Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although integration generally is considered a critical step in the retrovirus life cycle, it has been reported that visna virus, which causes degenerative neurologic disease in sheep, can productively infect sheep choroid plexus cells without detectable integration. To ascertain whether the integrase (IN) of visna virus is an inherently defective enzyme and to create tools for further study of integration of the phylogenetically related human immunodeficiency virus type 1 (HIV-1), we purified visna virus IN by using a bacterial expression system and applied various in vitro oligonucleotide-based assays to studying this protein. We found that visna virus IN demonstrates the full repertoire of in vitro functions characteristic of retroviral integrases. In particular, visna virus IN exhibits site-specific endonuclease activity following the invariant CA found two nucleotides from the 3' ends of viral DNA (processing activity), joins processed oligonucleotides to various sites on other oligonucleotides (strand transfer or integration activity), and reverses the integration reaction by resolving a complex that mimics one end of viral DNA integrated into host DNA (disintegration activity). In addition, although it has been reported that purified HIV-1 IN cannot specifically nick visna virus DNA ends, purified visna virus IN does specifically process and integrate HIV-1 DNA ends.
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PMID:In vitro activities of purified visna virus integrase. 818 95

Evidence supports the premise that a pro-oxidant condition exists in HIV-seropositive patients, a result of an overabundance in production of reactive oxygen forms combined with a multilevel deficiency in nutritional and metabolic sources of antioxidants. Apoptosis (a programmed cell death) is recognized as a possible pathway of immune cell loss in patients with HIV infection and AIDS. The cascade of events that results from 'oxidative stress' (OS) is markedly similar to that which can initiate apoptosis and includes oxidation of cellular membranes, alteration of metabolic pathways, disruption of electron transport systems, depletion of cellular ATP production, loss of Ca2+ homeostasis, endonuclease activation and DNA/chromatin fragmentation. Downstream events secondary to these effects may also play a role in activation of latent virus and subsequent viral replication. Primary and secondary metabolites found in plants act as synergistic antioxidants, and can protect plants from oxidation-induced cell death. Experiments have shown that some of these same metabolites can inhibit cell killing by HIV. Can these compounds be useful in inhibiting viral activation and the death of immune cells in HIV/AIDS through their synergistic antioxidant properties? A brief review of the evidence for OS in HIV is presented and the potential basis for OS playing a role in the initiation of cell death and viral replication is explored. The functional antioxidant activities of plant metabolites are illustrated and the use of these synergistic antioxidants from plants are proposed as a mechanism by which viral replication and cell killing in HIV infection can be inhibited.
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PMID:Could oxidative stress initiate programmed cell death in HIV infection? A role for plant derived metabolites having synergistic antioxidant activity. 819 35

FIV is a lentivirus infection of cats which induces an immunodeficiency syndrome associated with early qualitative defects in antigen-specific T cell function and with late quantitative defects in CD4+ T lymphocytes. We have observed that peripheral blood mononuclear cells (PBMC) from FIV-infected cats have impaired survival in culture. The mechanism of this in vitro dysfunction and depletion is not known. We have proposed that inappropriate induction of programmed cell death (apoptosis) could account for these in vitro defects. Here, we report that PBMC from FIV-infected cats, with impaired T cell blastogenesis and impaired survival in vitro, undergo an active cell death upon culture, which has the morphological and biochemical characteristics of programmed cell death (PCD). Apoptosis occurred in all six asymptomatic FIV-infected cats, and in none of the nine uninfected cats, which were studied. Changes in cell morphology under both light and electron microscopy, and fragmentation of genomic DNA were characteristic for apoptosing cells. Cell death was spontaneous and occurred in the absence of any stimuli, and culture with the T cell mitogen, concanavalin A (Con A), did not significantly enhance cell death. Activation-induced cell death was inhibited, in a dose-dependent manner, by addition to the incubation medium of zinc, which has been shown to inhibit the action of endonuclease responsible for the characteristic fragmentation of DNA. Since apoptosis has recently been implicated in AIDS pathogenesis, FIV infection may prove useful to study this aspect of retroviral, in particular HIV, infection.
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PMID:Programmed cell death (apoptosis) as a mechanism of cell death in peripheral blood mononuclear cells from cats infected with feline immunodeficiency virus (FIV). 839 41

Analogues of the 2',5'-linked adenylate trimer 5'-monophosphates (p5'A2'p5'A2'p5'A) containing 8-hydroxyadenosine and 8-mercaptoadenosine in the first, second, and third nucleotide positions were tested for their ability to bind to and activate RNase L of mouse L cells. The ability of p5'ASH2'p5'ASH2'p5'ASH (pASH3) (1c) to bind 2-5A dependent endonuclease was markedly decreased. On the other hand, an analogue of the 2',5'-linked adenylate trimer monophosphate substituted by 8-hydroxyadenosine in the first, second, and third nucleotide positions bound almost as well as parent 2-5A [pppA(2'p5'A)2] (p3A3) (1d) to RNase L. The 8-substituted analogues of 2-5A were more resistant to the degradation by the (2',5') phosphodiesterase. Of particular interest is monophosphate, pASH3 (1c) which possessed higher anti-HIV activity than pA3 (1a) or pAOH3 (1b).
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PMID:Characterization and biological activity of 8-substituted analogues of 2',5'-oligoadenylates. 846 24

Primer extension analysis was evaluated as a means to identify PCR-amplified DNA from HIV-1. Solution hybridization with radioactive labeled oligonucleotides followed by an extension reaction with Klenow fragment of Escherichia coli DNA polymerase I and subsequent separation on denaturing polyacrylamide gels reveals single stranded DNA products of the predicted size. The specificity of the reaction is further demonstrated by specific endonuclease digestion. The analysis is more sensitive than Southern blotting and about equally sensitive as Slot blot analysis when double-stranded DNA probes are used for hybridization. With end-labeled oligonucleotide probes, primer extension analysis proved an order of magnitude more sensitive than membrane hybridization. The analysis also allows quantitation of amplified DNA from 1 pg to about 1 ng of DNA product. Under the conditions described for amplification, primer extension analysis is capable of detecting a single HIV-1 plasmid DNA molecule in the presence of 1 microgram of total DNA. 3'-end mismatching of the oligonucleotide probe does not result in a significantly altered detection limit. Primer extension analysis can also be carried out with at least three different PCR-amplified DNAs in the same reaction tube.
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PMID:Primer extension analysis provides a sensitive tool for the identification of PCR-amplified DNA from HIV-1. 851 44

Although human immunodeficiency virus (HIV) infection is progressive, the rate of decline in CD4+ lymphocyte counts varies. The role of immune system components in limiting HIV infection has yet to be defined, but a previous report on the U.S. Navy HIV Seropositive Cohort reported that strong reactivity in the anti-p55 (core precursor), p24 (core) and p53 (reverse transcriptase) Western blot bands was associated with higher CD4+ lymphocyte counts at the first clinical evaluation for HIV. The previous report examined the cross-sectional association between Western blot banding patterns and initial CD4+ lymphocyte counts. This report examines the association between these banding patterns in individuals who progressed rapidly as compared with patterns of patients who did not, based on their trends in repeated CD4+ lymphocyte counts as a marker of progression. Rapid and slower progressors were identified from a cohort of 3414 Navy and Marine Corps personnel who had a first positive HIV Western blot during 1986-1991. For purposes of this study, rapid progressors were defined as individuals whose CD4+ lymphocyte counts declined to < 500 cells/mm3 within 1 year of seroconversion. A total of 325 individuals met these criteria. A comparison group of 63 slower progressors also was identified; this group consisted of those whose CD4+ lymphocyte counts remained at > or = 500 cells/mm3 for a minimum of 5 years of follow-up after their first positive Western blot. Rapid progressors were slightly younger than slower progressors and were more likely to be never married but did not differ significantly from slower progressors in race or sex. Rapid progressors had weaker reactivity in the anti-p55 core precursor (P < 0.0001), p15 core (P < 0.01), gp41 transmembrane (P < 0.01) and p31 endonuclease (P < 0.05) bands on the Western blot. The odds ratio for rapid progressor status associated with weak or absent reactivity was 7.8 in the anti-p55 band and ranged from 2.0 to 3.2 in the anti-p31, p15, and gp41 bands. These associations remained significant after adjustment for age, race, and sex. The p55 association persisted in repeated Western blots during routine clinical evaluation during a period of 5 years after the first positive Western blot. It was concluded that several possible explanations may account for the weaker reactivity of rapid progressors: (i) weak anti-p55 reactivity might have been a marker of early immune system damage; (ii) high concentrations of p55 or related proteins in the serum may have bound the available anti-p55 antibodies in rapid progressors, making them difficult to identify on the Western blot; or (iii) lack of anti-p55, p15, gp41, or p31 reactivity might have allowed more rapid progression.
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PMID:Western blot banding patterns of HIV rapid progressors in the U.S. Navy Seropositive Cohort: implications for vaccine development. Navy Retroviral Working Group. 887 45

A new restriction endonuclease, named Splase, was constructed by genetically fusing the DNA-cleavage domain of the restriction endonuclease Fok1 with the zinc-finger DNA-binding domain of the transcription factor Sp1. The resulting protein was expressed in Escherichia coli., partially purified, and shown to selectively digest plasmid DNA harboring consensus Sp1 sites. Splase was also shown to selectively digest the long terminal repeat of the HIV-1 DNA at Sp1 sites. Splase recognizes a 10-bp DNA sequence and hydrolyzes phosphodiester bonds upstream of the binding sequence. The binding specificity of Splase makes this a "rare cutter" restriction enzyme which could be valuable in creating large DNA fragments for genome sequencing projects. The result also presents the opportunity to create other restriction enzymes by altering the binding specificity of the zinc-finger recognition helix.
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PMID:Splase: a new class IIS zinc-finger restriction endonuclease with specificity for Sp1 binding sites. 889 94

The simultaneous presence of multiple HIV-1 subtypes has become common in communities with the growth of the pandemic. As a consequence, the potentiality for an increased frequency of HIV-1 mixed infections caused by viruses of distinct subtypes could be expected. Thus, there is a need to estimate the prevalence and geographic distribution of infections caused by viruses of a singular subtype as well as coinfections caused by two or more HIV-1 strains of distinct subtypes. To address this need, we have developed a genetic method based on restriction fragment length polymorphism (RFLP) to screen for these two types of infections within infected populations. In this assay, restriction enzymes may be used to predict the phylogroup of HIV-1 infected samples. A 297 bp pol fragment spanning the entire viral protease gene and a 311 bp fragment of the p24 gag region are used for this analysis. The viral regions are amplified by nested PCR using DNA templates from uncultured peripheral blood mononuclear cells (PBMC) or virus culture. Classification of HIV-1 strains to well defined subtypes B, D, F, and A/C is done by sequential endonuclease restriction analysis of a PCR amplified-protease gene followed by analysis of the p24 gag region. The electrophoretic migration patterns visualized by ethidium bromide staining or by radiolabeled probes are then determined on a 10% polyacrylamide gel. In infections caused by viruses of a singular subtype, a single restriction pattern is detected, whereas in multiple infections caused by two or more viral strains of different subtypes, the combination of different digestion patterns are observed in infected individuals. Using this methodology we have screened for genetic variations in HIV-1 proviral DNA from thirty-three Brazilian samples. Our RFLP procedure classified thirty-two samples as single infections caused by viruses of subtypes B (31) and F (1), and one sample as dual infection caused by distinct viral strains. Subsequent sequence and phylogenetic analysis of the viral protease gene in lymphocytes of all these patients confirmed our RFLP findings in single infections, and demonstrated the existence of two distinct HIV-1 strains of subtypes F and D in a patient which lymphocytes showed the simultaneous presence of two different digestion patterns. As up to now, single infections caused by subtype D variants were not identified in Brazil, our data provide the first evidence of subtype D HIV-1 in this country. Because sequencing of HIV proviral DNA is not particularly practical for large-scale molecular epidemiological studies, the protease/gag-based RFLP screening method will be useful to predict the phylogroup of HIV-1, and to identify multiple infections caused by HIV-1 strains of distinct subtypes. We believe that this information is crucial for both evaluation of the HIV-1/AIDS pandemic and intervention strategies.
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PMID:Identification of single and dual infections with distinct subtypes of human immunodeficiency virus type 1 by using restriction fragment length polymorphism analysis. 893 82

The control of gene transcription by antigene oligonucleotides rests upon the specific recognition of double-helical DNA by triplex-forming oligonucleotides. The development of the antigene strategy requires access to the targeted DNA sequence within the chromatin structure of the cell nucleus. In this sudy we have used HIV-1 chronically infected cells containing the HIV provirus as endogenous genes to demonstrate that the integrated HIV-1 proviral genome is accessible to triplex-forming oligonucleotides within cell nuclei. An oligonucleotide-psoralen conjugate targeted to the polypurine tract (PPT) of the HIV-1 proviral sequence was used as a tool to convert the noncovalent triple-helical complex into a covalent lesion on genomic DNA after UV irradiation of cells. Triplex-derived adducts were analyzed using two different methods. The photo-induced psoralen cross-link prevented cleavage of the target sequence by DraI restriction endonuclease, and the sequence-specific inhibition of cleavage was revealed and quantitated by Southern blot analysis. A quantitative analysis of cross-linking efficiency was also carried out by a competitive PCR-based assay. These two approaches allowed us to demonstrate that a triplex-forming oligonucleotide can recognize and bind specifically to a 15-bp sequence within the chromatin structure of cell nuclei.
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PMID:Accessibility of nuclear DNA to triplex-forming oligonucleotides: the integrated HIV-1 provirus as a target. 899 Jan 64

Experiments undertaken with commercially available recombinantly produced human immunodeficiency virus Type 1 (HIV-1) gp120 demonstrated that the resuspended lyophilized protein, a product of the baculovirus expression system, had intrinsic nuclease activity. This nuclease activity was distinguishable from the molecular-grade bovine serum albumin that it was constituted in. The activity was thermolabile in that if the preparation was heated to 100 degrees C for 10 min, the activity was abolished, although this did not happen when it was stored at -20 degrees C. The nuclease activity was also Ca+2- and Mg+2-dependent, and had endonuclease as opposed to exonuclease activity. Zn+2 ions were found to inhibit the enzyme. The intensity of nuclease activity varied from batch to batch. A lyophilized homogenate of Sf9 insect cells expressing the Rho baculovirus-derived red blood cell protein 4.2 (Pallidin) was also found to have nuclease activity on reconstitution. In contrast, most, though not all E. coli-produced recombinant proteins were found to be free of nuclease activity. The use of activity gels to identify the size of the nuclease contained in the gp120 preparation was limited, because despite the use of many renaturation methods, the enzyme in the gp120 preparation could not be functionally resuscitated following sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunoprecipitation studies were useful to demonstrate that nuclease activity in the gp120 preparation was functionally distinguishable from the gp120 itself. When mononuclear cells transformed with anti-CD3 were concurrently incubated with gp120 (5-40 micrograms/mL), internucleosomal DNA fragments characteristic of apoptosis were demonstrated in the supernatant by DNA gel electrophoresis. In the context of HIV-1 and AIDS, where the depletion of CD4+ T-cells has been found to be associated with apoptosis, nuclease activity intrinsic to the gp120 preparation used in experimentation may potentially alter experimental results.
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PMID:Identification of endonuclease activity in HIV-1 gp120 preparations produced using baculovirus expression systems. 926 86


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