PubMed Health⌕ Search

Biomedical subjects

J L Gallarda

Publications and source records attributed to J L Gallarda.

8 recordsLinked to original sources
PubMed2000

Blood screening by nucleic acid amplification technology: current issues, future challenges.

BACKGROUND: Nucleic acid amplification technology (NAT) is presently being evaluated in US clinical trials to determine the safety and efficacy of mini-pool testing for human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNA in the blood-donor population. Although the risk for transfusion-transmitted HIV and HCV infection is extremely low, there is still a small chance that blood donated by infected individuals before seroconversion can escape detection by current antibody-based assays. METHODS: This report describes the amplification technologies being used and reviews several issues surrounding NAT-based blood screening. The performance features of NAT and current enzyme immunoassay technologies are compared, and the benefits of NAT in reducing transfusion-transmitted infections are discussed. CONCLUSIONS: The current US clinical trials of mini-pool NAT testing for HIV and HCV RNA have successfully identified preseroconversion infectious blood units. Although the current NAT-based screening systems are semiautomated, mini-pool testing represents an unprecedented innovation among government and nongovernment agencies in the highly regulated blood transfusion industry. Despite cost-effectiveness issues, based on the public perception of infectious diseases acquired through blood transfusion, NAT-based screening of the blood supply is expected to become a standard in transfusion medicine.

Automation↗
PubMed1992

Early detection of antibody to human immunodeficiency virus type 1 by using an antigen conjugate immunoassay correlates with the presence of immunoglobulin M antibody.

Sequential plasma samples obtained from 16 individuals who seroconverted were tested for the presence of antibody to human immunodeficiency virus type 1 (HIV-1) by an antigen conjugate enzyme immunoassay (EIA) and a conventional antibody conjugate assay. In 11 of these individuals, the antigen conjugate assay detected antibody to HIV-1 2 to 11 days (mean, 5.5 days) earlier than the antibody conjugate assay. In 11 individuals, HIV-1 p24 antigen was detected a median of 6.5 days (range, 3 to 14 days) prior to positivity by the antigen conjugate EIA. Using class-specific probes, we determined the profiles of immunoglobulin M (IgM), IgG, and IgA antibodies for each individual and correlated these profiles with the EIA signals from both assays. In general, the appearance of IgM exhibited a peak at about 1 week postseroconversion, which was followed by gradually declining levels. Absorbance levels for IgG antibody, however, rose steadily and reached a plateau after 3 to 5 weeks. The levels of IgA were generally low and variable. In contrast to the progressive increase in EIA absorbance observed by the antibody conjugate assay, the antigen conjugate assay displayed a rapid early rise in absorbance which generally coincided with the transient expression of IgM antibody. The subsequent gradual increase coincided with rising levels of IgG. Because the configuration of the antigen conjugate EIA allows for an increased sensitivity for IgM compared with that for other classes of immunoglobulins, these results suggest that earlier detection of antibody to HIV-1 is due to the detection of IgM antibody during the early phase of seroconversion.

Biomarkers↗
PubMed1989

The beta-globin stage selector element factor is erythroid-specific promoter/enhancer binding protein NF-E4.

The analysis of transcriptional regulatory proteins is often hampered because such factors are present in cells in only sparing abundance. Although direct biochemical purification has been successfully applied to the analysis of many of these factors, such methods are labor intensive and expensive. We have developed an alternative strategy to identify and characterize such trans-acting factors and have used it to analyze the proteins that interact with the chicken adult beta-globin gene enhancer and promoter. The methodology involves (1) a sensitive 'reverse' radioimmunoassay used for the identification of antibodies to sequence-specific DNA-binding proteins, and (2) a monoclonal antibody-based DNase I footprint selection technique, which unambiguously identifies proteins responsible for particular footprints. Because this methodology relies on the isolation of antibodies to sequence-specific DNA-binding proteins, it should be of general utility in studying any trans-acting regulatory factor for which a specific DNA-binding sequence can be identified. In the present analysis, we report the identification of a 65-kD protein that is present only in mature definitive (adult) chicken erythroid cells. We show that this protein (termed NF-E4) binds to closely related sequences present in both the beta-globin promoter and enhancer. Biochemical analysis of extracts prepared from both nonerythroid and a variety of erythroid cell types suggests that NF-E4 is the trans-acting factor that confers definitive erythrocyte stage-specific transcriptional activation to the adult beta-globin gene.

Animals↗
PubMed1988

Partial molecular genetic map of the rabbit VH chromosomal region.

Thirty VH-containing cosmid clones, isolated from rabbit germ-line DNA libraries, were restriction mapped and shown to contain approximately 100 VH genes in 765-kb of DNA. Twenty-two of the cosmid clones were grouped into seven distinct clusters. The VH genes were separated by an average of 8 kb, although some were separated by less than 3 kb. Comparison of the nucleotide sequences of two of these VH genes with the sequences of another 11 VH genes showed that they were all generally more than 80% homologous suggesting that rabbit VH genes are members of one highly homologous gene family. Most rabbit Ig molecules have the VH allotypic specificities a1, a2, or a3 and are designated VHa-positive. A small number (less than 30%) of Ig molecules lack these VHa allotypic specificities and are designated VHa-negative. The VH containing cosmid clones were hybridized with synthetic oligomer probes designed to be specific for genes encoding VHa-positive or VHa-negative molecules. At least 50% of the germ-line VH genes hybridized with the VHa-negative oligomer and thus presumably encode VHa-negative molecules; as few as 15% of the genes could be identified as encoding VHa-positive molecules based on hybridization with the VHa-positive oligomer. Approximately 35% of the VH genes did not hybridize with either oligomer and could not be classified as VHa-negative or VHa-positive. We propose that the predominance of serum VHa-positive molecules, in contrast to the predominance of VHa-negative encoding germ-line genes, may reflect preferential usage of a few germline VH genes. The implications of this idea toward explaining the allelic inheritance of VHa allotypes are discussed.

Amino Acid Sequence↗
PubMed1987

Expression of cytoskeletal protein 4.1 during avian erythroid cellular maturation.

We have isolated a cDNA clone encoding part of protein 4.1, an integral component of the erythrocyte cytoskeleton. The recombinant was isolated by immunological screening of a chicken erythroid lambda gt11 cDNA library using a monoclonal antibody directed against protein 4.1. DNA blot analysis shows that the gene is present as a single copy per haploid chicken genome, while RNA blot analysis reveals the presence of a single mRNA of 7 kilobases in reticulocytes. Message of the same size (in reduced amounts) is also present in an erythroleukemic cell line transformed by avian erythroblastosis virus and is also present in vastly reduced quantities in nonerythroid hemopoietic cells. Immunoblotting and immunofluorescence experiments show that a subset of the chicken 4.1 variant proteins is preferentially expressed during in vitro differentiation of chicken erythroleukemic cells. These data indicate that the gene is both actively transcribed and translated during early erythroid cellular maturation.

Animals↗
PubMed1985

Organization of rabbit immunoglobulin genes. I. Structure and multiplicity of germ-line VH genes.

Two rabbit germ-line VH gene segments have been isolated from a recombinant phage DNA library. Nucleotide sequence analysis indicates that both of the genes share structural and regulatory features common to mouse and human VH genes, although one appears to be a pseudogene. Comparison of the protein sequences encoded by these genes to the protein sequences of rabbit immunoglobulin V regions indicates that both genes encode VH a-negative-like molecules. Quantitative genomic blot analysis with a VH probe capable of recognizing most, if not all, germ-line VH genes indicates that there are approximately 100 VH genes in the haploid genome of rabbits. The average spacing between the germ-line VH genes was determined to be approximately 6 kb. The molecular basis for the allelic inheritance of rabbit VH allotypes is discussed in view of the structural organization of germ-line VH genes.

Amino Acid Sequence↗