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D P Barbis

Publications and source records attributed to D P Barbis.

9 recordsLinked to original sources
PubMed1995

The complete cDNA and deduced amino acid sequence of equine IgE.

The cDNA from a transcript encoding the complete heavy chain of the equine immunoglobulin IgE has been cloned and sequenced. A fragment of the equine epsilon gene was amplified from cDNA using PCR and this fragment was then used to probe a horse cDNA library prepared from peripheral blood lymphocytes. A recombinant clone containing the cDNA encoding the complete horse epsilon chain and its associated V-D-J and leader, was subsequently isolated and sequenced. Comparison of the deduced amino acid sequence of equine IgE with the C epsilon heavy chains of other species indicates it to be most similar to human C epsilon (54%), followed by sheep C epsilon (52%); the greatest sequence similarity was found in the C epsilon 3 and C epsilon 4 domains among the species compared. The associated V-region had greatest similarity in FR1 to those reported for sheep and the cow (> 80%) but less than 60% to the VH sequences of other species in FR3.

Amino Acid Sequence↗
PubMed1994

Correlation between monoclonal antibody reactivity and expression of CD4 and CD8 alpha genes in the horse.

Equine peripheral blood lymphocytes (PBL) were enriched by positive selection using panning with a mixture of monoclonal antibodies against putative equine CD4 (Equine Leucocyte Antigen Workshop antibodies WS 1 and WS 72), or CD8 molecules (Workshop antibodies WS 12, WS 49, and WS 74). RNA was extracted from CD4 enriched cells (99% purity), from CD8 enriched cells (69% purity), from peripheral blood lymphocytes, and from neonatal equine thymus. RNA extracted from equine granulocytes and from equine kidney served as negative control. The RNA was electrophoresed in agarose and transferred to nylon membranes. Northern blots were hybridized with human and mouse cDNA probes for CD4 and CD8 alpha. The human CD4 probe detected a 2.9 kb RNA transcript in equine PBL, CD4 enriched lymphocytes, and thymocytes. The human CD8 alpha probe detected a 2.0 kb transcript in RNA from equine CD8 alpha enriched lymphocytes and thymocytes, but not from PBL or CD4 enriched lymphocytes. Mouse cDNA probes for CD4 and CD8 did not react with equine RNA. Results of hybridizations using the human probes support the assignment of the CD4 and CD8 specificities to the antibodies listed above. The results also suggest that the equine CD4 and CD8 alpha genes are more closely related to the human than to the murine counterparts.

Animals↗
PubMed1994

Variation in expression of MHC class II antigens on horse lymphocytes determined by MHC haplotype.

A panel of monoclonal antibodies was used to characterize the expression of equine Major Histocompatibility Complex (MHC) class II antigens on lymphocytes of horses of different MHC types. MHC class II antigen expression was compared between adult horses and foals, and the level of expression of MHC class II antigens on horse T cell subpopulations was also determined. Peripheral blood lymphocytes (PBL) from young and adult healthy horses of different MHC haplotypes were labeled with the antibodies and assayed by single- and two-color immunofluorescence flow cytometry. A variation in the expression of equine MHC class II antigens on PBL was identified that was determined by MHC haplotype. Both young and old animals carrying the MHC class II haplotype D3 expressed lower levels of MHC class II antigens on their PBL than did horses of other MHC class II types. D3 heterozygotes had an intermediate level of expression. Neonates and foals of any MHC haplotype had lower levels of expression of MHC class II antigens than did adults. MHC class II antigens were identified on surface immunoglobulin positive (sIg+) B cells as well as on T cells, with the B cells staining brightest. Dual staining for MHC class II antigens and markers for equine T cell subsets demonstrated that both EqCD4+ and EqCD8+ T cells expressed MHC class II molecules. These results suggest that the relatively low levels of MHC class II antigen expression on foal and neonate lymphocytes may be developmentally regulated, and not determined by environmental factors. The cause of the low expression of MHC class II antigens linked to the MHC class II haplotype D3 is not known, but it appears to reflect a decreased expression by all lymphocyte subpopulations.

Animals↗
PubMed1994

Characterization of canine parvovirus (CPV) interactions with 3201 T cells: involvement of GPI-anchored protein(s) in binding and infection.

Binding of canine parvovirus (CPV) to the susceptible feline T cell line 3201 was quantitated by fluorescence-activated cell sorter (FACS) analysis. CPV bound to the cells in a dose-dependent manner, while no binding to the non-permissive MSB-1 avian lymphoma cell line was detected. Binding could be competitively inhibited by addition of excess unlabeled empty capsids, or by pre-incubation of virus with a CPV-specific monoclonal antibody. To characterize the biochemical nature of this binding, live cells were treated with a variety of enzymes prior to use in the binding assay. Treatment with neuraminidase removed a significant proportion of the wild-type virus binding activity, while both proteinase K and phosphatidylinositol-specific phospholipase C (PI-PLC) prevented binding of a non-hemagglutinating (non-HA), non-sialic acid binding mutant to 3201 cells. This suggests that CPV binds to sialic acid expressed on host cells as well as erythrocyte membranes, and that it also binds a protein moiety which is glycosylphosphatidylinositol (GPI)-anchored. The role of these components in CPV infection was also examined by pretreating cells with neuraminidase or PI-PLC prior to inoculating them with either wild-type CPV or the non-hemagglutinating mutant. Neuraminidase treatment had no effect on the ability of CPV to infect the cells, while infectivity was severely compromised by pretreating the cells with either proteinase K or PI-PLC. GPI-anchored proteins on 3201 cells were further characterized by Triton X-114 extraction and reactivity to anti-CRD after PI-PLC treatment.

Animals↗
PubMed1992

Mutations adjacent to the dimple of the canine parvovirus capsid structure affect sialic acid binding.

The erythrocyte receptor on rhesus macaque erythrocytes used by canine parvovirus (CPV) for binding in hemagglutination (HA) was examined. Erythrocyte membrane proteins were electrophoresed and blotted to nitrocellulose and probed with [125I]-labeled CPV capsids, showing seven virus-binding proteins. Treatment of erythrocytes or isolated membranes with Clostridium perfringens neuraminidase virtually abolished virus binding. Binding was also affected by treatment with potassium periodate and inhibited by wheat germ agglutinin, but was not significantly affected by concanavalin A, peanut agglutinin, or soluble N-acetyl-neuraminlactose. A non-HA mutant of CPV failed to bind to erythrocytes or to blotted erythrocyte membrane proteins. The mutation was a single Arg-Lys difference of VP2 amino acid residue 377. The pH dependence of binding of the closely related feline panleukopenia virus was shown to result from a decreased binding in buffers with pH values of 6.8 or greater. The VP2 residues responsible for that difference have been shown to be 323 and 375. The sequences affecting binding were all adjacent to the dimple in the capsid, implicating that region of the capsid as the sialic acid binding site. The role of sialic acid in virus-host cell interactions was not defined, but the plaque sizes of the non-HA mutant and wild type CPV were indistinguishable.

Animals↗
PubMed1986

Molecular approaches to the study of nucleotide excision repair in eukaryotes.

Very little is known about the molecular mechanism of nucleotide excision repair in eukaryotes. Studies on human cells have been stimulated by the availability of excision repair-defective cell lines from patients suffering from the autosomal recessive disease xeroderma pigmentosum (XP). Such studies have contributed significantly to an understanding of the genetic complexity of excision repair in human cells. However, to date, no human excision repair genes or gene products known to complement the repair defect in XP cells have been isolated. The yeast Saccharomyces cerevisiae is an interesting model for exploring the molecular mechanism of nucleotide excision repair in eukaryotic cells. As is true in human cells, multiple yeast genes are involved and at least five genes are required for the specific incision of UV-irradiated DNA in vivo. These five genes have been isolated by molecular cloning and the nucleotide sequences of four of them have been determined. Each of these cloned genes is being used for overexpression of protein.

Base Sequence↗
PubMed1986

Isolation and partial characterization of virus-transformed cell lines representing the A, G and variant complementation groups of xeroderma pigmentosum.

We have established viral-transformed, apparently permanent (immortalized) cell lines from diploid fibroblasts representative of normal and xeroderma pigmentosum (XP) A, G and variant individuals. The XP-G and XP-variant cells represent complementation groups not previously available as permanent lines. All the new permanent cell lines exhibit SV40 T-antigen expression. They are also aneuploid and have growth characteristics typical of viral transformants. They have retained the phenotypes of UV sensitivity, reduced repair synthesis or defective 'postreplication repair' appropriate to the XP complementation group they represent. Additionally, the new cell lines are all transfectable with the selectable plasmid pRSVneo. The XP-G and XP-variant cell lines show enhanced transfection with UV-irradiated plasmid DNA; a phenomenon previously reported for normal immortalized cells and for immortalized cells from the A and F complementation groups of XP.

Antigens, Viral, Tumor↗
PubMed1985

Studies on gene transfer and reversion to UV resistance in xeroderma pigmentosum cells.

We have examined several parameters which address the feasibility of complementing the UV-sensitive phenotype of xeroderma pigmentosum (XP) fibroblasts by gene transfer. We present a comparative study which demonstrates that, relative to immortalized cells, human diploid cells are poor recipients for gene transfer. As measured by both transient and stable expression assays, diploid fibroblasts were completely refractory to DNA transfer by calcium phosphate coprecipitation and exhibited substantially reduced levels of expression following gene transfer by fusion with E. coli protoplasts. We also examined the significance of reversion of the phenotype of UV sensitivity in SV40-immortalized XP-A cell lines. In addition to confirming a previous report of reversion to wild-type levels of UV resistance at a frequency of approximately 10(-7), we have attempted to facilitate the identification of XP-A cells complemented with genomic DNA by employing less stringent selection schemes and cotransfection of a selectable marker. Under these conditions, we observed an increased frequency of reversion and were unable to identify true transfectants.

Cell Line↗