigen extracted. To develop a bioactive scaffold for enhancing bone repair and evaluate its biological property, rhBMP-2 loaded with antigen-free bovine cancellous bone was used to treat tibial bone defect. Twenty-four healthy adult goats were chosen to establish goat defects model and randomly divided into four groups. The goats were treated with rhBMP-2/antigen-free bovine cancellous bone scaffolds (group A), autogenous cancellous bone graft (group B), porous tricalciumphosphate scaffolds (group C) and nothing (group D). Animals were evaluated with radiological and histological methods at 4, 8 and 12 weeks after surgery. The gray value of radiographs was used to evaluate the healing of the defects, which revealed that the group A had a better outcome of defect healing compared with group C at 4, 8 and 12 weeks, respectively (p < 0.05), while the difference between groups A and B was without significance at each time (p > 0.05). The newly formed bone area was calculated from histological sections, and the results indicated that the amount of new bone in group A increased significantly compared with that in group C (p < 0.05) but was similar to that in group B (p > 0.05) at 4, 8 and 12 weeks, respectively. In addition, the expression of collagen I and vascular endothelial growth factor by real-time polymerase chain reaction at 12 weeks in group A was significantly higher than that in group C (p = 0.034, p = 0.032, respectively), but no significant differences were found when compared with that in group B (p = 0.36, p = 0.54, respectively). At the same time, group C presented better results than group D on bone defects healing. Therefore, the composites of antigen-free bovine cancellous bone loaded with rhBMP-2 have a good osteoinductive activity and capacity to promote the repair of bone defects. PMID:26801475

  13. Immunorelevant proteins for the diagnosis of bovine staphylococcal mastitis.

    PubMed

    Fabres-Klein, M H; Klein, R C; De Paula, S O; Ribon, A O B

    2013-07-01

    Staphylococcus aureus is a leading cause of bovine mastitis, a condition in which the udder of the cow is inflamed, reducing the quality and quantity of milk produced. Staphylococcal mastitis is a common infection that can develop into a chronic form. The segregation of infected animals is an important preventive practice but relies on an effective diagnostic method. For this purpose, we constructed a genomic library of S. aureus, and a screening step was conducted with antiserum produced using the total protein extract of the pathogen. The nucleotide sequences of the immunoselected clones were aligned with the genome of bovine S. aureus RF122, which enabled the identification of 65 different loci, including proteins related to metabolism, adhesion and cell wall production, toxins, regulatory proteins, and hypothetical proteins. The subcellular location of the immunoreactive polypeptides was also determined. Fifty-two percent were cytoplasmic, 34 % were located in areas exposed to the host's immune system, and for 14 %, the location could not be determined. In silico analysis of the presence of these proteins in mastitis pathogens showed that Fib, ClfA, and the hypothetical protein SAB0166 were the only proteins specific for S. aureus. Therefore, these proteins are promising candidates for the serodiagnosis of staphylococcal mastitis. PMID:23386318

  14. Envelope proteins of bovine herpesvirus 1: immunological and biochemical studies

    SciTech Connect

    Rodriguez Roque, L.L.

    1986-01-01

    The authors studied immunological and biochemical properties of the bovid herpesvirus 1 (BHV-1) envelope proteins in order to understand the pathogenesis of BHV-1 infection and to provide basic information for the production of effective subunit vaccines against BHV-1. Ten glycoproteins MW 180, 150, 130, 115, 97, 77, 74, 64, 55, and 45 kilodaltons (K), and a single non-glycosylated 108 K protein were quantitatively removed from purified BHV-1 virions by detergent treatment. These glycoproteins were present on the virion envelope and on the surface of BHV-1 infected cells. The quantitative removal from virions by treatment with nonionic detergents and their presence on the surface of infected cells indicate that 180/97, 150/77, and 130/74/55 K are major components of the BHV-1 envelope and are also the targets of virus neutralizing humoral immune response. Envelope glycoproteins of herpes simplex type 1 (HSV-1) bind immunoglobulin by the Fc end and it is suggested this may increase pathogenicity of this virus. They searched for a similar function in BVH-1 by measuring the ability of BHV-1 infected cells and viral envelope proteins to bind radiolabelled rabbit and bovine IgG. Binding activity for rabbit IgG or bovine IgG-Fc could not be demonstrated by BHV-1 infected MDBK cells, whereas, MDBK cells infected with HSV-1 bound rabbit IgG and bovine IgG-Fc. None of the three major envelope proteins of BHV-1 bound to rabbit or bovine IgG. The results of this study indicate that BHV-1, unlike some other herpesviruses, lack Fc binding activity.

  15. Crystallization of Proteins from Crude Bovine Rod Outer Segments?

    PubMed Central

    Baker, Bo Y.; Gulati, Sahil; Shi, Wuxian; Wang, Benlian; Stewart, Phoebe L.; Palczewski, Krzysztof

    2015-01-01

    Obtaining protein crystals suitable for X-ray diffraction studies comprises the greatest challenge in the determination of protein crystal structures, especially for membrane proteins and protein complexes. Although high purity has been broadly accepted as one of the most significant requirements for protein crystallization, a recent study of the Escherichia coli proteome showed that many proteins have an inherent propensity to crystallize and do not require a highly homogeneous sample (Totir et al., 2012). As exemplified by RPE65 (Kiser, Golczak, Lodowski, Chance, & Palczewski, 2009), there also are cases of mammalian proteins crystallized from less purified samples. To test whether this phenomenon can be applied more broadly to the study of proteins from higher organisms, we investigated the protein crystallization profile of bovine rod outer segment (ROS) crude extracts. Interestingly, multiple protein crystals readily formed from such extracts, some of them diffracting to high resolution that allowed structural determination. A total of seven proteins were crystallized, one of which was a membrane protein. Successful crystallization of proteins from heterogeneous ROS extracts demonstrates that many mammalian proteins also have an intrinsic propensity to crystallize from complex biological mixtures. By providing an alternative approach to heterologous expression to achieve crystallization, this strategy could be useful for proteins and complexes that are difficult to purify or obtain by recombinant techniques. PMID:25950977

  16. Immunocytochemical localization of secretory proteins in bovine pancreatic exocrine cells.

    PubMed

    Kraehenbuhl, J P; Racine, L; Jamieson, J D

    1977-02-01

    The bovine exocrine pancreatic cell produces a variety of enzymes and proenzymes for export. Biochemical studies by Greene L.J., C.H. Hirs, and G.E. Palade (J. Biol. Chem. 1963. 238:2054) have shown that the mass proportions of several of these proteins in resting pancreatic juice and zymogen granule fractions are identical. In this study we have used immunocytochemical techniques at the electron microscope level to determine whether regional differences exist in the bovine gland with regard to production of individual secretory proteins and whether specialization of product handling occurs at the subcellular level. The technique used is a modification of one previously reported (McLean, J.D., and S.J. Singer. 1970. Proc. Natl. Acad. Sci U.S.A. 69:1771) in which immunocytochemical reagents are applied to thin sections of bovine serum albumin-imbedded tissue and zymogen granule fractions. A double antibody technique was used in which the first step consisted of rabbit F(ab')2 antibovine secretory protein and the detection step consisted of sheep (F(ab')2 antirabbit F(ab')2 conjugated to ferritin. The results showed that all exocrine cells in the gland, and all zymogen granules and Golgi cisternae in each cell, were qualitatively alike with regard to their content of secretory proteins examined (trypsinogen, chymotrypsinogen A, carboxypeptidase A, RNase, and DNase). The data suggest that these secretory proteins are transported through the cisternae of the Golgi complex where they are intermixed before copackaging in zymogen granules; passage through the Golgi complex is apparently obligatory for these (and likely all) secretory proteins, and is independent of extent of glycosylation, e.g., trypsinogen, a nonglycoprotein vs. DNase, a glycoprotein. PMID:319100

  17. Stratification of chondroitin sulfate binding sites in 3D-model of bovine testicular hyaluronidase and effective size of glycosaminoglycan coat of the modified protein.

    PubMed

    Maksimenko, A V; Turashev, A D; Beabealashvili, R S

    2015-03-01

    A 3D-model of bovine testicular hyaluronidase (BTH) was constructed based on established tertiary structure of human hyaluronidase Hyal1 using a molecular homological modeling method in silico. The analysis of the BTH 3D-model demonstrated lysine residue stratification during enzyme modification. The 3D-model of chondroitin sulfate (CHS)-modified hyaluronidase (BTH-CHS) was obtained by modeling covalent binding of lysine residues with benzoquinone-activated CHS. The degree of enzyme modification and the length of CHS chains were varied during 3D modeling. The importance of deep BTH modification degree for the formation of active and stable enzyme derivatives was shown, as determined earlier experimentally. The effective size of the CHS coat for productive BTH modification was confirmed. It is theoretically achieved at the increase in molecular mass of BTH-CHS derivative to approximately 140-180kDa and can be practically obtained, according to experimental data, using CHS of different molecular mass (30-50 as well as 120-140kDa). PMID:25761683

  18. Structural and functional characteristics of bovine milk protein glycosylation.

    PubMed

    O'Riordan, Noelle; Kane, Marian; Joshi, Lokesh; Hickey, Rita M

    2014-03-01

    Most secreted and cell membrane proteins in mammals are glycosylated. Many of these glycoproteins are also prevalent in milk and play key roles in the biomodulatory properties of milk and ultimately in determining milk's nutritional quality. Although a significant amount of information exists on the types and roles of free oligosaccharides in milk, very little is known about the glycans associated with milk glycoproteins, in particular, the biological properties that are linked to their presence. The main glycoproteins found in bovine milk are lactoferrin, the immunoglobulins, glycomacropeptide, a glycopeptide derived from ?-casein, and the glycoproteins of the milk fat globule membrane. Here, we review the glycoproteins present in bovine milk, the information currently available on their glycosylation and the biological significance of their oligosaccharide chains. PMID:24398766

  19. Bone morphogenetic protein 4 and retinoic acid trigger bovine VASA homolog expression in differentiating bovine induced pluripotent stem cells.

    PubMed

    Malaver-Ortega, Luis F; Sumer, Huseyin; Jain, Kanika; Verma, Paul J

    2016-02-01

    Primordial germ cells (PGCs) are the earliest identifiable and completely committed progenitors of female and male gametes. They are obvious targets for genome editing because they assure the transmission of desirable or introduced traits to future generations. PGCs are established at the earliest stages of embryo development and are difficult to propagate in vitro-two characteristics that pose a problem for their practical application. One alternative method to enrich for PGCs in vitro is to differentiate them from pluripotent stem cells derived from adult tissues. Here, we establish a reporter system for germ cell identification in bovine pluripotent stem cells based on green fluorescent protein expression driven by the minimal essential promoter of the bovine Vasa homolog (BVH) gene, whose regulatory elements were identified by orthologous modelling of regulatory units. We then evaluated the potential of bovine induced pluripotent stem cell (biPSC) lines carrying the reporter construct to differentiate toward the germ cell lineage. Our results showed that biPSCs undergo differentiation as embryoid bodies, and a fraction of the differentiating cells expressed BVH. The rate of differentiation towards BVH-positive cells increased up to tenfold in the presence of bone morphogenetic protein 4 or retinoic acid. Finally, we determined that the expression of key PGC genes, such as BVH or SOX2, can be modified by pre-differentiation cell culture conditions, although this increase is not necessarily mirrored by an increase in the rate of differentiation. Mol. Reprod. Dev. 83: 149-161, 2016. 2015 Wiley Periodicals, Inc. PMID:26660942

  20. The bovine patella as a model of early osteoarthritis

    PubMed Central

    Hargrave-Thomas, E J; Thambyah, A; McGlashan, S R; Broom, N D

    2013-01-01

    The bovine patella model has been used extensively for studying important structure–function aspects of articular cartilage, including its degeneration. However, the degeneration seen in this model has, to our knowledge, never been adequately compared with human osteoarthritis (OA). In this study, bovine patellae displaying normal to severely degenerate states were compared with human tissue displaying intact cartilage to severe OA. Comparisons of normal and OA features were made with histological scoring, morphometric measurements, and qualitative observations. Differential interference contrast microscopy was used to image early OA changes in the articular cartilage matrix and to investigate whether this method provided comparable quality of visualisation of key structural features with standard histology. The intact bovine cartilage was found to be similar to healthy human cartilage and the degenerate bovine cartilage resembled the human OA tissues with regard to structural disruption, cellularity changes, and staining loss. The extent of degeneration in the bovine tissues matched the mild to moderate range of human OA tissues; however, no bovine samples exhibited late-stage OA. Additionally, in both bovine and human tissues, cartilage degeneration was accompanied by calcified cartilage thickening, tidemark duplication, and the advancement of the cement line by protrusions of bony spicules into the calcified cartilage. This comparison of degeneration in the bovine and human tissues suggests a common pathway for the progression of OA and thus the bovine patella is proposed to be an appropriate model for investigating the structural changes associated with early OA. PMID:24111904

  1. A bovine babesiosis model with dispersion.

    PubMed

    Friedman, Avner; Yakubu, Abdul-Aziz

    2014-01-01

    Bovine Babesiosis (BB) is a tick borne parasitic disease with worldwide over 1.3 billion bovines at potential risk of being infected. The disease, also called tick fever, causes significant mortality from infection by the protozoa upon exposure to infected ticks. An important factor in the spread of the disease is the dispersion or migration of cattle as well as ticks. In this paper, we study the effect of this factor. We introduce a number, [Formula: see text], a "proliferation index," which plays the same role as the basic reproduction number [Formula: see text] with respect to the stability/instability of the disease-free equilibrium, and observe that [Formula: see text] decreases as the dispersion coefficients increase. We prove, mathematically, that if [Formula: see text] then the tick fever will remain endemic. We also consider the case where the birth rate of ticks undergoes seasonal oscillations. Based on data from Colombia, South Africa, and Brazil, we use the model to determine the effectiveness of several intervention schemes to control the progression of BB. PMID:24257900

  2. Photo selective protein immobilization using bovine serum albumin

    NASA Astrophysics Data System (ADS)

    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

    2012-11-01

    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.

  3. Short communication: interaction of bovine milk protein with chlorpyrifos.

    PubMed

    Lv, Ying; Li, Xuefen; Wang, Zongyi; Zheng, Han; Zhang, Qi; Huo, Ran; Chen, Xiangning; Han, Tao

    2014-01-01

    Dairy products are considered as nutrient-dense foods and consumed by many people in western countries, as well as an increasing number of Asian people. Excessive and frequent application of pesticides on vegetables and fruits leads to a potential health hazard to consumers. The organophosphate insecticide chlorpyrifos has been reported to bind with human and bovine serum albumin. Thus, it is necessary to explore the interaction between food protein and chlorpyrifos. In this study, equilibrium dialysis and fluorescence spectra were used to demonstrate binding of milk proteins to chlorpyrifos. The amount of milk protein bound was 0.030.01mg/g. Moreover, the milk protein-chlorpyrifos complexes were stable at pH 3.5to 9.5 and ion concentrations from 0.1 to 1.0M. The amount of chlorpyrifos bound to milk proteins decreased to 50% after being in vitro digested by pepsin and trypsin. The results showed that the interaction between food proteins and the pesticide might partially remove the insecticide and reduce the concentration of pesticide absorbed into the blood and, thus, alleviate the corresponding toxicity. PMID:24534502

  4. Purification and characterization of bovine mannan-binding protein.

    PubMed

    Holmskov, U; Holt, P; Reid, K B; Willis, A C; Teisner, B; Jensenius, J C

    1993-04-01

    Bovine mannan-binding protein (bMBP) was observed in serum by its Ca(2+)-dependent binding to mannan and by an M(r) of 28 kDa under reducing conditions on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The lectin was isolated by precipitation with polyethyleneglycol (PEG), affinity chromatography on mannan-Sepharose eluted with EDTA, and absorption on Sepharose 4B rabbit anti-bovine Ig to remove anti-mannan antibodies. Fractions containing the lectin were reapplied to mannan-Sepharose and eluted first with N-acetyl-D-glucosamine (GlcNAc) to remove conglutinin, and then with mannose to elute the 28 kDa lectin. Further purification was achieved by ion-exchange chromatography on Mono-Q and by mannose-gradient elution from a mannan-Sepharose column. SDS-PAGE of the purified lectin showed three high molecular weight bands under non-reducing conditions. The reduced protein gave a single band of 28 kDa. On gel permeation chromatography under non-dissociating conditions, the protein emerged at a volume corresponding to M(r) approximately 750 kDa. Amino acid analysis showed the presence of hydroxyproline and hydroxylysine, and a high glycine content (17.7%), suggesting the presence of a collagen-like structure. This was supported by the susceptibility of the protein to collagenase digestion. The N-terminal 26 amino acids showed 62% identity with human MBP, when three gaps were allowed in the alignment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8490241

  5. ?-Cyclodextrin preventing protein aggregation in foam fractionation of bovine serum albumin.

    PubMed

    Li, Rui; Wu, Zhaoliang; Wang, Yanji; Ding, Linlin; Wang, Yanyan

    2016-02-20

    In this work, ?-cyclodextrin was developed to prevent protein aggregation in foam fractionation using bovine serum albumin (BSA) as a model protein. The role of ?-cyclodextrin in preventing the aggregation of BSA induced by the gas-liquid interface was studied at the molecular level. The results indicate that by holding the exposed phenylalanine residues in its hydrophobic cavity, ?-cyclodextrin effectively prevented the aggregation of BSA induced by the gas-liquid interface in foam fractionation. Furthermore, ?-cyclodextrin could be effectively separated from BSA in the foamate due to their weak association. PMID:26778542

  6. Pilot study on binding of bovine salivary proteins to grit silicates and plant phytoliths.

    PubMed

    Mau, Marcus; M Kaiser, Thomas; Sdekum, Karl-Heinz

    2013-06-01

    Mostly fed with grass in fresh or conserved form, cattle and other livestock have to cope with silicate defence bodies from plants (phytoliths) and environmental silicates (grit), which abrade tooth enamel and could additionally interact with various salivary proteins. To detect potential candidates for silicate-binding proteins, bovine whole saliva was incubated with grass-derived phytoliths and silicates. Interactions of salivary proteins with pulverized bovine dental enamel and dentine were additionally analysed. After intense washing, the powder fractions were loaded onto 1D-polyacrylamide gels, most prominent adhesive protein bands were cut out and proteins were identified by mass spectrometry within three independent replicates. All materials were mainly bound by bovine odorant-binding protein, bovine salivary protein 3010(3) and carbonic anhydrase VI. The phytolith/silicate fraction showed additional stronger interaction with haemoglobin ? and lactoperoxidase. Conceivably, the binding of these proteins to the surfaces may contribute to biological processes occurring on them. PMID:23776006

  7. Control of domain swapping in bovine odorant-binding protein.

    PubMed Central

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

    2002-01-01

    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. PMID:11931632

  8. Bovine Model of Doxorubicin-Induced Cardiomyopathy

    PubMed Central

    Bartoli, Carlo R.; Brittian, Kenneth R.; Giridharan, Guruprasad A.; Koenig, Steven C.; Hamid, Tariq; Prabhu, Sumanth D.

    2011-01-01

    Left ventricular assist devices (LVADs) constitute a recent advance in heart failure (HF) therapeutics. As the rigorous experimental assessment of LVADs in HF requires large animal models, our objective was to develop a bovine model of cardiomyopathy. Male calves (n = 8) were used. Four animals received 1.2 mg/kg intravenous doxorubicin weekly for seven weeks and four separate animals were studied as controls. Doxorubicin-treated animals were followed with weekly echocardiography. Target LV dysfunction was defined as an ejection fraction ?35%. Sixty days after initiating doxorubicin, a terminal study was performed to determine hemodynamic, histological, biochemical, and molecular parameters. All four doxorubicin-treated animals exhibited significant (P < 0.05) contractile dysfunction, with target LV dysfunction achieved in three animals. Doxorubicin-treated hearts exhibited significantly reduced coronary blood flow and interstitial fibrosis and significantly increased apoptosis and myocyte size. Gene expression of atrial natriuretic factor increased more than 3-fold. Plasma norepinephrine and epinephrine levels were significantly increased early and late during the development of cardiomyopathy, respectively. We conclude that sequential administration of intravenous doxorubicin in calves induces a cardiomyopathy with many phenotypic hallmarks of the failing human heart. This clinically-relevant model may be useful for testing pathophysiologic responses to LVADs in the context of HF. PMID:21253525

  9. Bovine model of doxorubicin-induced cardiomyopathy.

    PubMed

    Bartoli, Carlo R; Brittian, Kenneth R; Giridharan, Guruprasad A; Koenig, Steven C; Hamid, Tariq; Prabhu, Sumanth D

    2011-01-01

    Left ventricular assist devices (LVADs) constitute a recent advance in heart failure (HF) therapeutics. As the rigorous experimental assessment of LVADs in HF requires large animal models, our objective was to develop a bovine model of cardiomyopathy. Male calves (n = 8) were used. Four animals received 1.2 mg/kg intravenous doxorubicin weekly for seven weeks and four separate animals were studied as controls. Doxorubicin-treated animals were followed with weekly echocardiography. Target LV dysfunction was defined as an ejection fraction ? 35%. Sixty days after initiating doxorubicin, a terminal study was performed to determine hemodynamic, histological, biochemical, and molecular parameters. All four doxorubicin-treated animals exhibited significant (P < 0.05) contractile dysfunction, with target LV dysfunction achieved in three animals. Doxorubicin-treated hearts exhibited significantly reduced coronary blood flow and interstitial fibrosis and significantly increased apoptosis and myocyte size. Gene expression of atrial natriuretic factor increased more than 3-fold. Plasma norepinephrine and epinephrine levels were significantly increased early and late during the development of cardiomyopathy, respectively. We conclude that sequential administration of intravenous doxorubicin in calves induces a cardiomyopathy with many phenotypic hallmarks of the failing human heart. This clinically-relevant model may be useful for testing pathophysiologic responses to LVADs in the context of HF. PMID:21253525

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

    PubMed

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

    2010-03-24

    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

  11. Dicarbonyl L-Xylulose Reductase (DCXR), a Moonlighting Protein in the Bovine Epididymis

    PubMed Central

    Akintayo, Ayodl; Lgar, Christine; Sullivan, Robert

    2015-01-01

    During maturation and the acquisition of their fertilization potential, male germ cells are subjected to various sequential modifications that occur in the epididymis. Protein addition, reorganization or withdrawal, comprise some of these modifications. Dicarbonyl L-xylulose reductase (DCXR), a multifunctional protein involved in various enzymatic and protein interaction processes in different physiological systems, is one of the proteins added to spermatozoa in the epididymis. DCXR is a well-conserved protein with multiple characteristics including enzymatic activities and mediation of cell-cell interaction. In this study, we characterized the DCXR gene and protein expression in the bovine epididymis. Dicarbonyl L-xylulose reductase mRNA is differentially expressed in the caput, corpus, and cauda epididymide epithelial cells with a higher level observed in the cauda region. Tissue protein expression follows the same pattern as the corresponding mRNA expression with a cytoplasmic and apical distribution in the corpus and cauda epithelial cells, respectively. The protein can also be found with a nuclear localization in cauda epididymidis epithelial cells. Dicarbonyl L-xylulose reductase is secreted in the epididymis luminal compartment in the soluble fraction and is associated with microvesicular elements named epididymosomes. In spermatozoa, the DCXR protein was found in the cytoplasmic and membranous fractions. Expression of the DCXR protein is higher on caput spermatozoa but finally shows a weak detection in semen. These data describe DCXR in the bovine epididymis and reveal that its behavior differs from that found in humans. It seems that, in this model, the DCXR protein might have a questionable involvement in the fertilization process. PMID:25815750

  12. Phosphorylation of bovine interphotoreceptor retinoid-binding protein (IRBP)

    SciTech Connect

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

    1986-05-01

    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.

  13. Modeling Bovine Respiratory Syncytial Virus Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine respiratory disease (BRDC) is a leading cause of morbidity and mortality in calves in U.S. beef feedlots. Furthermore, it is the leading cause of weaned dairy heifer mortality and one of the primary causes of mortality in pre-weaned dairy calves. While the mechanisms leading to BRDC remain to...

  14. Oral administration of encapsulated bovine lactoferrin protein nanocapsules against intracellular parasite Toxoplasma gondii

    PubMed Central

    Anand, Namrata; Sehgal, Rakesh; Kanwar, Rupinder Kaur; Dubey, Mohan Lal; Vasishta, Rakesh Kumar; Kanwar, Jagat Rakesh

    2015-01-01

    Toxoplasma gondii is a deadly intracellular parasite known to reside in every nucleated cell and known to cause severe complications in immunocompromised host. Standard drugs are cost effective and cause side effects, therefore, there is a necessity for a new drug molecule with immunomodulatory potential. Lactoferrin (Lf) is a natural milk protein, which has shown antimicrobial properties in its nanoformulation using alginate chitosan calcium phosphate bovine lactoferrin nanocapsules (AEC-CCo-CP-bLf-NCs). The present study was aimed to analyze and compare the effect of bovine Lf (bLf) in its native as well as nanoformulation (AEC-CCo-CP-bLf-NC) against coccidian parasite T. gondii. In vitro analysis has shown a significant increase in nitric oxide production and low parasitemia in in vitro cell culture model. In vivo BALB/c mice model have been used to develop human toxoplasmosis model. After treatment with NCs it has substantially increased the bioavailability of the protein and showed comparatively increased levels of reactive oxygen species, nitric oxide production, and Th1 cytokine which helped in parasite clearance. The mechanism of action of NCs has been clarified by immunoreactivity analysis, which showed accumulation of Lf in macrophages of various visceral organs, which is the site of parasite multiplication. Effect of NCs has significantly decreased (P<0.05) the parasite load in various organs and helped survival of mice till day 25 postinfection. Fe metabolism inside the mice has been found to be maintained even after administration of mono form of Lf, this indicates novelty of Lf protein. From the present study we concluded that nanoformulation did not reduce the therapeutic potential of Lf protein; however, nanoformulation has enhanced the stability of the protein and shown anti-toxoplasmal activity. Our study presents for the first time nanoformulation of Lf protein against Toxoplasma, which has advantages over the standard drug therapy without any side effects. PMID:26504384

  15. Oral administration of encapsulated bovine lactoferrin protein nanocapsules against intracellular parasite Toxoplasma gondii.

    PubMed

    Anand, Namrata; Sehgal, Rakesh; Kanwar, Rupinder Kaur; Dubey, Mohan Lal; Vasishta, Rakesh Kumar; Kanwar, Jagat Rakesh

    2015-01-01

    Toxoplasma gondii is a deadly intracellular parasite known to reside in every nucleated cell and known to cause severe complications in immunocompromised host. Standard drugs are cost effective and cause side effects, therefore, there is a necessity for a new drug molecule with immunomodulatory potential. Lactoferrin (Lf) is a natural milk protein, which has shown antimicrobial properties in its nanoformulation using alginate chitosan calcium phosphate bovine lactoferrin nanocapsules (AEC-CCo-CP-bLf-NCs). The present study was aimed to analyze and compare the effect of bovine Lf (bLf) in its native as well as nanoformulation (AEC-CCo-CP-bLf-NC) against coccidian parasite T. gondii. In vitro analysis has shown a significant increase in nitric oxide production and low parasitemia in in vitro cell culture model. In vivo BALB/c mice model have been used to develop human toxoplasmosis model. After treatment with NCs it has substantially increased the bioavailability of the protein and showed comparatively increased levels of reactive oxygen species, nitric oxide production, and Th1 cytokine which helped in parasite clearance. The mechanism of action of NCs has been clarified by immunoreactivity analysis, which showed accumulation of Lf in macrophages of various visceral organs, which is the site of parasite multiplication. Effect of NCs has significantly decreased (P<0.05) the parasite load in various organs and helped survival of mice till day 25 postinfection. Fe metabolism inside the mice has been found to be maintained even after administration of mono form of Lf, this indicates novelty of Lf protein. From the present study we concluded that nanoformulation did not reduce the therapeutic potential of Lf protein; however, nanoformulation has enhanced the stability of the protein and shown anti-toxoplasmal activity. Our study presents for the first time nanoformulation of Lf protein against Toxoplasma, which has advantages over the standard drug therapy without any side effects. PMID:26504384

  16. 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...

  17. Association of bovine ?-casein protein variant I with milk production and milk protein composition.

    PubMed

    Visker, M H P W; Dibbits, B W; Kinders, S M; van Valenberg, H J F; van Arendonk, J A M; Bovenhuis, H

    2011-04-01

    The aim of this study was to detect new polymorphisms in the bovine ?-casein (?-CN) gene and to evaluate association of (new) ?-CN protein variants with milk production traits and milk protein composition. Screening of the ?-CN gene in genomic DNA from 72 Holstein Friesian (HF) bulls resulted in detection of 19 polymorphisms and revealed the presence of ?-CN protein variant I in the Dutch HF population. Studies of association of ?-CN protein variants with milk composition usually do not discriminate protein variant I from variant A2. Association of ?-CN protein variants with milk composition was studied in 1857 first-lactation HF cows and showed that associations of protein variants A2 and I were quite different for several traits. ?-CN protein variant I was significantly associated with protein percentage and protein yield, and with ?s1 -casein (?s1 -CN), ?s2 -casein (?s2 -CN), ?-casein (?-CN), ?-lactalbumin (?-LA), ?-lactoglobulin (?-LG), casein index and casein yield. Inferring ?-?-CN haplotypes showed that ?-CN protein variant I occurred only with ?-CN variant B. Consequently, associations of ?-?-CN haplotype IB with protein percentage, ?-CN, ?-LA, ?-LG and casein index are likely resulting from associations of ?-CN protein variant B, while associations of ?-?-CN haplotype IB with ?s1 -CN and ?s2 -CN seem to be resulting from associations of ?-CN variant I. PMID:24725229

  18. Mycobacterium avium Subspecies paratuberculosis Recombinant Proteins Modulate Antimycobacterial Functions of Bovine Macrophages

    PubMed Central

    Bannantine, John P.; Stabel, Judith R.; Laws, Elizabeth; D. Cardieri, Maria Clara; Souza, Cleverson D.

    2015-01-01

    It has been shown that Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) activates the Mitogen Activated Protein Kinase (MAPK) p38 pathway, yet it is unclear which components of M. paratuberculosis are involved in the process. Therefore, a set of 42 M. paratuberculosis recombinant proteins expressed from coding sequences annotated as lipoproteins were screened for their ability to induce IL-10 expression, an indicator of MAPKp38 activation, in bovine monocyte-derived macrophages. A recombinant lipoprotein, designated as MAP3837c, was among a group of 6 proteins that strongly induced IL-10 gene transcription in bovine macrophages, averaging a 3.1-fold increase compared to non-stimulated macrophages. However, a parallel increase in expression of IL-12 and TNF-? was only observed in macrophages exposed to a subset of these 6 proteins. Selected recombinant proteins were further analyzed for their ability to enhance survival of M. avium within bovine macrophages as measured by recovered viable bacteria and nitrite production. All 6 IL-10 inducing MAP recombinant proteins along with M. paratuberculosis cells significantly enhanced phosphorylation of MAPK-p38 in bovine macrophages. Although these proteins are likely not post translationally lipidated in E. coli and thus is a limitation in this study, these results form the foundation of how the protein component of the lipoprotein interacts with the immune system. Collectively, these data reveal M. paratuberculosis proteins that might play a role in MAPK-p38 pathway activation and hence in survival of this organism within bovine macrophages. PMID:26076028

  19. Bovine recombinant lipopolysaccharide binding protein (BRLBP) regulated apoptosis and inflammation response in lipopolysaccharide-challenged bovine mammary epithelial cells (BMEC).

    PubMed

    Sun, Yu; Li, Lian; Wu, Jie; Yu, Pan; Li, Chengmin; Tang, Juan; Li, Xiaojuan; Huang, Shuai; Wang, Genlin

    2015-06-01

    Lipopolysaccharide-binding protein (LBP) is an acute-phase protein involved in host response to Gram-negative and Gram-positive pathogens. It has been reported to exert diverse biological activities, such as anti-inflammatory effects. However, what effects it has on bovine mastitis has not been investigated. The aim of this study was to verify the anti-inflammatory properties of LBP on the inflammatory response of primary bovine mammary epithelial cells (BMEC) induced by lipopolysaccharide (LPS), and to determine the underlying mechanism. Bovine mammary epithelial cells were treated with various concentrations of LPS (1, 10, 20, and 100 ?g/mL) for 3, 6, 12, and 24h. The results showed that LPS significantly inhibited cell viability in a dose-dependent manner. When cells were treated with LPS (10 ?g/mL) for 12 h, the permeability of the cell membrane increased significantly. This promoted apoptosis. Various concentrations (10 and 20 ?g/mL) of bovine recombinant lipopolysaccharide binding protein (BRLBP) could weaken the inflammation injury of BMEC induced by LPS without cytotoxicity. Toll-like receptor 4 (TLR4), nuclear factor ?B (NF-?B), IL-1?, and tumor necrosis factor ? (TNF-?) from BMEC were decreased. TLR4 and NF-?B P65 protein levels were down-regulated, and nuclear transcription factor ?B activity was also weakened. All these results indicated that the protective effects of high concentrations of BRLBP on LPS-induced inflammation injury in BMEC were at least partially achieved by the decreased production of pro-inflammatory cytokines. BRLBP was found to directly inhibit LPS/TLR4-mediated NF-?B activation. One possible anti-inflammatory mechanism can be attributed to the negative role of BRLBP in suppressing TLR4/NF-?B activation mediated by LPS. These findings suggested that BRLBP may be a useful agent to treat LPS-induced mastitis. PMID:25700343

  20. Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis.

    PubMed

    Lee, Jae Eun; Lee, Jae Young; Kim, Hong Rye; Shin, Hyun Young; Lin, Tao; Jin, Dong Il

    2015-06-01

    Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum. PMID:25925056

  1. Bovine respiratory disease model based on dual infections with infection with bovine viral diarrhea virus and bovine corona virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine respiratory disease complex (BRDC) is the leading cause of economic loss in the U.S. cattle industry. BRDC likely results from simultaneous or sequential infections with multiple pathogens including both viruses and bacteria. Bovine viral diarrhea virus (BVDV) and bovine corona virus (BoCV...

  2. Glass transitions in aqueous solutions of protein (bovine serum albumin).

    PubMed

    Shinyashiki, Naoki; Yamamoto, Wataru; Yokoyama, Ayame; Yoshinari, Takeo; Yagihara, Shin; Kita, Rio; Ngai, K L; Capaccioli, Simone

    2009-10-29

    Measurements by adiabatic calorimetry of heat capacities and enthalpy relaxation rates of a 20% (w/w) aqueous solution of bovine serum albumin (BSA) by Kawai, Suzuki, and Oguni [Biophys. J. 2006, 90, 3732] have found several enthalpy relaxations at long times indicating different processes undergoing glass transitions. In a quenched sample, one enthalpy relaxation at around 110 K and another over a wide temperature range (120-190 K) were observed. In a sample annealed at 200-240 K after quenching, three separated enthalpy relaxations at 110, 135, and above 180 K were observed. Dynamics of processes probed by adiabatic calorimetric data are limited to long times on the order of 10(3) s. A fuller understanding of the processes can be gained by probing the dynamics over a wider time/frequency range. Toward this goal, we performed broadband dielectric measurements of BSA-water mixtures at various BSA concentrations over a wide frequency range of thirteen decades from 2 mHz to 1.8 GHz at temperatures from 80 to 270 K. Three relevant relaxation processes were detected. For relaxation times equal to 100 s, the three processes are centered approximately at 110, 135, and 200 K, in good agreement with those observed by adiabatic calorimetry. We have made the following interpretation of the molecular origins of the three processes. The fastest relaxation process having relaxation time of 100 or 1000 s at ca. 110 K is due to the secondary relaxation of uncrystallized water (UCW) in the hydration shell. The intermediate relaxation process with 100 s relaxation time at ca. 135 K is due to ice. The slowest relaxation process having relaxation time of 100 s at ca. 200 K is interpreted to originate from local chain conformation fluctuations of protein slaved by water. Experimental evidence supporting these interpretations include the change of temperature dependence of the relaxation time of the UCW at approximately T(gBSA) approximately = 200 K, the glass transition temperature of protein in the hydration shell, similar to that found for the secondary relaxation of water in a mixture of myoglobin in glycerol and water [Swenson et al. J. Phys.: Condens. Matter 2007, 19, 205109; Ngai et al. J. Phys. Chem. B 2008, 112, 3826]. The data all indicate in hydrated BSA or other proteins that the secondary relaxation of water and the conformation fluctuations of the protein in the hydration shell are inseparable or symbiotic processes. PMID:19799444

  3. A specific binding protein for cardiac glycosides exists in bovine serum.

    PubMed

    Antolovic, R; Kost, H; Mohadjerani, M; Linder, D; Linder, M; Schoner, W

    1998-06-26

    Searching for a binding protein in blood, which may be involved in the specific transport of cardiac glycosides to their receptor sites on the sodium pump, we isolated a cardiac glycoside-binding protein (CGBG) of 26 kDa from the globulin fraction of bovine serum by affinity chromatography and on a ouabain-Sepharose 4B column by a purification factor of 5000. The cardiac glycoside-binding globulin was labeled specifically and covalently by the protein-reactive digoxigenin derivative HDMA (N-hydroxysuccimidyldigoxigenin-3-O-methylcarbonyl-epsilon-+ ++aminocapro ate). Even very high concentrations of other steroids, such as estrogen, testosterone, progesterone, and cortisone, did not prevent HDMA-labeling (at 5 and 100 nM) of CGBG, but the cardenolides ouabain and digoxin or the bufadienolide proscillaridin A did so. CGBG is a homodimer of two 26-kDa subunits forming disulfide bonds, since HDMA labeling of a protein of 53 kDa was observed in SDS-polyacrylamide gel electrophoresis when beta-mercaptoethanol was absent during SDS denaturation. The N-terminal amino acid sequence K-D-V-Y-R-A-P-D-G-T-Q-S-A showed no sequence similarity with proteins recorded in gene and protein sequence data banks. A 90-kDa cytosolic CGBG exists in bovine kidneys and reacts with antibodies against CGBG. Binding of ouabain to the cardiac glycoside-binding globulin was monitored by quenching of intrinsic tryptophan fluorescence. Such studies reveal two negatively cooperative ouabain binding sites with Kd' of 1.52 nM and Kd' = 75 nM and with an interaction factor of 50 using a Koshland-Nmethy-Filmer model. The demonstration of a cardiac glycoside-binding globulin in plasma is consistent with the recent finding of endogenous cardiac glycosides in mammals. PMID:9632685

  4. Bovine serum albumin adsorption onto functionalized polystyrene lattices: A theoretical modeling approach and error analysis

    NASA Astrophysics Data System (ADS)

    Beragoui, Manel; Aguir, Chadlia; Khalfaoui, Mohamed; Enciso, Eduardo; Torralvo, Maria Jos; Duclaux, Laurent; Reinert, Laurence; Vayer, Marylne; Ben Lamine, Abdelmottaleb

    2015-03-01

    The present work involves the study of bovine serum albumin adsorption onto five functionalized polystyrene lattices. The adsorption measurements have been carried out using a quartz crystal microbalance. Poly(styrene-co-itaconic acid) was found to be an effective adsorbent for bovine serum albumin molecule adsorption. The experimental isotherm data were analyzed using theoretical models based on a statistical physics approach, namely monolayer, double layer with two successive energy levels, finite multilayer, and modified Brunauer-Emmet-Teller. The equilibrium data were then analyzed using five different non-linear error analysis methods and it was found that the finite multilayer model best describes the protein adsorption data. Surface characteristics, i.e., surface charge density and number density of surface carboxyl groups, were used to investigate their effect on the adsorption capacity. The combination of the results obtained from the number of adsorbed layers, the number of adsorbed molecules per site, and the thickness of the adsorbed bovine serum albumin layer allows us to predict that the adsorption of this protein molecule can also be distinguished by monolayer or multilayer adsorption with end-on, side-on, and overlap conformations. The magnitudes of the calculated adsorption energy indicate that bovine serum albumin molecules are physisorbed onto the adsorbent lattices.

  5. The major proteins of bovine seminal plasma interact with caseins and whey proteins of milk extender.

    PubMed

    Lusignan, Marie-France; Bergeron, Annick; Lafleur, Michel; Manjunath, Puttaswamy

    2011-09-01

    Milk has been used routinely as an extender for sperm preservation. Caseins, the major proteins in milk, are proposed to be the protective constituents of milk during sperm preservation. It is unclear whether the whey proteins in milk are also implicated in the protection of sperm. Our previous studies have shown that the major proteins of bovine seminal plasma (recently named as binder of sperm or BSP, which comprises BSP1, BSP3, and BSP5 proteins) mediate a continuous phospholipid and cholesterol efflux from the sperm plasma membrane that is detrimental for sperm preservation. In this study, we investigated whether the protective effect of milk could be due to an interaction between BSP proteins and milk proteins. The binding of BSP proteins to milk proteins was demonstrated by gel filtration chromatography. Milk was fractionated into three fractions: the first containing whey protein aggregates and kappa-casein, the second containing all milk proteins, and the third containing small peptides, salts, and sugars. BSP1 has a higher affinity for the milk proteins in the milk fractions as compared to BSP3 and BSP5. The binding of BSP proteins to milk proteins was further characterized by isothermal titration calorimetry. We demonstrated that BSP1 binds to caseins and the titration could be simulated with a Scatchard approach, leading to an affinity constant (K(a)) of 350 mM(-1) and a stoichiometric parameter for the association (n) of 4.5 BSP1 per casein. The association between BSP1 and alpha-lactalbumin was characterized by a K(a) of 240 mM(-1) and an n value of 0.8. These results indicate the existence of an interaction between BSP proteins and milk proteins that could be the origin of the protection of sperm during preservation in milk. PMID:21593483

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

    SciTech Connect

    Flavin, M.P.; Parton, L.A.; Bowman, C.M. )

    1990-09-01

    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.

  7. Engineered Lactobacillus rhamnosus GG expressing IgG-binding domains of protein G: Capture of hyperimmune bovine colostrum antibodies and protection against diarrhea in a mouse pup rotavirus infection model.

    PubMed

    Günaydın, Gökçe; Zhang, Ran; Hammarström, Lennart; Marcotte, Harold

    2014-01-16

    Rotavirus-induced diarrhea causes more than 500,000 deaths annually in the world, and although vaccines are being made available, new effective treatment strategies should still be considered. Purified antibodies derived from hyperimmune bovine colostrum (HBC), from cows immunized with rotavirus, were previously used for treatment of rotavirus diarrhea in children. A combination of HBC antibodies and a probiotic strain of Lactobacillus (L. rhamnosus GG) was also found to be more effective than HBC alone in reducing diarrhea in a mouse model of rotavirus infection. In order to further improve this form of treatment, L. rhamnosus GG was engineered to display surface expressed IgG-binding domains of protein G (GB1, GB2, and GB3) which capture HBC-derived IgG antibodies (HBC-IgG) and thus target rotavirus. The expression of IgG-binding domains on the surface of the bacteria as well as their binding to HBC-IgG and to rotavirus (simian strain RRV) was demonstrated by Western blot, flow cytometry, and electron microscopy. The prophylactic effect of engineered L. rhamnosus GG and anti-rotaviral activity of HBC antibodies was evaluated in a mouse pup model of RRV infection. The combination therapy with engineered L. rhamnosus GG (PG3) and HBC was significantly more effective in reducing the prevalence, severity, and duration of diarrhea in comparison to HBC alone or a combination of wild-type L. rhamnosus GG and HBC. The new therapy reduces the effective dose of HBC between 10 to 100-fold and may thus decrease treatment costs. This antibody capturing platform, tested here for the first time in vivo, could potentially be used to target additional gastrointestinal pathogens. PMID:24291196

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

    SciTech Connect

    Dianoux, A.C.; Stasia, M.J.; Vignais, P.V. )

    1989-01-24

    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.

  9. Expression of Go alpha mRNA and protein in bovine tissues.

    PubMed

    Price, S R; Tsai, S C; Adamik, R; Angus, C W; Serventi, I M; Tsuchiya, M; Moss, J; Vaughan, M

    1989-05-01

    Go alpha is a 39-kDa guanine nucleotide-binding protein (G protein) similar in structure and function to Gs alpha and Gi alpha of the adenylate cyclase complex and to transducin (Gt alpha) of the retinal photon receptor system. Although expression of Go alpha protein has been reported to be tissue-specific, other workers have found Go alpha mRNA in all rat tissues examined. In order to clarify this contradiction, studies to verify the distribution of Go alpha mRNA and protein in bovine and rat tissues were performed. Tissues were screened for the presence of Go alpha mRNA by use of a series of restriction fragments of a bovine retinal cDNA clone, lambda GO9, and oligonucleotide probes complementary to sequences specific among G alpha subunits for the 5' untranslated and coding regions of Go alpha. These probes hybridized predominantly with mRNA of 4.0 and 3.0 kb in bovine brain and retina. A 2.0-kb mRNA in retina also hybridized strongly with the cDNA but weakly with the oligonucleotide probes. In bovine lung, two mRNAs of 1.6 and 1.8 kb hybridized with the cDNA while only the 1.6-kb species hybridized with the coding-region oligonucleotide. In bovine heart, only a 4.0-kb mRNA was detected and in amounts much less than those in the other tissues. A similar distribution of Go alpha mRNAs was seen in rat tissues. In bovine tissues, Go alpha protein was identified with rabbit polyclonal antibodies directed against purified bovine brain Go alpha. An immunoreactive 39-kDa membrane protein was found principally in retina and brain, and in a lesser amount in heart.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2502171

  10. Computational modelling of bovine ovarian follicle development

    PubMed Central

    2013-01-01

    Background The development of ovarian follicles hinges on the timely exposure to the appropriate combination of hormones. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) are both produced in the pituitary gland and are transported via the blood circulation to the thecal layer surrounding the follicle. From there both hormones are transported into the follicle by diffusion. FSH-receptors are expressed mainly in the granulosa while LH-receptors are expressed in a gradient with highest expression in the theca. How this spatial organization is achieved is not known. Equally it is not understood whether LH and FSH trigger distinct signalling programs or whether the distinct spatial localization of their G-protein coupled receptors is sufficient to convey their distinct biological function. Results We have developed a data-based computational model of the spatio-temporal signalling processes within the follicle and (i) predict that FSH and LH form a gradient inside the follicle, (ii) show that the spatial distribution of FSH- and LH-receptors can arise from the well known regulatory interactions, and (iii) find that the differential activity of FSH and LH may well result from the distinct spatial localisation of their receptors, even when both receptors respond with the same intracellular signalling cascade to their ligand. Conclusion The model integrates the large amount of published data into a consistent framework that can now be used to better understand how observed defects translate into failed follicle maturation. PMID:23856357

  11. Bovine viral diarrhea viruses differentially alter the expression of the protein kinases and related proteins affecting the development of infection and anti-viral mechanisms in bovine monocytes.

    PubMed

    Pinchuk, George V; Lee, Sang-Ryul; Nanduri, Bindu; Honsinger, Kelly L; Stokes, John V; Pinchuk, Lesya M

    2008-09-01

    Using a proteomics approach, we evaluated the effect of cytopathic (cp), and non-cytopathic (ncp) bovine viral diarrhea viruses (BVDV) on the expression of protein kinases and related proteins in bovine monocytes. Proteins were isolated from membrane and cytosolic fractions with the differential detergent fractionation (DDF) method and identified with 2D-LC ESI MS2. Of approximately 10,000 proteins identified, 378 proteins had homology with known protein kinases or related proteins. Eighteen proteins involved in cell differentiation and activation, migration, anti-viral mechanisms (interferon/apoptosis), biosynthesis, sugar metabolism and oncogenic transformation were significantly altered in BVDV-infected monocytes compared to the uninfected controls. Six proteins, mostly related to cell migration, anti-viral mechanisms, sugar metabolism and possibly tumor resistance were differentially expressed between the ncp and cp BVDV-infected monocytes. Particularly, the expression of the receptor of activated C kinase (RACK), of pyridoxal kinase (PK), diacyglycerol kinase (DGK) and Brutons tyrosine kinase (BTK) was decreased in monocytes infected with cp BVDV compared to ncp BVDV, possibly contributing to the cytopathic effect of the virus. This and other findings are discussed in view of the possible role the identified proteins play in the development of viral infection and oncogenic transformation of cells. PMID:18570900

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

    PubMed

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

    2012-05-01

    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

  13. BOVINE SOMATOTROPIN AND RUMEN UNDEGRADABLE PROTEIN EFFECTS ON SKELETAL GROWTH IN PREPUBERTAL DAIRY HEIFERS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were to determine the effects of dietary rumen-undegradable protein (RUP) and bovine somatotropin (bST) during the period from weaning until puberty on body weight (BW) and skeletal growth rates. Fifty-one Holstein heifers at 90 d of age were randomly assigned to treatme...

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2011-09-01

    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.

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

    SciTech Connect

    Stasia, M.J.; Dianoux, A.C.; Vignais, P.V. )

    1989-12-12

    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.

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

    PubMed

    Srensen, Jesper; Palmer, David S; Qvist, Karsten Bruun; Schitt, Birgit

    2011-05-25

    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

  18. Guanosine 3',5'-cyclic nucleotide binding proteins of bovine retina identified by photoaffinity labeling.

    PubMed Central

    Thompson, D A; Khorana, H G

    1990-01-01

    Cyclic GMP-binding proteins present in membrane fractions of bovine retina and, in particular, rod outer segments (ROS) were identified by photoaffinity labeling with 8-azido-[32P]cGMP. Two soluble proteins and two membrane-associated proteins were specifically labeled. The soluble proteins, 93 and 72 kDa, corresponded respectively to the alpha subunit of ROS cGMP phosphodiesterase and cGMP-dependent protein kinase. One of the two membrane-associated proteins, 53 kDa, was present in all particulate retinal fractions. Its function is unknown. It is distinct from cAMP-dependent protein kinase or the 63-kDa cGMP-activated channel from ROS. The second membrane-associated protein, 37 kDa, was present only in fractions that did not contain ROS. The molecular mass of this protein was similar to that of a cGMP-binding protein previously attributed to rod cells. Images PMID:2156264

  19. Theoretical modeling of the kinetics of fibrilar aggregation of bovine ?-lactoglobulin at pH 2

    NASA Astrophysics Data System (ADS)

    Arnaudov, Luben N.; de Vries, Renko

    2007-04-01

    The authors propose a kinetic model for the heat-induced fibrilar aggregation of bovine ?-lactoglobulin at pH 2.0. The model involves a nucleation step and a simple addition reaction for the growth of the fibrils, as well as a side reaction leading to the irreversible denaturation and inactivation of a part of the protein molecules. For the early stages of the aggregation reaction, the authors obtain an analytical solution of the model. In agreement with their experimental results, the model predicts a critical protein concentration below where almost no fibrils are formed. The model agrees well with their experimental data from in situ light scattering. By fitting the experimental data with the model, the authors obtain the ionic strength dependent kinetic rate constants for ?-lactoglobulin fibrilar aggregation and the size of the critical nucleus.

  20. Protein Hydrolysates from Non-bovine and Plant Sources Replaces Tryptone in Microbiological Media

    NASA Astrophysics Data System (ADS)

    Ranganathan, Yamini; Patel, Shifa; Pasupuleti, Vijai K.; Meganathan, R.

    Tryptone (pancreatic digest of casein) is a common ingredient in laboratory and fermentation media for growing wild-type and genetically modified microorganisms. Many of the commercially manufactured products such as human growth hormone, antibiotics, insulin, etc. are produced by recombinant strains grown on materials derived from bovine sources. With the emergence of Bovine Spongiform Encephalopathy (BSE) and the consequent increase in Food and Drug Administration (FDA) regulations, elimination of materials of bovine origin from fermentation media is of paramount importance. To achieve this objective, a number of protein hydrolysates derived from non-bovine animal and plant sources were evaluated. Tryptone in Luria-Bertani (LB) broth was replaced with an equal quantity of alternate protein hydrolysates. Four of the six hydrolysates (one animal and three from plants) were found to efficiently replace the tryptone present in LB-medium as measured by growth rate and growth yield of a recombinant Escherichia coli strain. In addition, we have determined plasmid stability, inducibility and activity of the plasmid encoded β-galactosidase in the recombinant strain grown in the presence of various protein hydrolysates.

  1. Tissue- and age-dependent expression of the bovine DEFB103 gene and protein.

    PubMed

    Mirabzadeh-Ardakani, Ali; Solie, Jay; Gonzalez-Cano, Patricia; Schmutz, Sheila M; Griebel, Philip J

    2016-02-01

    Beta-defensin 103 (DEFB103) shares little homology with 8 other members of the bovine beta-defensin family and in other species DEFB103 protein has diverse functions, including antimicrobial activity, a chemoattractant for dendritic cells, enhancing epithelial wound repair and regulating hair colour. Expression of the bovine DEFB103 gene was surveyed in 27 tissues and transcript was most abundant in tissues with stratified squamous epithelium. Oral cavity epithelial tissues and nictitating membrane consistently expressed high levels of DEFB103 gene transcript. An age-dependent decrease (P < 0.05) in DEFB103 gene expression was only observed for buccal epithelium when comparing healthy 10- to 14-day-old and 10- to 12-month-old calves. A bovine herpesvirus-1 respiratory infection did, however, significantly (P < 0.05) up-regulate DEFB103 gene expression in the buccal epithelium of 6- to 8-month-old calves. Finally, DEFB103 transcript was low in lymph nodes draining the skin and at the limit of detection in other internal organs such as lung, intestine and kidney. Affinity-purified rabbit antisera to bovine DEFB103 was used to identify cells expressing DEFB103 protein within tissues with stratified squamous epitheliums. DEFB103 protein was most abundant in basal epithelial cells and was present in these cells prior to birth. Beta-defensins have been identified as regulators of dendritic cell (DC) chemokine responses and we observed a close association between DCs and epithelial cells expressing DEFB103 in both the fetus and newborn calf. In conclusion, bovine DEFB103 gene expression is most abundant in stratified squamous epithelium with DEFB103 protein localised to basal epithelial cells. These observations are consistent with proposed roles for DEFB103 in DC recruitment and repair of stratified squamous epithelium. PMID:26299200

  2. A bovine model for polycystic ovary syndrome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polycystic ovary syndrome (PCOS) results in the greatest single cause of anovulatory infertility in reproductive age women (affecting 5-10%). Previously, research groups have created animal models utilizing non-human primates and sheep to better understand the mechanisms involved in PCOS. However, c...

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

    SciTech Connect

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

    1988-05-15

    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.

  4. Campylobacter fetus subspecies venerealis surface array protein from bovine isolates in Brazil.

    PubMed

    de Vargas, Agueda Castagna; Costa, Mateus Matiuzzi; Vainstein, Marilene Henning; Kreutz, Luiz Carlos; Neves, Jairo Pereira

    2002-08-01

    The electrophoretic patterns of 31 Campylobacter fetus subspecies venerealis capsular Surface Array Protein (SAP) isolated from bovines in reproduction from different regions of Brazil were analyzed. The persistence of the bacteria in the reproductive tract of naturally infected bovines and the dynamic of SAP expression were also evaluated. Cervical mucous and prepucial aspirates from five animals naturally infected were cultured for isolation of Campylobacter fetus and the SAPs extracted from the bacteria isolated were analyzed by SDS-PAGE. Ten different patterns of SAP expression were demonstrated by the identification of proteins with molecular mass of 97, 100, 127, and 149 kDa, respectively. The most prevalent identified protein had a molecular mass of 100 kDa (41.9%). Taking into consideration the time during which the five animals were evaluated, it was possible to conclude that one of these animals persisted with the etiological agent up to 171 days. The five naturally infected bovines analyzed presented variation on their surface protein pattern during the period of this study. C. fetus subspecies venerealis persisted in the reproductive tract of naturally infected animals. In natural condition of infection C. fetus subspecies venerealis persisted in an intermittent condition and an alteration of the protein surface was shown. PMID:12070688

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

    SciTech Connect

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

    1988-07-12

    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.

  6. Engineering Glycoprotein B of Bovine Herpesvirus 1 To Function as Transporter for Secreted Proteins: a New Protein Expression Approach

    PubMed Central

    Keil, Gnther M.; Hhle, Constanze; Giesow, Katrin; Knig, Patricia

    2005-01-01

    Glycoprotein B (gB) of bovine herpesvirus 1 (BHV-1) is essential for BHV-1 replication and is required for membrane fusion processes leading to virus penetration into the target cell and direct spreading of BHV-1 from infected to adjacent noninfected cells. Like many of the herpesvirus gB homologs, BHV-1 gB is proteolytically processed by furin, an endoproteinase localized in the trans-Golgi network. Cleavage by furin is a common mechanism for the activation of a number of viral fusion (F) proteins. Among these, the F proteins of both human and bovine respiratory syncytial virus (RSV) have the so-far unique feature that cleavage of the respective F protein precursors occurs at two furin recognition sites, resulting in the release of a 27-amino-acid intervening peptide which is secreted into the extracellular space. We showed recently that the intervening peptide of bovine RSV can be replaced by bovine interleukins which are secreted into the medium of cells infected with the respective bovine RSV recombinants (P. Knig, K. Giesow, K. Schuldt, U. J. Buchholz, and G. M. Keil, J. Gen. Virol. 85:1815-1824, 2004). To elucidate whether the approach to transport heterologous proteins as furin-excisable polypeptides functions in principle also in glycoproteins which are cleaved by furin only once, we inserted a second furin cleavage site into BHV-1 gB and integrated a 16-amino-acid peptide sequence, the 246-amino-acid green fluorescent protein (GFP), or the 167 amino acids for mature bovine alpha interferon (boIFN-?) as an intervening polypeptide. The resulting gB variants rescued gB-negative BHV-1 mutants, the resulting BHV-1 recombinants were fully infectious, and infected cells secreted biologically active GFP and boIFN-?, respectively. In contrast to the gB2Fu and gB2FuGFP precursor molecules, which were efficiently cleaved at both furin sites, the majority of pgB2FuIFN-? was not cleaved at the site between the amino-terminal (NH2) subunit and boIFN-?, whereas cleavage at the newly introduced site was normal. This resulted in virus particles that also contain the NH2-subunit/boIFN-? fusion protein within their envelopes. Our results demonstrate that BHV-1 gB can be used as a transporter for peptides and proteins which could be important for development of novel vaccines. In addition, the general principle might be useful for other applications, e.g., in gene therapy and also in nonviral systems. PMID:15613307

  7. Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells.

    PubMed

    Lin, C; Agnes, J T; Behrens, N; Shao, M; Tagawa, Y; Gershwin, L J; Corbeil, L B

    2016-01-01

    Our previous studies showed that bovine respiratory syncytial virus (BRSV) followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2) epithelial cells with H. somni concentrated culture supernatant (CCS) stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2-RSAD2) and ISG15 (IFN-stimulated gene 15-ubiquitin-like modifier) were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT) upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide) but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt) does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo. PMID:26859677

  8. Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells

    PubMed Central

    Lin, C.; Agnes, J. T.; Behrens, N.; Tagawa, Y.; Gershwin, L. J.; Corbeil, L. B.

    2016-01-01

    Our previous studies showed that bovine respiratory syncytial virus (BRSV) followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2) epithelial cells with H. somni concentrated culture supernatant (CCS) stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2—RSAD2) and ISG15 (IFN-stimulated gene 15—ubiquitin-like modifier) were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT) upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide) but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt) does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo. PMID:26859677

  9. Sequence of membrane-associated thyroid hormone binding protein from bovine liver: its identity with protein disulphide isomerase.

    PubMed

    Yamauchi, K; Yamamoto, T; Hayashi, H; Koya, S; Takikawa, H; Toyoshima, K; Horiuchi, R

    1987-08-14

    The complementary DNAs of the bovine liver membrane-associated 3,5,3'-triiodo-L-thyronine binding protein with 55 k-dalton (T3BP) were cloned and the nucleotide sequences were determined. Monospecific antibodies against T3BP were used to screen a bovine liver cDNA library in lambda gtll. We analyzed the sequences of two cloned T3BP cDNAs. The clones encoded a polypeptide of 510 amino acid residues, including a signal peptide of 20 amino acid. Northern blot analysis of bovine and human RNA showed that the mRNAs encoding T3BP are 2.7 kilobase in length. Amino acid sequence of N-terminal and other three peptides isolated from purified T3BP were found in the predicted amino acid sequence from the cDNA sequence. The predicted amino acid sequence is closely homologous (93%) with that of rat protein disulphide isomerase (EC 5.3.4.1), which catalyzes the isomerization of the protein disulphide bonds and has been shown to play an important role in post-translational regulation. The results suggest that T3BP and protein disulphide isomerase should be the same protein. PMID:3619939

  10. Cross-Reactivity Between the Soybean Protein P34 and Bovine Caseins

    PubMed Central

    Smaldini, Paola Lorena; Curciarello, Renata; Cauerhff, Ana; Fossati, Carlos Alberto

    2015-01-01

    Purpose Soy-based formulas are widely used as dairy substitutes to treat milk allergy patients. However, reactions to soy have been reported in a small proportion of patients with IgE-mediated milk allergies. The aim of this work was to explore whether P34, a mayor soybean allergen, is involved in this cross-reactivity. Methods In vitro recognition of P34 was evaluated by immunoblotting, competitive ELISA and basophil activation tests (BAT) using sera from allergic patients. In vivo cross-reactivity was examined using an IgE-mediated milk allergy mouse model. Results P34 was recognized by IgE antibodies from the sera of milk allergic patients, casein-specific monoclonal antibodies, and sera from milk-allergic mice. Spleen cells from sensitized mice incubated with milk, soy or P34 secreted IL-5 and IL-13, while IFN-? remained unchanged. In addition, the cutaneous test was positive with cow's milk proteins (CMP) and P34 in the milk allergy mouse model. Moreover, milk-sensitized mice developed immediate symptoms following sublingual exposure to P34. Conclusions Our results demonstrate that P34 shares epitopes with bovine casein, which is responsible for inducing hypersensitivity symptoms in milk allergic mice. This is the first report of the in vivo cross-allergenicity of P34. PMID:25553264

  11. Production and properties of health-promoting proteins and peptides from bovine colostrum and milk.

    PubMed

    Korhonen, H J

    2013-01-01

    The high nutritive value and diverse functional properties of milk proteins are well known. Beyond these qualities, milk proteins have attracted growing scientific and commercial interest as a source of biologically active molecules. Such proteins are found in abundance in colostrum which is the initial milk secreted by mammalian species during late pregnancy and the first few days after birth of the offspring. The best characterized colostrum-based bioactive proteins include alpha-lactalbumin, beta-lactoglobulin, immunoglobulins, lactoferrin, lactoperoxidase and growth factors. All of them can nowadays be enriched and purified on an industrial scale from bovine colostral whey or cheese whey. These native proteins exhibit a wide range of biological activities that are known to affect the digestive function, metabolic responses to absorbed nutrients, growth and development of organs and disease resistance. Also, some of these proteins may prove beneficial in reduction of the risks of chronic human diseases reflected by the metabolic syndrome. It is speculated that such potentially beneficial effects are partially attributed to bioactive peptides derived from intact proteins. These peptides can be liberated during gastrointestinal digestion or fermentation of milk by starter cultures. The efficacy of a few peptides has been established in animal and human studies and the number of commercial products supplemented with specific milk peptides is envisaged to increase on global markets. Bovine colostrum appears as a highly potential source of biologically active native proteins and peptide fractions for inclusion as health-promoting ingredients in various food applications. PMID:24200017

  12. Protein degradation in bovine milk caused by Streptococcus agalactiae.

    PubMed

    Åkerstedt, Maria; Wredle, Ewa; Lam, Vo; Johansson, Monika

    2012-08-01

    Streptococcus (Str.) agalactiae is a contagious mastitis bacterium, often associated with cases of subclinical mastitis. Different mastitis bacteria have been evaluated previously from a diagnostic point of view, but there is a lack of knowledge concerning their effect on milk composition. Protein composition is important in achieving optimal yield and texture when milk is processed to fermented products, such as cheese and yoghurt, and is thus of great economic value. The aim of this in vitro study was to evaluate protein degradation mainly caused by exogenous proteases originating from naturally occurring Str. agalactiae. The samples were incubated at 37°C to imitate degradation caused by the bacteria in the udder. Protein degradation caused by different strains of Str. agalactiae was also investigated. Protein degradation was observed to occur when Str. agalactiae was added to milk, but there were variations between strains of the bacteria. Caseins, the most economically important proteins in milk, were degraded up to 75% in milk inoculated with Str. agalactiae in relation to sterile ultra-high temperature (UHT) milk, used as control milk. The major whey proteins, α-lactalbumin and β-lactoglobulin, were degraded up to 21% in relation to the sterile control milk. These results suggest that different mastitis bacteria but also different strains of mastitis bacteria should be evaluated from a milk quality perspective to gain knowledge about their ability to degrade the economically important proteins in milk. PMID:22850579

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

    PubMed Central

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

    2008-01-01

    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. PMID:19165789

  14. Antihypertensive peptides derived from bovine casein and whey proteins.

    PubMed

    Saito, Tadao

    2008-01-01

    Peptides play an important primary role as a supply of essential amino acids and a source of nitrogen. Recent studies have 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. Various physiologically functional peptides, such as opioid, immunostimulating, mineral carrier, ACE inhibitory, antihypertensive, and antimicrobial peptides, have been derived from milk protein: both caseins and whey proteins (Meisel, 1998; Korhonen & Pihlanto-Leppälä, 2001). Milk is known to be a rich source for the supply of bioactive peptides compared to other protein sources such as animal and fish meat, wheat, and soybean proteins. Among the bioactive peptides, ACE inhibitory peptides and antihypertensive peptides have been extensively researched worldwide, because hypertension is a major risk factor in cardiovascular disease, such as heart disease (FitzGerald & Meisel, 2000; Kitts & Weiler, 2003). We discuss the isolation, utilization, and application of bioactive peptides, especially ACE inhibitory peptides and antihypertensive peptides including our recent human studies on their use as a functional food material. PMID:18183935

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

    PubMed Central

    2011-01-01

    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. PMID:22035425

  16. Protein solubility modeling.

    PubMed

    Agena, S M; Pusey, M L; Bogle, I D

    1999-07-20

    A thermodynamic framework (UNIQUAC model with temperature dependent parameters) is applied to model the salt-induced protein crystallization equilibrium, i.e., protein solubility. The framework introduces a term for the solubility product describing protein transfer between the liquid and solid phase and a term for the solution behavior describing deviation from ideal solution. Protein solubility is modeled as a function of salt concentration and temperature for a four-component system consisting of a protein, pseudo solvent (water and buffer), cation, and anion (salt). Two different systems, lysozyme with sodium chloride and concanavalin A with ammonium sulfate, are investigated. Comparison of the modeled and experimental protein solubility data results in an average root mean square deviation of 5.8%, demonstrating that the model closely follows the experimental behavior. Model calculations and model parameters are reviewed to examine the model and protein crystallization process. PMID:10397850

  17. Protein solubility modeling

    NASA Technical Reports Server (NTRS)

    Agena, S. M.; Pusey, M. L.; Bogle, I. D.

    1999-01-01

    A thermodynamic framework (UNIQUAC model with temperature dependent parameters) is applied to model the salt-induced protein crystallization equilibrium, i.e., protein solubility. The framework introduces a term for the solubility product describing protein transfer between the liquid and solid phase and a term for the solution behavior describing deviation from ideal solution. Protein solubility is modeled as a function of salt concentration and temperature for a four-component system consisting of a protein, pseudo solvent (water and buffer), cation, and anion (salt). Two different systems, lysozyme with sodium chloride and concanavalin A with ammonium sulfate, are investigated. Comparison of the modeled and experimental protein solubility data results in an average root mean square deviation of 5.8%, demonstrating that the model closely follows the experimental behavior. Model calculations and model parameters are reviewed to examine the model and protein crystallization process. Copyright 1999 John Wiley & Sons, Inc.

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

    PubMed

    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

    2010-12-01

    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

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

    PubMed Central

    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

    2010-01-01

    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

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

    PubMed

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

    2011-04-01

    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 03-140 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 (1710(9) ; 3110(9) ?l/mol), and lesser affinity to beta-casein (4010(8) ; 7010(7) ?l/mol) and insulin (2610(8) ; 1610(8) ?l/mol). No children with milk-reactive IAA developed autoantibodies to other islet autoantigens or diabetes (median follow-up 98 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

  1. Association analysis of bovine bactericidal/permeability-increasing protein gene polymorphisms with somatic cell score in Holstein cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bactericidal/permeability-increasing (BPI) protein is expressed primarily in bovine neutrophils and epithelial cells and functions as a binding protein of bacterial lipopolysaccharide produced by Gram-negative bacteria. The protein is important in host defense against bacterial infections and may pl...

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

    PubMed Central

    Choi, Sangho

    2012-01-01

    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. PMID:22705734

  3. Cathepsin S from bovine spleen. Purification, distribution, intracellular localization and action on proteins.

    PubMed Central

    Kirschke, H; Wiederanders, B; Brmme, D; Rinne, A

    1989-01-01

    Cathepsin S was detected in bovine kidney, spleen, lymph nodes and lung by immunochemical methods. The immunostaining of cathepsin S in kidney was concentrated to the cells of the proximal tubule, where the enzyme was present in cytoplasmic granules. The purification method for cathepsin S from bovine spleen involved (NH4)2SO4 fractionation, chromatography on CM-Sephadex C-50, gel filtration on Sephacryl S-200 and chromatofocusing (pH 8.0-6.0). The enzyme was partially destroyed by autolysis of the homogenate at pH 4.2. The isoelectric point of cathepsin S was 7.0. Cathepsin S was found to hydrolyse proteins at a similar rate to cathepsin L below pH 7.0. At pH values of 7.0-7.5 cathepsin S retained most of its activity, whereas cathepsin L was completely inactive. Images Fig. 1. Fig. 2. Fig. 3. Fig. 6. PMID:2690828

  4. Protein structure modeling with MODELLER.

    PubMed

    Webb, Benjamin; Sali, Andrej

    2014-01-01

    Genome sequencing projects have resulted in a rapid increase in the number of known protein sequences. In contrast, only about one-hundredth of these sequences have been characterized at atomic resolution using experimental structure determination methods. Computational protein structure modeling techniques have the potential to bridge this sequence-structure gap. In this chapter, we present an example that illustrates the use of MODELLER to construct a comparative model for a protein with unknown structure. Automation of a similar protocol has resulted in models of useful accuracy for domains in more than half of all known protein sequences. PMID:24573470

  5. Protein structure modeling with MODELLER.

    PubMed

    Eswar, Narayanan; Eramian, David; Webb, Ben; Shen, Min-Yi; Sali, Andrej

    2008-01-01

    Genome sequencing projects have resulted in a rapid increase in the number of known protein sequences. In contrast, only about one-hundredth of these sequences have been characterized using experimental structure determination methods. Computational protein structure modeling techniques have the potential to bridge this sequence-structure gap. This chapter presents an example that illustrates the use of MODELLER to construct a comparative model for a protein with unknown structure. Automation of similar protocols (correction of protcols) has resulted in models of useful accuracy for domains in more than half of all known protein sequences. PMID:18542861

  6. Sequence and pattern of expression of a bovine homologue of a human mitochondrial transport protein associated with Grave's disease.

    PubMed

    Fiermonte, G; Runswick, M J; Walker, J E; Palmieri, F

    1992-01-01

    A human cDNA has been isolated previously from a thyroid library with the aid of serum from a patient with Grave's disease. It encodes a protein belonging to the mitochondrial metabolite carrier family, referred to as the Grave's disease carrier protein (GDC). Using primers based on this sequence, overlapping cDNAs encoding the bovine homologue of the GDC have been isolated from total bovine heart poly(A)+ cDNA. The bovine protein is 18 amino acids shorter than the published human sequence, but if a frame shift requiring the removal of one nucleotide is introduced into the human cDNA sequence, the human and bovine proteins become identical in their C-terminal regions, and 308 out of 330 amino acids are conserved over their entire sequences. The bovine cDNA has been used to investigate the expression of the GDC in various bovine tissues. In the tissues that were examined, the GDC is most strongly expressed in the thyroid, but substantial amounts of its mRNA were also detected in liver, lung and kidney, and lesser amounts in heart and skeletal muscle. PMID:1457817

  7. Study on the interaction between bovine serum albumin and 4?-azido-2?-deoxyfluoroarabinocytidine or analogs by spectroscopy and molecular modeling

    NASA Astrophysics Data System (ADS)

    Wang, Ruiyong; Wang, Xiaogai; Li, Zhigang; Xie, Yuanzhe; Yang, Lingling; Shi, Jie; Chang, Junbiao

    2014-11-01

    The binding of 4?-azido-2?-deoxyfluoroarabinocytidine (FNC) or analogs (cytidine and 5?-cytidylate monophosphate) to bovine serum albumin (BSA) was investigated by fluorescence, UV-vis absorption spectroscopy and molecular modeling. The three compounds quenched the intrinsic fluorescence of BSA and the results revealed the presence of static quenching mechanism. The positive ?H and positive ?S for the systems suggested that the hydrophobic forces stabilized the interaction between the compounds and protein. Results also showed that FNC was the weakest quencher.

  8. Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

    NASA Astrophysics Data System (ADS)

    Lemaire-Vieille, Catherine; Schulze, Tobias; Podevin-Dimster, Valérie; Follet, Jérome; Bailly, Yannick; Blanquet-Grossard, Françoise; Decavel, Jean-Pierre; Heinen, Ernst; Cesbron, Jean-Yves

    2000-05-01

    The expression of the cellular form of the prion protein (PrPc) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrPc is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrPc, a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5' untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrPc at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.

  9. Distribution of G/sub o. cap alpha. / mRNA and protein in bovine tissues

    SciTech Connect

    Price, S.R.; Tsai, S.C.; Adamik, R.; Angus, C.W.; Van Meurs, K.P.; Czarnecki, S.; Bruckwick, E.C.; Moss, J.; Vaughan, M.

    1987-05-01

    G/sub o..cap alpha../ is a 39 kDa guanyl nucleotide-binding protein similar in structure and function to G/sub s..cap alpha../ and G/sub i..cap alpha../ in the adenylate cyclase complex and transducin (G/sub t..cap alpha../) in the retinal photon receptor system. A bovine retinal cDNA clone, lambdaG09, that encodes the complete amino acid sequence of G/sub o..cap alpha../ has been isolated. Nick-translated lambdaG09 cDNA and a 5' end-labeled oligonucleotide probe complementary to a 24 base sequence unique to G/sub o..cap alpha../ were used as probes for Northern analysis of poly(A)/sup +/ RNA from bovine tissues. A major 4.0 kb mRNA was detected in brain and retina and in lesser amounts in heart. Several smaller mRNAs also hybridized with both probes in these tissues and in liver and lung. G/sub o..cap alpha../ protein was identified using rabbit polyclonal antibodies directed against purified bovine G/sub o..cap alpha../ and pertussis toxin-catalyzed (/sup 32/P)ADP-ribosylation. Soluble and membrane proteins were incubated with toxin and (/sup 32/P)NAD and then separated by gel electrophoresis before transfer to nitrocellulose for immunoreaction and subsequent autoradiography. A radiolabeled and immunoreactive 39 kDa membrane protein was found principally in retina and brain, and to a lesser extent, in heart. Thus, in the tissues examined, distribution of the 4.0 kb mRNA parallels that of the immunoreactive G/sub o..cap alpha../ with relatively small amounts in heart and larger amounts in brain and retina.

  10. Computer Models of Proteins

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Dr. Marc Pusey (seated) and Dr. Craig Kundrot use computers to analyze x-ray maps and generate three-dimensional models of protein structures. With this information, scientists at Marshall Space Flight Center can learn how proteins are made and how they work. The computer screen depicts a proten structure as a ball-and-stick model. Other models depict the actual volume occupied by the atoms, or the ribbon-like structures that are crucial to a protein's function.

  11. Serum-derived bovine immunoglobulin/protein isolate: postulated mechanism of action for management of enteropathy

    PubMed Central

    Petschow, Bryon W; Burnett, Bruce; Shaw, Audrey L; Weaver, Eric M; Klein, Gerald L

    2014-01-01

    The health and performance of the gastrointestinal tract is influenced by the interaction of a variety of factors, including diet, nutritional status, genetics, environment, stress, the intestinal microbiota, immune status, and gut barrier. Disruptions in one or more of these factors can lead to enteropathy or intestinal disorders that are known to occur in concert with certain disease states or conditions such as irritable bowel syndrome or human immunodeficiency virus (HIV) infection. Nutritional support in the form of a medical food along with current therapies could help manage the adverse effects of enteropathy, which include effects on nutrient digestion, absorption, and metabolism, as well as utilization of nutrients from foodstuffs. Numerous studies have demonstrated that oral administration of plasma- or serum-derived protein concentrates containing high levels of immunoglobulins can improve weight management, normalize gut barrier function, and reduce the severity of enteropathy in animals. Recent trials in humans provide preliminary evidence that a serum-derived bovine immunoglobulin/protein isolate is safe and improves symptoms, nutritional status, and various biomarkers associated with enteropathy in patients with HIV infection or diarrhea-predominant irritable bowel syndrome. This review summarizes data from preclinical and clinical studies with immunoglobulin-containing plasma/serum protein concentrates, with a focus on the postulated mode of action of serum-derived bovine immunoglobulin/protein isolate for patients with enteropathy. PMID:24904221

  12. Identification of the glycosylation pattern of meat proteins in tissues from bovine myelin.

    PubMed

    Katsiferis, Theofanis; Wiethlter, Horst

    2010-08-01

    A variant form of Creutzfeld-Jacob disease in humans is associated with the consumption of food contaminated with the bovine central neural system. This has focused attention on the need for procedures to detect tissues of the neural system in meat and meat products. A method was developed for the identification of myelin glycoproteins in bovine neural tissue. The glycosylated structures of glycoproteins in different protein mixtures from central nervous system (CNS) and peripheral nervous system (PNS) isolated from bovine myelin were identified by the specific lectin-glycoprotein reactions. Digoxigenin-labeled lectins bonded to the terminal glycoconjugate sequences of nine CNS glycoproteins ranging from 15 to 200 kDa and four PNS glycoproteins ranging from 22 to 105 kDa. Datura stramonium (DSA) recognized the epitope Galbeta1-4GlcNAc by two CNS and three PNS glycoproteins. Maackia amurensis (MAA) recognized the epitope NeuAcalpha2-3Gal by four CNS and two PNS glycoproteins. The peanut Arachis hypogaea (PNA) reacted with the high molecular CNS glycoprotein (200 kDa) with the sequence Galbeta1-3GalNAc. Galanthus nivalis (GNA) bonded to mannose subunits linked alpha1-3 and alpha1-6 by six CNS glycoproteins with molecular weights between 17 and 200 kDa. Four of these glycoproteins were recognized from monoclonal antibodies against the "HNK-1 epitope". PMID:20513571

  13. Prolonging hypothermic storage (4 C) of bovine embryos with fish antifreeze protein.

    PubMed

    Ideta, Atsushi; Aoyagi, Yoshito; Tsuchiya, Kanami; Nakamura, Yuuki; Hayama, Kou; Shirasawa, Atsushi; Sakaguchi, Kenichiro; Tominaga, Naomi; Nishimiya, Yoshiyuki; Tsuda, Sakae

    2015-01-01

    Embryos obtained via superovulation are necessary for mammalian artificial reproduction, and viability is a key determinant of success. Nonfreezing storage at 4 C is possible, but currently used storage solutions can maintain embryo viability for only 24-48 h. Here we found that 10 mg/ml antifreeze protein (AFP) dissolved in culture medium 199 with 20% (v/v) fetal bovine serum and 25 mM HEPES could keep bovine embryos alive for 10 days at 4 C. We used a recombinant AFP isolated from the notched-fin eelpout (Zoarces elongatus Kner). Photomicroscopy indicated that the AFP-embryo interaction was enhanced at 37 C. Embryos pre-warmed with the AFP solution at 37 C for 60 min maintained high viability, whereas those that were not pre-warmed could live no longer than 7 days. Thus, short-term storage of bovine embryos was achieved by a combination of AFP-containing medium and controlled pre-warming. PMID:25311466

  14. Prolonging hypothermic storage (4 C) of bovine embryos with fish antifreeze protein

    PubMed Central

    IDETA, Atsushi; AOYAGI, Yoshito; TSUCHIYA, Kanami; NAKAMURA, Yuuki; HAYAMA, Kou; SHIRASAWA, Atsushi; SAKAGUCHI, Kenichiro; TOMINAGA, Naomi; NISHIMIYA, Yoshiyuki; TSUDA, Sakae

    2014-01-01

    Embryos obtained via superovulation are necessary for mammalian artificial reproduction, and viability is a key determinant of success. Nonfreezing storage at 4 C is possible, but currently used storage solutions can maintain embryo viability for only 24–48 h. Here we found that 10 mg/ml antifreeze protein (AFP) dissolved in culture medium 199 with 20% (v/v) fetal bovine serum and 25 mM HEPES could keep bovine embryos alive for 10 days at 4 C. We used a recombinant AFP isolated from the notched-fin eelpout (Zoarces elongatus Kner). Photomicroscopy indicated that the AFP–embryo interaction was enhanced at 37 C. Embryos pre-warmed with the AFP solution at 37 C for 60 min maintained high viability, whereas those that were not pre-warmed could live no longer than 7 days. Thus, short-term storage of bovine embryos was achieved by a combination of AFP-containing medium and controlled pre-warming. PMID:25311466

  15. Qualitative Analyses of Protein Phosphorylation in Bovine Pluripotent Stem Cells Generated from Embryonic Fibroblasts.

    PubMed

    Nong, W; Xie, T S; Li, L Y; Lu, A G; Mo, J; Gou, Y F; Lan, G; Jiang, H; Len, J; Li, M M; Jiang, Q Y; Huang, B

    2015-12-01

    Pluripotent stem cells (PSCs) generated from somatic cells via ectopic expression of specific transcription factors provide an unlimited cell resource for regenerative medicine and transgenic breeding. Here, we describe the successful generation of bovine induced PSCs (biPSCs) from foetal fibroblasts by lentivirus-mediated delivery of bovine pluripotency reprogramming factors (PRFs) OCT3/4, SOX2, KLF4, c-MYC, NANOG and LIN28. The generated biPSCs resembled embryonic stem cells (ESCs) in their gene expression profiles, self-renewal capabilities and proliferation, as well as maintenance of a normal karyotype and differentiation into diverse cell types of all three germ layers both in vitro and in vivo. Qualitative phosphoproteomics of biPSCs revealed a large number of phosphorylated proteins, which might be related to the control of biPSCs status. The successful generation of biPSCs and the analysis of their phosphoproteome would further our understanding of the epigenetic mechanisms underlying iPSC pluripotency, thus promoting their application in bovine transgenic breeding and marking avenues for future research. PMID:26493745

  16. Sequestration of bovine seminal plasma proteins by different assemblies of phosphatidylcholine: A new technical approach.

    PubMed

    Le Guillou, J; Ropers, M-H; Gaillard, C; David-Briand, E; van Leeuwen-Ibarrola, J; Desherces, S; Schmitt, E; Bencharif, D; Amirat-Briand, L; Anton, M; Tainturier, D

    2016-04-01

    Binder of SPerm (BSP) proteins, the main proteins from bovine seminal plasma, are known to partially intercalate into the outer leaflet of the spermatozoa membrane and bind to choline-containing lipids being present therein. This insertion generates a negative effect on semen quality after cryopreservation by inducing an early-stage capacitation of spermatozoa. The assumption of surface properties exhibited by BSP proteins was checked by tensiometry measurements: BSP proteins are highly surface active. This suggests that BSP proteins can reach the interface covered by phospholipids not only by interactions between one and each other but also due to their own surface activity. The insertion of BSP proteins into the lipid domains outer leaflet of spermatozoa was reproduced on a biomimetic system such as Langmuir monolayers. The insertion of BSP proteins can be performed in the compressible fluid domains which contain choline-bearing lipids. Monolayer films were used as well to study the complexation of BSP proteins by two phospholipid assemblies: low density lipoprotein (LDLs) from egg yolk or liposomes produced from egg phospholipids. Irrespective of the phospholipid structure (lipoprotein or liposome), BSP was hindered to alter the structure of the membrane. Only the overall ratio BSP proteins:phosphatidylcholine was important. The difference between the two sequestering agents lies on their surface properties: LDL have a strong tendency to merge with the outer layer whereas liposomes mainly remain in the bulk on the same time scale. PMID:26628332

  17. Identification and quantification of major bovine milk proteins by liquid chromatography.

    PubMed

    Bordin, G; Cordeiro Raposo, F; de la Calle, B; Rodriguez, A R

    2001-08-31

    In the field of food quality, bovine milk products are of particular interest due to the social and economic importance of the dairy products market. However, the risk of fraudulent manipulation is high in this area, for instance, replacing milk powder by whey is very interesting from an economic point of view. Therefore, there is a need to have suitable analytical methods available for the determination of all milk components, which is currently not the case, especially for the main proteins. The detection of potential manipulations requires then a clear analytical characterisation of each type of bovine milk, what constitutes the goal of this work. The separation of the major milk proteinic components has been carried out by ion-pair reversed-phase HPLC with photodiode array detection, using a C4 column. The overall optimisation has been achieved using a statistical experimental design procedure. The identification of each protein was ascertained using retention times, peak area ratios and second derivative UV spectra. Quantification was based on calibration curves drawn using purified proteins. Major sources of uncertainty were identified and the full uncertainty budget was established. The procedure was initially developed using the skimmed milk powder certified reference material CRM 063R and then applied to various types of commercial milks as well as to raw milk. The method is able to separate and quantify the seven major proteins (K-casein, alphas2-casein, alphas1-casein, beta-casein, alpha-lactalbumin, beta-lactoglobulin B and beta-lactoglobulin A) in one run and also to provide precise determinations of the total protein concentration. These are important results towards the further development of a reference method for major proteins in milk. In addition, the use of a certified material reference is suggested in order to make comparisons of method performances possible. PMID:11589472

  18. Study of the protein-bound fraction of calcium, iron, magnesium and zinc in bovine milk

    NASA Astrophysics Data System (ADS)

    Silva, Fernando V.; Lopes, Gisele S.; Nbrega, Joaquim A.; Souza, Gilberto B.; Nogueira, Ana Rita A.

    2001-10-01

    Two approaches were used to study the interaction of Ca, Fe, Mg and Zn with bovine milk proteins by inductively coupled plasma optical emission spectrometry (ICPOES). Selective separations in bovine milk samples were accomplished employing an acid protein precipitation using 100 g l -1 trichloroacetic acid (TCA), and an enzymatic protein hydrolysis using 50 g l -1 pepsin (PEP) solution, respectively. The results were compared with total mineral contents determined after microwave-assisted acid digestion. The results obtained by enzymatic and acid precipitation evidenced the different interaction forms of Ca, Fe, Mg and Zn in the system formed by milk components. Iron was not solubilized by the TCA treatment, but was recovered completely after the enzymatic treatment. Quantitative recoveries of Ca, Mg and Zn were obtained using both approaches, showing that these analytes were bound to milk compounds affected by either treatment. Calcium, Mg and Zn are mainly associated with colloidal calcium phosphate and Fe is bound to the backbone of the casein polypeptide chain, cleaved by pepsin enzyme. The proposed approaches could be used to assess the complexity of these chemical interactions.

  19. The application of the high-performance ion-exchange chromatography for the analysis of bovine milk proteins.

    PubMed

    Bican, P

    1985-07-15

    A high-performance ion-exchange chromatographic (HPIEC) method for the analysis of milk proteins is described. The technique provides an alternative to other separation methods. The resolution obtained for both the proteins of the casein group and bovine lactoserum is comparable with that of electrophoretic analysis on polyacrylamide gels and low-pressure chromatography. PMID:4007137

  20. Factors regulating the bovine, caprine, rat and human ovarian aromatase promoters in a bovine granulosa cell model.

    PubMed

    Sahmi, Fatiha; Nicola, Edmir S; Zamberlam, Gustavo O; Gonalves, Paulo D B; Vanselow, Jens; Price, Christopher A

    2014-05-01

    The ovarian promoter of the primate and rodent genes encoding cytochrome P450 aromatase (CYP19A1) are robustly responsive to forskolin in luteinized cell models, whereas the ruminant ovarian promoter is minimally active. We explored this discrepancy by investigating the activity of the bovine ovarian promoter in two bovine granulosa cell models, luteinizing and non-luteinizing cells in vitro. In non-luteinizing cells, both FSH and IGF1 increased abundance of transcripts derived from the ovarian promoter. Comparison of the activity of promoters of several species in response to transcription factors (forskolin, NR5A2, FOXL2) in luteinizing cells demonstrated that a rat ovarian promoter-luciferase reporter was regulated mainly by forskolin (18-fold increase over basal expression) and addition of NR5A2 or FOXL2 had no further effect. Activity of a human promoter was significantly increased by NR5A2 plus forskolin (153-fold) compared with forskolin alone (71-fold over basal); addition of FOXL2 did not significantly increase promoter activity. Forskolin alone provoked minor activation of caprine and bovine promoter reporters (3-fold over basal), and addition of NR5A2 increased activity (7- to 11-fold). When forskolin, NR5A2 and FOXL2 treatments were combined, the activity of the caprine and bovine promoters increased to 20- and 34-fold, respectively. These data suggest that a major reason why CYP19A1 is not expressed in luteinized cells (and the corpus luteum) of ruminants may be the stimulatory effect of FOXL2, which does not appear to be the case in the human and rat. PMID:24556528

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

    SciTech Connect

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

    1988-12-01

    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.

  2. Expression of the ABC transport proteins MDR1 (ABCB1) and BCRP (ABCG2) in bovine rumen.

    PubMed

    Haslam, I S; Simmons, N L

    2014-07-01

    Rumen fermentation of plant-based forage in bovines is the major site for generation and absorption of short-chain fatty acids. Consequentially, the rumen is also the site for initial exposure to toxins released from diet. Accordingly, we have investigated the expression of bovine ABC transporters in the rumen associated with cytoprotection against xenobiotic exposure, namely MDR1 (ABCB1), MRP2 (ABCC2) and BCRP (ABCG2). Bovine rumen samples from the ventral sac were obtained post-mortem from a commercial slaughterhouse after humane killing. Rumen papilla samples were then prepared for total RNA isolation for RT-PCR, SDS-PAGE/Western blotting and immunohistochemistry. PCR products of the predicted size were observed for both MDR1 and BCRP, but not for MRP2 using bovine-specific primers. ?-actin was used as a control transcript. Western blot analysis using C219 primary monoclonal antibody revealed MDR1 protein expression in bovine rumen (Mapp, of ~170-180kD). Immunolocalisation of MDR1 using UIC2 monoclonal antibody within cryosections of bovine rumen showed extensive membrane staining in the cells of the stratum granulosum, stratum spinosum and stratum basale. MDR1 expression was absent from outer stratum corneum. Protein expression and immunolocalisation were also confirmed for BCRP, with prevalent staining in the stratum basale, becoming weaker in the stratum spinosum and stratum granulosum. PMID:24747985

  3. Identification of bovine sperm acrosomal proteins that interact with a 32-kDa acrosomal matrix protein.

    PubMed

    Nagdas, Subir K; Smith, Linda; Medina-Ortiz, Ilza; Hernandez-Encarnacion, Luisa; Raychoudhury, Samir

    2016-03-01

    Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction comprises three major (54, 50, and 45 kDa) and several minor (38-19 kDa) polypeptides. The set of minor polypeptides (38-19 kDa) termed "OMCrpf polypeptides" is selectively solubilized by high-pH extraction (pH 10.5), while the three major polypeptides (55, 50, and 45 kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32 kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38-19-kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent-soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment, whereas IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidylcholine (LPC)-induced acrosome reaction, whereas the IZUMO1 and lactadherin polypeptides remain associated to the particulate fraction. Almost entire population of bovine sperm IZUMO1 relocates to the equatorial segment during the LPC-induced acrosome reaction. We propose that the interaction of OMC32 matrix polypeptide with detergent-soluble acrosomal proteins regulates the release of hydrolases/other acrosomal protein(s) during the acrosome reaction. PMID:26897631

  4. 87-kDa protein, a major specific substrate for protein kinase C: purification from bovine brain and characterization

    SciTech Connect

    Albert, K.A.; Nairn, A.C.; Greengard, P.

    1987-10-01

    The 87-kDa protein, a major specific substrate for protein kinase C, has been purified 500-fold to apparent homogeneity from bovine forebrain supernatant. The purification procedure included batch adsorption to DE-52 (DEAE-cellulose), (NH/sub 4/)/sub 2/SO/sub 4/ precipitation, and chromatography on DEAE-Sephacel, Bio-Gel HTP (hydroxylapatite), Sephacryl S-400, and fast protein liquid chromatography ProRPC. The amino acid composition was notable for its high proportion of alanine (28.6 mol%) and its enrichment in glutamate/glutamine (18.1 mol%), glycine (12.6 mol%), and proline (11.3 mol%). The partial specific volume was 0.702 ml/g; the Stokes radius and sedimentation coefficient were 85 A and 2.11 S, respectively. Although the relative molecular mass of the protein on NaDodSO/sub 4//8% PAGE was 87-90 kDa, the molecular mass as determined from the above values was 68 kDa. The frictional ratio was 3.2, and the axial ratio was 60, indicating that the 87-kDa protein is an extremely elongated monomer. The purified 87-kDa protein was phosphorylated by purified protein kinase C to a stoichiometry of 2.2 mol of /sup 32/P per mol of 87-kDa protein (calculated using a value of 68 kDa for the molecular mass). Phosphorylation was exclusively on serine residues.

  5. Nicotinic Acid Increases Adiponectin Secretion from Differentiated Bovine Preadipocytes through G-Protein Coupled Receptor Signaling

    PubMed Central

    Kopp, Christina; Hosseini, Afshin; Singh, Shiva P.; Regenhard, Petra; Khalilvandi-Behroozyar, Hamed; Sauerwein, Helga; Mielenz, Manfred

    2014-01-01

    The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum) is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA) is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A) ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX) to verify the involvement of GPR signaling, and treated with 10 or 15 M NA for 12 or 24 h. NA increased adiponectin concentrations (p ? 0.001) and the mRNA abundances of GPR109A (p ? 0.05) and chemerin (p ? 0.01). Pre-incubation with PTX reduced the adiponectin response to NA (p ? 0.001). The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows. PMID:25411802

  6. A new bovine conjunctiva model shows that Listeria monocytogenes invasion is associated with lysozyme resistance.

    PubMed

    Warren, Jessica; Owen, A Rhys; Glanvill, Amy; Francis, Asher; Maboni, Grazieli; Nova, Rodrigo J; Wapenaar, Wendela; Rees, Catherine; Ttemeyer, Sabine

    2015-08-31

    Listerial keratoconjunctivitis ('silage eye') is a wide spread problem in ruminants causing economic losses to farmers and impacts negatively on animal welfare. It results from direct entry of Listeria monocytogenes into the eye, often following consumption of contaminated silage. An isolation protocol for bovine conjunctival swabbing was developed and used to sample both infected and healthy eyes bovine eyes (n=46). L. monocytogenes was only isolated from one healthy eye sample, and suggests that this organism can be present without causing disease. To initiate a study of this disease, an infection model was developed using isolated conjunctiva explants obtained from cattle eyes post slaughter. Conjunctiva were cultured and infected for 20 h with a range of L. monocytogenes isolates (n=11), including the healthy bovine eye isolate and also strains isolated from other bovine sources, such as milk or clinical infections. Two L. monocytogenes isolates (one from a healthy eye and one from a cattle abortion) were markedly less able to invade conjunctiva explants, but one of those was able to efficiently infect Caco2 cells indicating that it was fully virulent. These two isolates were also significantly more sensitive to lysozyme compared to most other isolates tested, suggesting that lysozyme resistance is an important factor when infecting bovine conjunctiva. In conclusion, we present the first bovine conjunctiva explant model for infection studies and demonstrate that clinical L. monocytogenes isolates from cases of bovine keratoconjunctivitis are able to infect these tissues. PMID:25778543

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

    PubMed Central

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

    1998-01-01

    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. PMID:9499124

  8. Photoimmunological properties of borage in bovine neutrophil in vitro model.

    PubMed

    Asadollahi, Firouzeh; Mehrzad, Jalil; Chaichi, Mohammad Javad; Taghavi Razavizadeh, Alireza

    2015-10-01

    Borage (Echium amoenum fisch) is one of the most commonly used medicinal plants, and has long been used as a traditional herbal medicine for many (non)infectious diseases in Iran. Study on photoredox and photoimmunology of borage is little. Natural immunomodulatory plants with minimal adverse/toxic effects could help boost animal health and, ultimately, public health. To determine the effect of borage on the functions of key circulating innate immune cells, effects of borage extract (BE) on bovine neutrophils (PMN) photoredox and phagocytosis events were evaluated using an in vitro model system. Blood PMN isolated from healthy high yielding dairy cows (n = 8/treatment) were pre-incubated with BE and the impact on phagocytosis-dependent-and-independent cellular chemiluminescence (CL), phagocytosis, killing of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), fluorescence-based PMN H2O2 production and necrosis were assessed. Relative to control (no BE) PMN, treatment with BE significantly increased phagocytosis-dependent-and-independent PMN CL (>10-15% increase). While BE also led to increased PMN H2O2 production, necrosis was also surprisingly higher in these cells. Phagocytosis and killing of both E. coli and S. aureus by PMN treated with BE was substantially higher than that by control PMN. The increased photoimmunobiological events especially intracellular CL, intracellular H2O2 formation, and phagocytic capacity of BE-treated PMN support the potential immunotherapeutic implications of borage and its components for particularly immunocompromised animals and humans. PMID:26334939

  9. Studies on the protein-bound chondroitin sulphate of bovine cortical bone

    PubMed Central

    Herring, G. M.

    1968-01-01

    A fraction containing chondroitin sulphate, isolated from bovine cortical bone under mild conditions, was separated by ion-exchange chromatography into three fractions with apparent homogeneity on electrophoresis and ultracentrifugation. Two of these appeared to consist of chondroitin sulphate bound to a glycoprotein `core' that had similarities to the bone sialoprotein described previously. The differences in composition of the two fractions were considered to be due to variation in the number or lengths of the polysaccharide chains. The presence of xylose and the alkali-lability of the bond between protein and polysaccharide suggested the presence of a xylosylserine linkage. The third fraction had the properties of a relatively pure chondroitin sulphate which contained a small amount of peptide. These fractions differed considerably from the proteinpolysaccharide complexes of epiphysial and other cartilages, and their relevance to the possible role of glycosaminoglycans is discussed. ImagesFig. 5. PMID:5642623

  10. Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

    PubMed Central

    Sudiman, Jaqueline; Sutton-McDowall, Melanie L.; Ritter, Lesley J.; White, Melissa A.; Mottershead, David G.; Thompson, Jeremy G.; Gilchrist, Robert B.

    2014-01-01

    Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/? FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/? FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.73.9%, 43.54.2%) compared to controls (43.32.4%, 28.93.7%) and to mature GDF9+FSH (36.13.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies. PMID:25058588

  11. Charged Gold Nanoparticles with Essentially Zero Serum Protein Adsorption in Undiluted Fetal Bovine Serum

    PubMed Central

    Murthy, Avinash K.; Stover, Robert J.; Hardin, William G.; Schramm, Robert; Nie, Golay D.; Gourisankar, Sai; Truskett, Thomas M.; Sokolov, Konstantin V.; Johnston, Keith P.

    2013-01-01

    The adsorption of even a single serum protein molecule on a gold nanosphere used in biomedical imaging may increase the size too much for renal clearance. Herein, we design charged ~5 nm Au nanospheres coated with binary mixed charge ligand monolayers that do not change in size upon incubation in pure fetal bovine serum (FBS). This lack of protein adsorption is unexpected given the Au surface is moderately charged. The mixed charge monolayers are comprised of anionic citrate ligands modified by place exchange with naturally-occurring amino acids: either cationic lysine or zwitterionic cysteine ligands. The zwitterionic tips of either the lysine or cysteine ligands interact weakly with the proteins and furthermore increase the distance between the buried charges closer to the Au surface and the interacting sites on the protein surface. The ~5 nm nanospheres were assembled into ~20 nm diameter nanoclusters with strong NIR absorbance (of interest in biomedical imaging and therapy) with a biodegradable polymer, PLA(1k)-b-PEG(10k)-b-PLA(1k). Upon biodegradation of the polymer in acidic solution, the nanoclusters dissociated into primary ~5 nm Au nanospheres, which also did not adsorb any detectable serum protein in undiluted FBS. PMID:23565806

  12. The Quaternary Structure of the Recombinant Bovine Odorant-Binding Protein Is Modulated by Chemical Denaturants

    PubMed Central

    Stepanenko, Olga V.; Stepanenko, Olesya V.; Staiano, Maria; Kuznetsova, Irina M.; Turoverov, Konstantin K.; DAuria, Sabato

    2014-01-01

    A large group of odorant-binding proteins (OBPs) has attracted great scientific interest as promising building blocks in constructing optical biosensors for dangerous substances, such as toxic and explosive molecules. Native tissue-extracted bovine OBP (bOBP) has a unique dimer folding pattern that involves crossing the ?-helical domain in each monomer over the other monomers ?-barrel. In contrast, recombinant bOBP maintaining the high level of stability inherent to native tissue bOBP is produced in a stable native-like state with a decreased tendency for dimerization and is a mixture of monomers and dimers in a buffered solution. This work is focused on the study of the quaternary structure and the folding-unfolding processes of the recombinant bOBP in the absence and in the presence of guanidine hydrochloride (GdnHCl). Our results show that the recombinant bOBP native dimer is only formed at elevated GdnHCl concentrations (1.5 M). This process requires re-organizing the protein structure by progressing through the formation of an intermediate state. The bOBP dimerization process appears to be irreversible and it occurs before the protein unfolds. Though the observed structural changes for recombinant bOBP at pre-denaturing GdnHCl concentrations show a local character and the overall protein structure is maintained, such changes should be considered where the protein is used as a sensitive element in a biosensor system. PMID:24409322

  13. Mesoporous calcium silicate for controlled release of bovine serum albumin protein.

    PubMed

    Xue, Weichang; Bandyopadhyay, Amit; Bose, Susmita

    2009-06-01

    The purpose of this study is to synthesize mesoporous calcium silicate (CS or wollastonite, CaSiO(3)) and evaluate its possible application in protein/drug delivery. First, calcium silicate was synthesized by wet chemical method and then mesoporosity was created by acid modification of the synthesized CS particle using hydrochloric acid at pH 7, 4.5, and 0.5. The results showed that a hydrated silica gel with abundant Si-OH functional group formed on the surface of calcium silicate due to acid modification. This surface layer had mesoporous structure, with pore diameter between 4 and 5 nm. BET specific average surface area increased to 221, 333, and 356 m(2) g(-1) due to acid modification at pH 7, 4.5, and 0.5, respectively, whereas the surface area for unmodified CS particles was 65 m(2) g(-1). Protein adsorption studies indicated that mesoporous CS has higher ability to adsorb bovine serum albumin and lysozyme compared to unmodified particles. The release kinetics showed that proteins on mesoporous CS released sequentially over one week, whereas the proteins on unmodified particle followed burst release kinetics within a few hours. Human osteoblast cell-material interaction study showed that these materials were biocompatible and promoted excellent bone cell proliferation. In summary, this work has demonstrated the potential to produce mesoporous CS as a carrier for protein/drug delivery for bone regeneration and other biomedical applications. PMID:19249262

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

    SciTech Connect

    Darchen, F.; Hammel, F.; Monteils, M.P.; Scherman, D. ); Zahraoui, A.; Tavitian, A. )

    1990-08-01

    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.

  15. In vivo delivery of bovine viral diahorrea virus, E2 protein using hollow mesoporous silica nanoparticles

    NASA Astrophysics Data System (ADS)

    Mahony, D.; Cavallaro, A. S.; Mody, K. T.; Xiong, L.; Mahony, T. J.; Qiao, S. Z.; Mitter, N.

    2014-05-01

    Our work focuses on the application of mesoporous silica nanoparticles as a combined delivery vehicle and adjuvant for vaccine applications. Here we present results using the viral protein, E2, from bovine viral diarrhoea virus (BVDV). BVDV infection occurs in the target species of cattle and sheep herds worldwide and is therefore of economic importance. E2 is a major immunogenic determinant of BVDV and is an ideal candidate for the development of a subunit based nanovaccine using mesoporous silica nanoparticles. Hollow type mesoporous silica nanoparticles with surface amino functionalisation (termed HMSA) were characterised and assessed for adsorption and desorption of E2. A codon-optimised version of the E2 protein (termed Opti-E2) was produced in Escherichia coli. HMSA (120 nm) had an adsorption capacity of 80 μg Opti-E2 per mg HMSA and once bound E2 did not dissociate from the HMSA. Immunisation studies in mice with a 20 μg dose of E2 adsorbed to 250 μg HMSA was compared to immunisation with Opti-E2 (50 μg) together with the traditional adjuvant Quillaja saponaria Molina tree saponins (QuilA, 10 μg). The humoral responses with the Opti-E2/HMSA nanovaccine although slightly lower than those obtained for the Opti-E2 + QuilA group demonstrated that HMSA particles are an effective adjuvant that stimulated E2-specific antibody responses. Importantly the cell-mediated immune responses were consistently high in all mice immunised with Opti-E2/HMSA nanovaccine formulation. Therefore we have shown the Opti-E2/HMSA nanoformulation acts as an excellent adjuvant that gives both T-helper 1 and T-helper 2 mediated responses in a small animal model. This study has provided proof-of-concept towards the development of an E2 subunit nanoparticle based vaccine.Our work focuses on the application of mesoporous silica nanoparticles as a combined delivery vehicle and adjuvant for vaccine applications. Here we present results using the viral protein, E2, from bovine viral diarrhoea virus (BVDV). BVDV infection occurs in the target species of cattle and sheep herds worldwide and is therefore of economic importance. E2 is a major immunogenic determinant of BVDV and is an ideal candidate for the development of a subunit based nanovaccine using mesoporous silica nanoparticles. Hollow type mesoporous silica nanoparticles with surface amino functionalisation (termed HMSA) were characterised and assessed for adsorption and desorption of E2. A codon-optimised version of the E2 protein (termed Opti-E2) was produced in Escherichia coli. HMSA (120 nm) had an adsorption capacity of 80 μg Opti-E2 per mg HMSA and once bound E2 did not dissociate from the HMSA. Immunisation studies in mice with a 20 μg dose of E2 adsorbed to 250 μg HMSA was compared to immunisation with Opti-E2 (50 μg) together with the traditional adjuvant Quillaja saponaria Molina tree saponins (QuilA, 10 μg). The humoral responses with the Opti-E2/HMSA nanovaccine although slightly lower than those obtained for the Opti-E2 + QuilA group demonstrated that HMSA particles are an effective adjuvant that stimulated E2-specific antibody responses. Importantly the cell-mediated immune responses were consistently high in all mice immunised with Opti-E2/HMSA nanovaccine formulation. Therefore we have shown the Opti-E2/HMSA nanoformulation acts as an excellent adjuvant that gives both T-helper 1 and T-helper 2 mediated responses in a small animal model. This study has provided proof-of-concept towards the development of an E2 subunit nanoparticle based vaccine. Electronic supplementary information (ESI) available: Desorption studies of Opti-E2 bound HMSA. See DOI: 10.1039/c4nr01202j

  16. A bovine whey protein extract can enhance innate immunity by priming normal human blood neutrophils.

    PubMed

    Rusu, Daniel; Drouin, Réjean; Pouliot, Yves; Gauthier, Sylvie; Poubelle, Patrice E

    2009-02-01

    Bovine milk-derived products, in particular whey proteins, exhibit beneficial properties for human health, including the acquired immune response. However, their effects on innate immunity have received little attention. Neutrophils are key cells of innate defenses through their primary functions of chemotaxis, phagocytosis, oxidative burst, and degranulation. A whey protein extract (WPE) purified from bovine lactoserum was evaluated for its direct and indirect effects on these primary functions of normal human blood neutrophils in vitro. Although WPE had no direct effects on primary functions, a 24-h pretreatment of neutrophils with WPE was associated with a significant and dose-dependent increase of their chemotaxis, superoxide production, and degranulation in response to N-formyl-methionine-leucine-phenylalanine, as well as of their phagocytosis of bioparticles. The pretreatment increased the surface expression of CD11b, CD16B, and CD32A receptors. The major WPE protein components beta-lactoglobulin (beta-LG) and alpha-lactalbumin (alpha-LA) were the main active fractions having an additive effect on human neutrophils that became more responsive to a subsequent stimulation. This effect on NADPH oxidase activity was associated with translocation of p47(phox) to plasma membrane. Glycomacropeptide, a peptide present in measurable amounts in WPE products, was able to enhance the individual effect of beta-LG or alpha-LA on neutrophils. The present data suggest that WPE, through beta-LG and alpha-LA, has the capacity to enhance or "prime" human neutrophil responses to a subsequent stimulation, an effect that could be associated with increased innate defenses in vivo. PMID:19106313

  17. Molecular Dynamics Simulations Capture the Misfolding of the Bovine Prion Protein at Acidic pH

    PubMed Central

    Cheng, Chin Jung; Daggett, Valerie

    2014-01-01

    Bovine spongiform encephalopathy (BSE), or mad cow disease, is a fatal neurodegenerative disease that is transmissible to humans and that is currently incurable. BSE is caused by the prion protein (PrP), which adopts two conformers; PrPC is the native innocuous form, which is ?-helix rich; and PrPSc is the ?-sheet rich misfolded form, which is infectious and forms neurotoxic species. Acidic pH induces the conversion of PrPC to PrPSc. We have performed molecular dynamics simulations of bovine PrP at various pH regimes. An acidic pH environment induced conformational changes that were not observed in neutral pH simulations. Putative misfolded structures, with nonnative ?-strands formed in the flexible N-terminal domain, were found in acidic pH simulations. Two distinct pathways were observed for the formation of nonnative ?-strands: at low pH, hydrophobic contacts with M129 nucleated the nonnative ?-strand; at mid-pH, polar contacts involving Q168 and D178 facilitated the formation of a hairpin at the flexible N-terminus. These mid- and low pH simulations capture the process of nonnative ?-strand formation, thereby improving our understanding of how PrPC misfolds into the ?-sheet rich PrPSc and how pH factors into the process. PMID:24970211

  18. Molecular dynamics simulations capture the misfolding of the bovine prion protein at acidic pH.

    PubMed

    Cheng, Chin Jung; Daggett, Valerie

    2014-01-01

    Bovine spongiform encephalopathy (BSE), or mad cow disease, is a fatal neurodegenerative disease that is transmissible to humans and that is currently incurable. BSE is caused by the prion protein (PrP), which adopts two conformers; PrPC is the native innocuous form, which is ?-helix rich; and PrPSc is the ?-sheet rich misfolded form, which is infectious and forms neurotoxic species. Acidic pH induces the conversion of PrPC to PrPSc. We have performed molecular dynamics simulations of bovine PrP at various pH regimes. An acidic pH environment induced conformational changes that were not observed in neutral pH simulations. Putative misfolded structures, with nonnative ?-strands formed in the flexible N-terminal domain, were found in acidic pH simulations. Two distinct pathways were observed for the formation of nonnative ?-strands: at low pH, hydrophobic contacts with M129 nucleated the nonnative ?-strand; at mid-pH, polar contacts involving Q168 and D178 facilitated the formation of a hairpin at the flexible N-terminus. These mid- and low pH simulations capture the process of nonnative ?-strand formation, thereby improving our understanding of how PrPC misfolds into the ?-sheet rich PrPSc and how pH factors into the process. PMID:24970211

  19. Response of the rabbit metaphysis to implants of bovine bone morphogenetic protein (bBMP)

    SciTech Connect

    Nilsson, O.; Urist, M.R.

    1985-05-01

    The response of protodifferentiated and differentiated bone cells to bovine bone morphogenetic protein (bBMP) was observed in implants in the adult rabbit distal femoral metaphysis. Bovine serum albumin and denatured bBMP were implanted in the contralateral femur of controls. The changes of the bone marrow reflected the reaction of protodifferentiated cells. The changes in preexisting trabecular bone tissue reflected the reaction of differentiated cells to bBMP. /sup 45/Ca radioisotope quantitative methods demonstrated that the bone morphogenetic response was superimposed upon the reaction to the injury of surgical implantation. By the end of the fourth week, roentgenograms and histologic sections showed larger deposits of intrametaphyseal cartilage and bone in bBMP than in control implanted femurs. By the end of the eighth week, bone formation was associated with remodeling of the entire distal femur and expansion of the external diameter of the metaphysis. These observations indicate the need for investigation of perisinusoid and perivascular cells of periosteum, endosteum, and marrow stroma that are undifferentiated with respect to cartilage and bone but are principal target tissues for BMP.

  20. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    PubMed Central

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-01-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene. Images PMID:3039499

  1. Removal of Available Decorin Core-Protein from Powdered Bovine Hide by Treatments used to Process Intact Hides into Leather

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using a modification of a previously developed sandwich Elisa procedure to measure decorin core-protein (DCP), we determined the available decorin content of a sample of raw powdered bovine hide before and after treatment with the reagents used in the early steps of the process for converting a hide...

  2. BOVINE SOMATOTROPIN AND RUMEN UNDERGRADABLE PROTEIN EFFECTS ON ORGAN AND TISSUE WEIGHTS AND COMPONENTS OF GROWTH OF PREPUBERTAL HEIFERS.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were to determine the effects of recombinant bovine somatotropin (bST) and added dietary rumen undegradable protein (RUP) on organ and tissue weights and body composition in growing dairy heifers. Twenty-four heifers (6 per treatment group) were randomly assigned to tre...

  3. Isothermal titration calorimetric studies on the interaction of the major bovine seminal plasma protein, PDC-109 with phospholipid membranes.

    PubMed

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

    2011-01-01

    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. PMID:22022488

  4. Evaluation of Cocktails with Recombinant Proteins of Mycobacterium bovis for a Specific Diagnosis of Bovine Tuberculosis

    PubMed Central

    Mon, María Laura; Moyano, Roberto Damián; Viale, Mariana Noelia; Colombatti Olivieri, María Alejandra; Gamietea, Ignacio José; Montenegro, Valeria Noely; Alonso, Bernardo; Santangelo, María de la Paz; Singh, Mahavir; Duran, Rosario; Romano, María Isabel

    2014-01-01

    The Delayed type hypersensitivity skin test (DTH) and interferon-gamma assay are used for the diagnosis of bovine tuberculosis (TBB). The specificity of these diagnoses, however, is compromised because both are based on the response against purified protein derivative of Mycobacterium bovis (PPD-B). In this study, we assessed the potential of two cocktails containing M. bovis recombinant proteins: cocktail 1 (C1): ESAT-6, CFP-10 and MPB83 and cocktail 2 (C2): ESAT-6, CFP-10, MPB83, HspX, TB10.3, and MPB70. C1, C2, and PPD-B showed similar response by DTH in M. bovis-sensitized guinea pigs. Importantly, C1 induced a lower response than PPD-B in M. avium-sensitized guinea pigs. In cattle, C1 displayed better performance than PPD-B and C2; indeed, C1 showed the least detection of animals either vaccinated or Map-infected. To optimize the composition of the cocktails, we obtained protein fractions from PPD-B and tested their immunogenicity in experimentally M. bovis-infected cattle. In one highly reactive fraction, seven proteins were identified. The inclusion of FixB in C1 enhanced the recognition of M. bovis-infected cattle without compromising specificity. Our data provide a promising basis for the future development of a cocktail for TBB detection without interference by the presence of sensitized or infected animals with other mycobacteria. PMID:25110654

  5. Requirement for nuclear autoantigenic sperm protein mRNA expression in bovine preimplantation development.

    PubMed

    Nagatomo, Hiroaki; Kohri, Nanami; Akizawa, Hiroki; Hoshino, Yumi; Yamauchi, Nobuhiko; Kono, Tomohiro; Takahashi, Masashi; Kawahara, Manabu

    2016-03-01

    Nuclear autoantigenic sperm protein (NASP) is associated with DNA replication, cell proliferation, and cell cycle progression through its specific binding to histones. The aim of this study was to examine the roles of NASP in bovine preimplantation embryonic development. Using NASP gene knockdown (KD), we confirmed the reduction of NASP messenger RNA (mRNA) expression during preimplantation development. NASP KD did not affect cleavage but significantly decreased development of embryos into the blastocyst stage. Furthermore, blastocyst hatching was significantly decreased in NASP KD embryos. Cell numbers in the inner cell mass of NASP KD blastocysts were also decreased compared to those of controls. These results suggest that NASP mRNA expression is required for preimplantation development into the blastocyst stage in cattle. PMID:26690724

  6. Investigation of the protein-peptide composition of the secretory granules of bovine lactogenic hormone

    SciTech Connect

    Nagornaya, L.V.; Kizim, E.A.; Kravtsov, G.M.; Tsibezov, V.V.; Vinogradov, V.A.

    1987-02-20

    By a combination of methods of gel chromatography, fluorometric analysis, and electrophoresis in polyacrylamide gel with immunochemical identification, they investigated the protein-peptide composition of the secretory granules of lactogenic hormone (LTH), isolated from the bovine anterior pituitary. It was established that the content of peptides in the granules is less than 3% of the content of immunoreactive LTH. Using gel chromatography, the monomer of the hormone and two immunoreactive forms with molecular weight 42 and 65 kilodaltons were detected in the secondary granules. The content of the forms with respect to immunoreactivity was 90, 3, and 7%, respectively. Four immunoreactive forms of LTH were detected in the secretory granules by the method of electrophoresis in polyacrylamide gel followed by immunochemical identification.

  7. Non-covalent nanodiamondpolymer dispersions and electrostatic immobilization of bovine serum albumin protein

    NASA Astrophysics Data System (ADS)

    Skaltsas, T.; Pispas, S.; Tagmatarchis, N.

    2015-11-01

    Nanodiamonds (NDs) lack efficient dispersion, not only in solvents but also in aqueous media. The latter is of great importance, considering the inherent biocompatibility of NDs and the plethora of suitable strategies for immobilizing functional biomolecules. In this work, a series of polymers was non-covalently interacted with NDs, forming NDpolymer ensembles, and their dispersibility and stability was examined. Dynamic light scattering gave valuable information regarding the size of the ensembles in liquid phase, while their morphology was further examined by high-resolution transmission electron microscopy imaging. In addition, thermal analysis measurements were applied to collect information on the thermal behavior of NDs and their ensembles and to calculate the amount of polymer interacting with the NDs, as well as the dispersibility values of the NDpolymer ensembles. Finally, the bovine serum albumin protein was electrostatically bound to a NDpolymer ensemble in which the polymeric moiety was carrying quaternized pyridine units.

  8. Grafting of bovine serum albumin proteins on plasma-modified polymers for potential application in tissue engineering

    NASA Astrophysics Data System (ADS)

    Kaslkov, Nikola Slepi?kov; Slepi?ka, Petr; Kolsk, Zde?ka; Hoda?ov, Petra; Ku?kov, t?pnka; vor?k, Vclav

    2014-04-01

    In this work, an influence of bovine serum albumin proteins grafting on the surface properties of plasma-treated polyethylene and poly- l-lactic acid was studied. The interaction of the vascular smooth muscle cells with the modified polymer surface was determined. The surface properties were characterized by X-ray photoelectron spectroscopy, atomic force microscopy, nano-LC-ESI-Q-TOF mass spectrometry, electrokinetic analysis, and goniometry. One of the motivations for this work is the idea that by the interaction of the cell with substrate surface, the proteins will form an interlayer between the cell and the substrate. It was proven that when interacting with the plasma-treated high-density polyethylene and poly- l-lactic acid, the bovine serum albumin protein is grafted on the polymer surface. Since the proteins are bonded to the substrate surface, they can stimulate cell adhesion and proliferation.

  9. Alterations in bovine platelet function and acute phase proteins induced by Pasteurella haemolytica A1.

    PubMed Central

    Cheryk, L A; Hooper-McGrevy, K E; Gentry, P A

    1998-01-01

    Platelet function was assessed by aggregometry in 10 Holstein calves before and after exposure to Pasteurella haemolytica (biotype A, serotype 1) by intrabronchial challenge. At 24 h after exposure the platelets had become more reactive to stimulation with known platelet agonists such as adenosine diphosphate (ADP) and platelet-activating factor (PAF) and the platelet aggregates that formed were more resistant to disaggregation. The activation of platelets was an early response in the challenged calves as platelet function had returned to pretreatment levels 72 h after exposure to the bacteria while the acute phase reactant proteins, haptoglobin and fibrinogen, were approaching their peak values and alpha 2-macroglobulin levels had also risen significantly (P < 0.05) at this time. The plasma levels of these proteins were still elevated and albumin levels were depressed 6 d post-treatment. At post-mortem all calves exhibited pneumonic tissue damage. When P. haemolytica leukotoxin was added directly to bovine platelet suspensions both spontaneous aggregation and an increase in the aggregation response to ADP and PAF stimulation were observed. The morphological appearance of the platelet aggregates exhibited the typical pattern for bovine platelets with 2 distinct zones of cells being visible within each aggregate. One zone contained platelets in which the cytoplasmic granules were still evident and the other zone contained irregularly shaped platelets devoid of granular content. In the latter zone, discrete gaps, or pores, were evident in the plasma membrane of numerous platelets. This pore formation is characteristic of leukotoxin action and is not observed in ADP or PAF induced aggregates. Images Figure 2. PMID:9442932

  10. A residue level protein-protein interaction model in electrolyte solutions

    NASA Astrophysics Data System (ADS)

    Song, Xueyu

    2014-03-01

    The osmotic second virial coefficients B2 are directly related to the solubility of protein molecules in electrolyte solutions and can be useful to narrow down the search parameter space of protein crystallization conditions. Using a residue level model of protein-protein interaction in electrolyte solutions B2 of bovine pancreatic trypsin inhibitor and lysozyme in various solution conditions such as salt concentration, pH and temperature are calculated using an extended Fast Multipole Methods in combination with the boundary element formulation. Overall, the calculated B2 are well correlated with the experimental observations for various solution conditions. In combination with our previous work on the binding affinity calculations of protein complexes it is demonstrated that our residue level model can be used as a reliable model to describe protein-protein interaction in solutions.

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

    PubMed Central

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

    2002-01-01

    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. PMID:12379688

  12. Study on the interaction between bovine serum albumin and 4'-azido-2'-deoxyfluoroarabinocytidine or analogs by spectroscopy and molecular modeling.

    PubMed

    Wang, Ruiyong; Wang, Xiaogai; Li, Zhigang; Xie, Yuanzhe; Yang, Lingling; Shi, Jie; Chang, Junbiao

    2014-11-11

    The binding of 4'-azido-2'-deoxyfluoroarabinocytidine (FNC) or analogs (cytidine and 5'-cytidylate monophosphate) to bovine serum albumin (BSA) was investigated by fluorescence, UV-vis absorption spectroscopy and molecular modeling. The three compounds quenched the intrinsic fluorescence of BSA and the results revealed the presence of static quenching mechanism. The positive ?H and positive ?S for the systems suggested that the hydrophobic forces stabilized the interaction between the compounds and protein. Results also showed that FNC was the weakest quencher. PMID:24971719

  13. Influence of blood proteins in the in vitro adhesion of Staphylococcus epidermidis to teflon, polycarbonate, polyethylene and bovine pericardium.

    PubMed

    Carballo, J; Ferreirs, C M; Criado, M T

    1991-12-01

    The influence of human plasma proteins (fibrinogen, albumin and fibronectin) on the adherence of Staphylococcus epidermis to teflon, polyethylene, polycarbonate and bovine pericardium was studied in an in vitro quantitative assay by scintillation counting. Bacterial adhesion was generally reduced by the presence of protein during the adherence assay except in the case of bovine pericardium, in which adherence remained almost unaffected. The effect of these plasma proteins on bacterial surface properties resulted in strong increases of surface charge as measured by ion-exchange chromatography and with no effect on hydrophobicity, estimated as contact angles. Adherence was not found to be correlated with these two properties, suggesting that bacteria-surface interactions must not be simplified to the influence of interfacial forces. PMID:1812542

  14. Anaplasma marginale major surface protein 1a: a marker of strain diversity with implications for control of bovine anaplasmosis.

    PubMed

    Cabezas-Cruz, Alejandro; de la Fuente, Jos

    2015-04-01

    Classification of bacteria is challenging due to the lack of a theory-based framework. In addition, the adaptation of bacteria to ecological niches often results in selection of strains with diverse virulence, pathogenicity and transmission characteristics. Bacterial strain diversity presents challenges for taxonomic classification, which in turn impacts the ability to develop accurate diagnostics and effective vaccines. Over the past decade, the worldwide diversity of Anaplasma marginale, an economically important tick-borne pathogen of cattle, has become apparent. The extent of A. marginale strain diversity, formerly underappreciated, has contributed to the challenges of classification which, in turn, likely impacts the design and development of improved vaccines. Notably, the A. marginale surface protein 1a (MSP1a) is a model molecule for these studies because it serves as a marker for strain identity, is both an adhesin necessary for infection of cells and an immuno-reactive protein and is also an indicator of the evolution of strain diversity. Herein, we discuss a molecular taxonomic approach for classification of A. marginale strain diversity. Taxonomic analysis of this important molecule provides the opportunity to understand A. marginale strain diversity as it relates geographic and ecological factors and to the development of effective vaccines for control of bovine anaplasmosis worldwide. PMID:25802034

  15. Cloning and characterization of two recombinant Neospora protein fragments and their use in serodiagnosis of bovine neosporosis.

    PubMed Central

    Louie, K; Sverlow, K W; Barr, B C; Anderson, M L; Conrad, P A

    1997-01-01

    Bovine neosporosis causes fetal abortion and/or congenital neurologic disease in cattle. For the serodiagnosis of this parasitic disease, two immunodominant clones from a bovine Neospora lambda gt11 library were identified, characterized, and expressed as recombinant proteins for the development of an enzyme-linked immunosorbent assay (ELISA). These two clones, designated N54 and N57, were 29 and 20 kDa, respectively, when expressed as histidine fusion proteins from the pRSET expression vector. Antibodies to recombinant protein N54 recognized five major bands from a Neospora tachyzoite lysate with molecular masses of 97, 87, 77, 67, and 64 kDa. Antibodies to recombinant protein N57 recognized four primary bands with molecular masses of 34, 31, 30, and 28 kDa. When a defined "gold standard" panel of bovine sera from confirmed Neospora-positive and Neospora-negative cattle were characterized by immunoblotting, 57 of the 60 Neospora-positive serum samples recognized proteins with the molecular masses of the N54 heptuplet. Binding to the N57 quadruplet was more variable. The same gold standard panel was used to evaluate and compare an N54-based ELISA, an N57-based ELISA, and a whole-tachyzoite lysate-based ELISA. The sensitivities and specificities were 95 and 96% (N54 ELISA), 82 and 93% (N57 ELISA), and 74 and 93% (lysate ELISA). Thus, compared to the whole-tachyzoite lysate-based ELISA, both recombinant-protein-based ELISAs had higher sensitivities and higher or the same specificities and can be used to replace the whole-tachyzoite lysate ELISA for the serodiagnosis of bovine neosporosis. PMID:9384291

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

    PubMed

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

    2011-08-31

    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

  17. An Impedance Network Model for the Electrical Properties of a Single-Protein Nanodevice

    NASA Astrophysics Data System (ADS)

    Akimov, V.; Alfinito, E.; Pennetta, C.; Reggiani, L.; Minic, J.; Gorojankina, T.; Pajot-Augy, E.; Salesse, R.

    A simple impedance network model to mimic the electrical properties of a single protein molecule nanodevice is presented. Within this model two sensing proteins of the GPCR family (bovine light-sensing rhodopsin and rat 17 olfactory receptor) in ground and activated states are studied. We predict a detectable impedance difference between ground and activated states of both proteins so proteins of this family are promising candidates as nanobiosensors.

  18. Analysis of the binding of bovine and human fibrinogen to ferritin: evidence that fibrinogen is a common ferritin-binding protein in mammals.

    PubMed

    Okada, Akiko; Yoshikawa, Yasunaga; Watanabe, Kiyotaka; Orino, Koichi

    2015-08-01

    Both human and horse fibrinogen are heme-binding proteins, and horse fibrinogen also exhibits heme-mediated ferritin binding. This study found that bovine and human fibrinogen are heme-mediated ferritin-binding proteins and demonstrated direct binding of bovine ferritin to protoporphyrin (PPIX) and its derivatives or to Zn ions. Binding of bovine and human fibrinogen to bovine spleen ferritin coated on microtiter plate wells was detected using an anti-human fibrinogen antibody, and this binding was inhibited in a dose-dependent manner by hemin (iron-PPIX) and also inhibited by Zn-PPIX. PPIX showed less of an inhibitory effect on the binding of bovine and human fibrinogen to bovine ferritin. The inhibitory effect of Sn-PPIX was similar to that of PPIX, but with respect to human fibrinogen, PPIX did not inhibit the binding of human fibrinogen to ferritin. Bovine fibrinogen immobilized on CNBr-activated Sepharose 4B beads showed affinity for hemin, Sn-PPIX, Zn-PPIX, and iron-free PPIX in the order Sn-PPIX < iron-free PPIX < hemin < Zn-PPIX. The fibrinogen beads also directly bound to zinc ions. These results suggest that bovine fibrinogen is a heme- and zinc-binding protein and that binding of circulating mammalian fibrinogen to ferritin is heme mediated. PMID:25860295

  19. Recombinant Mycoplasma mycoides proteins elicit protective immune responses against contagious bovine pleuropneumonia.

    PubMed

    Nkando, Isabel; Perez-Casal, Jose; Mwirigi, Martin; Prysliak, Tracy; Townsend, Hugh; Berberov, Emil; Kuria, Joseph; Mugambi, John; Soi, Reuben; Liljander, Anne; Jores, Joerg; Gerdts, Volker; Potter, Andrew; Naessens, Jan; Wesonga, Hezron

    2016-03-01

    Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a devastating respiratory disease mainly affecting cattle in sub-Saharan Africa. The current vaccines are based on live-attenuated Mmm strains and present problems with temperature stability, duration of immunity and adverse reactions, thus new vaccines are needed to overcome these issues. We used a reverse vaccinology approach to identify 66 Mmm potential vaccine candidates. The selection and grouping of the antigens was based on the presence of specific antibodies in sera from CBPP-positive animals. The antigens were used to immunize male Boran cattle (Bos indicus) followed by a challenge with the Mmm strain Afadé. Two of the groups immunized with five proteins each showed protection after the Mmm challenge (Groups A and C; P<0.05) and in one group (Group C) Mmm could not be cultured from lung specimens. A third group (Group N) showed a reduced number of animals with lesions and the cultures for Mmm were also negative. While immunization with some of the antigens conferred protection, others may have increased immune-related pathology. This is the first report that Mmm recombinant proteins have been successfully used to formulate a prototype vaccine and these results pave the way for the development of a novel commercial vaccine. PMID:26964722

  20. Preparation of quadri-subtype influenza virus-like particles using bovine immunodeficiency virus gag protein.

    PubMed

    Tretyakova, Irina; Hidajat, Rachmat; Hamilton, Garrett; Horn, Noah; Nickols, Brian; Prather, Raphael O; Tumpey, Terrence M; Pushko, Peter

    2016-01-01

    Influenza VLPs comprised of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins have been previously used for immunological and virological studies. Here we demonstrated that influenza VLPs can be made in Sf9 cells by using the bovine immunodeficiency virus gag (Bgag) protein in place of M1. We showed that Bgag can be used to prepare VLPs for several influenza subtypes including H1N1 and H10N8. Furthermore, by using Bgag, we prepared quadri-subtype VLPs, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specific antisera. The content and co-localization of each HA subtype within the quadri-subtype VLP were evaluated. Electron microscopy showed that Bgag-based VLPs resembled influenza virions with the diameter of 150-200nm. This is the first report of quadri-subtype design for influenza VLP and the use of Bgag for influenza VLP preparation. PMID:26529299

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

    PubMed Central

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

    2002-01-01

    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 α-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. PMID:12097596

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

    TOXLINE Toxicology Bibliographic Information

    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 JC; Heremans H; Kettmann R

    1998-04-30

    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.

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

    PubMed

    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

    1998-04-30

    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

  4. Comparative proteome profiling of bovine and human Staphylococcus epidermidis strains for screening specifically expressed virulence and adaptation proteins.

    PubMed

    Siljamki, Pia; Varmanen, Pekka; Kankainen, Matti; Pyrl, Satu; Karonen, Taru; Iivanainen, Antti; Auvinen, Petri; Paulin, Lars; Laine, Pia K; Taponen, Suvi; Simojoki, Heli; Sukura, Antti; Nyman, Tuula A; Savijoki, Kirsi

    2014-08-01

    The present study reports a comparative proteome cataloging of a bovine mastitis and a human-associated Staphylococcus epidermidis strain with a specific focus on surfome (cell-wall bound and extracellular) proteins. Protein identification by 1DE coupled with LC-MS/MS analyses resulted in 1400 and 1287 proteins from the bovine (PM221) and human (ATCC12228) strains, respectively, covering over 50% of all predicted and more than 30% of all predicted surfome proteins in both strains. Comparison of the identification results suggests elevated levels of proteins involved in adherence, biofilm formation, signal transduction, house-keeping functions, and immune evasion in PM221, whereas ATCC12228 was more effective in expressing host defense evasion proteases, skin adaptation lipases, hemagglutination, and heavy-metal resistance proteins. Phenotypic analyses showed that only PM221 displays protein- and DNA-mediated adherent growth, and that PM221 was more efficient in cleaving tributyrin, a natural compound of milk fat under low CO2 conditions. These findings are in line with the identification data and suggest that distinct expression of lipases and adhesive surfome proteins could lead to the observed phenotypes. This study is the first extensive survey of S. epidermidis proteomes to date, providing several protein candidates to be examined for their roles in adaptation and virulence in vivo. All MS data have been deposited in the ProteomeXchange with identifier PXD000404 (http://proteomecentral.proteomexchange.org/dataset/PXD000404). PMID:24909406

  5. 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

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

    2005-09-01

    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 waters 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.

  6. Stability strengths and weaknesses in protein structures detected by statistical potentials: Application to bovine seminal ribonuclease.

    PubMed

    De Laet, Marie; Gilis, Dimitri; Rooman, Marianne

    2016-01-01

    We present an in silico method to estimate the contribution of each residue in a protein to its overall stability using three database-derived statistical potentials that are based on inter-residue distances, backbone torsion angles and solvent accessibility, respectively. Residues that contribute very unfavorably to the folding free energy are defined as stability weaknesses, whereas residues that show a highly stabilizing contribution are called stability strengths. Strengths and/or weaknesses on residues that are in spatial contact are clustered into 3-dimensional (3D) stability patches. The identification and analysis of strength- and weakness-containing regions in a protein may reveal structural or functional characteristics, and/or interesting spots to introduce mutations. To illustrate the power of our method, we apply it to bovine seminal ribonuclease. This enzyme catalyzes the degradation of RNA strands, and has the peculiarity of undergoing 3D domain swapping in physiological conditions. The weaknesses and strengths were compared among the monomeric, dimeric and swapped dimeric forms. We identified weaknesses among the catalytic residues and a mixture of weaknesses and strengths among the substrate-binding residues in the three forms. In the regions involved in 3D swapping, we observed an accumulation of weaknesses in the monomer, which disappear in the dimer and especially in the swapped dimer. Moreover, monomeric homologous proteins were found to exhibit less weaknesses in these regions, whereas mutants known to favor unswapped dimerization appear stabilized in this form. Our method has several perspectives for functional annotation, rational prediction of targeted mutations, and mapping of stability changes upon conformational rearrangements. Proteins 2016; 84:143-158. 2015 Wiley Periodicals, Inc. PMID:26573727

  7. Strain rate-dependent viscohyperelastic constitutive modeling of bovine liver tissue.

    PubMed

    Roan, Esra; Vemaganti, Kumar

    2011-04-01

    The mechanical response of most soft tissue is considered to be viscohyperelastic, making the development of accurate constitutive models a challenging task. In this article, we present a constitutive model for bovine liver tissue that utilizes a viscous dissipation potential, and use it to model the response of bovine liver tissue at strain rates ranging from 0.001 to 0.04 s(-1). On the material modeling front of this study, the free energy is assumed to depend on the right Cauchy-Green deformation tensor, whereas a separate rate-dependent viscous potential is posited to characterize viscoelasticity. This viscous dissipation component is a function of the time rate of change of the right Cauchy-Green deformation tensor. On the experimental front, no-slip uniaxial compression experiments are conducted on bovine liver tissue at various strain rates. A numerical correction approach is used to account for the no-slip edge conditions, and the constitutive model is fit to the resulting corrected stress-strain data. The complete derivation of the material model, its implementation in the finite element software package ABAQUS, and a validation study are presented in this article. The results show that bovine liver tissue exhibits a strong strain-rate dependence even at the low strain rates considered here and that the proposed constitutive model is able to accurately describe this response. PMID:21052853

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

    PubMed

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

    2009-03-01

    The effects of beta-lactoglobulin (beta-LG), beta-casein (beta-CN), and kappa-CN variants and beta-kappa-CN haplotypes on the relative concentrations of the major milk proteins alpha-lactalbumin (alpha-LA), beta-LG, alpha(S1)-CN, alpha(S2)-CN, beta-CN, and kappa-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 16 years. In addition, genetic variants and casein haplotypes have a major impact on the protein composition of milk and explain a considerable part of the genetic variation in milk protein composition. The beta-LG genotype was associated with the relative concentrations of beta-LG (A > B) and of alpha-LA, alpha(S1)-CN, alpha(S2)-CN, beta-CN, and kappa-CN (B > A) but not with any milk production trait. The beta-CN genotype was associated with the relative concentrations of beta-CN and alpha(S2)-CN (A(2) > A(1)) and of alpha(S1)-CN and kappa-CN (A(1) > A(2)) and with protein yield (A(2) > A(1)). The kappa-CN genotype was associated with the relative concentrations of kappa-CN (B > E > A), alpha(S2)-CN (B > A), alpha-LA, and alpha(S1)-CN (A > B) and with protein percentage (B > A). Comparing the effects of casein haplotypes with the effects of single casein variants can provide better insight into what really underlies the effect of a variant on protein composition. We conclude that selection for both the beta-LG genotype B and the beta-kappa-CN haplotype A(2)B will result in cows that produce milk that is more suitable for cheese production. PMID:19233813

  9. The bovine kidney as an experimental model in urology: external gross anatomy.

    PubMed

    Carvalho, Francismar S; Bagetti Filho, Hlio J S; Henry, Robert W; Pereira-Sampaio, Marco A

    2009-01-01

    The objective of this work was to obtain and record detailed and accurate measurements of the bovine kidney and to compare these new data with findings in humans. Thirty-eight bovine kidneys were used. The total number of lobes, along with the number of lobes located in the cranial polar, caudal polar and hilar regions, were recorded. Several measurements of the kidneys were made and evaluated. The hilar region presents the greatest length (mean of 76.87 mm) of the 3 renal regions of the kidney. The large area of the bovine renal hilus could make access to hilar structures easier than in the human kidney. The coefficient of variation for renal length was small (8.14%), while the coefficient of variation for the lobar number was high (26.82%). The number of renal lobes ranged from 13 to 35, with a mean of 20.62. The hilar region presents the highest number of lobes, while the cranial pole presents the lowest. The number of lobes in the cranial and caudal poles increases with the width of these regions. This is different from the hilar region, in which the lobar number increases with the length of the hilus. These data indicate that the adult bovine kidney can be used as a model for certain urologic procedures, but researchers must be aware that there are some major differences between the adult bovine kidney and the human kidney, as indicated by the data reported in this paper. PMID:18824839

  10. DNA Bending is Induced in an Enhancer by the DNA-Binding Domain of the Bovine Papillomavirus E2 Protein

    NASA Astrophysics Data System (ADS)

    Moskaluk, Christopher; Bastia, Deepak

    1988-03-01

    The E2 gene of bovine papillomavirus type 1 has been shown to encode a DNA-binding protein and to trans-activate the viral enhancer. We have localized the DNA-binding domain of the E2 protein to the carboxyl-terminal 126 amino acids of the E2 open reading frame. The DNA-binding domain has been expressed in Escherichia coli and partially purified. Gel retardation and DNase I ``footprinting'' on the bovine papillomavirus type 1 enhancer identify the sequence motif ACCN6GGT (in which N = any nucleotide) as the E2 binding site. Using electrophoretic methods we have shown that the DNA-binding domain changes conformation of the enhancer by inducing significant DNA bending.

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

    PubMed

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

    1997-04-15

    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

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

    PubMed

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

    2002-11-01

    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

  13. Critical Examination of the Colloidal Particle Model of Globular Proteins

    PubMed Central

    Sarangapani, Prasad S.; Hudson, Steven D.; Jones, Ronald L.; Douglas, Jack F.; Pathak, Jai A.

    2015-01-01

    Recent studies of globular protein solutions have uniformly adopted a colloidal view of proteins as particles, a perspective that neglects the polymeric primary structure of these biological macromolecules, their intrinsic flexibility, and their ability to sample a large configurational space. While the colloidal perspective often serves as a useful idealization in many cases, the macromolecular identity of proteins must reveal itself under thermodynamic conditions in which the native state is no longer stable, such as denaturing solvents and high protein concentrations where macromolecules tend to have screened excluded volume, charge, and hydrodynamic interactions. Under extreme pH conditions, charge repulsion interactions within the protein chain can overcome the attractive hydrogen-bonding interactions, holding it in its native globular state. Conformational changes can therefore be expected to have great significance on the shear viscosity and other rheological properties of protein solutions. These changes are not envisioned in conventional colloidal protein models and we have initiated an investigation of the scattering and rheological properties of model proteins. We initiate this effort by considering bovine serum albumin because it is a globular protein whose solution properties have also been extensively investigated as a function of pH, temperature, ionic strength, and concentration. As we anticipated, near-ultraviolet circular dichroism measurements and intrinsic viscosity measurements clearly indicate that the bovine serum albumin tertiary structure changes as protein concentration and pH are varied. Our findings point to limited validity of the colloidal protein model and to the need for further consideration and quantification of the effects of conformational changes on protein solution viscosity, protein association, and the phase behavior. Small-angle Neutron Scattering measurements have allowed us to assess how these conformational changes influence protein size, shape, and interprotein interaction strength. PMID:25650939

  14. The p66Shc Adaptor Protein Controls Oxidative Stress Response in Early Bovine Embryos

    PubMed Central

    Betts, Dean H.; Bain, Nathan T.; Madan, Pavneesh

    2014-01-01

    The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 24 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 24 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos. PMID:24475205

  15. Identification of a nuclear export signal sequence for bovine papillomavirus E1 protein

    SciTech Connect

    Rosas-Acosta, German; Wilson, Van G.

    2008-03-30

    Recent studies have demonstrated nuclear export by papillomavirus E1 proteins, but the requisite export sequence(s) for bovine papillomavirus (BPV) E1 were not defined. In this report we identify three functional nuclear export sequences (NES) present in BPV E1, with NES2 being the strongest in reporter assays. Nuclear localization of BPV1 E1 was modulated by over- or under-expression of CRM1, the major cellular exportin, and export was strongly reduced by the CRM1 inhibitor, Leptomycin B, indicating that E1 export occurs primarily through a CRM1-dependent process. Consistent with the in vivo functional results, E1 bound CRM1 in an in vitro pull-down assay. In addition, sumoylated E1 bound CRM1 more effectively than unmodified E1, suggesting that E1 export may be regulated by SUMO modification. Lastly, an E1 NES2 mutant accumulated in the nucleus to a greater extent than wild-type E1, yet was defective for viral origin replication in vivo. However, NES2 exhibited no intrinsic replication defect in an in vitro replication assay, implying that nucleocytoplasmic shuttling may be required to maintain E1 in a replication competent state.

  16. Activated platelet-derived growth factor ? receptor and Ras-mitogen-activated protein kinase pathway in natural bovine urinary bladder carcinomas.

    PubMed

    Corteggio, Annunziata; Di Geronimo, Ornella; Roperto, Sante; Roperto, Franco; Borzacchiello, Giuseppe

    2012-03-01

    Bovine papillomavirus types 1 or 2 (BPV-1/2) are involved in the aetiopathogenesis of bovine urinary bladder cancer. BPV-1/2 E5 activates the platelet-derived growth factor ? receptor (PDGF?R). The aim of this study was to analyse the Ras/mitogen-activated protein kinase (MAPK) pathway in relation to activation of PDGF?R in natural bovine urinary bladder carcinomas. Co-immunoprecipitation and Western blot analysis demonstrated that recruitment of growth factor receptor bound protein 2 (GRB-2) and Sos-1 to the activated PDGF?R was increased in carcinomas compared to normal tissues. Higher grade bovine urinary bladder carcinomas were associated with activation of Ras, but not with activation of downstream mitogen-activated protein kinase/extracellular signal-regulated kinase (Mek 1/2) or extracellular signal-regulated kinase (Erk 1/2). PMID:21546288

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

    PubMed Central

    Martignani, Eugenio; Eirew, Peter; Accornero, Paolo

    2010-01-01

    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. PMID:20976049

  18. Protections of bovine serum albumin protein from damage on functionalized graphene-based electrodes by flavonoids.

    PubMed

    Sun, Bolu; Gou, Yuqiang; Xue, Zhiyuan; Zheng, Xiaoping; Ma, Yuling; Hu, Fangdi; Zhao, Wanghong

    2016-05-01

    A sensitive electrochemical sensor based on bovine serum albumin (BSA)/poly (diallyldimethylammonium chloride) (PDDA) functionalized graphene nanosheets (PDDA-G) composite film modified glassy carbon electrode (BSA/PDDA-G/GCE) had been developed to investigate the oxidative protein damage and protections of protein from damage by flavonoids. The performance of this sensor was remarkably improved due to excellent electrical conductivity, strong adsorptive ability, and large effective surface area of PDDA-G. The BSA/PDDA-G/GCE displayed the greatest degree of BSA oxidation damage at 40min incubation time and in the pH5.0 Fenton reagent system (12.5mM FeSO4, 50mM H2O2). The antioxidant activities of four flavonoids had been compared by fabricated sensor based on the relative peak current ratio of SWV, because flavonoids prevented BSA damage caused by Fenton reagent and affected the BSA signal in a solution containing Co(bpy)3(3+). The sensor was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM). UV-vis spectrophotometry and FTIR were also used to investigate the generation of hydroxyl radical and BSA damage, respectively. On the basis of results from electrochemical methods, the order of the antioxidant activities of flavonoids is as follows: (+)-catechin>kaempferol>apigenin>naringenin. A novel, direct SWV analytical method for detection of BSA damage and assessment of the antioxidant activities of four flavonoids was developed and this electrochemical method provided a simple, inexpensive and rapid detection of BSA damage and evaluation of the antioxidant activities of samples. PMID:26952415

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

    SciTech Connect

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

    1987-06-05

    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.

  20. Age-related changes in protein conformation in bovine lens crystallins.

    PubMed

    Liang, J N; Bose, S K; Chakrabarti, B

    1985-03-01

    In order to investigate the conformational changes associated with the aging process, circular dichroism (CD), absorption and fluorescence measurements of bovine lens crystallins isolated from the nucleus of old (cow) and young (calf) animals are reported. Results show considerable differences in spectroscopic parameters between the young and old alpha-crystallin; however, no such changes were observed for beta- and gamma-crystallins. Age-related changes include an increased absorption in near-u.v. and decreased intensity in the far-u.v. region; near-u.v. circular dichroism shows a considerable difference, whereas the dichroism in far-u.v. remains the same. The decrease in tryptophan fluorescence of old alpha-crystallin is of the same magnitude as is the increase in non-tryptophan fluorescence. The fluorescence of the sulfhydryl (SH) specific probe, 2-(4'-maleimidylanilino) naphthalene-6-sulfonate, indicates that accessible (to the probe) SH groups of cow alpha-crystallin are fewer than those of calf, and they are also in a more polar environment. This study demonstrates that, with aging, alpha-crystallin undergoes a change in the tertiary structure involving tryptophan, tyrosine and cysteine residues. This conformational change has been explained by the suggestion that a large portion of the protein unfolds during the aging process, resulting in a change in interaction properties between the aromatic amino acid residues and between the residues and the peptide backbone. The unfolding is also associated with the accessibility, reactivity and spatial arrangement of these residues, including the cysteine by which aggregation or cross-linking of the protein is likely to occur. PMID:4065237

  1. Fetal bovine serum xenoproteins modulate human monocyte adhesion and protein release on biomaterials in vitro

    PubMed Central

    Schmidt, David; Joyce, Evan James; Kao, Weiyuan John

    2010-01-01

    Monocyte-derived macrophages are critical in the host foreign body response to biomaterials and have been studied extensively in various culture conditions in vitro such as medium supplemented with fetal bovine serum (FBS) or autologous human serum (AHS). Since monocyte maturation into macrophages is highly plastic and may vary considerably depending on the surface, isolation procedures, and in vitro culture conditions, we hypothesize that variations in protein adsorption and serum type will greatly impact monocyte behavior in a surface-dependent manner. The impact of xenoproteins on monocyte-surface interaction is not well studied methodically and the use of AHS rather than FBS for macrophage-biomaterials studies in vitro is far from universal. The commonly used reference materials: tissue culture polystyrene (TCPS), polyethylene glycol (PEG), and poly-dimethylsiloxane (PDMS) were employed in this study and we found a 3-fold higher adherent monocyte density on TCPS when AHS was used versus FBS-supplemented medium. On PEG hydrogels, an 8-10 fold higher adhesion density was observed when AHS was employed versus FBS, while on PDMS no difference in adhesion density was observed between the two sera conditions. Additionally, the presence of lipopolysaccharide abrogated the serum-dependent effect on cell adhesion on TCPS. Significant differential variations in protein release were observed between the serum conditions on these surfaces, in particular there was a 100-fold higher concentration of growth-related oncogene for the AHS condition on PDMS even though the adhesion levels were comparable between the two serum conditions. These results emphasize the combined impact of the surface type and FBS xenoproteins in mediating the observed monocyte response to biomaterials in vitro. PMID:20837169

  2. A simple pharmacokinetic model linking plasma progesterone concentrations with the hormone released from bovine intravaginal inserts.

    PubMed

    Mariano, R N; Turino, L N; Cabrera, M I; Scndolo, D E; Maciel, M G; Grau, R J A

    2010-10-01

    On the basis of pharmacokinetic modeling, this study provides some insights into predicting in vivo plasma progesterone concentrations when using bovine intravaginal inserts for systemic progesterone delivery. More significantly, this contribution is the first attempt to build a simple pharmacokinetic model that links plasma progesterone concentrations with the hormone released from bovine intravaginal inserts. After evaluating three rival pharmacokinetic models and considering some phenomena involved in the intravaginal administration of progesterone, a primary pharmacokinetic model having a good data fitting capability with only two adjustable parameters is proposed to the above mentioned task. Kinetic parameters are given for lactating Holstein dairy cows with two levels of daily milk yields; and non-pregnant, non-lactating Holstein-Friesian cattle. Model predictions indicate the occurrence of a preferential distribution of the intravaginally administered progesterone dose through a first uterine pass effect. PMID:20307896

  3. Bovine papillomavirus type 4 L1 gene transfection in a Drosophila S2 cell expression system: absence of L1 protein expression

    PubMed Central

    Ges, Luiz Gustavo Bentim; de Freitas, Antonio Carlos; Ferraz, Oilita Pereira; Rieger, Tania Tassinari; dos Santos, Jos Ferreira; Pereira, Alexandre; Beak, Willy; Lindsey, Charles J.; de Cassia Stocco, Rita

    2008-01-01

    The development of a bovine papillomavirus (BPV) vaccine is an outstanding challenge. BPV protein L1 gene transfection in the Drosophila melanogaster S2 cell expression system failed to produce L1 protein notwithstanding correct L1 gene insertion. Severe genetic inbalance in the host cell line, including cytogenetic alterations, may account for the lack of protein expression. PMID:24031166

  4. Early Results of Novel Bovine Pericardial Patch Using Comprehensive Anticalcification Procedure in a Swine Model.

    PubMed

    Choi, Jae-Woong; Kim, Dong Jin; Kim, Dong Jung; Baik, Sun Jung; Lee, Jeong Sang; Kim, Jun Sung; Park, Kay-Hyun; Lim, Cheong; Kim, Yong Jin

    2016-01-01

    We evaluated the short-term safety and effectiveness of our comprehensive anticalcification procedure in swine model. Our comprehensive anticalcification procedure consisted of four steps, including decellularization with sodium dodecyl sulfate and tritonX-100, space filler treatment with polyethylene glycol (PEG), glutaraldehyde cross-linking with organic solvent, and detoxification with glycine. We simultaneously implanted both the commercially available bovine pericardial patch (Supple Peri-Guard) and novel bovine pericardial patch processed by the comprehensive anticalcification procedure into the main pulmonary artery in seven pigs. Every pig underwent a cardiac angiography and was killed on the postoperative day 28. The extracted patches were stained with hematoxylin and eosin. All pigs survived for 4 weeks without any complication. Cardiac angiography showed the absence of leakage and structural problem. Neointimas were formed evenly without intimal hyperplasia. There were no significant differences in the degree of inflammation, necrosis, and calcification between the novel and commercially available patch (p = 0.450, p = 0.317, p = 0.999). Novel bovine pericardial patch using comprehensive anticalcification procedure was similar to existing cardiovascular patch in early surgical results in a swine model. The comprehensive anticalcification procedure could facilitate appropriate bioprosthetic properties of the bovine pericardium. PMID:26479466

  5. Suppression and treatment of experimental allergic encephalitis in guinea-pigs with the bovine spinal cord protein (BSCP).

    PubMed Central

    MacPherson, C F

    1980-01-01

    Experimental allergic encephalitis (EAE) was suppressed in guinea-pigs that had been sensitized with 50 micrograms of bovine myelin basic protein (MyBP) in Freund's complete adjuvant (FCA) by daily injections of 500 micrograms of the bovine spinal cord protein (BSCP) in saline between day 8 (D 8) and D 23 after sensitization. Injections of 500 micrograms of bovine serum albumin in saline, or saline alone, did not suppress disease. Reversal of clinical disease was achieved with doses of 750 micrograms of BSCP in saline when treatment was begun within a day after the first sign of disease was observed. Six injections of 500 micrograms of BSCP in Freund's incomplete adjuvant (FIA) were required to suppress EAE whether BSCP-FIA injections were begun on D 0 or as late as D 8 when T cells sensitized to MyBP had invaded the central nervous system. When administration of BSCP-FIA was withheld until after disease onset, only three or four injections were needed to reverse clinical signs. BSCP appeared to be as effective quantitatively as MyBP in the suppression or treatment of disease. Because there is no cross-reaction between BSCP and MyBP at the antibody level, the present results raise the possibility that the suppressive activity of BSCP may be due to an amino acid sequence in BSCP and MyBP that is recognized only by T cells. When antigens were injected in FIA at 4 day intervals after sensitization, the onset of disease was delayed up to 5 days. Moreover, even bovine gamma-globulin appeared to have suppressive activity when injected three or four times in FIA. The findings suggest that assessments of the suppressive capacity of an antigen may be inaccurate if the antigen is injected in FIA frequently during the interval between sensitization and onset of disease. PMID:6159309

  6. Effects of metformin on bovine granulosa cells steroidogenesis: possible involvement of adenosine 5' monophosphate-activated protein kinase (AMPK).

    PubMed

    Tosca, Lucie; Chabrolle, Christine; Uzbekova, Svetlana; Dupont, Jolle

    2007-03-01

    In mammals, IGFs are important for the proliferation and steroidogenesis of ovarian cells. Metformin is an insulin sensitizer molecule used for the treatment of the infertility of women with polycystic ovary syndrome. It is, however, unclear whether metformin acts on ovarian cells. Adenosine 5' monophosphate-activated protein kinase (AMPK) is involved in metformin action in various cell types. We investigated the effects of metformin on bovine granulosa cell steroidogenesis in response to IGF1 and FSH, and studied AMPK in bovine ovaries. In granulosa cells from small follicles, metformin (10 mM) reduced production of both progesterone and estradiol and decreased the abundance of HSD3B, CYP11A1, and STAR proteins in presence or absence of FSH (10(-8) M) and IGF1 (10(-8) M). In cows, the different subunits of AMPK are expressed in various ovarian cells including granulosa and theca cells, corpus luteum, and oocytes. In bovine granulosa cells from small follicles, metformin, like AICAR (1 mM) a pharmaceutical activator of AMPK, increased phosphorylation of both Thr172 of AMPK alpha and Ser 79 of ACACA (Acetyl-CoA Carboxylase). Both metformin and AICAR treatment reduced progesterone and estradiol secretion in presence or absence of FSH and IGF1. Metformin decreased phosphorylation levels of MAPK3/MAPK1 and MAPK14 in a dose- and time-dependent manner. The adenovirus-mediated production of dominant negative AMPK abolished the effects of metformin on secretion of progesterone and estradiol and on MAPK3/MAPK1 phosphorylation but not on MAPK14 phosphorylation. Thus, in bovine granulosa cells, metformin decreases steroidogenesis and MAPK3/MAPK1 phosphorylation through AMPK activation. PMID:17123942

  7. Bovine somatotropin and rumen-undegradable protein effects on skeletal growth in prepubertal dairy heifers.

    PubMed

    Moallem, U; Dahl, G E; Duffey, E K; Capuco, A V; Erdman, R A

    2004-11-01

    The objectives of this study were to determine the effects of dietary rumen-undegradable protein (RUP) and bovine somatotropin (bST) during the period from weaning until puberty on body weight (BW) and skeletal growth rates and age at puberty. Fifty-one Holstein heifers at 90 d of age were randomly assigned to 4 treatment groups consisting of 0.1 mg/kg BW per day of bST and 2% added dietary RUP (dry matter basis) applied in a 2 x 2 factorial design (n = 13 per group, except bST with no RUP group, n = 12). From 90 to 314 d, bST increased average daily gain (ADG) by 0.07 kg/d and BW by 16.2 kg, while added RUP increased ADG by 0.10 kg/d and BW by 21.4 kg. Both bST and added RUP effects on BW and ADG were additive. Skeletal growth rates, as measured by withers height (WH) and hip height (HH) were increased by both bST and added RUP. Somatotropin and RUP increased WH by 1.8 and 2.7 cm and hip height by 2.5 and 4.0 cm, respectively, at 314 d of age. Growth curves showed that added RUP effects on rates of BW, WH, and HH growth were greatest from 90 to 150 d age and diminished thereafter, suggesting that protein was limiting during this time period. Conversely, bST effects tended to be greater as the heifers approached puberty, but only in the presence of added RUP. Age at puberty was not affected by treatment, averaging 314 d of age across treatments. From 314 to 644 d of age, rates of BW, WH, and HH growth were similar among treatment groups. However, treatment differences present at 314 d of age persisted through 644 d of age, more than 10 mo after treatments ceased. These results suggest that protein during the early postweaning period and bST during the 200 to 300 d of age period just prior to puberty could be used to accelerate simultaneous increases in both BW and skeletal growth rates in dairy heifers without reducing age at puberty. PMID:15483172

  8. Comparative protein structure modeling using MODELLER.

    PubMed

    Eswar, Narayanan; Webb, Ben; Marti-Renom, Marc A; Madhusudhan, M S; Eramian, David; Shen, Min-Yi; Pieper, Ursula; Sali, Andrej

    2007-11-01

    Functional characterization of a protein sequence is a common goal in biology, and is usually facilitated by having an accurate three-dimensional (3-D) structure of the studied protein. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3-D model for a protein that is related to at least one known protein structure. Comparative modeling predicts the 3-D structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and discusses all four steps of comparative modeling, frequently observed errors, and some applications. Modeling lactate dehydrogenase from Trichomonas vaginalis (TvLDH) is described as an example. The download and installation of the MODELLER software is also described. PMID:18429317

  9. Comparative Protein Structure Modeling Using MODELLER.

    PubMed

    Webb, Benjamin; Sali, Andrej

    2014-01-01

    Functional characterization of a protein sequence is one of the most frequent problems in biology. This task is usually facilitated by accurate three-dimensional (3-D) structure of the studied protein. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3-D model for a protein that is related to at least one known protein structure. Comparative modeling predicts the 3-D structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and discusses all four steps of comparative modeling, frequently observed errors, and some applications. Modeling lactate dehydrogenase from Trichomonas vaginalis (TvLDH) is described as an example. The download and installation of the MODELLER software is also described. PMID:25199792

  10. Comparative protein structure modeling using Modeller.

    PubMed

    Eswar, Narayanan; Webb, Ben; Marti-Renom, Marc A; Madhusudhan, M S; Eramian, David; Shen, Min-Yi; Pieper, Ursula; Sali, Andrej

    2006-10-01

    Functional characterization of a protein sequence is one of the most frequent problems in biology. This task is usually facilitated by accurate three-dimensional (3-D) structure of the studied protein. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3-D model for a protein that is related to at least one known protein structure. Comparative modeling predicts the 3-D structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and discusses all four steps of comparative modeling, frequently observed errors, and some applications. Modeling lactate dehydrogenase from Trichomonas vaginalis (TvLDH) is described as an example. The download and installation of the MODELLER software is also described. PMID:18428767

  11. Comparative Protein Structure Modeling Using Modeller

    PubMed Central

    Eswar, Narayanan; Marti-Renom, Marc A.; Madhusudhan, M.S.; Eramian, David; Shen, Min-yi; Pieper, Ursula

    2014-01-01

    Functional characterization of a protein sequence is one of the most frequent problems in biology. This task is usually facilitated by accurate three-dimensional (3-D) structure of the studied protein. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3-D model for a protein that is related to at least one known protein structure. Comparative modeling predicts the 3-D structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and discusses all four steps of comparative modeling, frequently observed errors, and some applications. Modeling lactate dehydrogenase from Trichomonas vaginalis (TvLDH) is described as an example. The download and installation of the MODELLER software is also described. PMID:18428767

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

    PubMed

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

    2006-01-01

    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

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

    PubMed Central

    2013-01-01

    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. PMID:24129059

  14. Processing of the Bovine Spongiform Encephalopathy-Specific Prion Protein by Dendritic Cells

    PubMed Central

    Rybner-Barnier, Catherine; Jacquemot, Catherine; Cuche, Céline; Doré, Grégory; Majlessi, Laleh; Gabellec, Marie-Madeleine; Moris, Arnaud; Schwartz, Olivier; Di Santo, James; Cumano, Ana; Leclerc, Claude; Lazarini, Françoise

    2006-01-01

    Dendritic cells (DC) are suspected to be involved in transmissible spongiform encephalopathies, including bovine spongiform encephalopathy (BSE). We detected the disease-specific, protease-resistant prion protein (PrPbse) in splenic DC purified by magnetic cell sorting 45 days after intraperitoneal inoculation of BSE prions in immunocompetent mice. We showed that bone marrow-derived DC (BMDC) from wild-type or PrP-null mice acquired both PrPbse and prion infectivity within 2 h of in vitro culture with a BSE inoculum. BMDC cleared PrPbse within 2 to 3 days of culture, while BMDC infectivity was only 10-fold diminished between days 1 and 6 of culture, suggesting that the infectious unit in BMDC is not removed at the same rate as PrPbse is removed from these cells. Bone marrow-derived plasmacytoid DC and bone marrow-derived macrophages (BMM) also acquired and degraded PrPbse when incubated with a BSE inoculum, with kinetics very similar to those of BMDC. PrPbse capture is probably specific to antigen-presenting cells since no uptake of PrPbse was observed when splenic B or T lymphocytes were incubated with a BSE inoculum in vitro. Lipopolysaccharide activation of BMDC or BMM prior to BSE infection resulted in an accelerated breakdown of PrPbse. Injected by the intraperitoneal route, BMDC were not infectious for alymphoid recombination-activated gene 20/common cytokine γ chain-deficient mice, suggesting that these cells are not capable of directly propagating BSE infectivity to nerve endings. PMID:16641258

  15. Bovine leukemia virus nucleocapsid protein is an efficient nucleic acid chaperone

    SciTech Connect

    Qualley, Dominic F. Sokolove, Victoria L.; Ross, James L.

    2015-03-13

    Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two other retroviral NCs: human immunodeficiency virus type 1 (HIV-1) NC, which is structurally similar to BLV NC but from a different retrovirus genus, and human T-cell leukemia virus type 1 (HTLV-1) NC, which possesses several key structural differences from BLV NC but is from the same genus. Our data show that BLV and HIV-1 NCs bind to NAs with stronger affinity in relation to HTLV-1 NC, and that they also accelerate the annealing of complementary stem-loop structures to a greater extent. Analysis of kinetic parameters derived from the annealing data suggests that while all three NCs stimulate annealing by a two-step mechanism as previously reported, the relative contributions of each step to the overall annealing equilibrium are conserved between BLV and HIV-1 NCs but are different for HTLV-1 NC. It is concluded that while BLV and HTLV-1 belong to the same genus of retroviruses, processes that rely on NC may not be directly comparable. - Highlights: • BLV NC binds strongly to DNA and RNA. • BLV NC promotes mini-TAR annealing as well as HIV-1 NC. • Annealing kinetics suggest a low degree of similarity between BLV NC and HTLV-1 NC.

  16. First Principles Predictions of the Structure and Function of G-Protein-Coupled Receptors: Validation for Bovine Rhodopsin

    PubMed Central

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

    2004-01-01

    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 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 CRMS from the crystal structure for the ligand atoms. This predicted conformation has the carbonyl O only 2.82 from the N of Lys-296. Making this Schiff base bond and minimizing leads to a final conformation only 0.62 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 from the N of Lys-296. Making this Schiff base bond and minimizing leads to a final conformation only 2.92 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

  17. Regulation and Function of Phosphorylation on VP8, the Major Tegument Protein of Bovine Herpesvirus 1

    PubMed Central

    Zhang, Kuan; Afroz, Sharmin; Brownlie, Robert; Snider, Marlene

    2015-01-01

    ABSTRACT The major tegument protein of bovine herpesvirus 1 (BoHV-1), VP8, is essential for virus replication in cattle. VP8 is phosphorylated in vitro by casein kinase 2 (CK2) and BoHV-1 unique short protein 3 (US3). In this study, VP8 was found to be phosphorylated in both transfected and infected cells but was detected as a nonphosphorylated form in mature virions. This suggests that phosphorylation of VP8 is strictly controlled during different stages of the viral life cycle. The regulation and function of VP8 phosphorylation by US3 and CK2 were further analyzed. An in vitro kinase assay, site-directed mutagenesis, and liquid chromatography-mass spectrometry were used to identify the active sites for US3 and CK2. The two kinases phosphorylate VP8 at different sites, resulting in distinct phosphopeptide patterns. S16 is a primary phosphoreceptor for US3, and it subsequently triggers phosphorylation at S32. CK2 has multiple active sites, among which T107 appears to be the preferred residue. Additionally, CK2 consensus motifs in the N terminus of VP8 are essential for phosphorylation. Based on these results, a nonphosphorylated VP8 mutant was constructed and used for further studies. In transfected cells phosphorylation was not required for nuclear localization of VP8. Phosphorylated VP8 appeared to recruit promyelocytic leukemia (PML) protein and to remodel the distribution of PML in the nucleus; however, PML protein did not show an association with nonphosphorylated VP8. This suggests that VP8 plays a role in resisting PML-related host antiviral defenses by redistributing PML protein and that this function depends on the phosphorylation of VP8. IMPORTANCE The progression of VP8 phosphorylation over time and its function in BoHV-1 replication have not been characterized. This study demonstrates that activation of S16 initiates further phosphorylation at S32 by US3. Additionally, VP8 is phosphorylated by CK2 at several residues, with T107 having the highest level of phosphorylation. Evidence for a difference in the phosphorylation status of VP8 in host cells and mature virus is presented for the first time. Phosphorylation was found to be a critical modification, which enables VP8 to attract and to redistribute PML protein in the nucleus. This might promote viral replication through interference with a PML-mediated antiviral defense. This study provides new insights into the regulation of VP8 phosphorylation and suggests a novel, phosphorylation-dependent function for VP8 in the life cycle of BoHV-1, which is important in view of the fact that VP8 is essential for virus replication in vivo. PMID:25673708

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

    PubMed Central

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

    1994-01-01

    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 PMID:8057465

  19. Serum-neutralizing antibody to VP4 and VP7 proteins in infants following vaccination with WC3 bovine rotavirus.

    PubMed Central

    Ward, R L; Knowlton, D R; Greenberg, H B; Schiff, G M; Bernstein, D I

    1990-01-01

    Serum specimens from infants 2 to 12 months old vaccinated with the WC3 bovine rotavirus were analyzed to determine the relative concentrations of neutralizing antibody to the VP4 and VP7 proteins of the vaccine virus. To do this, reassortant rotaviruses that contained the WC3 genome segment for only one of these two neutralization proteins were made. The segment for the other neutralization protein in these reassortants was from heterotypic rotaviruses that were serotypically distinct from WC3. Sera were examined from 31 infants who had no evidence of a previous rotavirus infection and the highest postvaccination WC3-neutralizing antibody titers (i.e., 160 to 600) of the 103 subjects administered the vaccine. A reassortant (3/17) that contained both neutralization proteins from the heterotypic rotaviruses, i.e., EDIM (EW strain of mouse rotavirus) VP7 and rhesus rotavirus VP4, was not neutralized by these sera (geometric mean titer [GMT], less than 20). A reassortant (E19) that contained EDIM VP7 and WC3 VP4 was also very poorly neutralized by these antisera (GMT = 20). In contrast, antibody titers to a reassortant (R20) that contained WC3 VP7 and rhesus rotavirus VP4 were higher than those against WC3 (GMTs of 458 and 313, respectively). Thus, VP7 appeared to be the dominant immunogen for production of neutralizing antibody after intestinal infection of previously uninfected infants vaccinated with WC3 bovine rotavirus. Images PMID:2159538

  20. Leucine and histidine independently regulate milk protein synthesis in bovine mammary epithelial cells via mTOR signaling pathway*

    PubMed Central

    Gao, Hai-na; Hu, Han; Zheng, Nan; Wang, Jia-qi

    2015-01-01

    The aim of this study is to investigate the effects of leucine (Leu) and histidine (His) on the expression of both the mammalian target of rapamycin (mTOR) signaling pathway-related proteins and caseins in immortalized bovine mammary epithelial cells (CMEC-H), using a single supplement through Western blotting. The Earles balanced salt solution (EBSS) was set as the control group and other treatment groups, based on the EBSS, were added with different concentrations of Leu or His, respectively. The results showed that, compared with the control group, the expression of caseins and the phosphorylation of mTOR (Ser2481), Raptor (Ser792), eIF4E (Ser209), and eEF2 (Thr56) increased with the Leu concentrations ranging from 0.45 to 10.80 mmol/L (P<0.01). The P-4EBP1 (Thr37) at 10.80 mmol/L Leu, and P-RPS6 (Ser235/236) at 5.40 to 10.80 mmol/L Leu all decreased. Similarly, the His supplementation from 0.15 to 9.60 mmol/L increased the expression of ?s2-casein, ?-casein, ?-casein, P-mTOR (Ser2481), P-Raptor (Ser792), P-S6K1 (Thr389), P-4EBP1 (Thr37), P-eIF4E (Ser209), and P-eEF2 (Thr56) (P<0.01) in CMEC-H, whereas the ?s1-casein expression was only reduced at 9.60 mmol/L His, G protein ? subunit-like protein (G?L) at 0.15 and 9.60 mmol/L His, and P-RPS6 at 4.80 to 9.60 mmol/L His. Our linear regression model assay suggested that the ?s1-casein expression was positively correlated with P-mTOR (P<0.01), P-S6K1 (P<0.01), and P-eEF2 (P<0.01) for the addition of Leu, while the expressions of ?-casein (P<0.01) and ?-casein (P<0.01) were positively correlated with P-eEF2 for the addition of His. In conclusion, the milk protein synthesis was up-regulated through activation of the mTOR pathway with the addition of Leu and His in CMEC-H. PMID:26055918

  1. Sequence-specific sup 1 H NMR assignments and structural characterization of bovine seminal fluid protein PDC-109 domain b

    SciTech Connect

    Constantine, K.L.; Ramesh, V.; Llinas, M. ); Banyai, L.; Trexler, M.; Patthy, L. )

    1991-02-12

    Sequence-specific resonance assignments for the isolated second or b domain of the bovine seminal fluid protein PDC-109 have been obtained from analysis of two-dimensional {sup 1}H NMR experiments recorded at 500 MHz. These assignments include the identification of all aromatic and most aliphatic amino acid resonances. Stereospecific assignment of resonances stemming from the Val{sup 2}CH{sub 3}{sup {gamma},{gamma}{prime}} groups and from seven CH{sup {beta},{beta}{prime}} geminal pairs has been accomplished by analysis of {sup 3}J{sub {alpha}{beta}} coupling constants in conjunction with patterns of cross-peak intensities observed in two-dimensional nuclear Overhauser effect (NOESY) spectra. Analysis of NOESY and {sup 3}J{sub {alpha}NH} data reveals a small antiparallel {beta}-sheet involving stretches containing residues 25-28 and 39-42, a cis-proline residue (Pro{sup 4}), antiparallel strands consisting of residues 1-3, 5-7, and 10-13, and an aromatic cluster composed of Tyr{sup 7}, Trp{sup 26}, and Tyr{sup 33}. The results of distance geometry and restrained molecular dynamics calculations indicate that the global fold of the PDC-109 b domain, a type 2 module related to those found in fibronectin, is somewhat different from that predicted by modeling the structure on the basis of homology between type 2 and kringle units. A shallow depression in the molecular surface which presents a solvent-exposed hydrophobic area-a potential ligand-binding site-is identified in the NMR-based models.

  2. Modeling Protein Self Assembly

    ERIC Educational Resources Information Center

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

    2004-01-01

    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…

  3. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

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

    2007-01-01

    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…

  4. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

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

    2007-01-01

    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

  5. Bovine binder-of-sperm protein BSP1 promotes protrusion and nanotube formation from liposomes

    SciTech Connect

    Lafleur, Michel; Courtemanche, Lesley; Karlsson, Goeran; Edwards, Katarina; Schwartz, Jean-Louis; Manjunath, Puttaswamy

    2010-08-27

    Research highlights: {yields} Binder-of-sperm protein 1 (BSP1) modifies the morphology of lipidic vesicles inducing bead necklace-like and thread-like structures. {yields} In the presence of multilamellar liposomes, BSP1 leads to the formation of long nanotubes. {yields} The insertion of BSP1 in the external lipid leaflet of membranes induces local changes in bilayer curvature. -- Abstract: Binder-of-sperm (BSP) proteins interact with sperm membranes and are proposed to extract selectively phosphatidylcholine and cholesterol from these. This change in lipid composition is a key step in sperm capacitation. The present work demonstrates that the interactions between the protein BSP1 and model membranes composed with phosphatidylcholine lead to drastic changes in the morphology of the lipidic self-assemblies. Using cryo-electron microscopy and fluorescence microscopy, we show that, in the presence of the protein, the lipid vesicles elongate, and form bead necklace-like structures that evolve toward small vesicles or thread-like structures. In the presence of multilamellar vesicles, where a large reservoir of lipid is available, the presence of BSP proteins lead to the formation of long nanotubes. Long spiral-like threads, associated with lipid/protein complexes, are also observed. The local curvature of lipid membranes induced by the BSP proteins may be involved in lipid domain formation and the extraction of some lipids during the sperm maturation process.

  6. Induced thermotolerance in bovine two-cell embryos and the role of heat shock protein 70 in embryonic development.

    PubMed

    Al-Katanani, Y M; Hansen, P J

    2002-06-01

    Induced thermotolerance is a phenomenon whereby exposure to a mild heat shock can induce heat shock proteins (HSP) and other cellular changes to make cells more resistant to a subsequent, more severe heat shock. Given that the 2-cell bovine embryo is very sensitive to heat shock, but can also produce HSP70 in response to elevated temperature, experiments were conducted to test whether 2-cell embryos could be made to undergo induced thermotolerance. Another objective was to test the role of the heat-inducible form of heat shock protein 70 (HSP70i) in development and sensitivity of bovine embryos to heat shock. To test for induced thermotolerance, 2-cell bovine embryos were first exposed to a mild heat shock (40 degrees C for 1 hr, or 41 degrees C or 42 degrees C for 80 min), allowed to recover at 38.5 degrees C and 5% (v/v) CO2 for 2 hr, and then exposed to a severe heat shock (41 degrees C for 4.5, 6, or 12 hr). Regardless of the conditions, previous exposure to mild heat shock did not reduce the deleterious effect of heat shock on development of embryos to the blastocyst stage. The role of HSP70i in embryonic development was tested in two experiments by culturing embryos with a monoclonal antibody to the inducible form of HSP70. At both 38.5 degrees C and 41 degrees C, the proportion of 2-cell embryos that developed to blastocyst was reduced (P < 0.05) by addition of anti-HSP70i to the culture medium. In contrast, sensitivity to heat shock was not generally increased by addition of antibody. In conclusion, bovine 2-cell embryos appear incapable of induced thermotolerance. Lack of capacity for induced thermotolerance could explain in part the increased sensitivity of 2-cell embryos to heat shock as compared to embryos at later stages of development. Results also implicate a role for HSP70i in normal development of bovine embryos. PMID:11984827

  7. Interaction study on bovine serum albumin physically binding to silver nanoparticles: Evolution from discrete conjugates to protein coronas

    NASA Astrophysics Data System (ADS)

    Guo, Jun; Zhong, Ruibo; Li, Wanrong; Liu, Yushuang; Bai, Zhijun; Yin, Jun; Liu, Jingran; Gong, Pei; Zhao, Xinmin; Zhang, Feng

    2015-12-01

    The nanostructures formed by inorganic nanoparticles together with organic molecules especially biomolecules have attracted increasing attention from both industries and researching fields due to their unique hybrid properties. In this paper, we systemically studied the interactions between amphiphilic polymer coated silver nanoparticles and bovine serum albumins by employing the fluorescence quenching approach in combination with the Stern-Volmer and Hill equations. The binding affinity was determined to 1.30 107 M-1 and the interaction was spontaneously driven by mainly the van der Waals force and hydrogen-bond mediated interactions, and negatively cooperative from the point of view of thermodynamics. With the non-uniform coating of amphiphilic polymer, the silver nanoparticles can form protein coronas which can become discrete protein-nanoparticle conjugates when controlling their molar ratios of mixing. The protein's conformational changes upon binding nanoparticles was also studied by using the three-dimensional fluorescence spectroscopy.

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

    SciTech Connect

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

    2012-05-09

    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.

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

    SciTech Connect

    Lubienski, Andreas Bitsch, Rudi G.; Lubienski, Katrin; Kauffmann, Guenter; Duex, Markus

    2006-12-15

    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.

  10. Alternative pathway fof bovine complement Immunochemical studies on factor B-like serum protein and its conversion product B gamma 2.

    PubMed Central

    Tabel, H

    1981-01-01

    Rabbits produced antibodies to a factor B-like serum protein (factor Bbov), its conversion product B gamma 2 and some other bovine serum proteins after repeated immunization with zymosan which previously had been incubated with fresh bovine serum. Such antisera were used to monitor purification of B gamma 2 from fresh bovine sera incubated with zxymosan. Subsequently, antisera specific for factor Bbov and B gamma 2 were produced. Antiserum produced against B gamma 2 cross-reacted with factor Bbov. Functional assays for factor Bbov were carried out in a hemolytic system with guinea pig erythrocytes in EGTA buffer. Heat inactivation (56 degrees C/5 min) of bovine serum destroyed the antigenicity of factor Bbov but not that of B gamma 2. Factor Bbov had an apparent molecular weight of 95,000 and B gamma 2 a molecular weight of 40,000 daltons. Conversion of factor Bbov to B gamma 2 was determined qualitatively by immunoelectrophoresis and quantitatively by radial immunodiffusion. Conversion of factor Bbov to B gamma 2 in bovine serum, in the presence of zymosan or cobra venom factor (CoVF) required Mg++ but not Ca++, did not occur in heat inactivated (56 degrees C/5 min) serum and was maximal, but not complete, when fresh bovine serum was incubated with zymosan (20 mg/mL) at 37 for two hours. Images Fig. 1. Fig. 2 Fig. 3. Fig. 4. Fig. 6. Fig. 7. PMID:6176300

  11. 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...

  12. Characterization of a 60-kDa Thermally Stable Antigenic Protein as a Marker for the Immunodetection of Bovine Plasma-Derived Food Ingredients.

    PubMed

    Ofori, Jack A; Hsieh, Yun-Hwa P

    2015-08-01

    A sandwich enzyme-linked immunosorbent assay (sELISA) based on 2 monoclonal antibodies (Bb3D6 and Bb6G12) that recognize a 60-kDa antigenic protein in bovine blood was previously developed for detecting bovine blood in animal feed for the prevention of mad cow disease. This study sought to establish the identity of this 60-kDa antigenic protein and consequently determine the suitability of the sELISA for detecting bovine plasma-derived food ingredients (BPFIs), which are widely used in dietary products without explicit labeling. Results from western blot confirmed the 60-kDa protein to be present in the plasma fraction of bovine blood. Further proteomic analyses involving 2-dimensional gel electrophoresis (2-D GE) and amino acid sequencing revealed the 60-kDa protein to be bovine serum albumin (BSA). The sELISA proved capable of detecting BPFIs in all the commercial dietary supplements tested, including those that were formulated with hydrolyzed BPFIs. The assay could also detect 0.01% and 0.5% of different BPFIs in spiked raw and cooked ground beef, respectively. This assay based on the detection of BSA therefore has the potential to become a valuable analytical tool to protect consumers who avoid consuming BPFIs for religious, health, or ethical reasons. PMID:26172875

  13. Effect of high-pressure treatment at various temperatures on indigenous proteolytic enzymes and whey protein denaturation in bovine milk.

    PubMed

    Moatsou, Golfo; Bakopanos, Constantinos; Katharios, Dimitis; Katsaros, George; Kandarakis, Ioannis; Taoukis, Petros; Politis, Ioannis

    2008-08-01

    The objective of the present study was to determine the effect of high pressure (HP) processing (200, 450 and 650 MPa) at various temperatures (20, 40 and 55 degrees C) on the total plasmin plus plasminogen-derived activity (PL), plasminogen activator(s) (PA) and cathepsin D activities and on denaturation of major whey proteins in bovine milk. Data indicated that transfer of both PL and PA from the casein micelles to milk serum occurred at all pressures utilized at room temperature (20 degrees C). In addition to the transfer of PL and PA from micelles, there were reductions in activities of PL (16-18%) and PA (38-62%) for the pressures 450 and 650 MPa, at room temperature. There were synergistic negative effects between pressure and temperature on residual PL activity at 450 and 650 MPa and on residual PA activity only at 450 MPa. Cathepsin D activity in the acid whey from HP-treated milk was in general baroresistant at room temperature. The residual activity of cathepsin D decreased significantly at 650 MPa and 40 degrees C and at the pressures 450 and 650 MPa at 55 degrees C. Synergistic negative effects on the amount of native beta-lactoglobulin were observed at 450 and 650 MPa and on the amount of native alpha-lactalbumin at 650 MPa. There were significant correlations between enzymatic activities (PL, PA and cathepsin D) and the residual native beta-lactoglobulin and alpha-lactalbumin in bovine milk. In conclusion, HP significantly affected the activity of indigenous proteolytic enzymes and whey protein denaturation in bovine milk. Reduction in activity of indigenous enzymes (PL, PA and cathepsin D) and transfer of PL and PA from the casein to milk serum induced by HP is expected to have a profound effect on cheese yield, proteolysis during cheese ripening and quality of UHT milk during storage. PMID:18513457

  14. Infection of the upper respiratory tract of hamsters by the bovine parainfluenza virus type 3 BN-1 strain expressing enhanced green fluorescent protein.

    PubMed

    Ohkura, Takashi; Minakuchi, Moeko; Sagai, Mami; Kokuho, Takehiro; Konishi, Misako; Kameyama, Ken-Ichiro; Takeuchi, Kaoru

    2015-02-01

    Bovine parainfluenza virus type 3 (BPIV3) is an important pathogen associated with bovine respiratory disease complex (BRDC). We have generated a recombinant BPIV3 expressing enhanced green fluorescent protein (rBPIV3-EGFP) based on the BN-1 strain isolated in Japan. After intranasal infection of hamsters with rBPIV3-EGFP, EGFP fluorescence was detected in the upper respiratory tract including the nasal turbinates, pharynx, larynx, and trachea. In the nasal turbinates, rBPIV3-EGFP attained high titers (>10(6) TCID50/g of tissue) 2-4 days after infection. Ciliated epithelial cells in the nasal turbinates and trachea were infected with rBPIV3-EGFP. Histopathological analysis indicated that mucosal epithelial cells in bronchi were shed by 6 days after infection, leaving non-ciliated cells, which may have increased susceptibility to bacterial infection leading to the development of BRDC. These data indicate that rBPIV3-EGFP infection of hamsters is a useful small animal model for studying the development of BPIV3-associated BRDC. PMID:25543964

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

    PubMed Central

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

    2012-01-01

    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

  16. Comparative role of proteins in transport of HCH-isomers in desert locust, Schistocerca gregaria Forskal and bovines.

    PubMed

    Mahajan, Sapna; Patyal, S K; Ahmed, S B; Nath, A

    2003-06-01

    The studies on binding of hexachlorocyclohexane (HCH) with carrier proteins were carried out to establish the role of proteins in the transport of insecticides in insects. Sephadex G-200 column chromatography resolved haemolymph of adult male desert locust, Schistocerca gregaria into three major protein peaks. There was significant binding of gamma-HCH with first protein peak (F1). Two classes of binding sites were observed on first protein peak for gamma-HCH. However low level of binding was observed with the third protein peak (F3) of the haemolymph. Bindings of HCH-isomers (alpha, beta and gamma) with bovine serum albumin (BSA) were not related to their water solubilities. Moderate to low affinities (1.4 -1.84 x 10(6) M(-1)) of HCH-isomers for BSA were observed. The present studies showed that more HCH binds to haemolymph lipoprotein of locust as compared to BSA. This indicates a significant role of haemolymph proteins in the transport of insecticides in insects. PMID:15266906

  17. Keratin and S100 calcium-binding proteins are major constituents of the bovine teat canal lining.

    PubMed

    Smolenski, Grant A; Cursons, Ray T; Hine, Brad C; Wheeler, Thomas T

    2015-01-01

    The bovine teat canal provides the first-line of defence against pathogenic bacteria infecting the mammary gland, yet the protein composition and host-defence functionality of the teat canal lining (TCL) are not well characterised. In this study, TCL collected from six healthy lactating dairy cows was subjected to two-dimensional electrophoresis (2-DE) and mass spectrometry. The abundance and location of selected identified proteins were determined by western blotting and fluorescence immunohistochemistry. The variability of abundance among individual cows was also investigated. Two dominant clusters of proteins were detected in the TCL, comprising members of the keratin and S100 families of proteins. The S100 proteins were localised to the teat canal keratinocytes and were particularly predominant in the cornified outermost layer of the teat canal epithelium. Significant between-animal variation in the abundance of the S100 proteins in the TCL was demonstrated. Four of the six identified S100 proteins have been reported to have antimicrobial activity, suggesting that the TCL has additional functionality beyond being a physical barrier to invading microorganisms. These findings provide new insights into understanding host-defence of the teat canal and resistance of cows to mastitis. PMID:26407704

  18. Molecular modelling of protein-protein/protein-solvent interactions

    NASA Astrophysics Data System (ADS)

    Luchko, Tyler

    The inner workings of individual cells are based on intricate networks of protein-protein interactions. However, each of these individual protein interactions requires a complex physical interaction between proteins and their aqueous environment at the atomic scale. In this thesis, molecular dynamics simulations are used in three theoretical studies to gain insight at the atomic scale about protein hydration, protein structure and tubulin-tubulin (protein-protein) interactions, as found in microtubules. Also presented, in a fourth project, is a molecular model of solvation coupled with the Amber molecular modelling package, to facilitate further studies without the need of explicitly modelled water. Basic properties of a minimally solvated protein were calculated through an extended study of myoglobin hydration with explicit solvent, directly investigating water and protein polarization. Results indicate a close correlation between polarization of both water and protein and the onset of protein function. The methodology of explicit solvent molecular dynamics was further used to study tubulin and microtubules. Extensive conformational sampling of the carboxy-terminal tails of 8-tubulin was performed via replica exchange molecular dynamics, allowing the characterisation of the flexibility, secondary structure and binding domains of the C-terminal tails through statistical analysis methods. Mechanical properties of tubulin and microtubules were calculated with adaptive biasing force molecular dynamics. The function of the M-loop in microtubule stability was demonstrated in these simulations. The flexibility of this loop allowed constant contacts between the protofilaments to be maintained during simulations while the smooth deformation provided a spring-like restoring force. Additionally, calculating the free energy profile between the straight and bent tubulin configurations was used to test the proposed conformational change in tubulin, thought to cause microtubule destabilization. No conformational change was observed but a nucleotide dependent 'softening' of the interaction was found instead, suggesting that an entropic force in a microtubule configuration could be the mechanism of microtubule collapse. Finally, to overcome much of the computational costs associated with explicit soIvent calculations, a new combination of molecular dynamics with the 3D-reference interaction site model (3D-RISM) of solvation was integrated into the Amber molecular dynamics package. Our implementation of 3D-RISM shows excellent agreement with explicit solvent free energy calculations. Several optimisation techniques, including a new multiple time step method, provide a nearly 100 fold performance increase, giving similar computational performance to explicit solvent.

  19. Bovine liver slices: A multifunctional in vitro model to study the prohormone dehydroepiandrosterone (DHEA).

    PubMed

    Rijk, Jeroen C W; Bovee, Toine F H; Peijnenburg, Ad A C M; Groot, Maria J; Rietjens, Ivonne M C M; Nielen, Michel W F

    2012-09-01

    Biotransformation of inactive prohormones like dehydroepiandrosterone (DHEA) can lead to the formation of potent androgens and subsequent androgenic responses in target tissues. In the present study, precision-cut bovine liver slices were used to study the effects of DHEA on the metabolite, transcript and androgenic activity level. Bovine liver slices were exposed for 6h to various concentrations of DHEA. Changes in androgenic activity of the DHEA containing cell culture media were measured using a yeast androgen bioassay and metabolites were identified using ultra performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOFMS), while gene expression in the DHEA-treated liver slices was examined using bovine microarrays and compared with the profile as obtained with 17ß-testosterone (17ß-T). An increase in androgenic activity was observed in the bioassay upon testing of samples from incubations of DHEA with liver slices and the formation of 4-androstenedione (4-AD), 5-androstene-3ß,17ß-diol, 17ß-T, 7α-hydroxy-DHEA, 7-keto-DHEA and 17α-T could be confirmed by UPLC-TOFMS analysis. Exposure of liver slices to DHEA and the strong androgen 17ß-T resulted in the identification of significantly up- and down-regulated genes and revealed similar gene expression profiles for both compounds. The results indicate that DHEA itself is biologically not very active, but is rapidly converted by the liver slices into the more androgen active compounds 4-AD and 17ß-T. Moreover, the present data highlight the multi-functionality of bovine liver slices as an in vitro bioactivation model, allowing the assessment of androgen activity or gene expression as effect-based endpoints for prohormone exposure. PMID:22640920

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

    PubMed Central

    Lefèbvre, Laurent; Vanderplasschen, Alain; Ciminale, Vincenzo; Heremans, Hubertine; Dangoisse, Olivier; Jauniaux, Jean-Claude; Toussaint, Jean-François; Zelnik, Vlado; Burny, Arsène; Kettmann, Richard; Willems, Luc

    2002-01-01

    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 α-helix rich in arginine residues. Subtle mutation of this α-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. PMID:11773414

  1. Two distinct anti-allergic drugs, amlexanox and cromolyn, bind to the same kinds of calcium binding proteins, except calmodulin, in bovine lung extract.

    PubMed

    Oyama, Y; Shishibori, T; Yamashita, K; Naya, T; Nakagiri, S; Maeta, H; Kobayashi, R

    1997-11-17

    In order to explore candidates for proteins required in exocytosis, we used two anti-allergic drugs, amlexanox and cromolyn, which inhibit IgE mediated degranulation of mast cells and basophils, as molecular probes in affinity chromatography. These two drugs chiefly bound to the same kinds of calcium binding proteins in bovine lung. These proteins were as follows: bovine calgranulin C homolog, an 8-kDa unknown protein, S-100L, calgranulin B, calcyphosine, and annexins I-V. The homologous affinity of the two drugs to these proteins is in accord with the similar anti-allergic property of both drugs. From these findings it is presumed that these drugs interact with these proteins and affect pharmacologically the degranulation. PMID:9388479

  2. Electrolyte effect on the phase behavior of silica nanoparticles with lysozyme and bovine-serum-albumin proteins

    NASA Astrophysics Data System (ADS)

    Yadav, Indresh; Aswal, V. K.; Kohlbrecher, J.

    2015-05-01

    Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) studies have been carried out to investigate the effect of an electrolyte on the phase behavior of anionic silica nanoparticles with two globular proteinscationic lysozyme [molecular weight (MW) 14.7 kDa] and anionic bovine serum albumin (MW 66.4 kDa). The results are compared with our earlier published work on similar systems without any electrolyte [I. Yadav, S. Kumar, V. K. Aswal, and J. Kohlbrecher, Phys. Rev. E 89, 032304 (2014), 10.1103/PhysRevE.89.032304]. Both the nanoparticle-protein systems transform to two phase at lower concentration of protein in the presence of an electrolyte. The autocorrelation function in DLS suggests that the diffusion coefficient (D) of a nanoparticle-protein system decreases in approaching two phase with the increase in protein concentration. This variation in D can be attributed to increase in attractive interaction and/or overall increase in the size. Further, these two contributions (interaction and structure) are determined from the SANS data. The changes in the phase behavior of nanoparticle-protein systems in the presence of an electrolyte are explained in terms of modifications in both the repulsive and attractive components of interaction between nanoparticles. In a two-phase system individual silica nanoparticles coexist along with their fractal aggregates.

  3. Mapping of nuclear import signal and importin {alpha}3 binding regions of 52K protein of bovine adenovirus-3

    SciTech Connect

    Paterson, Carolyn P.; Ayalew, Lisanework E.; Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada ; Tikoo, Suresh K.

    2012-10-10

    The L1 region of bovine adenovirus (BAdV)-3 encodes a non-structural protein designated 52K. Anti-52K serum detected a protein of 40 kDa, which localized to the nucleus but not to the nucleolus in BAdV-3-infected or transfected cells. Analysis of mutant 52K proteins suggested that three basic residues ({sup 105}RKR{sup 107}) of the identified domain (amino acids {sup 102}GMPRKRVLT{sup 110}) are essential for nuclear localization of 52K. The nuclear import of a GST-52K fusion protein utilizes the classical importin {alpha}/{beta}-dependent nuclear transport pathway. The 52K protein is preferentially bound to the cellular nuclear import receptor importin {alpha}3. Although deletion of amino acid 102-110 is sufficient to abrogate the nuclear localization of 52K, amino acid 90-133 are required for interaction with importin-{alpha}3 and localizing a cytoplasmic protein to the nucleus. These results suggest that 52K contains a bipartite NLS, which preferentially utilize an importin {alpha}3 nuclear import receptor-mediated pathway to transport 52K to the nucleus.

  4. Electrolyte effect on the phase behavior of silica nanoparticles with lysozyme and bovine-serum-albumin proteins.

    PubMed

    Yadav, Indresh; Aswal, V K; Kohlbrecher, J

    2015-05-01

    Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) studies have been carried out to investigate the effect of an electrolyte on the phase behavior of anionic silica nanoparticles with two globular proteins-cationic lysozyme [molecular weight (MW) 14.7 kDa] and anionic bovine serum albumin (MW 66.4 kDa). The results are compared with our earlier published work on similar systems without any electrolyte [I. Yadav, S. Kumar, V. K. Aswal, and J. Kohlbrecher, Phys. Rev. E 89, 032304 (2014)]. Both the nanoparticle-protein systems transform to two phase at lower concentration of protein in the presence of an electrolyte. The autocorrelation function in DLS suggests that the diffusion coefficient (D) of a nanoparticle-protein system decreases in approaching two phase with the increase in protein concentration. This variation in D can be attributed to increase in attractive interaction and/or overall increase in the size. Further, these two contributions (interaction and structure) are determined from the SANS data. The changes in the phase behavior of nanoparticle-protein systems in the presence of an electrolyte are explained in terms of modifications in both the repulsive and attractive components of interaction between nanoparticles. In a two-phase system individual silica nanoparticles coexist along with their fractal aggregates. PMID:26066176

  5. The molecular cloning of the complementary deoxyribonucleic acid for bovine vitamin D-dependent calcium-binding protein: structure of the full-length protein and evidence for homologies with other calcium-binding proteins of the troponin-C superfamily of proteins.

    PubMed

    Kumar, R; Wieben, E; Beecher, S J

    1989-02-01

    We have cloned the cDNA for bovine intestinal vitamin D-dependent calcium-binding protein and, based on the sequence of the DNA, have deduced the structure of the full-length protein. The sequence of the cDNA clone predicts a protein comprised of 78 amino acids with a mol wt of 8788. The mRNA for the protein in bovine duodenum is about 500-600 bases in length. The protein sequence of bovine intestinal calcium-binding protein is 87% homologous with the sequence of porcine intestinal vitamin D-dependent calcium-binding protein and 81% homologous with the sequence of rat intestinal vitamin D-dependent calcium-binding protein. Hydrophilicity plots of the proteins noted above show that despite differences in amino acid sequence the proteins have similar patterns. In addition, the predicted secondary structure of the proteins is similar. Bovine intestinal calcium-binding protein shows 48.6% homology with the alpha-chain and 38.2% homology with the beta-chain of bovine S-100 protein and a similar high degree of homology with the beta-chain of human S-100 protein. The protein also demonstrates 36-43% homology with parvalbumin alpha and beta from various species and with troponin-C. There is some homology with the 28K vitamin D-dependent calcium-binding proteins. Vitamin D-dependent bovine intestinal calcium-binding protein is closely related to other mammalian intestinal calcium-binding proteins and to the S-100 proteins, parvalbumins, and troponin-C. PMID:2710141

  6. Protein-calixarene interactions: complexation of Bovine Serum Albumin by sulfonatocalix[n]arenes.

    PubMed

    Memmi, L; Lazar, A; Brioude, A; Ball, V; Coleman, A W

    2001-12-01

    The complexation of Bovine Serum Albumin with sulfonatocalix[n]arenes has been demonstrated by means of electrospray mass spectrometry, dynamic light scattering and atomic force microscopy; with sulfonatocalix[4]arene one strong and two weaker binding sites are detected; the effects on the structure of thin films formed by surface deposition of BSA show that the sulfonatocalix[n]arenes act to reticulate the films and produce essentially planar systems. PMID:12240021

  7. Increasing the X-ray Diffraction Power of Protein Crystals by Dehydration: The Case of Bovine Serum Albumin and a Survey of Literature Data

    PubMed Central

    Krauss, Irene Russo; Sica, Filomena; Mattia, Carlo Andrea; Merlino, Antonello

    2012-01-01

    Serum albumin is one of the most widely studied proteins. It is the most abundant protein in plasma with a typical concentration of 5 g/100 mL and the principal transporter of fatty acids in plasma. While the crystal structures of human serum albumin (HSA) free and in complex with fatty acids, hemin, and local anesthetics have been characterized, no crystallographic models are available on bovine serum albumin (BSA), presumably because of the poor diffraction power of existing hexagonal BSA crystals. Here, the crystallization and diffraction data of a new BSA crystal form, obtained by the hanging drop method using MPEG 5K as precipitating agent, are presented. The crystals belong to space group C2, with unit-cell parameters a = 216.45 , b = 44.72 , c = 140.18 , ? = 114.5. Dehydration was found to increase the diffraction limit of BSA crystals from ~8 to 3.2 , probably by improving the packing of protein molecules in the crystal lattice. These results, together with a survey of more than 60 successful cases of protein crystal dehydration, confirm that it can be a useful procedure to be used in initial screening as a method of improving the diffraction limits of existing crystals. PMID:22489183

  8. A novel murine model for evaluating bovine papillomavirus prophylactics/therapeutics for equine sarcoid-like tumours.

    PubMed

    Bogaert, Lies; Woodham, Andrew W; Da Silva, Diane M; Martens, Ann; Meyer, Evelyne; Kast, W Martin

    2015-09-01

    Equine sarcoids are highly recurrent bovine papillomavirus (BPV)-induced fibroblastic neoplasms that are the most common skin tumours in horses. In order to facilitate the study of potential equine sarcoid prophylactics or therapeutics, which can be a slow and costly process in equines, a murine model for BPV-1 protein-expressing equine sarcoid-like tumours was developed in mice through stable transfection of BPV-1 E5 and E6 in a murine fibroblast tumour cell line (K-BALB). Like equine sarcoids, these murine tumour cells (BPV-KB) were of fibroblast origin, were tumorigenic and expressed BPV-1 proteins. As an initial investigation of the preclinical potential of this tumour model for equine sarcoids prophylactics, mice were immunized with BPV-1 E5E6 Venezuelan equine encephalitis virus replicon particles, prior to BPV-KB challenge, which resulted in an increased tumour-free period compared with controls, indicating that the BPV-KB murine model may be a valuable preclinical alternative to equine clinical trials. PMID:26044793

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

    PubMed Central

    2010-01-01

    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. PMID:21092319

  10. Glucose Availability and AMP-Activated Protein Kinase Link Energy Metabolism and Innate Immunity in the Bovine Endometrium

    PubMed Central

    Turner, Matthew L.; Cronin, James G.; Noleto, Pablo G.; Sheldon, I. Martin

    2016-01-01

    Defences against the bacteria that usually infect the endometrium of postpartum cattle are impaired when there is metabolic energy stress, leading to endometritis and infertility. The endometrial response to bacteria depends on innate immunity, with recognition of pathogen-associated molecular patterns stimulating inflammation, characterised by secretion of interleukin (IL)-1β, IL-6 and IL-8. How metabolic stress impacts tissue responses to pathogens is unclear, but integration of energy metabolism and innate immunity means that stressing one system might affect the other. Here we tested the hypothesis that homeostatic pathways integrate energy metabolism and innate immunity in bovine endometrial tissue. Glucose deprivation reduced the secretion of IL-1β, IL-6 and IL-8 from ex vivo organ cultures of bovine endometrium challenged with the pathogen-associated molecular patterns lipopolysaccharide and bacterial lipopeptide. Endometrial inflammatory responses to lipopolysaccharide were also reduced by small molecules that activate or inhibit the intracellular sensor of energy, AMP-activated protein kinase (AMPK). However, inhibition of mammalian target of rapamycin, which is a more global metabolic sensor than AMPK, had little effect on inflammation. Similarly, endometrial inflammatory responses to lipopolysaccharide were not affected by insulin-like growth factor-1, which is an endocrine regulator of metabolism. Interestingly, the inflammatory responses to lipopolysaccharide increased endometrial glucose consumption and induced the Warburg effect, which could exacerbate deficits in glucose availability in the tissue. In conclusion, metabolic energy stress perturbed inflammatory responses to pathogen-associated molecular patterns in bovine endometrial tissue, and the most fundamental regulators of cellular energy, glucose availability and AMPK, had the greatest impact on innate immunity. PMID:26974839

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

    PubMed Central

    2013-01-01

    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 eight weeks with/without stimulation with transforming growth factor-?1 (TGF-?1. 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-?1-stimulated cartilage discs displayed a preserved structural and functional integrity of the chondrocytes and surrounding matrix, remained vital in long-term culture (eight 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 to 5 ?m). 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-?1 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

  12. Irreversible aggregation of recombinant bovine granulocyte-colony stimulating factor (bG-CSF) and implications for predicting protein shelf life.

    PubMed

    Roberts, Christopher J; Darrington, Richard T; Whitley, Maureen B

    2003-05-01

    The kinetics of irreversible aggregation of bovine Granulocyte-Colony Stimulating Factor (bG-CSF) in solution were investigated as a function of temperature (T), concentration, and pH, and analyzed in terms of an Extended Lumry-Eyring model of protein aggregation proceeding via a non-native conformational state. In the spirit of classic Lumry-Eyring models, the observed kinetics are separated into contributions from thermodynamic or conformational stability of unaggregated native and non-native states, and the intrinsic aggregation kinetics of non-native molecules. It is found that a detailed treatment of the intrinsic kinetics coupled with a two-state approximation of the reversible unfolding transition is sufficient to allow quantitative prediction of low-T stability from high-T data despite highly non-Arrhenius kinetics. Accounting for shifts in conformational equilibrium quantitatively captures the non-Arrhenius T dependence, without requiring the assumption of a change in the rate-determining step with T. From a more general perspective, the observed aggregation behavior of bG-CSF is consistent with the rate-determining step being aggregation at T below a crossover temperature T(x) that is inversely related to initial protein concentration. Above T(x), irreversible unfolding is presumably the rate-determining step. The results illustrate that protein aggregation kinetics can, in principle, be predicted quantitatively from so-called accelerated data provided the thermodynamic and kinetic components can be separately extrapolated to longer term storage conditions. PMID:12712430

  13. 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

    Lee, K-B.; La Mar, G.N.; Kehres, L.A.; Fujinari, E.M.; Smith, K.M. ); Pochapsky, T.C.; Sligar, S.G. )

    1990-10-01

    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.

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

    PubMed Central

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

    2011-01-01

    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 = 0.600.61) 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. PMID:21282583

  15. Dietary Compound Kaempferol Inhibits Airway Thickening Induced by Allergic Reaction in a Bovine Serum Albumin-Induced Model of Asthma

    PubMed Central

    Shin, Daekeun; Park, Sin-Hye; Choi, Yean-Jung; Kim, Yun-Ho; Antika, Lucia Dwi; Habibah, Nurina Umy; Kang, Min-Kyung; Kang, Young-Hee

    2015-01-01

    Asthma is characterized by aberrant airways including epithelial thickening, goblet cell hyperplasia, and smooth muscle hypertrophy within the airway wall. The current study examined whether kaempferol inhibited mast cell degranulation and prostaglandin (PG) release leading to the development of aberrant airways, using an in vitro model of dinitrophenylated bovine serum albumin (DNP-BSA)-sensitized rat basophilic leukemia (RBL-2H3) mast cells and an in vivo model of BSA-challenged asthmatic mice. Nontoxic kaempferol at 10–20 μM suppressed β-hexosaminidase release and cyclooxygenase 2 (COX2)-mediated production of prostaglandin D2 (PGD2) and prostaglandin F2α (PGF2α) in sensitized mast cells. Oral administration of ≤20 mg/kg kaempferol blocked bovine serum albumin (BSA) inhalation-induced epithelial cell excrescence and smooth muscle hypertrophy by attenuating the induction of COX2 and the formation of PGD2 and PGF2α, together with reducing the anti-α-smooth muscle actin (α-SMA) expression in mouse airways. Kaempferol deterred the antigen-induced mast cell activation of cytosolic phospholipase A2 (cPLA2) responsive to protein kinase Cμ (PKCμ) and extracellular signal-regulated kinase (ERK). Furthermore, the antigen-challenged activation of Syk-phospholipase Cγ (PLCγ) pathway was dampened in kaempferol-supplemented mast cells. These results demonstrated that kaempferol inhibited airway wall thickening through disturbing Syk-PLCγ signaling and PKCμ-ERK-cPLA2-COX2 signaling in antigen-exposed mast cells. Thus, kaempferol may be a potent anti-allergic compound targeting allergic asthma typical of airway hyperplasia and hypertrophy. PMID:26694364

  16. Dietary Compound Kaempferol Inhibits Airway Thickening Induced by Allergic Reaction in a Bovine Serum Albumin-Induced Model of Asthma.

    PubMed

    Shin, Daekeun; Park, Sin-Hye; Choi, Yean-Jung; Kim, Yun-Ho; Antika, Lucia Dwi; Habibah, Nurina Umy; Kang, Min-Kyung; Kang, Young-Hee

    2015-01-01

    Asthma is characterized by aberrant airways including epithelial thickening, goblet cell hyperplasia, and smooth muscle hypertrophy within the airway wall. The current study examined whether kaempferol inhibited mast cell degranulation and prostaglandin (PG) release leading to the development of aberrant airways, using an in vitro model of dinitrophenylated bovine serum albumin (DNP-BSA)-sensitized rat basophilic leukemia (RBL-2H3) mast cells and an in vivo model of BSA-challenged asthmatic mice. Nontoxic kaempferol at 10-20 ?M suppressed ?-hexosaminidase release and cyclooxygenase 2 (COX2)-mediated production of prostaglandin D2 (PGD2) and prostaglandin F2? (PGF2?) in sensitized mast cells. Oral administration of ?20 mg/kg kaempferol blocked bovine serum albumin (BSA) inhalation-induced epithelial cell excrescence and smooth muscle hypertrophy by attenuating the induction of COX2 and the formation of PGD2 and PGF2?, together with reducing the anti-?-smooth muscle actin (?-SMA) expression in mouse airways. Kaempferol deterred the antigen-induced mast cell activation of cytosolic phospholipase A2 (cPLA2) responsive to protein kinase C? (PKC?) and extracellular signal-regulated kinase (ERK). Furthermore, the antigen-challenged activation of Syk-phospholipase C? (PLC?) pathway was dampened in kaempferol-supplemented mast cells. These results demonstrated that kaempferol inhibited airway wall thickening through disturbing Syk-PLC? signaling and PKC?-ERK-cPLA2-COX2 signaling in antigen-exposed mast cells. Thus, kaempferol may be a potent anti-allergic compound targeting allergic asthma typical of airway hyperplasia and hypertrophy. PMID:26694364

  17. Molecular characterization of the major capsid protein VP6 of bovine group B rotavirus and its use in seroepidemiology.

    PubMed

    Tsunemitsu, Hiroshi; Kamiyama, Mariko; Kawashima, Kenji; Katsuda, Ken; Kohmoto, Mariko; Saif, Linda J; Shouji, Tomotaro; Onodera, Toshiyuki

    2005-09-01

    The major inner capsid protein (VP6) gene of the bovine group B rotavirus (GBR) Nemuro strain is 1269 nt in length and contains one open reading frame encoding 391 aa. Nucleotide and amino acid sequence identities of the Nemuro VP6 gene compared with the published corresponding human and rodent GBR genes were respectively 66-67 and 70-72 %, which are notably lower than those between human and rodent viruses (72-73 and 83-84 %, respectively). Overall identities of VP6 genes among GBRs were substantially lower than those among both group A rotaviruses (GARs) and group C rotaviruses (GCRs) derived from different species of mammals. These results demonstrate that bovine GBR is remarkably distinct from other GBRs and that GBRs from different species may have had a longer period of divergence than GARs and GCRs. Recombinant VP6 was generated with a baculovirus expression system and used for an ELISA to detect GBR antibodies. All 13 paired sera from adult cows with GBR-induced diarrhoea in the field showed antibody responses in the ELISA. In serological surveys of GBR infection using the ELISA, 47 % of cattle sera were positive for GBR antibodies, with a higher antibody prevalence in adults than in young cattle. In pigs, a high prevalence of GBR antibodies (97 %) was detected in sera from sows. These results suggest that GBR infection is common in cattle and pigs, notwithstanding the scarcity of reports of GBR detection in these species to date. PMID:16099916

  18. Analyzing models for interactions of aptamers to proteins

    NASA Astrophysics Data System (ADS)

    Silva, Dilson; Missailidis, Sotiris

    2014-10-01

    We have devised an experimental and theoretical model, based on fluorescent spectroscopy and molecular modelling, to describe the interaction of aptamer (selected against various protein targets) with proteins and albumins in particular. This model, described in this work, has allowed us to decipher the nature of the interactions between aptamers and albumins, the binding site of the aptamers to albumins, the potential role of primer binding to the albumin and expand to the ability of albumin to carry aptamers in the bloodstream, providing data to better understand the level of free aptamer for target binding. We are presenting the study of a variety of aptamers, including those against the MUC1 tumour marker, heparanase and human kallikrein 6 with bovine and human serum albumins and the effect these interactions may have on the bioavailability of the aptamer for target-specific binding and therapeutic activity.

  19. Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells.

    PubMed

    Zhang, Jin-You; Wu, Yi; Zhao, Shuan; Liu, Zhen-Xing; Zeng, Shen-Ming; Zhang, Gui-Xue

    2015-09-15

    Progesterone is an important steroid hormone in the regulation of the bovine estrous cycle. The steroidogenic acute regulatory protein (StAR) is an indispensable component for transporting cholesterol to the inner mitochondrial membrane, which is one of the rate-limiting steps for progesterone synthesis. Low-density lipoprotein (LDL) supplies cholesterol precursors for progesterone formation, and the lysosomal degradation pathway of LDL is essential for progesterone biosynthesis in granulosa cells after ovulation. However, it is currently unknown how LDL and lysosomes coordinate the expression of the StAR gene and progesterone production in bovine granulosa cells. Here, we investigated the role of lysosomes in LDL-treated bovine granulosa cells. Our results reported that LDL induced expression of StAR messenger RNA and protein as well as expression of cholesterol side-chain cleavage cytochrome P-450 (CYP11A1) messenger RNA and progesterone production in cultured bovine granulosa cells. The number of lysosomes in the granulosa cells was also significantly increased by LDL; whereas the lysosomal inhibitor, chloroquine, strikingly abolished these LDL-induced effects. Our results indicate that LDL promotes StAR expression, synthesis of progesterone, and formation of lysosomes in bovine granulosa cells, and lysosomes participate in the process by releasing free cholesterol from hydrolyzed LDL. PMID:26100237

  20. Proteomic analysis of the temporal expression of bovine milk proteins during coliform mastitis and label-free relative quantification.

    PubMed

    Boehmer, J L; Ward, J L; Peters, R R; Shefcheck, K J; McFarland, M A; Bannerman, D D

    2010-02-01

    The discovery of biomarkers in milk indicative of local inflammation or disease in the bovine mammary gland has been hindered by the extreme biological complexity of milk, the dynamic range of proteins in the matrix that renders the identification of low-abundance proteins difficult, and the challenges associated with quantifying changes during disease in the abundance of proteins for which no antibody exists. The objectives of the current study were to characterize the temporal expression of milk proteins following Escherichia coli challenge and to evaluate change in relative abundance of identified proteins using a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) label-free semiquantitative approach. Liquid chromatography-MS/MS conducted on whey from milk samples collected just before infusion with E. coli and at 12, 18, 24, 36, 48, and 60h following infection resulted in the identification of the high- to medium-abundance proteins alpha(S1)-, alpha(S2)- beta-, and kappa-caseins and the whey proteins serum albumin, beta-lactoglobulin, and alpha-lactalbumin. Additionally, a select number of lower abundance markers of inflammation were also identified, including lactoferrin, transferrin, apolipoprotein AI, fibrinogen, glycosylation-dependent cell adhesion molecule-1, peptidoglycan recognition receptor protein, and cyclic dodecapeptide-1. Normalized peptide counts for each protein identified were used to evaluate temporal changes in milk proteins following infection. For comparison with relative protein abundance determined using proteomic-based methods, changes in serum albumin, lactoferrin, and transferrin in milk during disease were also measured using ELISA. Label-free, proteomic-based quantification revealed relative changes in milk proteins that corresponded to expression profiles generated by ELISA. The results indicate that label-free LC-MS/MS methods are a viable means of tracking changes in relative protein abundance in milk during disease. Despite the identification of primarily abundant milk proteins, the results indicate that, with further refinement, LC-MS/MS could be used to evaluate temporal changes in proteins related to host response for which no antibody or ELISA currently exists. PMID:20105531

  1. Bovine adipose triglyceride lipase is not altered and adipocyte fatty acid binding protein is increased by dietary flaxseed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this paper, we report the full length coding sequence of bovine ATGL cDNA are reported and analyze its expression in bovine tissues. Similar to human, mouse, and pig ATGL sequences, bovine ATGL has a highly conserved patatin domain that is necessary for lipolytic function in mice and humans. Thi...

  2. Molecular Aspects of the Interaction of Iminium and Alkanolamine Forms of the Anticancer Alkaloid Chelerythrine with Plasma Protein Bovine Serum Albumin.

    PubMed

    Bhuiya, Sutanwi; Pradhan, Ankur Bikash; Haque, Lucy; Das, Suman

    2016-01-14

    The interaction between a quaternary benzophenanthridine alkaloid chelerythrine (herein after, CHL) and bovine serum albumin (herein after, BSA) was probed by employing various spectroscopic tools and isothermal titration calorimetry (ITC). Fluorescence studies revealed that the binding affinity of the alkanolamine form of the CHL is higher compared to the iminium counterpart. This was further established by fluorescence polarization anisotropy measurement and ITC. Fluorescence quenching study along with time-resolved fluorescence measurements establish that both forms of CHL quenched the fluorescence intensity of BSA through the mechanism of static quenching. Site selective binding and molecular modeling studies revealed that the alkaloid binds predominantly in the BSA subdomain IIA by electrostatic and hydrophobic forces. From Forster resonance energy transfer (FRET) studies, the average distances between the protein donor and the alkaloid acceptor were found to be 2.71 and 2.30 nm between tryptophan (Trp) 212 (donor) and iminium and alkanolamine forms (acceptor), respectively. Circular dichroism (CD) study demonstrated that the ?-helical organization of the protein is reduced due to binding with CHL along with an increase in the coiled structure. This is indicative of a small but definitive partial unfolding of the protein. Thermodynamic parameters obtained from ITC experiments revealed that the interaction is favored by negative enthalpy change and positive entropy change. PMID:26653994

  3. A three-dimensional model of primary bovine endometrium using an electrospun scaffold.

    PubMed

    MacKintosh, S B; Serino, L P; Iddon, P D; Brown, R; Conlan, R S; Wright, C J; Maffeis, T G G; Raxworthy, M J; Sheldon, I M

    2015-06-01

    Endometrial stromal and epithelial cell function is typically studied in vitro using standard two-dimensional monocultures, but these cultures fail to reflect the complex three-dimensional (3D) architecture of tissue. A 3D model of bovine endometrium that reflects the architectural arrangement of in vivo tissue would beneficially assist the study of tissue function. An electrospun polyglycolide (PGA) scaffold was selected to grow a 3D model of primary bovine endometrial epithelial and stromal cells, that reflects the architecture of the endometrium for the study of pathophysiology. Electrospun scaffolds were seeded with stromal and epithelial cells, and growth was assessed using histological techniques. Prostaglandin E2 and prostaglandin F2? responsiveness of endometrial scaffold constructs was tested using oxytocin plus arachidonic acid (OT + AA) or lipopolysaccharide (LPS). Stromal and epithelial cells growing on the electrospun scaffold had an architectural arrangement that mimicked whole tissue, deposited fibronectin, had appropriate expression of vimentin and cytokeratin and were responsive to OT + AA and LPS, as measured by prostaglandin accumulation. In conclusion, a functional 3D model of stromal and epithelial cells was developed using a PGA electrospun scaffold which may be used to study endometrial pathophysiology. PMID:26019144

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

    SciTech Connect

    Perkins, J; Parida, S; Clavijo, A

    2007-05-14

    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.

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

    PubMed Central

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

    2013-01-01

    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? 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

  6. Protein crystal growth in microgravity review of large scale temperature induction method: bovine insulin, human insulin and human alpha interferon

    NASA Astrophysics Data System (ADS)

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

    1996-10-01

    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 the PCF's first seven flights on the Space Shuttle, the last with laser light scattering instrumentation. The PCF's objective is twofold: (1) 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 to continue productions trials aimed at the development of a processing facility for crystalline recombinant alpha interferon.

  7. Development of an in vitro model of the early-stage bovine tuberculous granuloma using Mycobacterium bovis-BCG.

    PubMed

    Garza-Cuartero, Laura; McCarthy, Elaine; Brady, Joseph; Cassidy, Joseph; Hamilton, Clare; Sekiya, Mary; NcNair, Jim; Mulcahy, Grace

    2015-12-15

    Mycobacterium bovis causes 3.1% of human tuberculosis cases, as described by the World Health Organisation. In cattle, this organism causes bovine tuberculosis (BTB) which can have a prevalence of up to 39.5% in some developing countries. In developed countries, although the prevalence of BTB has been reduced through eradication programmes, complete eradication has in some cases proved elusive, with prevalences in cattle of 0.5% in the Republic of Ireland and of 4.3% in the UK. As the tuberculous granuloma is the fundamental lesion that reflects the pathogenesis, immune control and progression of BTB, we aimed to develop an in vitro model of the early-stage bovine tuberculous granuloma, in order to model the early stages of BTB, while also reducing the use of experimentally infected animals. In vitro models of human and ovine mycobacterial granulomas have previously been developed; however, so far, there is no model for the BTB granuloma. As the disease in cattle differs in a number of ways from that in other species, we consider this to be a significant gap in the tools available to study the pathogenesis of BTB. By combining bovine monocyte-derived macrophages infected with M. bovis-BCG and autologous lymphocytes we have developed an early-stage tuberculous bovine granuloma model. In the model, 3D cell aggregations formed a spherical-shape that grew for up to 11 days post-infection. This bovine tuberculous granuloma model can aid in the study of such lesion development, and in comparative studies of pathogenesis, such as, for example, the question of mycobacterial latency in bovine tuberculosis. PMID:26553300

  8. An abundant and novel protein of 22 kDa (SM22) is widely distributed in smooth muscles. Purification from bovine aorta.

    PubMed Central

    Lees-Miller, J P; Heeley, D H; Smillie, L B

    1987-01-01

    Using a rabbit polyclonal-antibody preparation directed against the chicken gizzard protein, we demonstrated by immunoblotting the presence of the 22 kDa protein (SM22) in a variety of chicken smooth-muscle-containing organs, including uterus, intestine, gizzard, oesophagus and aorta. Protein SM22 was present in only trace amounts in brain, liver and heart, and could not be detected in chicken breast muscle. The antibody preparation did not cross-react with extracts of bovine aorta. However, the presence of SM22 as a major component in bovine aorta and pig carotid was demonstrated by its co-migration with the purified chicken gizzard protein on one- and two-dimensional polyacrylamide electrophoretic gels. Its molar abundance relative to actin was estimated to be 0.9:6.0 and 1.4:6.0 for bovine aorta and pig carotid respectively. Like the chicken gizzard protein, it separates on pH-gradient electrophoresis into at least three variants, alpha, beta and gamma, with similar apparent Mr. Purification of the aorta SM22 showed it to have a similar amino acid composition to the chicken gizzard protein. We conclude that SM22 is widely distributed and an abundant and unique protein component of smooth-muscle tissues of birds and mammals. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3446186

  9. Expression of cytokine and apoptosis-related genes in bovine peripheral blood mononuclear cells stimulated with Brucella abortus recombinant proteins.

    PubMed

    Im, Young Bin; Jung, Myunghwan; Shin, Min-Kyoung; Kim, Suk; Yoo, Han Sang

    2016-01-01

    Brucellosis is a clinically and economically important disease. Therefore, eradication programs of the disease have been implemented in several countries. One hurdle in these programs is the detection of infected animals at the early stage. Although the protein antigens as diagnostic antigens have recently received attention, the exact mechanisms at the beginning of immune responses are not yet known. Therefore, genes encoding five B. abortus cellular proteins were cloned and the expressed recombinant proteins were purified. The expression of several cytokine genes (IL-1β, IL-4, IL-6, IL-12p40, IFN-γ, TNF-α, and iNOS) was analyzed in bovine peripheral blood mononuclear cells (bPBMC) after stimulation with the recombinant proteins. Three apoptosis-related genes, Bax, Bcl-2, and TLR4, were also included in the analysis to find out the adverse effects of the proteins to the cells. Each protein induced different patterns of cytokine expression depending on the stimulation time and antigen dose. Expression of IL-6, IL-12p40, and IFN-γ was induced with all of the proteins while IL-1β, IL-4, TNF-α, and iNOS gene expression was not. Expression of apoptosis-related genes was not altered except TLR4. These results suggest that the cellular antigens of B. abortus induce both humoral and cellular immunity via the production of IL-6, IL-12p40, and IFN-γ in bPBMC without exerting any adverse effects on the cells. PMID:26864657

  10. Mutant bovine odorant-binding protein: Temperature affects the protein stability and dynamics as revealed by infrared spectroscopy and molecular dynamics simulations.

    PubMed

    Marabotti, Anna; Lefvre, Thierry; Staiano, Maria; Crescenzo, Roberta; Varriale, Antonio; Rossi, Mos; Pzolet, Michel; D'Auria, Sabato

    2008-08-01

    Bovine odorant-binding protein (bOBP), a member of the lipocalin family, presents the so-called 3D "domain-swapped" protein structure. In fact, in solution, it appears as a dimer in which each monomer is composed by the classical lipocalin fold, with a central beta-barrel followed by a stretch of residues and the alpha-helix domain protruding out of the barrel and crossing the dimer interface. Recently, a deswapped mutant form of bOBP was obtained, in which a Gly residue was inserted after position 121 and the two residues in position 64 and 156 were replaced by Cys residues for restoring the disulfide bridge common to the lipocalin family. In this work, we used Fourier transform infrared spectroscopy and molecular dynamics simulations to investigate the effect of temperature on the structural stability and conformational dynamics of the mutant bOBP. The spectroscopic and molecular simulation data pointed out that the hydrophobic regions of the protein matrix appear to be an important factor for the protein stability and integrity. In addition, it was also found that the mutant bOBP is significantly stabilized by the binding of the ligand, which may have an impact on the biological function of bOBP. The obtained results will allow for a better use of this protein as probe for the design of advanced protein-based biosensors for the detection of compounds used in the fabrication of explosive powders. PMID:18260099

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

    PubMed Central

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

    2011-01-01

    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. PMID:21811561

  12. Carotenoid binding to proteins: Modeling pigment transport to lipid membranes.

    PubMed

    Reszczynska, Emilia; Welc, Renata; Grudzinski, Wojciech; Trebacz, Kazimierz; Gruszecki, Wieslaw I

    2015-10-15

    Carotenoid pigments play numerous important physiological functions in human organism. Very special is a role of lutein and zeaxanthin in the retina of an eye and in particular in its central part, the macula lutea. In the retina, carotenoids can be directly present in the lipid phase of the membranes or remain bound to the protein-pigment complexes. In this work we address a problem of binding of carotenoids to proteins and possible role of such structures in pigment transport to lipid membranes. Interaction of three carotenoids, beta-carotene, lutein and zeaxanthin with two proteins: bovine serum albumin and glutathione S-transferase (GST) was investigated with application of molecular spectroscopy techniques: UV-Vis absorption, circular dichroism and Fourier transform infrared spectroscopy (FTIR). Interaction of pigment-protein complexes with model lipid bilayers formed with egg yolk phosphatidylcholine was investigated with application of FTIR, Raman imaging of liposomes and electrophysiological technique, in the planar lipid bilayer models. The results show that in all the cases of protein and pigment studied, carotenoids bind to protein and that the complexes formed can interact with membranes. This means that protein-carotenoid complexes are capable of playing physiological role in pigment transport to biomembranes. PMID:26361975

  13. Dynamic tensile properties of bovine periodontal ligament: A nonlinear viscoelastic model.

    PubMed

    Oskui, Iman Z; Hashemi, Ata

    2016-03-21

    As a support to the tooth, the mechanical response of the periodontal ligament (PDL) is complex. Like other connective tissues, the PDL exhibits non-linear and time-dependent behavior. The viscoelasticity of the PDL plays a significant role in low and high loading rates. Little information, however, is available on the short-term viscoelastic behavior of the PDL. Also, due to the highly non-linear stress-strain response, it was hypothesized that the dynamic viscoelastic properties of the PDL would be greatly dependent on the preload. Therefore, the present study was designed to explore the dynamic tensile properties of the bovine PDL as a function of loading frequency and preload. The in vitro dynamic tensile tests were performed over a wide range of frequencies (0.01-100Hz) with dynamic force amplitude of 1N and different preloads of 3, 5 and 10N. The generalized Maxwell model was utilized to describe the non-linear viscoelastic behavior of the PDL. The low loss factor of the bovine PDL, measured between 0.04 and 0.08, indicates low energy dissipation due to the high content of collagen fibers. Moreover, the influence of viscous components in the linear region of the stress-strain curve (10N preload) was lower than those of the toe region (3N preload). The data reported in this study could be used in developing accurate computational models of the PDL. PMID:26920510

  14. Determination of an ensemble of structures representing the denatured state of the bovine acyl-coenzyme a binding protein.

    PubMed

    Lindorff-Larsen, Kresten; Kristjansdottir, Sigridur; Teilum, Kaare; Fieber, Wolfgang; Dobson, Christopher M; Poulsen, Flemming M; Vendruscolo, Michele

    2004-03-17

    The denatured state of a protein contains important information about the determinants of the folding process. By combining site-directed spin-labeling NMR experiments and restrained computer simulations, we have determined ensembles of conformations that represent the denatured state of the bovine acyl-coenzyme A binding protein (ACBP) at three different concentrations of guanidine hydrochloride. As the experimentally determined distance information corresponds to weighted averages over a broad ensemble of structures, we applied the experimental restraints to a system of noninteracting replicas of the protein by using a Monte Carlo sampling scheme. This procedure permits us to sample ensembles of conformations that are compatible with the experimental data and thus to obtain information regarding the distribution of structures in the denatured state. Our results show that the denatured state of ACBP is highly heterogeneous. The high sensitivity of the computational method that we present, however, enabled us to identify long-range interactions between two regions, located near the N- and C-termini, that include both native and non-native elements. The preferential formation of these contacts suggests that the sequence-dependent patterns of helical propensity and hydrophobicity are important determinants of the structure in the denatured state of ACBP. PMID:15012160

  15. Progesterone effect mediated by the voltage-dependent calcium channel and protein kinase C on noncapacitated cryopreserved bovine spermatozoa.

    PubMed

    Crdoba, M; Beconi, M T

    2001-03-01

    An increase in intracellular calcium is essential to trigger capacitation and the acrosome reaction. The aim of this study was to determine the progesterone effect mediated by the voltage-dependent calcium channel and protein kinase C on heparin-capacitated and noncapacitated spermatozoa. Protein kinase C was activated by 1-oleoyl-2-acetyl glycerol, a membrane-permeant diacyl-glycerol, and inhibited by GF-109203X. The percentage of true acrosome reaction was evaluated using differential-interferential optical contrast microscopy and trypan blue stain. The calcium concentration was evaluated by FURA-2AM and methoxyverapamil was used as a voltage-dependent calcium channel inhibitor. A rapid calcium increase and acrosome reaction were induced by progesterone in capacitated and noncapacitated spermatozoa, a higher intracellular calcium increase being observed in capacitated than in noncapacitated samples (P < 0.05). The calcium increase and acrosome reaction were blocked significantly by GF-109203X in noncapacitated and capacitated spermatozoa by the addition of progesterone and/or 1-oleoyl-2-acetylglycerol. Methoxyverapamil blocked calcium influx in samples treated with progesterone and heparin/progesterone, but not in those treated with 1-oleoyl-2-acetyl glycerol. Progesterone induces the acrosome reaction in noncapacitated cryopreserved bovine spermatozoa through intracellular mechanisms dependent on protein kinase C and the voltage-dependent calcium channel. PMID:11350374

  16. Adsorption of bovine serum albumin on CoCrMo surface: effect of temperature and protein concentration.

    PubMed

    Valero Vidal, C; Olmo Juan, A; Igual Muñoz, A

    2010-10-01

    The adsorption of bovine serum albumin (BSA) onto CoCrMo surface has been studied as a function of concentration of BSA and temperature by electrochemical techniques. The electrochemical impedance spectroscopy (EIS) technique was used to investigate the interfacial behaviour of BSA at open circuit potential (OCP). The charge transfer resistance was very sensitive to the amount of adsorbed protein, indicating that the adsorption process was accompanied by the transfer of charge and influenced the mechanism and kinetics of the corrosion reaction. At all the temperatures studied, adsorption of BSA onto the CoCrMo surface was successfully described with a Langmuir adsorption isotherm. EIS study was also carried out for determine the surface charge density, resulting from protein adsorption, and it was shown to be directly proportional to the amount of adsorbed protein (surface concentration). Thermodynamic data of adsorption was obtained for analyzing the adsorption of BSA onto CoCrMo surface. Gibbs free energy of adsorption, DeltaG(ADS) values, for BSA in the investigated temperature range (-51kJmol(-1)) showed that the molecules have a strong affinity for the CoCrMo surface. Enthalpy (DeltaH(ADS)) and entropy (DeltaS(ADS)) of adsorption suggested that the adsorption process of BSA onto the CoCrMo surface is an endothermic process and the molecule suffers structural changes when adsorbing on the metallic surface. PMID:20554436

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

    SciTech Connect

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

    1987-07-01

    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.

  18. Highly sensitive detection of small ruminant bovine spongiform encephalopathy within transmissible spongiform encephalopathy mixes by serial protein misfolding cyclic amplification.

    PubMed

    Gough, Kevin C; Bishop, Keith; Maddison, Ben C

    2014-11-01

    It is assumed that sheep and goats consumed the same bovine spongiform encephalopathy (BSE)-contaminated meat and bone meal that was fed to cattle and precipitated the BSE epidemic in the United Kingdom that peaked more than 20 years ago. Despite intensive surveillance for cases of BSE within the small ruminant populations of the United Kingdom and European Union, no instances of BSE have been detected in sheep, and in only two instances has BSE been discovered in goats. If BSE is present within the small ruminant populations, it may be at subclinical levels, may manifest as scrapie, or may be masked by coinfection with scrapie. To determine whether BSE is potentially circulating at low levels within the European small ruminant populations, highly sensitive assays that can specifically detect BSE, even within the presence of scrapie prion protein, are required. Here, we present a novel assay based on the specific amplification of BSE PrP(Sc) using the serial protein misfolding cyclic amplification assay (sPMCA), which specifically amplified small amounts of ovine and caprine BSE agent which had been mixed into a range of scrapie-positive brain homogenates. We detected the BSE prion protein within a large excess of classical, atypical, and CH1641 scrapie isolates. In a blind trial, this sPMCA-based assay specifically amplified BSE PrP(Sc) within brain mixes with 100% specificity and 97% sensitivity when BSE agent was diluted into scrapie-infected brain homogenates at 1% (vol/vol). PMID:25143565

  19. Purification and characterization of a bovine serum lectin (CL-43) with structural homology to conglutinin and SP-D and carbohydrate specificity similar to mannan-binding protein.

    PubMed

    Holmskov, U; Teisner, B; Willis, A C; Reid, K B; Jensenius, J C

    1993-05-15

    A previously undescribed bovine serum lectin (designated CL-43) was identified by its Ca(2+)-dependent binding to mannan and by its molecular mass of 43 kDa under reducing conditions on SDS-PAGE. The lectin was isolated by polyethylene glycol precipitation, affinity chromatography on mannan-Sepharose (followed by elution with EDTA), and absorption on Sepharose-4B-coupled rabbit anti-bovine Ig (to remove anti-mannan antibodies). Fractions containing the lectin were reapplied to mannan-Sepharose. Bound conglutinin was eluted with GlcNAc, and then the 43-kDa lectin, together with mannan-binding protein (MBP), was eluted with mannose. The 43-kDa lectin was separated from MBP by ion exchange chromatography on Mono-Q. On SDS-PAGE under nonreducing conditions the lectin showed a molecular mass of 120 kDa. On gel chromatography under nondissociating conditions the protein was eluted at a volume corresponding to a molecular mass of approximately 750 kDa. Amino acid analysis showed the presence of hydroxyproline and hydroxylysine and a high content of glycine (24.3%) indicating the presence of a collagen-like structure. This was supported by the susceptibility of the protein to collagenase digestion. The designation CL-43 was chosen since this molecule appears to belong to the collectins, i.e. proteins with collagen structure and lectin activity. The N-terminal sequence (27 amino acids) showed 56% identity with bovine SP-D and 44% identity to bovine conglutinin. An inhibition assay with biotinylated CL-43, using solid-phase mannan as ligand, revealed the following carbohydrate inhibition pattern: mannose and ManNAc > fucose > GlcNAc > glucose and maltose > galactose > lactose > GalNAc. We conclude that CL-43 is a circulating lectin, with structural similarities to bovine conglutinin and SP-D, and a ligand binding profile resembling that of MBP. PMID:8486682

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

    SciTech Connect

    Talhouk, R.S.

    1988-01-01

    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.

  1. Selective binding of proteins on functional nanoparticles via reverse charge parity model: an in vitro study

    NASA Astrophysics Data System (ADS)

    Ghosh, Goutam; Panicker, Lata; Barick, K. C.

    2014-03-01

    The conformation of proteins absorbed on nanoparticles surface plays a crucial role in applications of nanoparticles in biomedicine. The surface protein conformation depends on several factors, namely, nature of protein-nanoparticles interaction, chemical composition of the surface of nanoparticles etc. A model of the electrostatic binding of proteins on charged surface nanoparticles has been proposed earlier (Ghosh et al 2013 Colloids Surf. B 103 267). Also, the irreversible denaturation of the protein conformation due to binding of counterions was reported. In this paper, we have used this model, involving reverse charge parity, to show selective binding of proteins on charged surface iron oxide nanoparticles (IONPs). IONPs were surface functionalized with cetylpyridinium chloride (CPC), cetyl(trimethyl)ammonium bromide (CTAB) and cetylpyridinium iodide (CPI). The effect of counterions (Cl-, Br- and I-) on protein conformation has also been investigated. Several proteins such as ?-lactalbumin (ALA), ?-lactoglobulin (BLG), ovalbumin (OVA), bovin serum albumin (BSA) and HEWL were chosen for this investigation.

  2. Homology Modeling Study of Bovine μ-Calpain Inhibitor-Binding Domains

    PubMed Central

    Chai, Han-Ha; Lim, Dajeong; Lee, Seung-Hwan; Chai, Hee-Yeoul; Jung, Eunkyoung

    2014-01-01

    The activated mammalian CAPN-structures, the CAPN/CAST complex in particular, have become an invaluable target model using the structure-based virtual screening of drug candidates from the discovery phase to development for over-activated CAPN linked to several diseases, such as post-ischemic injury and cataract formation. The effect of Ca2+-binding to the enzyme is thought to include activation, as well as the dissociation, aggregation, and autolysis of small regular subunits. Unfortunately, the Ca2+-activated enzyme tends to aggregate when provided as a divalent ion at the high-concentration required for the protease crystallization. This is also makes it very difficult to crystallize the whole-length enzyme itself, as well as the enzyme-inhibitor complex. Several parameters that influence CAPN activity have been investigated to determine its roles in Ca2+-modulation, autoproteolysis, phosphorylation, and intracellular distribution and inhibition by its endogenous inhibitor CAST. CAST binds and inhibits CAPN via its CAPN-inhibitor domains (four repeating domains 1–4; CAST1–4) when CAPN is activated by Ca2+-binding. An important key to understanding CAPN1 inhibition by CAST is to determine how CAST interacts at the molecular level with CAPN1 to inhibit its protease activity. In this study, a 3D structure model of a CAPN1 bound bovine CAST4 complex was built by comparative modeling based on the only known template structure of a rat CAPN2/CAST4 complex. The complex model suggests certain residues of bovine CAST4, notably, the TIPPKYQ motif sequence, and the structural elements of these residues, which are important for CAPN1 inhibition. In particular, as CAST4 docks near the flexible active site of CAPN1, conformational changes at the interaction site after binding could be directly related to CAST4 inhibitory activity. These functional interfaces can serve as a guide to the site-mutagenesis in research on bovine CAPN1 structure-function relationships for the design of small molecules inhibitors to prevent uncontrolled and unspecific degradation in the proteolysis of key protease substrates. PMID:24806345

  3. Inducible expression of green fluorescent protein in porcine tracheal epithelial cells by the bovine tracheal antimicrobial peptide promoter.

    PubMed

    Dyce, Paul W; DeVries, Robert J; Walton, John; Hacker, Roger R; Li, Julang

    2003-11-01

    Tracheal antimicrobial peptides (TAP) are expressed primarily in respiratory epithelial cells of cattle. The TAP expression is inducible upon challenge with bacteria and bacterial lipopolysaccharide (LPS). In pigs, a promoter that can be activated by bacterial infection has yet to be identified. The objective of this study was to use green fluorescent protein (GFP) as a reporter gene to determine the function and inducibility of the bovine TAP promoter in porcine primary tracheal epithelial cells. Thus, evaluating the feasibility of using this promoter to direct transgene expression in porcine cells. The percentage of GFP expressing cells increased in response to LPS challenge in both a dose-dependent and time-dependent manner (p < 0.05). Moreover, when the intensity of the GFP fluorescence was measured, it was observed that the percentage of cells that have a high intensity of GFP fluorescence, also increased gradually as LPS dose increased, the difference between the unchallenged (control) and challenged group become statistically significant at the concentration of 100 ng/mL after 36 h LPS challenge (p < 0.05). The level of induced-expression driven by the TAP promoter was 67.8 +/-12.2% that of the cytomegalovirus (CMV) promoter. The intensity of GFP fluorescence by the TAP promoter was 39.8 +/- 7.6% when compared to the expression driven by the CMV promoter. These data suggest the TAP promoter functions at a lower, but comparable, level to the strong CMV promoter. Our data demonstrated that the bovine TAP promoter was functional in porcine primary tracheal epithelial cells. The ability of the TAP promoter to control gene expression in an inducible manner in the porcine respiratory tract presents an important application potential in transgenic animal studies. PMID:12968291

  4. Reactivity of bovine whey proteins, peptides, and amino acids toward triplet riboflavin as studied by laser flash photolysis.

    PubMed

    Cardoso, Daniel R; Franco, Douglas W; Olsen, Karsten; Andersen, Mogens L; Skibsted, Leif H

    2004-10-20

    The reaction between the triplet excited state of riboflavin and amino acids, peptides, and bovine whey proteins was investigated in aqueous solution in the pH range from 4 to 9 at 24 degrees C using nanosecond laser flash photolysis. Only tyrosine and tryptophan (and their peptides) were found to compete with oxygen in quenching the triplet state of riboflavin in aqueous solution, with second-order rate constants close to the diffusion limit, 1.75 x 10(9) and 1.40 x 10(9) L mol(-1) s(-1) for tyrosine and tryptophan, respectively, with beta-lactoglobulin and bovine serum albumin having comparable rate constants of 3.62 x 10(8) and 2.25 x 10(8) L mol(-1) s(-1), respectively. Tyrosine, tryptophan, and their peptides react with the photoexcited triplet state of riboflavin by electron transfer from the tyrosine and tryptophan moieties followed by a fast protonation of the resulting riboflavin anion rather than by direct H-atom abstraction, which could be monitored by time-resolved transient absorption spectroscopy as a decay of triplet riboflavin followed by a rise in riboflavin anion radical absorption. For cysteine- and thiol-containing peptides, second-order rate constants depend strongly on pH, for cysteine corresponding to pKaRSH = 8.35. H-atom abstraction seems to operate at low pH, which with rising pH gradually is replaced by electron transfer from the thiol anion. From the pH dependence of the second-order rate constant, the respective values for the H-atom abstraction (k = 1.64 x 10(6) L mol(-1) s(-1)) and for the electron transfer (k = 1.20 x 10(9) L mol(-1) s(-1)) were determined. PMID:15479029

  5. Characterization of the Interaction between Eupatorin and Bovine Serum Albumin by Spectroscopic and Molecular Modeling Methods

    PubMed Central

    Xu, Hongliang; Yao, Nannan; Xu, Haoran; Wang, Tianshi; Li, Guiying; Li, Zhengqiang

    2013-01-01

    This study investigated the interaction between eupatorin and bovine serum albumin (BSA) using ultraviolet-visible (UV-vis) absorption, fluorescence, synchronous fluorescence, circular dichroism (CD) spectroscopies, and molecular modeling at pH 7.4. Results of UV-vis and fluorescence spectroscopies illustrated that BSA fluorescence was quenched by eupatorin via a static quenching mechanism. Thermodynamic parameters revealed that hydrophobic and electrostatic interactions played major roles in the interaction. Moreover, the efficiency of energy transfer, and the distance between BSA and acceptor eupatorin, were calculated. The effects of eupatorin on the BSA conformation were analyzed using UV-vis, CD, and synchronous fluorescence. Finally, the binding of eupatorin to BSA was modeled using the molecular docking method. PMID:23839090

  6. Evaluation of comparative protein modeling by MODELLER.

    PubMed

    Sali, A; Potterton, L; Yuan, F; van Vlijmen, H; Karplus, M

    1995-11-01

    We evaluate 3D models of human nucleoside diphosphate kinase, mouse cellular retinoic acid binding protein I, and human eosinophil neurotoxin that were calculated by MODELLER, a program for comparative protein modeling by satisfaction of spatial restraints. The models have good stereochemistry and are at least as similar to the crystallographic structures as the closest template structures. The largest errors occur in the regions that were not aligned correctly or where the template structures are not similar to the correct structure. These regions correspond predominantly to exposed loops, insertions of any length, and non-conserved side chains. When a template structure with more than 40% sequence identity to the target protein is available, the model is likely to have about 90% of the mainchain atoms modeled with an rms deviation from the X-ray structure of approximately 1 A, in large part because the templates are likely to be that similar to the X-ray structure of the target. This rms deviation is comparable to the overall differences between refined NMR and X-ray crystallography structures of the same protein. PMID:8710825

  7. A dynamic model of bovine tuberculosis spread and control in Great Britain.

    PubMed

    Brooks-Pollock, Ellen; Roberts, Gareth O; Keeling, Matt J

    2014-07-10

    Bovine tuberculosis (TB) is one of the most complex, persistent and controversial problems facing the British cattle industry, costing the country an estimated 100 million per year. The low sensitivity of the standard diagnostic test leads to considerable ambiguity in determining the main transmission routes of infection, which exacerbates the continuing scientific debate. In turn this uncertainty fuels the fierce public and political disputes on the necessity of controlling badgers to limit the spread of infection. Here we present a dynamic stochastic spatial model for bovine TB in Great Britain that combines within-farm and between-farm transmission. At the farm scale the model incorporates stochastic transmission of infection, maintenance of infection in the environment and a testing protocol that mimics historical government policy. Between-farm transmission has a short-range environmental component and is explicitly driven by movements of individual cattle between farms, as recorded in the Cattle Tracing System. The resultant model replicates the observed annual increase of infection over time as well as the spread of infection into new areas. Given that our model is mechanistic, it can ascribe transmission pathways to each new case; the majority of newly detected cases involve several transmission routes with moving infected cattle, reinfection from an environmental reservoir and poor sensitivity of the diagnostic test all having substantive roles. This underpins our findings on the implications of control measures. Very few of the control options tested have the potential to reverse the observed annual increase, with only intensive strategies such as whole-herd culling or additional national testing proving highly effective, whereas controls focused on a single transmission route are unlikely to be highly effective. PMID:25008532

  8. Specific interaction between the bovine papillomavirus E5 transforming protein and the beta receptor for platelet-derived growth factor in stably transformed and acutely transfected cells.

    PubMed Central

    Petti, L; DiMaio, D

    1994-01-01

    The E5 protein of bovine papillomavirus is a 44-amino-acid membrane protein which induces morphologic and tumorigenic transformation of fibroblasts. We previously showed that the E5 protein activates and forms a complex with the endogenous beta receptor for platelet-derived growth factor (PDGF) in transformed rodent fibroblasts and that the PDGF beta receptor can mediate tumorigenic transformation by the E5 protein in a heterologous cell system. Other workers have identified the receptor for epidermal growth factor (EGF) as a potential target of the E5 protein in NIH 3T3 cells. Here, we investigate the specificity of the interaction of the E5 protein with various growth factor receptors, with particular emphasis on the PDGF beta receptor and the EGF receptor. Under conditions where both the PDGF beta receptor and the EGF receptor are stably expressed in E5-transformed mouse and bovine fibroblasts and in E5-transformed epithelial cells, the E5 protein specifically forms a complex with and activates the PDGF receptor and not the EGF receptor. Under conditions of transient overexpression in COS cells, the E5 protein has the potential to associate with several growth factor receptors, including the EGF receptor. However, upon coexpression of PDGF beta receptors and EGF receptors in COS cells, the E5 protein preferentially forms a complex with the PDGF receptor. Therefore, we conclude that the PDGF beta receptor is the primary target for the E5 protein in a variety of cell types, including bovine fibroblasts. Images PMID:8189497

  9. Proteins of bovine viral diarrhea virus: characterization, biotype-specific differences, and immunological properties

    SciTech Connect

    Donis, R.O.

    1987-01-01

    Virus-specific polypeptides in bovine viral diarrhea-mucosal disease (BVD) virus-infected bovine cells were studied by radiolabeling. A total of 12 polypeptides with apparent Mr of 165, 135, 118, 80, 75, 62, 56-58, 48, 37, 32, 25 and 19 kilodaltons (k) were identified in infected cells. Five glycoproteins were detected in infected cells. Two abundant species had apparent Mr of 48 k and 56-58 k while the minor species had masses of 118, 75 and 65 k. When cells were radiolabeled with L-(/sup 35/S)-methionine in the presence of tunicamycin the 56-58 k migrated with apparent masses of 54 k and 48-50 K in PAGE. Endoglycosidase F digestion of virus-induced polypeptides caused a 4-6 K reduction in the apparent molecular mass of the 56-58 k yielding a 52 k digested product. Tunicamycin caused a drastic reduction in the yield of infectious virus indicating that the carbohydrate moieties serve a vital role in the infection cycle of BVD virus. The noncytopathic biotype BVD (NCB-BVD) virus isolates can be consistently differentiated from cytopathic biotype BVD (CB-BVD) isolates on the basis of unique polypeptide profiles they induce in the infected cell: the most abundant polypeptide in CB-BVD infected cells is the 80 kD polypeptide while NCB-BVD lack this polypeptide and induce a predominant 118 k polypeptide. A panel of 25 murine monoclonal antibodies (Mabs) against the two major glycoproteins of BVD virus was produced. Based on their viral polypeptide specificity and on their ability to neutralize viral infectivity the Mabs in the panel were divided into 3 classes: Class 1 Mabs reacted with the 56-58 k glycoprotein and neutralized the virus, Class 2 Mabs recognized the 56-58 k glycoprotein but were not neutralizing and Class 3 Mabs reacted with the 48 k glycoprotein and did not neutralize the virus. These results identify the 56-58 k as one of the envelope glycoproteins of BVD virus.

  10. Generation of the bovine viral diarrhea virus e0 protein in transgenic astragalus and its immunogenicity in sika deer.

    PubMed

    Gao, Yugang; Zhao, Xueliang; Zang, Pu; Liu, Qun; Wei, Gongqing; Zhang, Lianxue

    2014-01-01

    The bovine viral diarrhea virus (BVDV), a single-stranded RNA virus, can cause fatal diarrhea syndrome, respiratory problems, and reproductive disorders in herds. Over the past few years, it has become clear that the BVDV infection rates are increasing and it is likely that an effective vaccine for BVDV will be needed. In this study, transgenic Astragalus was used as an alternative productive platform for the expression of glycoprotein E0. The immunogenicity of glycoprotein E0 expressed in transgenic Astragalus was detected in deer. The presence of pBI121-E0 was confirmed by polymerase chain reaction (PCR), transcription was verified by reverse transcription- (RT-) PCR, and recombinant protein expression was confirmed by ELISA and Western blot analyses. Deer that were immunized subcutaneously with the transgenic plant vaccine developed specific humoral and cell-mediated immune responses against BVDV. This study provides a new method for a protein with weak immunogenicity to be used as part of a transgenic plant vaccine. PMID:24963321

  11. Generation of the Bovine Viral Diarrhea Virus E0 Protein in Transgenic Astragalus and Its Immunogenicity in Sika Deer

    PubMed Central

    Gao, Yugang; Zang, Pu; Liu, Qun; Wei, Gongqing

    2014-01-01

    The bovine viral diarrhea virus (BVDV), a single-stranded RNA virus, can cause fatal diarrhea syndrome, respiratory problems, and reproductive disorders in herds. Over the past few years, it has become clear that the BVDV infection rates are increasing and it is likely that an effective vaccine for BVDV will be needed. In this study, transgenic Astragalus was used as an alternative productive platform for the expression of glycoprotein E0. The immunogenicity of glycoprotein E0 expressed in transgenic Astragalus was detected in deer. The presence of pBI121-E0 was confirmed by polymerase chain reaction (PCR), transcription was verified by reverse transcription- (RT-) PCR, and recombinant protein expression was confirmed by ELISA and Western blot analyses. Deer that were immunized subcutaneously with the transgenic plant vaccine developed specific humoral and cell-mediated immune responses against BVDV. This study provides a new method for a protein with weak immunogenicity to be used as part of a transgenic plant vaccine. PMID:24963321

  12. 60S ribosomal protein L35 regulates ?-casein translational elongation and secretion in bovine mammary epithelial cells.

    PubMed

    Jiang, Nan; Hu, Lijun; Liu, Chaonan; Gao, Xueli; Zheng, Shimin

    2015-10-01

    60S ribosomal protein L35 (RPL35) is an important component of the 60S ribosomal subunit and has a role in protein translation and endoplasmic reticulum (ER) docking. However, few studies have investigated RPL35 in eukaryotes and much remains to be learned. Here, we analyzed the function of RPL35 in ?-casein (CSN2) synthesis and secretion in bovine mammary epithelial cells (BMECs). We found that methionine (Met) could promote the expressions of CSN2 and RPL35. Analysis of overexpression and inhibition of RPL35 confirmed that it could mediate the Met signal and regulate CSN2 expression. The mechanism of CSN2 regulation by RPL35 was analyzed by coimmunoprecipitation (Co-IP), colocalization, fluorescence resonance energy transfer (FRET) and gene mutation. We found that RPL35 could control ribosome translational elongation during synthesis of CSN2 by interacting with eukaryotic translational elongation factor 2 (eEF2), and that eEF2 was the signaling molecule downstream of RPL35 controlling this process. RPL35 could also control the secretion of CSN2 by locating it to the ER. Taken together, these results revealed that, RPL35 was an important positive regulatory factor involving in the Met-mediated regulation of CSN2 translational elongation and secretion. PMID:26297660

  13. Influences of different thermal processings in milk, bovine meat and frog protein structure.

    PubMed

    Coura Oliveira, Tatiana; Lopes Lima, Samuel; Bressan, Josefina

    2013-01-01

    Several studies have associated the digestibility of proteins to its imunogenic potential. Though, it was objectified to evaluate the impact of the thermal processing with high and low temperatures on the proteins structure of three types of foods, by means of the digestibility in vitro and electroforesis en gel de poliacrilamida. The pasteurize was observed in such a way, firing 95 C during 15 minutes, how much freeze dried causes qualitative and quantitative modifications of constituent proteins of the food. The most sensible proteins to the increasing thermal processing order were beef, frog meat, and the last, cow milk. PMID:23848117

  14. Interleukin-6 inhibits adrenal androgen release from bovine adrenal zona reticularis cells by inhibiting the expression of steroidogenic proteins.

    PubMed

    McIlmoil, S; Call, G B; Barney, M; Strickland, J; Judd, A M

    2015-10-01

    Interleukin-6 (IL-6) is secreted by adrenocortical cells and modifies cortisol secretion. In this study, the effects of IL-6 on adrenal androgen release were investigated. The zona reticularis (ZR) was generally isolated from bovine adrenal glands by dissection. In select experiments, the intact adrenal cortex (ie, all 3 adrenocortical zones) was dissected from the adrenal glands. For androgen release experiments, ZR and intact adrenocortical cubes were dispersed into isolated cells, the cells cultured and exposed to IL-6 and/or adrenocorticotropic hormone (ACTH), and androgen release determined by radioimmunoassay. Basal and ACTH-stimulated androgen release from the ZR was inhibited by IL-6 in a concentration-dependent (10-1000 pg/mL) and time-dependent (4-24h) manner (P < 0.01 by 1-way analysis of variance and the Bonferroni test). In contrast, IL-6 increased basal and ACTH-stimulated androgen release from mixed adrenocortical cells (P < 0.01). The mechanism of IL-6 inhibition of androgen release was investigated by exposing ZR strips to IL-6 and measuring the expression of the messenger RNA (mRNA) and protein of steroidogenic factors. Basal and ACTH-stimulated expression of the mRNA and protein for steroidogenic acute regulatory protein, cholesterol side chain cleavage enzyme, 3-?-hydroxysteroid dehydrogenase type 2, steroid 17-?-hydroxylase/17,20 lyase/17,20 desmolase, and the nuclear factor steroidogenic factor 1 (SF-1), that stimulates steroidogenesis, were decreased by IL-6 (P < 0.01). In contrast IL-6 increased the mRNA and protein for dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1), a nuclear factor that inhibits steroidogenesis (P < 0.01). In summary, IL-6 decreased androgen release and the expression of steroidogenic factors in the ZR, and this decrease may be mediated in part through increasing DAX-1 and decreasing SF-1. PMID:26218834

  15. Validation of bovine glycomacropeptide as an intestinal anti-inflammatory nutraceutical in the lymphocyte-transfer model of colitis.

    PubMed

    Ortega-Gonzlez, Mercedes; Capitn-Caadas, Fermn; Requena, Pilar; Ocn, Borja; Romero-Calvo, Isabel; Aranda, Carlos; Surez, Mara Dolores; Zarzuelo, Antonio; Snchez de Medina, Fermn; Martnez-Augustin, Olga

    2014-04-14

    Milk ?-casein-derived bovine glycomacropeptide (GMP) exerts immunomodulatory effects. It exhibits intestinal anti-inflammatory activity in chemically induced models of colitis. However, to validate its clinical usefulness as a nutraceutical, it is important to assess its effects in a model with a closer pathophysiological connection with human inflammatory bowel disease. Therefore, in the present study, we used the lymphocyte-transfer model of colitis in mice and compared the effects of GMP in this model with those obtained in the dextran sulphate sodium (DSS) model. GMP (15 mg/d) resulted in higher body-weight gain and a reduction of the colonic damage score and myeloperoxidase (MPO) activity in Rag1(-/-) mice with colitis induced by the transfer of nave T cells. The colonic and ileal weight:length ratio was decreased by approximately 25%, albeit non-significantly. GMP treatment reduced the percentage of CD4? interferon (IFN)-?? cells in mesenteric lymph nodes (MLN). The basal production of IL-6 by MLN obtained from the GMP-treated mice ex vivo was augmented. However, concanavalin A-evoked production was similar. The colonic expression of regenerating islet-derived protein 3?, S100A8, chemokine (C-X-C motif) ligand 1 and IL-1? was unaffected by GMP, while that of TNF-? and especially IFN-? was paradoxically increased. In the DSS model, GMP also reduced the activity of colonic MPO, but it failed to alter weight gain or intestinal weight:length ratio. GMP augmented the production of IL-10 by MLN cells and was neutral towards other cytokines, except exhibiting a trend towards increasing the production of IL-6. The lower effect was attributed to the lack of the effect of GMP on epithelial cells. In conclusion, GMP exerts intestinal anti-inflammatory effects in lymphocyte-driven colitis. PMID:24229852

  16. Proteomic analysis of differentially expressed proteins in bovine milk during experimentally induced Escherichia coli mastitis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of the current study were to profile changes in protein composition using 2-dimensional gel electrophoresis (2D-GE) on whey samples from a group of 8 cows prior to and 18 hours after infection with Escherichia coli, and to identify differentially expressed milk proteins by peptide seq...

  17. Protein Bioavailability in Animal Model.

    PubMed

    2016-01-01

    All the nutrients in food are not absorbed by our system. The relative amount of nutrition from an administered dosage through which it enters the system of circulation and the rate at which the level of nutrition appears in the systemic circulation is called 'bioavailability'. Although plant foods have high nutrient contents our digestion system cannot absorb it fully. Hence, it becomes necessary to determine the bioavailability of the administered sample to check the overall effect on the living system. Animal food preparation, protein bioavailability in animal model, food efficiency, apparent digestibility, protein digestibility tests, etc., are mentioned in well explained methodologies. PMID:26939286

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

    SciTech Connect

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

    1985-11-01

    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.

  19. Protein profiles of bovine placenta derived from somatic cell nuclear transfer.

    PubMed

    Kim, Hong Rye; Kang, Jae Ku; Yoon, Jong Taek; Seong, Hwan Hoo; Jung, Jin Kwan; Lee, Hong Mie; Sik Park, Chang; Jin, Dong Il

    2005-11-01

    Practical application of animal cloning by somatic cell nuclear transfer (SCNT) has been hampered by an extremely low success rate. To address whether placental dysfunction in SCNT causes fetal loss during pregnancy, we have used a global proteomics approach using 2-DE and MS to analyze the differential protein patterns of three placentae from the afterbirth of cases of postnatal death, derived from SCNT of Korean Native cattle, and three normal placentae obtained from the afterbirth of fetuses derived from artificial insemination. Proteins within a pI range of 4.0-7.0 and 6.0-9.0 were analyzed separately by 2-DE in triplicate. A total of approximately 2000 spots were detected in placental 2-DE gels stained with CBB. In the comparison of normal and SCNT samples, 60 spots were identified as differentially expressed proteins, of which 33 spots were up-regulated proteins in SCNT placentae, while 27 spots were down-regulated proteins. Most of the proteins identified in this analysis appeared to be related with protein repair or protection, cytoskeleton, signal transduction, immune system, metabolism, extracellular matrix and remodeling, transcription regulation, cell structure or differentiation and ion transport. One of up-regulated proteins in SCNT was TIMP-2 protein known to be related to extracellular matrix and remodeling during pregnancy. Western blot analysis showed an increased level of TIMP-2 in SCNT placenta compared to normal. Our results revealed composite profiles of key proteins involved in abnormal placenta derived from SCNT, and suggested expression abnormality of these genes in SCNT placenta, resulting in fetal losses following SCNT. PMID:16196098

  20. 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) ...

  1. Mathematical Models for Estimating the Risks of Bovine Spongiform Encephalopathy (BSE).

    PubMed

    Al-Zoughool, Mustafa; Cottrell, David; Elsaadany, Susie; Murray, Noel; Oraby, Tamer; Smith, Robert; Krewski, Daniel

    2015-01-01

    When the bovine spongiform encephalopathy (BSE) epidemic first emerged in the United Kingdom in the mid 1980s, the etiology of animal prion diseases was largely unknown. Risk management efforts to control the disease were also subject to uncertainties regarding the extent of BSE infections and future course of the epidemic. As understanding of BSE increased, mathematical models were developed to estimate risk of BSE infection and to predict reductions in risk in response to BSE control measures. Risk models of BSE-transmission dynamics determined disease persistence in cattle herds and relative infectivity of cattle prior to onset of clinical disease. These BSE models helped in understanding key epidemiological features of BSE transmission and dynamics, such as incubation period distribution and age-dependent infection susceptibility to infection with the BSE agent. This review summarizes different mathematical models and methods that have been used to estimate risk of BSE, and discusses how such risk projection models have informed risk assessment and management of BSE. This review also provides some general insights on how mathematical models of the type discussed here may be used to estimate risks of emerging zoonotic diseases when biological data on transmission of the etiological agent are limited. PMID:26158300

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

    PubMed

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

    1996-05-01

    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

  3. Development of a single nucleotide polymorphism genotyping microarray platform for the identification of bovine milk protein genetic polymorphisms.

    PubMed

    Chessa, S; Chiatti, F; Ceriotti, G; Caroli, A; Consolandi, C; Pagnacco, G; Castiglioni, B

    2007-01-01

    The objective of this study was to develop and validate a fast method for typing the main mutations of bovine milk protein genes by using microarray technology. An approach based on the ligation detection reaction (LDR) and a universal array (UA) was used. Polymorphisms in both the coding and noncoding sequences of alpha(S1)-casein, beta-casein, kappa-casein, and beta-lactoglobulin genes were considered because of their well-known effects on milk composition and cheese production. A total of 22 polymorphic sites, corresponding to 21 different variants, were included in the diagnostic microarray. First, a multiplex PCR was developed to amplify all the DNA target sequences simultaneously. Second, the LDR-UA assay was implemented. The method was validated by analyzing 100 Italian Friesian DNA samples, which were also genotyped by conventional methods both at the protein level by means of milk isoelectrofocusing and at the molecular level using PCR-RFLP and PCR-single strand conformation polymorphism techniques. The genotypes obtained using the LDR-UA approach were in full agreement with those obtained by the conventional analyses. An important result of the LDR-UA assay was a more accurate genotyping of the different milk protein alleles than was found with conventional typing methods. At the kappa-casein gene, in fact, 4 samples were heterozygous (3 reference samples and 1 validation sample) for an allele coding for Thr(136) and Ala(148). This variant, which can be considered as the wild type of the genus Bos, is not usually identifiable by the conventional typing methods used. The multiplex PCR-LDR-UA approach developed provides for an accurate, inexpensive, and high-throughput assay that does not exhibit false positive or false negative signals, thus making it highly suitable for animal genotyping. PMID:17183114

  4. Whole genome scan to detect quantitative trait loci for bovine milk protein composition.

    PubMed

    Schopen, G C B; Koks, P D; van Arendonk, J A M; Bovenhuis, H; Visker, M H P W

    2009-08-01

    The objective of this study was to perform a whole genome scan to detect quantitative trait loci (QTL) for milk protein composition in 849 Holstein-Friesian cows originating from seven sires. One morning milk sample was analysed for the major milk proteins using capillary zone electrophoresis. A genetic map was constructed with 1341 single nucleotide polymorphisms, covering 2829 centimorgans (cM) and 95% of the cattle genome. The chromosomal regions most significantly related to milk protein composition (P(genome) < 0.05) were found on Bos taurus autosomes (BTA) 6, 11 and 14. The QTL on BTA6 was found at about 80 cM, and affected alpha(S1)-casein, alpha(S2)-casein, beta-casein and kappa-casein. The QTL on BTA11 was found at 124 cM, and affected beta-lactoglobulin, and the QTL on BTA14 was found at 0 cM, and affected protein percentage. The proportion of phenotypic variance explained by the QTL was 3.6% for beta-casein and 7.9% for kappa-casein on BTA6, 28.3% for beta-lactoglobulin on BTA11, and 8.6% for protein percentage on BTA14. The QTL affecting alpha(S2)-casein on BTA6 and 17 showed a significant interaction. We investigated the extent to which the detected QTL affecting milk protein composition could be explained by known polymorphisms in beta-casein, kappa-casein, beta-lactoglobulin and DGAT1 genes. Correction for these polymorphisms decreased the proportion of phenotypic variance explained by the QTL previously found on BTA6, 11 and 14. Thus, several significant QTL affecting milk protein composition were found, of which some QTL could partially be explained by polymorphisms in milk protein genes. PMID:19397519

  5. 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...

  6. Modelling the spread of Bovine Viral Diarrhoea Virus (BVDV) in a managed metapopulation of cattle herds.

    PubMed

    Courcoul, Aurlie; Ezanno, Pauline

    2010-04-21

    In numerous epidemiological models developed within a metapopulation framework, it is assumed that a single infected individual introduced into a patch infects the whole patch and that the proportion of infected individuals into infected patches is consistent over time and among patches. If this approach is relevant for rapidly spreading pathogens, it is less appropriate for moderately spreading pathogens, like the Bovine Viral Diarrhoea Virus (BVDV), characterized by a variability in within-patch prevalence. Our objective is to study the respective influence of neighbouring relationships and animal movements on the spread of BVDV in a managed metapopulation of 100 cattle herds. Infection dynamics is represented by two coupled stochastic compartmental models in discrete-time: a within-herd and a between-herd models. Animal movements are mechanistically modelled. They largely influence the BVDV persistence, the prevalence in infected herds and the epidemic size. Neighbouring relationships only influence epidemic size. Whatever the neighbouring relationships, the infection does not persist in the metapopulation without animal movement between herds. The proposed model can be easily adapted for different herd contact structures. PMID:19875250

  7. Bovine tuberculosis: within-herd transmission models to support and direct the decision-making process.

    PubMed

    Alvarez, Julio; Bezos, Javier; de la Cruz, Maria Luisa; Casal, Carmen; Romero, Beatriz; Domínguez, Lucas; de Juan, Lucía; Pérez, Andrés

    2014-10-01

    Use of mathematical models to study the transmission dynamics of infectious diseases is becoming increasingly common in veterinary sciences. However, modeling chronic infectious diseases such as bovine tuberculosis (bTB) is particularly challenging due to the substantial uncertainty associated with the epidemiology of the disease. Here, the methodological approaches used to model bTB and published in the peer-reviewed literature in the last decades were reviewed with a focus on the impact that the models' assumptions may have had on their results, such as the assumption of density vs. frequency-dependent transmission, the existence of non-infectious and non-detectable stages, and the effect of extrinsic sources of infection (usually associated with wildlife reservoirs). Although all studies suggested a relatively low rate of within-herd transmission of bTB when test-and-cull programs are in place, differences in the estimated length of the infection stages, sensitivity and specificity of the tests used and probable type of transmission (density or frequency dependent) were observed. Additional improvements, such as exploring the usefulness of contact-networks instead of assuming homogeneous mixing of animals, may help to build better models that can help to design, evaluate and monitor control and eradication strategies against bTB. PMID:24875061

  8. Covariance structures of fat and protein influence the estimation of IgG in bovine colostrum.

    PubMed

    Lkke, Mette Marie; Engelbrecht, Rikke; Wiking, Lars

    2016-02-01

    On-farm instruments for assessing colostrum quality are needed in order to ensure that the calf is supplied with enough IgG to avoid failure of passive transfer. The aim of this study was to evaluate methods for estimating the IgG concentration in cows' colostrum. This research included 126 colostrum samples from 21 Danish farms with different breeds, ensuring a broad variation pattern in IgG, total protein and fat concentration. Approximately one third of the samples did not fulfil the recommendation of >50 g IgG/l colostrum, and the IgG concentration decreased with time from calving to milking. The ratio of IgG to total protein varied from 6 to 61%, however IgG and total protein were correlated with r 2 = 070. The variation in fat was independent of variations in protein and IgG. The IgG concentration was measured by ELISA and compared to fast measurements by specific gravity by colostrometer, Brix by refractometer and prediction from infrared spectroscopy. The three fast methods were all correlated to the total protein concentration of colostrum; however specific gravity was also influenced by the fat concentration. Furthermore, specific gravity generally overestimated the IgG concentration, and the cut-off level should be raised to 1050 in order to ensure adequate IgG in colostrum. None of the methods estimated IgG concentration better than the correlation of total protein and IgG, meaning that they all depended on the indirect correlation between total protein and IgG. The results suggest that using a refractometer for quality control of colostrum is an easy and feasible method, and a cut-off level of Brix 22 seems sufficient to assure adequate IgG concentration in colostrum fed to the calf. PMID:26869112

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

    SciTech Connect

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

    2012-08-15

    Purpose: To compare the effectiveness of microwave (MW) ablation with radiofrequency (RF) ablation for treating breast tissue in a nonperfused ex vivo model of healthy bovine udder tissue. 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 antenna. RF ablations were performed with a bipolar RF system with 2- and 3-cm active tip electrodes. Tissue temperatures were continuously monitored during ablation. Results: The mean short-axis diameters of the coagulation zones were 1.34 {+-} 0.14, 1.45 {+-} 0.13, and 1.74 {+-} 0.11 cm for MW ablation at outputs of 25W, 35W, and 45W. For RF ablation, the corresponding values were 1.16 {+-} 0.09 and 1.26 {+-} 0.14 cm with electrodes having 2- and 3-cm active tips, respectively. The mean coagulation volumes were 2.27 {+-} 0.65, 2.85 {+-} 0.72, and 4.45 {+-} 0.47 cm{sup 3} for MW ablation at outputs of 25W, 35W, and 45W and 1.18 {+-} 0.30 and 2.29 {+-} 0.55 cm{sup 3} got RF ablation with 2- and 3-cm electrodes, respectively. MW ablations at 35W and 45W achieved significantly longer short-axis diameters than RF ablations (P < 0.05). The highest tissue temperature was achieved with MW ablation at 45W (P < 0.05). On histological examination, the extent of the ablation zone in MW ablations was less affected by tissue heterogeneity than that in RF ablations. Conclusion: MW ablation appears to be advantageous with respect to the volume of ablation and the shape of the margin of necrosis compared with RF ablation in an ex vivo bovine udder.

  10. Bovine serum albumin surface imprinted polymer fabricated by surface grafting copolymerization on zinc oxide rods and its application for protein recognition.

    PubMed

    Li, Xiangjie; Zhou, Jingjing; Tian, Lei; Li, Wei; Zhang, Baoliang; Zhang, Hepeng; Zhang, Qiuyu

    2015-10-01

    A novel bovine serum albumin (BSA) surface imprinted polymer based on ZnO rods was synthesized by surface grafting copolymerization. It exhibited an excellent recognition performance to bovine serum albumin. The adsorption capacity and imprinting factor of bovine serum albumin could reach 89.27 mg/g and 2.35, respectively. Furthermore, the fluorescence property of ZnO was used for tracing the process of protein imprinting and it implied the excellent optical sensing property of this material. More importantly, the hypothesis that the surface charge of carrier could affect the imprinting process was confirmed. That is, ZnO with positive surface charge could not only improve the recognition specificity of binding sites to template proteins (pI < 7), but also deteriorate the bindings between sites and non-template proteins (pI > 7). It was also important that the reusability of ZnO@BSA molecularly imprinted polymers was satisfactory. This implied that the poor mechanical/chemical stability of traditional zinc oxide sensors could be solved by the introduction of surface grafting copolymerization. These results revealed that the ZnO@BSA molecularly imprinted polymers are a promising optical/electrochemical sensor element. PMID:26226935

  11. Some nutritional effects of folate-binding protein in bovine milk on the bioavailability of folate to rats

    SciTech Connect

    Tani, M.; Iwai, K.

    1984-04-01

    The excretions of folate compounds into both the urine and bile were investigated in rats after the administration of pteroylglutamic acid (PteGlu) with or without the folate-binding protein (FBP) prepared from bovine milk. When the sample solution, containing either free or bound (/sup 3/H)PteGlu (i.e., bound to the FBP from milk), was delivered to rats intragastrically via oral intubation, the amounts of (/sup 3/H)PteGlu excreted into the feces did not change. On the other hand, the urinary excretion of /sup 3/H-labeled folate compounds, especially (/sup 3/H)5-methyltetrahydrofolic acid (5-CH/sub 3/-H/sub 4/PteGlu), after the administration of bound (/sup 3/H)PteGlu was significantly lower (P less than 0.01) than that after the administration of free (/sup 3/H)PteGlu. The urinary excretion of (/sup 3/H)5-CH/sub 3/-H/sub 4/PteGlu was directly proportional to the initial amount of free (/sup 3/H)PteGlu administered. The similar effect of FBP was also observed when the biliary excretion of /sup 3/H-labeled folate compounds was investigated in situ. Furthermore, the incorporation of (/sup 3/H)PteGlu into folate-requiring intestinal microorganisms was considerably reduced when it was bound to FBP. These results suggest that milk FBP has some nutritional effects on the bioavailability of folate in vivo.

  12. Identification of alternatively spliced mRNAs encoding potential new regulatory proteins in cattle infected with bovine leukemia virus.

    PubMed Central

    Alexandersen, S; Carpenter, S; Christensen, J; Storgaard, T; Viuff, B; Wannemuehler, Y; Belousov, J; Roth, J A

    1993-01-01

    The polymerase chain reaction was used to detect and characterize low-abundance bovine leukemia virus (BLV) mRNAs. In infected cattle we could detect spliced mRNA with a splice pattern consistent with a Tax/Rex mRNA, as well as at least four alternatively spliced RNAs. Two of the alternatively spliced mRNAs encoded hitherto unrecognized BLV proteins, designated RIII and GIV. The Tax/Rex and alternatively spliced mRNAs could be detected at their highest levels in BLV-infected cell cultures; the next highest levels were found in samples from calves experimentally infected at 6 weeks postinoculation. Alternatively spliced mRNAs were also expressed, albeit at lower levels, in naturally infected animals; they were detected by a nested polymerase chain reaction. Interestingly, the GIV mRNA was specifically detected in naturally infected cows with persistent lymphocytosis and in two of five calves at 6 months after experimental infection with BLV. Furthermore, the calf with the strongest signal for GIV had the highest lymphocyte counts. These data may suggest a correlation between expression of the GIV product and development of persistent lymphocytosis. Some of the donor and acceptor sites in the alternatively spliced mRNAs were highly unusual. The biological mechanisms and significance of such a choice of unexpected splice sites are currently unknown. Images PMID:8380084

  13. Synthesis of nano-bioactive glass-ceramic powders and its in vitro bioactivity study in bovine serum albumin protein

    NASA Astrophysics Data System (ADS)

    Nabian, Nima; Jahanshahi, Mohsen; Rabiee, Sayed Mahmood

    2011-07-01

    Bioactive glasses and ceramics have proved to be able to chemically bond to living bone due to the formation of an apatite-like layer on its surface. The aim of this work was preparation and characterization of bioactive glass-ceramic by sol-gel method. Nano-bioglass-ceramic material was crushed into powder and its bioactivity was examined in vitro with respect to the ability of hydroxyapatite layer to form on the surface as a result of contact with bovine serum albumin (BSA) protein. The obtained nano-bioactive glass-ceramic was analyzed before and after contact with BSA solution. This study used scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray powder diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analysis to examine its morphology, crystallinity and composition. The TEM images showed that the NBG particles size were 10-40 nm. Bioactivity of nanopowder was confirmed by SEM and XRD due to the presence of a rich bone-like apatite layer. Therefore, this nano-BSA-bioglass-ceramic composite material is promising for medical applications such as bone substitutes and drug carriers.

  14. Analysis of immune responses to recombinant proteins from strains of Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia.

    PubMed

    Perez-Casal, Jose; Prysliak, Tracy; Maina, Teresa; Wang, Yejun; Townsend, Hugh; Berverov, Emil; Nkando, Isabel; Wesonga, Hezron; Liljander, Anne; Jores, Joerg; Naessens, Jan; Gerdts, Volker; Potter, Andrew

    2015-11-15

    Current contagious bovine pleuropneumonia (CBPP) vaccines are based on live-attenuated strains of Mycoplasma mycoides subsp. mycoides (Mmm). These vaccines have shortcomings in terms of efficacy, duration of immunity and in some cases show severe side effects at the inoculation site; hence the need to develop new vaccines to combat the disease. Reverse vaccinology approaches were used and identified 66 candidate Mycoplasma proteins using available Mmm genome data. These proteins were ranked by their ability to be recognized by serum from CBPP-positive cattle and thereafter used to inoculate nave cattle. We report here the inoculation of cattle with recombinant proteins and the subsequent humoral and T-cell-mediated immune responses to these proteins and conclude that a subset of these proteins are candidate molecules for recombinant protein-based subunit vaccines for CBPP control. PMID:26384697

  15. 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

    2011-01-01

    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. PMID:21214909

  16. Protein models: the Grand Challenge of protein docking.

    PubMed

    Anishchenko, Ivan; Kundrotas, Petras J; Tuzikov, Alexander V; Vakser, Ilya A

    2014-02-01

    Characterization of life processes at the molecular level requires structural details of protein-protein interactions (PPIs). The number of experimentally determined protein structures accounts only for a fraction of known proteins. This gap has to be bridged by modeling, typically using experimentally determined structures as templates to model related proteins. The fraction of experimentally determined PPI structures is even smaller than that for the individual proteins, due to a larger number of interactions than the number of individual proteins, and a greater difficulty of crystallizing protein-protein complexes. The approaches to structural modeling of PPI (docking) often have to rely on modeled structures of the interactors, especially in the case of large PPI networks. Structures of modeled proteins are typically less accurate than the ones determined by X-ray crystallography or nuclear magnetic resonance. Thus the utility of approaches to dock these structures should be assessed by thorough benchmarking, specifically designed for protein models. To be credible, such benchmarking has to be based on carefully curated sets of structures with levels of distortion typical for modeled proteins. This article presents such a suite of models built for the benchmark set of the X-ray structures from the Dockground resource (http://dockground.bioinformatics.ku.edu) by a combination of homology modeling and Nudged Elastic Band method. For each monomer, six models were generated with predefined C(?) root mean square deviation from the native structure (1, 2, , 6 ). The sets and the accompanying data provide a comprehensive resource for the development of docking methodology for modeled proteins. PMID:23934791

  17. Mycobacterium avium subspecies paratuberculosis recombinant proteins modulate antimycobacterial functions of bovine macrophages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    It has been shown that Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) activates the Mitogen Activated Protein Kinase (MAPK) p38 pathway, yet it is unclear which components of M. paratuberculosis are involved in the process. Therefore, a set of 42 M. paratuberculosis recombinan...

  18. Genetic influences on the bovine acute phase protein response following an endotoxin challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previously we reported that the pro-inflammatory cytokine response following an endotoxin challenge differs between diverse Bos taurus breeds [i.e., Angus (AG) and Romosinuano (RO)]. Our current objective was to elucidate potential genetic differences in the acute phase protein (APP) response follow...

  19. Investigating the Use of Protein Saver Cards for Storage and Subsequent Detection of Bovine Anti-Brucella abortus Smooth Lipopolysaccharide Antibodies and Gamma Interferon

    PubMed Central

    Commander, Nicola J.; Erdenlig, Sevil; McGiven, John A.; Stack, Judy

    2013-01-01

    Brucella abortus, a smooth strain of the genus Brucella, is the causative agent of bovine brucellosis. To support the ongoing development of diagnostic tests for bovine brucellosis, the use of Protein Saver cards (Whatman) for bovine blood serum and plasma sample collection has been evaluated. These cards offer significant logistical and safety alternatives to transporting and storing liquid samples and may aid in diagnostic programs and validation studies. To evaluate the utility of these cards, 204 bovine blood serum samples from Brucella-infected and noninfected animals were stored on and eluted from the Protein Saver cards. Anti-Brucella smooth lipopolysaccharide (sLPS) antibody titers for the serum eluates were compared to those of the unprocessed original serum samples by indirect enzyme-linked immunosorbent assay (ELISA). The results showed a highly significant correlation between titers from the serum eluates and the unprocessed sera. Therefore, under these circumstances, serum eluates and unprocessed serum samples may be used interchangeably. Blood plasma from 113 mitogen-stimulated whole-blood samples was added to and eluted from the Protein Saver cards. The gamma interferon (IFN-?) titers in the plasma eluates were compared to those of the unprocessed plasma samples obtained by IFN-? ELISA. The results showed a significant correlation between the plasma eluates and the unprocessed plasma samples. To derive a signal in the plasma eluate, it was necessary to develop a novel and highly sensitive ELISA for the detection of IFN-?. The serum samples stored on cards at room temperature over a 10-day period showed little variation in antibody titers. However, the plasma eluates showed a progressive loss of IFN-? recovery over 10 days when stored at room temperature. PMID:23986318

  20. Computational modeling of membrane proteins

    PubMed Central

    Leman, Julia Koehler; Ulmschneider, Martin B.; Gray, Jeffrey J.

    2014-01-01

    The determination of membrane protein (MP) structures has always trailed that of soluble proteins due to difficulties in their overexpression, reconstitution into membrane mimetics, and subsequent structure determination. The percentage of MP structures in the protein databank (PDB) has been at a constant 1-2% for the last decade. In contrast, over half of all drugs target MPs, only highlighting how little we understand about drug-specific effects in the human body. To reduce this gap, researchers have attempted to predict structural features of MPs even before the first structure was experimentally elucidated. In this review, we present current computational methods to predict MP structure, starting with secondary structure prediction, prediction of trans-membrane spans, and topology. Even though these methods generate reliable predictions, challenges such as predicting kinks or precise beginnings and ends of secondary structure elements are still waiting to be addressed. We describe recent developments in the prediction of 3D structures of both α-helical MPs as well as β-barrels using comparative modeling techniques, de novo methods, and molecular dynamics (MD) simulations. The increase of MP structures has (1) facilitated comparative modeling due to availability of more and better templates, and (2) improved the statistics for knowledge-based scoring functions. Moreover, de novo methods have benefitted from the use of correlated mutations as restraints. Finally, we outline current advances that will likely shape the field in the forthcoming decade. PMID:25355688

  1. The prion protein gene polymorphisms associated with bovine spongiform encephalopathy susceptibility differ significantly between cattle and buffalo.

    PubMed

    Zhao, Hui; Du, Yanli; Chen, Shunmei; Qing, Lili; Wang, Xiaoyan; Huang, Jingfei; Wu, Dongdong; Zhang, Yaping

    2015-12-01

    Prion protein, encoded by the prion protein gene (PRNP), plays a crucial role in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Several polymorphisms within the PRNP are known to be associated with influencing bovine spongiform encephalopathy (BSE) susceptibility in cattle, namely two insertion/deletion (indel) polymorphisms (a 23-bp indel in the putative promoter and a 12-bp indel in intron 1), the number of octapeptide repeats (octarepeats) present in coding sequence (CDS) and amino acid polymorphisms. The domestic buffaloes, Bubalus bubalis, are a ruminant involved in various aspects of agriculture. It is of interest to ask whether the PRNP polymorphisms differ between cattle and buffalo. In this study, we analyzed the previously reported polymorphisms associated with BSE susceptibility in Chinese buffalo breeds, and compared these polymorphisms in cattle with BSE, healthy cattle and buffalo by pooling data from the literature. Our analysis revealed three significant findings in buffalo: 1) extraordinarily low deletion allele frequencies of the 23- and 12-bp indel polymorphisms; 2) significantly low allelic frequencies of six octarepeats in CDS and 3) the presence of S4R, A16V, P54S, G108S, V123M, S154N and F257L substitutions in buffalo CDSs. Sequence alignments comparing the buffalo coding sequence to other species were analyzed using the McDonald-Kreitman test to reveal five groups (Bison bonasus, Bos indicus, Bos gaurus, Boselaphus tragocamelus, Syncerus caffer caffer) with significantly divergent non-synonymous substitutions from buffalo, suggesting potential divergence of buffalo PRNP and others. To the best of our knowledge this is the first study of PRNP polymorphisms associated with BSE susceptibility in Chinese buffalo. Our findings have provided evidence that buffaloes have a unique genetic background in the PRNP gene in comparison with cattle. PMID:26319996

  2. Insulin-like growth factor binding proteins in follicular fluid from morphologically distinct healthy and atretic bovine antral follicles.

    PubMed

    Irving-Rodgers, H F; Catanzariti, K D; Master, M; Grant, P A; Owens, P C; Rodgers, R J

    2003-01-01

    In bovine follicles 2-5 mm in diameter, two morphologically distinct types of healthy follicles and two types of atretic follicles have been described recently. Healthy follicles either have columnar basal granulosa cells with follicular basal lamina composed of many layers or 'loops' or they have rounded basal cells with a conventional single-layered, aligned follicular basal lamina. In atretic follicles, cell death either commences at the basal layer and progresses to the antrum (basal atresia) with macrophage penetration of the membrana granulosa or death progresses from the antrum in a basal direction (antral atresia). Little is known about how these different phenotypes develop. To determine whether insulin-like growth factor binding protein (IGFBP) levels in follicular fluid differ between these different types of follicles, we measured IGFBP levels in fluids from these follicles. A total of 61 follicles were assessed by light microscopy and characterized by morphological analysis as either healthy, with columnar or rounded basal granulosa cells, or as undergoing antral or basal atresia. The IGFBP concentration in the follicular fluid of individual follicles from the four groups (n = 12-20 per group) was identified by Western ligand blots using (125)I-insulin-like growth factor (IGF)-II as a probe. Insulin-like growth factor binding proteins 2, 3 (44 and 40 kDa), 4 (glycosylated and non-glycosylated) and 5 were observed. The levels (per volume of fluid) of IGFBPs 2, 4 and 5 were greater in atretic follicles than in healthy follicles. However, there were no statistical differences in levels of each IGFBP between either the two types of healthy follicle or between the two types of atretic follicles. Thus, IGFBP levels are not related to the different types of healthy or atretic follicles. PMID:12921699

  3. Presence of ecto-protein tyrosine phosphatase activity is vital for survival of Setaria cervi, a bovine filarial parasite.

    PubMed

    Singh, Neetu; Heneberg, Petr; Rathaur, Sushma

    2014-10-01

    The ecto protein tyrosine phosphatases (PTP) are known to play a crucial role in the pathogenesis and survival of the intracellular parasites. However, their presence and role in filarial parasites is still unknown. We found a significant amount of tyrosine phosphatase activity in the surface antigen fraction extracted from Setaria cervi (S. cervi), a bovine filarial parasite. An antibody designed against the conserved catalytic core of human protein tyrosine phosphatases, PTP1B cross reacted with a 63 kDa band in the surface antigen. We detected a significant amount of PTP activity in the intact S. cervi adult parasites as well as microfilariae in this study for the first time. This PTP may be localized on the surface of the parasite with an exposed active site available for the external substrates. The PTP activity was also inhibited by sodium orthovanadate and phenyl arsine oxide, specific inhibitors of PTP in both the life stages. The Km and Vmax for PTP in the adult parasites and microfilariae were determined to be 2.574??0.14 mM; 206.3??2.75 ?M Pi/h/two parasites and 5.510??0.59 mM; 62.27??2.27 ?M Pi/h/10(6) parasites respectively using O-P-L-Tyrosine as substrate. Interestingly, a positive correlation was observed between the inhibition in PTP activity and reduction in the motility/ viability of the parasites when they were subjected to the specific PTP inhibitors (Orthovanadate and Phenyl arsine oxide) for 4 h in the KRB maintenance medium. The activity was also significantly inhibited in the parasites exposed to antifilarial drug/compounds for e.g. Diethylcarbamazine, Acetylsalicylic Acid and SK7, a methyl chalcone. Therefore suggesting a possible role played by PTP in the survival of the parasite, its interaction with the host as well as in the screening of newly synthesized antifilarials/drugs. PMID:25028209

  4. Milk protein genetic variants and isoforms identified in bovine milk representing extremes in coagulation properties.

    PubMed

    Jensen, H B; Holland, J W; Poulsen, N A; Larsen, L B

    2012-06-01

    A gel-based proteomic approach consisting of 2-dimensional gel electrophoresis coupled with mass spectrometry was applied for detailed protein characterization of a subset of individual milk samples with extreme rennet coagulation properties. A milk subset with either good or poor coagulation abilities was selected from 892 Danish Holstein-Friesian and Jersey cows. Screening of genetic variants of the major milk proteins resulted in the identification of common genetic variants of ?-casein (CN; A(1), A(2), B), ?-CN (A, B), and ?-lactoglobulin (LG; A, B), as well as a low frequency variant, ?-CN variant E, and variants not previously reported in Danish breeds (i.e., ?-CN variant I and ?-LG variant C). Clear differences in the frequencies of the identified genetic variants were evident between breeds and, to some extent, between coagulation groups within breeds, indicating that an underlying genetic variation of the major milk proteins affects the overall milk coagulation ability. In milk with good coagulation ability, a high prevalence of the B variants of all 3 analyzed proteins were identified, whereas poorly coagulating milk was associated with the ?-CN variant A(2), ?-CN variant A or E, and ?-LG variant A or C. The ?-CN variant I was identified in milk with both good and poor coagulation ability, a variant that has not usually been discriminated from ?-CN variant A(2) in other studied cow populations. Additionally, a detailed characterization of ?-CN isoforms was conducted. Six ?-CN isoforms varying in phosphorylation and glycosylation levels from each of the genetic variants of ?-CN were separated and identified, along with an unmodified ?-CN form at low abundance. Relative quantification showed that around 95% of total ?-CN was phosphorylated with 1 or 2 phosphates attached, whereas approximately 35% of the identified ?-CN was glycosylated with 1 to 3 tetrasaccharides. Comparing isoforms from individual samples, we found a very consistent ?-CN isoform pattern, with only minor differences in relation to breed, ?-CN genetic variant, and milk coagulation ability. PMID:22612926

  5. Gaussian model of protein folding

    NASA Astrophysics Data System (ADS)

    Erman, Burak; Dill, Ken

    2000-01-01

    The thermodynamics and kinetics of protein folding depend on the sequence of monomer units along the chain. To explore the sequence dependence, current modeling—all-atom simulations and lattice models, for example—require time-consuming computer simulations. There are currently no analytical models by which folding properties can be computed directly from the monomer sequence. Here we introduce a simple analytical model of folding, based on assuming springlike forces for covalent and noncovalent interactions. Thermodynamic and kinetic properties of folding can be obtained directly for specific sequences in Go-like models. Remarkably, although it is a continuum model, some choices of parameters give the same stable conformations as in the corresponding lattice model. The main point of elasticity-based folding models is that their properties can be understood in complete detail, and with little computational investment. This may be useful for understanding how the shapes of energy landscapes, including stable states and kinetic barriers, depend on amino acid sequence.

  6. From explanation to prediction: a model for recurrent bovine tuberculosis in Irish cattle herds.

    PubMed

    Wolfe, Dianna M; Berke, Olaf; Kelton, David F; White, Paul W; More, Simon J; O'Keeffe, James; Martin, S Wayne

    2010-05-01

    There is a good understanding of factors associated with bovine tuberculosis (BTB) risk in Irish herds. As yet, however, this knowledge has not been incorporated into predictive models with the potential for improved, risk-based surveillance. The goal of the study was to enhance the national herd scoring system for BTB risk, thus leading to improved identification of cattle herds at high risk of recurrent BTB episodes. A retrospective cohort study was conducted to develop a statistical model predictive of recurrent bovine tuberculosis episodes in cattle herds in the Republic of Ireland. Herd-level disease history data for the previous 12 years, the previous 3 years, the previous episode, and the current-episode were used in survival analyses to determine the aspects of disease history that were predictive of a recurrent breakdown within 3 years of a cleared BTB episode. Relative to herds with 0-1 standard reactors in the current BTB episode, hazard ratios increased to 1.3 and 1.6 for herds with 2-5 and >5 standard reactors, respectively. Compared to herds with <30 animals, hazard ratios increased from 1.8 to 2.5 and then to 3.1 for herds with 30-79, 80-173, and >174 animals respectively. Relative to herds with <4 herd-level tests in the previous 3 years, herds with 4-5 and >5 tests had 1.1 and 1.4 times greater hazard of a BTB breakdown. Herds that did not have a BTB episode in the 5 years prior to their 2001 episode were 0.8 times less likely to breakdown in the next 3 years than herds that did. Herds breaking down in the spring or summer were 0.8 times less likely to suffer a recurrent breakdown than herds breaking down in autumn or winter (this was likely due to seasonality in testing regimes). The presence of a confirmed BTB lesion was not predictive of increased risk of recurrent BTB. Despite the availability of detailed disease history, the predictive ability of the model was poor. One explanation for this was that herds suffering a recurrence of BTB on their first test after clearing a BTB episode were different from herds that broke down later in the period at risk. Future research might need to include additional variables to identify which subsets of herd BTB episodes, if any, have identifiable features that are predictive of recurrent breakdowns. PMID:20236717

  7. Protein--non-ionic detergent interaction. Interaction of bovine serum albumin with alkyl glucosides studied by equilibrium dialysis and infrared spectroscopy.

    PubMed

    Wasylewski, Z; Kozik, A

    1979-03-15

    The binding isotherms of bovine serum albumin with octylglucoside and decyl glucoside were determined at 7 degrees C and 25 degrees C at pH 7.4 and ionic strength 0.1 M. The average number of detergent molecules bound was found to increase with increasing hydrocarbon chain length. Competitive binding indicates that alkylglycosides combine with the same sites as alkyl sulphates. Native bovine serum albumin has about 12 and 10 sites for non-ionic ligands at 7 degrees C and about 15 and 13 sites at 25 degrees C for octyl and decyl glucosides respectively. The values for standard free energy changes--delta G0, were calculated from the intrinsic association constants. Fourier-transformed infrared spectroscopy was used to study the effects of alkyl glucosides on the conformation of albumin. The results obtained indicate that there are no significant changes in protein structure. PMID:456344

  8. Bone temperature during cementation with a heatsink: a bovine model pilot study

    PubMed Central

    2014-01-01

    Background Bone cement is an effective means of supporting implants, but reaches high temperatures while undergoing polymerisation. Bone has been shown to be sensitive to thermal injury with osteonecrosis reported after one minute at 47C. Necrosis during cementing may lead to loosening of the prosthesis. Some surgeons fill the joint cavity with cool irrigation fluid to provide a heatsink during cementing, but this has not been supported by research. This paper assesses a simple technique to investigate the efficacy of this method. Findings We used a model acetabulum in a bovine humerus to allow measurement of bone temperatures in cementing. Models were prepared with a 50mm diameter acetabulum and three temperature probe holes; two as close as possible to the acetabular margin at half the depth of the acetabulum and at the full depth of the acetabulum, and one 10mm from the acetabular rim. Four warmed models were cemented with Palacos RG using a standard mixing system and a 10mm polyethylene disc to represent an acetabular component. Two of the acetabular models were filled with room temperature water to provide a heatsink. An electronic probe measured temperature at 5second intervals from the moment of cementing. In the models with no heatsink, peak temperature was 40.3C. The mean temperature rise was 10.9C. In the models with a heatsink, there was an average fall in the bone temperature during cementing of 4.4C. Conclusions These results suggest that using a heatsink while cementing prostheses may reduce the peak bone temperature. This study demonstrates a simple, repeatable technique which may be useful for larger trials. PMID:25099248

  9. Bovine model of chronic ischemic cardiomyopathy: implications for ventricular assist device research.

    PubMed

    Bartoli, Carlo R; Sherwood, Leslie C; Giridharan, Guruprasad A; Slaughter, Mark S; Wead, William B; Prabhu, Sumanth D; Koenig, Steven C

    2013-12-01

    Ventricular assist devices (VADs) have emerged as a successful treatment option for advanced heart failure. The objective of this study was to develop a clinically relevant model of chronic ischemic cardiomyopathy to investigate functional, histological, and molecular changes during mechanical circulatory support. In calves (n?=?17, 94??7?kg), 90??m microspheres were injected percutaneously into the left coronary artery. Serial echocardiography was performed weekly to evaluate cardiac function. Sixty days after coronary microembolization, a terminal study was performed via thoracotomy to measure hemodynamics. Regional myocardial and end-organ blood flows were quantified with 15-?m fluorescent-labeled microspheres. Myocardial fibrosis, myocyte size, and myocardial apoptosis were quantified with histological stains. Eleven animals survived coronary microembolization and exhibited clinical and statistically significant echocardiographic and hemodynamic signs of severe systolic dysfunction. Statistically significant decreases in regional myocardial blood flow and increases in myocardial fibrosis, myocyte size, total myocardial apoptosis, and cardiac myocyte-specific apoptosis were observed. End-organ hypoperfusion was observed. Coronary microembolization induced stable and reproducible chronic left ventricular failure in calves. The anatomical size and physiology of the bovine heart and thorax are appropriate to study novel interventions for the clinical management of heart failure. This model is an appropriate physiological substrate in which to test VAD and adjunctive biological therapies. PMID:23876076

  10. Experimentally-based multiscale model of the elastic moduli of bovine trabecular bone and its constituents.

    PubMed

    Hamed, Elham; Novitskaya, Ekaterina; Li, Jun; Jasiuk, Iwona; McKittrick, Joanna

    2015-09-01

    The elastic moduli of trabecular bone were modeled using an analytical multiscale approach. Trabecular bone was represented as a porous nanocomposite material with a hierarchical structure spanning from the collagen-mineral level to the trabecular architecture level. In parallel, compression testing was done on bovine femoral trabecular bone samples in two anatomical directions, parallel to the femoral neck axis and perpendicular to it, and the measured elastic moduli were compared with the corresponding theoretical results. To gain insights on the interaction of collagen and minerals at the nanoscale, bone samples were deproteinized or demineralized. After such processing, the treated samples remained as self-standing structures and were tested in compression. Micro-computed tomography was used to characterize the hierarchical structure of these three bone types and to quantify the amount of bone porosity. The obtained experimental data served as inputs to the multiscale model and guided us to represent bone as an interpenetrating composite material. Good agreement was found between the theory and experiments for the elastic moduli of the untreated, deproteinized, and demineralized trabecular bone. PMID:26046284

  11. Herpes simplex (HSV-1) infection of bovine aorta smooth muscle cells (SMC) inhibits matrix protein synthesis

    SciTech Connect

    Lashgari, M.S.; Friedman, H.M.; Kefalides, N.A.

    1986-03-01

    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.

  12. Evaluation of L-selectin expression and assessment of protein tyrosine phosphorylation in bovine polymorphonuclear neutrophil leukocytes around parturition.

    PubMed

    Monfardini, Erica; Paape, Max J; Wang, Yan; Capuco, Anthony V; Husheem, Michael; Wood, Larry; Burvenich, Christian

    2002-01-01

    Impaired polymorphonuclear neutrophil leukocyte (PMN) function around parturition has been associated with increased clinical mastitis in dairy cows. Rolling and attachment of PMN to the endothelium is the first step in the recruitment process and is accomplished by interaction between L-selectin on PMN and its ligand on endothelial cells. Furthermore, tyrosine phosphorylation is involved in the initiation of many PMN functions. The objective of this work was to determine changes in expression of L-selectin and tyrosine phosphorylation in the perinatal period. Eight clinically healthy Holstein cows were used as PMN donors at d-21, -14, -7,0 (calving), +1, +2, +7, +14, +28. Evaluation of L-selectin expression was carried out on activated and resting PMN. Anti-bovine L-selectin monoclonal antibody (MAB) and flow cytometric analysis were used to measure the percentage of PMN fluorescing and receptor expression (log mean fluorescent channel, LMFC). Activated and resting PMN showed similar trends in % PMN fluorescence and LM FC. The percentage of PMN fluorescing tended to decrease at parturition, followed by a significant increase at d +14 and +28 (P < 0.02). For LMFC a decrease was observed on d +1 followed by an increase through d +28 (P < 0.01). Protein tyrosine phosphorylation of lysates prepared from PMN isolated throughout the study was detected by electrophoresis and western blotting using anti-phosphotyrosine MAB. Several protein bands were tyrosine phosphorylated. Two of these bands (42-44 kDa and 90 kDa) varied in intensity over time. The intensity of the 42-44 kDa band gradually increased from d -7, peaked at d +7 (P < 0.03), and steadily decreased to d +28 (P < 0.02). Antibody to activated mitogen protein kinase reacted with the 42-44 kDa band. Reduced PMN function during the periparturient period could be related to reduced L-selectin adhesion molecules on the cell surface, and to modulation in the phosphorylation of functionally important molecules. PMID:12056478

  13. Genotyping bovine milk proteins using allele discrimination by primer length and automated DNA sizing technology.

    PubMed

    Lindersson, M; Lundn, A; Andersson, L

    1995-04-01

    A method for genotyping kappa-casein (A, B, E), beta-casein (A1, A2, A3, A5, B) and beta-lactoglobulin (A, B) simultaneously by the use of allele discrimination by primer length combined with automated detection of fragments with a sequencing instrument is described. Seven different mutations within the milk protein genes were analysed in order to distinguish between the alleles examined. The samples were amplified in two separate multiplex polymerase chain reactions (PCRs), which were then pooled and separated according to size in a single lane on the gel. By using stringent PCR conditions, we have been able to achieve allele-specific amplifications and minimize amplification of mis-matched primer for all seven mutations. PMID:7733509

  14. Identification and characterization of a bovine sperm acrosomal matrix protein and its mechanism of interaction with acrosomal hydrolases.

    PubMed

    Nagdas, Subir K; Smith, Linda; Mcnamara, Allen; Hernandez-Encarnacion, Luisa; Medina-Ortiz, Ilza

    2015-12-01

    Fertilization, the union of male and female gametes to create offspring, is an intricate biological process dependent upon several biochemical and physiological events. Our understanding of the functions of protein constituents of the outer acrosomal membrane-associated matrix complex (OMC) is limited. A highly purified OMC fraction isolated from bovine cauda sperm heads comprised 54, 50, 45, and 38-19 kDa polypeptides. The objective of this study is to identify and characterize the 45 kDa (OMC45) polypeptide, to define its role in binding acrosomal hydrolases, and to examine the fate of OMC45 polypeptide during the acrosome reaction. We isolated OMC45 polypeptide from the high-pH insoluble fraction of OMC. Proteomic analysis of OMC45 by MALDI-TOF-TOF yielded eight peptides that matched the NCBI database sequence of Tektin 3 (TEKT3). Triton X-100-permeabilized cauda sperm exhibited intense staining of the acrosomal segment with anti-OMC45 and anti-TEKT3. The OMC45 polypeptide was solubilized by radio-immunoprecipitation assay buffer extraction. The solubilized fraction was subjected to immunoprecipitation analysis. The OMC45 polypeptide was recovered in the anti-OMC45 immunoprecipitation pellet. An identical blot stained with anti-TEKT3 exhibited the presence of TEKT3 polypeptide in the anti-OMC45 pellet. Our immunofluorescence and biochemical studies confirm the proteomics identification of OMC45 polypeptide and that it exhibits a sequence similarity to TEKT3. OMC45 glycoprotein possesses both N-linked and O-linked oligosaccharides. Deglycosylated OMC45 revealed a significant reduction in both acrosin and N-acetylglucosaminidase (NAGA) binding in comparison with acrosin and NAGA binding to a native OMC45 polypeptide, demonstrating the important role of oligosaccharides in hydrolase binding. OMC45 polypeptide is not released during the acrosome reaction but remains in the particulate cell subfraction, associated with the hybrid membrane complex. PMID:26268136

  15. Effect of bovine somatotropin and rumen-undegradable protein on mammary growth of prepubertal dairy heifers and subsequent milk production.

    PubMed

    Capuco, A V; Dahl, G E; Wood, D L; Moallem, U; Erdman, R E

    2004-11-01

    Rapid body growth during the prepubertal period may be associated with reductions in mammary parenchymal growth and subsequent milk yield. The objective of this study was to test effects of dietary rumen-undegradable protein (RUP) and administration of recombinant bovine somatotropin (bST) during the prepubertal period on mammary growth and milk yield of dairy heifers. Seventy-two Holstein heifers were used in the experiment. At 90 d of age, 8 heifers were slaughtered before initiation of treatment. Remaining heifers were assigned randomly to 1 of 4 treatments. Treatments consisted of a control diet (5.9% RUP, 14.9% CP, DM basis) or RUP-supplemented diet (control diet plus 2% added RUP) with or without 0.1 mg of bST/kg of BW per day applied in a 2 x 2 factorial design. A total of 6 heifers per treatment (3 each at 5 and 10 mo of age) were slaughtered for mammary tissue analysis. Remaining heifers were bred to evaluate impact of treatment on subsequent milk yield and composition. Mammary parenchymal growth was not affected by RUP or bST treatment. Total parenchymal mass increased from 16 to 364 g, and parenchymal DNA from 58 to 1022 mg from 3 to 10 mo of age, respectively. Furthermore, number of mammary epithelial cells likely was not affected by diet or bST because the epithelial cell proliferation index, assessed by Ki-67 labeling, was not affected by treatment, nor was total parenchymal DNA and lipid content. Neither deleterious effects of increased rates of gain nor positive effects of bST were evident in prepubertal mammary growth. Subsequent milk production and composition was not different among treatments. PMID:15483159

  16. Noncytopathic bovine viral diarrhea virus 2 impairs virus control in a mouse model.

    PubMed

    Seong, Giyong; Lee, Jin-Sol; Lee, Kyung-Hyun; Shin, Seung-Uk; Yoon, Ji Young; Choi, Kyoung-Seong

    2016-02-01

    Bovine viral diarrhea virus (BVDV) is an economically important pathogen that causes development of mild to severe clinical signs in wild and domesticated ruminants. We previously showed that mice could be infected by BVDV. In the present study, we infected mice intraperitoneally with non-cytopathic (ncp) BVDV1 or ncp BVDV2, harvested the blood and organs of the infected mice at days 4, 7, 10 and 14 postinfection (pi), and performed immunohistochemical analyses to confirm BVDV infection. Viral antigens were detected in the spleens of all infected mice from days 4 through 14 and were also found in the mesenteric lymph nodes, gut-associated lymphoid tissue (GALT), heart, kidney, intestine, and bronchus-associated lymphoid tissue (BALT) of some infected mice. In ncp BVDV2-infected mice, flow cytometric analysis revealed markedly fewer CD4(+) and CD8(+) T lymphocytes and lower expression of costimulatory molecules CD80 (B7-1) and CD86 (B7-2) and major histocompatibility complex (MHC) class II (I-A/I-E) than those in ncp BVDV1-infected mice. Production of the cytokines interleukin (IL)-6 and monocyte chemotactic protein (MCP)-1 was higher in the plasma of ncp BVDV2-infected mice than that in that of ncp BVDV1-infected mice. Our results demonstrate that ncp BVDV1 and ncp BVDV2 interact differently with the host innate immune response in vivo. These findings highlight an important distinction between ncp BVDV1 and ncp BVDV2 and suggest that ncp BVDV2 impairs the host's ability to control the infection and enhances virus dissemination. PMID:26586332

  17. Eliminating bovine tuberculosis in cattle and badgers: insight from a dynamic model.

    PubMed

    Brooks-Pollock, Ellen; Wood, James L N

    2015-06-01

    Bovine tuberculosis (BTB) is a multi-species infection that commonly affects cattle and badgers in Great Britain. Despite years of study, the impact of badgers on BTB incidence in cattle is poorly understood. Using a two-host transmission model of BTB in cattle and badgers, we find that published data and parameter estimates are most consistent with a system at the threshold of control. The most consistent explanation for data obtained from cattle and badger populations includes within-host reproduction numbers close to 1 and between-host reproduction numbers of approximately 0.05. In terms of controlling infection in cattle, reducing cattle-to-cattle transmission is essential. In some regions, even large reductions in badger prevalence can have a modest impact on cattle infection and a multi-stranded approach is necessary that also targets badger-to-cattle transmission directly. The new perspective highlighted by this two-host approach provides insight into the control of BTB in Great Britain. PMID:25972466

  18. Modeling of bovine type-I collagen fibrils: interaction with pickling and retanning agents.

    PubMed

    Bulo, Rosa E; Siggel, Lorenz; Molnar, Ferenc; Weiss, Horst

    2007-02-12

    Bovine Type I collagen was investigated, building on a large scale computer model of a collagen fibril in water, and focusing on two stages of the leather manufacturing process. The effects of different salts (NaCl, CaCl(2), and Na(2)SO(4)) on the swelling behavior of collagen at low pH (the pickling process) were studied. The salts suppress the swelling of the fibrils at low pH and we find specific stabilizing influences for CaCl(2) and Na(2)SO(4), due to weak Ca(2+)/Cl(-) and strong SO(4) (2-)/lysine/arginine interactions, respectively. Using state-of-the-art sampling techniques, such as the metadynamics algorithm, to allow an efficient exploration of configuration space, we were able to investigate the effect of polyacrylate and poly(methyl acrylate) - two polymeric retanning agents - on the fibril. Both polymers interact with the ammonium groups on the surface, but polyacrylate shows significantly stronger interactions. We suggest that it is this stronger interaction that contributes to the reduced suitability of PAA as a tanning agent. PMID:17295396

  19. Effect of bovine lactoferrin in a therapeutic hamster model of hepatic amoebiasis.

    TOXLINE Toxicology Bibliographic Information

    Ordaz-Pichardo C; León-Sicairos N; Hernández-Ramírez VI; Talamás-Rohana P; de la Garza M

    2012-06-01

    Entamoeba histolytica is the causative agent of amoebiasis, a disease that produces dysentery as a result of the perforation of the large intestine. This parasite often invades other organs, primarily the liver, leading to an amoebic liver abscess (ALA), which can cause death. Metronidazole is the drug of choice for the treatment of ALA; however, it produces toxic side effects in patients. Lactoferrin (Lf) is a glycoprotein of the innate immune response that sequesters iron in the mucosae. Lf possesses immune-regulatory properties, such as antiinflammatory and antioxidant activities. Moreover, the microbicidal activity of apoLf, which lacks bound iron, has been shown. In this study, we evaluated the therapeutic effect of bovine Lf (bLf) against ALA in a model of hepatic amoebiasis in hamsters. Interestingly, hamsters treated intragastrically with Lf (2.5 mg/100 g mass) over a period of 8 days showed no clinical signs of disease and ALA was effectively decreased, with only 0.63% detectable lesion, compared with 63% in untreated animals. Furthermore, liver function and blood cells approached normal levels among those receiving bLf treatment. These results suggest that bLf may aid in the therapy of amoebiasis, likely without producing undesirable effects in patients.

  20. Effect of bovine lactoferrin in a therapeutic hamster model of hepatic amoebiasis.

    PubMed

    Ordaz-Pichardo, Cynthia; León-Sicairos, Nidia; Hernández-Ramírez, Verónica Ivonne; Talamás-Rohana, Patricia; de la Garza, Mireya

    2012-06-01

    Entamoeba histolytica is the causative agent of amoebiasis, a disease that produces dysentery as a result of the perforation of the large intestine. This parasite often invades other organs, primarily the liver, leading to an amoebic liver abscess (ALA), which can cause death. Metronidazole is the drug of choice for the treatment of ALA; however, it produces toxic side effects in patients. Lactoferrin (Lf) is a glycoprotein of the innate immune response that sequesters iron in the mucosae. Lf possesses immune-regulatory properties, such as antiinflammatory and antioxidant activities. Moreover, the microbicidal activity of apoLf, which lacks bound iron, has been shown. In this study, we evaluated the therapeutic effect of bovine Lf (bLf) against ALA in a model of hepatic amoebiasis in hamsters. Interestingly, hamsters treated intragastrically with Lf (2.5 mg/100 g mass) over a period of 8 days showed no clinical signs of disease and ALA was effectively decreased, with only 0.63% detectable lesion, compared with 63% in untreated animals. Furthermore, liver function and blood cells approached normal levels among those receiving bLf treatment. These results suggest that bLf may aid in the therapy of amoebiasis, likely without producing undesirable effects in patients. PMID:22332957

  1. Eliminating bovine tuberculosis in cattle and badgers: insight from a dynamic model

    PubMed Central

    Brooks-Pollock, Ellen; Wood, James L. N.

    2015-01-01

    Bovine tuberculosis (BTB) is a multi-species infection that commonly affects cattle and badgers in Great Britain. Despite years of study, the impact of badgers on BTB incidence in cattle is poorly understood. Using a two-host transmission model of BTB in cattle and badgers, we find that published data and parameter estimates are most consistent with a system at the threshold of control. The most consistent explanation for data obtained from cattle and badger populations includes within-host reproduction numbers close to 1 and between-host reproduction numbers of approximately 0.05. In terms of controlling infection in cattle, reducing cattle-to-cattle transmission is essential. In some regions, even large reductions in badger prevalence can have a modest impact on cattle infection and a multi-stranded approach is necessary that also targets badger-to-cattle transmission directly. The new perspective highlighted by this two-host approach provides insight into the control of BTB in Great Britain. PMID:25972466

  2. Preparation and Characterization of 125I Labeled Bovine Serum Albumin

    PubMed Central

    Ashwitha Rai, K. S.; Jyothi; Rasmi, R. R.; Sarnaik, Jayula; Kadwad, V. B.; Shenoy, K. B.; Somashekarappa, H. M.

    2015-01-01

    Bovine serum albumin is a model protein, which has been conventionally used as protein standard and in many areas of biochemistry, pharmacology and medicine. Radioiodination procedure for bovine serum albumin employing chloramine-T as an oxidant with slight modification was evaluated critically to establish the optimal conditions for the preparation of radiolabeled tracer (125I-BSA) with required specific activity without impairing the immune reactivity and biological activity. Optimized radioiodination procedure involving 10 g of chloramine-T along with 20 g of sodium metabisulphite with 60 seconds incubation at 2 yielded 125I-BSA with high integrity. PMID:25767326

  3. Preparation and characterization of (125)i labeled bovine serum albumin.

    PubMed

    Ashwitha Rai, K S; Jyothi; Rasmi, R R; Sarnaik, Jayula; Kadwad, V B; Shenoy, K B; Somashekarappa, H M

    2015-01-01

    Bovine serum albumin is a model protein, which has been conventionally used as protein standard and in many areas of biochemistry, pharmacology and medicine. Radioiodination procedure for bovine serum albumin employing chloramine-T as an oxidant with slight modification was evaluated critically to establish the optimal conditions for the preparation of radiolabeled tracer ((125)I-BSA) with required specific activity without impairing the immune reactivity and biological activity. Optimized radioiodination procedure involving 10 g of chloramine-T along with 20 g of sodium metabisulphite with 60 seconds incubation at 2 yielded (125)I-BSA with high integrity. PMID:25767326

  4. Discriminating scrapie and bovine spongiform encephalopathy isolates by infrared spectroscopy of pathological prion protein.

    PubMed

    Thomzig, Achim; Spassov, Sashko; Friedrich, Manuela; Naumann, Dieter; Beekes, Michael

    2004-08-01

    For the surveillance of transmissible spongiform encephalopathies (TSEs) in animals and humans, the discrimination of different TSE strains causing scrapie, BSE, or Creutzfeldt-Jakob disease constitutes a substantial challenge. We addressed this problem by Fourier transform-infrared (FT-IR) spectroscopy of pathological prion protein PrP27-30. Different isolates of hamster-adapted scrapie (263K, 22A-H, and ME7-H) and BSE (BSE-H) were passaged in Syrian hamsters. Two of these agents, 22A-H and ME7-H, caused TSEs with indistinguishable clinical symptoms, neuropathological changes, and electrophoretic mobilities and glycosylation patterns of PrP27-30. However, FT-IR spectroscopy revealed that PrP27-30 of all four isolates featured different characteristics in the secondary structure, allowing a clear distinction between the passaged TSE agents. FT-IR analysis showed that phenotypic information is mirrored in beta-sheet and other secondary structure elements of PrP27-30, also in cases where immunobiochemical typing failed to detect structural differences. If the findings of this study hold true for nonexperimental TSEs in animals and humans, FT-IR characterization of PrP27-30 may provide a versatile tool for molecular strain typing without antibodies and without restrictions to specific TSEs or mammalian species. PMID:15155741

  5. Influence of non ionizing radiation of base stations on the activity of redox proteins in bovines

    PubMed Central

    2014-01-01

    Background The influence of electromagnetic fields on the health of humans and animals is still an intensively discussed and scientifically investigated issue (Prakt Tierarzt 11:15-20, 2003; Umwelt Medizin Gesellschaft 17:326-332, 2004; J Toxicol Environment Health, Part B 12:572597, 2009). We are surrounded by numerous electromagnetic fields of variable strength, coming from electronic equipment and its power cords, from high-voltage power lines and from antennas for radio, television and mobile communication. Particularly the latter causes controversy, as everyone likes to have good mobile reception at anytime and anywhere, whereas nobody wants to have such a basestation antenna in their proximity. Results In this experiment, the NIR has resulted in changes in the enzyme activities. Certain enzymes were disabled, others enabled by NIR. Furthermore, individual behavior patterns were observed. While certain cows reacted to NIR, others did not react at all, or even inversely. Conclusion The present results coincide with the information from the literature, according to which NIR leads to changes in redox proteins, and that there are individuals who are sensitive to radiation and others that are not. However, the latter could not be distinctly attributed there are cows that react clearly with one enzyme while they do not react with another enzyme at all, or even the inverse. The study approach of testing ten cows each ten times during three phases has proven to be appropriate. Future studies should however set the post-exposure phase later on. PMID:24946856

  6. Stochastic simulation modeling to determine time to detect Bovine Viral Diarrhea antibodies in bulk tank milk.

    PubMed

    Foddai, Alessandro; Ene, Claes; Krogh, Kaspar; Stockmarr, Anders; Halasa, Tariq

    2014-11-01

    A stochastic simulation model was developed to estimate the time from introduction of Bovine Viral Diarrhea Virus (BVDV) in a herd to detection of antibodies in bulk tank milk (BTM) samples using three ELISAs. We assumed that antibodies could be detected, after a fixed threshold prevalence of seroconverted milking cows was reached in the herd. Different thresholds were set for each ELISA, according to previous studies. For each test, antibody detection was simulated in small (70 cows), medium (150 cows) and large (320 cows) herds. The assays included were: (1) the Danish blocking ELISA, (2) the SVANOVIR()BVDV-Ab ELISA, and (3) the ELISA BVD/MD p80 Institute Pourquier. The validation of the model was mainly carried out by comparing the predicted incidence of persistently infected (PI) calves and the predicted detection time, with records from a BVD infected herd. Results showed that the SVANOVIR, which was the most efficient ELISA, could detect antibodies in the BTM of a large herd 280 days (95% prediction interval: 218; 568) after a transiently infected (TI) milking cow has been introduced into the herd. The estimated time to detection after introduction of one PI calf was 111 days (44; 605). With SVANOVIR ELISA the incidence of PIs and dead born calves could be limited and the impact of the disease on the animal welfare and income of farmers (before detection) could be minimized. The results from the simulation modeling can be used to improve the current Danish BVD surveillance program in detecting early infected herds. PMID:25081944

  7. Modeling T1 and T2 relaxation in bovine white matter

    NASA Astrophysics Data System (ADS)

    Barta, R.; Kalantari, S.; Laule, C.; Vavasour, I. M.; MacKay, A. L.; Michal, C. A.

    2015-10-01

    The fundamental basis of T1 and T2 contrast in brain MRI is not well understood; recent literature contains conflicting views on the nature of relaxation in white matter (WM). We investigated the effects of inversion pulse bandwidth on measurements of T1 and T2 in WM. Hybrid inversion-recovery/Carr-Purcell-Meiboom-Gill experiments with broad or narrow bandwidth inversion pulses were applied to bovine WM in vitro. Data were analysed with the commonly used 1D-non-negative least squares (NNLS) algorithm, a 2D-NNLS algorithm, and a four-pool model which was based upon microscopically distinguishable WM compartments (myelin non-aqueous protons, myelin water, non-myelin non-aqueous protons and intra/extracellular water) and incorporated magnetization exchange between adjacent compartments. 1D-NNLS showed that different T2 components had different T1 behaviours and yielded dissimilar results for the two inversion conditions. 2D-NNLS revealed significantly more complicated T1/T2 distributions for narrow bandwidth than for broad bandwidth inversion pulses. The four-pool model fits allow physical interpretation of the parameters, fit better than the NNLS techniques, and fits results from both inversion conditions using the same parameters. The results demonstrate that exchange cannot be neglected when analysing experimental inversion recovery data from WM, in part because it can introduce exponential components having negative amplitude coefficients that cannot be correctly modeled with nonnegative fitting techniques. While assignment of an individual T1 to one particular pool is not possible, the results suggest that under carefully controlled experimental conditions the amplitude of an apparent short T1 component might be used to quantify myelin water.

  8. A bovine mammary endothelial/epithelial cell culture model of the blood/milk barrier.

    PubMed Central

    Guidry, A J; O'Brien, C N; Douglass, L W

    1998-01-01

    The complex nature of the mammary gland has hampered in-depth studies of the relationship of the circulatory system to cells lining the teat ducts and alveoli of the gland. This study reports an in vitro model of endothelial and epithelial cells separated by a subcellular matrix that simulates the blood milk barrier of the bovine mammary gland. Dual chamber culture dishes with a porous membrane separating the upper and lower chamber were used. Endothelial and epithelial cells were cultured on opposite sides of the porous membrane. A collagen and fibroblast subcellular matrix, separating the 2 cell layers, simulated the in vivo interstitial tissue. Changes in surface binding of anti-bodies to polymorphonuclear neutrophils (PMN) following their migration from the upper to the lower chamber simulated the passage of PMN from blood to milk. Changes in the binding of antibodies to PMN agreed with results observed following the migration of PMN from blood to milk in vivo. This gives credence to the model's potential value for studies where more direct observation of the blood/milk barrier is required. The model will be further tested for its usefulness as an assay for determining: 1) antibiotic diffusion from milk to blood and from blood to milk, 2) cytotoxicity of prophylactic and therapeutic mammary infusion products, 3) factors affecting bacterial adhesion and penetration of mammary epithelial tissue, 4) effectiveness of antibodies present in lacteal secretions in preventing bacterial adhesion, and 5) the feasibility of gene constructs to induce synthesis and secretion of mastitis-preventing compounds and prophylactic and therapeutic compounds for treatment of human disorders. PMID:9553710

  9. Herbal adaptogens combined with protein fractions from bovine colostrum and hen egg yolk reduce liver TNF-? expression and protein carbonylation in Western diet feeding in rats

    PubMed Central

    2014-01-01

    Background We examined if a purported anti-inflammatory supplement (AF) abrogated Western-diet (WD)-induced liver pathology in rats. AF contained: 1) protein concentrates from bovine colostrum and avian egg yolk; 2) herbal adaptogens and antioxidants; and 3) acetyl-L-carnitine. Methods Nine month-old male Brown Norway rats were allowed ad libitum access to WD for 4143 days and randomly assigned to WD?+?AF feeding twice daily for the last 3133 days (n?=?8), or WD and water-placebo feeding twice daily for the last 3133 days (n?=?8). Rats fed a low-fat/low-sucrose diet (CTL, n?=?6) for 4143 days and administered a water-placebo twice daily for the last 3133 days were also studied. Twenty-four hours following the last gavage-feed, liver samples were analyzed for: a) select mRNAs (via RT-PCR) as well as genome-wide mRNA expression patterns (via RNA-seq); b) lipid deposition; and, c) protein carbonyl and total antioxidant capacity (TAC). Serum was also examined for TAC, 8-isoprostane and clinical chemistry markers. Results WD?+?AF rats experienced a reduction in liver Tnf-? mRNA (-2.8-fold, p?protein carbonyl content differed between WD?+?AF versus WD rats, although liver protein carbonyls tended to be lower in WD?+?AF versus CTL rats (p?=?0.08). RNA-seq revealed that 19 liver mRNAs differed between WD?+?AF versus WD when both groups were compared with CTL rats (+/- 1.5-fold, p?

  10. Expression of insulin-like growth factor binding protein (IGFBP)-3, and the effects of IGFBP-2 and -3 in the bovine corpus luteum.

    PubMed

    Brown, T A; Braden, T D

    2001-04-01

    The present study was conducted to gain insight into the insulin-like growth factor (IGF) system in the bovine corpus luteum (CL). Specific aims were to measure the levels of IGF binding protein-3 (IGFBP-3) and RNA encoding IGFBP-3 in the CL throughout diestrus, and to investigate the effects of IGFBP-2 and -3 on IGF-I-stimulated progesterone (P4) production and IGF-I-receptor binding. Bovine CL were collected from a local abattoir and classified according to stage of diestrus based on anatomical characteristics. Corpora lutea from early, mid and late diestrus were each analyzed for the presence of IGFBP-3 by ligand blot analysis, and for RNA encoding IGFBP-3 by Northern blot analysis. Dissociated cells from mid-cycle CL were treated with IGF-I, IGFBP-2 or -3, or a combination of IGF-I and IGFBP-2 or -3. The effect of IGFBP-2 and IGFBP-3 on [(125)I] IGF-I binding to its receptor on CL plasma membranes also was investigated. IGFBP-3 protein and RNA expression were higher in early CL, compared to mid or late CL (p < 0.05). IGF-I stimulated P4 production in a dose-dependant manner (p < 0.05). IGFBP-2 and -3 blocked the stimulatory effect of IGF-I on P4 production (p < 0.05). Both IGFBP-2 and -3 inhibited [(125)I]-IGF-I binding to its receptor in a dose-dependant manner. These results demonstrate that IGFBP-3 protein and RNA are expressed predominantly during early diestrus in the bovine CL. Moreover, both IGFBP-2 and -3 can modulate IGF-I actions in the CL by interfering with binding of IGF-I to its receptor. PMID:11438401

  11. Evaluation of the recombinant 10-kilodalton immunodominant region of the BP26 protein of Brucella abortus for specific diagnosis of bovine brucellosis.

    PubMed

    Tiwari, Arvind Kumar; Kumar, Subodh; Pal, Vijai; Bhardwaj, Bhupendra; Rai, Ganga Prasad

    2011-10-01

    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. PMID:21852548

  12. Spatio-temporal expression patterns of aurora kinases a, B, and C and cytoplasmic polyadenylation-element-binding protein in bovine oocytes during meiotic maturation.

    PubMed

    Uzbekova, Svetlana; Arlot-Bonnemains, Yannick; Dupont, Jolle; Dalbis-Tran, Rozenn; Papillier, Pascal; Pennetier, Sophie; Thlie, Aurore; Perreau, Christine; Mermillod, Pascal; Prigent, Claude; Uzbekov, Rustem

    2008-02-01

    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

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

    PubMed

    Barnes, Richard; Lehman, Clarence

    2013-06-01

    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 rle 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

  14. Multiple reaction monitoring-based determination of bovine ?-lactalbumin in infant formulas and whey protein concentrates by ultra-high performance liquid chromatography-tandem mass spectrometry using tryptic signature peptides and synthetic peptide standards.

    PubMed

    Zhang, Jingshun; Lai, Shiyun; Zhang, Yu; Huang, Baifen; Li, Duo; Ren, Yiping

    2012-05-21

    The determination of ?-lactalbumin in various dairy products attracts wide attention in multidiscipline fields because of its nutritional and biological functions. In the present study, we quantified the bovine ?-lactalbumin in various infant formulas and whey protein concentrates using ultra-high performance liquid chromatography coupled to tandem mass spectrometer in multiple reaction monitoring mode. Bovine ?-lactalbumin was quantified by employing the synthetic internal standard based on the molar equivalent relationship among the internal standard, bovine ?-lactalbumin and their signature peptides. This study especially focused on the recovery rates of the sample preparation procedure and robust quantification of total bovine ?-lactalbumin in its native and thermally denatured form with a synthetic internal standard KILDKVGINNYWLAHKALCSE. The observed recovery rates of bovine ?-lactalbumin ranged from 95.8 to 100.6% and the reproducibility was excellent (RSD<6%) at different spiking levels. The limit of quantitation is 10 mg/100 g for infant formulas and whey protein concentrates. In order to validate the applicability of the method, 21 brands of infant formulas were analyzed. The acquired contents of bovine ?-lactalbumin were 0.67-1.84 g/100g in these infant formulas in agreement with their label claimed values. The experiment of heat treatment time showed that the loss of native ?-lactalbumin enhanced with an increasing intensity of heat treatment. Comparing with Ren's previous method by analysis of only native bovine ?-lactalbumin, the present method at the peptide level proved to be highly suitable for measuring bovine ?-lactalbumin in infant formulas and whey protein concentrates, avoiding forgoing the thermally induced denatured ?-lactalbumin caused by the technological processing. PMID:22541822

  15. Metabolism of soluble rapeseed meal (Brassica rapa L.) protein during incubations with buffered bovine rumen contents in vitro.

    PubMed

    Stefa?ski, T; Ahvenjrvi, S; Huhtanen, P; Shingfield, K J

    2013-01-01

    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

  16. Formation of complexes between 125I-labelled human or bovine somatotropins and binding proteins in vivo in rat liver and kidney.

    PubMed Central

    Bonifacino, J S; Roguin, L P; Paladini, A C

    1983-01-01

    At 5 min after intravenous injection, both 125I-labelled human somatotropin and 125I-labelled bovine somatotropin were concentrated in rat liver and kidney. When the labelled hormones were administered along with an excess of the corresponding unlabelled hormone, a significant decrease of the uptake was observed in the liver, but not in the kidney. Study of the subcellular distribution of radioiodinated somatotropins in liver revealed that most of the radioactivity was specifically concentrated in the microsomal fraction. In contrast, the kidney fraction that accounted for most of the radioactivity was the 100 000 g supernatant. After solubilization, with 1% (w/v) Triton X-100, of the microsomal fractions obtained from both organs, the radioactive material was analysed by gel filtration on Sepharose CL-6B. By using this approach, it was demonstrated that both 125I-labelled human somatotropin and 125I-labelled bovine somatotropin bind in vivo to proteins present in liver. A small proportion of 125I-labelled human somatotropin was also shown to form complexes with proteins present in kidney. The present results demonstrate that the liver uptake is mainly due to binding of somatotropins to specific proteins, in contrast with the kidney, in which binding to specific sites contributes minimally to the overall uptake. PMID:6615460

  17. Effects of Rhodomyrtus tomentosa Leaf Extract on Staphylococcal Adhesion and Invasion in Bovine Udder Epidermal Tissue Model

    PubMed Central

    Mordmuang, Auemphon; Shankar, Shiv; Chethanond, Usa; Voravuthikunchai, Supayang Piyawan

    2015-01-01

    Bovine mastitis is one of the most important infectious diseases in dairy herds, and staphylococci are the most important etiologic agents of this disease. Antibiotics and chemical agents used in livestock for prevention and cure of the disease can accumulate in milk and give rise to food safety concerns. Rhodomyrtus tomentosa leaf extract was studied as an alternative approach to reduce the bacterial infections. The ethanolic extract of this plant demonstrated antibacterial activity with minimum inhibitory concentration (MIC) values as low as 16–64 μg/mL against staphylococcal isolates. In addition, the extract had an effect on the bacterial cell surface properties by increasing its hydrophobicity in a concentration dependent manner. To further extend the antibacterial efficacy, silver nanoparticles synthesized with the extract, a pure rhodomyrtone, and liposomal encapsulated rhodomyrtone were applied and their inhibitory effects on bacterial adhesion and invasion were determined by ex vivo study in a bovine udder epidermal tissue model. These agents exerted remarkable antibacterial activity against staphylococci and decreased the adhesion of the bacterial cells to the tissues. These results supported that R. tomentosa ethanolic extract could be applied as an alternative agent for bovine udder care in dairy farms. PMID:26501314

  18. Effects of Rhodomyrtus tomentosa Leaf Extract on Staphylococcal Adhesion and Invasion in Bovine Udder Epidermal Tissue Model.

    PubMed

    Mordmuang, Auemphon; Shankar, Shiv; Chethanond, Usa; Voravuthikunchai, Supayang Piyawan

    2015-10-01

    Bovine mastitis is one of the most important infectious diseases in dairy herds, and staphylococci are the most important etiologic agents of this disease. Antibiotics and chemical agents used in livestock for prevention and cure of the disease can accumulate in milk and give rise to food safety concerns. Rhodomyrtus tomentosa leaf extract was studied as an alternative approach to reduce the bacterial infections. The ethanolic extract of this plant demonstrated antibacterial activity with minimum inhibitory concentration (MIC) values as low as 16-64 μg/mL against staphylococcal isolates. In addition, the extract had an effect on the bacterial cell surface properties by increasing its hydrophobicity in a concentration dependent manner. To further extend the antibacterial efficacy, silver nanoparticles synthesized with the extract, a pure rhodomyrtone, and liposomal encapsulated rhodomyrtone were applied and their inhibitory effects on bacterial adhesion and invasion were determined by ex vivo study in a bovine udder epidermal tissue model. These agents exerted remarkable antibacterial activity against staphylococci and decreased the adhesion of the bacterial cells to the tissues. These results supported that R. tomentosa ethanolic extract could be applied as an alternative agent for bovine udder care in dairy farms. PMID:26501314

  19. Regional distribution of bovine Neospora caninum infection in the German state of Rhineland-Palatinate modelled by Logistic regression.

    PubMed

    Schares, G; Brwald, A; Staubach, C; Ziller, M; Klss, D; Wurm, R; Rauser, M; Labohm, R; Drger, K; Fasen, W; Hess, R G; Conraths, F J

    2003-12-01

    To obtain a rapid overview over the distribution of bovine Neospora caninum-infections in the German state of Rhineland-Palatinate, an ELISA to determine specific bovine antibodies against a p38 surface antigen of N. caninum tachyzoites was modified to examine bulk milk samples from cattle herds. Experimental bulk milk samples were used to demonstrate that the seroprevalence in a group of animals can be estimated with this ELISA. A cut-off was selected for the specific detection of herds having a seroprevalence > or =10%. About 90% of the dairy herds located in Rhineland-Palatinate were examined. An overall prevalence of bulk milk-positive herds of 7.9% (95% confidence interval 7.0-8.9%), respectively, was determined. Major regional differences in the distribution of bulk milk-positive herds were observed. Prevalences were higher in regions with an increased degree of urbanisation. Logistic regression was applied to model the prevalence of bulk milk-positive herds on a district and city level. Variables describing the dog density, mean temperature in July, mean temperature in January and the total yearly precipitation in districts and cities were able to explain most of the observed variability in the regional prevalences. Our results provide evidence that in addition to risk factors related to individual farms also risk factors related to the farm location such as dog density in the surrounding and climate factors are important in the epidemiology of bovine neosporosis. PMID:14636679

  20. A three-dimensional cell culture model for bovine endometrium: regeneration of a multicellular spheroid using ascorbate.

    PubMed

    Yamauchi, N; Yamada, O; Takahashi, T; Imai, K; Sato, T; Ito, A; Hashizume, K

    2003-01-01

    The development of a multicellular spheroid comprising bovine endometrial epithelial cells (BEE) and bovine endometrial stromal cells (BES) is described in this study. The BES were cultured to confluence in medium with L -ascorbic acid phosphate magnesium salt n -hydrate (AsA-P) which stimulates collagen synthesis in BES. The BEE were co-cultured on a BES cell-sheet for 24h before detachment of the cell-sheet to generate a hetero-spheroid. After EDTA treatment and agitating with pipette, the floating cell-sheet shrank and became an aggregated cell mass in a few days; it finally formed a round-shaped hetero-spheroid composed of BES and BEE. Histological examination found that hetero-spheroids were covered with BEE on the outer layer. When cell viability was examined with TUNEL (terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling), no positive signal was detected in the spheroid for up to 10 days. Immunofluorescence observations showed that spheroids contained abundant extracellular matrices, including type-I, -III, -IV collagen, fibronectin, and laminin. PGF(2alpha) produced by hetero-spheroids in response to oxytocin was significantly higher than those produced by monolayer cultured BEE (P< 0.05). MMPs were not detected in media from spheroids cultured for 5 days after detachment of the cell sheet. These results indicate that bovine endometrial cells have the capacity to regenerate as a multicellular spheroid after treatment with ascorbate in vitro. The spheroid displays an endometrium-mimic feature. Thus, we conclude that spheroids formed by BES and BEE are a useful in vitro model of bovine endometrium. PMID:12566253

  1. Speciation of trace elements in proteins in human and bovine serum by size exclusion chromatography and inductively coupled plasma-mass spectrometry with a magnetic sector mass spectrometer.

    PubMed

    Wang, J; Houk, R S; Dreessen, D; Wiederin, D R

    1999-10-01

    Proteins are separated by size exclusion chromatography while atomic ions from the inorganic elements are detected on-line by inductively coupled plasma-mass spectrometry. A double focusing mass analyzer provides very high sensitivity, low background, and sufficient spectral resolution to separate the atomic ions of interest from most polyatomic ions at the same nominal m/z value. The chromatograms show the distribution of the elements of interest between protein-bound and free fractions and provide the approximate molecular weights of those protein fractions that contain the elements monitored. The distribution of various elements, including V, Mo, Fe, Co, Mn, and lanthanides, in human or bovine serum samples are shown. Alkali metals and Tl are present primarily as free metal ions and are not bound to proteins. Inorganic elements spiked into the serum samples can be followed into various proteins. EDTA does not remove Fe, Pb, Sn, or Th from the proteins but does extract Mn from some proteins. Procedures for determining the effects of breaking disulfide linkages on the metal binding characteristics of proteins are also described. PMID:10550683

  2. Evaluation of Serum-Derived Bovine Immunoglobulin Protein Isolate in Subjects with Diarrhea-Predominant Irritable Bowel Syndrome

    PubMed Central

    Wilson, Dale; Evans, Malkanthi; Weaver, Eric; Shaw, Audrey L.; Klein, Gerald L.

    2013-01-01

    BACKGROUND There is increased interest in combining nutritional modalities with pharmacological therapies for managing patients with diarrhea-predominant IBS (IBS-D). AIM A randomized, double-blind, placebo-controlled study to evaluate the impact of oral serum-derived bovine immunoglobulin/protein isolate (SBI) on gastrointestinal symptom scores and quality of life (QoL) in subjects with IBS-D. METHODS Study subjects previously diagnosed with IBS-D according to ROME II criteria were recruited from London, Ontario, Canada and assigned to receive 5 g/day SBI, 10 g/day SBI, or placebo for 6 weeks. Daily symptom frequency and severity scores and a modified IBS-36 questionnaire assessed the impact of nutritional intervention. Laboratory assessments were performed at screening and end of treatment (EOT) to evaluate safety. Within-group comparisons of changes in number of days per week with symptoms and symptom severity were conducted on the per-protocol population of subjects using a t-test. RESULTS Subjects who received SBI at 10 g/day (N = 15) had statistically significant within-group reductions in abdominal pain (p < 0.01), loose stools (p < 0.01), bloating (p < 0.05), flatulence (p < 0.01), urgency (p < 0.05) and any symptom (p < 0.01) at EOT vs. baseline. Subjects receiving 5 g/day of SBI (N = 15) realized statistically significant within-group reductions in days with flatulence (p < 0.035), incomplete evacuation (p < 0.05), and any symptom (p < 0.01). There were no significant changes in QoL scores or in hematology or clinical chemistry among treatment groups. CONCLUSIONS This pilot study showed that nutritional therapy with either 10 g/day or 5 g/day of SBI in 30 patients was well tolerated and resulted in statistically significant within group improvements in both symptom days and in daily symptom scores in subjects with IBS-D. Additional studies are underway with larger numbers of subjects to validate these findings. PMID:24833942

  3. Chemical modification of lysozyme, glucose 6-phosphate dehydrogenase, and bovine eye lens proteins induced by peroxyl radicals: role of oxidizable amino acid residues.

    PubMed

    Arenas, Andrea; Lpez-Alarcn, Camilo; Kogan, Marcelo; Lissi, Eduardo; Davies, Michael J; Silva, Eduardo

    2013-01-18

    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

  4. Effects of butyrate on the expression of insulin-like growth factor binding proteins in bovine kidney epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sodium butyrate induces cell cycle arrest and apoptosis in bovine kidney epithelial cells primarily via down-regulating cell cycle-related gene expression and enhancing expression of pro-apoptotic genes. The insulin-like growth factor (IGF) system plays an essential role in these processes as well a...

  5. Antiviral Efficacy of a Respiratory Syncytial Virus (RSV) Fusion Inhibitor in a Bovine Model of RSV Infection.

    PubMed

    Jordan, Robert; Shao, Matt; Mackman, Richard L; Perron, Michel; Cihlar, Tomas; Lewis, Sandy A; Eisenberg, Eugene J; Carey, Anne; Strickley, Robert G; Chien, Jason W; Anderson, Mark L; McEligot, Heather A; Behrens, Nicole E; Gershwin, Laurel J

    2015-08-01

    Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants. Effective treatment for RSV infection is a significant unmet medical need. While new RSV therapeutics are now in development, there are very few animal models that mimic the pathogenesis of human RSV, making it difficult to evaluate new disease interventions. Experimental infection of Holstein calves with bovine RSV (bRSV) causes a severe respiratory infection that is similar to human RSV infection, providing a relevant model for testing novel therapeutic agents. In this model, viral load is readily detected in nasal secretions by quantitative real-time PCR (qRT-PCR), and cumulative symptom scoring together with histopathology evaluations of infected tissue allow for the assessment of disease severity. The bovine RSV model was used to evaluate the antiviral activity of an RSV fusion inhibitor, GS1, which blocks virus entry by inhibiting the fusion of the viral envelope with the host cell membrane. The efficacy of GS1, a close structural analog of GS-5806 that is being developed to treat RSV infection in humans was evaluated in two randomized, blind, placebo-controlled studies in bRSV-infected calves. Intravenous administration of GS1 at 4 mg/kg of body weight/day for 7 days starting 24 h or 72 h postinoculation provided clear therapeutic benefit by reducing the viral load, disease symptom score, respiration rate, and lung pathology associated with bRSV infection. These data support the use of the bovine RSV model for evaluation of experimental therapeutics for treatment of RSV. PMID:26055364

  6. Antiviral Efficacy of a Respiratory Syncytial Virus (RSV) Fusion Inhibitor in a Bovine Model of RSV Infection

    PubMed Central

    Jordan, Robert; Shao, Matt; Mackman, Richard L.; Perron, Michel; Cihlar, Tomas; Lewis, Sandy A.; Eisenberg, Eugene J.; Carey, Anne; Strickley, Robert G.; Chien, Jason W.; Anderson, Mark L.; McEligot, Heather A.; Behrens, Nicole E.

    2015-01-01

    Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants. Effective treatment for RSV infection is a significant unmet medical need. While new RSV therapeutics are now in development, there are very few animal models that mimic the pathogenesis of human RSV, making it difficult to evaluate new disease interventions. Experimental infection of Holstein calves with bovine RSV (bRSV) causes a severe respiratory infection that is similar to human RSV infection, providing a relevant model for testing novel therapeutic agents. In this model, viral load is readily detected in nasal secretions by quantitative real-time PCR (qRT-PCR), and cumulative symptom scoring together with histopathology evaluations of infected tissue allow for the assessment of disease severity. The bovine RSV model was used to evaluate the antiviral activity of an RSV fusion inhibitor, GS1, which blocks virus entry by inhibiting the fusion of the viral envelope with the host cell membrane. The efficacy of GS1, a close structural analog of GS-5806 that is being developed to treat RSV infection in humans was evaluated in two randomized, blind, placebo-controlled studies in bRSV-infected calves. Intravenous administration of GS1 at 4 mg/kg of body weight/day for 7 days starting 24 h or 72 h postinoculation provided clear therapeutic benefit by reducing the viral load, disease symptom score, respiration rate, and lung pathology associated with bRSV infection. These data support the use of the bovine RSV model for evaluation of experimental therapeutics for treatment of RSV. PMID:26055364

  7. Endometrial transcriptional profiling of a bovine fertility model by Next-Generation Sequencing

    PubMed Central

    Mesquita, F.S.; Ramos, R.S.; Pugliesi, G.; Andrade, S.C.S.; Van Hoeck, V.; Langbeen, A.; Oliveira, M.L.; Gonella-Diaza, A.M.; Gasparin, G.; Fukumasu, H.; Pulz, L.H.; Membrive, C.M.; Coutinho, L.L.; Binelli, M.

    2015-01-01

    Studying the multitude of molecular networks and pathways that are potentially involved in a complex trait such as fertility requires an equally complex and broad strategy. Here, we used Next-Generation Sequencing for the characterization of the transcriptional signature of the bovine endometrial tissue. Periovulatory endocrine environments were manipulated to generate two distinctly different fertility phenotypes. Cycling, non-lactating, multiparous Nelore cows were manipulated to ovulate larger (> 13 mm; LF group; high fertility phenotype) or smaller (< 12 mm; SF group) follicles. As a result, greater proestrus estrogen concentrations, corpora lutea and early diestrus progesterone concentrations were also observed in LF group in comparison to SF group. Endometrial cell proliferation was estimated by the protein marker MKI67 on tissues collected 4 (D4) and 7 (D7) days after induction of ovulation. Total RNA extracts from D7 were sequenced and compared according to the transcriptional profile of each experimental group (LF versus SF). Functional enrichment analysis revealed that LF and SF endometria were asynchronous in regards to their phenotype manifestation. Major findings indicated an LF endometrium that was switching phenotypes earlier than the SF one. More specifically, a proliferating SF endometrium was observed on D7, whereas the LF tissue, which expressed a proliferative phenotype earlier at D4, seemed to have already shifted towards a biosynthetically and metabolically active endometrium on D7. Data on MKI67 support the transcriptomic results. RNA-Seq-derived transcriptional profile of the endometrial tissue indicated a temporal effect of the periovulatory endocrine environment, suggesting that the moment of the endometrial exposure to the ovarian steroids, E2 and P4, regulates the timing of phenotype manifestation. Gene expression profiling revealed molecules that may be targeted to elucidate ovarian steroid-dependent mechanisms that regulate endometrial tissue receptivity. Data was deposited in the SRA database from NCBI (SRA Experiment SRP051330) and are associated with the Bio-Project (PRJNA270391). An overview of the gene expression data has been deposited in NCBI's Gene Expression Omnibus (GEO) and is accessible through GEO Series accession number GSE65450. Further assessment of the data in combination with other data sets exploring the transcriptional profile of the endometrial tissue during early diestrus may potentially identify novel molecular mechanisms and/or markers of the uterine receptivity. PMID:26981354

  8. Endometrial transcriptional profiling of a bovine fertility model by Next-Generation Sequencing.

    PubMed

    Mesquita, F S; Ramos, R S; Pugliesi, G; Andrade, S C S; Van Hoeck, V; Langbeen, A; Oliveira, M L; Gonella-Diaza, A M; Gasparin, G; Fukumasu, H; Pulz, L H; Membrive, C M; Coutinho, L L; Binelli, M

    2016-03-01

    Studying the multitude of molecular networks and pathways that are potentially involved in a complex trait such as fertility requires an equally complex and broad strategy. Here, we used Next-Generation Sequencing for the characterization of the transcriptional signature of the bovine endometrial tissue. Periovulatory endocrine environments were manipulated to generate two distinctly different fertility phenotypes. Cycling, non-lactating, multiparous Nelore cows were manipulated to ovulate larger (> 13 mm; LF group; high fertility phenotype) or smaller (< 12 mm; SF group) follicles. As a result, greater proestrus estrogen concentrations, corpora lutea and early diestrus progesterone concentrations were also observed in LF group in comparison to SF group. Endometrial cell proliferation was estimated by the protein marker MKI67 on tissues collected 4 (D4) and 7 (D7) days after induction of ovulation. Total RNA extracts from D7 were sequenced and compared according to the transcriptional profile of each experimental group (LF versus SF). Functional enrichment analysis revealed that LF and SF endometria were asynchronous in regards to their phenotype manifestation. Major findings indicated an LF endometrium that was switching phenotypes earlier than the SF one. More specifically, a proliferating SF endometrium was observed on D7, whereas the LF tissue, which expressed a proliferative phenotype earlier at D4, seemed to have already shifted towards a biosynthetically and metabolically active endometrium on D7. Data on MKI67 support the transcriptomic results. RNA-Seq-derived transcriptional profile of the endometrial tissue indicated a temporal effect of the periovulatory endocrine environment, suggesting that the moment of the endometrial exposure to the ovarian steroids, E2 and P4, regulates the timing of phenotype manifestation. Gene expression profiling revealed molecules that may be targeted to elucidate ovarian steroid-dependent mechanisms that regulate endometrial tissue receptivity. Data was deposited in the SRA database from NCBI (SRA Experiment SRP051330) and are associated with the Bio-Project (PRJNA270391). An overview of the gene expression data has been deposited in NCBI's Gene Expression Omnibus (GEO) and is accessible through GEO Series accession number GSE65450. Further assessment of the data in combination with other data sets exploring the transcriptional profile of the endometrial tissue during early diestrus may potentially identify novel molecular mechanisms and/or markers of the uterine receptivity. PMID:26981354

  9. Stochastic model for protein flexibility analysis

    NASA Astrophysics Data System (ADS)

    Xia, Kelin; Wei, Guo-Wei

    2013-12-01

    Protein flexibility is an intrinsic property and plays a fundamental role in protein functions. Computational analysis of protein flexibility is crucial to protein function prediction, macromolecular flexible docking, and rational drug design. Most current approaches for protein flexibility analysis are based on Hamiltonian mechanics. We introduce a stochastic model to study protein flexibility. The essential idea is to analyze the free induction decay of a perturbed protein structural probability, which satisfies the master equation. The transition probability matrix is constructed by using probability density estimators including monotonically decreasing radial basis functions. We show that the proposed stochastic model gives rise to some of the best predictions of Debye-Waller factors or B factors for three sets of protein data introduced in the literature.

  10. Bioeconomic modeling of lactational antimicrobial treatment of new bovine subclinical intramammary infections caused by contagious pathogens.

    PubMed

    van den Borne, B H P; Halasa, T; van Schaik, G; Hogeveen, H; Nielen, M

    2010-09-01

    This study determined the direct and indirect epidemiologic and economic effects of lactational treatment of new bovine subclinical intramammary infections (IMI) caused by contagious pathogens using an existing bioeconomic model. The dynamic and stochastic model simulated the dynamics of Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, and Escherichia coli during lactation and the dry period in a 100-cow dairy herd during 1 quota year. Input parameters on cure were obtained from recent Dutch field data. The costs of clinical IMI, subclinical IMI, and intervention were calculated into the combined total annual net costs of IMI per herd. The cost effectiveness of 4 scenarios with lactational intervention was determined; scenarios included no intervention, treatment after 1 mo of infection, treatment after 2 mo of infection, and treatment after 1 mo of infection and culling of uncured cows after 2 mo of infection. Model behavior was observed for variation in parameter input values. Compared with no lactational intervention, lactational intervention of new subclinical IMI resulted in fewer clinical flare ups, less transmission within the herd, and much lower combined total annual net costs of IMI in dairy herds. Antimicrobial treatment of IMI after 1 mo of infection and culling of uncured cows after 2 mo of infection resulted in the lowest costs, whereas treatment after 2 mo of infection was associated with the highest costs between the scenarios with intervention. Changing the probability of cure resulted in a nonlinear change in the cumulative incidence of IMI cases and associated costs. Lactational treatment was able to prevent IMI epidemics in dairy herds at high transmission rates of Strep. uberis, Strep. dysgalactiae, and E. coli. Lactational treatment did not limit the spread of Staph. aureus at high transmission rates, although the associated costs were lower compared with no intervention. To improve udder health in a dairy herd, lactational treatment of contagious subclinical IMI must therefore be preceded by management measures that lower the transmission rate. Lactational treatment of environmental subclinical IMI seemed less cost effective. Detection of subclinical IMI needs improvement to be able to most effectively treat subclinical IMI caused by contagious pathogens during lactation. PMID:20723677

  11. Impact of external sources of infection on the dynamics of bovine tuberculosis in modelled badger populations

    PubMed Central

    2012-01-01

    Background The persistence of bovine TB (bTB) in various countries throughout the world is enhanced by the existence of wildlife hosts for the infection. In Britain and Ireland, the principal wildlife host for bTB is the badger (Meles meles). The objective of our study was to examine the dynamics of bTB in badgers in relation to both badger-derived infection from within the population and externally-derived, trickle-type, infection, such as could occur from other species or environmental sources, using a spatial stochastic simulation model. Results The presence of external sources of infection can increase mean prevalence and reduce the threshold group size for disease persistence. Above the threshold equilibrium group size of 6–8 individuals predicted by the model for bTB persistence in badgers based on internal infection alone, external sources of infection have relatively little impact on the persistence or level of disease. However, within a critical range of group sizes just below this threshold level, external infection becomes much more important in determining disease dynamics. Within this critical range, external infection increases the ratio of intra- to inter-group infections due to the greater probability of external infections entering fully-susceptible groups. The effect is to enable bTB persistence and increase bTB prevalence in badger populations which would not be able to maintain bTB based on internal infection alone. Conclusions External sources of bTB infection can contribute to the persistence of bTB in badger populations. In high-density badger populations, internal badger-derived infections occur at a sufficient rate that the additional effect of external sources in exacerbating disease is minimal. However, in lower-density populations, external sources of infection are much more important in enhancing bTB prevalence and persistence. In such circumstances, it is particularly important that control strategies to reduce bTB in badgers include efforts to minimise such external sources of infection. PMID:22738118

  12. [Exposure of the major immunodominant epitope of the gp51 envelope protein of bovine leukosis virus on the surface of the hepatitis B core antigen capsid].

    PubMed

    Ul'rikh, R; Borisova, G P; Siakkou, Kh; Plattser, K; Ose, V P; Berzin', I G; Dre?linia, D E; Pushko, P M; Tsibinogin, V V; Pumpen, P P

    1991-01-01

    Insertion of 48 amino acid long sequence of envelope protein gp51 of bovine leukemia virus (BLV), located from position 56 till 103 of mature protein, into Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids. These capsids preserve morphology of intact HBcAg but expose on their outer surface BLV epitopes which are localised in the inserted gp51 fragment and responsible for the recognition of chimeras by monoclonal anti-gp51 antibodies MAK14. The anti-genicity of gp51 epitopes within chimeric capsids is not disturbed after shortening of C terminal part of inserted gp51 fragment by deletion of amino acids 73-103. The resulting chimeras show the same capsid-forming ability as well as HBcAg and gp51 antigenic properties. PMID:1715509

  13. Protein fragment reconstruction using various modeling techniques.

    PubMed

    Boniecki, Michal; Rotkiewicz, Piotr; Skolnick, Jeffrey; Kolinski, Andrzej

    2003-11-01

    Recently developed reduced models of proteins with knowledge-based force fields have been applied to a specific case of comparative modeling. From twenty high resolution protein structures of various structural classes, significant fragments of their chains have been removed and treated as unknown. The remaining portions of the structures were treated as fixed - i.e., as templates with an exact alignment. Then, the missed fragments were reconstructed using several modeling tools. These included three reduced types of protein models: the lattice SICHO (Side Chain Only) model, the lattice CABS (Calpha + Cbeta + Side group) model and an off-lattice model similar to the CABS model and called REFINER. The obtained reduced models were compared with more standard comparative modeling tools such as MODELLER and the SWISS-MODEL server. The reduced model results are qualitatively better for the higher resolution lattice models, clearly suggesting that these are now mature, competitive and complementary (in the range of sparse alignments) to the classical tools of comparative modeling. Comparison between the various reduced models strongly suggests that the essential ingredient for the sucessful and accurate modeling of protein structures is not the representation of conformational space (lattice, off-lattice, all-atom) but, rather, the specificity of the force fields used and, perhaps, the sampling techniques employed. These conclusions are encouraging for the future application of the fast reduced models in comparative modeling on a genomic scale. PMID:15072433

  14. Silica Vesicle Nanovaccine Formulations Stimulate Long-Term Immune Responses to the Bovine Viral Diarrhoea Virus E2 Protein

    PubMed Central

    Mody, Karishma T.; Mahony, Donna; Cavallaro, Antonino S.; Zhang, Jun; Zhang, Bing; Mahony, Timothy J.; Yu, Chengzhong; Mitter, Neena

    2015-01-01

    Bovine Viral Diarrhoea Virus (BVDV) is one of the most serious pathogen, which causes tremendous economic loss to the cattle industry worldwide, meriting the development of improved subunit vaccines. Structural glycoprotein E2 is reported to be a major immunogenic determinant of BVDV virion. We have developed a novel hollow silica vesicles (SV) based platform to administer BVDV-1 Escherichia coli-expressed optimised E2 (oE2) antigen as a nanovaccine formulation. The SV-140 vesicles (diameter 50 nm, wall thickness 6 nm, perforated by pores of entrance size 16 nm and total pore volume of 0.934 cm3g-1) have proven to be ideal candidates to load oE2 antigen and generate immune response. The current study for the first time demonstrates the ability of freeze-dried (FD) as well as non-FD oE2/SV140 nanovaccine formulation to induce long-term balanced antibody and cell mediated memory responses for at least 6 months with a shortened dosing regimen of two doses in small animal model. The in vivo ability of oE2 (100 μg)/SV-140 (500 μg) and FD oE2 (100 μg)/SV-140 (500 μg) to induce long-term immunity was compared to immunisation with oE2 (100 μg) together with the conventional adjuvant Quil-A from the Quillaja saponira (10 μg) in mice. The oE2/SV-140 as well as the FD oE2/SV-140 nanovaccine generated oE2-specific antibody and cell mediated responses for up to six months post the final second immunisation. Significantly, the cell-mediated responses were consistently high in mice immunised with oE2/SV-140 (1,500 SFU/million cells) at the six-month time point. Histopathology studies showed no morphological changes at the site of injection or in the different organs harvested from the mice immunised with 500 μg SV-140 nanovaccine compared to the unimmunised control. The platform has the potential for developing single dose vaccines without the requirement of cold chain storage for veterinary and human applications. PMID:26630001

  15. Exploring the affinity binding of alkylmaltoside surfactants to bovine serum albumin and their effect on the protein stability: A spectroscopic approach.

    PubMed

    Hierrezuelo, J M; Carnero Ruiz, C

    2015-08-01

    Steady-state and time-resolved fluorescence together with circular dichroism (CD) spectroscopic studies was performed to examine the interactions between bovine serum albumin (BSA) and two alkylmaltoside surfactants, i.e. n-decyl-β-D-maltoside (β-C10G2) and n-dodecyl-β-D-maltoside (β-C12G2), having identical structures but different tail lengths. Changes in the intrinsic fluorescence of BSA from static as well as dynamic measurements revealed a weak protein-surfactant interaction and gave the corresponding binding curves, suggesting that the binding mechanism of surfactants to protein is essentially cooperative in nature. The behavior of both surfactants is similar, so that the differences detected were attributed to the more hydrophobic nature of β-C12G2, which favors the adsorption of micelle-like aggregates onto the protein surface. These observations were substantially demonstrated by data derived from synchronous, three-dimensional and anisotropy fluorescence experiments. Changes in the secondary structure of the protein induced by the interaction with surfactants were analyzed by CD to determine the contents of α-helix and β-strand. It was noted that whereas the addition of β-C10G2 appears to stabilize the secondary structure of the protein, β-C12G2 causes a marginal denaturation of BSA for a protein:surfactant molar ratio as high as 1 to 100. PMID:26042703

  16. An activity coefficient model for proteins.

    PubMed

    Agena, S M; Bogle, I D; Pessoa, F L

    1997-07-01

    Modeling of the properties of biochemical components is gaining increasing interest due to its potential for further application within the area of biochemical process development. Generally protein solution properties such as protein solubility are expressed through component activity coefficients which are studied here. The original UNIQUAC model is chosen for the representation of protein activity coefficients and, to the best of our knowledge, this is the first time it has been directly applied to protein solutions. Ten different protein-salt-water systems with four different proteins, serum albumin, alphacymotrypsin, beta-lactoglobulin and ovalbumin, are investigated. A root-mean-squared deviation of 0.54% is obtained for the model by comparing calculated protein activity coefficients and protein activity coefficients deduced from osmotic measurements through virial expansion. Model predictions are used to analyze the effect of salt concentrations, pH, salt types, and temperature on protein activity coefficients and also on protein solubility and demonstrate consistency with results from other references. PMID:18636445

  17. A soft and transparent handleable protein model

    NASA Astrophysics Data System (ADS)

    Kawakami, Masaru

    2012-08-01

    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.

  18. Transdisciplinary habitat models for elk and cattle as a proxy for bovine tuberculosis transmission risk.

    PubMed

    Brook, Ryan K; McLachlan, Stéphane M

    2009-10-01

    Zoonotic diseases such as bovine tuberculosis (TB) that infect wildlife and livestock are particularly difficult to eradicate where wild animals make extensive use of agricultural landscapes. Transmission of TB between cattle (Bos taurus) and wild elk (Cervus elaphus) in southwestern Manitoba, Canada remains poorly understood but there is a risk when commingling occurs on summer pasture. Elk use of cattle summer pastures was assessed using ecological data (187 VHF and 25 GPS collared elk monitored over four years representing 8% of the elk population). Local knowledge was documented by conducting interviews and participatory mapping exercises with 86 cattle producers (98% of those within the study area). Of the 294 cattle pastures mapped by farmers, 13% were used by radio-collared elk, 38% were reported by farmers as being used by elk, and 42% were identified as used by elk when both when all datasets were combined. Cattle pastures that had been used by elk and those that had no elk were compared using binary logistic regression based on each of the three datasets (i.e. farmer observations, radio-collared elk on pasture, and combined dataset). For all three datasets, distance to protected area and proportion of forest cover on the cattle pasture were identified as the most and second most important predictor variables, respectively. There was strong agreement among the relative probabilities of elk occurrence on each pasture derived from the resource selection function (RSF) models developed using farmer interviews and elk collaring data. The farmer interview and collar datasets were then combined to generate a final integrated RSF map summarizing the probability of elk-cattle commingling and were contrasted over each of four cattle grazing seasons (spring, early summer, late summer, and autumn). These predictive maps indicate that use of cattle pastures by elk is extensive, particularly in spring and early summer. Farmer observations indicate that elk and cattle share water sources and livestock mineral supplements on pasture. Local knowledge and conventional ecological data complement and validate one another and help us better understand the temporospatial aspects of shared space use among wildlife and livestock and more generally the risks of disease transmission in agricultural landscapes. PMID:19541377

  19. Evaluation of recombinant 28 kDa outer membrane protein of Brucella abortus for the clinical diagnosis of bovine brucellosis in Korea.

    PubMed

    Lim, Jeong Ju; Kim, Dong Hyeok; Lee, Jin Ju; Kim, Dae Geun; Min, Wongi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee; Kim, Suk

    2012-06-01

    Brucella spp. are Gram-negative, facultative, intracellular coccobacilli that are pathogenic to a variety of mammals, including ruminants and humans. The conventional serological test for diagnosing brucellosis in cattle in Korea is the standard tube agglutination test. However, agglutination tests sometimes give false-positive results due to cross-reactions with other pathogens. The outer membrane proteins of Brucella species have been extensively studied for their immunogenicity and serodiagnostic applications. However, an application of B. abortus OMPs for serodiagnosis has not been successfully established. In this study, cloning and expression of B. abortus Omp28, a group 3 antigen, were accomplished by PCR amplification cloning into a pMAL expression system, and purification of a recombinant Omp28 (rOmp28). The immunogenicity of rOmp28 was confirmed by Western blot analysis with Brucella-positive bovine serum. To determine whether rOmp2 has a potential benefit for use in the serodiagnosis of bovine brucellosis, rOmp28-based ELISA and latex bead agglutination test were performed. B. abortus positive (n=122) or negative (n=88) from TAT were positive (118/122, 96.7%) or negative (84/88, 95.4%) in ELISA and were positive (94/122, 77%) or negative (71/88, 81.7%) in that the latex bead agglutination test, respectively. The sensitivity, specificity and accuracy were 96.7, 95.4, 96.2% in ELISA and 77, 80.6, 78.5% in latex bead agglutination test, respectively. These findings suggest that the rOmp28 of B. abortus might be a good candidate for developing serological diagnostic tools for bovine brucellosis. PMID:22214857

  20. Characterization of Bovine NANOG5?-flanking Region during Differentiation of Mouse Embryonic Stem Cells

    PubMed Central

    Jang, Hye-Jeong; Park, Hwan Hee; Linh, Tran Thi Thuy; Lee, Hak-Kyo; Song, Ki-Duk; Lee, Woon Kyu

    2015-01-01

    Embryonic stem cells (ESCs) have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (?420/+181) bovine NANOG 5?-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP) as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (?420/+181) promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells. PMID:26580439

  1. Characterization of Bovine NANOG5'-flanking Region during Differentiation of Mouse Embryonic Stem Cells.

    PubMed

    Jang, Hye-Jeong; Park, Hwan Hee; Linh, Tran Thi Thuy; Lee, Hak-Kyo; Song, Ki-Duk; Lee, Woon Kyu

    2015-12-01

    Embryonic stem cells (ESCs) have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (-420/+181) bovine NANOG 5'-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP) as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (-420/+181) promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells. PMID:26580439

  2. Statistical Mechanics Model for Protein Folding

    NASA Astrophysics Data System (ADS)

    Yakubovich, Alexander; Solov'yov, Andrey V.; Greiner, Walter

    2009-12-01

    We present a statistical mechanics formalism for the theoretical description of the process of protein folding?unfolding transition in water environment. The presented formalism is based on the construction of the partition function of a protein obeying two-stage-like folding kinetics. Than, using the statistical mechanics model of solvation of hydrophobic hydrocarbons we obtain the partition function of infinitely diluted solution of proteins in water environment. The results of the presented theoretical model for protein folding is compared with the experimental measurements of the heat capacity on temperature dependencies of staphylococcal nuclease and metmyoglobin.

  3. In vitro development of bovine embryos in conditioned media from bovine granulosa cells and vero cells cultured in exogenous protein- and amino acid-free chemically defined human tubal fluid medium.

    PubMed

    Maeda, J; Kotsuji, F; Negami, A; Kamitani, N; Tominaga, T

    1996-04-01

    We have investigated the effect of protein- and amino acid-free simple human tubal fluid (HTF) medium conditioned with bovine granulosa cells (BGC) or Vero cells on the development of early bovine embryos to the blastocyst stage. Serum-containing medium (SCM) and serum-free medium (CM) conditioned by BGC (BGC-SCM, BGC-CM) and by Vero cells (VC-SCM, VC-CM) were prepared. Early embryos (1-cell stage and 5- to 8-cell stage) were obtained by in vitro maturation and fertilization of oocytes from slaughtered cows. Embryos were randomly divided into 7 culture groups as follows: culture with BGC-SCM, BGC-CM, VC-SCM, or VC-CM; coculture with BGC or Vero cells; or culture with fresh HTF medium without serum. The proportion of 5- to 8-cell embryos developing to the blastocyst stage in BGC-CM (16%) and VC-CM (12%) was significantly lower (p < 0.05) than in BGC-SCM (41%) and in VC-SCM (29%) and after coculture with BGC (48%) and Vero cells (30%). Similarly, the percentages of 1-cell embryos developing to blastocyst in BGC-CM and VC-CM were significantly lower than in BGC-SCM and VC-SCM and after coculture. Cell numbers per blastocyst developed from 5- to 8-cell embryos in BGC-CM (96.8 cells) and in VC-CM (94.0 cells) were somewhat lower than those in BGC-SCM (128.5 cells) and VC-SCM (117.1 cells) and after coculture with BGC (124.2 cells) and Vero cells (115.3 cells). These results suggest that BGC and Vero cells cultured in a protein- and amino acid-free simple HTF medium synthesize and secrete factor(s) promoting blastocyst formation in vitro. Physiochemical analysis indicated that the embryotrophic substances in BGC-CM were distributed in two molecular size ranges, one between 10 kDa and 30 kDa and another greater than 30 kDa. PMID:8924514

  4. Measurements of bovine albumin as a model system for the development of a hand-held ellipsometer for ophthalmic applications

    NASA Astrophysics Data System (ADS)

    Miran Baygi, M. H.; Payne, P. A.

    2000-06-01

    Scattering ellipsometry is a measurement technique that can extract the characteristics of particles with relative ease. For some while, we have been interested in the use of scattering ellipsometry to examine the particles in the anterior chamber of the human eye. These particles are globular proteins, typically some 6 or 7 nm in diameter and measurements conducted at a wavelength of 670 nm confirm this. Particles or particle aggregates much greater in size will give rise to characteristic patterns in the ellipsometric results and analysis of these patterns could lead to a complete description of the physical properties of such particles. Examples of measurements of this nature made on latex particles are included. The results obtained from the measurements described confirm that bovine serum albumin is suitable as a calibrant for a scattering ellipsometer.

  5. Models of globular proteins in aqueous solutions

    NASA Astrophysics Data System (ADS)

    Wentzel, Nathaniel James

    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.

  6. Effect of a bovine lung surfactant protein isolate (SP-B/C) on egg phosphatidylglycerol acyl chain order in a lipid mixture with dipalmitoylphosphatidylcholine and palmitic acid.

    PubMed

    Krill, S L; Gupta, S L

    1994-04-01

    Dynamic surface tension measurements of films of a d62 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine:L-alpha-phosphatidyl-DL - glycerol:d31 palmitic acid (d62-DPPC:EggPG:d31-PA) lipid matrix in the presence of a bovine pulmonary surfactant protein isolate (SP-B/C) demonstrate the improved surface activity over that of the lipids alone. Thus, significant interaction of the proteins with the lipid matrix is demonstrated. The effect of SP-B/C on the acyl chain order of the negatively charged EggPG within a d62-DPPC:EggPG:d31-PA lipid matrix in D2O saline was investigated in thermal perturbation Fourier transform IR spectroscopic studies. The EggPG thermotropic phase behavior was determined independently of the other lipid components with perdeuterated lipids and D2O. The data demonstrate the high degree of EggPG acyl chain disorder in the absence of the protein isolate. A broad transition occurs between 30 and 40 degrees C. The addition of the protein isolate did not alter the acyl chain order at 0.281 and 1.46 mg/mL of protein. However, alterations in the lipid carbonyl vibrational mode were observed. PMID:8046609

  7. Comparative evaluation of recombinant LigB protein and heat-killed antigen-based latex agglutination test with microscopic agglutination test for diagnosis of bovine leptospirosis.

    PubMed

    Nagalingam, Mohandoss; Thirumalesh, Sushma Rahim Assadi; Kalleshamurthy, Triveni; Niharika, Nakkala; Balamurugan, Vinayagamurthy; Shome, Rajeswari; Sengupta, Pinaki Prasad; Shome, Bibek Ranjan; Prabhudas, Krishnamsetty; Rahman, Habibur

    2015-10-01

    This study aimed to develop latex agglutination test (LAT) using recombinant leptospiral immunoglobulin-like protein (LigB) (rLigB) antigen and compare its diagnostic efficacy with LAT using conventional heat-killed leptospiral antigen and microscopic agglutination test (MAT) in diagnosing bovine leptospirosis. The PCR-amplified 1053-bp ligB gene sequences from Leptospira borgpetersenii Hardjo serovar were cloned in pET 32 (a) vector at EcoRI and NotI sites and expressed in BL21 E. coli cells as fusion protein with thioredoxin (-57 kDa) and characterized by SDS-PAGE and immunoblot. Out of 390 serum samples [cattle (n = 214), buffaloes (n = 176)] subjected to MAT, 115 samples showed reciprocal titre≥100 up to 1600 against one or more serovars. For recombinant LigB protein/antigen-based LAT, agglutination was observed in the positive sample, while no agglutination was observed in the negative sample. Similarly, heat-killed leptospiral antigen was prepared from and used in LAT for comparison with MAT. A two-sided contingency table was used for analysis of LAT using both the antigens separately against MAT for 390 serum samples. The sensitivity, specificity and positive and negative predictive values of recombinant LigB LAT were found to be 75.65, 91.27, 78.38 and 89.96 %, respectively, and that of heat-killed antigen-based LAT were 72.17, 89.82, 74.77 and 88.53 %, respectively, in comparison with MAT. This developed test will be an alternative/complementary to the existing battery of diagnostic assays/tests for specific detection of pathogenic Leptospira infection in bovine population. PMID:26065562

  8. Effects of Saturated Long-chain Fatty Acid on mRNA Expression of Genes Associated with Milk Fat and Protein Biosynthesis in Bovine Mammary Epithelial Cells

    PubMed Central

    Qi, Lizhi; Yan, Sumei; Sheng, Ran; Zhao, Yanli; Guo, Xiaoyu

    2014-01-01

    This study was conducted to determine the effects of saturated long-chain fatty acids (LCFA) on cell proliferation and triacylglycerol (TAG) content, as well as mRNA expression of αs1-casein (CSN1S1) and genes associated with lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows, and were passaged twice. Then cells were cultured with different levels of palmitate or stearate (0, 200, 300, 400, 500, and 600 μM) for 48 h and fetal bovine serum in the culture solution was replaced with fatty acid-free BSA (1 g/L). The results showed that cell proliferation tended to be increased quadratically with increasing addition of stearate. Treatments with palmitate or stearate induced an increase in TAG contents at 0 to 600 μM in a concentration-dependent manner, and the addition of 600 μM was less effective in improving TAG accumulation. The expression of acetyl-coenzyme A carboxylase alpha, fatty acid synthase and fatty acid-binding protein 3 was inhibited when palmitate or stearate were added in culture medium, whereas cluster of differentiation 36 and CSN1S1 mRNA abundance was increased in a concentration-dependent manner. The mRNA expressions of peroxisome proliferator-activated receptor gamma, mammalian target of rapamycin and signal transducer and activator of transcription 5 with palmitate or stearate had no significant differences relative to the control. These results implied that certain concentrations of saturated LCFA could stimulate cell proliferation and the accumulation of TAG, whereas a reduction may occur with the addition of an overdose of saturated LCFA. Saturated LCFA could up-regulate CSN1S1 mRNA abundance, but further studies are necessary to elucidate the mechanism for regulating milk fat and protein synthesis. PMID:25049969

  9. Bovine somatotropin and rumen-undegradable protein effects in prepubertal dairy heifers: effects on body composition and organ and tissue weights.

    PubMed

    Moallem, U; Dahl, G E; Duffey, E K; Capuco, A V; Wood, D L; McLeod, K R; Baldwin, R L; Erdman, R A

    2004-11-01

    The objectives of this study were to determine the effect of recombinant bovine somatotropin (bST) and added dietary rumen undegradable protein (RUP) on organ and tissue weights and body composition in growing dairy heifers. Thirty-two Holstein heifers were in the experiment, 8 killed initially at 3 mo of age, with the remaining 24 Holstein heifers randomly assigned to treatments (n = 6) consisting of 0.1 mg/kg of body weight per day of bST and 2% added dietary RUP (dry matter basis) applied in a 2 x2 factorial design. A total of 6 heifers per treatment group (3 each at 5 and 10 mo of age), were slaughtered to determine body composition and organ masses. Feed intake measured from group intakes were increased by 0.25 and 0.35 kg/d with bST and RUP, respectively. Administration of bST tended to increase the weights of visceral organs including heart, kidney, and spleen by 16, 16, and 38%, respectively. At 10 mo of age, there was a trend for increased empty body weights (EBW) and non-carcass components for heifers treated with bST, but there were no effects of RUP. Body components and organ weights, expressed as a percentage of BW were not affected by RUP or bST. Somatotropin increased ash weight at 10 mo without affecting amounts of protein, fat, and energy. Rates of ash deposition between 3 and 10 mo of age were increased 7 and 4 g/d by bST and RUP, respectively. There were no treatment effects on rates of body fat, protein, and energy deposition. Bovine somatotropin and RUP altered the metabolism of growing heifers in a manner that was consistent with increased rates of skeletal growth. This suggests that nutritional and endocrine manipulations could increase growth rates of skeletal tissues without increasing fat deposition in prepubertal dairy heifers. PMID:15483171

  10. Water and bovine serum lubrication compared in simulator PTFE/CoCr wear model.

    PubMed

    Good, V D; Clarke, I C; Anissian, L

    1996-01-01

    Controversy surrounds wear data from laboratory hip simulator studies, whether derived from water-based or serum-based studies or whether a major design parameter such as the size of the femoral head has an effect on the volume of wear particulate released. To investigate these relationships, we studied cup wear in water- and serum-based lubricants using as our standard the polytetrafluoroethylene (PTFE) data derived by Charnley. To model Charnley's clinical experience, PTFE acetabular cups were used in sets of three each of four sizes of CoCr femoral heads: 22.25-, 28-, 32-, and 42-mm diameters. Six criteria were used to evaluate the performance of the lubricants against clinical accuracy and scientific methods. The PTFE wear data from the serum-based tests was consistently linear with the duration of the test, exhibited a precision within +/-3% about the average for each set of three cups, and copious amounts of wear debris were clearly seen circulating and settling to the bottom of the wear chambers. The wear data clearly demonstrated Charnley's thesis that volume of wear increases with regard to the size of the femoral head in a linear manner. This increase was considered satisfactory at 9%/ mm. However, in terms of clinical accuracy, the simulator wear rates averaged 3 to 4 times greater than the comparable clinical data for wear magnitude. Thus, the serum-based tests satisfied three of the six criteria used. The PTFE wear data from the water-based tests was generally nonlinear, continually increasing with test duration. These wear trends were examined in three discrete phases to estimate the changing wear rates. By the end of the tests, the wear rates had increased from 1.3 to 3.9 times, with the 42-mm heads showing the greatest change. The resulting precision was never better than +/-26% and deteriorated to +/-70%. In terms of clinical accuracy, the water-based wear rates varied from 2 to 7 times less than the Charnley PTFE wear magnitudes, averaging 4 times less. The water-based data did not satisfactorily model the relationship between increased wear with increased head size. Minimal PTFE wear debris was observed, and what did emerge after thousands of wear cycles appeared as streamers up to 30 mm long and up to 5 mm wide. When these detached, they floated up to the surface where they could be separated into smaller particulates. A similar phenomenon was noted for polyethylene wear tests conducted with water lubrication. Thus the water-based tests satisfied none of the six validation criteria evaluated. These data raise serious doubts as to the validity of testing implant and material combinations in water as a predictor of clinical performance. Bovine serum was not totally satisfactory, but the wear data did model some of the important clinical characteristics of hip joint behavior. PMID:8953393

  11. Effect of epidermal growth factor (EGF) on the phosphorylation of mitogen-activated protein kinase (MAPK) in the bovine oviduct in vitro: Alteration by heat stress

    PubMed Central

    WIJAYAGUNAWARDANE, Missaka P. B.; HAMBRUCH, Nina; HAEGER, Jan-Dirk; PFARRER, Christiane

    2015-01-01

    Epidermal growth factor (EGF) has been shown to be involved in control of the oviductal microenvironment. To elucidate the potential mechanisms responsible for the detrimental effect of heat stress and to identify the relation with the endocrine status, the effects of EGF on the level of phosphorylated mitogen-activated-protein kinase (MAPK) and proliferation of bovine oviductal epithelial cells (OECs) exposed to different cyclic ovarian steroidal environments (luteal phase (LP), follicular phase (FP) and postovulatory phase (PO)) and temperatures (mild heat stress (40 C) and severe heat stress (43 C)) were investigated. Western blot was performed to evaluate phosphorylated MAPK, while proliferation was analyzed by MTT assay. Stimulation of OECs with EGF alone or with EGF in the PO and FP environments significantly increased the amount of phosphorylated MAPK, with MAPK 44 phosphorylation being highest during exposure to PO conditions. These effects were not observed in the LP. Heat treatment completely blocked effects of EGF on phosphorylated MAPK. Additionally, severe heat stress led to a significantly lower basal level of phosphorylated MAPK. PD98059 (MAPK inhibitor) completely abolished EGF-stimulated MAPK phosphorylation and OECs proliferation. Overall the results indicate that EGF has the potential to increase the amount of phosphorylated MAPK in OECs and therefore could be involved in regulation of the bovine oviductal microenvironment. However, these regulatory mechanisms may be compromised in the presence of heat stress (high ambient temperature), leading to low fertility rates and impaired embryo survival. PMID:26050642

  12. Deciphering Supramolecular Structures with Protein-Protein Interaction Network Modeling

    PubMed Central

    Tsuji, Toshiyuki; Yoda, Takao; Shirai, Tsuyoshi

    2015-01-01

    Many biological molecules are assembled into supramolecules that are essential to perform complicated functions in the cell. However, experimental information about the structures of supramolecules is not sufficient at this point. We developed a method of predicting and modeling the structures of supramolecules in a biological network by combining structural data of the Protein Data Bank (PDB) and interaction data in IntAct databases. Templates for binary complexes in IntAct were extracted from PDB. Modeling was attempted by assembling binary complexes with superposed shared subunits. A total of 3,197 models were constructed, and 1,306 (41% of the total) contained at least one subunit absent from experimental structures. The models also suggested 970 (25% of the total) experimentally undetected subunit interfaces, and 41 human disease-related amino acid variants were mapped onto these model-suggested interfaces. The models demonstrated that protein-protein interaction network modeling is useful to fill the information gap between biological networks and structures. PMID:26549015

  13. Adipogenesis of bovine perimuscular preadipocytes

    SciTech Connect

    Taniguchi, Masaaki; Le Luo Guan; Zhang Bing; Dodson, Michael V.; Okine, Erasmus; Moore, Stephen S.

    2008-02-01

    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.

  14. Interleukin 6 increases the in vitro expression of key proteins associated with steroidogenesis in the bovine adrenal zona fasciculata.

    PubMed

    McIlmoil, S; Strickland, J; Judd, A M

    2016-04-01

    In this study, the in vitro effects of interleukin 6 (IL-6) on the messenger RNAs (mRNAs) and proteins for key steroidogenic factors in the bovine adrenal zona fasciculata (ZF) were determined. Bovine adrenal glands were obtained from an abattoir, and the ZF was isolated. Strips of ZF were then exposed to different concentration of murine IL-6 and/or adrenocorticotropic hormone (ACTH) for various intervals, the protein and mRNA extracted, and the mRNA and protein expression determined by real-time polymerase chain reaction and Western blots. Exposure (1 h) to IL-6 increased in a concentration-dependent manner (10-pg IL-6/mL, P < 0.05 vs control; 100-pg IL-6/mL, P < 0.01 vs control) the relative expression of the mRNAs and proteins for steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), 3β hydroxysteroid dehydrogenase type 2 (3β HSD), 17α-hydroxylase/17,20-lyase/17,20-desmolase (P450 17OH), steroid 21-hydroxylase (P450 21OH), steroid 11-β-hydroxylase type 1 (P450 11βOH), and steroidogenic factor 1 (SF-1), a nuclear factor that increases StAR and steroidogenic enzymes (SEs) expression. Similarly, IL-6 (10 pg/mL) increased the relative expression of proteins and mRNAs for StAR, P450scc, 3β HSD, P450 17OH, P450 21 OH, P450 11βOH, and SF-1 in a time-dependent manner (30 min, P < 0.05 vs control; 60, 120, and 240 min, P < 0.01 vs control). In contrast, IL-6 decreased in a concentration-dependent (P < 0.01 vs control for 1, 10, and 100 pg IL-6/mL) and time-dependent (P < 0.05 vs control for 30, 60,120, and 240 min of 10 pg IL-6/mL) manner the relative expression of the mRNA and protein for adrenal hypoplasia congenita-like protein (DAX-1), a nuclear factor that decreases expression of StAR and SEs. Incubation (1 h) of ZF with 100-nM ACTH increased (P < 0.05 vs control) the relative expression of StAR, P450scc, 3β HSD, P450 17OH, P450 21OH, P450 11βOH, and SF-1 and decreased (P < 0.01 vs control) the relative expression of DAX-1. Murine IL-6 (10 pg/mL) augmented (P < 0.05 vs ACTH) both the stimulatory and inhibitory effects of ACTH. Bovine IL-6 (100 pg/mL, 1-h incubation) also increased (P < 0.01 vs control) the relative expression of the proteins for StAR, P450scc, and SF-1 and decreased (P < 0.01 vs control) the relative expression of DAX-1. In summary, IL-6 increased ZF expression of StAR and 5 SEs, which may be mediated in part by decreasing DAX-1 expression and increasing SF-1 expression. PMID:26700094

  15. A Mesoscopic Model for Protein-Protein Interactions in Solution

    PubMed Central

    Lund, Mikael; Jönsson, Bo

    2003-01-01

    Protein self-association may be detrimental in biological systems, but can be utilized in a controlled fashion for protein crystallization. It is hence of considerable interest to understand how factors like solution conditions prevent or promote aggregation. Here we present a computational model describing interactions between protein molecules in solution. The calculations are based on a molecular description capturing the detailed structure of the protein molecule using x-ray or nuclear magnetic resonance structural data. Both electrostatic and van der Waals interactions are included and the salt particles are explicitly treated allowing investigations of systems containing mono-, di-, and trivalent ions. For three different proteins—lysozyme, α-chymotrypsinogen, and calbindin D9k—we have investigated under which conditions (salt concentration, ion valency, pH, and/or solvent) the proteins are expected to aggregate via evaluation of the second virial coefficient. Good agreement is found with experimental data where available. Calbindin is investigated in more detail, and it is demonstrated how changes in solvent and/or counterion valency lead to attractive ion-ion correlation effects. For high valency counterions we have found abnormal trends in the second virial coefficient. With trivalent counterions, attraction of two negatively charged protein molecules can be favored because the repulsive term is decreased for entropic reasons due to the low number of particles present. PMID:14581196

  16. Bovine Pericardium Patch Wrapping Intestinal Anastomosis Improves Healing Process and Prevents Leakage in a Pig Model

    PubMed Central

    Testini, Mario; Gurrado, Angela; Portincasa, Piero; Scacco, Salvatore; Marzullo, Andrea; Piccinni, Giuseppe; Lissidini, Germana; Greco, Luigi; De Salvia, Maria Antonietta; Bonfrate, Leonilde; Debellis, Lucantonio; Sardaro, Nicola; Staffieri, Francesco; Carrat, Maria Rosaria; Crovace, Antonio

    2014-01-01

    Failure of intestinal anastomosis is a major complication following abdominal surgery. Biological materials have been introduced as reinforcement of abdominal wall hernia in contaminated setting. An innovative application of biological patch is its use as reinforcement of gastrointestinal anastomosis. The aim of study was to verify whether the bovine pericardium patch improves the healing of anastomosis, when in vivo wrapping the suture line of pig intestinal anastomosis, avoiding leakage in the event of deliberately incomplete suture. Forty-three pigs were randomly divided: Group 1 (control, n?=?14): hand-sewn ileo-ileal and colo-colic anastomosis; Group 2 (n?=?14): standard anastomosis wrapped by pericardium bovine patch; Group 3 (n?=?1) and 4 (n?=?14): one suture was deliberately incomplete and also wrapped by patch in the last one. Intraoperative evaluation, histological, biochemical, tensiometric and electrophysiological studies of intestinal specimens were performed at 48 h, 7 and 90 days after. In groups 2 and 4, no leak, stenosis, abscess, peritonitis, mesh displacement or shrinkage were found and adhesion rate decreased compared to control. Biochemical studies showed mitochondrial function improvement in colic wrapped anastomosis. Tensiometric evaluations suggested that the patch preserves the colic contractility similar to the controls. Electrophysiological results demonstrated that the patch also improves the mucosal function restoring almost normal transport properties. Use of pericardium bovine patch as reinforcement of intestinal anastomosis is safe and effective, significantly improving the healing process. Data of prevention of acute peritonitis and leakage in cases of iatrogenic perforation of anastomoses, covered with patch, is unpublished. PMID:24489752

  17. Bovine pericardium patch wrapping intestinal anastomosis improves healing process and prevents leakage in a pig model.

    PubMed

    Testini, Mario; Gurrado, Angela; Portincasa, Piero; Scacco, Salvatore; Marzullo, Andrea; Piccinni, Giuseppe; Lissidini, Germana; Greco, Luigi; De Salvia, Maria Antonietta; Bonfrate, Leonilde; Debellis, Lucantonio; Sardaro, Nicola; Staffieri, Francesco; Carrat, Maria Rosaria; Crovace, Antonio

    2014-01-01

    Failure of intestinal anastomosis is a major complication following abdominal surgery. Biological materials have been introduced as reinforcement of abdominal wall hernia in contaminated setting. An innovative application of biological patch is its use as reinforcement of gastrointestinal anastomosis. The aim of study was to verify whether the bovine pericardium patch improves the healing of anastomosis, when in vivo wrapping the suture line of pig intestinal anastomosis, avoiding leakage in the event of deliberately incomplete suture. Forty-three pigs were randomly divided: Group 1 (control, n = 14): hand-sewn ileo-ileal and colo-colic anastomosis; Group 2 (n = 14): standard anastomosis wrapped by pericardium bovine patch; Group 3 (n = 1) and 4 (n = 14): one suture was deliberately incomplete and also wrapped by patch in the last one. Intraoperative evaluation, histological, biochemical, tensiometric and electrophysiological studies of intestinal specimens were performed at 48 h, 7 and 90 days after. In groups 2 and 4, no leak, stenosis, abscess, peritonitis, mesh displacement or shrinkage were found and adhesion rate decreased compared to control. Biochemical studies showed mitochondrial function improvement in colic wrapped anastomosis. Tensiometric evaluations suggested that the patch preserves the colic contractility similar to the controls. Electrophysiological results demonstrated that the patch also improves the mucosal function restoring almost normal transport properties. Use of pericardium bovine patch as reinforcement of intestinal anastomosis is safe and effective, significantly improving the healing process. Data of prevention of acute peritonitis and leakage in cases of iatrogenic perforation of anastomoses, covered with patch, is unpublished. PMID:24489752

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

    NASA Astrophysics Data System (ADS)

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

    1997-01-01

    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.

  19. Protein crystal growth in microgravity review of large scale temperature induction method: Bovine insulin, human insulin and human {alpha}-interferon

    SciTech Connect

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

    1997-01-10

    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.

  20. Use of a Standardized Bovine Serum Panel To Evaluate a Multiplexed Nonstructural Protein Antibody Assay for Serological Surveillance of Foot-and-Mouth Disease?

    PubMed Central

    Perkins, Julie; Parida, Satya; Clavijo, Alfonso

    2007-01-01

    Liquid array technology has previously been used to show proof of principle of a multiplexed nonstructural protein serological assay to differentiate foot-and-mouth disease virus-infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B, and 3D and the recombinant protein signature 3ABC in combination with four controls. To determine the 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 by using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright, United Kingdom. This serum panel has been used to assess the performance of other singleplex enzyme-linked immunosorbent assay (ELISA)-based nonstructural protein antibody assays. The 3ABC signature in the multiplexed assay showed performance comparable to that of a commercially available nonstructural protein 3ABC ELISA (Cedi test), and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex was acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promote further assay development and optimization to generate an assay for routine use in foot-and-mouth disease serological surveillance. PMID:17913861

  1. Cultured C2C12 cell lines as a model for assessment of bacterial attachment to bovine primary muscle cells.

    PubMed

    Zulfakar, Siti Shahara; White, Jason D; Ross, Tom; Tamplin, Mark L

    2013-06-01

    The mechanisms of bacterial attachment to meat tissues need to be understood to enhance meat safety interventions. However, little is known about attachment of foodborne pathogens to meat muscle cells. In this study, attachment of six Escherichia coli and two Salmonella strains to primary bovine muscle cells and a cultured muscle cell line, C2C12, was measured, including the effect of temperature. At 37C, all but one strain (EC623) attached to C2C12 cells, whereas only five of eight strains (M23Sr, H10407, EC473, Sal1729a and Sal691) attached to primary cells. At 10 C, two strains (H10407 and EC473) attached to C2C12 cells, compared to four strains (M23Sr, EC614, H10407 and Sal1729a) of primary cells. Comparing all strains at both temperatures, EC614 displayed the highest CFU per C2C12 cell (4.602.02CFU/muscle cell at 37 C), whereas greater numbers of M23Sr attached per primary cell (51.8839.43CFU/muscle cell at 37 C). This study indicates that primary bovine muscle cells may provide a more relevant model system to study bacterial attachment to beef carcasses compared to cell lines such as C2C12. PMID:23501253

  2. The carboxy-terminal domain shared by the bovine papillomavirus E2 transactivator and repressor proteins contains a specific DNA binding activity.

    PubMed Central

    McBride, A A; Schlegel, R; Howley, P M

    1988-01-01

    The E2 open reading frame of bovine papilloma virus 1 (BPV-1) has been shown to encode both positive and negative acting transcriptional regulatory factors. The DNA binding properties of these factors were analysed to investigate the mechanism by which they might regulate viral gene expression. Polypeptides corresponding to the full-length E2 product and a shorter protein thought to represent the repressor function were synthesized in vitro by translation of T7 polymerase generated transcripts. Using rabbit antisera generated against synthetic peptides from the E2 open reading frame, it was possible to immunoprecipitate each of these products and show that each was capable of binding the same specific sequence located at several sites in the BPV-1 genome. This DNA binding property was mapped to a conserved carboxy-terminal domain of 101 amino acids by analysis of truncated polypeptides synthesized from the E2 open reading frame. Images PMID:2835232

  3. Reactivity of monoclonal antibodies against a tryptophan-riboflavin adduct toward irradiated and non-irradiated bovine-eye-lens protein fractions: an indicator of in vivo visible-light-mediated phototransformations.

    PubMed

    Mancini, M; Edwards, A M; Becker, M I; de Ioannes, A; Silva, E

    2000-03-01

    We describe here the reactivity toward the soluble protein of bovine eye lens of anti-tryptophan-riboflavin (anti-Trp-RF) adduct monoclonal antibodies, which recognize the hapten tryptophan-riboflavin generated by irradiation of a solution of bovine serum albumin in the presence of riboflavin. It is demonstrated that five different anti-Trp-RF adduct monoclonal antibodies, all belonging to the IgG1 isotype, react with the total soluble proteins of bovine eye lens. The components of the soluble protein are separated by Sephadex G-200 chromatography and the isolated fractions analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). All the separated protein fractions also react by a direct ELISA with the monoclonal antibodies; this reaction is more intense when the isolated fractions have been previously irradiated with visible light in the presence of riboflavin under an atmosphere of oxygen or nitrogen. Irradiation of the total soluble protein with visible light in the presence of riboflavin produces the appearance of new bands, corresponding to compounds of higher molecular weight. Riboflavin-sensitized irradiation of the protein fractions with visible light under an oxygen or nitrogen atmosphere is accompanied by a concomitant decrease of the tryptophan fluorescence. It is postulated that the action of visible light in the presence of either the endogenous riboflavin or its derivatives could be partly responsible for the protein aggregation observed during aging. PMID:10877061

  4. A proteomic study of the differential protein expression in MDBK cells after bovine herpesvirus type 1 infection (BHV-1) strain treatment

    PubMed Central

    Guo, Li; Yang, Yanling; Liu, Linna; Liao, Peng; Wen, Yongjun; Wu, Hua; Cheng, Shipeng

    2015-01-01

    Different BHV-1 strains, such as the virulent IBRV LN01/08 strains and the attenuated vaccine strain IBRV LNM, produces different clinical immune responses; however, the study of the differential protein expression in Madin-Darby bovine kidney (MDBK) cells after BHV-1-infection still remains unclear. Here, we applied a comparative proteomic strategy, based on 2D and MALDI-TOF/MS platforms, to examine the differential expression of proteins in MDBK cells that were treated and not treated with virulent IBRV LN01/08 and attenuated IBRV LNM strains. A total of eight differential proteins, including pyruvate kinase, heat shock protein (HSP) 90 (HSP90AA1 and HSP90AB1), annexin A, albumin (ALB), scinderin (SCIN), tubulin (alpha 1a) and vimentin (VIM), were identified. Among these proteins, pyruvate kinase, and HSP90 (HSP90AB1), tubulin and vimentin were identified in the virulent IBRV LN01/08 strain group, but were not identified in the attenuated IBRV LNM group. These results play an important role in tumor formation and development, cell migration, tumor cell line apoptosis, cell invasion and viral infection. The HSP90 (HSP90AA1) protein was identified in the control group and the attenuated IBRV LNM-infected group. Most studies have shown that HSP90 proteins were more of a cancer gene target, and inhibiting its function would result to oncogene degradation during cancer treatment. On the other hand, ALB is associated to cell differentiation, apoptosis, necrosis, cell death, viral infection, autophagy, interstitial tissue inflammation, and cell survival. These results provide a theoretical basis for the systematic understanding of BHV-1-infection mechanisms and BHV-1-induced immune responses. PMID:26064331

  5. A Recombinant Human Parainfluenza Virus Type 3 (PIV3) in Which the Nucleocapsid N Protein Has Been Replaced by That of Bovine PIV3 Is Attenuated in Primates

    PubMed Central

    Bailly, Jane E.; McAuliffe, Josephine M.; Durbin, Anna P.; Elkins, William R.; Collins, Peter L.; Murphy, Brian R.

    2000-01-01

    The shipping fever (SF) and Kansas (Ka) strains of bovine parainfluenza virus type 3 (BPIV3) are restricted in their replication in rhesus monkeys 100- to 1,000-fold compared to human parainfluenza virus type 3 (HPIV3), and the Ka strain also was shown to be attenuated in humans. To initiate an investigation of the genetic basis of the attenuation of BPIV3 in primates, we produced viable chimeric HPIV3 recombinants containing the nucleoprotein (N) open reading frame (ORF) from either BPIV3 Ka or SF in place of the HPIV3 N ORF. These chimeric recombinants were designated cKa-N and cSF-N, respectively. Remarkably, cKa-N and cSF-N grew to titers comparable to those of their HPIV3 and BPIV3 parents in LLC-MK2 monkey kidney and Madin-Darby bovine kidney cells. Thus, the heterologous nature of the N protein did not impede replication in vitro. However, cKa-N and cSF-N were each restricted in replication in rhesus monkeys to a similar extent as Ka and SF, respectively. This identified the BPIV3 N protein as a determinant of the host range restriction of BPIV3 in primates. These chimeras thus combine the antigenic determinants of HPIV3 with the host range restriction and attenuation phenotype of BPIV3. Despite their restricted replication in rhesus monkeys, the chimeric viruses induced a level of resistance to HPIV3 challenge in these animals which was indistinguishable from that conferred by immunization with HPIV3. The infectivity, attenuation, and immunogenicity of these BPIV3/HPIV3 chimeras suggest that the modified Jennerian approach described in the present report represents a novel method to design vaccines to protect against HPIV3-induced disease in humans. PMID:10708435

  6. Determination by photoaffinity labelling of the hydrophobic part of the binding site for acyl-CoA esters on acyl-CoA-binding protein from bovine liver.

    PubMed Central

    Hach, M; Pedersen, S N; Brchers, T; Hjrup, P; Knudsen, J

    1990-01-01

    Acyl-CoA esters containing the photoreactive acids 12-(4'-azido-2'-nitrophenoxy)[1-14C]dodecanoic acid ([14C]AND-acid) or N-(4'-azido-2'-nitro-[3'-5'-3H]phenyl)-12-aminododecanoic acid ([3H]NANPA-acid) were synthesized. The photoreactive acyl-CoA esters could be bound to bovine acyl-CoA-binding protein (ACBP) and photocrosslinked to the protein. The photocrosslinked acyl-CoA-ACBP complex was separated from unlabelled ACBP on reverse-phase h.p.l.c. and the purified complex was digested with trypsin, Staphylococcus aureus V8 proteinase or endoproteinase Asp-N. By four independent peptide maps it was shown that the amino acids taking part in forming the hydrophobic binding site for acyl-CoA esters in bovine ACBP are located on the peptide segment from Asp21 to Asp38. Both photoreactive acyl-CoA esters used in this study labelled strongly in the segment from Tyr28 to Ala34. 12-(4'-Azido-2'-nitrophenoxy)[1-14C]-dodecanoyl-CoA ([14C]AND-CoA) also introduced a label at position Asp38, but o labelling was found before Ser29. In contrast, N-(4'-azido-2'-nitro[3',5'-3H]phenyl)-12-aminododecanoyl-CoA [3H]NANPA-CoA) also labelled the segment from Asp21 to Tyr28. The difference in labelling by the two photoreactive ligands is most likely caused by different mobility of the arylazido group when linked to the fatty acid either through a phenolic O- or an anilinic N- bond. PMID:2222414

  7. Phosphoproteome analysis of sarcoplasmic and myofibrillar proteins in bovine longissimus muscle in response to postmortem electrical stimulation.

    PubMed

    Li, Chunbao; Zhou, Guanghong; Xu, Xinglian; Lundström, Kerstin; Karlsson, Anders; Lametsch, René

    2015-05-15

    Protein phosphorylation changes of the sarcoplasmic and myofibrillar proteins in beef longissimus muscle in response to electrical stimulation (ES) was investigated. Sarcoplasmic and myofibrillar proteins purified from muscle samples taken at 0, 3 and 10h after ES were separated on SDS-PAGE and stained with phosphorous and protein specific stains. There was a significant effect of ES on phosphorylation of total sarcoplasmic and myofibrillar proteins (P<0.05). However, although there an instant effect of ES on the phosphorylation level of the myofibrillar proteins, the ES effect on the sarcoplasmic proteins (P<0.05) was first observed after 3h. Several protein bands were analyzed by LC-MS/MS, revealing that the major glycolytic proteins, including glycogen debranching enzyme, glycogen phosphorylase and 6-phosphofructokinase probably are affected by ES together with different heat shock proteins. This work gives an insight into the regulation of the glycolytic enzymes and muscle contraction on application of electrical stimulation. PMID:25577070

  8. Evaluation of an animal model system for cryptosporidiosis: therapeutic efficacy of paromomycin and hyperimmune bovine colostrum-immunoglobulin.

    PubMed Central

    Tzipori, S; Rand, W; Griffiths, J; Widmer, G; Crabb, J

    1994-01-01

    Several immunodeficient rodent models currently exist in which persistent, largely asymptomatic, Cryptosporidium parvum infections can be established. Piglets, in contrast, develop a self-limiting diarrheal illness. We have consequently developed an animal model system in which scid mice were used to screen drugs for inhibitory activity against C. parvum, after which the drugs' therapeutic potential was evaluated with piglets. Paromomycin and hyperimmune bovine colostrum-immunoglobulin were selected to evaluate this system. C. paravum infections in suckling scid mice tended to be associated with villus surfaces, while in weaned and in older scid mice infections were more commonly localized in abscessed crypts. Rates of oocyst shedding in suckling scid mice were 50 to 200 times higher than in weaned mice and therefore made suckling mice a considerably more sensitive model for drug testing. Paromomycin given in high doses over 9 to 10 days was not toxic to either scid mice (3,000 mg/kg of body weight per day) or piglets (500 mg/kg/day). Paromomycin treatment was very effective against villus surface infections in suckling mice and considerably less effective against infections in inaccessible sites such as abscessed crypts and stomach pits seen in weaned and adult scid mice. The therapeutic efficacy of paromomycin in piglets depended on the severity of the diarrheal illness. Mild to moderate diarrhea and infection were cleared after paromomycin treatment of piglets infected with one C. parvum isolate. However, paromomycin had no impact on severely affected piglets infected with a second isolate, presumably because of a rapid transit time through the gut. In contrast to paromomycin hyperimmune bovine colostrum-immunoglobulin treatment reduced the rate of C. parvum infection moderately in scid mice and only slightly in piglets, again probably because of a rapid transit time through the gut and inactivation in the stomach. It was also clear that the impact of effective drugs against C. parvum can be detected within 5 days after the onset of treatment in either model. Images PMID:8556484

  9. 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.

    PubMed

    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

    2011-12-01

    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 μM, P = 0.017; 10 μM, P = 0.001; 25 μM, P = 0.008; and 50 μM, 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

  10. Conserved Cysteine Residue in the DNA-Binding Domain of the Bovine Papillomavirus Type 1 E2 Protein Confers Redox Regulation of the DNA- Binding Activity in Vitro

    NASA Astrophysics Data System (ADS)

    McBride, Alison A.; Klausner, Richard D.; Howley, Peter M.

    1992-08-01

    The bovine papillomavirus type 1 E2 open reading frame encodes three proteins involved in viral DNA replication and transcriptional regulation. These polypeptides share a carboxyl-terminal domain with a specific DNA-binding activity; through this domain the E2 polypeptides form dimers. In this study, we demonstrate the inhibition of E2 DNA binding in vitro by reagents that oxidize or otherwise chemically modify the free sulfydryl groups of reactive cysteine residues. However, these reagents had no effect on DNA-binding activity when the E2 polypeptide was first bound to DNA, suggesting that the free sulfydryl group(s) may be protected by DNA binding. Sensitivity to sulfydryl modification was mapped to a cysteine residue at position 340 in the E2 DNA-binding domain, an amino acid that is highly conserved among the E2 proteins of different papillomaviruses. Replacement of this residue with other amino acids abrogated the sensitivity to oxidation-reduction changes but did not affect the DNA-binding property of the E2 protein. These results suggest that papillomavirus DNA replication and transcriptional regulation could be modulated through the E2 proteins by changes in the intracellular redox environment. Furthermore, a motif consisting of a reactive cysteine residue carboxyl-terminal to a lysine residue in a basic region of the DNA-binding domain is a feature common to a number of transcriptional regulatory proteins that, like E2, are subject to redox regulation. Thus, posttranslational regulation of the activity of these proteins by the intracellular redox environment may be a general phenomenon.

  11. Evaluation of protein-protein docking model structures using all-atom molecular dynamics simulations combined with the solution theory in the energy representation

    NASA Astrophysics Data System (ADS)

    Takemura, Kazuhiro; Guo, Hao; Sakuraba, Shun; Matubayasi, Nobuyuki; Kitao, Akio

    2012-12-01

    We propose a method to evaluate binding free energy differences among distinct protein-protein complex model structures through all-atom molecular dynamics simulations in explicit water using the solution theory in the energy representation. Complex model structures are generated from a pair of monomeric structures using the rigid-body docking program ZDOCK. After structure refinement by side chain optimization and all-atom molecular dynamics simulations in explicit water, complex models are evaluated based on the sum of their conformational and solvation free energies, the latter calculated from the energy distribution functions obtained from relatively short molecular dynamics simulations of the complex in water and of pure water based on the solution theory in the energy representation. We examined protein-protein complex model structures of two protein-protein complex systems, bovine trypsin/CMTI-1 squash inhibitor (PDB ID: 1PPE) and RNase SA/barstar (PDB ID: 1AY7), for which both complex and monomer structures were determined experimentally. For each system, we calculated the energies for the crystal complex structure and twelve generated model structures including the model most similar to the crystal structure and very different from it. In both systems, the sum of the conformational and solvation free energies tended to be lower for the structure similar to the crystal. We concluded that our energy calculation method is useful for selecting low energy complex models similar to the crystal structure from among a set of generated models.

  12. Modeling protein synthesis from a physicist's perspective: A toy model

    NASA Astrophysics Data System (ADS)

    Basu, Aakash; Chowdhury, Debashish

    2007-10-01

    Proteins are polymers of amino acids. These macromolecules are synthesized by intracellular machines called ribosomes. Although the experimental investigation of protein synthesis has been a traditional area of research in molecular cell biology, important quantitative models of protein synthesis have been reported in research journals devoted to statistical physics and related interdisciplinary topics. From the perspective of a physicist, protein synthesis is the classical transport of interacting ribosomes on a messenger RNA (mRNA) template that dictates the sequence of the amino acids on the protein. We discuss appropriate simplification of the models and methods. In particular, we develop and analyze a simple toy model using some elementary techniques of nonequilibrium statistical mechanics and predict the average rate of protein synthesis and the spatial organization of the ribosomes in the steady state.

  13. Choline and acetylcholine detection based on peroxidase-like activity and protein antifouling property of platinum nanoparticles in bovine serum albumin scaffold.

    PubMed

    He, Shao-Bin; Wu, Gang-Wei; Deng, Hao-Hua; Liu, Ai-Lin; Lin, Xin-Hua; Xia, Xing-Hua; Chen, Wei

    2014-12-15

    Platinum nanoparticles (PtNPs) in the scaffold of bovine serum albumin (BSA) through biomineralization are found to possess excellent peroxidase-like activity that can catalyze N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS) coupled with 4-amino-antipyrine (4-AAP) by the action of hydrogen peroxide to give an obvious purple product. Based on this phenomenon, acetylcholinesterase (AChE) and choline oxidase (ChOx) are used to catalyze ACh and choline to form the active product H2O2 and the as-produced H2O2 is detected optically. Owning to the protection effect of the protein shell, BSA-PtNPs turn out to be very stable and preserve the catalytic activity in the presence of protein and even in the real plasma samples. This protein antifouling property makes the BSA-PtNPs suitable for a wide range of applications in sensors for biological samples. Choline in infant formula and ACh in plasma have been successfully detected. PMID:25038538

  14. Cloning of the Bovine Immunodeficiency Virus gag Gene and Development of a Recombinant-Protein-Based Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Zheng, Ling; Swanson, Michelle; Liao, Jinghua; Wood, Charles; Kapil, Sanjay; Snider, Ron; Loughin, Thomas A.; Minocha, Harish C.

    2000-01-01

    An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific bovine immunodeficiency virus (BIV) antibodies in cattle, using recombinant Gag protein as an antigen. The gag coding region from BIV was cloned into an expression vector, pQE32, which expressed high levels of recombinant protein from Escherichia coli. The ELISA was standardized by a checkerboard titration against known BIV-positive and -negative sera from cattle and a monoclonal antibody to the Gag protein. A total of 139 cattle serum samples, from the diagnostic laboratory at Kansas State University, Manhattan, and from the Dairy Station, Louisiana State University, Baton Rouge, were compared by ELISA and immunoblot assays for the detection of BIV-specific antibodies. Of 26 cattle sera samples which tested positive using the immunoblot assay, 23 were positive by ELISA, thus establishing a strong correlation between the two tests. The sensitivity and specificity of ELISA relative to immunoblotting were 0.88 and 0.93, respectively. ELISA proved to be as specific as immunoblotting but was much less time-consuming and easier to perform. PMID:10882651

  15. Antibody response to Brucella outer membrane proteins in bovine brucellosis: immunoblot analysis and competitive enzyme-linked immunosorbent assay using monoclonal antibodies.

    PubMed Central

    Cloeckaert, A; Kerkhofs, P; Limet, J N

    1992-01-01

    Sera from Brucella-infected bovines were analyzed by immunoblotting by using sonicated cell extracts of B. melitensis or B. abortus and a competitive enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies against outer membrane proteins (OMPs) with molecular masses of 10, 16.5, 19, 25 to 27, 36 to 38, and 89 kDa. Antibody responses against OMPs were compared with antibody responses against smooth lipopolysaccharide. Immunoblot analysis indicated that the antibody response in infected animals was largely different from one animal to another. The antigens of concern were OMPs with molecular masses of 10, 16.5, 19, 25 to 27, 36 to 38, and 89 kDa and other proteins with molecular masses of between 40 and 80 kDa. According to the specificity of the competitive ELISA, OMPs useful for the detection of infected animals are the OMPs of 10, 16.5, 19, 25 to 27, and 36 to 38 kDa. A competitive ELISA with the anti-89 kDa monoclonal antibody was not specific. Results of the competitive ELISA confirmed the individual variability of the humoral immune response against OMPs. It therefore seems that a combination of several protein antigens is necessary for the development of an immunoassay with a sensitivity comparable to that of the smooth lipopolysaccharide ELISA. Images PMID:1452700

  16. Theoretical model to investigate the alkyl chain and anion dependent interactions of gemini surfactant with bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Vishvakarma, Vijay K.; Kumari, Kamlesh; Patel, Rajan; Dixit, V. S.; Singh, Prashant; Mehrotra, Gopal K.; Chandra, Ramesh; Chakrawarty, Anand Kumar

    2015-05-01

    Surfactants are used to prevent the irreversible aggregation of partially refolded proteins and they also assist in protein refolding. We have reported the design and screening of gemini surfactant to stabilize bovine serum albumin (BSA) with the help of computational tool (iGEMDOCK). A series of gemini surfactant has been designed based on bis-N-alkyl nicotinate dianion via varying the alkyl group and anion. On changing the alkyl group and anion of the surfactant, the value of Log P changes means polarity of surfactant can be tuned. Further, the virtual screening of the gemini surfactant has been carried out based on generic evolutionary method. Herein, thermodynamic data was studied to determine the potential of gemini surfactant as BSA stabilizer. Computational tools help to find out the efficient gemini surfactant to stabilize the BSA rather than to use the surfactant randomly and directionless for the stabilization. It can be confirmed through the experimental techniques. Previously, researcher synthesized one of the designed and used gemini surfactant to stabilize the BSA and their interactions were confirmed through various techniques and computational docking. But herein, the authors find the most competent gemini surfactant to stabilize BSA using computational tools on the basis of energy score. Different from the single chain surfactant, the gemini surfactants exhibit much stronger electrostatic and hydrophobic interactions with the protein and are thus effective at much lower concentrations. Based on the present study, it is expected that gemini surfactants may prove useful in the protein stabilization operations and may thus be effectively employed to circumvent the problem of misfolding and aggregation.

  17. Strain activation of bovine aortic smooth muscle cell proliferation and alignment: study of strain dependency and the role of protein kinase A and C signaling pathways

    NASA Technical Reports Server (NTRS)

    Mills, I.; Cohen, C. R.; Kamal, K.; Li, G.; Shin, T.; Du, W.; Sumpio, B. E.

    1997-01-01

    Smooth muscle cell (SMC) phenotype can be altered by physical forces as demonstrated by cyclic strain-induced changes in proliferation, orientation, and secretion of macromolecules. However, the magnitude of strain required and the intracellular coupling pathways remain ill defined. To examine the strain requirements for SMC proliferation, we selectively seeded bovine aortic SMC either on the center or periphery of silastic membranes which were deformed with 150 mm Hg vacuum (0-7% center; 7-24% periphery). SMC located in either the center or peripheral regions showed enhanced proliferation compared to cells grown under the absence of cyclic strain. Moreover, SMC located in the center region demonstrated significantly (P < 0.005) greater proliferation as compared to those in the periphery. In contrast, SMC exposed to high strain (7-24%) demonstrated alignment perpendicular to the strain gradient, whereas SMC in the center (0-7%) remained aligned randomly. To determine the mechanisms of these phenomena, we examined the effect of cyclic strain on bovine aortic SMC signaling pathways. We observed strain-induced stimulation of the cyclic AMP pathway including adenylate cyclase activity and cyclic AMP accumulation. In addition, exposure of SMC to cyclic strain caused a significant increase in protein kinase C (PKC) activity and enzyme translocation from the cytosol to a particulate fraction. Further study was conducted to examine the effect of strain magnitude on signaling, particularly protein kinase A (PKA) activity as well as cAMP response element (CRE) binding protein levels. We observed significantly (P < 0.05) greater PKA activity and CRE binding protein levels in SMC located in the center as compared to the peripheral region. However, inhibition of PKA (with 10 microM Rp-cAMP) or PKC (with 5-20 ng/ml staurosporine) failed to alter either the strain-induced increase in SMC proliferation or alignment. These data characterize the strain determinants for activation of SMC proliferation and alignment. Although strain activated both the AC/cAMP/PKA and the PKC pathways in SMC, singular inhibition of PKA and PKC failed to prevent strain-induced alignment and proliferation, suggesting either their lack of involvement or the multifactorial nature of these responses.

  18. A network model to correlate conformational change and the impedance spectrum of single proteins

    NASA Astrophysics Data System (ADS)

    Alfinito, Eleonora; Pennetta, Cecilia; Reggiani, Lino

    2008-02-01

    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, and the enzyme acetylcholinesterase (AChE), whose inhibition is one of the most qualified treatments of Alzheimer's disease. Both these proteins exert their function starting with a conformational change of their native structure. Our guess is that such a change should be accompanied with a detectable variation of their electrical properties. To investigate this conjecture, we present an impedance network model of proteins, able to estimate the different impedance spectra associated with the different configurations. The distinct types of conformational change of rhodopsin and AChE agree with their dissimilar electrical responses. In particular, for rhodopsin the model predicts variations of the impedance spectra up to about 30%, while for AChE the same variations are limited to about 10%, which supports the existence of a dynamical equilibrium between its native and complexed states.

  19. Hyperoxia-induced ciliary loss and oxidative damage in an in vitro bovine model: The protective role of antioxidant vitamins E and C

    SciTech Connect

    Al-Shmgani, Hanady S.; Moate, Roy M.; Sneyd, J. Robert; Macnaughton, Peter D.; Moody, A. John

    2012-12-14

    Highlights: Black-Right-Pointing-Pointer A new bovine bronchial model for studying hyperoxia-induced cilia loss is presented. Black-Right-Pointing-Pointer Hyperoxia-induced cilia loss was associated with increased sloughing of cells. Black-Right-Pointing-Pointer Hyperoxia led to higher epithelial glutathione levels, evidence of oxidative stress. Black-Right-Pointing-Pointer Hyperoxia led to increased DNA damage (Comet), and lipid peroxidation (TBARS). Black-Right-Pointing-Pointer Vitamins C and E partially protected against hyperoxia-induced cilia loss. -- Abstract: Although elevated oxygen fraction is used in intensive care units around the world, pathological changes in pulmonary tissue have been shown to occur with prolonged exposure to hyperoxia. In this work a bovine bronchus culture model has been successfully used to evaluate the effects of hyperoxia on ciliated epithelium in vitro. Samples were cultured using an air interface method and exposed to normoxia, 21% O{sub 2} or hyperoxia, 95% O{sub 2}. Cilial coverage was assessed using scanning electron microscopy (SEM). Tissue damage (lactate dehydrogenase, LDH, in the medium), lipid peroxidation (thiobarbituric acid reactive substances, TBARS), DNA damage (comet assay), protein oxidation (OxyBlot kit) and antioxidant status (total glutathione) were used to assess whether the hyperoxia caused significant oxidative stress. Hyperoxia caused a time-dependent decline (t{sub Vulgar-Fraction-One-Half} = 3.4 d compared to 37.1 d under normoxia) in cilial coverage (P < 0.0001). This was associated with a significant increase in the number of cells (2.80 {+-} 0.27 Multiplication-Sign 10{sup 6} compared to 1.97 {+-} 0.23 Multiplication-Sign 10{sup 6} ml{sup -1} after 6 d), many apparently intact, in the medium (P < 0.05); LDH release (1.06 {+-} 0.29 compared to 0.83 {+-} 0.36 {mu}mol min{sup -1} g{sup -1} after 6 d; P < 0.001); lipid peroxidation (352 {+-} 16 versus 247 {+-} 11 {mu}mol MDA g{sup -1} for hyperoxia and normoxia, respectively); % tail DNA (18.7 {+-} 2.2 versus 11.1 {+-} 1.5); protein carbonyls (P < 0.05); and total glutathione (229 {+-} 20 {mu}mol g{sup -1} versus 189 {+-} 15 {mu}mol g{sup -1}). Vitamins E (10{sup -7} M) and C (10{sup -6} or 10{sup -7} M) alone or in combination (10{sup -7} M and 10{sup -6} M, respectively) had a significant protective effect on the hyperoxia-induced reduction in percentage cilial coverage (P < 0.05). In conclusion, hyperoxia caused damage to cultured bovine bronchial epithelium and denudation of cilia. The antioxidant vitamins E and C significantly protected against hyperoxia-induced cilia loss.

  20. Passive oral immunization with bovine immunoglobulins: enterpathogenic Escherichia coli from infants and bovine anti-E. coli lactoserum assayed in the rabbit ileal loop model.

    PubMed

    Zinkernagel, R M; Colombini, A

    1975-12-30

    The effect of immune bovine lactoserum (BLS) antipolyvalent enteropathogenic Escherichia coli on bacterial growth, viability and bacteria-induced fluid accumulation was examined in rabbit ileal loops. Human enteropathogenic E. coli strains 0125:K70 (B15), 0111:K58 (B4) and 055:K59 (B5) (1-3 X 10(9) per inoculum) induced secretion of 4-6 ml fluid per 10 cm loop. This effect was inhibited effectively by BLS (corresponding to 50 mg IgG 1 per loop) while the viability of bacteria counts decreased 2-25 fold compared with bovine serum albumin. E. coli 026:K60 (B6), 0126:K71 (B16) and 0127:K63 (B8) caused moderate secretion (2-3 ml/10 cm loop) that was significantly neutralized by BLS. E. coli 086:K61 (B7) and 0128:K67 (B12) did not give positive loops. The fluid secretion was shown to be dose dependent for E. coli 0125:K70 (B15) over the range from 2.5 X 10(9) to 8 X 10(7) bacteria/loop. The titration of the effect of BLS on fluid secretion caused by the same strain revealed a dose dependent decrease. The best inhibition was obtained with 100 mg BLS/loop, the highest dose tested. PMID:765710

  1. Pathogenicity of yeasts and algae isolated from bovine mastitis secretions in a murine model.

    PubMed

    Jensen, H E; Aalbaek, B

    1994-01-01

    The pathogenicity of strains of yeasts and algae isolated from bovine mastitis secretions was evaluated in immunosuppressed mice. Strains of Candida tropicalis (n = 3) were the most pathogenic, but all strains of Geotrichum capitatum (n = 5) and the colourless alga Prototheca zopfii (n = 5) were also lethal to mice at the highest dose of 1 x 10(7) CFU per mouse. Reisolation of the inoculated micro-organisms and the occurrence of histopathological lesions within organs of mice challenged with 1 x 10(3) to 1 x 10(7) CFU per animal revealed strains of C. krusei (n = 17), C. kefyr (n = 6) and C. rugosa (n = 6) to have a moderate pathogenicity, whereas strains of C. valida (n = 1) and C. catenulata (n = 2) were weakly pathogenic and non-pathogenic respectively. PMID:7845414

  2. Analysis of Biobanked Serum from a Mycobacterium avium subsp paratuberculosis Bovine Infection Model Confirms the Remarkable Stability of Circulating miRNA Profiles and Defines a Bovine Serum miRNA Repertoire

    PubMed Central

    Riepema, Karel; Bakker, Douwe; Gordon, Stephen V.

    2015-01-01

    Johne’s Disease (JD) is a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Current disease control strategies are hampered by the lack of sensitive and specific diagnostic modalities. Therefore, novel diagnostic and prognostic tools are needed, and circulating microRNAs (miRNAs) may hold potential in this area. The aims of this study were twofold: (i) to address the stability of miRNA in bovine sera from biobanked samples, and (ii) to assess the potential of miRNAs as biomarkers for JD disease progression. To address these aims we used bovine sera from an experimental MAP infection model that had been stored at -20°C for over a decade, allowing us to also assess the stability of miRNA profiles in biobanked serum samples through comparison with fresh sera. Approximately 100–200 intact miRNAs were identified in each sample with 83 of these being consistently detected across all 57 samples. The miRNA profile of the biobanked sera stored at -20°C for over 10 years was highly similar to the profile of <1 year-old sera stored at -80°C, with an overlap of 73 shared miRNAs. IsomiR analysis also indicated a distinct bovine serum-specific isomiR profile as compared to previously reported bovine macrophage miRNA profiles. To explore the prognostic potential of miRNA profiles cattle defined as seropositive for anti-MAP antibodies (n = 5) were compared against seronegative cattle (n = 7). No significant differential expressed miRNAs were detected at either the early (6 months) or late (43, 46 and 49 months) intervals (FDR≤0.05, fold-change≥1.5) across seropositive or seronegative animals. However, comparing pre-infection sera to the early and late time-points identified increased miR-29a and miR-92b abundance (2-fold) that may be due to blood-cell population changes over time (P<0.001). In conclusion our study has demonstrated that bovine circulating miRNAs retain their integrity under long-term sub-optimal storage temperatures opening the way for increased miRNA analyses from biobanked samples for a range of infectious and non-infectious diseases. PMID:26675426

  3. Associations of a polymorphic AP-2 binding site in the 5'-flanking region of the bovine beta-lactoglobulin gene with milk proteins.

    PubMed

    Kuss, A W; Gogol, J; Geidermann, H

    2003-06-01

    Studies on a polymorphic position (R10) in an Activator-Protein-2 (AP-2) binding site of the bovine beta-Lactoglobulin (beta-Lg) gene promoter region and quantitative traits of individual milk proteins were based on material from 79 German Holstein Friesian (HF) and 61 Simmental (Sm) cows. At least four milk samples per cow were analyzed with alkaline Urea-PAGE in combination with densitometry for quantification of individual milk proteins. The two alleles of the R10 single nucleotide polymorphism (SNP) carry either G or C in position -435 bp of the beta-Lg promoter region. G- and C-alleles were found in Sm with nearly equal frequencies, while in HF the C-allele frequency was higher (0.73) than that of the G-allele. In both breeds, the R10 G-homozygotes had higher (P < 0.001) amounts of beta-Lg secreted per day and proportion of beta-Lg in milk protein compared with the C-homozygotes. A similar association was found for alpha-lactalbumin, whereas the relative proportions and daily secreted amounts of caseins (alphaS1, beta, kappa) showed lower values in beta-Lg R10 G-homozygotes. A positive association (P < 0.001) of R10 CC with milk yield has also been observed and indicates a close proximity of the beta-Lg locus to a candidate gene for this trait. The association between the SNP in the AP-2 binding site of the beta-Lg gene and its gene product can be explained as the result of differences in protein binding activity, and, therefore, allele specific differences in gene expression. Thus, our study clearly links a DNA polymorphism of molecular function very closely with in vivo expression parameters of the same locus. PMID:12836958

  4. Mathematical analysis of a model for the growth of the bovine corpus luteum.

    PubMed

    Prokopiou, Sotiris A; Byrne, Helen M; Jeffrey, Mike R; Robinson, Robert S; Mann, George E; Owen, Markus R

    2014-12-01

    The corpus luteum (CL) is an ovarian tissue that grows in the wound space created by follicular rupture. It produces the progesterone needed in the uterus to maintain pregnancy. Rapid growth of the CL and progesterone transport to the uterus require angiogenesis, the creation of new blood vessels from pre-existing ones, a process which is regulated by proteins that include fibroblast growth factor 2 (FGF2). In this paper we develop a system of time-dependent ordinary differential equations to model CL growth. The dependent variables represent FGF2, endothelial cells (ECs), luteal cells, and stromal cells (like pericytes), by assuming that the CL volume is a continuum of the three cell types. We assume that if the CL volume exceeds that of the ovulated follicle, then growth is inhibited. This threshold volume partitions the system dynamics into two regimes, so that the model may be classified as a Filippov (piecewise smooth) system. We show that normal CL growth requires an appropriate balance between the growth rates of luteal and stromal cells. We investigate how angiogenesis influences CL growth by considering how the system dynamics depend on the dimensionless EC proliferation rate, ??. We find that weak (low ??) or strong (high ??) angiogenesis leads to 'pathological' CL growth, since the loss of CL constituents compromises progesterone production or delivery. However, for intermediate values of ??, normal CL growth is predicted. The implications of these results for cow fertility are also discussed. For example, inadequate angiogenesis has been linked to infertility in dairy cows. PMID:24337679

  5. The Escherichia coli O157:H7 cattle immunoproteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine rectoanal junction squamous epithelial (RSE) cells.

    PubMed

    Kudva, Indira T; Krastins, Bryan; Torres, Alfredo G; Griffin, Robert W; Sheng, Haiqing; Sarracino, David A; Hovde, Carolyn J; Calderwood, Stephen B; John, Manohar

    2015-06-01

    Building on previous studies, we defined the repertoire of proteins comprising the immunoproteome (IP) of Escherichia coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (O157 IP), a β-adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics-based platform proteome mining tool for antigen discovery, called "proteomics-based expression library screening" (PELS; Kudva et al., 2006). The E. coli O157 IP (O157-IP) comprised 91 proteins, and included those identified previously using proteomics-based expression library screening, and also proteins comprising DMEM and bovine rumen fluid proteomes. Outer membrane protein A (OmpA), a common component of the above proteomes, and reportedly a contributor to E. coli O157 adherence to cultured HEp-2 epithelial cells, was interestingly found to be a modulator rather than a contributor to E. coli O157 adherence to bovine rectoanal junction squamous epithelial cells. Our results point to a role for yet to be identified members of the O157-IP in E. coli O157 adherence to rectoanal junction squamous epithelial cells, and additionally implicate a possible role for the outer membrane protein A regulator, TdcA, in the expression of such adhesins. Our observations have implications for the development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract. PMID:25643951

  6. Modelling bovine babesiosis: a tool to simulate scenarios for pathogen spread and to test control measures for the disease.

    PubMed

    Hoch, Thierry; Goebel, Julien; Agoulon, Albert; Malandrin, Laurence

    2012-09-15

    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

  7. MicroRNA regulation of bovine monocyte inflammatory and metabolic networks in an in vivo infection model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine mastitis is an inflammation-driven disease of the bovine mammary gland that costs the global dairy industry several billion dollars per annum. Because disease susceptibility is a multi-factorial complex phenotype, a multi-omic integrative biology approach is required to dissect the multilayer...

  8. Modelling of DNA-protein recognition

    NASA Technical Reports Server (NTRS)

    Rein, R.; Garduno, R.; Colombano, S.; Nir, S.; Haydock, K.; Macelroy, R. D.

    1980-01-01

    Computer model-building procedures using stereochemical principles together with theoretical energy calculations appear to be, at this stage, the most promising route toward the elucidation of DNA-protein binding schemes and recognition principles. A review of models and bonding principles is conducted and approaches to modeling are considered, taking into account possible di-hydrogen-bonding schemes between a peptide and a base (or a base pair) of a double-stranded nucleic acid in the major groove, aspects of computer graphic modeling, and a search for isogeometric helices. The energetics of recognition complexes is discussed and several models for peptide DNA recognition are presented.

  9. Screening of Mycobacterium avium subsp. paratuberculosis mutants for attenuation in a bovine monocyte-derived macrophage model.

    PubMed

    Lamont, Elise A; Talaat, Adel M; Coussens, Paul M; Bannantine, John P; Grohn, Yrjo T; Katani, Robab; Li, Ling-ling; Kapur, Vivek; Sreevatsan, Srinand

    2014-01-01

    Vaccination remains a major tool for prevention and progression of Johne's disease, a chronic enteritis of ruminants worldwide. Currently there is only one licensed vaccine within the United States and two vaccines licensed internationally against Johne's disease. All licensed vaccines reduce fecal shedding of Mycobacterium avium subsp. paratuberculosis (MAP) and delay disease progression. However, there are no available vaccines that prevent disease onset. A joint effort by the Johne's Disease Integrated Program (JDIP), a USDA-funded consortium, and USDA-APHIS/VS sought to identify transposon insertion mutant strains as vaccine candidates in part of a three phase study. The focus of the Phase I study was to evaluate MAP mutant attenuation in a well-defined in vitro bovine monocyte-derived macrophage (MDM) model. Attenuation was determined by colony forming unit (CFUs) counts and slope estimates. Based on CFU counts alone, the MDM model did not identify any mutant that significantly differed from the wild-type control, MAP K-10. Slope estimates using mixed models approach identified six mutants as being attenuated. These were enrolled in protection studies involving murine and baby goat vaccination-challenge models. MDM based approach identified trends in attenuation but this did not correlate with protection in a natural host model. These results suggest the need for alternative strategies for Johne's disease vaccine candidate screening and evaluation. PMID:25072030

  10. Evaluation of reference genes for quantitative real-time PCR to investigate protein disulfide isomerase transcription pattern in the bovine lungworm Dictyocaulus viviparus.

    PubMed

    Strube, Christina; Buschbaum, Sandra; Wolken, Sonja; Schnieder, Thomas

    2008-12-01

    Quantitative real-time PCR (qPCR) is the most sensitive technique for transcript quantification provided that gene transcription patterns are normalized to an evaluated reference gene. For Dictyocaulus viviparus, the housekeeping genes beta-tubulin, beta-actin, elongation factor 1alpha (ef-1alpha), glyceraldehyde-3-phosphatase dehydrogenase (gapdh), and 60S ribosomal protein L37a (60S rpL37a) were characterized and evaluated. Evaluation using the geNorm software revealed ef-1alpha and beta-tubulin as the most suitable reference genes, whereas the coefficient of variation approach resulted in ef-1alpha and 60S rpL37a as transcripts with the least variation among 12 developmental lungworm stages. The critical influence of reference genes on qPCR data analysis, with the possible consequence of erroneous, misleading results due to inappropriate reference genes used for data normalization, is shown for protein disulfide isomerase 2 (pdi-2) transcription patterns. Proper normalization of pdi-2 transcription using ef-1alpha and beta-tubulin as reference genes resulted in a more than 7-fold enriched pdi-2 transcription in L1 compared to that in eggs, and a dramatic decrease in L3. Following an increase in the L5 stage there is again a decrease of pdi-2 transcription in adult lungworms. These fluctuations in the transcription levels reflect the requirement of cuticule collagen during bovine lungworm development. PMID:18761062

  11. Facile synthesis of nano-sized agarose based amino acid-Its pH-dependent protein-like behavior and interactions with bovine serum albumin.

    PubMed

    Chudasama, Nishith A; Siddhanta, A K

    2015-11-19

    In a facile synthesis agarose was amphoterically functionalized to afford nano-sized agarose amino acids, aminoagarose succinate half-esters (AAE) containing one pendant carboxyl group. Nano-sized AAEs (<10 nm; DLS) were characterized and they had three various degrees of substitution [overall DSs 0.88, 0.89 and 0.96], both the amino and half-ester groups were placed on C-6 positions of the 1,3 beta-d-galactopyranose moieties of agarose backbone ((13)C NMR). AAEs performed like large protein molecules exhibiting pH-responsive structural variations (optical rotatory dispersion), presenting a mixed solubility pattern like random coil (soluble) and aggregate (precipitation) formations. Circular dichroism studies showed their pH-dependent associative interactions with bovine serum albumin, which indicated complexation at acidic and basic pHs, and decomplexation at pH 6.8 with AAE (DS 0.96). Thus, these nano-sized AAE based systems may be of potential utility in the domains demanding the merits of preferential protein bindings e.g. pH-responsive cationic/anionic drug carrier, separations or chiral sensing applications. PMID:26413976

  12. Expression and purification of the 26 kDa periplasmic protein of Brucella abortus: a reagent for the diagnosis of bovine brucellosis.

    PubMed

    Kumar, Sanjay; Tuteja, Urmil; Kumar, Ashok; Batra, Harsh Vardhan

    2008-03-01

    Development of a single diagnostic test for brucellosis in animals is the top priority of present-day research in the field. There is currently a battery of serological tests relying mainly on the use of LPS (lipopolysaccharide) as an antigen, culminating in false positives due to serological cross-reactivity. Other problems include difficulties in antigen production and the associated biohazard risk. This has prompted the need to develop an alternative antigen to replace LPS. In the present study, we cloned and expressed a BP26 (26 kDa periplasmic protein) antigen gene (bp26) of Brucella abortus. The recombinant periplasmic protein [rBP26 (recombinant BP26)] was expressed to high levels in Escherichia coli and purified in a single step. The purified rBP26 was examined for its binding activity with antibodies in a serum derived from a rabbit immunized intramuscularly with whole-cell lysate of B. abortus, as well as with commercial Brucella antibody (Difco). The purified rBP26 was used to develop an in-house plate ELISA and was further tested with a panel of 75 bovine brucellosis sera samples characterized previously by conventional serological tests. The results of both were in excellent agreement. The results show that rBP26 has potential use in the diagnosis of brucellosis, both in the laboratory and in field-based conditions with high levels of sensitivity and specificity. PMID:17685896

  13. Occurrence of genes coding for MSCRAMM and biofilm-associated protein Bap in Staphylococcus spp. isolated from bovine subclinical mastitis and relationship with somatic cell counts.

    PubMed

    Zuniga, Eveline; Melville, Priscilla A; Saidenberg, André B S; Laes, Marco A; Gonsales, Fernanda F; Salaberry, Sandra R S; Gregori, Fabio; Brandão, Paulo E; Dos Santos, Franklin G B; Lincopan, Nilton E; Benites, Nilson R

    2015-12-01

    This study aimed to elucidate aspects of the epidemiology of bovine subclinical mastitis through the assessment of genes encoding MSCRAMM (microbial surface components recognizing adhesive matrix molecules - a group of adhesins) and protein Bap (implicated in biofilm formation), in coagulase-positive (CPS) and coagulase-negative (CNS) Staphylococcus isolated from subclinical mastitis. Milk samples were collected for microbiological exams, somatic cell count (SCC) and a survey of the genes coding for MSCRAMM (cna, eno, ebpS, fnbA, fnbB and fib) and biofilm-associated protein Bap (bap) in 106 Staphylococcus spp. isolates using PCR. The frequencies of occurrence of eno (82.1%), fnbA (72.6%), fib (71.7%) and bap (56.6%) were higher (P < 0.0001) compared with the other assessed genes (cna, ebpS and fnbB). The higher frequency of occurrence (P < 0.005) of the bap gene in CNS compared with CPS suggests that in these species biofilm formation is an important mechanism for the persistence of the infection. The medians of the SCCs in the samples where eno, fnbA, fib and bap genes were detected were higher compared with Staphylococcus without the assessed genes (P < 0.05) and negative samples (P < 0.01), which indicated that the presence of these MSCRAMM may be related to a higher intensity of the inflammatory process. PMID:26318876

  14. Purification by Strep-Tactin affinity chromatography of a delete envelope gp51 protein of Bovine Leukaemia virus expressed in Sf21 insect cells.

    PubMed

    De Giuseppe, Antonio; Forti, Katia; Feliziani, Francesco; Severi, Giulio; Cagiola, Monica

    2010-04-01

    Bovine leukaemia virus (BLV) causes disease in cattle and it is related to human T lymphotrofic viruses HTLV-1 and HTLV-2. The objective of this study was to express and purify deleted and stable forms of the gp51 envelope glycoprotein of BLV using a baculovirus system. Two forms of the gp51 were synthesised: one comprised the gp51 N-terminal 174 amino acids and a single 6xHis tag (Delta(175-268)gp51-His) and the second form contained the same amino acid sequence and a C-terminal Strep-tag II in addition to the 6xHis tag (Delta(175-268)gp51-STH). The two proteins were expressed and purified by immobilized metal-affinity chromatography (IMAC) or by Strep-Tactin column. The Strep-Tactin technology was more efficient than IMAC method and achieved a high pure recombinant deleted gp51. In addition the Delta(175-268)gp51-STH protein was further concentrated by IMAC. This purified antigen could be used for the isolation of immunoreactive molecules and to develop a competitive ELISA test. PMID:20232124

  15. Food Protein Functionality-A New Model.

    PubMed

    Foegeding, E Allen

    2015-12-01

    Proteins in foods serve dual roles as nutrients and structural building blocks. The concept of protein functionality has historically been restricted to nonnutritive functions-such as creating emulsions, foams, and gels-but this places sole emphasis on food quality considerations and potentially overlooks modifications that may also alter nutritional quality or allergenicity. A new model is proposed that addresses the function of proteins in foods based on the length scale(s) responsible for the function. Properties such as flavor binding, color, allergenicity, and digestibility are explained based on the structure of individual molecules; placing this functionality at the nano/molecular scale. At the next higher scale, applications in foods involving gelation, emulsification, and foam formation are based on how proteins form secondary structures that are seen at the nano and microlength scales, collectively called the mesoscale. The macroscale structure represents the arrangements of molecules and mesoscale structures in a food. Macroscale properties determine overall product appearance, stability, and texture. The historical approach of comparing among proteins based on forming and stabilizing specific mesoscale structures remains valid but emphasis should be on a common means for structure formation to allow for comparisons across investigations. For applications in food products, protein functionality should start with identification of functional needs across scales. Those needs are then evaluated relative to how processing and other ingredients could alter desired molecular scale properties, or proper formation of mesoscale structures. This allows for a comprehensive approach to achieving the desired function of proteins in foods. PMID:26513109

  16. Fast loop modeling for protein structures

    NASA Astrophysics Data System (ADS)

    Zhang, Jiong; Nguyen, Son; Shang, Yi; Xu, Dong; Kosztin, Ioan

    2015-03-01

    X-ray crystallography is the main method for determining 3D protein structures. In many cases, however, flexible loop regions of proteins cannot be resolved by this approach. This leads to incomplete structures in the protein data bank, preventing further computational study and analysis of these proteins. For instance, all-atom molecular dynamics (MD) simulation studies of structure-function relationship require complete protein structures. To address this shortcoming, we have developed and implemented an efficient computational method for building missing protein loops. The method is database driven and uses deep learning and multi-dimensional scaling algorithms. We have implemented the method as a simple stand-alone program, which can also be used as a plugin in existing molecular modeling software, e.g., VMD. The quality and stability of the generated structures are assessed and tested via energy scoring functions and by equilibrium MD simulations. The proposed method can also be used in template-based protein structure prediction. Work supported by the National Institutes of Health [R01 GM100701]. Computer time was provided by the University of Missouri Bioinformatics Consortium.

  17. A bovine whey protein extract stimulates human neutrophils to generate bioactive IL-1Ra through a NF-kappaB- and MAPK-dependent mechanism.

    PubMed

    Rusu, Daniel; Drouin, Rjean; Pouliot, Yves; Gauthier, Sylvie; Poubelle, Patrice E

    2010-02-01

    Innate immunity depends on the efficiency of neutrophils to be activated rapidly to restore homeostasis. It can benefit from priming agents that enhance neutrophil capacity to respond more efficiently to a subsequent stimulation. Among natural products, a bovine whey protein extract (WPE) has been shown to prime normal human blood neutrophils by enhancing their chemotaxis, phagocytosis, oxidative burst, and degranulation. These leukocytes are also an important source of cytokines, some of which have antiinflammatory functions. We investigated the role of WPE, as well as its mechanisms of action, on the production of interleukin (IL)-1 receptor antagonist (IL-1Ra) by neutrophils in vitro. WPE dose-dependently stimulated de novo synthesis and release of IL-1Ra by normal human blood neutrophils. Among the major proteins present in WPE, beta-lactoglobulin (beta-LG) and alpha-lactalbumin (alpha-LA) were the only active components. They had additive effects that exactly reproduced those of WPE. Similarly to WPE, they also stimulated the accumulation of IL-1beta, IL-8, IL-6, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and tumor necrosis factor-alpha. However, neutrophils incubated with WPE, beta-LG, and alpha-LA produced IL-1Ra in excess of IL-1beta and the ratio IL-1Ra:IL-1beta increased linearly. The amounts of IL-1Ra stimulated by WPE or beta-LG + alpha-LA significantly reduced the IL-1 activity in EL4 cells. Inhibitors of p38 and extracellular signal-regulated kinases (ERK)1/2 mitogen-activated protein kinase, and nuclear factor-kappaB cascades reduced neutrophil production of IL-1Ra. Our data suggest that WPE, through beta-LG + alpha-LA, has immunomodulatory properties and the potential to increase host defenses. PMID:20032479

  18. Mitochondrial integrity in a neonatal bovine model of right ventricular dysfunction.

    PubMed

    Bruns, Danielle R; Brown, R Dale; Stenmark, Kurt R; Buttrick, Peter M; Walker, Lori A

    2015-01-15

    Right ventricular (RV) function is a key determinant of survival in patients with both RV and left ventricular (LV) failure, yet the mechanisms of RV failure are poorly understood. Recent studies suggest cardiac metabolism is altered in RV failure in pulmonary hypertension (PH). Accordingly, we assessed mitochondrial content, dynamics, and function in hearts from neonatal calves exposed to hypobaric hypoxia (HH). This model develops severe PH with concomitant RV hypertrophy, dilation, and dysfunction. After 2 wk of HH, pieces of RV and LV were obtained along with samples from age-matched controls. Comparison with control assesses the effect of hypoxia, whereas comparison between the LV and RV in HH assesses the additional impact of RV overload. Mitochondrial DNA was unchanged in HH, as was mitochondrial content as assessed by electron microscopy. Immunoblotting for electron transport chain subunits revealed a small increase in mitochondrial content in HH in both ventricles. Mitochondrial dynamics were largely unchanged. Activity of individual respiratory chain complexes was reduced (complex I) or unchanged (complex V) in HH. Key enzymes in the glycolysis pathway were upregulated in both HH ventricles, alongside upregulation of hypoxia-inducible factor-1? protein. Importantly, none of the changes in expression or activity were different between ventricles, suggesting the changes are in response to HH and not RV overload. Upregulation of glycolytic modulators without chamber-specific mitochondrial dysfunction suggests that mitochondrial capacity and activity are maintained at the onset of PH, and the early RV dysfunction in this model results from mechanisms independent of the mitochondria. PMID:25416385

  19. Mitochondrial integrity in a neonatal bovine model of right ventricular dysfunction

    PubMed Central

    Bruns, Danielle R.; Brown, R. Dale; Stenmark, Kurt R.; Buttrick, Peter M.

    2014-01-01

    Right ventricular (RV) function is a key determinant of survival in patients with both RV and left ventricular (LV) failure, yet the mechanisms of RV failure are poorly understood. Recent studies suggest cardiac metabolism is altered in RV failure in pulmonary hypertension (PH). Accordingly, we assessed mitochondrial content, dynamics, and function in hearts from neonatal calves exposed to hypobaric hypoxia (HH). This model develops severe PH with concomitant RV hypertrophy, dilation, and dysfunction. After 2 wk of HH, pieces of RV and LV were obtained along with samples from age-matched controls. Comparison with control assesses the effect of hypoxia, whereas comparison between the LV and RV in HH assesses the additional impact of RV overload. Mitochondrial DNA was unchanged in HH, as was mitochondrial content as assessed by electron microscopy. Immunoblotting for electron transport chain subunits revealed a small increase in mitochondrial content in HH in both ventricles. Mitochondrial dynamics were largely unchanged. Activity of individual respiratory chain complexes was reduced (complex I) or unchanged (complex V) in HH. Key enzymes in the glycolysis pathway were upregulated in both HH ventricles, alongside upregulation of hypoxia-inducible factor-1α protein. Importantly, none of the changes in expression or activity were different between ventricles, suggesting the changes are in response to HH and not RV overload. Upregulation of glycolytic modulators without chamber-specific mitochondrial dysfunction suggests that mitochondrial capacity and activity are maintained at the onset of PH, and the early RV dysfunction in this model results from mechanisms independent of the mitochondria. PMID:25416385

  20. Single Particle Dynamic Imaging and Fe3+ Sensing with Bright Carbon Dots Derived from Bovine Serum Albumin Proteins

    PubMed Central

    Yang, Qingxiu; Wei, Lin; Zheng, Xuanfang; Xiao, Lehui

    2015-01-01

    In this work, we demonstrated a convenient and green strategy for the synthesis of highly luminescent and water-soluble carbon dots (Cdots) by carbonizing carbon precursors, i.e., Bovine serum albumin (BSA) nanoparticles, in water solution. Without post surface modification, the as-synthesized Cdots exhibit fluorescence quantum yield (Q.Y.) as high as 34.8% and display superior colloidal stability not only in concentrated salt solutions (e.g. 2 M KCl) but also in a wide range of pH solutions. According to the FT-IR measurements, the Cdots contain many carboxyl groups, providing a versatile route for further chemical and biological functionalization. Through conjugation of Cdots with the transacting activator of transcription (TAT) peptide (a kind of cell penetration peptide (CPP)) derived from human immunodeficiency virus (HIV), it is possible to directly monitor the dynamic interactions of CPP with living cell membrane at single particle level. Furthermore, these Cdots also exhibit a dosage-dependent selectivity toward Fe3+ among other metal ions, including K+, Na+, Mg2+, Hg2+, Co2+, Cu2+, Pb2+ and Al3+. We believed that the Cdots prepared by this strategy would display promising applications in various areas, including analytical chemistry, nanomedicine, biochemistry and so on. PMID:26634992

  1. Single Particle Dynamic Imaging and Fe(3+) Sensing with Bright Carbon Dots Derived from Bovine Serum Albumin Proteins.

    PubMed

    Yang, Qingxiu; Wei, Lin; Zheng, Xuanfang; Xiao, Lehui

    2015-01-01

    In this work, we demonstrated a convenient and green strategy for the synthesis of highly luminescent and water-soluble carbon dots (Cdots) by carbonizing carbon precursors, i.e., Bovine serum albumin (BSA) nanoparticles, in water solution. Without post surface modification, the as-synthesized Cdots exhibit fluorescence quantum yield (Q.Y.) as high as 34.8% and display superior colloidal stability not only in concentrated salt solutions (e.g. 2 M KCl) but also in a wide range of pH solutions. According to the FT-IR measurements, the Cdots contain many carboxyl groups, providing a versatile route for further chemical and biological functionalization. Through conjugation of Cdots with the transacting activator of transcription (TAT) peptide (a kind of cell penetration peptide (CPP)) derived from human immunodeficiency virus (HIV), it is possible to directly monitor the dynamic interactions of CPP with living cell membrane at single particle level. Furthermore, these Cdots also exhibit a dosage-dependent selectivity toward Fe(3+) among other metal ions, including K(+), Na(+), Mg(2+), Hg(2+), Co(2+), Cu(2+), Pb(2+) and Al(3+). We believed that the Cdots prepared by this strategy would display promising applications in various areas, including analytical chemistry, nanomedicine, biochemistry and so on. PMID:26634992

  2. Monolith immuno-affinity enrichment liquid chromatography tandem mass spectrometry for quantitative protein analysis of recombinant bovine somatotropin in serum.

    PubMed

    Smits, Nathalie G E; Blokland, Marco H; Wubs, Klaas L; Nessen, Merel A; van Ginkel, Leen A; Nielen, Michel W F

    2015-08-01

    The use of recombinant bovine somatotropin (rbST) to enhance milk production is approved in several countries, but it is prohibited in the European Union. According to EU legislation, it is necessary to confirm positive screening results prior to enforcement. Although adequate screening assays are available nowadays, development of liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory methods to detect low levels of rbST is still a challenge. Here, we present a novel approach using immuno-affinity enrichment on monolithic micro-columns in combination with state-of-the-art ultra-high pressure LC-MS/MS (UHPLC-MS/MS) detection. The developed approach enables detection and confirmation of rbST in serum at a decision limit (CC?) concentration of 0.8ngmL(-1). Furthermore, the method is easy to handle, robust and reproducible. We successfully applied the confirmatory method to serum samples from rbST treated cows that were found suspect after immunoassay-based screening. The use of rbST could be confirmed over 1week after treatment, and the developed method demonstrated the sensitivity needed for effective control. Graphical Abstract Graphical summary of the workflow, for serum preparation, enrichment with monolith microcolumns and LC-MS/MS measurement of rbST. PMID:26077745

  3. Single Particle Dynamic Imaging and Fe3+ Sensing with Bright Carbon Dots Derived from Bovine Serum Albumin Proteins

    NASA Astrophysics Data System (ADS)

    Yang, Qingxiu; Wei, Lin; Zheng, Xuanfang; Xiao, Lehui

    2015-12-01

    In this work, we demonstrated a convenient and green strategy for the synthesis of highly luminescent and water-soluble carbon dots (Cdots) by carbonizing carbon precursors, i.e., Bovine serum albumin (BSA) nanoparticles, in water solution. Without post surface modification, the as-synthesized Cdots exhibit fluorescence quantum yield (Q.Y.) as high as 34.8% and display superior colloidal stability not only in concentrated salt solutions (e.g. 2 M KCl) but also in a wide range of pH solutions. According to the FT-IR measurements, the Cdots contain many carboxyl groups, providing a versatile route for further chemical and biological functionalization. Through conjugation of Cdots with the transacting activator of transcription (TAT) peptide (a kind of cell penetration peptide (CPP)) derived from human immunodeficiency virus (HIV), it is possible to directly monitor the dynamic interactions of CPP with living cell membrane at single particle level. Furthermore, these Cdots also exhibit a dosage-dependent selectivity toward Fe3+ among other metal ions, including K+, Na+, Mg2+, Hg2+, Co2+, Cu2+, Pb2+ and Al3+. We believed that the Cdots prepared by this strategy would display promising applications in various areas, including analytical chemistry, nanomedicine, biochemistry and so on.

  4. Effect of culling and vaccination on bovine tuberculosis infection in a European badger (Meles meles) population by spatial simulation modelling.

    PubMed

    Abdou, Marwa; Frankena, Klaas; O'Keeffe, James; Byrne, Andrew W

    2016-03-01

    The control of bovine tuberculosis (bTB) in cattle herds in the Republic of Ireland (ROI) is partially hindered by spill-back infection from wild badgers (Meles meles). The aim of this study was to determine the relative effects of interventions (combinations of culling and/or vaccination) on bTB dynamics in an Irish badger population. A spatial agent-based stochastic simulation model was developed to evaluate the effect of various control strategies for bovine tuberculosis in badgers: single control strategies (culling, selective culling, vaccination, and vaccine baits), and combined strategies (Test vaccinate/cull (TVC)), split area approaches using culling and vaccination, or selective culling and vaccination, and mixed scenarios where culling was conducted for five years and followed by vaccination or by a TVC strategy. The effect of each control strategy was evaluated over a 20-year period. Badger control was simulated in 25%, 50%, and 75% area (limited area strategy) or in the entire area (100%, wide area strategy). For endemic bTB, a culling strategy was successful in eradicating bTB from the population only if applied as an area-wide strategy. However, this was achieved only by risking the extinction of the badger population. Selective culling strategies (selective culling or TVC) mitigated this negative impact on the badger population's viability. Furthermore, both strategies (selective culling and TVC) allowed the badger population to recover gradually, in compensation for the population reduction following the initial use of removal strategies. The model predicted that vaccination can be effective in reducing bTB prevalence in badgers, when used in combination with culling strategies (i.e. TVC or other strategies). If fecundity was reduced below its natural levels (e.g. by using wildlife contraceptives), the effectiveness of vaccination strategies improved. Split-area simulations highlighted that interventions can have indirect effects (e.g. on population size) in non-treatment areas. Our model suggests that mixed control strategies could maintain infection prevalence to a low level for a considerable period even with a growing population. The model supported the hypothesis that culling strategies appeared to be the most effective method for the control of bTB in badgers using parameters, where available, from ROI, either singly or in combination with other strategies. In this model, the success of a vaccination strategy depended partially upon population density and the proportion of the population infected, therefore an initial culling program (to reduce density and/or remove infected badgers) followed by long-term vaccination may be effective in controlling bTB in badgers. PMID:26774448

  5. Mathematical Modelling of the Transmission Dynamics of Contagious Bovine Pleuropneumonia Reveals Minimal Target Profiles for Improved Vaccines and Diagnostic Assays

    PubMed Central

    Ssematimba, Amos; Jores, Joerg; Mariner, Jeffrey C.

    2015-01-01

    Contagious bovine pleuropneumonia (CBPP) is a cattle disease that has hampered the development of the livestock sector in sub-Saharan Africa. Currently, vaccination with a live vaccine strain is its recommended control measure although unofficial antimicrobial use is widely practiced. Here, modelling techniques are used to assess the potential impact of early elimination of infected cattle via accurate diagnosis on CBPP dynamics. A herd-level stochastic epidemiological model explicitly incorporating test sensitivity and specificity is developed. Interventions by annual vaccination, annual testing and elimination and a combination of both are implemented in a stepwise manner and their effectiveness compared by running 1000 simulations per intervention over ten years. The model predicts that among the simulated interventions, the ones likely to eliminate the disease from an isolated herd all involved annual vaccination of more than 75% of the animals with a vaccine that protects for at least 18 months combined with annual testing (and elimination of positive reactors) of 75% of the animals every six months after vaccination. The highest probability of disease elimination was 97.5% and this could occur within a median of 2.3 years. Generally, our model predicts that regular testing and elimination of positive reactors using improved tests will play a significant role in minimizing CBPP burden especially in the current situation where improved vaccines are yet to be developed. PMID:25668725

  6. SWISS-MODEL: An automated protein homology-modeling server.

    PubMed

    Schwede, Torsten; Kopp, Jrgen; Guex, Nicolas; Peitsch, Manuel C

    2003-07-01

    SWISS-MODEL (http://swissmodel.expasy.org) is a server for automated comparative modeling of three-dimensional (3D) protein structures. It pioneered the field of automated modeling starting in 1993 and is the most widely-used free web-based automated modeling facility today. In 2002 the server computed 120 000 user requests for 3D protein models. SWISS-MODEL provides several levels of user interaction through its World Wide Web interface: in the 'first approach mode' only an amino acid sequence of a protein is submitted to build a 3D model. Template selection, alignment and model building are done completely automated by the server. In the 'alignment mode', the modeling process is based on a user-defined target-template alignment. Complex modeling tasks can be handled with the 'project mode' using DeepView (Swiss-PdbViewer), an integrated sequence-to-structure workbench. All models are sent back via email with a detailed modeling report. WhatCheck analyses and ANOLEA evaluations are provided optionally. The reliability of SWISS-MODEL is continuously evaluated in the EVA-CM project. The SWISS-MODEL server is under constant development to improve the successful implementation of expert knowledge into an easy-to-use server. PMID:12824332

  7. Evidence of a pro-apoptotic effect of specific antibodies in a bovine macrophage model of infection with Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Jolly, Ana; Lomparda, Silvina; Hajos, Silvia E; Mundo, Silvia L

    2016-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic granulomatous enteritis in ruminants. Understanding the protective immune response following infection is crucial to improve the diagnosis and the development of vaccines against this disease. The goal of this work was to assess whether specific antibodies were able to modulate the macrophage response to MAP infection by evaluating apoptosis and TNF-? secretion in an in vitro model. Sera from healthy (n=2), MAP-infected (n=3) and lipoarabinomannan (LAM)-immunized (n=3) bovines were evaluated. LAM was chosen as immunogen due to its relevant role in mycobacterial pathogenesis. We demonstrated by two different techniques (Acridine Orange/Ethidium Bromide microscopy and Annexin V/7-Amino-Actinomycin D flow cytometry) that the immune sera from both, MAP-infected and LAM-immunized bovines, significantly increased macrophage apoptosis in infected cultures. Comparable levels of apoptosis were detected when MAP was pre-incubated with purified specific antibodies instead of whole serum. Furthermore, this effect was accompanied by a significantly higher secretion of TNF-?. These results strongly suggest that specific antibodies could limit the impact of MAP on the apoptosis of bovine cells. This work would contribute to elucidate the role of the specific antibody response in bovine JD and its prevention. PMID:26827838

  8. Fold assessment for comparative protein structure modeling.

    PubMed

    Melo, Francisco; Sali, Andrej

    2007-11-01

    Accurate and automated assessment of both geometrical errors and incompleteness of comparative protein structure models is necessary for an adequate use of the models. Here, we describe a composite score for discriminating between models with the correct and incorrect fold. To find an accurate composite score, we designed and applied a genetic algorithm method that searched for a most informative subset of 21 input model features as well as their optimized nonlinear transformation into the composite score. The 21 input features included various statistical potential scores, stereochemistry quality descriptors, sequence alignment scores, geometrical descriptors, and measures of protein packing. The optimized composite score was found to depend on (1) a statistical potential z-score for residue accessibilities and distances, (2) model compactness, and (3) percentage sequence identity of the alignment used to build the model. The accuracy of the composite score was compared with the accuracy of assessment by single and combined features as well as by other commonly used assessment methods. The testing set was representative of models produced by automated comparative modeling on a genomic scale. The composite score performed better than any other tested score in terms of the maximum correct classification rate (i.e., 3.3% false positives and 2.5% false negatives) as well as the sensitivity and specificity across the whole range of thresholds. The composite score was implemented in our program MODELLER-8 and was used to assess models in the MODBASE database that contains comparative models for domains in approximately 1.3 million protein sequences. PMID:17905832

  9. Elucidation of intermediate (mobile) and slow (solidlike) protein motions in bovine lens homogenates by carbon-13 NMR spectroscopy

    SciTech Connect

    Morgan, C.F.; Schleich, T.; Caines, G.H.; Farnsworth, P.N. )

    1989-06-13

    The motional dynamics of lens cytoplasmic proteins present in calf lens homogenates were investigated by two {sup 13}C nuclear magnetic resonance (NMR) techniques sensitive to molecular motion to further define the organizational differences between the cortex and nucleus. For the study of intermediate (mobile) protein rotational reorientation motion time scales (rotational correlation time ({tau}{sub 0}) range of 1-500 ns), the authors employed {sup 13}C off-resonance rotating frame spin-lattice relaxation, whereas for the study of slow (solidlike) motions ({tau}{sub 0} {ge} 10 {mu}s) they used the solid-state NMR techniques of dipolar decoupling and cross-polarization. The frequency dependence of the peptide bond carbonyl off-resonance rotating frame spectral intensity ratio of the lens proteins present in native calf nuclear homogenate at 35{degree}C indicates the presence of a polydisperse mobile protein fraction with a {tau}{sub 0,eff} (mean) value of 57 ns. Lowering the temperature to 1{degree}C, a temperature which produces the cold cataract, results in an overall decrease in {tau}{sub 0,eff} to 43 ns, suggesting a selective removal of {beta}{sub H}-, LM-, and possibly {gamma}{sub s}-crystallins from the mobile lens protein population. The presence of solidlike or motionally restricted protein species was established by dipolar decoupling and cross-polarization. Comparison of proton dipolar-decoupled and nondecoupled {sup 13}C NMR spectra of native cortical homogenate at 20{degree}C indicates the absence of significant contributions from slowly tumbling, motionally restricted species. These studies establish the presence of both mobile and solidlike protein phases in calf lens nuclear homogenate, whereas for the native cortical homogenate, within the detection limits of NMR, the protein phase is mobile, except at low temperature where a small fraction of solidlike protein phase is present.

  10. A model of consumers' risk perceptions toward recombinant bovine growth hormone (rbGH): the impact of risk characteristics.

    PubMed

    Grobe, D; Douthitt, R; Zepeda, L

    1999-08-01

    This study estimates the effect risk characteristics, described as outrage factors by Hadden, have on consumers' risk perceptions toward the food-related biotechnology, recombinant bovine growth hormone (rbGH). The outrage factors applicable to milk from rbGH treated herds are involuntary risk exposure, unfamiliarity with the product's production process, unnatural product characteristics, lack of trust in regulator's ability to protect consumers in the marketplace, and consumers' inability to distinguish milk from rbGH treated herds compared to milk from untreated herds. An empirical analysis of data from a national survey of household food shoppers reveals that outrage factors mediate risk perceptions. The results support the inclusion of outrage factors into the risk perception model for the rbGH product, as they add significantly to the explanatory power of the model and therefore reduce bias compared to a simpler model of attitudinal and demographic factors. The study indicates that outrage factors which have a significant impact on risk perceptions are the lack of trust in the FDA as a food-related information source, and perceiving no consumer benefits from farmers' use of rbGH. Communication strategies to reduce consumer risk perceptions therefore could utilize agencies perceived as more trustworthy and emphasize the benefits of rbGH use to consumers. PMID:10765429

  11. Hydration dynamics near a model protein surface

    SciTech Connect

    Russo, Daniela; Hura, Greg; Head-Gordon, Teresa

    2003-09-01

    The evolution of water dynamics from dilute to very high concentration solutions of a prototypical hydrophobic amino acid with its polar backbone, N-acetyl-leucine-methylamide (NALMA), is studied by quasi-elastic neutron scattering and molecular dynamics simulation for both the completely deuterated and completely hydrogenated leucine monomer. We observe several unexpected features in the dynamics of these biological solutions under ambient conditions. The NALMA dynamics shows evidence of de Gennes narrowing, an indication of coherent long timescale structural relaxation dynamics. The translational water dynamics are analyzed in a first approximation with a jump diffusion model. At the highest solute concentrations, the hydration water dynamics is significantly suppressed and characterized by a long residential time and a slow diffusion coefficient. The analysis of the more dilute concentration solutions takes into account the results of the 2.0M solution as a model of the first hydration shell. Subtracting the first hydration layer based on the 2.0M spectra, the translational diffusion dynamics is still suppressed, although the rotational relaxation time and residential time are converged to bulk-water values. Molecular dynamics analysis shows spatially heterogeneous dynamics at high concentration that becomes homogeneous at more dilute concentrations. We discuss the hydration dynamics results of this model protein system in the context of glassy systems, protein function, and protein-protein interfaces.

  12. Immediate-early RNA 2.9 and early RNA 2.6 of bovine herpesvirus 1 are 3' coterminal and encode a putative zinc finger transactivator protein.

    PubMed Central

    Wirth, U V; Fraefel, C; Vogt, B; Vlcek, C; Paces, V; Schwyzer, M

    1992-01-01

    Bovine herpesvirus 1 (BHV-1) contains three major immediate-early (IE) genes involved in regulation of the productive cycle of replication. Two spliced IE RNAs, IER4.2 (4.2 kb) and IER2.9 (2.9 kb), are under the control of a single promoter; IER1.7 (1.7 kb) is transcribed from a different promoter in the opposite direction. Examining the kinetics of transcription, we found that the IER4.2/2.9 promoter was turned off at the end of the IE period. An alternative promoter became active, directing synthesis of an unspliced early RNA, ER2.6 (2.6 kb), which was colinear with the second exon of IER2.9 except for its 5' end in the intron about 10 bases upstream of the splice site. Sequence analysis revealed a single open reading frame common to IER2.9 and ER2.6 with a coding potential of 676 amino acids. The putative protein, named p135, contained a cysteine-rich zinc finger domain near the N terminus with homology to ICP0 of herpes simplex virus type 1, to protein 61 of varicella-zoster virus, to early protein 0 of pseudorabies virus, and to other viral and cellular proteins. The remaining parts of p135 exhibited only limited homology, mainly with pseudorabies virus protein 0, but the entire sequence was highly conserved between two strains of BHV-1 (K22 and Jura). The latency-related antisense transcript covered a large portion of ER2.6 excluding the zinc finger coding region. In transient expression assays, p135 activated a variety of promoters, including that for ER2.6, but repressed the IER1.7 promoter. Thus, p135 combines functional characteristics of ICP0, a strong transactivator, and of protein 61, a repressor. BHV-1 seems to have evolved a subtle mechanism to ensure the continued synthesis of p135 while turning off IER4.2, which encodes p180, the herpes simplex virus type 1 ICP4 homolog. Images PMID:1313901

  13. Regeneration of bovine and octopus opsins in situ with natural and artificial retinals

    SciTech Connect

    Koutalos, Y.; Ebrey, T.G.; Tsuda, M.; Odashima, K.; Lien, T.; Park, M.H.; Shimizu, N.; Derguini, F.; Nakanishi, K.; Gilson, H.R.; Honig, B. )

    1989-03-21

    The authors consider the problem of color regulation in visual pigments for both bovine rhodopsin and octopus rhodopsin. Both pigments have 11-cis-retinal as their chromophore. These rhodopsins were bleached in their native membranes, and the opsins were regenerated with natural and artificial chromophores. Both bovine and octopus opsins were regenerated with the 9-cis- and 11-cis-retinal isomers, but the octopus opsin was additionally regenerated with the 13-cis and all-trans isomers. Titration of the octopus opsin with 11-cis-retinal gave an extinction coefficient for octopus rhodopsin of 27,000 {plus minus} 3,000 M{sup {minus}1} cm{sup {minus}1} at 475 nm. The absorption maxima of bovine artificial pigments formed by regenerating opsin with the 11-cis dihydro series of chromophores support a color regulation model for bovine rhodopsin in which the chromophore-binding site of the protein has two negative charges: one directly hydrogen bonded to the Schiff base nitrogen and another near carbon-13. Formation of octopus artificial pigments with both all-trans and 11-cis dihydro chromophores leads to a similar model for octopus rhodopsin and metarhodopsin: there are two negative charges in the chromophore-binding site, one directly hydrogen bonded to the Schiff base nitrogen and a second near carbon-13. The interaction of this second charge with the chromophore in octopus rhodopsin is weaker than in bovine, while in metarhodopsin it is as strong as in bovine.

  14. Penetrable silica microspheres for immobilization of bovine serum albumin and their application to the study of the interaction between imatinib mesylate and protein by frontal affinity chromatography.

    PubMed

    Ma, Liyun; Li, Jing; Zhao, Juan; Liao, Han; Xu, Li; Shi, Zhi-Guo

    2016-01-01

    In the current study, novel featured silica, named penetrable silica, simultaneously containing macropores and mesopores, was immobilized with bovine serum albumin (BSA) via Schiff base method. The obtained BSA-SiO2 was employed as the high-performance liquid chromatographic (HPLC) stationary phase. Firstly, D- and L-tryptophan were used as probes to investigate the chiral separation ability of the BSA-SiO2 stationary phase. An excellent enantioseparation factor was obtained up to 4.3 with acceptable stability within at least 1month. Next, the BSA-SiO2 stationary phase was applied to study the interaction between imatinib mesylate (IM) and BSA by frontal affinity chromatography. A single type of binding site was found for IM with the immobilized BSA, and the hydrogen-bonding and van der Waals interactions were expected to be contributing interactions based on the thermodynamic studies, and this was a spontaneous process. Compared to the traditional silica for HPLC stationary phase, the proposed penetrable silica microsphere possesseda larger capacity to bond more BSA, minimizing column overloading effects and enhancing enantioseparation ability. In addition, the lower running column back pressure and fast mass transfer were meaningful for the column stability and lifetime. It was a good substrate to immobilize biomolecules for fast chiral resolution and screening drug-protein interactions. Graphical Abstract The preparation of BSA-SiO2 column based on the penetrable silica and its application in chiral separation and frontal analysis. PMID:26573171

  15. High antibody titres against predicted Mycoplasma surface proteins do not prevent sequestration in infected lung tissue in the course of experimental contagious bovine pleuropneumonia.

    PubMed

    Schieck, Elise; Liljander, Anne; Hamsten, Carl; Gicheru, Nimmo; Scacchia, Massimo; Sacchini, Flavio; Heller, Martin; Schnee, Christiane; Sterner-Kock, Anja; Hlinak, Andreas; Naessens, Jan; Poole, Jane; Persson, Anja; Jores, Joerg

    2014-08-01

    Contagious bovine pleuropneumonia (CBPP), a severe respiratory disease of cattle caused by Mycoplasma mycoides subsp. mycoides (Mmm) is endemic in many African countries due to fragmented veterinary services and the lack of an efficient vaccine and sensitive diagnostics. More efficient tools to control the disease are needed, but to develop the tools, a better understanding of host-pathogen interactions is necessary. The aim of this study was to characterize the kinetics of the humoral immune response against 65 Mmm surface antigens for an extended period in cattle that survived a primary infection with Mmm. We describe clinical and haematological outcomes, and dissect the humoral immune response over time, to specific antigens and compared the antibody responses between different pathomorphological outcomes. No antigen-specific antibodies correlating with protection were identified. Interestingly we found that animals that developed Mycoplasma-containing sequestra had significantly higher antibody levels against proteins comprising the surface proteome than the animals that cleared Mycoplasma from their lungs. Based on these data we suggest that high antibody titres might play a role in the establishment of pathomorphological changes, such as vasculitis, which should be investigated in future studies. Beneficial antibody specificities and cellular immune responses need to be identified in order to foster the development of an improved vaccine in the future. PMID:24880898

  16. Insulin-like growth factor and insulin-like growth factor-binding proteins in the bovine uterus throughout the oestrous cycle.

    PubMed

    Costello, Lisa M; O'Boyle, Padraic; Diskin, Michael G; Hynes, Ailish C; Morris, Dermot G

    2014-01-01

    The aims of the present study were to assess several components of the insulin-like growth factor (IGF) system in bovine uterine flushings across different days of the oestrous cycle and to examine the relationship between the IGF system and systemic progesterone concentrations. Uterine flushings and plasma were collected from cows on Days 3, 7, 11 and 15 of the oestrous cycle. The IGF-1 concentration was more than 5-fold higher in the uterus compared with plasma on Days 7 and 11 of the cycle, with values similar on Days 3 and 15. Similarly, uterine concentrations of IGF-binding protein (IGFBP)-2 and IGFBP-3 were up to 10- and 4-fold higher than in plasma, respectively, suggesting synthesis and/or transportation of the IGFBPs into the uterus. In addition, concentrations of IGFBP-2 and IGFBP-3 were higher in the uterine horns, ipsilateral to the corpus luteum, on Day 15. This difference could indicate a local controlling mechanism with progesterone possibly playing a role in regulating the concentration of IGFBPs between the uterine horns. There was no significant relationship between systemic progesterone concentrations and IGFBP concentrations on Day 7 of the oestrous cycle. The present study shows that uterine concentrations of IGFBPs are cycle stage specific and also suggests IGF-dependent and -independent functions for IGFBPs during a time of major change in the developing embryo. PMID:23607981

  17. Mas-related G-protein-coupled receptor c agonist bovine adrenal medulla 8-22 attenuates bone cancer pain in mice

    PubMed Central

    Sun, Yu-E; Lu, Cui-E; Lei, Yishan; Liu, Yue; Ma, Zhengliang; Gu, Xiaoping

    2015-01-01

    Objectives: The aim of this study is to investigate the effects of Mas-related G-protein-coupled receptor C (MrgC) agonist bovine adrenal medulla 8-22 (BAM8-22) on bone cancer pain and mirror-image pain. Methods: Bone cancer pain was induced by intramedullary injection of NC2472 fibrosarcoma cells in the mice. BAM8-22 and/or anti-MrgC antibody were injected intrathecally at day 14 after bone cancer induction and their effects on pain behaviors were detected. The pain behaviours were assessed by the number of spontaneous foot lifts and paw withdrawal mechanical threshold (PWMT) tests. MrgC expression was detected using western blot analysis and immunofluorescence assay. Results: There were increased bone cancer pain and mirror-image pain in the tumor-bearing mice while not in the sham-treated mice. BAM8-22 attenuated bone cancer pain in mice dose dependently with the highest effects at 2 hr after BAM8-22 administration, and anti-MrgC antibody reversed the effects of BAM8-22. However, intrathecal administration of BAM8-22 did not affect the mirror-image pain. Furthermore, BAM8-22 stimulated the expression of MrgC in the spinal dorsal horn. Conclusions: MrgC agonist BAM8-22 could attenuate bone cancer pain in mice. This study may provide a novel strategy for the treatment of bone cancer pain. PMID:26884930

  18. Distinct composition of bovine milk from Jersey and Holstein-Friesian cows with good, poor, or noncoagulation properties as reflected in protein genetic variants and isoforms.

    PubMed

    Jensen, H B; Poulsen, N A; Andersen, K K; Hammershj, M; Poulsen, H D; Larsen, L B

    2012-12-01

    The objective of this study was to examine variation in overall milk, protein, and mineral composition of bovine milk in relation to rennet-induced coagulation, with the aim of elucidating the underlying causes of milk with impaired coagulation abilities. On the basis of an initial screening of 892 milk samples from 42 herds with Danish Jersey and Holstein-Friesian cows, a subset of 102 samples was selected to represent milk with good, poor, or noncoagulating properties (i.e., samples that within each breed represented the most extremes in regard to coagulation properties). Milk with good coagulation characteristics was defined as milk forming a strong coagulum based on oscillatory rheology, as indicated by high values for maximum coagulum strength (G'(max)) and curd firming rate (CFR) and a short rennet coagulation time. Poorly coagulating milk formed a weak coagulum, with a low G'(max) and CFR and a long rennet coagulation time. Noncoagulating milk was defined as milk that failed to form a coagulum, having G'(max) and CFR values of zero at measurements taken within 1h after addition of rennet. For both breeds, a lower content of total protein, total casein (CN) and ?-CN, and lower levels of minerals (Ca, P, Mg) were identified in poorly coagulating and noncoagulating milk in comparison with milk with good coagulation properties. Liquid chromatography/electrospray ionization-mass spectrometry revealed the presence of a great variety of genetic variants of the major milk proteins, namely, ?(S1)-CN (variants B and C), ?(S2)-CN (A), ?-CN (A(1), A(2), B, I, and F), ?-CN (A, B, and E), ?-lactalbumin (B), and ?-lactoglobulin (A, B, and C). In poorly coagulating and noncoagulating milk samples of both breeds, the predominant composite genotype of ?(S1)-, ?-, and ?-CN was BB-A(2)A(2)-AA, which confirmed a genetic contribution to impaired milk coagulation. Interestingly, subtle variations in posttranslational modification of CN were observed between the coagulation classes in both breeds. Poorly coagulating and noncoagulating milk contained a lower fraction of the least phosphorylated ?(S1)-CN form, ?(S1)-CN 8P, relative to total ?(S1)-CN, along with a lower fraction of glycosylated ?-CN relative to total ?-CN. Thus, apparent variation was observed in the milk and protein composition, in the genetic makeup of the major milk proteins, and in the posttranslational modification level of CN between milk samples with either good or impaired coagulation ability, whereas the composition of poorly coagulating and noncoagulating milk was similar. PMID:23040012

  19. 77 FR 29914 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-21

    ... RIN 0579-AC68 Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products AGENCY... live bovines and products derived from bovines with regard to bovine spongiform encephalopathy. This... with regard to bovine spongiform encephalopathy. Comments on the proposed rule were required to......

  20. Relationship between single nucleotide polymorphisms in the bovine heat shock protein 70 gene and milk characteristics of beef cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heat shock proteins (HSPs) are induced by various stressors such as heat, cold, toxins, and oxygen deprivation. Our objective was to determine the genetic diversity in a segment of the HSP-70 gene of cattle. Genomic DNA was collected from 157 cows. The cows were Angus (n = 42), Brahman (n = 41), and...

  1. REMOVAL OF DECORIN CORE-PROTEIN FROM POWDERED BOVINE HIDES BY TREATMENTS USED TO PROCESS INTACT HIDES INTO LEATHER

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using a modification of a previously developed sandwich ELISA procedure, we determined the amount of decorin core-protein (DCP) content of raw powdered hide and powdered hide treated with the early steps of the tanning process. We found approximately 0.15 mg DCP/g hide in raw powdered hide. Treatm...

  2. Hydrolysis with Cucurbita ficifolia serine protease reduces antigenic response to bovine whey protein concentrate and αs-casein.

    PubMed

    Babij, Konrad; Bajzert, Joanna; Dąbrowska, Anna; Szołtysik, Marek; Zambrowicz, Aleksandra; Lubec, Gert; Stefaniak, Tadeusz; Willak-Janc, Ewa; Chrzanowska, Józefa

    2015-11-01

    In the present study the effect of hydrolysis with non-commercial Cucurbita ficifolia serine protease on a reduction of the IgE and IgG binding capacity of whey protein concentrate and αs-casein was investigated. The intensity of the protein degradation was analyzed by the degree of hydrolysis, the free amino groups content and RP-HPLC. The ability to bind the antibodies by native proteins and their hydrolysates was determined using a competitive ELISA test. Deep hydrolysis contributed to a significant reduction of immunoreactive epitopes present in WPC. In the case of IgE and IgG present in the serum pool of children with CMA, the lowest binding capacity was detected in the 24 h WPC hydrolysate, where the inhibition of the reaction with native WPC was ≤23 and ≤60 %, respectively. The analysis of the IgG reactivity in the antiserum of the immunized goat showed that the lowest antibody binding capacity was exhibited also by 24 h WPC hydrolysate at a concentration of 1000 μg/ml where the inhibition of the reaction with nWPC was ≤47 %. One-hour hydrolysis of α-casein was sufficient to significant reduction of the protein antigenicity, while the longer time (5 h) of hydrolysis probably lead to the appearance of new epitopes reactive with polyclonal. PMID:26036686

  3. THE MYCOBACTERIUM AVIUM SUPSPECIES PARATUBERCULOSIS 35 KDA PROTEIN PLAYS A ROLE IN INVASION OF BOVINE EPITHELIAL CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) enters intestinal epithelial cells of cattle and other ruminants via a mechanism that remains to be fully elucidated. In this study, we observed that a gene encoding the M. paratuberculosis 35-kDa major membrane protein (MMP) is ...

  4. Modification of the catalytic subunit of bovine heart cAMP-dependent protein kinase with affinity labels related to peptide substrates.

    PubMed

    Bramson, H N; Thomas, N; Matsueda, R; Nelson, N C; Taylor, S S; Kaiser, E T

    1982-09-25

    The modification and concomitant inactivation of the catalytic subunit of bovine heart cAMP-dependent protein kinase with affinity analogs of peptide substrates potentially capable of undergoing disulfide interchange with enzyme-bound sulfhydryl groups have been used to probe the active site associated with peptide binding. The regeneration of catalytic activity on treatment of the modified enzymes with dithiothreitol and the observation that prior reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) blocks the modification of the kinase by these reagents are consistent with the proposal that only thiol residues are reacting. The affinity analog Leu-Arg-Arg-Ala-Cys(3-nitro-2-pyridinesulfenyl)-Leu-Gly, 1, and the closely related peptide AcLeu-Arg-Arg-Ala-Cys(3-nitro-2-pyridinesulfenyl)-Leu-Gly-OEt, 3, react with a single sulfhydryl as shown by the stoichiometry of the release of the 3-nitro-2-pyridinesulfenyl group and the amount of label incorporated in the enzyme when the radioactively labeled peptide analog of 3 (peptide 4) is employed as the modifying agent. The kinetics of the reaction of 1 with 4.3 microM catalytic subunit was monophasic (employing substrate in excess conditions), yielding an apparent value of KI of approximately 40 microM and a k2 value of approximately 0.25 s-1. The low value of the observed KI, together with the observation that protein kinase substrates inhibit the modification reactions, suggest strongly that the cysteine residue undergoing reaction is in the vicinity of the active site. By trypsin-catalyzed degradation and identification of the peptide segment modified by covalent attachment of the peptide portion of the radioactive analog 4, the single cysteine modified was identified as cysteine-198. PMID:6286662

  5. Insulin-like growth factor-I and insulin-like growth factor binding proteins in the bovine mammary gland: Receptors, endogenous secretion, and appearance in milk

    SciTech Connect

    Campbell, P.G.

    1988-01-01

    This is the first study to characterize both insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) in bovine milk, to characterize the IGF-I receptor in the dry and lactating mammary gland, and to report de novo synthesis and secretion of IGF-I and IGFBP from normal mammary tissue. Immunoreactive IGF-I was principally associated with 45 kDa IGFBP in milk. Multiparous cows had a higher IGF-I concentration of 307 ng/ml than primiparous cows at 147 ng/ml. IGF-I concentration on day 56 of lactation was 34 ng/ml for combined parity groups. At parturition, IGF-I mass in blood and milk pools was 1.4 and 1.2 mg, respectively. Binding of {sup 125}I-IGF-I was specific for IGF-I with anIC{sub 50} of 2.2 ng which was a 10- and 1273-fold greater affinity than IGF-II and insulin, respectively. Association constants, as determined by Scatchard analysis, were similar for both pregnant and lactating cows at 3.5 and 4.0 L/nM, respectively. In addition, estimated mean receptor concentration was 0.25 and 0.23 pM/mg protein for pregnant and lactating cows, respectively. In a survey of mammary microscomes prepared from 48 cows, {sup 125}I-IGF-I binding declined with progressing lactation and a similar trend was observed during pregnancy.

  6. Bovine Serum Albumin-Catalyzed Deprotonation of [1-13C]-Glycolaldehyde: Protein Reactivity Toward Deprotonation of ?Hydroxy ?Carbonyl Carbon

    PubMed Central

    Go, Maybelle K.; Malabanan, M. Merced; Amyes, Tina L.; Richard, John P.

    2010-01-01

    Bovine serum albumin (BSA) in D2O at 25 C and pD 7.0 was found to catalyze the deuterium exchange reactions of [1-13C]-glycolaldehyde ([1-13C]-GA) to form [1-13C, 2-2H]-GA and [1-13C, 2,2-di-2H]-GA. The formation of [1-13C, 2-2H]-GA and [1-13C, 2,2-di-2H]-GA in a total yield of 51 3% was observed at early reaction times, and at latter times [1-13C, 2-2H]-GA was observed to undergo BSA-catalyzed conversion to [1-13C, 2,2-di-2H]-GA. The overall second-order rate constant for these deuterium exchange reactions is (kE)P = 0.25 M?1 s?1. By comparison, values of (kE)P = 0.04 M?1 s?1 (Go, M. K., Amyes, T. L., and Richard, J. P. (2009), Biochemistry 48, 57695778) and 0.06 M?1 s?1 (Go, M. K., Koudelka, A., Amyes, T. L., and Richard, J. P. (2010), Biochemistry 49, 53775389) have been determined, respectively, for the wildtype- and K12G mutant TIM-catalyzed deuterium exchange reactions of [1-13C]-GA to form [1-13C, 2,2-di-2H]-GA. These data show that TIM and BSA exhibit a modest catalytic activity towards deprotonation of ?-hydroxy ?-carbonyl carbon. It is suggested that this activity is intrinsic to many globular proteins, and that it must be enhanced to demonstrate successful de novo design of protein catalysts of reactions through enamine intermediates. PMID:20687575

  7. Protein Simulation Data in the Relational Model

    PubMed Central

    Simms, Andrew M.; Daggett, Valerie

    2011-01-01

    High performance computing is leading to unprecedented volumes of data. Relational databases offer a robust and scalable model for storing and analyzing scientific data. However, these features do not come without a costsignificant design effort is required to build a functional and efficient repository. Modeling protein simulation data in a relational database presents several challenges: the data captured from individual simulations are large, multi-dimensional, and must integrate with both simulation software and external data sites. Here we present the dimensional design and relational implementation of a comprehensive data warehouse for storing and analyzing molecular dynamics simulations using SQL Server. PMID:23204646

  8. Protein Simulation Data in the Relational Model.

    PubMed

    Simms, Andrew M; Daggett, Valerie

    2012-10-01

    High performance computing is leading to unprecedented volumes of data. Relational databases offer a robust and scalable model for storing and analyzing scientific data. However, these features do not come without a cost-significant design effort is required to build a functional and efficient repository. Modeling protein simulation data in a relational database presents several challenges: the data captured from individual simulations are large, multi-dimensional, and must integrate with both simulation software and external data sites. Here we present the dimensional design and relational implementation of a comprehensive data warehouse for storing and analyzing molecular dynamics simulations using SQL Server. PMID:23204646

  9. Protein viscoelastic dynamics: a model system.

    PubMed

    Fogle, Craig; Rudnick, Joseph; Jasnow, David

    2015-09-01

    A model system inspired by recent experiments on the dynamics of a folded protein under the influence of a sinusoidal force is investigated and found to replicate many of the response characteristics of such a system. The essence of the model is a strongly overdamped oscillator described by a harmonic restoring force for small displacements that reversibly yields to stress under sufficiently large displacement. This simple dynamical system also reveals unexpectedly rich behavior-exhibiting a series of dynamical transitions and analogies with equilibrium thermodynamic phase transitions. The effects of noise and of inertia are briefly considered and described. PMID:26465510

  10. A Bovine PeptideAtlas of milk and mammary gland proteomes

    PubMed Central

    Bislev, Stine L.; Deutsch, Eric W.; Sun, Zhi; Farrah, Terry; Aebersold, Ruedi; Moritz, Robert L.; Bendixen, Emke; Codrea, Marius C.

    2016-01-01

    Proteome information resources of farm animals are lagging behind those of the classical model organisms despite their important biological and economic relevance. Here we present a Bovine PeptideAtlas, representing a first collection of bovine proteomics datasets within the PeptideAtlas framework. This database was built primarily as a source of information for designing selected reaction monitoring assays for studying milk production and mammary gland health, but it has an intrinsic general value for the farm animal research community. The Bovine PeptideAtlas comprises 1921 proteins at 1.2% false discovery rate (FDR) and 8559 distinct peptides at 0.29% FDR identified in 107 samples from 6 tissues. The PeptideAtlas web interface has a rich set of visualization and data exploration tools, enabling users to interactively mine information about individual proteins and peptides, their genome mappings, and supporting spectral evidence. PMID:22837157

  11. Ultrastructure of bovine sperm chromatin.

    PubMed

    Filho, Romualdo Morandi; Beletti, Marcelo Emilio; de Oliveira, Fabio

    2015-12-01

    Mammalian semen chromatin comprises DNA, protamine, and, at lower levels, other proteins. This constitution confers intense compaction to the chromatin, helping to protect the DNA and causing the head of the sperm to be very small, facilitating the safe transport of its genetic contents. It is known that changes in the sperm chromatin compaction lead to fertility problems in bulls, justifying studies of this structure. Although there are theoretical models of sperm chromatin because of its high compaction, there is no morphological evidence of such models. The aim of this study was to demonstrate the ultrastructure of bovine sperm chromatin in an attempt to corroborate the theoretical chromatin models existing today. The isolated bull sperm heads had their chromatin partially unpacked by chemical treatment using sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) and were then embedded in Epon resin. Using an ultramicrotome, ultrathin sections were obtained, which were contrasted with uranyl acetate and lead citrate, and then viewed under transmission electron microscopy. The methodology used allowed the visualization of toroidal structures interconnected by a filamentous nuclear matrix, which is entirely consistent with the most current theoretical models. Microsc. Res. Tech. 78:1117-1120, 2015. 2015 Wiley Periodicals, Inc. PMID:26515508

  12. Molecular modeling and spectroscopic studies on the interaction of the chiral drug venlafaxine hydrochloride with bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Shahabadi, Nahid; Hadidi, Saba

    2014-03-01

    This study was designed to examine the interaction of racemic antidepressant drug "S,R-venlafaxine hydrochloride (VEN)" with bovine serum albumin (BSA) under physiological conditions. The mechanism of interaction was studied by spectroscopic techniques combination with molecular modeling. Stern-Volmer analysis of fluorescence quenching data shows the presence of the static quenching mechanism. The thermodynamic parameters indicated that the hydrogen bonding and weak van der Waals interactions are the predominant intermolecular forces stabilizing the complex. The number of binding sites (n) was calculated. Through the site marker competitive experiment, VEN was confirmed to be located in subdomain IIIA of BSA. The binding distance (r = 4.93 nm) between the donor BSA and acceptor VEN was obtained according to Förster's non-radiative energy transfer theory. According to UV-vis spectra and CD data binding of VEN leaded to conformational changes of BSA. Molecular docking simulations of S and R-VEN revealed that both isomers have similar interaction and the same binding sites, from this point of view S and R isomers are equal.

  13. Efficacy of a topical bovine-derived thrombin solution as a hemostatic agent in a rodent model of hepatic injury.

    PubMed

    Rosselli, Desiree D; Brainard, Benjamin M; Schmiedt, Chad W

    2015-10-01

    Hemorrhage is a major concern in patients undergoing hepatic surgery or in those with hepatic trauma. In these cases, employing traditional hemostatic strategies can be problematic due to the diffuse nature of hepatic hemorrhage and limited opportunities for direct hemostasis. This study assessed the efficacy of a bovine-derived thrombin solution, (BT), as a topical liquid agent to augment hemostasis and survival following severe hepatic hemorrhage in a rat model. Heart rate (HR), arterial blood pressure (ABP), packed cell volume (PCV), and overall survival were evaluated in 54 rats randomly assigned to receive topical application of BT, saline, or suture ligation applied immediately to a liver lobe following controlled laceration. Six additional rats received liver laceration with no applied treatment. Intravenous fluid resuscitation was initiated and HR and ABP were recorded for 60 min, after which survivors were recovered from anesthesia. Rats were then monitored for 72 h, after which survivors were euthanized. There was no significant difference in survival time, percentage survival, intra-operative ABP or HR, or post-operative PCV between treatment groups. There is insufficient evidence to recommend BT as the sole therapy using this delivery method for mitigating severe hemorrhage from liver injury. PMID:26424911

  14. Model systems to analyze the role of miRNAs and commensal microflora in bovine mucosal immune system development.

    PubMed

    Liang, Guanxiang; Malmuthuge, Nilusha; Guan, Le Luo; Griebel, Philip

    2015-07-01

    Information is rapidly accumulating regarding the role of miRNAs as key regulators of immune system development and function. It is also increasingly evident that miRNAs play an important role in host-pathogen interactions through regulation of both innate and acquired immune responses. Little is known, however, about the specific role of miRNAs in regulating normal development of the mucosal immune system, especially during the neonatal period. Furthermore, there is limited knowledge regarding the possible role the commensal microbiome may play in regulating mucosal miRNAs expression, although evidence is emerging that a variety of enteric pathogens influence miRNA expression. The current review focuses on recent information that miRNAs play an important role in regulating early development of the bovine mucosal immune system. A possible role for the commensal microbiome in regulating mucosal development by altering miRNA expression is also discussed. Finally, we explore the potential advantages of using the newborn calf as a model to determine how interactions between developmental programming, maternal factors in colostrum, and colonization of the gastrointestinal tract by commensal bacteria may alter mucosal miRNA expression and immune development. Identifying the key factors that regulate mucosal miRNA expression is critical for understanding how the balance between protective immunity and inflammation is maintained to ensure optimal gastrointestinal tract function and health of the whole organism. PMID:25467799

  15. Biological activities and physicochemical properties of Maillard reaction products in sugar-bovine casein peptide model systems.

    PubMed

    Jiang, Zhanmei; Wang, Lizhe; Wu, Wei; Wang, Yu

    2013-12-15

    The purpose of this study was to evaluate the biological activities and physicochemical properties of Maillard reaction products (MRPs), derived from aqueous reducing sugar (ribose, galactose and lactose) and bovine casein peptide (BCP) model systems. The fluorescence intensity of ribose-BCP MRPs reached the maximum value within 1h, while it took 3h for galactose-BCP MRPs. Size exclusion chromatography of all the MRPs indicated molecular rearrangements and production of new smaller molecules, as a function of the heating time. The consumption of ribose and amino groups was the highest in the ribose-BCP MRPs. BCP lost its known angiotensin-I-converting enzyme (ACE) inhibitory activity by the Maillard reaction with reducing sugars. Ribose-BCP MRPs had the lowest ACE inhibitory activity, but they showed the highest 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity and ferrous reducing power among all the MRPs. Galactose-BCP MRPs inhibited, slightly the growth of Caco-2 cells, while ribose-BCPand lactose-BCP MRPs had no cytotoxicity. PMID:23993556

  16. Enrofloxacin and Macrolides Alone or in Combination with Rifampicin as Antimicrobial Treatment in a Bovine Model of Acute Chlamydia psittaci Infection

    PubMed Central

    Prohl, Annette; Lohr, Markus; Ostermann, Carola; Liebler-Tenorio, Elisabeth; Berndt, Angela; Schroedl, Wieland; Rothe, Michael; Schubert, Evelyn; Sachse, Konrad; Reinhold, Petra

    2015-01-01

    Chlamydia psittaci is a zoonotic bacterium with a wide host range that can cause respiratory disease in humans and cattle. In the present study, effects of treatment with macrolides and quinolones applied alone or in combination with rifampicin were tested in a previously established bovine model of respiratory C. psittaci infection. Fifty animals were inoculated intrabronchially at the age of 6–8 weeks. Seven served as untreated controls, the others were assigned to seven treatment groups: (i) rifampicin, (ii) enrofloxacin, (iii) enrofloxacin + rifampicin, (iv) azithromycin, (v) azithromycin + rifampicin, (vi) erythromycin, and (vii) erythromycin + rifampicin. Treatment started 30 hours after inoculation and continued until 14 days after inoculation (dpi), when all animals were necropsied. The infection was successful in all animals and sufficient antibiotic levels were detected in blood plasma and tissue of the treated animals. Reisolation of the pathogen was achieved more often from untreated animals than from other groups. Nevertheless, pathogen detection by PCR was possible to the same extent in all animals and there were no significant differences between treated and untreated animals in terms of local (i.e. cell count and differentiation of BALF-cells) and systemic inflammation (i.e. white blood cells and concentration of acute phase protein LBP), clinical signs, and pathological findings at necropsy. Regardless of the reduced reisolation rate in treated animals, the treatment of experimentally induced respiratory C. psittaci infection with enrofloxacin, azithromycin or erythromycin alone or in combination with rifampicin was without obvious benefit for the host, since no significant differences in clinical and pathological findings or inflammatory parameters were detected and all animals recovered clinically within two weeks. PMID:25768665

  17. Ex vivo investigations on the potential of optical coherence tomography (OCT) as a diagnostic tool for reproductive medicine in a bovine model.

    PubMed

    Trottmann, Matthias; Klle, Sabine; Leeb, Regina; Doering, Daniel; Reese, Sven; Stief, Christian G; Dulohery, Kate; Leavy, Myles; Kuznetsova, Julia; Homann, Christian; Sroka, Ronald

    2016-01-01

    Routine infertility investigations in the male and female include imaging techniques such as ultrasonography and endoscopy (fertiloscopy). However, these techniques lack the resolution to localize vital sperm or to reveal detailed morphological analysis of the oviduct which is often the cause of infertility in females. Therefore we set out to evaluate the efficiency of optical coherence tomography (OCT) as a diagnostic imaging tool for micron-scale visualization of the male and female genital tract. Using the bovine as a model, the optical features of the Telesto(TM) , Ganymede(TM) (both Thorlabs) and Niris(TM) (Imalux) OCT imaging systems were compared. Comparative visualization of ex vivo bovine testicular tissue by the Telesto(TM) microscopic optical coherence tomography system (left) and corresponding H&E staining (right). PMID:25808935

  18. Study on protein adsorption kinetics to a dye-ligand adsorbent by the pore diffusion model.

    PubMed

    Zhang, Songping; Sun, Yan

    2002-07-26

    Adsorption kinetics of bovine serum albumin (BSA) and bovine hemoglobin (bHb) to Cibacron Blue 3GA (CB) modified Sepharose CL-6B has been studied. The effects of liquid-phase ionic strength and CB coupling density on the uptake rates of these two proteins in Tris-HCl buffer (pH 7.5) were evaluated by effective pore diffusivity derived from a pore diffusion model. The results showed that despite their similar molecular masses and sizes, the effects of aqueous-phase ionic strength and CB density on the effective pore diffusivities of BSA and bHb were distinctly different. The effective pore diffusivity of BSA to CB-Sepharose increased significantly with decreasing CB density and increasing liquid-phase ionic strength. This was considered due to the decrease in electrostatic repulsion between the BSA and CB molecules of like charge. That is, the increase in ionic strength and the decrease in CB coupling density reduced the electrostatic hindrance effect on BSA diffusion to CB-Sepharose, facilitating the hindered pore diffusion. In contrast, because of the higher isoelectric point of bHb (7.0) compared to BSA (4.7), bHb suffered little electrostatic hindrance effect during its diffusion to CB-Sepharose. Therefore, the effective pore diffusivity of bHb was unchanged with the change in liquid-phase ionic strength and CB coupling density. PMID:12198854

  19. Inelastic neutron scattering analysis of low frequency motion in proteins: A normal mode study of the bovine pancreatic trypsin inhibitor

    NASA Astrophysics Data System (ADS)

    Smith, Jeremy; Cusack, Stephen; Pezzeca, Ulrik; Brooks, Bernard; Karplus, Martin

    1986-09-01

    An improved understanding of the function of macromolecules of biological interest requires the characterization of their low-frequency internal fluctuations (?w?kBT where kBT200 cm-1 at room temperature), which dominate the atomic displacements. To explore the potential or inelastic neutron scattering for supplying information about such motions, we employ the formalism of Zemach and Glauber [A. C. Zemach and R. J. Glauber, Phys. Rev. 101, 118 (1956)] to calculate the one-phonon vibrational incoherent scattering function, Svibinc (q,w), of a small protein, BPTI. An in vacuo normal mode analysis of BPTI [B. Brooks and M. Karplus, Proc. Natl. Acad. Sci. U.S.A. 80, 6571 (1983)], is used for the calculation of Svibinc (q,w). The dependence of Svibinc (q,w) on energy transfer and momentum transfer is investigated and the results are placed in the context of present instrumental capabilities. Although the overall energy dependence of Svibinc (q,w) has a simple form, the variations in the hydrogen atom displacements in the different modes have a significant effect on the scattering. Mode anisotropy is strongly manifested in oriented spectra calculated without instrumental resolution broadening. Contributions from individual atoms and residues to the unbroadened whole molecule scattering are examined in detail. They are found to depend significantly on the atom or residue examined. This suggests that inelastic neutron scattering has considerable potential for the investigation of local dynamic variations in proteins by use of a deuterated protein with specifically protonated residues. Orientational averaging and instrumental resolution broadening of Svibinc (q,w) indicate that the one-phonon scattering detectable on the most suitable current instrument would show strong features arising principally from the frequency distribution of the vibrational modes. Multiphonon scattering is small at low momentum transfers in the low frequency range of most interest (<30 cm-1) but increases rapidly with increasing energy and momentum transfers. It is shown to originate from the lowest frequency modes. The momentum transfer dependence of the one-phonon Svibinc (q,w) is investigated. An expression is found which can be used to extrapolate the calculated one-phonon scattering to zero momentum transfer using experimentally obtainable ranges of momentum transfer. This permits the extraction of an amplitude-weighted frequency distribution function. At current resolutions this function is similar in form to the unweighted frequency distribution function, knowledge of which would be very useful for analysis of protein thermodynamics and dynamics.

  20. Frustration in protein elastic network models

    NASA Astrophysics Data System (ADS)

    Lezon, Timothy; Bahar, Ivet

    2010-03-01

    Elastic network models (ENMs) are widely used for studying the equilibrium dynamics of proteins. The most common approach in ENM analysis is to adopt a uniform force constant or a non-specific distance dependent function to represent the force constant strength. Here we discuss the influence of sequence and structure in determining the effective force constants between residues in ENMs. Using a novel method based on entropy maximization, we optimize the force constants such that they exactly reporduce a subset of experimentally determined pair covariances for a set of proteins. We analyze the optimized force constants in terms of amino acid types, distances, contact order and secondary structure, and we demonstrate that including frustrated interactions in the ENM is essential for accurately reproducing the global modes in the middle of the frequency spectrum.

  1. Markov state models of protein misfolding.

    PubMed

    Sirur, Anshul; De Sancho, David; Best, Robert B

    2016-02-21

    Markov state models (MSMs) are an extremely useful tool for understanding the conformational dynamics of macromolecules and for analyzing MD simulations in a quantitative fashion. They have been extensively used for peptide and protein folding, for small molecule binding, and for the study of native ensemble dynamics. Here, we adapt the MSM methodology to gain insight into the dynamics of misfolded states. To overcome possible flaws in root-mean-square deviation (RMSD)-based metrics, we introduce a novel discretization approach, based on coarse-grained contact maps. In addition, we extend the MSM methodology to include "sink" states in order to account for the irreversibility (on simulation time scales) of processes like protein misfolding. We apply this method to analyze the mechanism of misfolding of tandem repeats of titin domains, and how it is influenced by confinement in a chaperonin-like cavity. PMID:26897000

  2. PreImplantation factor (PIF) detection in maternal circulation in early pregnancy correlates with live birth (bovine model)

    PubMed Central

    2013-01-01

    Background Early identification of viable pregnancy is paramount for successful reproduction. Detection of specific signals from pre-implantation viable embryos in normal pregnancy circulation would indicate initiation of embryo-maternal interaction and create a continuum to accurately reflect embryo/fetal well-being post-implantation. Viable mammalian embryos secrete PreImplantation Factor (PIF), a biomarker which plays key, multi-targeted roles to promote implantation, trophoblast invasion and modulate maternal innate and adaptive immunity toward acceptance. Anti-PIF monoclonal antibody (mAb-based chemiluminescent ELISA) accurately detects PIF in singly cultured embryos media and its increased levels correlate with embryo development up to the blastocyst stage. Herein reported that PIF levels (ELISA) in early maternal serum correlate with pregnancy outcome. Methods Artificially inseminated (AI) blind-coded Angus cattle (N?=?21-23) serum samples (day10,15 & 20 post-AI) with known calf birth were blindly tested, using both non-pregnant heifers (N?=?30) and steer serum as negative controls. Assay properties and anti-PIF monoclonal antibody specificity were determined by examining linearity, spike and recovery experiments and testing the antibody against 234 different circulating proteins by microarray. Endogenous PIF was detected using <3kDa filter separation followed by anti-PIF mAb-based affinity chromatography and confirmed by ELISA and HPLC. PIF expression was established in placenta using anti-PIF mAb-based IHC. Results PIF detects viable pregnancy at day 10 post-AI with 91.3% sensitivity, reaching 100% by day 20 and correlating with live calf birth. All non-pregnant samples were PIF negative. PIF level in pregnant samples was a stringent 3?+?SD higher as compared to heifers and steer sera. Assay is linear and spike and recovery data demonstrates lack of serum interference. Anti-PIF mAb is specific and does not interact with circulating proteins. Anti-PIF based affinity purification demonstrates that endogenous PIF is what ELISA detects. The early bovine placenta expresses PIF in the trophoblast layer. Conclusion Data herein documents that PIF is a specific, reliable embryo-derived biomarker conveniently detectable in early maternal circulation. PIF ELISA emerges as practical tool to detect viable early pregnancy from day 20 post-AI. PMID:24238492

  3. Isolation and characterization of peptides with dipeptidyl peptidase-IV inhibitory activity from pepsin-treated bovine whey proteins.

    PubMed

    Lacroix, Isabelle M E; Li-Chan, Eunice C Y

    2014-04-01

    Inhibition of the enzyme dipeptidyl peptidase (DPP)-IV is one of the strategies used for the treatment of type 2 diabetes. In the present study, pepsin-treated whey protein isolate (WPI) and α-lactalbumin displaying DPP-IV inhibitory activity were fractionated by successive chromatographic steps and the resulting active fractions analyzed for their constituent peptides by liquid chromatography-electrospray ionization-tandem mass spectrometry. Among the identified sequences, 24 peptides derived from α-lactalbumin and 11 from β-lactoglobulin were synthesized and their effects on DPP-IV activity assessed. The most potent fragments, LKPTPEGDL and LKPTPEGDLEIL (IC50=45 and 57 μM, respectively), were found to inhibit DPP-IV in an un-competitive manner. Although several of the peptides tested showed some inhibitory activity, only two were as effective as the un-fractionated WPI hydrolysate and none were as potent as the un-fractionated α-lactalbumin hydrolysate. The peptides' structural features, including length and amino acid composition, were found to impact their inhibitory activity. This study provides new insights on the active components responsible for the DPP-IV inhibitory activity of pepsin-treated whey proteins. PMID:24440459

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

    PubMed Central

    Gatot, Jean-Stéphane; Callebaut, Isabelle; Van Lint, Carine; Demonté, Dominique; Kerkhofs, Pierre; Portetelle, Daniel; Burny, Arsène; Willems, Luc; Kettmann, Richard

    2002-01-01

    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 ligands. In particular, by affinity chromatography, we have shown that BLV SU protein specifically interacted with zinc ions. To identify the protein domains involved in binding, 16 peptides distributed along the sequence were tested. Two of them appeared to be able to interact with zinc. To unravel the role of these SU regions in the biology of the virus, mutations were introduced into the env gene of a BLV molecular clone in order to modify residues potentially interacting with zinc. The fusogenic capacity of envelope mutated within the first zinc-binding region (104 to 123) was completely abolished. Furthermore, the integrity of this domain was also required for in vivo infectivity. In contrast, mutations within the second zinc-binding region (218 to 237) did not hamper the fusogenic capacity; indeed, the syncytia were even larger. In sheep, mutations in region 218 to 237 did not alter infectivity or viral spread. Finally, we demonstrated that the envelope of the related HTLV-1 was also able to bind zinc. Interestingly, zinc ions were found to be associated with the receptor-binding domain (RBD) of Friend murine leukemia virus (Fr-MLV) SU glycoprotein, further supporting their relevance in SU structure. Based on the sequence similarities shared with the Fr-MLV RBD, whose three-dimensional structure has been experimentally determined, we located the BLV zinc-binding peptide 104-123 on the opposite side of the potential receptor-binding surface. This observation supports the hypothesis that zinc ions could mediate interactions of the SU RBD either with the C-terminal part of SU, thereby contributing to the SU structural integrity, or with a partner(s) different from the receptor. PMID:12134000

  5. The role of native bovine alpha-lactalbumin in bovine mammary epithelial cell apoptosis and casein expression.

    PubMed

    Riley, Lisa G; Wynn, Peter C; Williamson, Peter; Sheehy, Paul A

    2008-08-01

    Folding variants of alpha-lactalbumin (alpha-la) are known to induce cell death in a number of cell types, including mammary epithelial cells (MEC). The native conformation of alpha-la however has not been observed to exhibit this biological activity. Here we report that native bovine alpha-la reduced the viability of primary bovine mammary epithelial cells (BMEC) and induced caspase activity in mammospheres, which are alveolar-like structures formed by culturing primary BMEC on extracellular matrix in the presence of lactogenic hormones. These observations suggest a possible role for bovine alpha-la in involution and/or maintaining the luminal space in mammary alveoli during lactation. In addition, co-incubation of bovine alpha-la in an in-vitro mammosphere model resulted in decreased beta-casein mRNA expression and increased alphas1- and kappa-casein mRNA expression. This differential effect on casein expression levels is unusual and raises the possibility of manipulating expression levels of individual caseins to alter dairy processing properties. Manipulation of alpha-la levels could be further investigated for its potential to enhance milk protein expression and/or improve lactational persistency by influencing the balance between proliferation and apoptosis of BMEC, which has a major influence on the milk-producing capacity of the mammary gland. PMID:18680616

  6. Bacteriostatic effect of orally administered bovine lactoferrin on proliferation of Clostridium species in the gut of mice fed bovine milk.

    PubMed Central

    Teraguchi, S; Shin, K; Ozawa, K; Nakamura, S; Fukuwatari, Y; Tsuyuki, S; Namihira, H; Shimamura, S

    1995-01-01

    When milk-fed mice were orally inoculated with Clostridium ramosum C1, this strain proliferated in the gut and became the dominant component of the fecal microflora. In this experimental model, bovine lactoferrin (bLF) administered with milk suppressed the proliferation of this strain in vivo and decreased the numbers of C. ramosum and other bacteria in the feces. This bacteriostatic effect of bLF was dependent on the concentration of bLF, the duration of feeding, and the administered dose of C. ramosum C1. Compared with bovine serum albumin, ovalbumin, bovine whey protein isolate, or bovine casein, only bLF showed this specific activity. A similar effect of bLF was observed after oral inoculation with C. ramosum JCM 1298, C. paraputrificum VPI 6372, or C. perfringens ATCC 13124. A hydrolysate prepared by digestion of bLF with porcine pepsin showed the same inhibitory effect on proliferation of C. ramosum in vivo as occurred with undigested bLF. These results indicate that ingested bLF can exert a bacteriostatic effect against clostridia in the gut even after it has been digested to some extent. PMID:7574587

  7. Glucosylceramide, a neutral glycosphingolipid anticoagulant cofactor, enhances the interaction of human- and bovine-activated protein C with negatively charged phospholipid vesicles.

    PubMed

    Yegneswaran, Subramanian; Deguchi, Hiroshi; Griffin, John H

    2003-04-25

    The effect of glucosylceramide (GlcCer) on activated protein C (APC)-phospholipid interactions was examined using fluorescence resonance energy transfer. Human APC, labeled with either fluorescein (Fl-APC) or dansyl (DEGR-APC) donor, bound to phosphatidylcholine/phosphatidylserine (PC/PS, 9:1 w/w) vesicles containing octadecylrhodamine (OR) acceptor with a K(d) (app) = 16 micro g/ml, whereas Fl-APC (or DEGR-APC) bound to PC/PS/GlcCer(OR) (8:1:1) vesicles with a K(d) (app) = 3 micro g/ml. This 5-fold increase in apparent affinity was not species-specific since bovine DEGR-APC also showed a similar GlcCer-dependent enhancement of binding of APC to PC/PS vesicles. From the efficiency of fluorescence resonance energy transfer, distances of closest approach of approximately 63 and approximately 64 A were estimated between the dansyl on DEGR-APC and rhodamine in PC/PS/GlcCer(OR) and PC/PS(OR), respectively, assuming kappa(2) = 2/3. DEGR-APC bound to short chain C8-GlcCer with an apparent K(d) of 460 nm. The presence of C8-GlcCer selectively enhanced the binding of C16,6-NBD-phosphatidylserine but not C16,6-7-nitrobenz-2-oxa-1,3-diazole (NBD)-phosphatidylcholine to coumarin-labeled APC. These data suggest that APC binds to GlcCer, that PC/PS/GlcCer vesicles like PC/PS vesicles bind to the N-terminal gamma-carboxyglutamic acid domain of APC, and that one mechanism by which GlcCer enhances the activity of APC is by increasing its affinity for membrane surfaces containing negatively charged phospholipids. PMID:12560338