Alcohol-induced alterations in the palate epithelium of the rat fetus.

Maternal alcoholism (ethanol and sugar cane brandy) during gestation induces delayed cellular growth and differentiation in fetal rat palate epithelium, with increased nuclear, cytoplasmic and cellular volumes, increased epithelial and keratin thickness and decreased cellular numerical density.

Detection of leptospires in bovine semen by polymerase chain reaction

Objective: In view of the considerable importance of venereal transmission of bovine leptospirosis, the objective of the present study was to compare the polymerase chain reaction (PCR), culture/isolation and serology to detect leptospire infection in bovine semen. Design: Blood for serologic examination and semen for bacterial culture and PCR were collected from 20 bulls at artificial insemination centres in Brazil. Each animal was sampled twice for serology. Result: Forty-five percent (9/20) of the serum samples collected showed agglutinin titers to serovar hardjo in the first sample and 25% (5/20) had agglutinin titers to serovar hardjo in the second sample. Eighty percent (16/20) of semen samples were positive by PCR. Leptospires could not be isolated from any of the semen samples examined. Conclusion: Polymerase chain reaction can be a method of great potential for the detection of leptospires at artificial insemination centres.

Structure of tick anticoagulant peptide at 1.6 Å resolution complexed with bovine pancreatic trypsin inhibitor

The structure of tick anticoagulant peptide (TAP) has been determined by X-ray crystallography at t.6 Å resolution complexed with bovine pancreatic trypsin inhibitor (BPTI). The TAP-BPTI crystals are tetragonal, a = b = 46.87, c = 50.35 Å, space group P41, four complexes per unit cell. The TAP molecules are highly dipolar and form an intermolecular helical array along the c-axis with a diameter of about 45 Å. Individual TAP units interact in a head-to-tail fashion, the positive end of one molecule associating with the distal negative end of another, and vice versa. The BPTI molecules have a uniformly distributed positively charged surface that interacts extensively through 14 hydrogen bonds and two hydrogen bonded salt bridges with the helical groove around the helical TAP chains. Comparing the structure of TAP in TAP-BPTI with TAP bound to factor Xa(Xa) suggests a massive reorganization in the N-terminal tetrapeptide and the first disulfide loop of TAP (CyS5(T)- Cys 15(T)) upon binding to Xa. The Tyr1(T)OH atom of TAP moves 14.2 Å to interact with Asp189 of the S1 specificity site, Arg3(T)CZ moves 5.0 Å with the guanidinium group forming a cation-π-electron complex in the S4 subsite of Xa, while Lys7(T)NZ differs in position by 10.6 Å in TAP-BPTI and TAP-Xa, all of which indicates a different pre-Xa-bound conformation for the N- terminal of TAP in its native state. In contrast to TAP, the BPTI structure of TAP-BPTI is practically the same as all those of previously determined structures of BPTI, only arginine and lysine side-chain conformations showing significant differences.

Seroprevalence of bovine leptospirosis in Garanhuns municipal district, Pernambuco State, Brazil

The prevalence of Leptospira interrogans serovars in dairy cattle was determined by analyzing 464 serum samples from cows on 15 properties in Garanhuns municipal district, Pernambuco State, Brazil. A microscopic seroagglutination test including 12 serovars of Leptospira interrogans as antigens was used. Samples with titres 3 100 were considered positive. Two hundred and twenty-one (47.63%) of the samples were positive to one or more serovars. The prevalence of the serovars was hardjo (21.98%), bratislava (15.73%), castellonis(11.64%), tarassovi (10.56%), pyrogenes(1.72%), icterohaemorrhagiae (1.08%), pomona (0.86%), wolffi (0.86%), grippotyphosa (0.86%), djasiman (0.43%), canicola (0.21 %), and copenhageni (0.21 %).

A radiation hybrid map of bovine X Chromosome (BTAX)

We present a comprehensive radiation hybrid map of the bovine X chromosome (Chr) containing 20 new markers, including both microsatellites and expressed genes. This study was conducted with a 5000-rad whole genome RH cell panel consisting of 90 hybrid cell lines. Retention frequencies of individual markers range from 7.8% for XIST to 31.1% for TGLA325. Statistical analysis with RHMAPPER placed all the loci into five linkage groups under a LOD score criterion of 6.0. These groups could be oriented relative to each other because they included multiple microsatellite loci from the consensus linkage map of the X Chr. Markers included in both this RH map and the bovine cytogenetic map were in a consistent order. The comparative bovine-human map thus generated consists of five blocks of genes, the order of which is conserved, although in the opposite direction when presented as ideograms with p and q arms. Inversions of three blocks account for the difference in gene order across the entirety of the two X Chrs.

Growth hormone mRNA expression in the pituitary of Bos indicus and Bos taurus x Bos indicus crossbred young bulls treated with recombinant bovine somatotropin

The effects of breed and of recombinant bovine somatotropin (rbST) treatment on growth hormone gene expression were studied in young bulls. The experiment was completely randomized in a [2 × 2]-factorial arrangement, using two levels of rbst (0 or 250 mg/animal/14 days), and two breed groups (Nelore and Simmental x Nelore crossbred). A CDNA encoding Bos indicus growth hormone was cloned and sequenced for use as a probe in Northern and dot blot analyses. Compared to the Bos taurus structural gene, the Bos indicus CDNA was found to begin 21 bases downstream from the transcription initiation site and had only two discrepancies (C to T at position 144-His and T to C at position 354-Phe), without changes in the polypeptide sequence. However, two amino acid substitutions were found for Bubalus spp., which belong to the same tribe. The rbst treatment did not change any of the characteristics evaluated (body and pituitary gland weights, growth hormone MRNA expression level). Crossbred animals had significantly higher body weight and heavier pituitaries than Nelore cattle. Pituitary weight was proportional to body weight in both breed groups. Growth hormone MRNA expression in the pituitary was similar (P>0.075) for both breed and hormonal treatment groups, but was 31.9% higher in the pure Nelore group, suggesting that growth hormone gene transcription regulation differs among these breeds.

Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes

Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/ mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 μM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/ BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm. © 2003 American Society of Animal Science. All rights reserved.

Pheno and genotyping of Staphylococcus aureus, isolated from bovine milk samples from São Paulo State, Brazil

In the present study, 87 Staphylococcus aureus isolates obtained from milk samples of 87 cows with mastitis in 6 different municipal districts of 2 regions of São Paulo State, Brazil, were compared pheno and genotypically. Pulsed-field gel electrophoresis (PFGE) analysis of the strains was performed, and PCR was carried out to detect genes for a number of staphylococcal cell surface proteins, exoproteins, and 3 classes of agr genes. Nine distinct S. aureus lineages (LA-LI) were identified by PFGE. The lineages LA and LE, which accounted together for 63 strains (72.2%), were prevalent and had been collected from all of the 6 municipal districts, indicating a broad geographic distribution of these lineages; LB, LC, LD, LF, LG, LH, and LI, however, were isolated sporadically and accounted for 24 strains (27.8%). Some characteristics, like penicillin resistance and the presence of cap8 and agr class II genes, were associated with the prevalent lineages (LA and LE), and penicillin susceptibility and the presence of cna and cap5 genes were associated with sporadic lineages. According to the present results, some S. aureus lineages possess a combination of genes that confer the propensity to cause and disseminate infection, and only a limited number of clones are responsible for the cases of bovine mastitis on the various farms. © 2004 NRC Canada.

Comparative analysis of noncoding sequences of orthologous bovine and human gene pairs

Genomic sequence comparison across species has enabled the elucidation of important coding and regulatory sequences encoded within DNA. Of particular interest are the noncoding regulatory sequences, which influence gene transcriptional and posttranscriptional processes. A phylogenetic footprinting strategy was employed to identify noncoding conservation patterns of 39 human and bovine orthologous genes. Seventy-three conserved noncoding sequences were identified that shared greater than 70% identity over at least 100 bp. Thirteen of these conserved sequences were also identified in the mouse genome. Evolutionary conservation of noncoding sequences across diverse species may have functional significance, and these conserved sequences may be good candidates for regulatory elements.

Effects of cigarette smoke on the Meckel's cartilage of rat fetus : Morphologic, morphometric and stereologic study

The purpose of this study was to investigate the effects of cigarette smoke on the development of the embryo mandible (Meckel's) cartilage in rat fetuses. When inhaled by female Wistar rats between the 9th and the 12th day of pregnancy, cigarette smoke (5 cigarettes a day) caused intrauterine growth retardation, providing smaller fetuses and placentas. In fetuses from the experimental group, the histopathologic examination revealed a poorly developed Meckel' s cartilage with smaller chondroblasts showing a scanty cytoplasm with spherical and paler central nuclei, as well as more abundant cartilage matrix. Morphometric analysis revealed that Meckel's cartilage lacunae were smaller in the fetuses from the experimental group, although not showing any remarkable alteration in shape. The results suggested that inhalation of cigarette smoke by pregnant rats during the organogenic period induced growth retardation and delayed cellular differentiation in rat fetal Meckel's cartilage.

The effect of adhesive systems on the pullout strength of a fiberglass-reinforced composite post system in bovine teeth

Purpose: To evaluate the pullout strength of a glass fiber-reinforced composite post (glass FRC) cemented with three different adhesive systems and one resin cement. The null hypothesis was that pullout strengths yielded by the adhesive systems are similar. Materials and Methods: Thirty bovine teeth were selected. The size of the specimens was standardized at 16 mm by sectioning off the coronal portion and part of the root. The specimens were divided into three groups, according to the adhesive system, which were applied following the manufacturers' instructions: G1, ScotchBond Multi-Purpose Plus; G2, Single Bond; G3, Tyrian SPE/One-Step Plus. The glass FRCs (Reforpost) were etched with 37% H3PO4 for 1 min and silanized (Porcelain Primer). Thereafter, they were cemented with the dual resin cement En-Force. The specimens were stored for 24 h, attached to an adapted device, and submitted to the pullout test in a universal testing machine (1 mm/min). The data were submitted to the one-way ANOVA and Tukey's test (α = 0.05). Results: G1 (30.2 ± 5.8 Kgf) displayed the highest pullout strength (p < 0.001) when compared to G2 (18.6 ± 5.8 Kgf) and G3 (14.3 ± 5.8 Kgf), which were statistically similar. Analysis of the specimens revealed that all failures occurred between the adhesive system and the root dentin (pullout of the post cement), regardless of group. Conclusion: The multiple-bottle, total-etch adhesive system provided higher pullout strength of the glass FRC when compared to the single-bottle, total-etch, and single-step self-etching adhesive systems. The null hypothesis was rejected (p < 0.001).

Bovine serum albumin displays luciferase-like activity in presence of luciferyl adenylate: Insights on the origin of protoluciferase activity and bioluminescence colours

Luciferyl adenylate, the key intermediate in beetle bioluminescence, is produced through adenylation of D-luciferin by beetle luciferases and also by mealworm luciferase-like enzymes which produce a weak red chemiluminescence. However, luciferyl adenylate is only weakly chemiluminescent in water at physiological pH and it is unclear how efficient bioluminescence evolved from its weak chemiluminescent properties. We found that bovine serum albumin (BSA) and neutral detergents enhance luciferyl adenylate chemiluminescence by three orders of magnitude, simulating the mealworm luciferase-like enzyme chemiluminescence properties. These results suggest that the beetle protoluciferase activity arose as an enhanced luciferyl adenylate chemiluminescence in the protein environment of the ancestral AMP-ligase. The predominance of luciferyl adenylate chemiluminescence in the red region under most conditions suggests that red luminescence is a more primitive condition that characterized the original stages of protobioluminescence, whereas yellow-green bioluminescence may have evolved later through the development of a more structured and hydrophobic active site. Copyright © 2006 John Wiley & Sons, Ltd.

Determinação precoce do sexo fetal pela análise do DNA no plasma materno

Purpose: to verify the viability of early diagnosis of fetal gender in maternal plasma by the real-time polymerase chain reaction (real-time PCR) starting at the 5th week of pregnancy. Methods: peripheral blood was collected from pregnant women with single fetus starting at the 5th week of gestation. After centrifugation, 0.4 mL plasma was separated for fetal DNA extraction. The DNA was analyzed in duplicate by real-time PCR for two genomic regions, one of the Y chromosome and the other common to both sexes, through the TaqMan® method, which uses a pair of primers and a fluorescent probe. Patients who aborted were excluded. Results: a total of 79 determinations of fetal DNA in maternal plasma were performed in 52 pregnant women. The results of the determinations were compared to fetal gender after delivery. Accuracy according to gestational age was 92.6% (25 of 27 cases) at 5 weeks with 87% sensitivity, and 95.6% (22 of 23 cases) at 6 weeks with 92% sensitivity. Starting at the 7th week of pregnancy, accuracy was 100% (29 of 29 cases). Specificity was 100% regardless of gestational age. Conclusion: real-time PCR for the detection of fetal gender in maternal plasma starting at the 5th week of gestation has good sensitivity and excellent specificity. There was agreement of the results in 100% of the cases in which male gender was diagnosed, regardless of gestational age, and from the 7th week of gestation for female gender diagnosis.

Bio-safety technology in production of bovine herpesvirus type 5 (BoHV-5) using an alternative serum-free medium

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