ABSTRACT
Human immunodeficiency virus (HIV) infection results in a functional impairment of CD4(+) T cells long before a quantitative decline in circulating CD4(+) T cells is evident. The mechanism(s) responsible for this functional unresponsiveness and eventual depletion of CD4(+) T cells remains unclear. Both direct effects of cytopathic infection of CD4(+) cells and indirect effects in which uninfected "bystander" cells are functionally compromised or killed have been implicated as contributing to the immunopathogenesis of HIV infection. Because T-cell receptor engagement of major histocompatibility complex (MHC) molecules in the absence of costimulation mediated via CD28 binding to CD80 (B7-1) or CD86 (B7-2) can lead to anergy or apoptosis, we determined whether HIV type 1 (HIV-1) virions incorporated MHC class I (MHC-I), MHC-II, CD80, or CD86. Microvesicles produced from matched uninfected cells were also evaluated. HIV infection increased MHC-II expression on T- and B-cell lines, macrophages, and peripheral blood mononclear cells (PBMC) but did not significantly alter the expression of CD80 or CD86. HIV virions derived from all MHC-II-positive cell types incorporated high levels of MHC-II, and both virions and microvesicles preferentially incorporated CD86 compared to CD80. CD45, expressed at high levels on cells, was identified as a protein present at high levels on microvesicles but was not detected on HIV-1 virions. Virion-associated, host cell-derived molecules impacted the ability of noninfectious HIV virions to trigger death in freshly isolated PBMC. These results demonstrate the preferential incorporation or exclusion of host cell proteins by budding HIV-1 virions and suggest that host cell proteins present on HIV-1 virions may contribute to the overall pathogenesis of HIV-1 infection.
Subject(s)
Antigens, CD/metabolism , B7-1 Antigen/metabolism , Herpesvirus 1, Human/metabolism , Histocompatibility Antigens Class II/metabolism , Histocompatibility Antigens Class I/metabolism , Leukocyte Common Antigens/metabolism , Membrane Glycoproteins/metabolism , Virion/metabolism , B7-2 Antigen , Cell Line , Herpesvirus 1, Human/immunology , Herpesvirus 1, Human/pathogenicity , Humans , Protein Tyrosine Phosphatase, Non-Receptor Type 1ABSTRACT
STUDY OBJECTIVE: To assess the changes in viscosity of peritoneal fluid during laparoscopic exposure to CO2 insufflation. DESIGN: Analysis and mathematic modeling of peritoneal fluid viscosity in vivo and in vitro as a result of exposure to unconditioned CO2 (Canadian Task Force classification II-2). SETTING: Medical school university research laboratory and hospital. MATERIALS: Peritoneal fluid from 45 women. INTERVENTION: Peritoneal fluid was obtained at laparoscopy before insufflation and tested for viscosity after exposure to currently used raw dry unconditioned CO2. MEASUREMENTS AND MAIN RESULTS: Peritoneal fluid viscosity was tested by viscometric methods and mathematic modeling. Initial viscosity of peritoneal fluid before gas exposure was 1.425 centipoise (cP). Viscosity measurements were obtained at 20-second intervals for gas flows of 1 and 3 L/minute. Increases in viscosity occur rapidly, and by 200 seconds it was 59 cP and 98 cP for 1 and 3 L flow rates, respectively. CONCLUSION: Very dry CO2 for laparoscopy causes peritoneal fluid viscosity to increase dramatically. (J Am Assoc Gynecol Laparosc 8(1):117-123, 2001)
Subject(s)
Ascitic Fluid , Laparoscopy , Pneumoperitoneum, Artificial , Adult , Carbon Dioxide , Dehydration , Female , Humans , ViscosityABSTRACT
Our previous biochemical studies of HIV-1 and MuLV virions isolated and identified mature Gag products, HIV-1 p6(Gag) and MuLV p12(Gag), that were conjugated to a single ubiquitin. To study the importance of the monoubiquitination of Gag, a series of lysine to arginine mutants were constructed that eliminated ubiquitination at one or both of the lysines in HIV-1(NL4-3) p6(Gag) and both lysines in Moloney MuLV p12(Gag). HPLC and immunoblot analysis of the HIV-1 mutants demonstrated that either of the lysines in p6(Gag), K27 or K33, could be monoubiquitinated. However, infectivity assays showed that monoubiquitination of HIV-1 p6(Gag) or MuLV p12(Gag) is not required for viral replication in vitro. Pulse-chase radiolabeling of HIV-1-producing cells revealed that monoubiquitination of p6(Gag) does not affect the short-term release of virus from the cell, the maturation of Pr55(Gag), or the sensitivity of these processes to proteasome inhibitors. Experiments with protease-deficient HIV-1 showed that Pr55(Gag) can be monoubiquitinated, suggesting that p6(Gag) is first modified as a domain within Gag. Examination of the proteins inside an HIV-1 mutant found that free ubiquitin was incorporated into the virions in the absence of the lysines in p6(Gag), showing that the ubiquitin inside the virus is not initially brought in as a p6(Gag) conjugate. Although our results establish that monoubiquitination of p6(Gag) and p12(Gag) is not required for viral replication in vitro, this modification may be a by-product of interactions between Gag and cellular proteins during assembly and budding.
Subject(s)
HIV-1/metabolism , Moloney murine leukemia virus/metabolism , Ubiquitins/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Arginine/genetics , Cell Line , Chromatography, High Pressure Liquid , Gene Products, gag/analysis , Gene Products, gag/genetics , Gene Products, gag/metabolism , HIV Protease/deficiency , HIV Protease/genetics , HIV-1/enzymology , HIV-1/pathogenicity , Humans , Immunoblotting , Lysine/genetics , Mice , Molecular Sequence Data , Moloney murine leukemia virus/pathogenicity , Mutagenesis, Site-Directed , Protein Precursors/metabolism , Ubiquitins/analysis , Virus Replication , gag Gene Products, Human Immunodeficiency VirusABSTRACT
Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6(gag), a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6(gag). Consistent with this, viruses with mutations in PR or p6(gag) were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.
Subject(s)
Cysteine Endopeptidases/metabolism , Gene Products, gag/biosynthesis , Gene Products, gag/metabolism , HIV-1/physiology , HIV-2/physiology , Multienzyme Complexes/metabolism , Protease Inhibitors/pharmacology , Protein Precursors/metabolism , Cell Line , HIV-1/drug effects , HIV-1/genetics , HIV-2/drug effects , HIV-2/genetics , HeLa Cells , Human Immunodeficiency Virus Proteins , Humans , Kinetics , Proteasome Endopeptidase Complex , Protein Processing, Post-Translational , Ubiquitins/metabolism , Viral Regulatory and Accessory Proteins/metabolismABSTRACT
HLA class II DR is one of the most abundant cell surface proteins incorporated onto human immunodeficiency virus type 1 (HIV-1) during budding. The mechanism for HLA class II protein incorporation is not known and may involve a viral protein. To determine whether Env affects HLA class II protein incorporation, HIV-1 virions, either with or without Env on their surface, were produced from HLA class II-expressing cells and analyzed by whole-virus immunoprecipitation with antisera against HLA class II proteins. HLA class II proteins were detected on virions only when wild-type Env was incorporated, while similar experiments showed that HLA class I proteins were incorporated independent of Env packaging. Therefore, the packaging of HIV-1 Env protein is required for the efficient incorporation of HLA class II but not class I proteins into the virion. Analysis of two Env mutants revealed that the presence of a 43-amino-acid sequence between amino acids 708 and 750 in the gp41(TM) cytoplasmic tail was required for efficient incorporation of HLA class II proteins. These data show that HIV-1 actively incorporates HLA class II proteins in a process that, either directly or indirectly, requires Env.
Subject(s)
Gene Products, env/metabolism , HIV-1/metabolism , Histocompatibility Antigens Class II/metabolism , Endocytosis , Gene Products, env/genetics , HIV Envelope Protein gp41/genetics , HIV Envelope Protein gp41/metabolism , HIV-1/genetics , HIV-1/pathogenicity , Histocompatibility Antigens Class I/metabolism , Histocompatibility Antigens Class II/immunology , Humans , Immunoblotting , Precipitin Tests , Tyrosine/chemistry , Virion/immunology , Virion/metabolismABSTRACT
Host proteins are incorporated both on and inside human immunodeficiency virus type 1 (HIV-1) virions. To identify cellular proteins inside HIV-1, virion preparations were treated by a protease-digestion technique that removes external host proteins, allowing for the study of the proteins inside the virus. Treated HIV-1 preparations were analyzed by immunoblot, high-pressure liquid chromatography, and protein sequence analyses. These analyses identified several cellular proteins inside HIV-1: elongation factor 1alpha, glyceraldehyde-3-phosphate dehydrogenase, HS-1, phosphatidylethanolamine-binding protein, Pin1, Lck, Nm23-H1, and the C-terminal tail of CD43. Several of these proteins were found as fragments of their full-sized proteins that appear to be generated by our protease treatment of the virions, the HIV-1 protease, or a cellular protease. Recent advances in cell biology and biochemistry have identified some of these proteins as actin-binding proteins. These results support the hypothesis that actin filaments are incorporated into the virion and may provide additional clues for the understanding of the interaction between viral and cellular proteins during assembly and budding.
Subject(s)
Actins/metabolism , Androgen-Binding Protein , HIV-1/chemistry , Microfilament Proteins/analysis , Nucleoside-Diphosphate Kinase , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Blood Proteins/analysis , Blood Proteins/chemistry , Blood Proteins/metabolism , Carrier Proteins/analysis , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Chaperonin 60/analysis , Chaperonin 60/chemistry , Chaperonin 60/metabolism , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Glyceraldehyde-3-Phosphate Dehydrogenases/analysis , Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , HIV Protease/genetics , HIV Protease/metabolism , HIV-1/metabolism , Humans , Immunoblotting , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/analysis , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/chemistry , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Molecular Sequence Data , Monomeric GTP-Binding Proteins/analysis , Monomeric GTP-Binding Proteins/chemistry , Monomeric GTP-Binding Proteins/metabolism , NIMA-Interacting Peptidylprolyl Isomerase , NM23 Nucleoside Diphosphate Kinases , Peptide Elongation Factor 1/analysis , Peptide Elongation Factor 1/chemistry , Peptide Elongation Factor 1/metabolism , Peptidylprolyl Isomerase/analysis , Peptidylprolyl Isomerase/chemistry , Peptidylprolyl Isomerase/metabolism , Phosphatidylethanolamine Binding Protein , Phospholipid Transfer Proteins , Sequence Analysis, Protein , Subtilisin/metabolism , Transcription Factors/analysis , Transcription Factors/chemistry , Transcription Factors/metabolism , Virion/chemistry , Virion/metabolismABSTRACT
The presence of FK506-binding protein-12 was demonstrated in virions of HIV-1, although its concentration was lower than that of cyclophilin A. The effect of two inhibitors of the peptidyl-prolyl cis-trans isomerases FK506 and cyclosporin A (CsA) was studied in H9 cells that were chronically infected by HIV-1. Both drugs inhibited virus production in the infected cells in a concentration-dependent manner, by decreasing the number of the producing cells. FK506 did not have an effect on Gag processing, based on the p24 antigen content of virions produced in the presence of this drug. Furthermore, FK506 treatment of uninfected H9 cells did not diminish their susceptibility toward HIV-1 infection, whereas CsA treatment decreased the degree of HIV-1 infection with an IC(50) of 1-2 microg/ml. Also, pretreatment of the virus with CsA decreased its infectivity in HeLaCD4-LTR/beta-gal cells; in contrast, at concentrations up to 10 microg/ml, FK506 did not have an effect. Our findings on the antiviral activity of FK506 and CsA suggest that FK506 is effective only in chronically infected cells, by selectively inhibiting the growth of HIV-1 infected cells, whereas CsA has a specific effect on virus replication.
Subject(s)
Anti-HIV Agents/pharmacology , Cyclosporine/pharmacology , HIV-1/drug effects , Tacrolimus/pharmacology , Dose-Response Relationship, Drug , HIV Core Protein p24/metabolism , HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , HIV-1/physiology , HeLa Cells , Humans , Immunophilins/analysis , Tacrolimus Binding Proteins , Tumor Cells, CulturedABSTRACT
BACKGROUND AND OBJECTIVES: Explanations for laparoscopic-induced hypothermia fail to explain clinical observations. It is possible that water evaporation occurs from the jet stream of gas inflation resulting in tissue surface super-cooling leading to tissue damage and drying. METHODS: Theoretical calculations based on thermal conductivity, mass transfer effects and heat flux considerations correlated closely with synthetic and tissue experiments. Thermocouple measurements at a rate of 15 data points per second were performed. RESULTS: Cooling rates of 10 to 25 degrees centigrade per second for high flow rates were found based on gas flow rate and effective size of gas delivery site. These rapid temperature drops extended beyond a 2 cm2 diameter. CONCLUSIONS: Evaporative cooling accounts for significant hypothermia. The cooling is dependent on the lack of water vapor in the gases currently used during laparoscopy. Cooling rates are independent of height from tissue and geometry of delivery port. Heating and hydrating the gas to a physiologic condition eliminates hypothermia and tissue dessication.
Subject(s)
Hypothermia/etiology , Laparoscopy/adverse effects , Laparoscopy/methods , Culture Techniques , Epidermis , Humans , Hypothermia/physiopathology , Models, Theoretical , Sensitivity and SpecificityABSTRACT
The p6(Gag) protein of human immunodeficiency virus type 1 (HIV-1) is produced as the carboxyl-terminal sequence within the Gag polyprotein. The amino acid composition of this protein is high in hydrophilic and polar residues except for a patch of relatively hydrophobic amino acids found in the carboxyl-terminal 16 amino acids. Internal cleavage of p6(Gag) between Y36 and P37, apparently by the HIV-1 protease, removes this hydrophobic tail region from approximately 30% of the mature p6(Gag) proteins in HIV-1MN. To investigate the importance of this cleavage and the hydrophobic nature of this portion of p6(Gag), site-directed mutations were made at the minor protease cleavage site and within the hydrophobic tail. The results showed that all of the single-amino-acid-replacement mutants exhibited either reduced or undetectable cleavage at the site yet almost all were nearly as infectious as wild-type virus, demonstrating that processing at this site is not important for viral replication. However, one exception, Y36F, was 300-fold as infectious the wild type. In contrast to the single-substitution mutants, a virus with two substitutions in this region of p6(Gag), Y36S-L41P, could not infect susceptible cells. Protein analysis showed that while the processing of the Gag precursor was normal, the double mutant did not incorporate Env into virus particles. This mutant could be complemented with surface glycoproteins from vesicular stomatitis virus and murine leukemia virus, showing that the inability to incorporate Env was the lethal defect for the Y36S-L41P virus. However, this mutant was not rescued by an HIV-1 Env with a truncated gp41(TM) cytoplasmic domain, showing that it is phenotypically different from the previously described MA mutants that do not incorporate their full-length Env proteins. Cotransfection experiments with Y36S-L41P and wild-type proviral DNAs revealed that the mutant Gag dominantly blocked the incorporation of Env by wild-type Gag. These results show that the Y36S-L41P p6(Gag) mutation dramatically blocks the incorporation of HIV-1 Env, presumably acting late in assembly and early during budding.
Subject(s)
Gene Products, env/physiology , Gene Products, gag/physiology , HIV-1/physiology , Virus Assembly , Amino Acid Sequence , Gene Products, env/chemistry , Gene Products, gag/chemistry , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Structure-Activity Relationship , Virus ReplicationABSTRACT
The zinc finger structure that is found in the nucleocapsid protein of nearly all retroviruses has been proposed as a target for antiviral therapy. Since compounds that chemically attack the cysteines of the finger have been shown to inactivate both human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MuLV) in vitro, 14 of these compounds were tested in an MuLV-induced Friend disease model to assess their ability to inhibit retroviral replication in vivo. Of the 14 compounds tested, only Aldrithiol-2 clearly exhibited anti-retroviral activity as measured indirectly by the delay of Friend disease onset (P < 0.05). These results were confirmed by quantitative competitive polymerase chain reaction studies which monitored viral spread by measuring the level of viral DNA in the peripheral blood mononuclear cells of treated mice. Comparison of treated mice with untreated mice revealed that Aldrithiol-2 produced a greater than 2-log reduction in virus levels. These results functionally demonstrate that a zinc finger-attacking compound can inhibit viral replication in vivo. Since only 1 of the 14 compounds studied was effective, this study also shows the importance of in vivo testing of these types of antiviral compounds in an animal model. Given the strict conservation of the metal-coordinating cysteine structure within HIV-1 and MuLV zinc fingers, our results support the proposal that anti-retroviral drugs which target the nucleocapsid zinc finger may be clinically useful against HIV-1.
Subject(s)
Antiviral Agents/pharmacology , Friend murine leukemia virus/drug effects , Leukemia, Experimental/drug therapy , Nucleocapsid/drug effects , Retroviridae Infections/drug therapy , Thiazoles/pharmacology , Tumor Virus Infections/drug therapy , Virus Replication/drug effects , Zinc Fingers , Animals , Antiviral Agents/administration & dosage , Benzothiazoles , Data Interpretation, Statistical , Friend murine leukemia virus/genetics , Friend murine leukemia virus/growth & development , Leukemia, Experimental/virology , Mice , Retroviridae Infections/virology , Retroviridae Proteins/drug effects , Tumor Virus Infections/virologyABSTRACT
STUDY OBJECTIVE: To determine the distribution and concentration of aerosol particles caused by an ultrasonic (Harmonic) scalpel during simulated surgical use. DESIGN: Prospective experimental analysis (Canadian Task Force classification II-1). SETTING: Standard operating room. MATERIALS: Lean pork, lean beef, water, and blood, and the Harmonic scalpel with ball, curved scalpel, and cutting tips. INTERVENTIONS: Real-time sampling of airborne aerosols was performed over 6-second sampling periods. MEASUREMENTS AND MAIN RESULTS: Blood and tissue particles increased significantly during use of the Harmonic scalpel. Local exhaust evacuation methods diminished these concentrations. CONCLUSIONS: The Harmonic scalpel causes formation of bioaerosols that are composed of material of respirable size. When this device is used, a local exhaust system or smoke-evacuation method should be activated to reduce exposure to blood, blood by-products, and potentially infectious materials.
Subject(s)
Aerosols , Air Pollutants, Occupational , Surgical Instruments , Ultrasonics , Animals , Cattle , Occupational Exposure , Particle Size , SwineABSTRACT
Host proteins are incorporated into retroviral virions during assembly and budding. We have examined three retroviruses, human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and Moloney murine leukemia virus (Mo-MuLV), for the presence of ubiquitin inside each of these virions. After a protease treatment to remove exterior viral as well as contaminating cellular proteins, the proteins remaining inside the virion were analyzed. The results presented here show that all three virions incorporate ubiquitin molecules at approximately 10% of the level of Gag found in virions. In addition to free ubiquitin, covalent ubiquitin-Gag complexes were detected, isolated, and characterized from all three viruses. Our immunoblot and protein sequencing results on treated virions showed that approximately 2% of either HIV-1 or SIV p6Gag was covalently attached to a single ubiquitin molecule inside the respective virions and that approximately 2 to 5% of the p12Gag in Mo-MuLV virions was monoubiquitinated. These results show that ubiquitination of Gag is conserved among these retroviruses and occurs in the p6Gag portion of the Gag polyprotein, a region that is likely to be involved in assembly and budding.
Subject(s)
Gene Products, gag/metabolism , HIV-1/metabolism , Moloney murine leukemia virus/metabolism , Simian Immunodeficiency Virus/metabolism , Ubiquitins/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Cell Line , Chromatography, High Pressure Liquid , Haplorhini , Humans , Mice , Molecular Sequence Data , Tumor Cells, Cultured , Virion , gag Gene Products, Human Immunodeficiency VirusABSTRACT
OBJECTIVE: To assess the efficacy and safety of Insuflow (Georgia BioMedical, Inc.) filter heater hydrator device in reducing the incidence, severity and extent of hypothermia, length of recovery room stay and postoperative pain at the time of laparoscopy. DESIGN: Prospective, randomized, blinded, controlled multi-center study. Patients underwent gynecologic procedures via laparoscopy; surgeons, anesthesiologists and recovery room personnel assessed the results. SETTING: Seven North American institutions. PATIENTS: Seventy-two women for safety evaluation and efficacy studies. INTERVENTIONS: Intraoperative pre-conditioning of laparoscopic gas with the Insuflow device (treatment) or standard raw gas (control) during laparoscopic surgery and postoperatively. MAIN OUTCOME MEASURES: Incidence, severity and extent of hypothermia, postoperative pain perception and length of recovery room stay. RESULTS: The Insuflow group had significantly less intraoperative hypothermia, reduced length of recovery room stay and reduced postoperative pain. Pre-conditioning of laparoscopic gas by filtering heating and hydrating was well tolerated with no adverse effects. The safety profile of the Insuflow pre-conditioned gas showed significant benefits compared to currently used raw gas. CONCLUSIONS: Pre-conditioning laparoscopic gas by filtering heating and hydrating with the Insuflow device was significantly more effective than the currently used standard raw gas and was safe in reducing or eliminating laparoscopic-induced hypothermia, shortening recovery room length of stay and reducing postoperative pain.
Subject(s)
Hypothermia/prevention & control , Laparoscopy/adverse effects , Pain, Postoperative/prevention & control , Pneumoperitoneum, Artificial/instrumentation , Adolescent , Adult , Double-Blind Method , Equipment Safety , Female , Genital Diseases, Female/surgery , Humans , Hypothermia/etiology , Intraoperative Complications/prevention & control , Laparoscopes , Length of Stay , Middle Aged , Pain, Postoperative/etiology , Postoperative Period , Preoperative Care/methods , Prognosis , Prospective Studies , Recovery RoomABSTRACT
OBJECTIVE: The carbon monoxide (CO) smoke component from tissue pyrolysis was evaluated for peritoneal absorption in patients undergoing laparoscopy to determine its effects and ability to be detected in peripheral blood. SUMMARY BACKGROUND DATA: Previous studies have demonstrated changes in peripheral methemoglobin levels as a result of peritoneal absorption of laser smoke. METHODS: Fifty patients had preoperative, intraoperative, and postoperative levels of carboxyhemoglobin (COHb) and pulse oximetry evaluated. The control group (25) had no laser or cautery used and the study group (25) had carbon dioxide laser used during the laparoscopic procedures. RESULTS: The control group showed no change in COHb, or intra-abdominal CO levels, before, during, and after the procedures, and no change in blood CO or pulse oximetry reading. The laser smoke group showed a statistically significantly elevated (p < .05) peripheral blood COHb levels, a significant increase in intra-abdominal CO concentration, and a lack of correlation of pulse oximetry and blood oxygen saturation experiments. CONCLUSIONS: CO is created in extremely large quantities during laser use at laparoscopy and is absorbed through the peritoneal cavity. Symptoms of smoke poisoning can be seen with these elevations. Continuous or intermittent removal of smoke produced from laser use is recommended.
Subject(s)
Carbon Monoxide/pharmacokinetics , Carboxyhemoglobin/metabolism , Laparoscopy/adverse effects , Laser Therapy/adverse effects , Peritoneal Cavity , Smoke/adverse effects , Absorption , Adolescent , Adult , Air Pollutants/analysis , Air Pollutants/metabolism , Air Pollutants/pharmacokinetics , Carbon Monoxide/analysis , Carbon Monoxide/metabolism , Carbon Monoxide Poisoning/blood , Carbon Monoxide Poisoning/etiology , Endometriosis/surgery , Female , Humans , Hysterectomy/instrumentation , Laparoscopes , Middle AgedABSTRACT
Previous studies have suggested that the two conserved cysteines of the HIV-1 protease may be involved in regulating protease activity. Here, we examined diglutathionylated wild type protease (Cys-67-SSG, Cys-95-SSG) and the monoglutathionylated protease mutants (C67A, Cys-95-SSG and C95A, Cys-67-SSG) as potential substrates for thioltransferase (glutaredoxin). Time-dependent changes in the extent of deglutathionylation of each protein were assayed by reverse phase-high performance liquid chromatography. Glutathione alone was not an effective reductant, whereas thioltransferase displayed differential catalysis toward the Cys-95-SSG and Cys-67-SSG sites. At low thioltransferase concentrations (5 nM), deglutathionylation occurred almost exclusively at Cys-95-SSG. With substantially more thioltransferase (100 nM) Cys-67-SSG was partially deglutathionylated but only at 20% of the rate of Cys-95-SSG reduction. Treatment of the diglutathionylated protease with thioltransferase not only restored protease activity but generated an enzyme preparation that had a 3- to 5-fold greater specific activity relative to the fully reduced form. Immunoblot analysis of HIV-1MN virus with an antibody to thioltransferase detected a band co-migrating with recombinant thioltransferase that persisted following subtilisin treatment, indicating the presence of thioltransferase within HIV-1. Our results implicate thioltransferase in the regulation and/or maintenance of protease activity in HIV-1 infected cells.
Subject(s)
HIV Protease/metabolism , HIV-1/enzymology , Oxidoreductases , Proteins/analysis , Blotting, Western , Chromatography, High Pressure Liquid , Cysteine/metabolism , Escherichia coli , Glutaredoxins , Glutathione/metabolism , Structure-Activity Relationship , Subtilisins/metabolismABSTRACT
All retroviral nucleocapsid (NC) proteins, except those of spumaretroviruses, contain one or two zinc fingers, consisting of the sequence C-X2-C-X4-H-X4-C. Rice et al. (Science 270:1194-1197, 1995) have described a series of compounds which inactivate HIV-1 particles and oxidize the sulfur atoms in the NC zinc finger. We have characterized the effects of three such compounds on Moloney murine leukemia virus (MuLV). We find that, as with HIV-1, the compounds inactivate cell-free MuLV particles and induce disulfide cross-linking of NC in these particles. In contrast, the compounds have no effect on the infectivity of human foamy virus, a spumaretrovirus lacking zinc fingers in its NC protein. The resistance of foamy virus supports the hypothesis that the zinc fingers are the targets for inactivation of MuLV and HIV-1 by the compounds. The absolute conservation of the zinc finger motif among oncoretroviruses and lentiviruses, and the lethality of all known mutations altering the zinc-binding residues, suggest that only the normal, wild-type structure can efficiently perform all of its functions. This possibility would make the zinc finger an ideal target for antiretroviral agents.
Subject(s)
Antiviral Agents/pharmacology , Cross-Linking Reagents/pharmacology , HIV-1/physiology , Moloney murine leukemia virus/physiology , Nucleocapsid/chemistry , Spumavirus/physiology , Virus Replication/physiology , Amino Acid Sequence , Animals , Disulfides , Humans , Mice , Moloney murine leukemia virus/drug effects , Mutagenesis , Nucleocapsid/drug effects , Virus Replication/drug effects , Zinc FingersABSTRACT
OBJECTIVE: Laparoscopic procedures utilize a pneumoperitoneum to distend and separate the abdominal wall from the intra-abdominal structures. Carbon dioxide is commonly used for this purpose, although this study is inclusive of any gas used for abdominal distention. The gas is delivered from cylinders through a gas insufflation delivery system. The purpose of this study is to determine if laparoscopic gas delivery systems composed of gas cylinders and insufflators used for laparoscopy have microbes present. METHODS: Gas delivery systems were evaluated for the presence of microbial growth using standard techniques. External connection sites, gas cylinders and the internal conduit tubing of insufflators were cultured. Fifty two (52) insufflators and sixty (60) gas cylinders were evaluated. RESULTS: Twelve (12) of the sixty cylinders (20%) and fifty four (54) of the sixty insufflators (92.3%) were culture positive. The organisms identified are significant and a varied spectrum. CONCLUSIONS: Recognition that gas cylinders, insufflation attachments and internal components of insufflators quantitatively contain microbes is demonstrated. Reduction of microbial exposure from insufflation apparatus is achieved by cleansing external ports and use of a 0.3 micron filter for abdominal pneumoperitoneum.
Subject(s)
Equipment Contamination , Laparoscopes , Pneumoperitoneum, Artificial/instrumentation , Carbon Dioxide/administration & dosage , Humans , Insufflation/instrumentationABSTRACT
We have identified three types of cytoskeletal proteins inside human immunodeficiency virus type 1 (HIV-1) virions by analyzing subtilisin-digested particles. HIV-1 virions were digested with protease, and the treated particles were isolated by sucrose density centrifugation. This method removes both exterior viral proteins and proteins associated with microvesicles that contaminate virion preparations. Since the proteins inside the virion are protected from digestion by the viral lipid envelope, they can be isolated and analyzed after treatment. Experiments presented here demonstrated that this procedure removed more than 95% of the protein associated with microvesicles. Proteins in digested HIV-1(MN) particles from infected H9 and CEM(ss) cell lines were analyzed by high-pressure liquid chromatography, protein sequencing, and immunoblotting. The data revealed that three types of cytoskeletal proteins are present in virions at different concentrations relative to the molar level of Gag: actin (approximately 10 to 15%), ezrin and moesin (approximately 2%), and cofilin (approximately 2 to 10%). Our analysis of proteins within virus particles detected proteolytic fragments of alpha-smooth muscle actin and moesin that were cleaved at sites which might be recognized by HIV-1 protease. These cleavage products are not present in microvesicles from uninfected cells. Therefore, these processed proteins are most probably produced by HIV-1 protease digestion. The presence of these fragments, as well as the incorporation of a few specific cytoskeletal proteins into virions, suggests an active interaction between cytoskeletal and viral proteins.
Subject(s)
Actins/metabolism , HIV-1/metabolism , Microfilament Proteins/metabolism , Nerve Tissue Proteins/metabolism , Phosphoproteins/metabolism , Proteins/metabolism , Actin Depolymerizing Factors , Actins/chemistry , Amino Acid Sequence , Animals , Cell Line , Chromatography, High Pressure Liquid , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/metabolism , Humans , Mice , Microfilament Proteins/chemistry , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Phosphoproteins/chemistry , Proteins/chemistry , Sequence Homology, Amino Acid , Virion/metabolismABSTRACT
All retroviral nucleocapsid (NC) proteins, except those of spumaretroviruses, contain one or two copies of the conserved sequence motif C-X2-C-X4-H-X4-C. The conserved cysteine and histidine residues coordinate a zinc ion in each such motif. Rice et al. (W. G. Rice, J. G. Supko, L. Malspeis, R. W. Buckheit, Jr., D. Clanton, M. Bu, L. Graham, C. A. Schaeffer, J. A. Turpin, J. Domagala, R. Gogliotti, J. P. Bader, S. M. Halliday, L. Coren, R. C. Sowder II, L. 0. Arthur, and L. E. Henderson, Science 270:1194-1197, 1995) have described a series of compounds which inactivate human immunodeficiency virus type 1 (HIV-1) particles and oxidize the cysteine thiolates in the NC zinc finger. We have characterized the effects of three such compounds on Moloney murine leukemia virus (MuLV). We find that, as with HIV-1, the compounds inactivate cell-free MuLV particles and induce disulfide cross-linking of NC in these particles. The killed MuLV particles were found to be incapable of synthesizing full-length viral DNA upon infection of a new host cell. When MuLV particles are synthesized in the presence of one of these compounds, the normal maturational cleavage of the Gag polyprotein does not occur. The compounds have no effect on the infectivity of human foamy virus, a spumaretrovirus lacking zinc fingers in its NC protein. The resistance of foamy virus supports the hypothesis that the zinc fingers are the targets for inactivation of MuLV and HIV- I by the compounds. The absolute conservation of the zinc finger motif among oncoretroviruses and lentiviruses and the lethality of all known mutations altering the zinc-binding residues suggest that only the normal, wild-type structure can efficiently perform all of its functions. This possibility would make the zinc finger an ideal target for antiretroviral agents.