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A Golden

Publications and source records attributed to A Golden.

At least 37 records · Page 2Linked to original sources
PubMed1998

Detection and differentiation of HIV-1 group O sera from HIV-1 group M and HIV-2 using recombinant antigens and peptides.

Recombinant antigens and peptides were used to develop an HIV slot immunoblot assay to confirm and differentiate infection by HIV-1 group M, HIV-1 group O or HIV-2. Recombinant antigens from the gag, pol or env regions of HIV-1 and HIV-2, in addition to synthetic peptides from the immunodominant region (IDR) of transmembrane proteins gp41 (HIV-1) or gp36 (HIV-2), were blotted on nitrocellulose strips and used as a substitute for competitive Western blots. Evaluation of a large number of samples (N = 440) from various regions of the world, using the immunoblot, showed effective differentiation of HIV-1 group M, HIV-1 group O and HIV-2. The immunoblot identified correctly all (24/24) HIV-1 group O samples that were confirmed subsequently by PCR and sequence analysis. The immunoblot is a useful tool for identifying HIV-1 group O seropositive samples and has the potential to identify other serological HIV variants that may represent detection problems for HIV screening assays using HIV-1 group M subtype B reagents.

Amino Acid Sequence↗
PubMed1998

Recombinant mouse-human chimeric antibodies as calibrators in immunoassays that measure antibodies to Toxoplasma gondii.

In the present study, we examined the feasibility of using recombinant antibodies containing murine variable regions and human constant regions as calibrators or controls in immunoassays. As a model system, we chose the Abbott IMx Toxo immunoglobulin M (IgM) and Toxo IgG assays designed to detect antibodies to Toxoplasma gondii. Two mouse monoclonal antibodies were selected based on their reactivity to the T. gondii antigens P30 and P66. Heavy- and light-chain variable-region genes were cloned from both hybridomas and transferred into immunoglobulin expression vectors containing human kappa and IgG1 or IgM constant regions. The constructs were stably transfected into Sp2/0-Ag14 cells. In the IMx Toxo IgG assay, immunoreactivity of the anti-P30 chimeric IgG1 antibody paralleled that of the positive human plasma-derived assay calibrators. Signal generated with the anti-P66 chimeric IgG1 antibody was observed to plateau below the maximal reactivity observed for the assay calibrator. Examination of the IgM chimeric antibodies in the IMx Toxo IgM assay revealed that both the anti-P30 and anti-P66 antibodies matched the assay index calibrator manufactured with human Toxo IgM-positive plasma. When evaluated with patient samples, the correlation between results obtained with the chimeric antibody calibrators and the positive human plasma calibrators was > or =0.985. These data demonstrate that chimeric mouse-human antibodies are a viable alternative to high-titer positive human plasma for the manufacture of calibrators and controls for diagnostic assays.

Amino Acid Sequence↗
PubMed1998

A highly conserved centrosomal kinase, AIR-1, is required for accurate cell cycle progression and segregation of developmental factors in Caenorhabditis elegans embryos.

S. cerevisiae Ipl1, Drosophila Aurora, and the mammalian centrosomal protein IAK-1 define a new subfamily of serine/threonine kinases that regulate chromosome segregation and mitotic spindle dynamics. Mutations in ipl1 and aurora result in the generation of severely aneuploid cells and, in the case of aurora, monopolar spindles arising from a failure in centrosome separation. Here we show that a related, essential protein from C. elegans, AIR-1 (Aurora/Ipl1 related), is localized to mitotic centrosomes. Disruption of AIR-1 protein expression in C. elegans embryos results in severe aneuploidy and embryonic lethality. Unlike aurora mutants, this aneuploidy does not arise from a failure in centrosome separation. Bipolar spindles are formed in the absence of AIR-1, but they appear to be disorganized and are nucleated by abnormal-looking centrosomes. In addition to its requirement during mitosis, AIR-1 may regulate microtubule-based developmental processes as well. Our data suggests AIR-1 plays a role in P-granule segregation and the association of the germline factor PIE-1 with centrosomes.

Amino Acid Sequence↗
PubMed1997

High-level production of a secreted, heterodimeric alpha beta murine T-cell receptor in Escherichia coli.

For structural studies, high-level production of properly folded, disulfide-linked, unglycosylated protein in E. coli is an attractive alternative to production in eukaryotic systems. We describe here the production of heterodimeric, murine D10 T-cell receptor (sD10TCR) in E. coli as a secreted leucine zipper (LZ) fusion protein. Two genes, one (alpha-acid) encoding the alpha-chain variable and constant domains (V alpha and C alpha) of D10 TCR fused to an LZ 'acid' encoding sequence and the other (beta-base) encoding the beta-chain variable and constant domains (V beta and C beta) fused to an LZ 'base' encoding sequence, were co-expressed from a bacteriophage T7 promoter as a dicistronic message. Secreted alpha-acid and beta-base proteins formed proper inter- and intra-chain disulfide bonds in the periplasm, bypassing the need for in vitro protein refolding. Complementary LZ sequences facilitated the formation of alpha beta heterodimers. sD10TCR-LZ was purified by affinity chromotography using a D10 TCR clonotype-specific monoclonal antibody (mAb 3D3). Typical yields of purified protein were 4-5 mg/l of culture. Purified sD10TCR-LZ was reactive with a panel of conformationally sensitive TCR-specific monoclonal antibodies, consistent with its conformational integrity and appeared to be suitable for structural studies by X-ray crystallography or NMR spectroscopy.

Amino Acid Sequence↗
PubMed1997

Serologic and phylogenetic characterization of HIV-1 subtypes in Uganda.

OBJECTIVES: To determine the HIV genetic subtypes present in HIV-1-infected asymptomatic blood donors in Uganda and to evaluate serologic detection of infection by commercial immunoassays; to evaluate samples for HIV-1 group O infections. METHODS: Sixty-four HIV-seropositive plasma samples were collected from the Nakasero Blood Bank, Kampala, Uganda. The plasma were evaluated using commercial HIV enzyme immunoassays (EIA) and a research immunoblot. HIV-1 group M and O infections were identified on the basis of discordant seroreactivity in EIA and reactivity to group M and O antigens on the immunoblot. Regions of gag p24 and env gp41 were amplified using reverse transcriptase polymerase chain reaction, and genetic subtypes were determined by phylogenetic analysis. RESULTS: Serologic testing confirmed that 63 out of 64 plasma units were positive for HIV-1 group M infection and showed no evidence of HIV-1 group O infections. Genetic subtyping determined that 25 samples were subtype A, three subtype C, 22 subtype D, and nine were heterogeneous for subtypes A and D. CONCLUSIONS: Despite the sequence variation observed in Uganda, commercial EIA based on HIV-1 subtype B proteins detected all the infections. In contrast, a peptide-based assay failed to detect three infections by subtype D viruses. This emphasizes the negative impact of HIV genetic variation on assays that rely on peptides to detect HIV infections. The number of infections with heterogeneous subtype (due to mixed infections or recombinant viruses) is high and reflects the growing complexity of the HIV epidemic in endemic regions where multiple subtypes are present in the population.

Genotype↗
PubMed1995

Epitope mapping of anti-HIV and anti-HCV monoclonal antibodies and characterization of epitope mimics using a filamentous phage peptide library.

A large filamentous phage library (1 x 10(9) clones) displaying random 30-amino-acid (aa) sequences on the N terminus of the pIII coat protein was constructed and characterized. Clones in the library were affinity selected for binding to monoclonal antibodies (mAb) against two viral antigens, the HIV gp120 protein and the HCV core protein. The obtained aa sequences precisely identified the epitopes recognized by the mAb. Binding of peptide-carrying phages to the Ab was demonstrated by ELISA, Western blot and the surface plasmon resonance (SPR) method. The mAb-specific peptides were transferred via genetic techniques onto the N terminus of Escherichia coli alkaline phosphatase (AP). When fused to the enzyme, the peptides maintained their ability to bind their respective mAb, indicating that the peptides contained the necessary contact residues for binding. The affinity of the peptides was estimated to be 100 nM by SPR. A comparison is presented of the relative affinities of phage-derived peptides to the native viral epitopes also displayed on the AP scaffold. The approach of transferring epitopes from phage to AP for further evaluation should be applicable to many other mAb or receptors.

Acid Phosphatase↗
PubMed1993

Role of a raf proto-oncogene during Caenorhabditis elegans vulval development.

During Caenorhabditis elegans vulval induction, multipotent precursors respond to an inductive signal by generating vulval cells as opposed to non-specialized epidermal cells. Both classical and 'reverse' genetic approaches have revealed that a cascade of nematode homologues of mammalian proto-oncogenes is necessary for induction of the vulva. The inductive signal is a growth factor encoded by the lin-3 gene and its candidate receptor is a tyrosine kinase encoded by the let-23 gene. let-23 acts via a Ras protein encoded by the let-60 gene. A nematode homologue of mammalian raf family protein serine/threonine kinases has been cloned and found to be encoded by the lin-45 gene. Dominant negative lin-45 raf mutants prevent vulval induction. A recessive lin-45 raf mutation prevents the excessive vulval differentiation caused by activated ras, indicating that raf might act downstream of ras during vulval induction.

Animals↗
PubMed1993

C. elegans lin-45 raf gene participates in let-60 ras-stimulated vulval differentiation.

Vulval differentiation in Caenorhabditis elegans is controlled by intercellular signalling mediated by a receptor tyrosine kinase and a ras gene product. The lin-45 gene encodes a homologue of the raf family of serine/threonine kinases and is necessary for vulval differentiation. The lin-45 raf gene product appears to act downstream of the ras protein in this pathway. A proto-oncogene-mediated signalling pathway may be a common feature of metazoan development.

Amino Acid Sequence↗
PubMed1993

Bone mass among premenopausal women.

Bone mass in early adult life, or peak bone mass, is primarily under genetic control, although recent data suggest that alterations in nutrition and life style during growth may modify the genetic potential. Cross-sectional data indicated that upon completion of growth, there was a period of skeletal consolidation that resulted primarily from increased width of cortical bones. Data on cancellous bone are conflicting. Cross-sectional data suggest that cancellous bone mass reaches a maximum at about age 18 years. Bone mass in the spine appears stable until the beginning of menopause, unless there are other disturbances of ovarian function. On the basis of cross-sectional data, significant bone loss in the femoral neck in premenopausal women appears to occur prior to changes in ovarian function. This decline in hip density is also independent of body size, nutritional influences, or other variables thought to influence bone mass. Longitudinal data are not available to confirm the reduction in femoral neck bone mass. Preliminary data indicate that modest disruptions of ovarian function, insufficient to result in frank amenorrhea, might impact negatively on bone mass. A detailed longitudinal study of menstrual and other factors controlling bone mass in premenopausal women is needed. If bone mass can be modified, ensuring a maximal skeletal mass becomes a viable possibility for prevention of osteoporosis.

Adolescent↗
PubMed1990

Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins.

Treatment of platelets with thrombin was shown previously to induce rapid changes in tyrosine phosphorylation of several platelet proteins. In this report, we demonstrate that a variety of agonists which induce platelet aggregation also stimulate tyrosine phosphorylation of three proteins with apparent molecular masses of 84, 95, and 97 kD. Since platelet aggregation requires the agonist-induced activation of an integrin receptor (GP IIb-IIIa) as well as the binding of fibrinogen to this receptor, we examined the relationship between tyrosine phosphorylation and the function of GP IIb-IIIa. When platelets were examined under conditions that either precluded the activation of GP IIb-IIIa (prior disruption of the complex by EGTA at 37 degrees C) or the binding of fibrinogen (addition of RGDS or an inhibitory mAb), tyrosine phosphorylation of the 84-, 95-, and 97-kD proteins was not observed. However, although both GP IIb-IIIa activation and fibrinogen binding were necessary for tyrosine phosphorylation, they were not sufficient since phosphorylation was observed only under conditions in which the activated platelets were stirred and allowed to aggregate. In contrast, tyrosine phosphorylation was not dependent on another major platelet response, dense granule secretion. Furthermore, granule secretion did not require tyrosine phosphorylation of this set of proteins. These experiments demonstrate that agonist-induced tyrosine phosphorylation is linked to the process of GP IIb-IIIa-mediated platelet aggregation. Thus, tyrosine phosphorylation may be required for events associated with platelet aggregation or for events that follow aggregation.

Antibodies, Monoclonal↗
PubMed1989

Thrombin treatment induces rapid changes in tyrosine phosphorylation in platelets.

We previously demonstrated that platelets express high levels of the tyrosine protein kinase pp60c-src. By a quantitative immunoblot assay, it is shown in this report that pp60c-src represents 0.2-0.4% of total platelet protein. The expression of high levels of pp60c-src in platelets correlated with high levels of total cell phosphotyrosine. Unstimulated platelets were shown to possess numerous phosphotyrosine-containing proteins by immunoblot analysis using antibodies that specifically recognize phosphotyrosine residues. To examine whether the pattern of phosphotyrosine-containing proteins changes upon platelet activation, lysates from thrombin- and phorbol ester-treated platelets were subjected to immunoblot analysis. Novel phosphotyrosine-containing proteins were detected within seconds following platelet stimulation. These results suggest that tyrosine phosphorylation, perhaps mediated by pp60c-src, may be involved in events associated with platelet activation.

Animals↗
PubMed1986

Blood platelets express high levels of the pp60c-src-specific tyrosine kinase activity.

We have examined human and rabbit blood platelets for expression of pp60c-src, the normal cellular homolog of the transforming protein of Rous sarcoma virus. pp60c-src kinase activity was determined by an immune-complex kinase assay that uses enolase as the substrate, and pp60c-src protein levels were determined by an immunoblot assay. Lysates from platelets expressed high levels of pp60c-src-specific kinase activity and pp60c-src protein compared to the levels found in other tissues. pp60c-src was also found to be one of the major proteins phosphorylated in vitro in membranes isolated from platelets. Multiple protein species other than pp60c-src were also phosphorylated on tyrosine in the membrane phosphorylation reactions, and phosphotyrosine represented approximately equal to 80% of the total phosphoamino acid residues phosphorylated in the membranes. These results indicate that tyrosine kinases represent the major protein phosphorylating enzymes detected in isolated platelet membranes. Although the association of tyrosine kinase activity with many viral oncogene products and cellular growth hormone receptors has suggested a role for these enzymes in the regulation of cell proliferation, these results indicate that the expression of high levels of tyrosine kinase activity is not exclusively associated with proliferating cells.

Animals↗
PubMed1986

Investigations of the expression of the cellular src gene product.

We have examined the expression of the c-src gene product in a variety of embryonic and adult tissues, in peripheral blood cells, and in cells transformed by other tumor viruses, in an attempt to identify the types of cells in which pp60c-src might provide a specific function. These studies have indicated that c-src gene expression is regulated at multiple levels in different cell types and have suggested that pp60c-src is not exclusively involved in the regulation of cell proliferation. The lowest levels of pp60c-src were found in fibroblasts, which have previously served as the standard cell type for comparisons between pp60c-src and pp60v-src. The highest levels of pp60c-src-specific kinase activity were detected in three types of cells: neurons, platelets, and polyoma virus transformed cells. In this report, we will compare the expression of pp60c-src in fibroblasts to that in platelets, neurons, and polyoma virus transformed fibroblasts. In each of the three latter cell types, the c-src gene product displays a unique pattern of expression which can be distinguished from that in fibroblasts (see diagram Fig. 1).

Animals↗