Differential phosphorylation of IRS-1 by insulin and insulin-like growth factor I receptors in Chinese hamster ovary cells.

Insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) are closely related receptor tyrosine kinases. Despite their high degree of homology, recent evidence suggests that the two receptors have distinct biological roles. In several recent studies, the cytoplasmic tyrosine kinase domains of the two receptors have been shown to possess different signalling specificities. In this study, we examine the hypothesis that differential phosphorylation of insulin receptor substrate 1 (IRS-1) may contribute to these differences in signalling between the two receptors. Using Chinese hamster ovary (CHO) cells stably expressing human IR or IGF-IR and activated by their respective ligands, we show that there are differences between the two receptors with regard to the complement of SH2-containing proteins recruited to IRS-1. In particular, IGF-IR appears to couple IRS-1 preferentially to Grb2 whereas, in contrast, IR appears to couple IRS-1 preferentially to the p85 subunit of phosphatidyl inositol 3-kinase (PI3-kinase) and to Nck. The two receptors couple IRS-1 equally to the tyrosine phosphatase SHP2. We have also generated phosphospecific antibodies to three important tyrosine phosphorylation sites on IRS-1 (pY608, pY895 and pY1172). We used these antibodies to probe the phosphorylation status of these sites in intact CHO/IR and CHO/IGF-IR cells. In the case of pY608, these results also show evidence for differential phosphorylation of IRS-1 by the two receptors. Taken together, the results presented here support the notion that the cytoplasmic domains of IR and IGF-IR have differences in their intrinsic signalling potentials.

Impairment of phagosome-lysosome fusion in HIV-1-infected macrophages.

Phagosome-lysosome fusion is critical for intracellular killing of most organisms and is inhibited by some viruses, notably influenza. We explored the effects of infection in vitro with HIV-1 (IIIB or Ada-M) on phagosome-lysosome fusion in blood monocyte-derived macrophages. After 8 days of infection, fusion was assessed from the fluorescence change occurring up to 2 h after labeling the lysosome compartment with acridine orange and loading of phagosomes with opsonized yeast. Compared with mock-infected control macrophages, the proportion of cells showing fusion after infection was reduced from a mean of 70% to a mean of 47% (p = 0.0001). Inhibition was seen with heat-killed HIV-1 IIIB but not virus-free filtrate. It was mimicked by recombinant gp 120 and blocked by soluble CD4 or antibody to CD4 but not by a neutralizing antibody to the V3 loop of gp 120. The inhibitory effect was seen 8 days after the original, transient exposure to gp 120. These results suggest that a lasting abnormality of phagosome-lysosome fusion results from interaction between gp 120 and CD4, contributing, perhaps, to the increased susceptibility to opportunistic infections of people infected with HIV.

Poly A-linked colorimetric microtiter plate assay for HIV reverse transcriptase.

An assay for detection of the reverse transcriptase (RT) of the human immunodeficiency virus (HIV) was developed using poly A linked to microtiter plate with colorimetric detection of incorporated biotin deoxyuridine triphosphate (biotin-dUTP). During the RT reaction, biotin-dUTP was incorporated into oligodeoxythymidylic acid (oligo-dT) which had been hybridized with poly A. At the detection step, horseradish peroxidase conjugated streptavidin was added, followed by the reaction of a colorimetric substrate for this enzyme. This method was contrasted with the two standard isotopic RT assays. There was excellent correlation between the colorimetric RT assay and each of two isotopic RT assays for both detection and quantification of avian myoblastosis virus reverse transcriptase (AMV-RT) and of HIV RT in human lymphocytes infected in vitro with HIV-1. The total assay required for performing the colorimetric assay, including the RT reaction, was 40 min.

Detection of human immunodeficiency virus (HIV) by colorimetric assay for reverse transcriptase activity on magnetic beads.

A colorimetric assay for detection of reverse transcriptase (RT) of the human immunodeficiency virus (HIV) was developed using oligodeoxythymidylic acid (oligo-dT)-linked magnetic beads and digoxigenin-deoxyuridine triphosphate (dig-dUTP). During the RT reaction, dig-dUTP was incorporated into oligo-dT which had been hybridized to polyadenylic acid [poly (A)]. At the detection step, an alkaline phosphatase-conjugated antibody to digoxigenin was added, followed by the addition of a colorimetric substrate for this enzyme. This method showed excellent correlation with the isotopic RT assay, which used tritiated thymidine triphosphate ([3H]dTTP), for detection of purified avian-myeloblastosis-virus RT (AMV-RT). This assay also demonstrated close correlation with the isotopic RT assay using human peripheral-blood lymphocytes infected in vitro with HIV. This colorimetric RT assay offers important advantages over the conventional radioactive RT assays with respect to its simplicity, safety and cost. The total assay time, including the RT reaction step, was less than 1 h, and therefore provides a reliable rapid assay for detection and quantification of HIV.

Chemiluminescent enzyme-linked immunoassay for reverse transcriptase, illustrated by detection of HIV reverse transcriptase.

A chemiluminescent assay for reverse transcriptase (RT) of the human immunodeficiency virus 1 was developed using biotin-labeled oligodeoxythymidylic acid (biotin oligo-dT) and digoxigenin-deoxyuridine triphosphate instead of tritiated thymidine triphosphate. After the RT reaction, the newly polymerized strand from biotin oligo-dT contained digoxigenin labels. This nucleotide was bound to a streptavidin-coated microtiter plate by the reaction to biotin. At the detection step, an alkaline phosphatase-conjugated antibody to digoxigenin was added, followed by the reaction of a chemiluminescent substrate for this enzyme. This method shows very close correlation with the isotopic assay using purified avian myeloblastosis virus reverse transcriptase (RT). This assay was also compared with the isotopic RT assay using lymphocytes infected in vitro with HTLV-IIIB and again demonstrated a close correlation. The total assay time after the RT reaction step was less than 100 min.

Colorimetric reverse transcriptase assay for HIV-1.

A colorimetric assay for reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) was developed using a double labelled (biotin and digoxigenin) deoxyuridine triphosphate mixture instead of tritiated thymidine triphosphate. After the RT reaction, the newly polymerized strand from oligodeoxythymidylic acid (oligo-dT) contained both biotin and digoxigenin labels. This nucleotide was bound to streptavidin-magnetic beads by the reaction to biotin. At the detection step, an alkaline phosphatase conjugated antibody to digoxigenin was added, followed by the reaction of a colorimetric substrate for this enzyme. This RT assay was comparable to the isotopic RT assay using purified AMV-RT and two strains of HIV grown in cell lines. In addition it was equivalent to the isotopic RT assay for analysis of the time course of in vitro infection of human peripheral blood lymphocytes by HIV-1. The total assay time after the RT reaction step was less than one hour.