Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mutations were introduced by recombinant DNA techniques into 9 genes of an infectious molecular clone of human immunodeficiency virus type 1. The 24 mutants generated were characterized biochemically and biologically by transfection and infection experiments. None of the mutants which have mutations in gag (p17, p24, and p15 regions), pol (protease, reverse transcriptase, and endonuclease domains), env (gp120 region), tat, or rev were infectious, whereas vif, vpr, vpu, some of env (gp41) and nef mutants could grow in human CD4+ cells to various degrees. Of the non-infectious mutants, only endonuclease (pol) and gp41 mutants exhibited normal phenotypes with respect to the production of functional reverse transcriptase, the expression of gag, pol, and env proteins, and the generation of progeny virions, when examined in transient assays. All infectious mutants killed the CD4+ cells with the exception of a mutant carrying a defect in the vif gene.
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PMID:Generation and characterization of the human immunodeficiency virus type 1 mutants. 170 90

It has recently been suggested that endonuclease activation and/or apoptosis, possibly triggered by oxidant stress, are important pathogenetic mechanisms in oxygen deprivation/reoxygenation-induced proximal tubular cell death. To explore this possibility, DNA "laddering," a characteristic feature of these processes, was sought in: (1) postischemic rat kidneys (25- or 40-min arterial clamping; 0, 1, 4, 8, 24, and 48 h and 6 days reflow); (2) posthypoxic isolate rat proximal tubular segments and (3) cultured human kidney proximal tubular cells (HK-2) subjected either to energy depletion plus Ca2+ overload (antimycin A plus 2-deoxyglucose plus Ca2+ ionophore A23187), or to H2O2-induced cell death. DNA was subsequently extracted, electrophoresed through agarose gels, and visualized with ethidium bromide or Southern blotting. To maximize ladder detection, DNA samples were also end-labeled with 32P dideoxyadenosine triphosphate with terminal deoxynucleotidyl transferase (tdt), followed by electrophoresis. None of the postischemic DNA samples demonstrated any laddering by either ethidium bromide staining or Southern analysis (apoptotic lymphocyte DNA was a positive control). However, trace laddering was apparent by the tat technique, commencing at 1 h of reflow, peaking at 24 h, and resolving slowly thereafter. This finding correlated with the morphologic expression of tubular necrosis, not apoptosis. Hypoxia/reoxygenation caused proximal tubular segment death (44 to 64%), and HK-2 cells were slowly killed by both the H2O2 and the energy depletion/Ca(2+)-loading protocols. However, neither protocol induced ethidium bromide- or tdt-detectable DNA laddering. It was concluded that: (1) minimal DNA laddering develops postischemia, and this change is reliably detected only by the tdt method; (2) it correlates with the morphologic expression of tubular necrosis, not apoptosis; and (3) in vitro oxidative- and energy depletion-mediated proximal tubular cell death can be dissociated from DNA ladder formation.
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PMID:An evaluation of renal tubular DNA laddering in response to oxygen deprivation and oxidant injury. 789 95

Two commercially available expression vectors were modified to generate plasmids pGEXcPk and pQ9cPk. Proteins expressed from pGEXcPk and pQ9cPk had a short oligopeptide tag termed Pk at their carboxy termini and either glutathione S-transferase (GST) or a small histidine (His) tag, respectively, at their N termini. GST fusion proteins can be purified on immobilized glutathione and proteins coupled to the His tag selectively bind to Ni(2+)-NTA columns. The Pk tag is recognized by monoclonal antibody (MAb) SV5-P-k, previously produced in our laboratory. Thus proteins expressed from the pGEXcPk and pQ9cPk vectors can be purified in a two-step procedure, first via the N-terminal tag and second via the C-terminal tag. The combination of two affinity purification steps significantly improves the antigen purity and selects for full-size proteins. Moreover, by using the MAbSV5-P-k in the second purification step, Pk-linked antigens can be assembled directly into solid matrix-antibody-antigen (SMAA) complexes for use as vaccines. The genes for nef, endonuclease, p15, p17, p27, protease, Rev, reverse transcriptase (rt), tat, vif, vpr, and vpx of simian immunodeficiency virus (SIV mac 251) were cloned and expressed as both GST-SIV-Pk and His-SIV-Pk proteins. Multivalent SMAA complexes were made that contained His-p17-Pk, His-p27-Pk, His-rt-Pk, His-vpx-Pk, and His-vpr-Pk. Following two immunizations of mice with this mixture, antibodies could be detected to all five SIV antigens. When compared to single-protein immunizations, the immunogenicity of some of the proteins in this cocktail was either enhanced or decreased. Mice were also immunized with His-p17-Pk or His-p17-Pk-antibody complexes in the presence or absence of alum. The antibody-antigen complexes induced two- to four-fold higher antibody levels than antigen alone but did not appear to be more immunogenic in inducing lymphoproliferative responses. Sera from SIV-infected macaques were tested for the presence of antibodies reacting with the recombinant proteins by Western blot analysis. Antibodies to endonuclease, p15, p17, p27, rt, and vif were readily detected, antibodies against protease and vpx were present at much lower levels, but no antibodies were detected to nef, rev, tat, or vpr. Thus, we have developed a comprehensive range of reagents (available on request) that can be used to examine immune responses to SIV in both mice and monkeys.
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PMID:Expression and purification of nonglycosylated SIV proteins, and their use in induction and detection of SIV-specific immune responses. 807 30