Paula A. Sherman

Enzymatic assay for deoxyribonucleoside triphosphates using synthetic oligonucleotides as template primers.

The enzymatic assay for deoxyribonucleoside triphosphates has been improved by using synthetic oligonucleotides of a carefully defined sequence as template primers for DNA polymerase. High backgrounds, which limit the sensitivity of the assay when calf thymus DNA or alternating copolymers are used as template primers, were eliminated with these oligonucleotide template primers. Sensitivity was further increased by designing the template primer to incorporate multiple labeled deoxyribonucleotides per limiting unlabeled deoxyribonucleotide. Each of several DNA polymerases exhibited unique reaction characteristics with the oligonucleotide template primers, which was attributed to the differing exonuclease activities associated with these various enzymes. Assay optimization therefore included matching the polymerase with the template primer to obtain the lowest background reaction and highest sensitivity. This modified assay is particularly well suited for keeping cell sample size to a minimum in experimental protocols which generate large numbers of data points or require careful timing of sampling. With this technique, we measured the levels of all four deoxyribonucleoside triphosphates in extracts from as few as 2 x 10(4) cultured cells.

Transcriptional basis for hyporesponsiveness of the human inducible nitric oxide synthase gene to lipopolysaccharide/interferon-gamma.

The work reported here resolves, at the level of gene regulation, the controversy as to whether or not human monocytes/macrophages can produce nitric oxide (NO) when stimulated with lipopolysaccharide (LPS), with or without co‐stimulation by interferon‐γ(IFN‐γ). Studies included structural comparison of the promoters for human and mouse inducible NO synthase (iNOS) genes, transfection and assay of human and mouse iNOS promoter regions in response to LPS ± IFN‐γ, and electrophoretic mobility shift assays of KB response elements. Two explanations for hyporesponsiveness of the human iNOS promoter to LPS ± IFN‐γ were found: (1) multiple inactivating nucleotide substitutions in the human counterpart of the enhancer element that has been shown to regulate LPS/IFN‐γ‐induced expression of the mouse iNOS gene; and (2) an absence of one or more nuclear factors in human macrophages (e.g., an LPS‐inducible nuclear factor‐κB/Rel complex), that is (are) required for maximal expression of the gene. The importance of resolution of this controversy is that future research in this area should be directed toward the understanding of alternative mechanisms that can result in the successful production of NO.

Inhibition of HIV-1 integration protein by aurintricarboxylic acid monomers, monomer analogs, and polymer fractions.

Several aurintricarboxylic acid (ATA) monomers, monomer analogs, and polymer fractions have been tested as inhibitors of HIV-1 integration protein (IN). Both of the ATA monomers and all of the ATA polymer fractions inhibited a selective DNA cleavage reaction catalyzed by IN. The ATA monomer analogs were inactive or had low activity. The activities of the substances as inhibitors of HIV IN correlated in a positive way with their activities as inhibitors of the cytopathic effect of HIV-1 in CEM and HIV-2 in MT4 cells. These results suggest that inhibition of HIV IN may contribute to the antiviral activity of the ATA monomers and monomer analogs in cell culture.

The Protective Role of Nitric Oxide in a Neurotoxicant- Induced Demyelinating Model

Demyelination is often associated with acute inflammatory events involving the recruitment-activation of microglia/macrophage, astrocytes, and leukocytes. The ultimate role of inflammatory products in demyelinating disease and in the survival of oligodendrocytes, the myelin forming cells, is unresolved. The current study examines the role of inducible NO synthase (iNOS)-derived NO in a neurotoxicant-induced model of demyelination. NO levels were greatly elevated in the midline corpus callosum during demyelination in genetically intact C57BL/6 mice, and this NO was due solely to the induction of iNOS, as the correlates of NO were not found in mice lacking iNOS. C57BL/6 mice lacking iNOS exhibited more demyelination, but did not display an increased overall cellularity in the corpus callosum, attributable to an unimpeded microglia/macrophage presence. An enhanced course of pathology was noted in mice lacking iNOS. This was associated with a greater depletion of mature oligodendrocytes, most likely due to apoptosis of oligodendrocytes. Microglia and astrocytes did not undergo apoptosis during treatment. Our results suggest a moderately protective role for NO during acute inflammation-association demyelination.

Insights into the role of nitric oxide in inflammatory arthritis

Considerable evidence implicates NO as a mediator of inflammation and connective tissue injury in experimental models of arthritis as well as RA.

Potent inhibition of human neuronal nitric oxide synthase by NG-Nitro-L-Arginine methyl ester results from contaminating NG-NITRO-L-arginine

N(G)-Nitro-L-arginine methyl ester (L-NAME), inhibits the three isozymes of nitric oxide synthase (NOS) in vitro and in vivo. The mechanism of NOS inhibition by L-NAME is uncertain. L-NAME was a time-dependent inhibitor of neuronal NOS (nNOS). Concommitantly, L-NAME was hydrolyzed, non-enzymatically, to N(G)-Nitro-L-arginine (L-NA) during the enzyme assay. The time-dependent inhibition of nNOS by L-NAME was the result of this time-dependent formation of L-NA. Furthermore, N(G)-Nitro-L-arginine methyl amide, which is an isosteric analogue of L-NAME that is much less susceptible to hydrolysis, was a rapidly reversible weak inhibitor of NOS. These data suggested that L-NAME itself was a weak and rapidly reversible inhibitor of nNOS. Most of the inhibition of nNOS by a solution of L-NAME is the result of the formation of L-NA. L-NAME was a substrate for porcine liver esterase.

DNA Cleaving Activity of Purified Human Immunodeficiency Virus Integration Protein

An essential step in the life cycle of retroviruses is insertion of a double-stranded DNA copy of the viral RNA genome into the host cell DNA, to form the provirus. The insertion event depends on at least one viral protein, the integration protein (IN), which is a product of the viral pol gene. Mutations in the IN coding region of pol result in integration-negative retroviruses that will no longer replicate.1–3 The proviral DNA is identical to the precursor viral DNA except for the loss of two base pairs at each end, at the points of attachment to cellular DNA. One proposed function for IN is the removal of these two bases from the 3′-termini of both strands of the viral DNA, in preparation for integration.