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I Davidson

Publications and source records attributed to I Davidson.

At least 109 records · Page 6Linked to original sources
PubMed1991

Identification of viral proteins encoded by two DNA fragments of herpesvirus of turkeys (HVT).

Herpesvirus of turkeys (HVT) vaccine is used worldwide to immunize chickens against Marek's disease (MD). Polyclonal antiserum directed against one virus cross-reacts with proteins of the other, while only 5% homology at the DNA level was demonstrated between the two viruses. A partial library of HVT DNA fragments ranging from 1.5 to 13.5 kbp in size was constructed in pBR 322. Under stringent conditions of hybridization (low salt concentration, 10% dextran sulfate at 68 degrees C), two of the cloned HVT DNA fragments (13.5 and 11.0 kbp) hybridized to three MDV DNA fragments: BamHI-C, D, and G. The 13.5 kbp fragment detected two transcripts of 1.8 and 3.0 kb in RNA extracted from HVT-infected chicken embryo fibroblasts, while the 11.0 kbp fragment detected three transcripts of 1.6, 2.0, and 3.0 kb in the extracted RNA. Using hybrid selection, specific RNA was isolated by hybridization with the two cloned HVT DNA fragments and then was translated in vitro using rabbit reticulocyte lysate, and the resulting proteins were analyzed by NaDodSO4-PAGE. The RNA selected by the two HVT DNA fragments coded for a 30 kD protein and for several smaller proteins 10-20 kD in size, while the RNA selected by the 11.0 kb HVT DNA fragment was translated into a 40 kD protein. Immunoprecipitation of these in vitro synthesized proteins with hyperimmune anti-HVT chicken serum showed that they were of viral origin.

Animals↗
PubMed1991

Monospecific antibodies to Marek's disease virus antigen B dimer (200 kDa) and monomer (130 and 60 kDa) glycoproteins neutralize virus infectivity and detect the antigen B proteins in infected cell membranes.

Monospecific antibodies were prepared by nitrocellulose blot immunoaffinity to 3 polypeptide components of the host-membrane associated B antigen of Marek's disease herpesvirus (MDV) and to its soluble A antigen. The B antigen comprised a 200 kDa dimer which is 2-mercaptoethanol (2-ME) labile, a monomer of 130 kDa and a 60 kDa protein, both of which are 2-ME resistant. Cross-immunoblotting studies showed that the anti-dimer antibody recognized the dimer protein as well as the 130 and 60 kDa components. In contrast, the anti-130 kDa antibody gave the strongest signal on blots of reducing gels indicating that the monomer is largely formed by in vitro reduction with 2-ME. All four antibodies recognized membrane antigens on chicken embryo fibroblasts infected with MDV vaccine viruses representative of the three serotypes and in addition, neutralized the homologous MDV isolate. The anti-dimer antibody was greatest, the anti-monomer antibody was the weakest and the anti-60 kDa antibody intermediate in neutralizing efficacy to all four viruses. We conclude from these studies that the B antigen presents at least two classes of neutralizing epitopes: one is discontinuous and of broad specificity on the intact dimer molecule and the other, on the 130 and 60 kDa proteins, is continuous and of lower avidity.

Animals↗
PubMed1991

Use of an ELISA for differential diagnosis of Mycoplasma agalactiae and M mycoides subspecies mycoides (LC) in naturally infected goat herds.

Contagious agalactia is an ovine and caprine mycoplasmosis which manifests as mastitis, arthritis and keratoconjunctivitis. Mycoplasma agalactiae is recognised as a causal agent but M mycoides subspecies mycoides (LC), and M capricolum may also be responsible for this syndrome in goats. The clinical signs are not pathognomonic; diagnostic procedures are based on isolation of the organism from diseased animals or by detection of seroconversion. An ELISA specific for M agalactiae and M m mycoides (LC) is described. The specificity of the antigens was demonstrated by immunoblotting and by ELISA using monospecific hyperimmune rabbit sera. A correlation of ELISA activity with other serological tests and isolation of mycoplasmas was carried out in two goat herds under field conditions. Results indicate the ability to detect subclinical mycoplasma infection and individual carrier goats on the basis of ELISA, a finding which will assist control procedures.

Animals↗
PubMed1990

N-terminal amino acid sequence identity between a major allergen of Ascaris lumbricoides and Ascaris suum, and MHC-restricted IgE responses to it.

A protein allergen of the parasitic nematode Ascaris has been purified to homogeneity by immunoaffinity chromatography. It is the most abundant protein species in the parasite's body fluid and has been named ABA-1. The allergen's molecular weight (MW) has been previously estimated at 14,000, but this sizing is currently under re-evaluation. The immunological activity of the protein was intact after purification, as attested by immunoprecipitation and passive cutaneous anaphylaxis. The IgE response to ABA-1 was under major histocompatibility complex (MHC) restriction in the rat, in which only RT1u strains were found to respond following infection with the parasite. The tissue-invasive and intestinal stages of both Ascaris lumbricoides (of humans) and Ascaris suum (of pigs) have an antigen of similar MW to ABA-1 in their secretions or among their somatic antigens. These are antigenically indistinguishable; they were found to have similar amino acid compositions, and their N-terminal amino acid sequences were identical to 41 residues. Finally, the apparent MW, amino acid composition and isoelectric point of ABA-1 all argue for close similarity to the previously described Allergen A of the parasite.

Allergens↗
PubMed1989

The SV40 TC-II(kappa B) and the related H-2Kb enhansons exhibit different cell type specific and inducible proto-enhancer activities, but the SV40 core sequence and the AP-2 binding site have no enhanson properties.

The enhancer activity of the oligomerized SV40 TC-I and TC-II sequences has been investigated in lymphoid and non-lymphoid cell lines. While the TC-I sequence had no demonstrable enhanson activity, a class C enhanson (proto-enhancer), 5'-GGAAAGTCCCC-3', overlapping the TC-II sequence and the GT-I enhanson was identified. This TC-II enhanson, which is identical to the kappa B motif from the kappa chain enhancer, was active in both lymphoid and non-lymphoid cells, which contrasts with the previously reported lymphoid cell specificity of the kappa B motif. However, its activity in non-lymphoid cells is in agreement with our previous reports describing the effect of mutations in the 'TC region' within the total SV40 enhancer in lymphoid and non-lymphoid cells. The activity of the TC-II enhanson could be moderately increased in HeLa by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and cycloheximide treatment, indicating that the protein(s) mediating its activity may be partially repressed by the previously described inhibitor protein I kappa B. The TC-II related, H-2Kb element, 5'-TGGGGATTCCCCA-3', of the histocompatibility class I H-2Kb gene promoter is also a class C enhanson which is active in both lymphoid and non-lymphoid cells. However, in contrast to the TC-II enhanson, the H-2Kb enhanson exhibits a very low activity in HeLa cells, but can be strongly induced by TPA and/or cycloheximide treatments which suggests that its cognate factor is inactivated (repressed) by an inhibitor protein. Interestingly, cycloheximide, but not TPA treatment, could induce the activity of both the TC-II and H-2Kb enhansons in F9 embryonal carcinoma cells, suggesting that these cells lack some component(s) of the protein kinase C signal transduction pathway. We also show that oligomers of the SV40 'core' sequence, which overlaps the TC-II enhanson, had no enhanson activity in any of the cell types studied, which questions the possible role of the AP-3 protein in SV40 enhancer activity in these cell types. In addition, oligomers of the AP-2 binding sites which are present in the SV40 TC region and in the human metallothionein IIA promoter show no enhanson activity, irrespective of whether the cells are treated with TPA.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

The SV40 TC-II(kappa B) enhanson binds ubiquitous and cell type specifically inducible nuclear proteins from lymphoid and non-lymphoid cell lines.

We have characterized the complexes resulting from the specific binding in vitro of proteins present in nuclear extracts of several lymphoid and non-lymphoid cell lines to the TC-I and TC-II sequences of the simian virus 40 (SV40) enhancer. No proteins could be detected, binding selectively to the TC-I sequence, but two proteins TC-IIA and TC-IIB were identified interacting specifically with both the TC-II/kappa B enhanson, 5'-GGAAAGTCCCC-3' (important for the activity of the SV40 enhancer in vivo), and with the related H-2Kb enhanson, 5'-TGGGGATTCCCCA-3'. The binding of these two proteins to mutated TC-II enhansons correlates with the effect of these mutations in vivo, suggesting that both proteins may be important for SV40 enhancer activity. The TC-IIA binding activity was present in nuclear extracts of mature lymphoid B cells and was increased in pre-B cell nuclear extracts by lipopolysaccharide (LPS) and cycloheximide treatment. Furthermore, complex formation between the TC-IIA protein and the TC-II enhanson was efficiently competed by the kappa B motif from the kappa chain enhancer, indicating that TC-IIA is the NF-kappa B factor or a closely related protein. However, in contrast to previous reports, a TC-IIA/NF-kappa B-like protein whose properties could not be distinguished from those of the TC-IIA protein present in lymphoid B cells, was found in nuclear extracts of several untreated non-lymphoid cell lines, notably of HeLa cells, but not of undifferentiated F9 embryonal carcinoma (EC) cells [F9(ND)]. The TC-IIA binding activity which was moderately increased in HeLa cell nuclear extracts by 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or cycloheximide treatment could be induced in nuclear extracts of F9(ND) cells by cycloheximide, but not by TPA. Moreover, the TC-IIA binding activity could be induced in cytosolic fractions from F9(ND) cells by treatment with deoxycholate, indicating that these cells contain an inhibitor protein similar to the previously described NF-kappa B inhibitor, I kappa B. The second TC-II enhanson binding protein, TC-IIB, which could be clearly distinguished from the TC-IIA/NF-kappa B-like protein, by a number of differential properties, resembles the previously described KBF1/H2TF1 protein as it binds with a higher affinity to the H-2Kb enhanson than to the TC-II/kappa B enhanson, and its pattern of methylation interference on the H-2Kb and TC-II/kappa B enhansons is identical to that reported for the KBF1/H2TF1 protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Detection of specific protein binding to the SV40 early promoter in vivo.

The interactions in vivo between cellular proteins and the Simian Virus (SV40) early promoter region, contained in a plasmid capable of replicating in Cos cells, have been characterized by DNaseI and dimethyl sulfate (DMS) footprinting. The relative contribution of each GC-motif within the 21 bp repeat upstream element to transcription was first determined after transfection of Cos cells with either the wild type 21 bp repeats or recombinants where the GC-motifs were mutated either individually or in neighboring pairs. Mutation of GC-motifs III and VI was the most detrimental, mutation of GC-I, -IV and -V also decreased promoter activity but to a lesser extent, while mutation of GC-II had little effect on transcription. All six GC-motifs of the wild type 21 bp repeats were found protected from DNaseI nuclease attack in vivo though to varying degrees. Motifs GC-III, -V and -VI were more strongly protected than GC-I, -II and -IV. In vivo DNaseI footprinting of recombinants bearing mutations in the GC-motifs demonstrated the specificity of factor interaction and further indicated that, in agreement with the previously published in vitro results, the binding of factor(s) to each of the GC-motifs was independent. DMS protection experiments identified specific guanine (G) contacts characteristic of Sp1 binding to the GC-motifs and this in vivo pattern was compared to that obtained in vitro using a crude nuclear extract. These results indicate that the transcription factor Sp1 interacts in vivo with the GC-motifs of the SV40 early promoter.

Animals↗
PubMed1989

Cuff herniation.

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Equipment Failure↗
PubMed1988

The HeLa cell protein TEF-1 binds specifically and cooperatively to two SV40 enhancer motifs of unrelated sequence.

We have purified a protein (TEF-1) that specifically binds to two sequence unrelated motifs (GT-IIC and Sph) of the simian virus 40 (SV40) enhancer. TEF-1 binds cooperatively to templates containing tandem but not inverted or spaced repeats of its cognate motifs. This cooperative binding correlates with the ability of the tandem repeats to generate enhancer activity in vivo. In contrast, TEF-1 and a second SV40 enhancer binding protein, TEF-2, bind independently to templates containing the cognate motifs of both proteins (GT-I and either GT-IIC or Sph motifs) even though these motifs cooperate in enhancer activity in vivo. These results allow us to distinguish different classes of enhancer factors.

Allosteric Regulation↗
PubMed1988

Marek's disease virus serotype-1 antigens A and B and their unglycosylated precursors detected by Western blot analysis of infected cells.

The antigenic profile of cell cultures infected with Marek's disease virus (MDV) was determined by the immunoblotting method using convalescent immune serum obtained from chickens that survived infection with MDV strain GA5. The MDV antigen profile in infected cell lysates could be accurately determined since this method has advantages over the immunoprecipitation method used in other studies. We studied six very virulent MDV isolates and the prototype of serotype 1 MDV, the GA5 strain. Immunoblots of NaDodSO4-polyacrylamide gel electrophoresis (PAGE) performed under reducing conditions revealed a main viral antigen (B) of 120-130 kD, which was present in all cell lysates infected with MDV isolates. Analysis of infected cell proteins by NaDodSO4-PAGE performed under nonreducing conditions, revealed a 205 kD major MDV antigen, which, under reducing conditions, was identical to the 130 kD major antigen. The unglycosylated precursors of the major MDV antigens were elucidated. Two polypeptides of 43 and 45 kD were found to be the unglycosylated precursors of MDV antigen A (the glycosylated form of which appears in 4 distinct bands). The unglycosylated precursors of the MDV major antigen B were found to be three polypeptides of 80, 110, and 125 kD.

Animals↗
PubMed1987

One cell-specific and three ubiquitous nuclear proteins bind in vitro to overlapping motifs in the domain B1 of the SV40 enhancer.

We have used the gel retardation assay to investigate the binding of nuclear proteins to the domain B1 of the SV40 enhancer, which contains the GT-II motif. Four proteins (GT-IIA, GT-IIB alpha, GT-IIB beta and GT-IIC) were detected, three of which were present in nuclear extracts from several cell lines. The fourth protein (GT-IIC) showed a clear cell-specificity, being absent from the lymphoid cell extracts tested. The results of methylation interference assays and of the binding of the proteins to mutated templates indicate that the domain B1 contains three distinct, but overlapping, protein-binding motifs (GT-IIA, B and C). The cell-specific binding of protein GT-IIC in vitro correlates with the in vivo enhancer activity of its cognate motif, strongly suggesting that this protein acts as a positive trans-acting enhancer factor. Two of the proteins also recognize other enhancer motifs; protein GT-IIB alpha binds to the microE3 motif present in the immunoglobulin heavy chain enhancer; protein GT-IIC binds to an enhancer motif of the polyomavirus mutant PyEC9.1 adapted to growth in F9 embryonal carcinoma cells, but not to the corresponding wild-type sequence.

Base Sequence↗
PubMed1987

In vitro binding of cell-specific and ubiquitous nuclear proteins to the octamer motif of the SV40 enhancer and related motifs present in other promoters and enhancers.

We have used the gel retardation and DNase I footprinting assays to investigate the in vitro binding of nuclear proteins to the octamer motif present in domain A of the SV40 enhancer and in other enhancer and promoter elements. Three apparently cell-specific (oct-B1A, oct-B1B and oct-B2) and one ubiquitous (oct-B3) proteins were detected in various lymphoid and non-lymphoid cell extracts. We show that the previously described 'ubiquitous' NF-A1 factor may correspond in fact to two proteins, oct-B1A in HeLa cells and oct-B1B in lymphoid cells. Interestingly, the HeLa cell protein oct-B1A formed a complex with the SV40 octamer, which could be detected in gel retardation, but not in DNase I footprinting assays. This absence of protection from DNase I digestion correlates with the inactivity of the SV40 octamer in HeLa cells in vivo. We have also found that the in vitro interaction between the SV40 octamer motif and the lymphoid cell-specific protein oct-B2 was negatively modulated by a component present in the nuclear extracts from several lymphoid cell lines. The interactions between the multiple octamer-binding proteins and the related octamer motifs present in other promoter and enhancer elements were systematically compared and the possible role of these proteins in the control of transcription is discussed.

Base Sequence↗
PubMed1987

In vitro binding of several cell-specific and ubiquitous nuclear proteins to the GT-I motif of the SV40 enhancer.

We have investigated the specific in vitro binding of nuclear proteins from several cell lines to the GT-I motif of the SV40 enhancer which overlaps with the canonical enhancer "core" homology. The binding of three proteins (GT-IA, GT-IB, and GT-IC), one of which (GT-IC) exhibits cell specificity, was detected. Competition and direct binding experiments demonstrated that the two ubiquitous proteins also bind to the GC-rich motif III from the 21-bp repeat upstream element of the SV40 early promoter and that protein GT-IA is most probably the transcription factor Sp1. The third, cell-specific protein GT-IC exhibited a high affinity for both the GT-I motif and an upstream element in the promoter of the mouse beta-major-globin gene, suggesting that this protein can act both as an enhancer and an upstream element trans-acting factor. The good correlation between the known cell-specific in vivo activity of the wild-type and mutated GT-I motif and the cell-specific binding of protein GT-IC in vitro strongly supports the conclusion that this protein is an enhancer factor. Interestingly, its cognate recognition sequence does not coincide with the core homology.

Animals↗
PubMed1986

Detection of Marek's disease virus antigens and DNA in feathers from infected chickens.

Two novel tests, enzyme-linked immunosorbent assay (ELISA) and dot-blot hybridization, were developed to detect and quantify the antigens and DNA of Marek's disease virus (MDV) in feather tips from infected chickens. In both methods, buffered extracts of the feathers served as the same test material. The ELISA technique was compared to the conventional agar-gel precipitation (AGP) test, using the same convalescent serum from a MDV-infected bird. Of 86 feather samples tested, 34 were negative by both methods, while 6 out of 52 were ELISA positive but AGP negative. Viral antigen detection by the AGP and ELISA methods was compared with the detection of MDV DNA by the dot-blot DNA hybridization technique. At an ELISA reading (OD 405) of 0.3 and above, only 5 out of 48 DNA extracts failed to hybridize with the MDV-DNA probe. The use of the radioactively labelled MDV-DNA probe for hybridization with DNA extracts from feather tips of MDV-infected chickens was both sensitive and specific, and there was good correlation among the different tests.

Agar↗
PubMed1986

An improved ELISA method, using a streptavidin-biotin complex, for detecting Marek's disease virus antigens in feather-tips of infected chickens.

To improve sensitivity in the detection of Marek's disease virus (MDV) antigens in extracts of feather tips from infected chickens, we added a preformed streptavidin-biotin complex to the standard enzyme-linked immunosorbent assay (ELISA). Rabbit anti-chicken IgG-alkaline phosphatase that is used in the standard ELISA as the conjugate was replaced by a biotinylated rabbit anti-chicken IgG plus the streptavidin-biotin peroxidase complex (ABC) system. The ABC-ELISA system was correlated to the standard ELISA. There was increased sensitivity in the detection of MDV antigens present at low concentrations, while at the higher concentrations detection was similar to that in the standard ELISA. Both ELISA systems had the same increased sensitivity when compared with that of the agar gel precipitation (AGP) test.

Animals↗
PubMed1986

Laboratory assay methods for bovine tuberculin PPD (report of the Expert Group on Veterinary Sera and Vaccines of the European Pharmacopoeia Commission).

It had been found that preparations of bovine tuberculin PPD that appeared to be similar in potency when assayed in guinea-pigs sensitized with killed tubercle bacilli could differ markedly in potency when assayed in cattle. In an attempt to define a laboratory assay method that would reflect accurately the performance of bovine tuberculin PPD in cattle, the European Pharmacopoeia Group of Experts on Veterinary Sera and Vaccines carried out a collaborative study of various methods of sensitization. Seven laboratories in 6 countries participated. The only method of sensitization that gave results corresponding to those obtained in cattle was that using living, virulent, bovine, tubercle bacilli. The Group concluded that it was essential to use this method in laboratory assays of bovine tuberculin PPD.

Animals↗