Detection of infectious Bovine polyomavirus

Bovine polyomavirus (BPyV) is a member of the Polyomaviridae, a virus that was originally thought to be of simian origin but was later shown to be of bovine origin, the primate cultures having been contaminated through the use of foetal bovine serum. The significance of this agent to the biotechnology industry cannot be underestimated. The presence of BPyV in serum batches poses a serious risk for the contamination of human therapeutic products. The current PCR based assays provide a means of detecting virus sequences but give no indication as to the infectious nature of the virus. The communication reports the successful development of an assay to detect infectious BPyV using an in vitro amplification system followed by PCR. A lengthy culture period on bovine cells was required before replicating BPyV could be detected and distinguished from non-replicating virus in the cell culture supernatant. A mock-test assay using foetal bovine serum positive for BPyV showed that there was no evidence of replicating BPyV in the serum sample. The BPyV spiked serum control showed that replicating virus was present thus confirming that the serum itself did not inhibit replication of the virus. Cells harvested during the culture period were subjected to fixation, embedding and sectioning and examined by electron microscopy. Intact virus-like particles of approximately 40-50nm were observed in the nucleus of the bovine kidney cells, the site of polyomavirus replication.     

The effects of newborn calf serum and foetal bovine serum on the Na+ + K+-activated adenosine triphosphatase activity in 3T3 and SV3T3 cells grown to confluency

The effects of cell density and growth in 10% foetal bovine serum and 10% newborn calf serum on the activity of the enzyme (Na+ + K+)-ATPase were studied in 3T3 and SV3T3 cells. The enzyme activity decreases in 3T3 cells grown in foetal bovine serum as the cells approach confluency while in those grown in newborn calf serum the enzyme activity increases. The (Na+ + K+)-ATPase activity does not change with increase in cell density in SV3T3 cells grown in foetal bovine serum while the enzyme activity in those grown in newborn calf serum increases with increase in cells density up to about 1.35 x 10(5) cells/sq. cm. and then decreases with further increase in cell number. At confluency it was found that the enzyme activity is higher in the SV3T3 as compared to the 3T3 cells when the cells were grown in 10% foetal bovine serum, whereas in those grown in 10% newborn calf serum the enzyme activity is higher in the 3T3 as compared to the SV3T3 cells.     

Polyamine degradation in foetal and adult bovine serum.

1. Using protein-separative chromatographic procedures and assays specific for putrescine oxidase and spermidine oxidase, adult bovine serum was found to contain a single polyamine-degrading enzyme with substrate preferences for spermidine and spermine. Apparent Km values for these substrates were approx. 40 microM. The apparent Km for putrescine was 2 mM. With spermidine as substrate, the Ki values for aminoguanidine (AM) and methylglyoxal bis(guanylhydrazone) (MGBG) were 70 microM and 20 microM respectively. 2. Bovine serum spermidine oxidase degraded spermine to spermidine to putrescine and N8-acetylspermidine to N-acetylputrescine. Acrolein was produced in all these reactions and recovered in quantities equivalent to H2O2 recovery. 3. Spermidine oxidase activity was present in foetal bovine serum, but increased markedly after birth to levels in adult serum that were almost 100 times the activity in foetal bovine serum. 4. Putrescine oxidase, shown to be a separate enzyme from bovine serum spermidine oxidase, was present in foetal bovine serum but absent from bovine serum after birth. This enzyme displayed an apparent Km for putrescine of 2.6 microM. The enzyme was inhibited by AM and MGBG with Ki values of 20 nM. Putrescine, cadaverine and 1,3-diaminopropane proved excellent substrates for the enzyme compared with spermidine and spermine, and N-acetylputrescine was a superior substrate to N1- or N8-acetylspermidine.     

蚯蚓纤溶酶底物特异性

蚯蚓纤溶酶是具有较强溶栓作用的丝氨酸蛋白酶,为研究蚯蚓纤溶酶在溶栓的同时是否对其他血液蛋白有破坏作用,本实验以一些血液功能蛋白为底物,用蚯蚓纤溶酶在体外对其进行水解,采用SDS—PAGE检测水解前后底物成分的变化。结果显示,蚯蚓纤溶酶短时间内能完全水解纤维蛋白和纤维蛋白原,长时间作用下能部分水解牛血清白蛋白和人免疫球蛋白重链,不水解牛血红蛋白、牛血超氧化物歧化酶和牛凝血酶原。表明蚯蚓纤溶酶对血栓成分具有较强的识别和水解能力,而对其他血液蛋白作用微弱,说明蚯蚓纤溶酶作为药物使用时具有较强的溶栓和预防血栓形成的作用,且副作用较小。     

Serum is a rich source of ligands for the scavenger receptor of hepatic sinusoidal endothelial cells

The present study was prompted by findings in our laboratory showing that serum effectively inhibits scavenger receptor (SR)-mediated endocytosis in hepatic sinusoidal endothelial cells (SEC). Experiments with SEC in vitro showed that the presence of 20% human serum inhibited endocytosis of SR ligands, ¹²⁵I-formaldehyde treated bovine serum albumin (FSA) and ¹²⁵I-nidogen, by 30 and 50%, respectively, whereas pre-heated foetal bovine serum (10%) inhibited endocytosis of ¹²⁵I-FSA by as much as 56%. Human, bovine and rat serum had similar inhibitory effect on endocytosis in SEC. Fractionation of foetal bovine and human serum on anion exchange chromatography demonstrated that the inhibitory principle co-purified with macromolecules of high negative charge. The serum fraction that most effectively inhibited SR-mediated endocytosis of ¹²⁵I-FSA did not affect mannose receptor-mediated endocytosis of ¹²⁵I-mannan to the same extent. Trap-labelled negatively charged serum fraction administered intravenously to rats was eliminated almost exclusively by liver, with a blood decay of 50% over the first 3 min after injection. Isolation of liver cells showed that the populations of Kupffer cells and SEC contained 39 and 61% of liver radioactivity 30 min after injection of trap-labelled negatively charged fractions prepared from pre-heated (complement inactivated) foetal bovine sera. These findings suggest that the process of serum formation from native blood generates appreciable amounts of macromolecules that compete specifically with the SR for endocytosis in SEC. The inhibitory power of pre-heated serum is particularly great. For this reason pre-heated serum should be used with caution in studies of SR in SEC.     

Immunohistochemical studies on glucagon, glicentin and pancreatic polypeptide in human stomach: normal and pathological conditions

Summary Endocrine-like cells containing glucagon, glicentin or pancreatic polypeptide immunoreactivity in human foetal and adult stomach, with or without disease, were studied with the indirect immunoperoxidase method and mirror sectioning technique. In foetal and neonatal oxyntic mucosae, there were endocrine-like cells with glucagon and glicentin immunoreactivities and argyrophilia. Cells containing glicentin immunoreactivity alone were detected earlier than glucagon cells during foetal development, and were also distributed throughout foetal to neonatal life. Bovine pancreatic polypeptide immunoreactivity coexisted in a subpopulation of the glucagon-glicentin cells. These cells were absent from normal oxyntic mucosa in the postneonatal period and from normal antral mucosa throughout life. Hamartomatous polyp in adult oxyntic mucosa, hyperplastic oxyntic mucosa in Menetrier's disease and atrophic oxyntic mucosa in a remnant stomach with cancer showed scattered glucagon-glicentin cells, but few or no cells containing bovine pancreatic polypeptide. Intestinalized mucosa showed plentiful glicentin cells with occasional glucagon and/or bovine pancreatic polypeptide immunoreactivity. Some gastric cancer cells of both diffuse and adenoplastic types contained immunoreactive glicentin and, less frequently, glucagon. Bovine pancreatic polypeptide immunoreactivity was detected in a few adenoplastic cancer cells, but not in diffuse type cells. Three different anti-pancreatic polypeptide sera against bovine, porcine or human pancreatic polypeptide detected basically the same cells mentioned above, but pancreatic polypeptide cells lacking human pancreatic polypeptide immunoreactivity were also present in foetal oxyntic mucosa. Immunoabsorption tests revealed that the bovine pancreatic polypeptide immunoreactivity was remote from peptide YY and neuropeptide Y.     

Isolation and molecular characterization of bovine Rhesus-like transcripts and chromosome mapping of the relevant locus

The bovine erythrocyte membrane carries Rhesus (Rh)-like proteins. To obtain a bovine nucleotide probe, a cDNA library of foetal liver was constructed and screened with the human RhCE probe. Three clones (245 bp, 1012 bp and 1400 bp) were isolated and sequenced. They share a high degree of similarity (up to 73%) with Rh-like cDNAs of primates characterized so far and all of them were shown to contain a polymorphic microsatellite in their 3' untranslated region. Their sequences support the occurrence of different splicing isoforms transcribed from the same RH-like gene. One of the clones (1400 bp), which has a 134-nucleotide deletion causing a frameshift, is structurally similar to the human Rh4 cDNA isoform. Synteny mapping and genetic linkage analysis located the bovine RH-like locus on chromosome BTA2, on which none of the 10 previously mapped blood group systems are found. In situ hybridization mapped the RH-like locus to BTA2q45. No linkage was detected between the microsatellite and the only unmapped blood group system (locus F). These results strongly suggest that the putative bovine Rh-like polypeptides do not correspond to any previously described bovine blood group. Comparative studies of human and bovine maps clearly show that the human RH locus, which is located on HSA1p34-p36, and its bovine counterpart belong to a linkage group highly conserved between both species.     

Detection and identification of the atypical bovine pestiviruses in commercial foetal bovine serum batches

The recently emerging atypical bovine pestiviruses have been detected in commercial foetal bovine serum (FBS) of mainly South American origin so far. It is unclear how widely the viruses are presented in commercial FBS of different geographic origins. To further investigate the possible pestivirus contamination of commercially available FBS batches, 33 batches of FBS were obtained from ten suppliers and analysed in this study for the presence of both the recognised and the atypical bovine pestiviruses. All 33 batches of FBS were positive by real-time RT-PCR assays for at least one species of bovine pestiviruses. According to the certificate of analysis that the suppliers claimed for each batch of FBS, BVDV-1 was detected in all 11 countries and BVDV-2 was detected exclusively in the America Continent. The atypical pestiviruses were detected in 13 batches claimed to originate from five countries. Analysis of partial 5'UTR sequences showed a high similarity among these atypical bovine pestiviruses. This study has demonstrated, for the first time that commercial FBS batches of different geographic origins are contaminated not only with the recognised species BVDV-1 and BVDV-2, but also with the emerging atypical bovine pestiviruses.     

Effects of 17beta-oestradiol or oestrous stage-specific cow serum on the ability of bovine oviductal epithelial cell monolayers to prolong the viability of bull spermatozoa

The effect of 17beta-oestradiol and oestrous stage-specific cow serum on bovine oviductal epithelial cell monolayers to extend the viability of co-cultured bull spermatozoa was examined. Monolayers of cells from ampullary and isthmic segments were pre-treated with medium containing either oestrous cow serum, luteal-phase cow serum, 1 microg/ml 17beta-oestradiol + foetal bovine serum or foetal bovine serum alone (control) before the addition of motile frozen/thawed spermatozoa. Motility was visually assessed throughout a 48 h co-incubation period, while fertilising ability of spermatozoa was evaluated by adding in vitro matured bovine oocytes. Pre-treatment with 17beta-oestradiol or oestrous cow serum resulted in a higher percentage of motile spermatozoa after 18 h in isthmic and after 36 h in ampullary cultures compared with the control, but pre-treatment did not affect fertilisation rates. Only at 42 h in ampullary cultures was motility higher in luteal serum pre-treated cultures compared to the control. Motility was also assessed in medium conditioned by pre-treated monolayers. Pre-treatment with 17beta-oestradiol enhanced the ability of conditioned medium to prolong motility and medium conditioned with oestrous cow serum was superior to medium conditioned by luteal-phase serum at maintaining motility. In conclusion, the ability of oviductal epithelium to prolong the motility of spermatozoa is enhanced by 17beta-oestradiol.     

The suppression of endogenous protein degradation by fractions of foetal calf serum: dialysed serum is less able to suppress degradation in aged cells

We have studied the effect of various fractions of foetal bovine serum upon the endogenous degradation of long labelled proteins in cultured MRC5 cells, and upon other cellular functions. Only heat-inactivated serum was capable of suppressing protein degradation to a similar extent to complete serum. Acid-treated and delipidized sera were moderately effective. Albumin on its own was able to replace 40 per cent of the effect of serum, indicating the exogenous protein might compete with endogenous protein for degradation in lysosomes. Albumin was not capable of supporting DNA synthesis. Dialysed serum showed an age-related effect suppressing protein degradation to a lesser extent and being less effective in supporting DNA synthesis or cellular proliferation in aged cells. All the effects noted were related to lysosomal protein degradation. Serum diffusate did not suppress protein degradation.     

Selective maintenance of cultured epithelial cells from DMBA-induced mammary tumours by bovine colostrum supplement

Growth kinetics in cultures of DMBA-induced mammary tumours supplemented with bovine colostrum were compared with the kinetics of cultures maintained with the conventional supplement of foetal calf serum. Although the latter permitted a greater degree of cell proliferation, a substantial amount of the cell growth was due to the fibroblastic proliferation. In the presence of bovine colostrum, epithelial islands surrounded by a few solitary cells became established. Unlike the foetal calf serum supplemented cultures, these cultures frequently did not become completely confluent within 7 days. The absence of fibroblasts in colostrum supplemented cultures was confirmed by electron microscopy. Results from this study suggest that colostrum may be useful in selective maintenance of primary cultures of epithelial origin.     

Immunohistochemical characterization of purple acid phosphatase-containing leucocytes in the human placenta

Summary A tartrate-resistant acid phosphatase activity was detected in the human placenta. This enzyme displayed immunological properties similar to those of the group of purple acid phosphatases that can be demonstrated with a rabbit polyclonal antibody against bovine spleen purple acid phosphatase. The placental enzyme was mainly localized immunohistochemically to neutrophil granulocytes of the maternal blood between the placental villi and within foetal capillaries using the bovine spleen antibody and the commercial monoclonal antibody M1 directed against an antigen found on mature granulocytes. A minor activity was detected in decidual cells and the syncytiotrophoblast. The presence of purple acid phosphatase in placental granulocytes may be related to special immunological conditions of pregnancy.     

The adsorption of bovine blood proteins onto the surface of O-(carboxymethyl)chitin

Chitin was found to interact with bovine blood proteins and the affinities of these proteins for chitin tended to be decreased by the introduction of O-carboxymethyl (CM) groups onto the chitin surface, especially with fibrinogen. As the adsorption of blood proteins to the CM-chitin (d.s. 0.35) was assumed to follow an isothermal adsorption-curve, the adsorption coefficients were estimated by applying the Langmuir equation. Bovine serum albumin showed the highest affinity among the proteins applied in this experiment [KBSA (bovine serum albumin); 20.0, KB gamma G (bovine gamma globulin); 1.96, KBF (bovine fibrinogen); 1.20]. The binding site of BSA for CM-chitin was assumed to be regulated not only by the cationic groups of BSA but also by other factors such as the recognition capacity of BSA to bind to GlcNAc residues in CM-chitin.     

Cryopreservation of the sperm of the Japanese bitterling

Sperm of the Japanese bitterling Tanakia limbata that had been cryopreserved with 5 or 10% methanol plus 95 or 90% foetal bovine serum (FBS) showed higher percentage and longer duration of motility than those that had been cryopreserved with 90% FBS and 10% DMSO, glycerol, N,N-dimethylacetamide or N, N-dimethylformamide. Foetal bovine serum, used as extender, had some cryoprotective effects when spermatozoa were cooled either with 10% methanol or without methanol. Spermatozoa, cooled to −40° C and then immersed in liquid nitrogen, had greater post-thaw motility than those cooled to −20, −60, or −80° C. The post-thaw percentage of motile spermatozoa increased significantly ( P < 0·001) with decreases in the freezing rate from 60 to 5°C min⁻¹. These results indicate that 10% methanol plus 90% foetal bovine serum is a suitable diluent for cryopreservation of bitterling spermatozoa and that samples should be cooled to -40°C at a low freezing rate for effective storage.     

Effects of sera types and cell density on the concentration of cyclic nucleotides in normal and simian virus transformed mouse fibroblasts

The effects of serum and cell density on the concentration of cyclic AMP, cyclic GMP in normal mouse fibroblasts cells (3T3 cells) and their Simian Virus 40 transformed derivative (SV3T3 cells) were studied. 3T3 cells grown in 10% foetal bovine serum exhibit density dependent inhibition of growth and associated with this in an increase in the concentration of cyclic AMP, a decrease in the concentration of cyclic GMP and an increase in the ratio (cyclic AMP/cyclic GMP) of the cyclic nucleotides. 3T3 cells grown in 10% newborn calf serum exhibit a higher saturation density and this is associated with a low concentration of cyclic AMP and a high concentration of cyclic GMP. SV3T3 cells grown in either 10% foetal bovine serum or 10% newborn calf serum show high saturation densities and this is associated with a low and decreasing concentration of cyclic AMP and a high concentration of cyclic GMP. When the level of the cyclic AMP in both cell lines was artificially raised by adding dibutyryl cyclic AMP and theophylline to the growth media, the cells grew to low densities.     

Comparative study of the interaction of cephalosporins with blood serum proteins and organ homogenates

Interaction of 7 semisynthetic antibiotics (cephaloridine, cephalexin, cephradine, cephazolin, cephalotin, cephacetrile and cephapirin) with proteins of human, bovine and rabbit blood serum, as well as organ and tissue homogenates of rats was studied comparatively. The study showed that binding of the cephalosporins by the blood serum depended on both the chemical structure of the antibiotic and the species affiliation of the protein substrate. The binding lvels of cephazolin and cephalotin by the blood serum proteins (except bovine serum) were the highest, while the binding level of cephaloridine was the lowest. A significant decrease in the values of binding by the serum proteins of the drugs with high percentage of binding was observed when the drug concentrations in solution were increased. Binding of the cephalosporins by the blood serum proteins was in most cases completely reversible. The activity of the cephalosporins decreased in the presence of the rat organ and tissue homogenates. The levels of the activity decrease as compared to the theoretical ones were the highest with the use of cephalotin, cephacetrile and cephapirin. The lowest values of detection of these antibiotics were noted on their incubation with the liver, kidneys and lungs.     

Differentiation of Purkinje fibres and ordinary ventricular and atrial myocytes in the bovine heart: an immuno-and enzyme histochemical study

Summary The differentiation of Purkinje fibres and ordinary ventricular and atrial myocytes in bovine hearts was studied with specific antibodies against M-line proteins (MM-creatine kinase and myomesin) and with enzyme histochemistry (succinate dehydrogenase and mitochondrial glycerol-3-phosphate dehydrogenase). MM-creatine kinase was detected at an earlier stage in Purkinje fibres and atrial myocytes than in ordinary ventricular myocytes. The findings are in agreement with previous ultrastructural observations that an earlier appearance of a dense M-band occurs in Purkinje fibres than in ordinary ventricular myocytes. Myomesin was detected in all three cell types even at early foetal stages, in accordance with suggestions that it is an integral component of the myofibrillar structure. The activity of succinate dehydrogenase gradually increased in both ordinary ventricular and atrial myocytes, while the activity of mitochondrial glycerol-3-phosphate dehydrogenase was high at different stages of early foetal development in the two tissues, finally becoming low in the adult stage. The activity of succinate dehydrogenase and mitochondrial glycerol-3-phosphate dehydrogenase seemed to remain unchanged in the Purkinje fibres from early to late foetal stages. The present study shows that the Purkinje fibres are already different from ordinary ventricular myocytes at early foetal stages and that the two cell types differentiate in different ways. It is concluded that there are also developmental differences between ordinary ventricular and atrial myocytes.     

Modulation of albumin-like protein and lysozyme production by bovine tracheal gland serous cells. Dependence on culture conditions

Bovine tracheal submucosal gland serous cells were cultured in medium supplemented with either 10% fetal calf serum or 2% Ultroser G, a commercial serum substitute for cell culture. The proteins synthesized and secreted into the culture medium during [35S]methionine pulse, chase and isoproterenol-stimulated periods were analyzed. Marked differences in the patterns of secretory radiolabeled proteins with Mr values ranging from 15,000 to 95,000 were observed between pulse and chase media of cells cultured in fetal calf serum and Ultroser G. In the presence of Ultroser G, albumin-like protein production was inhibited 95% as compared to cultures incubated with fetal calf serum. A bovine lysozyme-type enzymatic activity was detected only in medium from stimulated cells cultured in Ultroser G. The results suggest that bovine tracheal serous cells synthesize different proteins according to the composition of culture medium and release certain proteins when adrenergically stimulated.     

Multilevel regulation of steroid synthesis and metabolism in the bovine placenta

Steroid hormones play critical roles in almost all physiological processes in male and female reproduction. In a normal pregnancy, the concentrations of steroid hormones in maternal and foetal blood vary with gestation in response to changing needs. The placenta plays a central role in producing the appropriate steroids to support the pregnancy by coordinating its own steroidogenic activity with that of the corpus luteum and responding to foetal signals. Although much is known about the steroidogenic potential of the bovine placenta, far less is known about how the placenta integrates the synthesis of steroids with their subsequent metabolism and clearance to achieve appropriate local and peripheral concentrations of steroids in maternal and foetal blood at each stage of gestation. This review focuses on the current knowledge of the temporal and spatial regulation and compartmentalization of the biochemical pathways by which potent steroid hormones are synthesized and metabolized in the bovine placenta. The aim is to increase our understanding of how the balance of synthesis and metabolism determines placental steroid output as it changes with development and differentiation, and how this is regulated in response to the variations in the foetal signals and luteal secretory activity. The review highlights knowledge gaps and suggests that mathematical modelling can help understand the effect of different levels of regulation on the steroidogenic output of an organ, such as the bovine placenta.     

Outer-membrane protein and lipopolysaccharide variation in Pasteurella haemolytica serotype A1 under different growth conditions.

Growth characteristics, as well as outer-membrane protein (OMP) and lipopolysaccharide (LPS) profiles in SDS-polyacrylamide gels, of two serotype A1 isolates of Pasteurella haemolytica were examined under different in vitro growth conditions. The two isolates were chosen as representatives of disease (S/C 82/1) and non-disease (W/D 83/4) isolates, respectively. The growth rates and final cell densities of both isolates increased as the degree of aeration increased. In particular, the final cell densities varied significantly according to the degree of aeration. Under anaerobic conditions, however, both the growth rate and final cell density were significantly reduced. There was reduced expression of a 40.5 kDa protein under anaerobic conditions in both isolates, whereas in S/C 82/1 expression of the 71, 77 and 100 kDa iron-regulated proteins increased as aeration decreased. There were also differences in low-molecular-mass components of LPS between cells grown anaerobically and those grown aerobically. Growth in the presence of 5% CO2 did not significantly alter the growth rate and had little, if any, affect on OMPs or LPS. Differences in the expression of certain proteins occurred as growth progressed from the exponential to the stationary phase. Growth in the presence of the iron chelators 2,2'-dipyridyl, ethylenediamine-dihydroxyphenylacetic acid (EDDA), desferrioxamine mesylate (desferal), ovotransferrin (conalbumin) and bovine transferrin was inhibited within a very narrow concentration range. In the presence of 2,2'-dipyridyl, EDDA or desferal, 71 and 100 kDa iron-regulated OMPs increased in both isolates whereas a 77 kDa protein increased in isolate S/C 82/1 only. In the presence of ovotransferrin or bovine transferrin there was, in both isolates, increased expression of the 71 kDa protein, a slight increase in expression of the 100 kDa protein but no expression of the 77 kDa protein; there was also increased production of the 40.5 kDa protein, and synthesis of two additional proteins of 23 and 26 kDa. Other differences occurred after growth in foetal and newborn calf sera. In foetal calf serum there was enhanced expression of the 71 but not of the 100 kDa protein. In newborn calf serum there was no enhanced expression of the 71, 77 or 100 kDa proteins, but expression of novel proteins of 97 and 98 kDa as well as a high-molecular-mass protein occurred. There was also slight quantitative differences in the LPS profiles of cells grown in foetal or newborn calf sera compared to those of cells grown in other media.(ABSTRACT TRUNCATED AT 400 WORDS)