FEBS JOURNAL, Issue 15 2001
The protein remains folded, can form the molten globule in acid solution
Bovine ,-lactalbumin (,-LA) is an ,/, protein which adopts partly folded states when dissolved at low pH (A-state), by removal of the protein-bound calcium at neutral pH and low salt concentration (apo-state), as well as in aqueous trifluoroethanol. Previous spectroscopic studies have indicated that the A-state of ,-LA at pH 2.0, considered a prototype molten globule, has a native-like fold in which the helical core is mostly retained, while the , subdomain is less structured. Here, we investigate the conformational features of three derivatives of ,-LA characterized by a single peptide bond fission or a deletion of 12 or 19/22 amino-acid residues of the , subdomain of the native protein (approximately from residue 34 to 57). These ,-LA derivatives were obtained by limited proteolysis of the protein in its partly folded state(s). A nicked ,-LA species consisting of fragments 1-,3,40 and 41,123 (nicked-LA) was prepared by thermolytic digestion of the 123-residue chain of ,-LA in 50% (v/v) aqueous trifluoroethanol. Two truncated or gapped protein species given by fragments 1,40 and 53,123 (des,1-LA) or fragments 1,34 and 54-,57,123 (des,2-LA) were obtained by digestion of ,-LA with pepsin in acid or with proteinase K at neutral pH in its apo-state, respectively. The two protein fragments of nicked or gapped ,-LA are covalently linked by the four disulfide bridges of the native protein. CD measurements revealed that, in aqueous solution at neutral pH and in the presence of calcium, the three protein species maintain the helical secondary structure of intact ,-LA, while the tertiary structure is strongly affected by the proteolytic cleavages of the chain. Temperature effects of CD signals in the far- and near-UV region reveal a much more labile tertiary structure in the ,-LA derivatives, while the secondary structure is mostly retained even upon heating. In acid solution at pH 2.0, the three ,-LA variants adopt a conformational state essentially identical to the molten globule displayed by intact ,-LA, as demonstrated by CD measurements. Moreover, they bind strongly the fluorescent dye 8-anilinonaphthalene-1-sulfonate, which is considered a diagnostic feature of the molten globule of proteins. Therefore, the , subdomain can be removed from the ,-LA molecule without impairing the capability of the rest of the chain to adopt a molten globule state. The results of this protein dissection study provide direct experimental evidence that in the ,-LA molten globule only the , domain is structured. [source]

Reversal of suppressed metabolism in prolonged cold preserved cartilage

JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2008
Tamara K. Pylawka
Abstract Chondrocytes in cold preserved cartilage are metabolically suppressed. The goal of this study was to address this metabolic suppression and seek ways to reverse it. Specifically, we examined the roles of rewarming protocols and nitric oxide (NO) in this metabolic suppression. Bovine and canine full-thickness articular cartilage explants were cultured under various temperature conditions, and NO production, proteoglycan (PG) synthesis, and cell viability were measured. Nitric oxide was shown to be negatively correlated with PG synthesis following abrupt rewarming of cold preserved osteochondral allografts. Gradual rewarming of the allograft tissue decreased NO production with higher PG synthesis. Inhibition of nitric oxide synthases (NOS) led to a decrease in NO production and a concomitant increase in PG synthesis. We were able to partially reverse metabolic suppression of cold preserved osteochondral allograft material with gradual rewarming and decrease NO production with NOS inhibition. Chondrocytes in cold preserved allograft material may be metabolically suppressed predisposing the graft to failure in vivo. Minimizing this loss of metabolic function by gradual graft rewarming and decreasing NO production by NOS inhibition at the time of graft implantation may have implications on graft survival in vivo. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:247,254, 2008 [source]

Analysis of trace amounts of bovine ,-lactoglobulin in infant formulas by capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection

JOURNAL OF SEPARATION SCIENCE, JSS, Issue 9-10 2005
María Teresa Veledo
Abstract Bovine ,-lactoglobulin (,LG) has been described by several authors as the main allergen present in cow's milk. It can induce allergic reactions even at the low concentration existing in hypoallergenic formulas based on hydrolyzed cow's milk proteins (generally lower than ,M). In this paper, the usefulness of a capillary electrophoresis method with on-capillary derivatization and laser-induced fluorescence detection for the analysis of trace amounts of ,LG in a commercial hypoallergenic formula has been demonstrated. To confirm the identity of the peak of ,LG based on migration time, an immunorecognition step employing an anti-,LG antibody was performed. ,LG was quantitated in the whey and casein fractions of the hypoallergenic formula. The concentration of ,LG in the whey fraction of the formula was about 3 orders of magnitude lower than the average value present in cow's milk. In the casein fraction of the formula, the concentration of ,LG was about 1 order of magnitude lower than in the whey fraction. The method developed was also used for the quality control of three cereal-based infant foods formulated without milk to test the presence or absence of ,LG as an indicator of milk contamination during the fabrication process. ,LG in a concentration of 10,7 M or higher was not observed in any of the cereal-based infant formulas analyzed. [source]

Influence of Maillard reaction conditions on the antigenicity of bovine ,-lactalbumin using response surface methodology

JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 14 2009
Guanhao Bu
Abstract BACKGROUND: Maillard reaction can modify functional properties of proteins. Bovine ,-lactalbumin (,-LA) is often supplemented to the new generation of infant formulae, but it is considered to be a main allergen. However, there is little information on the effect of Maillard reaction on ,-LA antigenicity. The objective of this study was to investigate the influence of Maillard reaction on the antigenicity of ,-LA in conjugates of whey protein isolate (WPI) with glucose under different conditions of protein/sugar weight ratio (0.17,7.83), temperature (40,60 °C) and time (24,120 h) using response surface methodology. RESULTS: Conjugation of WPI with glucose markedly reduced the antigenicity of ,-LA. This reduction in antigenicity could be controlled by regulating the three independent variables weight ratio, temperature and time. A model of optimal reaction conditions for lower antigenicity of ,-LA was established. According to the model, the minimum antigenicity of ,-LA was achieved at 52.8 °C, 78 h and 5.96:1 WPI/glucose weight ratio. WPI/glucose weight ratio had the greatest effect on the antigenicity of ,-LA, while reaction temperature influenced ,-LA antigenicity to a lesser extent. CONCLUSION: Well-controlled Maillard reaction between WPI and glucose is an efficient method to reduce ,-LA antigencity. Copyright © 2009 Society of Chemical Industry [source]

Ovine ooplasm directs initial nucleolar assembly in embryos cloned from ovine, bovine, and porcine cells

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2004
Hamish M. Hamilton
Abstract Here we present ultrastructural and immunocytochemical evidence that ovine ooplasm is directing the initial assembly of the nucleolus independent of the species of the nuclear donor. Intergeneric porcine,ovine somatic cell nuclear transfer (SCNT) and intrageneric ovine,ovine SCNT embryos were constructed and the nucleolus ultrastructure and nucleolus associated rRNA synthesis examined in 1-, 2-, 4-, early 8-, late 8-, and 16-cell embryos using transmission electron microscopy (TEM) and light microscopical autoradiography. In addition, immunocytochemical localization by confocal microscopy of nucleolin, a key protein involved in processing rRNA transcripts, was performed on early 8-, late 8-, and 16-cell embryos for both groups of SCNT embryos. Intergeneric porcine,ovine SCNT embryos exhibited nucleolar precursor bodies (NPBs) of an ovine (ruminant) ultrastructure, but no active rRNA producing fibrillo-granular nucleoli at any of the stages. Unusually, cytoplasmic organelles were located inside the nucleus of two porcine,ovine SCNT embryos. The ovine,ovine SCNT embryos, on the other hand, revealed fibrillo-granular nucleoli in 16-cell embryos. In parallel, autoradiographic labeling over the nucleoplasm, and in particular, the nulcleoli was detected. Bovine,ovine SCNT embryos at the eight-cell stage were examined for nucleolar morphology and exhibited ruminant-type NPBs as well as structures that appeared as fibrillar material surrounded by a rim of electron dense granules, perhaps formerly of nucleolar origin. Nucleolin was localized throughout the nucleoplasm and with particular intensity around the presumptive nucleolar compartments for all developmental stages examined in porcine,ovine and ovine,ovine SCNT embryos. In conclusion, this study suggests that factors within the ovine ooplasm are playing a role in the initial assembly of the embryonic nucleolus in intrageneric SCNT embryos. Mol. Reprod. Dev. 69: 117,125, 2004. © 2004 Wiley-Liss, Inc. [source]

NADPH-Diaphorase Activity and NO Synthase Expression in the Olfactory Epithelium of the Bovine

ANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 3 2010
S. Wenisch
With 2 figures Summary NADPH-diaphorase (NADPH-d) staining of the bovine olfactory epithelium was compared with the immunohistochemical localization of nitric oxide synthase (NOS), soluble guanylyl cyclase, and cGMP (cyclic guanosine 3,,5,-monophosphate). Out of the three isoforms, only the inducible NOS (NOS-II) was found at the epithelial surface correlating with the strong labelling for NADPH-d. In contrast, light diaphorase staining associated with deeper epithelial regions did not coincide with any NOS immunoreactivity. As there is overlapping expression of NOS-II, soluble guanylyl cyclase and cGMP at the luminal surface morphologically occupied by dendritic knobs of olfactory receptor neurons and microvillar endings of supporting cells, the nitric oxide (NO)/cGMP pathway is likely to be involved in modulating the odour signals during olfactory transduction. [source]

Neurotrophin Receptor-like Proteins in the Bovine (Bos taurus) Lymphoid Organs, with Special Reference to Thymus and Spleen

ANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 4 2001
M. B. Levanti
Increasing evidence suggests that neurotrophins could regulate immune functions acting directly or indirectly on immunocompetent cells. The indirect pathway involves stromal cells of the primary and secondary lymphoid organs. In the present study the occurrence of Trk proteins (TrkA, TrkB and TrkC), regarded as the high-affinity signal-transducing receptors for neurotrophins, was investigated in cow lymphoid organs using immunohistochemistry. The thymus and spleen of both fetal and adult animals, and the palatine tonsils, lymph nodes and Peyer's patches of adult animals, were analysed. Unidentified cells displaying TrkA-like immunoreactivity were found in the fetal thymus, whereas those expressing this protein in the adult gland were identified as epithelial cells. In the spleen, immunoreactive TrkA was observed in cells of the white pulp. TrkB immunoreactivity in both fetal and adult thymus and spleen was localized in monocyte/macrophage cells. As a rule, TrkC was absent from the thymus and the spleen independent of the animal's age. Different types of stromal cells, but never the lymphocytes themselves, displayed TrkA, TrkB, or TrkC immunoreactivity in the other lymphoid organs analysed. As in other vertebrate species, Trk proteins in the lymphoid organs of the cow were localized in the stromal, non-lymphoid cells, thus suggesting that neurotrophins might regulate the immune function acting indirectly on lymphocytes. [source]

New Series of N-Substituted Phenyl Ketone Oxime Ethers: Synthesis and Bovine ,3 -Adrenergic Agonistic Activities.

CHEMINFORM, Issue 20 2003
A. El Hadri
Abstract For Abstract see ChemInform Abstract in Full Text. [source]

Study on Interaction of Ginsenosides with Bovine or Human Serum Albumin Using Wavelength Modulation Surface Plasmon Resonance Biosensor

CHINESE JOURNAL OF CHEMISTRY, Issue 5 2006
Xia Liu
Abstract To use a newly developed wavelength modulation surface plasmon resonance (SPR) biosensor, an experimental protocol was developed to investigate the interaction of ginsenosides with serum albumin. With a known concentration of the ginsenosides, bound percentages of the ginsenosides with human serum albumin (HSA) or bovine serum albumin (BSA) were obtained. SPR technique could require no labeling and this method provided the detailed information on association and disassociation of molecules in real time. The results indicate that the sensitivity of wavelength modulation SPR biosensor is sufficient for detection and characterization of binding events involving low-molecular weight compounds and their immobilized protein targets. [source]

Early and Midterm Results of an Alternative Procedure to Homografts in Primary Repair of Truncus Arteriosus Communis

CONGENITAL HEART DISEASE, Issue 3 2010
Pedro Curi-Curi MD
ABSTRACT Background., Repair of truncus arteriosus communis (TAC) in the neonatal and early infant period has become a standard practice. We report our experience on primary repair of TAC with a bovine pericardial-valved woven Dacron conduit as an alternative procedure to homografts, with a focus on early and midterm results. Methods., From January 2001 to December 2007, 15 patients with mean age 1.5 years (range 3 months to 8 years), underwent primary repair of simple TAC. Cases with cardiogenic shock, complex-associated cardiac lesions, or adverse anatomy of the truncal valve were excluded. The Collett and Edwards anatomical type classification of TAC was as follows: type I, 13 (87%); and type II, 2 (13%). Right ventricular outflow tract was reconstructed in all the cases with a bovine pericardial-valved woven Dacron conduit. Results., Overall mortality was 6.6% (1 death due to severe pulmonary hypertension). At a mean follow-up of 31 months (range 6,51), there were no deaths (5-year actuarial survival 93.4%). Out of the 14 midterm survivors, three developed stenosis of the pericardial-valved woven Dacron conduit, but only one underwent interventional procedure including percutaneous balloon dilation with stenting for associated left pulmonary artery hypoplasia. The rate of patients with no surgical or percutaneous reinterventions performed because of obstruction of the right ventricular outflow tract reconstruction in the midterm (5 years) was 86%. Conclusions., Truncus arteriosus communis repair with a bovine pericardial-valved woven Dacron conduit can be performed with a very low perioperative mortality and satisfactory midterm morbidity, favorably compared with that reported for the use of homografts. Interventional cardiac catheterization may delay the time of reoperation for inevitable conduit replacement due to stenosis. [source]

Oestrogen receptor , is expressed in adult human skeletal muscle both at the mRNA and protein level

ACTA PHYSIOLOGICA, Issue 4 2003
A. Wiik
Abstract Aim:, There are two known oestrogen receptors (ER), oestrogen receptor , (ER,) and the recently cloned oestrogen receptor , (ER,). ER, mRNA has been detected in mouse, rat, bovine and human skeletal muscle. ER, mRNA has been detected in bovine skeletal muscle. To our knowledge, no study has investigated the expression of oestrogen receptor , in human skeletal muscle. Therefore, the primary aim of the present investigation was to study ER, mRNA and protein expression in human skeletal muscle. In addition the ER, expression was also studied. Methods:, Muscle biopsies were taken from vastus lateralis in six healthy adults (three women and three men). mRNA expression was detected with real-time PCR (TaqMan) and protein localization by immunohistochemistry. Results:, A clear expression of ER, and ER, mRNA was seen in skeletal muscle in all subjects. The ER, mRNA expression was 180 fold higher compared with that of ER, mRNA. Immunohistochemistry demonstrated positive staining for ER,, but not for ER,, with localization to the nuclei of skeletal muscle fibres. On average, 70% of all nuclei were ER, -positive. Conclusion:, The present study shows for the first time ER, mRNA and protein expression in human skeletal muscle tissue in both males and females. [source]

Preparative capillary zone electrophoresis using a dynamic coated wide-bore capillary

ELECTROPHORESIS, Issue 15 2006
Mahmoud M. Yassine
Abstract Preparative capillary zone electrophoresis separations of cytochrome,c from bovine and horse heart are performed efficiently in a surfactant-coated capillary. The surfactant, dimethylditetradecylammonium bromide (2C14DAB), effectively eliminated protein adsorption from the capillary surface, such that symmetrical peaks with efficiencies of 0.7,million plates/m were observed in 50-µm,id capillaries when low concentrations of protein were injected. At protein concentrations greater than 1,g/L, electromigration dispersion became the dominant source of band broadening and the peak shape distorted to triangular fronting. Matching of the mobility of the buffer co-ion to that of the cytochrome,c resulted in dramatic improvements in the efficiency and peak shape. Using 100,mM bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane phosphate buffer at pH,7.0 with a 100-µm,id capillary, the maximum sample loading capacity in a single run was 160,pmol (2.0,µg) of each protein. [source]

Neurogenesis in explants from the walls of the lateral ventricle of adult bovine brain: role of endogenous IGF-1 as a survival factor

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2003
M. Pérez-Martín
Abstract Previous studies have shown the existence of proliferating cells in explants from bovine (Bos Taurus) lateral ventricle walls that were maintained for several days in vitro in the absence of serum and growth factors. In this study we have characterized the nature of new cells and have assessed whether the insulin-like growth factor-1 (IGF-1) receptor regulates their survival and/or proliferation. The explants were composed of the ependymal layer and attached subependymal cells. Ependymal cells in culture were labelled with glial markers (S-100, vimentin, GFAP, BLBP, 3A7 and 3CB2) and did not incorporate bromodeoxiuridine when this molecule was added to the culture media. Most subependymal cells were immunoreactive for ,III-tubulin, a neuronal marker, and did incorporate bromodeoxiuridine. Subependymal neurons displayed immunoreactivity for IGF-1 and its receptor and expressed IGF-1 mRNA, indicating that IGF-1 is produced in the explants and may act on new neurons. Addition to the culture media of an IGF-1 receptor antagonist, the peptide JB1, did not affect the incorporation of bromodeoxiuridine to proliferating subependymal cells. However, JB1 significantly increased the number of TUNEL positive cells in the subependymal zone, suggesting that IGF-1 receptor is involved in the survival of subependymal neurons. In conclusion, these findings indicate that neurogenesis is maintained in explants from the lateral cerebral ventricle of adult bovine brains and that IGF-1 is locally produced in the explants and may regulate the survival of the proliferating neurons. [source]

Interaction of ostreolysin, a cytolytic protein from the edible mushroom Pleurotus ostreatus, with lipid membranes and modulation by lysophospholipids

FEBS JOURNAL, Issue 6 2003
Kristina Sep
Ostreolysin is a 16-kDa cytolytic protein specifically expressed in primordia and fruiting bodies of the edible mushroom Pleurotus ostreatus. To understand its interaction with lipid membranes, we compared its effects on mammalian cells, on vesicles prepared with either pure lipids or total lipid extracts, and on dispersions of lysophospholipids or fatty acids. At nanomolar concentrations, the protein lysed human, bovine and sheep erythrocytes by a colloid-osmotic mechanism, compatible with the formation of pores of 4 nm diameter, and was cytotoxic to mammalian tumor cells. A search for lipid inhibitors of hemolysis revealed a strong effect of lysophospholipids and fatty acids, occurring below their critical micellar concentration. This effect was distinct from the capacity of ostreolysin to bind to and permeabilize lipid membranes. In fact, permeabilization of vesicles occurred only when they were prepared with lipids extracted from erythrocytes, and not with lipids extracted from P. ostreatus or pure lipid mixtures, even if lysophospholipids or fatty acids were included. Interaction with lipid vesicles, and their permeabilization, correlated with an increase in the intrinsic fluorescence and ,-helical content of the protein, and with aggregation, which were not detected with lysophospholipids. It appears that either an unknown lipid acceptor or a specific lipid complex is required for binding, aggregation and pore formation. The inhibitory effect of lysophospholipids may reflect a regulatory role for these components on the physiological action of ostreolysin and related proteins during fruiting. [source]

Crystal structures of bovine ,-lactoglobulin in the orthorhombic space group C2221

FEBS JOURNAL, Issue 2 2001
Structural differences between genetic variants A, features of the Tanford transition
The crystal structures of ,-lactoglobulin genetic variants A and B have been determined in the orthorhombic space group C2221 (lattice Y) by X-ray diffraction at 2.0 Å and 1.95 Å resolution, respectively. The structural comparison shows that both variants exhibit the open conformation of the EF loop at the pH of crystallization (pH 7.9), in contrast to what has been reported for the same genetic variants at pH 7.1 in the trigonal space group P3221 (lattice Z) [Qin, B.Y., Bewley, M.C., Creamer, L.K., Baker, E.N. & Jameson, G.B. (1999) Protein Sci.8, 75,83]. Furthermore, it was found that the stereochemical environment of Tyr42 changes significantly with pH variation between pH 7 and pH 8. This may provide a structural explanation for an as yet unexplained feature of the Tanford transition, namely the increase in exposure of a tyrosine residue. [source]

Cloning and characterization of an immunoglobulin A Fc receptor from cattle

IMMUNOLOGY, Issue 2 2004
H. Craig Morton
Summary Here, we describe the cloning, sequencing and characterization of an immunoglobulin A (IgA) Fc receptor from cattle (bFc,R). By screening a translated EST database with the protein sequence of the human IgA Fc receptor (CD89) we identified a putative bovine homologue. Subsequent polymerase chain reaction (PCR) amplification confirmed that the identified full-length cDNA was expressed in bovine cells. COS-1 cells transfected with a plasmid containing the cloned cDNA bound to beads coated with either bovine or human IgA, but not to beads coated with bovine IgG2 or human IgG. The bFc,R cDNA is 873 nucleotides long and is predicted to encode a 269 amino-acid transmembrane glycoprotein composed of two immunoglobulin-like extracellular domains, a transmembrane region and a short cytoplasmic tail devoid of known signalling motifs. Genetically, bFc,R is more closely related to CD89, bFc,2R, NKp46, and the KIR and LILR gene families than to other FcRs. Moreover, the bFc,R gene maps to the bovine leucocyte receptor complex on chromosome 18. Identification of the bFc,R will aid in the understanding of IgA,Fc,R interactions, and may facilitate the isolation of Fc,R from other species. [source]

Identification of antigenic targets of paraproteins by expression cloning does not support a causal role of chronic antigenic stimulation in the pathogenesis of multiple myeloma and MGUS

INTERNATIONAL JOURNAL OF CANCER, Issue 2 2007
Klaus-Dieter Preuss
Abstract Antigenic targets of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) paraproteins have been suggested to play an important role as growth stimulators in the pathogenesis of these neoplasms. To identify such targets, we screened cDNA libraries from human testis, lung and breast cancer, bovine and porcine muscle and wheat germ for reactivity with paraproteins in the sera from 115 patients with MGUS and MM. Of >6 × 108 paraprotein,antigen interactions screened, an IgA paraprotein from a female patient bound to sperm-specific cylicin-2, and 3 IgG paraproteins bound to tripeptidyl-peptidase-II (TPP-2), insulin-like growth-factor binding-protein-2 (IGFBP-2) and porcine kinesin. Specificity was confirmed by reverse Western blots using recombinant antigens. The broad spectrum of auto-, allo- and heteroantigens as targets of human paraproteins in patients without signs of chronic antigenic stimulation renders a causal role of the antigenic stimulus in the pathogenesis of MGUS and MM unlikely. © 2007 Wiley-Liss, Inc. [source]

A study of the growth of Lactobacillus acidophilus in bovine, ovine and caprine milk

INTERNATIONAL JOURNAL OF DAIRY TECHNOLOGY, Issue 1 2003
Alexandra P Drakoularakou
The fermentation of reconstituted bovine, ovine and caprine milks with Lactobacillus acidophilus was investigated. Better growth of Lb. acidophilus was observed in ovine milk, but higher acidities developed in caprine milk. The high acidity produced after 12 h of fermentation (pH 3.9) created a hostile environment for the survival of the microorganism in caprine milk. [source]

Effect of milk source on the rheological properties of yogurt during the gelation process

INTERNATIONAL JOURNAL OF DAIRY TECHNOLOGY, Issue 3 2001
R Y Jumah
This paper presents research on the effect of milk source on the rheological properties of curd during the gelation process of yogurt. The highest value for viscosity was exhibited by ovine milk, followed by caprine, bovine and camel milks. For bovine, ovine and caprine milk, three different transient viscosity stages were identified and described by mathematical expressions, whereas camel milk showed no significant variation in viscosity during gelation. The chemical composition of milk, namely total solids and protein content, has a major effect on the rheological properties of curd. A power law model allows the determination of the flow behaviour index and the consistency coefficient of curd made from different milk sources. [source]

Gamma,delta T cell subsets are differentially associated with granuloma development and organization in a bovine model of mycobacterial disease

INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 6 2009
Brandon L. Plattner
Summary The characteristic lesion in bovine tuberculosis is well-organized respiratory granulomas. This is typically associated with a strong T-helper 1 biased cell-mediated immune response and eventual containment of the infection. In bovine paratuberculosis, the classic lesion is unorganized granulomatous intestinal inflammation. Clinical paratuberculosis is associated with a T-helper 2 biased humoral immune response and eventual death because of inability of the host to contain the infection. Recent reports have suggested that gamma,delta (,,) T cells play a significant role in granuloma development and/or maintenance during initial stages of infection and may influence the subsequent adaptive immune response. The objective of this study was to use an in vivo bovine model to evaluate ,, T cells during the early host immune response to mycobacterial infection. We used immunofluorescent staining, hyperspectral microscopy, and computerized assisted morphometry to evaluate staining and distribution of ,, T cells during development of organized and unorganized granulomas. Our data suggest that bovine ,, T cell subsets are differentially recruited to early infection sites, and may be instrumental during the initial antimycobacterial host immune response as well as for granuloma organization. [source]

From collagen chemistry towards cell therapy , a personal journey

INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2007
Michael E. Grant
Summary The Fell,Muir Award requires the recipient to deliver a lecture and a review manuscript which provides a personal overview of significant scientific developments in the field of matrix biology over the period of the recipient's career. In this context, this review considers the collagen family of structural proteins and the advances in biochemical, molecular biological and genetic techniques which led to the elucidation of the structure, synthesis and function of this important group of extracellular matrix constituents. Particular attention is focussed on early research on the identification and assembly of the soluble precursors of collagen types I and II, and the identification of the precursor of basement membrane collagen type IV. In subsequent studies investigating the maintenance of the chick chondrocyte phenotype in culture, the influence of the extracellular milieu was found to influence markedly both cell morphology and collagen gene expression. These studies led to the discovery of collagen type X whose expression is restricted to hypertrophic chondrocytes at sites of endochondral ossification. Such research provided a prelude to investigations of mammalian endochondral ossification which is known to be aberrant in a variety of human chondrodysplasias and is reactivated in bone fracture repair and in osteoarthritis. The cloning of bovine and then human collagen type X genes facilitated studies in relevant human diseases and contributed to the discovery of mutations in the COL10A1 gene in families with metaphyseal chondrodysplasia type Schmid. Clustering of mutations in the C-terminal domain of the type X collagen molecule has now been widely documented and investigations of the pathogenic mechanisms in animal models are beginning to suggest the prospect of novel treatment strategies. [source]

Molecular genetic characterization of Robertsonian translocations in cattle

JOURNAL OF ANIMAL BREEDING AND GENETICS, Issue 6 2001
H. Joerg
The chromosome fusion of acrocentric chromosomes, known as Robertsonian translocations, are the most common chromosome rearrangement in Bovidae. Cytogenetic studies revealed differences between the centromeres of Robertsonian translocations: the rob(1; 29) is called monocentric, whereas rob(14; 20) is a dicentric chromosome. To analyse the type of fusion, satellite sequences were hybridised to metaphase chromosomes of carriers of rob(1; 29) from different breeds and rob(14; 20) from the Simmental breed. A repeat element of the bovine 1.715 satellite was located in the centromeric regions of all 29 bovine acrocentric chromosomes. No signals were observed on either the X-,Y- or the rob(1; 29) chromosomes. In contrast, all rob(14; 20) chromosomes gave a distinct hybridisation signal. Microsatellite markers in the linkage group, originating from the fusion, revealed a characteristic allele combination for rob(1; 29) in all carriers and were able to confirm the screening of metaphases of 220 daughters of a heterozygote carrier of the rob(1; 29). The results indicate that rob(1; 29) lost parts of both centromeres and that the 1.715 satellite DNA is not necessary for the functioning of the centromere. Furthermore, rob(1; 29) appears to derive from the same mutation and is transmitted according to Mendelian law. Molekulargenetische Charakterisierung von Robertson'schen Translokationen beim Rind Die Fusion von akrozentrischen Chromosomen, bekannt als Robertson'sche Translokationen, sind die häufigsten Chromosomenveränderungen in der Rindergattung. Zytogenetische Studien zeigten Unterschiede in den Zentromeren von Robertson'schen Translokationen auf. Das Rob(1; 29) Chromosom wird als ein monozentrisches und das Rob(14/20) als ein dizentrisches Chromosom bezeichnet. Um die Fusionsarten zu analysieren, wurden Satellitensequenzen auf Metaphasenchromosomen von Trägern der Rob(1; 29) aus verschiedenen Rassen und von Trägern der Rob(14; 20) aus der Rasse der Simmentaler hybridisiert. Eine Sequenzwiederholung aus dem Rindersatelliten 1.715 wurde in den Zentromerregionen aller 29 akrozentrischer Rinderchromosomen nachgewiesen. Auf dem X, dem Y wie auch auf dem Rob(1; 29) Chromosom konnten jedoch keine Signale beobachtet werden, während alle Rob(14; 20) Chromosomen ein starkes Hybridisierungssignal aufwiesen. Die Mikrosatelliten in der Kopplungsgruppe, welche durch die Fusion entstanden ist, zeigten eine charakteristische Allelkombination für das Rob(1; 29) Chromsom und konnten Untersuchungen an den Metaphasen von 220 Töchtern eines heterozygoten Trägers bestätigen. Die Ergebnisse weisen darauf hin, dass Rob(1; 29) Chromosomen einen Teil beider Zentromere verloren haben und dass die 1.715 Satelliten DNA für ein funktionierendes Chromosom nicht notwendig ist. Die Rob(1; 29) Chromosomen scheinen eine identische Abstammung aufzuweisen und werden nach den Mendel'schen Regeln vererbt. [source]

Host range and lytic capability of four bacteriophages against bovine and clinical human isolates of Shiga toxin-producing Escherichia coli O157:H7

JOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2009
Y.D. Niu
Abstract Aims:, To evaluate host range and lytic capability of four bacteriophages (rV5, wV7, wV8 and wV11) against Escherichia coli O157:H7 (STEC O157:H7) from cattle and humans. Methods and Results:, Four hundred and twenty-two STEC O157:H7 isolates (297 bovine; 125 human) were obtained in Alberta, Canada. The four phages were serially diluted and incubated for 5 h with overnight cultures of STEC O157:H7 to estimate their multiplicity of infection (MOI). All bovine STEC O157:H7 were subjected to pulsed-field gel electrophoresis (PFGE) and phage typing (PT). Phage wV7 lysed all human and bovine isolates irrespective of PFGE genotype or PT phenotype and exhibited the lowest MOI (0·004,0·006, P < 0·0001) of all phages. Phages rV5 and wV11 exhibited a lower MOI (0·002,0·04, P < 0·0001) than did phage wV8 (25,29) and they had a narrower host range than wV7 or wV8. Phages rV5, wV11 and wV8 lysed 342 (81·0%), 321 (76·1%) and 407 (96·4%), respectively, of the 422 isolates. Susceptibility of bovine STEC O157:H7 to rV5, w11 and wV8 was influenced by PFGE genotype and/or PT phenotype. Conclusions:, Phages exhibited activity against the majority of bovine and human STEC O157:H7 isolates. PFGE genotype and/or PT phenotype of the host-target influenced their vulnerability to phage attack. Susceptibility of bovine STEC O157:H7 to phage may also differ among farms. Both lytic capability and host range should be considered in the selection of therapeutic phage for on-farm control of STEC O157:H7. Significance and Impact of the Study:, The present work indicates that a four-phage cocktail should be equally effective at mitigating STEC O157:H7 isolates both of bovine and of human origin. Given that some STEC O157:H7 exhibited resistance to some but not all phages, a phage cocktail is the logical approach to efficacious on-farm therapy. [source]

Comparison of Campylobacter jejuni genotypes from dairy cattle and human sources from the Matamata-Piako District of New Zealand

JOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2008
B.J. Gilpin
Abstract Aims:, To identify the prevalence and types of Campylobacter jejuni carried by dairy cattle and the extent of overlap of these types with those causing disease in humans. Methods and Results:, Faecal samples from 410 dairy cattle were collected from 36 farms in the Matamata-Piako district in New Zealand. Campylobacter jejuni was isolated on all 36 farms, with a prevalence of 51% (95% CI 45,57) in dairy cattle and 65% (95% CI 58,72) in calves. Eighty-nine of these isolates were typed using Penner serotyping and pulsed field gel electrophoresis and were compared with 58 human C. jejuni isolates from people resident within this study area. Conclusions:,Campylobacter jejuni were found in the faeces of over half of the dairy cows and calves examined. Twenty-one per cent of the bovine isolates and 43% of the human isolates formed indistinguishable clusters of at least one bovine and one human isolate. Significance and Impact of the Study:, While a direct link between bovine isolates and human cases was not demonstrated, the finding of indistinguishable genotypes among C. jejuni isolates from bovine and human sources confirms that dairy cows and calves are a potential source of human campylobacteriosis. Barriers to separate bovine faecal material from the general public are therefore important public health measures. [source]

Vascular smooth muscle cell growth-promoting factor/F-spondin inhibits angiogenesis via the blockade of integrin ,v,3 on vascular endothelial cells

JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2001
Yoshito Terai
Vascular smooth muscle cell growth-promoting factor (VSGP) was originally isolated from bovine ovarian follicular fluid as a stimulator of vascular smooth muscle cell proliferation. Homology searches indicate that bovine and human VSGPs are orthologs of rat F-spondin. Here, we examined whether recombinant human VSGP/F-spondin affected the biological activities of endothelial cells. VSGP/F-spondin did not affect the proliferation of human umbilical vein endothelial cells (HUVECs); however, it did inhibit VEGF- or bFGF-stimulated HUVEC migration. To clarify the mechanism of this inhibitory effect, we examined the adhesion of HUVECs to extracellular matrix proteins. VSGP/F-spondin specifically inhibited the spreading of HUVECs on vitronectin via the functional blockade of integrin ,v,3. As a result, VSGP/F-spondin inhibited the tyrosine phosphorylation of focal adhesion kinase (FAK) when HUVECs were plated on vitronectin. Moreover, VSGP/F-spondin inhibited the activation of Akt when HUVECs on vitronectin were stimulated with VEGF. VSGP/F-spondin inhibited tube formation by HUVECs in vitro and neovascularization in the rat cornea in vivo. These results indicate that VSGP/F-spondin inhibits angiogenesis at least in part by the blockade of endothelial integrin ,v,3. © 2001 Wiley-Liss, Inc. [source]

Levels of aloe mortality with and without elephants in the Thicket Biome of South Africa

AFRICAN JOURNAL OF ECOLOGY, Issue 2 2009
D. M. Parker
Abstract Studies concerning the influence of African elephants (Loxodonta africana) on vegetation have produced contradictory results; some show minimal or no effect while others report significant elephant-induced effects. Elephants are generalist megaherbivores but will selectively feed from preferred plant species. We investigated the mortality of aloe plants (highly preferred food items for elephants) at five sites with elephants (treatment) and five paired sites without elephants (control) in the Eastern Cape Province of South Africa. A significantly higher proportion of aloes were dead at treatment sites and significantly more aloes that had lost their crown (headless) were found at treatment sites compared with controls. We conclude that although the proportions of dead aloes at treatment sites were significantly higher, it remains unclear whether there is a need to be concerned with the potential small-scale extinction of aloes from parts of the Eastern Cape Province. The observed mortality may merely be an artefact of the loss of large herbivores through disease (e.g. rinderpest) and hunting in the past. Résumé Des études de l'influence des éléphants africains (Loxodonta africana) sur la végétation ont produit des résultats contradictoires: certaines montrent un effet minimal, voire nul, alors que d'autres signalent des effets significatifs. Les éléphants sont des grands herbivores généralistes, mais ils se nourrissent sélectivement de certaines parties des plantes. Nous avons étudié la mortalité de plants d'aloès (nourriture fortement privilégiée par les éléphants) à cinq sites avec éléphants (traitement) et à cinq autres sites sans éléphant (témoins), dans la Province du Cap oriental, en Afrique du Sud. Il y avait une proportion significativement plus grande d'aloès morts dans les sites du traitement, et ils étaient significativement plus nombreux à avoir perdu leur couronne (étêtés) que dans les sites témoins. Nous concluons que, bien que la proportion d'aloès morts soit significativement plus grande dans les sites du traitement, il n'est pas évident de savoir s'il faut s'inquiéter de cette possible extinction à petite échelle des aloès dans certaines parties de la Province du Cap oriental. La mortalité observée peut bien être simplement un artefact de la perte des grands herbivores en raison de maladies (ex. la peste bovine) et de la chasse dans le passé. [source]

PREPARATION AND CHARACTERIZATION OF PEPSIN-SOLUBILIZED TYPE I COLLAGEN FROM THE SCALES OF SNAKEHEAD (OPHIOCEPHALUS ARGUS)

JOURNAL OF FOOD BIOCHEMISTRY, Issue 1 2009
WENTAO LIU
ABSTRACT Pepsin-solubilized collagen prepared from the scales of snakehead (Ophiocephalus argus) was separated into two fractions, major and minor, by NaCl precipitation. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), amino acid composition, and secondary structure showed that the major collagen was typical type I collagen; in contrast, the minor collagen might be classified as type V collagen from the SDS-PAGE patterns and precipitation properties by NaCl. A sharp decrease in solubility of type I collagen was observed at the NaCl concentration of 40 g/L. The maximum and the minimum solubilities of collagen were observed at pH 3 and 8, respectively. Peptide maps of type I collagen digested by trypsin and V8 protease were different from those of calfskin and fish skin collagens. The imino acid content of type I collagen was lower than those of mammalian collagens and so did its denaturation temperature that was 30.3C obtained by viscosity measurement. PRACTICAL APPLICATIONS Collagen has been widely utilized as a material for foods, cosmetics, and pharmaceuticals. However, the use of collagen-derived products from land animals (e.g., bovine and pig) has been called into question because of foot-and-mouth disease crisis etc. Aquatic animal offals, which are readily available and inexpensive, seem to be safe sources for extraction of collagen. This work reports on preparation and characterization of collagen from snakehead scales, which will have potential in supplementing the skins and bones of land animals as an important collagen resource for use in functional food, biomedical, and cosmetic industries. [source]

Comparison of bovine and porcine ,-lactoglobulin: a mass spectrometric analysis

JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 6 2006
Gaetano Invernizzi
Abstract Nano-electrospray-ionization mass spectrometry (nano-ESI-MS) is applied to comparison of bovine and porcine ,-lactoglobulin (BLG and PLG). The conformational and oligomeric properties of the two proteins under different solvent and experimental conditions are analyzed. The pH-dependence of dimerization is described for the pH range 2,11. The results indicate maximal dimer accumulation at pH 6 for BLG and pH 4 for PLG, as well as a lower stability of the PLG dimer at pH 4 compared to BLG at pH 6. Conformational stability appears to be higher for BLG at acidic pH, but higher for PLG at basic pH. The higher stability of BLG at low pH is revealed by means of either chemical or thermal denaturation. Equilibrium folding intermediates of both proteins are detected. Finally, conditions are found that promote dissociation of the BLG dimer at pH 6 into folded monomers. Copyright © 2006 John Wiley & Sons, Ltd. [source]

Sequence analysis of genes encoding structural and nonstructural proteins of a human group B rotavirus detected in Calcutta, India

JOURNAL OF MEDICAL VIROLOGY, Issue 4 2001
Nobumichi Kobayashi
Abstract Nucleotide sequences of RNA segments encoding structural proteins(VP4, VP6, and VP7) and nonstructural proteins(NSP1 and NSP3) of a human group B rotavirus CAL-1, which was detected in Calcutta, India, were determined and their relatedness with cognate genes of other group B rotaviruses was analyzed. The CAL-1 genes showed generally high sequence identities (more than 90%) to those of human group B rotavirus, adult diarrheal rotavirus (ADRV) in China, while identities with bovine, murine, and ovine viruses were considerably lower (58,73%). Among RNA segments analyzed, sequence identity of the VP6 gene was relatively high compared with other gene segments. In the CAL-1 VP7 sequence, many characteristics were shared by ADRV, but not by other animal group B rotaviruses. In contrast, VP4 and NSP3 of CAL-1 were single animo acid and 23 amino acids longer than those of ADRV strain, respectively, due to differences of a few nucleotides. These findings suggested that human group B rotaviruses CAL-1 and ADRV might have originated from a common ancestral virus distinct from animal group B rotaviruses reported so far, while some notable sequence differences indicated the distinct nature of these viruses. J. Med. Virol. 64:583,588, 2001. © 2001 Wiley-Liss, Inc. [source]

Full-length bovine spp24 [spp24 (24-203)] inhibits BMP-2 induced bone formation,

JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2008
Chananit Sintuu
Abstract Secreted phosphoprotein 24 kDa (spp24) is a bone matrix protein. It contains a TGF-, receptor II homology 1 (TRH1) domain. A cyclic, synthetic 19 amino acid peptide (bone morphogenetic protein binding peptide or BBP) based on the sequence of the TRH1 domain enhances BMP-2 induced osteogenesis. Many observations suggest that different size forms of this protein have very different effects (inhibiting or enhancing) on BMP-2 induced osteogenesis. Using the stable recombinant Met(His)6 -tagged secretory form of full-length (fl) bovine spp24 [Met(His)6 -spp24 (residues 24,203)] and transgenic (TG) mice expressing fl bovine spp24 (residues 1,203), we have demonstrated that spp24 inhibits BMP-2 induced bone formation. The effects of Met(His)6 -spp24 (24,203) were determined in the ectopic bone-forming bioassay in male mice. Implantation of 5 µg of BMP-2 stimulated bone formation, assessed densitometrically as bone area and mineral content. When Met(His)6 -spp24 (24,203) was implanted with BMP-2, it elicited a dose-dependent decrease in BMP-2-medicated ectopic bone formation. When added at a 50-fold excess (w/w), Met(His)6 -spp24 (24,203) completely ablated the effects of BMP-2, while addition of a 10-fold excess had no effect. Constitutive expression of fl bovine spp24 (1,203) under the control of the osteocalcin promoter in TG female mice reduced femoral and vertebral bone mineral density at 3 months of age and reduced femoral BMD at 8 months of age, but had no effects in male mice, which can exhibit less osteocalcin-promoter driven gene transcription than females. Histomorphometric analysis demonstrated that bone volume and trabecular thickness were lower in TG female mice at 3 months of age than in sex- and age-matched wild type (WT) controls. Thus, fl spp24 and its secretory isoform (Met(His)6 -spp24 [24,203]), which contain a BMP-binding or TRH1 motif, inhibit ectopic bone formation in male mice and adversely affects BMD and histological parameters related to bone mass and formation in female mice expressing the human transgene. Under these conditions, fl spp24 acts as a BMP antagonist in vivo. © 2008 Orthopaedic Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:753,758, 2008 [source]