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Biomedical subjects

B Talbot

Publications and source records attributed to B Talbot.

At least 19 recordsLinked to original sources

Is the link between nutrients and foods understood? The case of fibre and folate.

OBJECTIVES: To assess and contrast awareness of the link between dietary fibre and folate and their major food sources (fruit, vegetables, bread and cereals). DESIGN: Mailed questionnaire investigating changes made to dietary intake of fibre, folate, fruit, vegetables, bread and cereals in the previous six months. SETTING: The survey was conducted between June and November 1998 in the Australian Capital Territory. SUBJECTS: One thousand one hundred and twenty-six adults randomly selected from the electoral roll. RESULTS: More women than men in both older (50+ years) and younger (18-49 years) age groups reported increasing their consumption of folate, fibre, fruit and vegetables in the prior six months. In contrast, more men than women reported increased consumption of bread, cereals, rice and pasta in the previous six months. For food categories and fibre, less than 4% of respondents were unsure about changes in these food habits. However, 26% of men and women were 'not sure' about changes to folate intake. Similar proportions of men and women (about 33%) reported consuming more fruit, vegetables or cereal-based foods over the prior six months, yet only 6% of these men and 14% of these women reported consuming more folate. In contrast, 44% of men and 51% of women who reported consuming more plant foods also reported consuming more dietary fibre. CONCLUSIONS: The results suggested that subjects, particularly the younger age group, had a poor understanding of the relationship between folate intake and its major food sources. The understanding of the relationship between fibre intake and its food sources appeared substantial, but confusion about specific food sources was still evident. These outcomes question the effectiveness of nutrition education used to date, particularly for the current priority of increasing folate intake in younger women in the new, 'health claims' environment.

Adolescent↗

Effects of enforcement of youth access laws on smoking prevalence.

Smoking is the primary preventable cause of death, and yet 3,000 adolescents become smokers each day. Most adult smokers begin this deadly habit when they are under the age of 18, which is the minimum legal age for the purchase of cigarettes. The majority of adolescent smokers are able to purchase cigarettes even though laws prohibit the sale of cigarettes to minors. In the late 1980s, Woodridge, IL, became one of the first towns in the nation to demonstrate a significant reduction in the ability of youth to purchase cigarettes. Almost 2 years after passage of this legislation, the percentage of regular smokers among 7th- and 8th-grade students had been reduced from 16 to 5%. Seven-year follow-up data in a sample of high school youths indicate that youths living in communities with regular enforcement had significantly less smoking than those living in communities without regular enforcement. In particular, rates of regular smoking were 8.1% in communities with regular enforcement versus 15.5% in communities without regular enforcement. It is possible that adolescents who had restricted access to tobacco products were less likely to become regular smokers. These findings have important public health implications, particularly in light of recent federal legislation mandating that all states develop programs to reduce access of youth to tobacco products.

Adolescent↗

Characterization of anti-peptide antibodies directed towards the automodification domain and apoptotic fragment of poly (ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a highly conserved nuclear enzyme present in higher eukaryotes. PARP is activated following DNA damage, is implicated in DNA repair, and its proteolysis has been shown to be an early marker of programmed cell death or apoptosis. In order to better understand the role of PARP in apoptosis and DNA repair and also to study PARP automodification, we have developed anti-peptide sera directed against four peptides from the conserved automodification domain of PARP. Four peptides were synthesized according to the four branched Multiple Antigenic Peptide (MAP) system and injected into rabbits. Immune sera were titrated by ELISA and analysed in Western blotting experiments on cell lines. The sera were also analysed for their capacity to inhibit PARP activity in an in vitro assay. Of the eight sera developed (two for each peptide), a serum directed against a peptide localized at the C-terminal part of the automodification domain of PARP (#422) appeared to be the best antibody to detect PARP from different species. All antipeptide antibodies were efficient in detecting the apoptotic fragment of PARP during programmed cell death in HL-60 apoptotic cells. None of the serum alone was able to completely inhibit PARP activity but combinations of the sera could significantly reduce automodification of PARP consistent with the localization of half of the automodification sites on bovine PARP. Sera were also used to map proteolysed purified PARP and to immunoprecipitate purified bovine PARP.

Amino Acid Sequence↗

Immune response to vaccination with DNA encoding the bovine viral diarrhea virus major glycoprotein gp53 (E2).

Bovine viral diarrhea virus (BVDV) is a worldwide pathogen in cattle which has not been controlled by classical vaccination. The region encoding the BVDV major glycoprotein gp53 (E2) known to possess virus-neutralizing activity was cloned into a mammalian expression vector under the human cytomegalovirus (CMV) intermediate early promoter. Intramuscular and intradermal administration of the recombinant plasmid DNA into BALB/c mice induced BVDV gp53-specific antibody responses to both biotypes (cytopathic and noncytopathic) of BVDV genotype 1, and to cytopathic BVDV genotype 2. BVDV-neutralizing antibodies were generated against BVDV genotype 1 strains and they also persisted 6 months after the last injection.

Animals↗

Restriction endonucleases whose sites are predictable from the amino acid sequence offer an improved strategy for typing bovine rotaviruses.

Variation in the third base of a codon hampers genotypic characterization, particularly of RNA viruses. Some restriction endonucleases, however, have a recognition site with a variable base at the third position and will always cleave when a certain amino acid pair occurs (such as glycine-proline for Sau96I and glutamic or aspartic acid followed by serine usually for HinfI). We developed a restriction fragment length polymorphism (RFLP) procedure based on these enzymes for P-typing bovine group A rotaviruses (BRV). Employing this procedure 20 BRV local strains, isolated in tissue culture as well as the original faecal sample, could be typed in one of three patterns. More variability was observed when restriction endonucleases were employed whose cleavage sites cannot be predicted from the amino acid sequence (TaqI and Tsp509I). These RFLP results agreed with the PCR-VP4 typing assay, neutralization tests, and nucleotide sequence analysis. RFLP with Sau96I and HinfI provided quick and objective P-typing of strains and could detect multiple genotypes in the same sample.

Animals↗

Antigenic variation among bovine viral diarrhea virus (BVDV) strains and the role of different cell fixation methods in immunoassays.

Antigenic variation among 13 Quebec isolates of bovine viral diarrhea virus (BVDV), 4 reference strains and 2 American isolates were studied by peroxidase-linked antibody assay (PLA assay) and neutralization test (NT). The Quebec strains consisted of 3 isolates before 1993 and 10 isolates from 1993. In the PLA assay, we compared 2 different fixatives, acetone and formalin. Acetone-fixation allowed us to identify 6 groups from amongst the viruses tested. All the Quebec isolates were different from the reference strains. In addition, antigenic variation was detected between Quebec isolates obtained before and during 1993. However, PLA assays performed after formalin fixation did not detect these antigenic variations. Neutralization tests were carried out with 2 polyclonal antibodies (PAb) and 6 monoclonal antibodies (MAb). They were used to classify BVDV strains and isolates into 4 groups and 7 subgroups respectively. In conclusion, we demonstrated that the BVDV isolates from the 1993 outbreak in Quebec are antigenically different from reference strains and from isolates existing in Quebec before 1993. In addition, we have shown that 2 internationally used fixation-methods in PLA assay give different results. The usefulness of each method is discussed.

Animals↗

Comparison of polymerase chain reaction and monoclonal antibodies for G-typing of group A bovine rotavirus directly from fecal material.

A reverse transcriptase and polymerase chain reaction (RT-PCR)-based assay for G-typing of bovine rotaviruses (BRV) was compared with a monoclonal antibody-based immunoassay (MAbs-ELISA) in the characterization of BRV field strains obtained from calves in different regions of Quebec between 1992 and 1994. The strains were analysed for two G types (G6 and G10) which are the most predominate BRV field strains. Fecal samples positive for BRV by polyacrylamide gel electrophoresis (n = 74) were typed by both methods revealing 77% correlation. RT-PCR detected 10 more G6 and 2 more G10 serotypes than MAbs-ELISA. Nine of the 12 discrepant samples could be cultivated and were confirmed as G6 (8) or G10 (1) by both methods. RT-PCR was able to efficiently detect artificial mixes of G6 and G10 and detected two mixed field infections. Four additional infections considered as mixed by MAbs-ELISA and as only G6 by RT-PCR were possibly MAbs-ELISA cross-reactions. RT-PCR provided a very sensitive method for typing BRV field isolates.

Animals↗

Persistence of a 1930 swine influenza A (H1N1) virus in Quebec.

Two antigenically distinct H1N1 influenza A viruses were isolated during an outbreak of respiratory disease in Quebec swine in 1990/91. Analysis of haemagglutinin and partial nucleoprotein sequences indicated that one was a variant of the swine H1N1 influenza virus circulating in the American Midwest whereas the other was very similar to virus isolated from swine in 1930. The existence of this latter isolate supports the concept that influenza viruses can be maintained for long periods in swine, perhaps in geographically limited pockets. Serological evidence indicates that these distinct strains continued to circulate widely in south-central Quebec until at least 1993.

Amino Acid Sequence↗

Recent H3N2 swine influenza virus with haemagglutinin and nucleoprotein genes similar to 1975 human strains.

Of the four pandemic strains of human influenza A virus observed this century, the 1977 virus strain was very similar in all genes to a 1950 isolate. Since mammalian influenza A viruses change annually by genetic drift, this reappearance could only be attributed at that time to conservation of the virus in a frozen state. We report here the isolation of swine influenza A viruses with haemagglutinin and nucleoprotein genes which are virtually identical to those of the human virus that circulated in 1975. We have also found serological evidence that this virus is circulating extensively in Quebec swine herds. We propose that human-like H3N2 influenza A strains may remain invariant for long periods in swine, which may serve as a reservoir for human pandemics.

Amino Acid Sequence↗

Bovine rotavirus type detection by neutralizing monoclonal antibodies.

A series of monoclonal antibodies were developed against bovine rotavirus Q17. Among the five high affinity antibodies characterized, two (RQ 31 and RQ 4) were able to neutralize type G 6 viruses and may be specific for PB 1 type virus. Seventy seven feces from diarrheic calves were tested by "double sandwich" ELISA using four monoclonal and one polyclonal anti-rotavirus antibodies. The combination of mono- and polyclonal antibodies thus appears to be a more efficient strategy for detection and typing of bovine rotaviruses.

Animals↗

Characterization of bovine rotavirus VP6 and VP7 as glycoproteins using monoclonal antibodies.

Bovine rotavirus proteins were analysed by a panel of monoclonal antibodies. Glycosylated epitopes were identified on both inner and outer capsid proteins (VP6 and VP7 respectively). VP7 possessed a periodate insensitive epitope which was, however, sensitive to endoglycosidase H, mixed glycosidases and to protease treatment. This epitope was not detected on viruses grown in the presence of 2-deoxy-D-glucose or tunicamycin. An epitope was detected on VP6 which was sensitive to periodate oxidation. The blotted protein reacted with a glycan assay kit; yet the epitope was not affected by endoglycosidase H and was found on viruses grown in the presence of 2-deoxy-D-glucose or tunicamycin. These results suggest that VP7 and VP6 epitopes are carbohydrate dependent. The VP7 epitope contains an N-linked carbohydrate moiety in contrast to the VP6 epitope which appears to contain O-linked glycosyl units.

Acetylglucosaminidase↗

Association of poly(ADP-ribose) polymerase with the nuclear matrix: the role of intermolecular disulfide bond formation, RNA retention, and cell type.

The recovery of the enzyme poly(ADP-ribose) polymerase (pADPRp) in the nuclease- and 1.6 M NaCl-resistant nuclear subfraction prepared from a number of different sources was assessed by Western blotting. When rat liver nuclei were treated with DNase I and RNase A followed by 1.6 M NaCl, approximately 10% of the nuclear pADPRp was recovered in the sedimentable fraction. The proportion of pADPRp recovered with the residual fraction decreased to less than 5% of the total nuclear polymerase when nuclei were prepared in the presence of the sulfhydryl blocking reagent iodoacetamide and increased to approximately 50% of the total nuclear pADPRp when nuclei were treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) prior to fractionation. To determine whether this effect of disulfide bond formation was unique to rat liver nuclei, nuclear matrix/cytoskeleton structures were prepared in situ by sequentially treating monolayers of tissue culture cells with Nonidet-P40, DNase I and RNase A, and 1.6 M NaCl (S.H. Kaufmann and J.H. Shaper (1991) Exp. Cell Res. 192, 511-523). When nuclear monolayers were prepared from HTC rat hepatoma cells, CaLu-1 human lung carcinoma cells, and CHO hamster ovary cells in the absence of NaTT, pADPRp was undetectable in the nuclease- and 1.6 M NaCl-resistant fraction. In contrast, when nuclear monolayers were isolated in the presence of NaTT, from 5% (CaLu-1) to 26% (HTC cells) of the total nuclear pADPRp was recovered with the nuclease- and salt-resistant fraction. Examination of these residual structures by SDS-polyacrylamide gel electrophoresis under nonreducing conditions suggested that pADPRp was present as a component of disulfide cross-linked complexes. Further analysis by immunofluorescence revealed that the pADPRp was diffusely distributed throughout the CaLu-1 or CHO nuclear matrix. In addition, when matrices were prepared in the absence of RNase A, pADPRp was also observed in the residual nucleoli. These observations reveal that the recovery of pADPRp with a nuclease- and salt-resistant nuclear subfraction is dependent on the source of the nuclei and on the conditions used to fractionate those nuclei. In addition, these observations raise the possibility that there might be different functional classes of pADPRp molecules within the nucleus.

Animals↗

Immunoassays for proteins alkylated by nicotine-derived N-nitrosamines.

Polyclonal antibodies recognizing the pyridyloxobutyl (POB) moiety of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were produced in rabbits immunized either with POB-bovine albumin or POB-Sepharose. The POB intermediates necessary to modify the protein were generated by alkaline (pH 9.0) treatment of the synthetic precursor 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone. In a competitive enzyme linked immunoabsorbent assay (ELISA), 70 pmole NNK inhibited 50% of the binding of the anti-POB antibodies to POB-protein absorbed on microtiterplates. This 50% inhibition varied from 70 pmole to 200 nmole using a series of NNK analogues, depending on the integrity of the POB moiety. Immunological techniques initiated in this study detect NNK-protein conjugates or measure the quantity of POB groups liberated upon alkaline or acid treatment of NNK modified protein.

Animals↗

Monoclonal antibodies to Quebec strain (Q17) of bovine rotavirus.

Fourteen monoclonal antibodies (MAbs) against "Quebec" strain (Q17) of bovine rotavirus were isolated and characterized. Four were specific for viral protein Vp7 and ten were specific for viral protein Vp6. Five different isotypes were represented by this group of antibodies. All of the anti-Vp6 and none of the anti-Vp7 antibodies were sensitive to the effects of periodate on their antigen. The antibodies could be separated into three groups based on their relative resistance to the dissociation of their antigen-antibody complex by thiocyanate. The MAbs cross-reacted with the proteins of porcine and human rotaviruses both by immunoprecipitation and immunoblot analyses. These techniques revealed the differences in Mw of the viral proteins from different serotypes.

Animals↗

Soybean L-(+)-lactate dehydrogenases: purification, characterization, and resolution of subunit structure.

Lactate dehydrogenase (LDH) (EC 1.1.1.27) from soybean (Glycine max) was purified 2360-fold to homogeneity using ion-exchange, hydroxyapatite, affinity, and hydrophobic chromatographies. The molecular weight of the holoenzyme is 150,000 +/- 5000. Two-dimensional (isoelectrofocusing-sodium dodecyl sulfate) gel electrophoresis reveals two polypeptides subunits of 5.9 and 6.5 pI and of 36,000 +/- 1000 and 37,000 +/- 1000 Mr, respectively. Nondissociating electrophoresis and isoelectric focusing of LDH resolved five tetrameric isoenzymes with pI's between 6.0 and 6.5. The data suggest that these LDH isoenzymes are derived from random association of the products of two different, but most probably related, genes. Kinetic measurements revealed substrate inhibition at high concentrations of lactic acid and biphasic kinetics with NAD.

Electrophoresis, Gel, Two-Dimensional↗

Detergent solubilised bovine viral diarrhea virus elicits a similar immune response as the inactivated virus.

The immune response to two detergent solubilised preparations of Bovine Viral Diarrhea Virus (BVDV) proteins were compared with the response to a chemically inactivated BVDV preparation. The antibody titres were measured with the standard immunofluorescence assay and with a newly developed enzyme linked immunosorbent assay (ELISA). The range of viral protein specificity of the antibodies was compared using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. The results clearly demonstrate that the detergent solubilised viral proteins are equally as effective immunogens as the killed BVDV vaccine both in terms of antibody titers and specificity.

Animals↗

Structural and functional analysis of poly(ADP ribose) polymerase: an immunological study.

Poly(ADP ribose) polymerase (EC 2.4.2.30) was studied using monoclonal antibodies for three different epitopes on the enzyme. The epitopes were mapped in relation to the functional domains of the protein and the inhibitory properties of the antibodies. The intranuclear and interspecies immunoreactivity of the enzyme was also investigated. The epitope of antibody 2 was mapped to the 17 kDa fragment generated by chymotryptic digestion of the C-terminal 54 kDa NAD-binding domain. Antibody 9 binds to the N-terminal 29 kDa fragment of the DNA binding domain and inhibits the enzyme activity by 80%. This antibody was used to purify poly(ADP ribose) polymerase by immunoaffinity chromatography. The third antibody binds to a central 36 kDa fragment that possesses part of the DNA-binding domain and the automodification domain. This antibody increases the enzymatic activity by 30%. An analysis of the species cross-reactivity of the antibodies was carried out by immunoblot analysis of nuclear proteins. Antibody 10 binding was detected in rat FR3T3 cells, Chinese hamster ovary cells (CHO) and epidermoid carcinoma lung human cells (CALU-1). The other two antibodies are specific for the human and bovine enzymes. Western blot analysis showed the association of poly(ADP ribose) polymerase with residual nuclear material obtained after nuclease treatment and high-salt extraction. Immunofluorescence studies with the three different monoclonals demonstrated that accessibility of the epitopes varies in the nucleus.

Animals↗