RESUMO
BACKGROUND: Little is known about the prevalence, characteristics, and outcomes of neonates needing emergent resuscitation within the level 4 neonatal intensive care units (NICU). Clinical factors prior to and following resuscitation event or characteristics of those who require chest compressions versus those without compressions has not been previously delineated. The aim of this study is to describe characteristics and outcomes of neonates who have compressive vs. non-compressive resuscitative events. METHODS: Retrospective observational study of neonates with a resuscitative event in the Children's Mercy Hospital level 4 NICU between January 2012 and April 2017. Data were derived from the NICU Code Blue database and the electronic medical record. Primary outcome was survival to hospital discharge. RESULT: Of the 641 resuscitative events, most were in the non-compressive group (nâ=â481). Those requiring chest compressions longer than 1 minute had significantly different clinical characteristics and decreased survival. There was no difference between groups in gestational age or birth weight. CONCLUSION: Non-compressive events are more common in the NICU setting than are compressive events. Neonates requiring chest compressions longer than 1 minute are more likely to have a higher respiratory severity score, need for vasopressors, worse renal function post-event, and decreased survival to discharge.
Assuntos
Unidades de Terapia Intensiva Neonatal , Ressuscitação , Criança , Hospitais Pediátricos , Humanos , Recém-Nascido , Alta do Paciente , Prevalência , Estudos RetrospectivosRESUMO
OBJECTIVE: Simulation training improves individual clinician confidence, performance and self-efficacy in resuscitation and procedural training experiences. The reality of resuscitation experiences in the neonatal intensive care unit (NICU) is that they are team-accomplished events. However, limited data exist on team-based simulation training (TBST) in the NICU. We report the experience of TBST over a 4-year period. STUDY DESIGN: This is a retrospective report of 65 TBST events in a 71-bed Level IV NICU at a regional subspecialty children's hospital. Participants were more than 500 NICU staff, including neonatal/cardiac/surgical attendings, neonatal fellows, neonatal nurse practitioners, pediatric residents, registered nurses and respiratory therapists. Background work, common case scenarios, training objectives and learning opportunities were reported, along with discipline-specific, and team and system areas for improvement. Qualitative, subjective data were tracked and efforts at collecting quantitative, objective data are ongoing. RESULTS: Seventy-five TBST events were scheduled from November 2010 through December 2014; 10 of these were canceled. TBST events occurred both night (n=23) and day (n=42), and also on weekends (n=19), using high-fidelity (n=42) and low-fidelity (n=23) systems. Resuscitation team participants at each TBST were 12-30 providers and staff. The duration of each TBST event was 30-65 min including debriefing. Systems issues were identified and corrected, including problems activating the code pathway, issues using a pager activation system and confusion over resuscitation team roles and responsibilities. Educational needs were addressed, focused on topic areas that included arrhythmias and use of extracorporeal cardiopulmonary resuscitation. CONCLUSION: With appropriate planning and implementation, TBST is feasible and realistic in a busy NICU.
Assuntos
Competência Clínica/normas , Neonatologia/economia , Equipe de Assistência ao Paciente/normas , Ressuscitação/educação , Treinamento por Simulação/estatística & dados numéricos , Hospitais Pediátricos , Humanos , Unidades de Terapia Intensiva Neonatal , Missouri , Projetos Piloto , Estudos Retrospectivos , Treinamento por Simulação/métodosRESUMO
We report here a semi-automated process by which mouse genome feature predictions and curated annotations (i.e., genes, pseudogenes, functional RNAs, etc.) from Ensembl, NCBI and Vertebrate Genome Annotation database (Vega) are reconciled with the genome features in the Mouse Genome Informatics (MGI) database (http://www.informatics.jax.org) into a comprehensive and non-redundant catalog. Our gene unification method employs an algorithm (fjoin--feature join) for efficient detection of genome coordinate overlaps among features represented in two annotation data sets. Following the analysis with fjoin, genome features are binned into six possible categories (1:1, 1:0, 0:1, 1:n, n:1, n:m) based on coordinate overlaps. These categories are subsequently prioritized for assessment of annotation equivalencies and differences. The version of the unified catalog reported here contains more than 59,000 entries, including 22,599 protein-coding coding genes, 12,455 pseudogenes, and 24,007 other feature types (e.g., microRNAs, lincRNAs, etc.). More than 23,000 of the entries in the MGI gene catalog have equivalent gene models in the annotation files obtained from NCBI, Vega, and Ensembl. 12,719 of the features are unique to NCBI relative to Ensembl/Vega; 11,957 are unique to Ensembl/Vega relative to NCBI, and 3095 are unique to MGI. More than 4000 genome features fall into categories that require manual inspection to resolve structural differences in the gene models from different annotation sources. Using the MGI unified gene catalog, researchers can easily generate a comprehensive report of mouse genome features from a single source and compare the details of gene and transcript structure using MGI's mouse genome browser.
Assuntos
Bases de Dados Genéticas , Genoma , Genômica/métodos , Software , Algoritmos , Animais , Genômica/estatística & dados numéricos , Internet , Camundongos , Modelos Genéticos , Anotação de Sequência Molecular , Fases de Leitura Aberta , Pseudogenes , RNA/genética , Terminologia como AssuntoRESUMO
The Mouse Genome Database (MGD) forms the core of the Mouse Genome Informatics (MGI) system (http://www.informatics.jax.org), a model organism database resource for the laboratory mouse. MGD provides essential integration of experimental knowledge for the mouse system with information annotated from both literature and online sources. MGD curates and presents consensus and experimental data representations of genotype (sequence) through phenotype information, including highly detailed reports about genes and gene products. Primary foci of integration are through representations of relationships among genes, sequences and phenotypes. MGD collaborates with other bioinformatics groups to curate a definitive set of information about the laboratory mouse and to build and implement the data and semantic standards that are essential for comparative genome analysis. Recent improvements in MGD discussed here include the enhancement of phenotype resources, the re-development of the International Mouse Strain Resource, IMSR, the update of mammalian orthology datasets and the electronic publication of classic books in mouse genetics.
Assuntos
Bases de Dados Genéticas , Genômica , Camundongos/genética , Animais , Genes , Genoma , Genótipo , Internet , Camundongos Mutantes , Fenótipo , Integração de Sistemas , Interface Usuário-ComputadorRESUMO
The Mouse Genome Database (MGD) is one component of the Mouse Genome Informatics (MGI) system (http://www.informatics.jax.org), a community database resource for the laboratory mouse. MGD strives to provide a comprehensive knowledgebase about the mouse with experiments and data annotated from both literature and online sources. MGD curates and presents consensus and experimental data representations of genetic, genotype (sequence) and phenotype information including highly detailed reports about genes and gene products. Primary foci of integration are through representations of relationships between genes, sequences and phenotypes. MGD collaborates with other bioinformatics groups to curate a definitive set of information about the laboratory mouse and to build and implement the data and semantic standards that are essential for comparative genome analysis. Recent developments in MGD discussed here include an extensive integration of the mouse sequence data and substantial revisions in the presentation, query and visualization of sequence data.
Assuntos
Biologia Computacional , Bases de Dados Genéticas , Genoma , Camundongos/genética , Animais , Genômica , Armazenamento e Recuperação da Informação , Internet , Biologia Molecular , Fenótipo , Terminologia como AssuntoRESUMO
Site specific amide hydrogen/deuterium content of oxidized and reduced Escherichia colithioredoxin, and alkylated derivatives, Cys-32-ethylglutathionylated and Cys-32-ethylcysteinylated thioredoxins are measured, after exposure for 20 s to D(2)O/phosphate buffer (pH 5.7), by electrospray mass spectrometry. The degree of deuteration of Oxi-TRX and Red-TRX correlated with the rates of H/D exchange measured previously by NMR. The ethylcysteinyl modification was shown to minimally perturb the active site of the reduced protein, but showed more global effects on structures of alpha-helices and beta-strands distant from the site of modification. In contrast, the larger ethylglutathionyl group had little effect on the protein's overall conformation, but significantly affected the structure of loops close to the active site. A molecular model of GS-ethyl-TRX derived from molecular simulation allowed the H/D exchange results to be interpreted in terms of specific interactions between the alkyl chain and the protein surface. The specific conformation of the ethylglutathione modification was predicted to be fixed by salt bridges between the carboxylates of the gamma-Glu and Gly of glutathione and the guanidinium of Arg-73 and epsilon-amino group of Lys-90 of the protein. Specific hydrogen bonding interactions between the glutathione carbonyl oxygens and the amide protons of thioredoxin residues Ile-75 and Ala-93 were predicted. The H/D exchange studies showed low levels of deuterium incorporation at backbone nitrogens of these residues. The data also provided evidence for an unusual amide proton-amide nitrogen hydrogen bond within the ethylglutathionylated chain. These same sets of electrostatic and hydrogen bonding interactions were not predicted or observed for the smaller alkyl modification in Cys-ethyl-TRX.
Assuntos
Escherichia coli/química , Hidrogênio/química , Tiorredoxinas/química , Alquilação , Cromatografia Líquida de Alta Pressão , Cisteína/química , Deutério/química , Glutationa/química , Cinética , Oxirredução , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Espectrometria de Massas por Ionização por Electrospray/métodos , Especificidade por Substrato , Tiorredoxinas/isolamento & purificaçãoRESUMO
Mass spectrometry as an analytical tool to study protein folding and structure by hydrogen/deuterium exchange is a relatively new approach. In this study, site-specific amide deuterium content was measured in oxidized and reduced E. coli thioredoxins by using the b(n) ions in electrospray ionization CID MS/MS experiments after 20-s incubation in D(2)O phosphate-buffered solution (pH 5.7). The deuterium levels correlated well with reported NMR-determined H/D exchange rate constants. The deuterium measured by y(n) ions, however, showed much less reliable correlation with rate exchange data. In general, residues in alpha helices and beta sheets, when measured by b(n) ions, showed low incorporation of deuterium while loops and turns had high deuterium levels. Most amide sites in the two protein forms showed similar deuterium levels consistent with the expected similarity of their structures, but there were some differences. The turn consisting of residues 18-22 in particular showed more variability in deuterium content consistent with reported structural differences in the two forms. The deuterium uptake by thioredoxins alkylated at Cys-32 by S-(2-chloroethyl)glutathione and S-(2-chloroethyl)cysteine, in peptides 1-24 and 45-58, was similar to that observed for oxidized and reduced thioredoxins, but several residues, particularly Leu-53 and Thr-54, showed slightly elevated deuterium levels, suggesting that structural changes had occurred from alkylation of the protein at Cys-32. It is concluded that b(n) ions are reliable for determining the extent of site-specific amide hydrogen isotope exchange and that mass spectrometry is useful as a complementary technique to NMR and other analytical methods for probing regional structural characteristics of proteins.
Assuntos
Escherichia coli/química , Tiorredoxinas/química , Sequência de Aminoácidos , Sítios de Ligação , Deutério/química , Deutério/metabolismo , Escherichia coli/metabolismo , Hidrogênio/química , Hidrogênio/metabolismo , Cinética , Dados de Sequência Molecular , Oxirredução , Conformação Proteica , Espectrometria de Massas por Ionização por Electrospray/métodos , Especificidade por Substrato , Tiorredoxinas/metabolismoRESUMO
1,2-Dibromoethane (DBE) is an environmental contaminant that is metabolized by glutathione S-transferases to a haloethane-glutathione conjugate. Since haloethane-glutathione conjugates are known to alkylate nuclear DNA and cytoplasmic proteins, these effects were investigated in isolated rat liver mitochondria exposed to DBE by measuring guanine adducts and several aspects of oxidative phosphorylation including respiratory control ratios, respiratory enzyme activity, and ATP levels. Mitochondrial large-amplitude swelling and glutathione status were assessed to evaluate mitochondrial membrane integrity and function. When exposed to DBE, mitochondria became uncoupled rapidly, yet no large-amplitude swelling or extramitochondrial glutathione was observed. Mitochondrial GSH was depleted to 2-53% of controls after a 60-min exposure to micromolar quantities of DBE; however, no extramitochondrial GSH or GSSG was detected. The depletion of mitochondrial glutathione corresponded to an increase of an intramitochondrial GSH-conjugate which, based on HPLC elution profiles and retention times, appeared to be S,S'-(1,2-ethanediyl)bis(glutathione). Activities of the NADH oxidase and succinate oxidase respiratory enzyme systems were inhibited 10-74% at micromolar levels of DBE, with succinate oxidase inactivation occurring at lower doses. ATP concentrations in DBE-exposed mitochondria in the presence of succinate were 5-90% lower than in the controls. The DNA adduct S-[2-(N(7)-guanyl)ethyl]glutathione was detected by HPLC in mtDNA isolated from DBE-exposed mitochondria. The results suggest that respiratory enzyme inhibition, glutathione depletion, decreased ATP levels, and DNA alkylation in DBE-exposed mitochondria occur via the formation of an S-(2-bromoethyl)glutathione conjugate, the precursor of the episulfonium ion alkylating species of DBE.
Assuntos
DNA Mitocondrial/efeitos dos fármacos , Dibrometo de Etileno/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Alquilação/efeitos dos fármacos , Animais , Adutos de DNA/análise , DNA Mitocondrial/metabolismo , Poluentes Ambientais/metabolismo , Poluentes Ambientais/farmacologia , Dibrometo de Etileno/metabolismo , Glutationa/deficiência , Glutationa/metabolismo , Guanina/metabolismo , Masculino , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/genética , Mitocôndrias Hepáticas/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
gamma-Glutamyltranspeptidase (GGT)-deficient mice (GGT(-/-)) display chronic glutathione (GSH) deficiency, growth retardation, and die at a young age (<20 weeks). Using livers from these mice, we investigated the relationship between GSH content, especially mitochondrial, and mitochondrial and cellular function. We found that the GSH content of isolated liver mitochondria was diminished by >/=50% in GGT(-/-) mice when compared with wild-type mice. Respiratory control ratios (RCRs) of GGT(-/-) mice liver mitochondria were =60% those of wild-type mice primarily as a result of impaired state 3 respiration. Mitochondrial adenine nucleotide content was decreased by >/=40% in mitochondria obtained from GGT(-/-) mice. We observed a strong correlation between mitochondrial GSH content and RCRs. Even moderate decreases (<50%) correlated with adverse effects with respect to respiration. Electron microscopy revealed that livers from GGT(-/-) knockout mice were deprived of fat and glycogen, and swollen mitochondria were observed in animals that were severely deprived of GSH. Thus, GGT(-/-) mice exhibit a loss of GSH homeostasis and impaired oxidative phosphorylation, which may be related to the rate of adenosine triphosphate (ATP) formation and subsequently leads to progressive liver injury, which characterizes the diseased state. We also found that supplementation of GGT(-/-) mice with N-acetylcysteine (NAC) partially restored liver GSH, but fully restored mitochondrial GSH and respiratory function. Electron microscopy revealed that the livers of NAC-supplemented GGT(-/-) mice contained fat and glycogen; however, slightly enlarged mitochondria were found in some livers. NAC supplementation did not have any beneficial effect on the parameters examined in wild-type mice.
Assuntos
Glutationa/metabolismo , Mitocôndrias Hepáticas/fisiologia , gama-Glutamiltransferase/deficiência , Difosfato de Adenosina/análise , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/biossíntese , Animais , Glutationa/análise , Fígado/citologia , Fígado/ultraestrutura , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Consumo de OxigênioRESUMO
Gamma-Glutamyltranspeptidase (GGT) catalyzes the first step in the extracellular hydrolysis of glutathione (GSH) and plays a critical role in GSH recycling; however, little is known about the impact of diminished GGT activity on immune function. We report here that GGT knockout (GGT(-/-)) mice have a 30 % decrease in splenic GSH and a 50 % reduction in thymus and spleen cellularity. The decreased cellularity was not selective for one population of cells, as each population was equivalently reduced. Following antigen challenge, GSH levels were reduced by 20-40 % in CD4+ and CD8+ T cells from GGT(-/-) mice when compared to T cells from wild-type mice. To test whether decreased GSH impairs immunity, we examined immune responsiveness following in vivo challenge with four different T cell-dependent stimuli. While there was no alteration in the antibody response to ovalbumin and sheep erythrocytes, cytotoxic T lymphocyte and alloantibody activity against P815 cells were decreased by 30 % and 65 %, respectively. Compared to wild-type littermates, anti-CD3-induced IL-2 and IL-6 production were also diminished in GGT(-/-) mice. These results demonstrate differential effects of decreased GSH on in vivo immune responsiveness to distinct stimuli, and suggest an important immunoregulatory role for GSH.
Assuntos
Glutationa/imunologia , Linfócitos T/imunologia , gama-Glutamiltransferase/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Complexo CD3/imunologia , Imunidade Celular/imunologia , Imunofenotipagem , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Imunológicos , Baço/citologia , Baço/enzimologia , Linfócitos T/classificação , Timo/citologia , Timo/enzimologia , gama-Glutamiltransferase/genéticaRESUMO
Recent studies have suggested that soluble forms of B7-1 and B7-2 may exist, but transcripts that code for these molecules have not been previously described. In this study, we report the cloning and characterization of an alternatively spliced soluble form of porcine B7-1 (sB7-1) that lacks exons coding for both the transmembrane and cytoplasmic domains. Northern blot analysis of RNA from alveolar macrophages revealed an approximate 3:1 ratio of the transmembrane form of B7-1 mRNA relative to sB7-1 mRNA. Porcine B7-1 was present on the surface of both B and T cells following stimulation with PMA/ionomycin. A histidine-tagged form of porcine sB7-1 (sB7-1-His) interacted with both CD28 and CTLA-4, and effectively blocked IL-2 production from human responder cells stimulated with PHA and either porcine or human stimulator cells. In addition, sB7-1-His inhibited human T cell proliferation in response to porcine or human peripheral blood leukocytes. This study is the first report of an alternatively spliced form of B7 that codes for a soluble protein. Furthermore, these data demonstrate that porcine B7-1 interacts with the human receptors CD28 and CTLA-4, suggesting a potential role for this molecule in pig to human xenotransplantation. Possible physiological functions for the soluble form of B7-1 are discussed.
Assuntos
Processamento Alternativo/imunologia , Antígeno B7-1/química , Antígeno B7-1/fisiologia , Imunoconjugados , Abatacepte , Adulto , Sequência de Aminoácidos , Animais , Antígenos CD , Antígenos de Diferenciação/metabolismo , Antígenos Heterófilos/imunologia , Antígeno B7-1/biossíntese , Antígeno B7-1/genética , Sequência de Bases , Northern Blotting , Antígenos CD28/metabolismo , Antígeno CTLA-4 , Células Cultivadas , Clonagem Molecular , Dimerização , Humanos , Imunossupressores/farmacologia , Interleucina-2/antagonistas & inibidores , Interleucina-2/biossíntese , Isoantígenos/imunologia , Células Jurkat , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Dados de Sequência Molecular , Ligação Proteica , Solubilidade , Suínos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Recent developments in basic immunology have revealed the importance of glutathione (GSH) and cellular redox balance in the generation of an immune response. In the liver, it has been shown that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters cellular GSH and reactive oxygen intermediate (ROI) production. We have tested the hypothesis that TCDD mediates the suppression of the cytotoxic T lymphocyte (CTL) response to alloantigen by increasing oxidative stress. Total cellular GSH, GSSG, and GSH-protein adducts were analyzed by HPLC. Changes in intracellular GSH and ROI were simultaneously measured in isolated hepatocytes and individual subpopulations of spleen cells (CD4+, CD8+, B220+, and Mac-1+) following in vivo exposure to TCDD and antigenic challenge with P815 mastocytoma cells. Monochlorobimane was utilized to measure GSH levels, and two fluorescent probes were used to evaluate ROI levels: dichlorofluoroscein diacetate to monitor peroxides and dihydroethidine to assess superoxide anion. In hepatocytes, in vivo treatment with TCDD resulted in a transient, 2-fold increase in GSH, a 50% decrease in peroxide levels and a small (20-40%) decrease in superoxide anion levels. Although alloantigen challenge resulted in increased GSH and peroxide in spleen cells, in vivo exposure to TCDD had no effect on splenic ROI levels, nor did it consistently alter GSH levels in any subpopulation of spleen cells examined. Moreover, in vivo treatment with the antioxidant N-acetyl cysteine failed to affect the immune suppression caused by TCDD. These results suggest to us that although TCDD perturbs cellular redox balance in the liver, it does not exacerbate or diminish the normal increased GSH and ROI which occur in the spleen in response to antigenic challenge.
Assuntos
Glutationa/fisiologia , Imunossupressores/toxicidade , Dibenzodioxinas Policloradas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Animais , Células Cultivadas , Feminino , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Estresse Oxidativo/efeitos dos fármacos , Baço/efeitos dos fármacos , Baço/metabolismoAssuntos
Dipeptidases/metabolismo , Leucotrieno C4/metabolismo , Animais , Cisteína/metabolismo , Glutationa/metabolismo , Cinética , Leucotrieno D4/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Knockout , Baço/metabolismo , Especificidade por Substrato , gama-Glutamiltransferase/deficiência , gama-Glutamiltransferase/genéticaRESUMO
Prenatal exposure to cocaine has been associated with adverse developmental effects and current data suggest cocaine induced malformations are caused by ischemic-reperfusion injury. This study was undertaken to assess a new in vitro model which uses a routine rat whole embryo culture system that incorporates a change in oxygen status, and to examine the effects of altered oxygen status and pretreatment with ellagic acid (EA), an anti-oxidant, after cocaine exposure. Embryos were evaluated by determining a developmental score and by measuring tissue reduced glutathione (GSH) levels. Following re-oxygenation with 95% O2 for the last 6 h of culture, embryos treated with cocaine had reduced developmental scores and GSH levels. Embryos treated with cocaine and not re-oxygenated with 95% O2 did not have reduced developmental scores. EA blocked the effects of cocaine on developmental score and GSH level. These data support ischemia-reperfusion injury as the mechanism of cocaine developmental toxicity.
Assuntos
Antioxidantes/farmacologia , Cocaína/toxicidade , Ácido Elágico/farmacologia , Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Oxigênio/farmacologia , Vasoconstritores/toxicidade , Animais , Feminino , Glutationa/análise , Glutationa/metabolismo , Técnicas de Cultura de Órgãos , Gravidez , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/induzido quimicamenteRESUMO
Human sera contain high levels of natural antibody (Ab) to Galalpha1-3Gal, a terminal glycosidic structure expressed on the surface of cells of mammals other than Old World primates. Incorporation of this determinant onto retroviral membranes by passage of viruses in cells encoding alpha-1-3-galactosyltransferase (GT) renders retroviruses sensitive to lysis by natural Ab and complement in normal human serum (NHS). Plasma membrane-budding viruses representing four additional virus groups were examined for their sensitivities to serum inactivation after passage through human cell lines that lack a functional GT or human cells expressing recombinant porcine GT. The inactivation of lymphocytic choriomeningitis virus (LCMV) by NHS directly correlated with host modification of the virus via expression of Galalpha1-3Gal and was blocked by incorporation of soluble Galalpha1-3Gal disaccharide into the inactivation assay. GT-deficient mice immunized to make high levels of Ab to Galalpha1-3Gal (anti-Gal Ab) were tested for resistance to LCMV passaged in GT-expressing cells. Resistance was not observed, but in vitro analyses of the mouse immune sera revealed that the antiviral activity of the sera was insufficient to eliminate LCMV infectivity on its natural targets of infection, macrophages, which express receptors for Ab and complement. Newcastle disease virus and vesicular stomatitis virus (VSV) were inactivated by NHS regardless of cell passage history, whereas Sindbis virus (SV) passaged in human cells resisted inactivation. Both VSV and SV passaged in Galalpha1-3Gal-expressing human cells incorporated this sugar moiety onto their major envelope glycoproteins. SV passaged in mouse cells expressing Galalpha1-3Gal was moderately sensitive to inactivation by NHS. These results indicate that enveloped viruses expressing Galalpha1-3Gal differ in their sensitivities to NHS and that a potent complement source, such as that in NHS, is required for efficient inactivation of sensitive viruses in vitro and in vivo.
Assuntos
Anticorpos Antivirais/imunologia , Formação de Anticorpos , Antígenos Virais/imunologia , Glicosídeos/imunologia , Herpesvirus Humano 1/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Especificidade de Anticorpos , Linhagem Celular , Epitopos/imunologia , Humanos , Camundongos , Proteínas do Envelope Viral/imunologiaRESUMO
The mechanism by which alpha-tocopherol (alpha-T) is secreted into the bile is not known; however, we have previously demonstrated that treatment with piperonyl butoxide (PIP, 1 g/kg) results in increased biliary output of both alpha-T and phosphatidylcholine within 3 h of ip injection in rats and that the biliary output of both substances was prevented by chemicals that disrupt microtubules (Toxicol. Appl. Pharmacol. 139, 411-417 (1996)). The P-glycoprotein (Pgp) encoded by the mdr2 gene has been shown to transport phosphatidylcholine into the bile; therefore, in the current study, we utilized the Pgp inhibitor verapamil to investigate the possible involvement of Pgps in the biliary secretion of alpha-T. When rats were iv injected with verapamil (4 mg/kg) 10 min prior to PIP treatment, verapamil prevented the PIP-induced increases in biliary alpha-T and phosphatidylcholine output and resulted in biliary alpha-T outputs that were significantly less than controls. Also, we determined that the biliary alpha-T levels in mdr2 knockout mice were 25% of those in wildtype mice; furthermore, mdr2 liver, lung, and kidney levels of alpha-T and glutathione differed from those of wildtype. To investigate the fate of biliary alpha-T, we injected 14C-labeled alpha-T into the bile duct cannulae of rats and determined that approximately 60% of the radioactivity was reabsorbed within 1 h. Our results indicate that alpha-T undergoes enterohepatic circulation and that the biliary secretion of alpha-T, basally and following chemical treatment, is dependent on the presence of a functioning mdr2 Pgp in rats and mice.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/fisiologia , Bile/química , Vitamina E/metabolismo , Animais , Ácidos e Sais Biliares/análise , Radioisótopos de Carbono/metabolismo , Feminino , Glutationa/análise , Dissulfeto de Glutationa/análise , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fosfatidilcolinas/metabolismo , Butóxido de Piperonila/farmacologia , Ratos , Ratos Sprague-Dawley , Verapamil/farmacologia , Vitamina E/análiseRESUMO
Retroviruses from various mammalian species, excluding humans, are effectively inactivated in normal human serum (NHS). Recent studies have shown that NHS inactivation of retroviruses occurs through natural Ab recognition of a terminal glycosidic moiety on the viral envelope that is acquired during replication in the host cell. This carbohydrate structure (the alpha-galactosyl epitope) is expressed on the cells of most mammals, with the exception of humans and other Old World primates. In this study, NHS sensitivity of HIV was assessed following viral propagation in human cells that were manipulated to express the alpha-galactosyl epitope. HUT-78 cells were transduced with an exogenous alpha1-3-galactosyl transferase gene, which codes for the terminal glycosyl transferase responsible for generation of the alpha-galactosyl epitope. The transduced HUT-78 cells expressed high levels of the alpha-galactosyl epitope on their membrane surface, rendering them sensitive to killing in NHS. Similarly, HIV passaged through these cells acquired the alpha-galactosyl epitope in association with the envelope glycoprotein gp120 and was also effectively inactivated in NHS. Viral inactivation was abolished by the addition of a synthetic disaccharide that contains the alpha-galactosyl epitope, indicating that virolysis is mediated by anti-alpha-galactosyl natural Ab. These results demonstrate that, like other retroviruses bearing the alpha-galactosyl epitope, HIV modified to express this epitope is inactivated in NHS. Furthermore, these data suggest that expression of the alpha-galactosyl epitope on the surface of viruses may have implications in the interspecies transmission of such viruses to humans.
Assuntos
Anticorpos Anti-HIV/imunologia , Antígenos HIV/imunologia , Soropositividade para HIV , HIV-1/imunologia , Epitopos Imunodominantes/imunologia , Animais , Linhagem Celular , Glicosilação , Anticorpos Anti-HIV/sangue , Humanos , Imunidade Celular , Epitopos Imunodominantes/genética , Suínos , TransfecçãoRESUMO
In order to investigate if hemoglobin might serve as a biomarker of exposure for 1,2-dichloroethane (DCE) encountered in the workplace, human hemoglobin was alkylated at physiologic pH by the episulfonium ion of S-(2-chloroethyl)glutathione (CEG). In vitro alkylation resulted in three alkylation products on the alpha chain and at least two alkylation products on the beta chain as determined directly by matrix-assisted laser desorption-ionization mass spectrometry. To ascertain if the site of alkylation was the reactive sulfhydryl present at cysteine-93 on the beta chain of hemoglobin (beta-93 Cys), a spectrophotometric assay using 4,4'-dithiodipyridine was used to measure the free sulfhydryl groups before and after treatment of hemoglobin with various amounts of CEG. Results indicate that the episulfonium ion did not react substantially at beta-93 Cys, as there was no measurable decrease in the sulfhydryl to hemoglobin ratio, even with a large excess of CEG. In contrast, iodoacetamide did react with the sulfhydryl groups and gave a dose-dependent decrease in the sulfhydryl to hemoglobin ratio as measured by this assay. CEG-treated hemoglobin was digested with Staphylococcus aureus endoproteinase Glu-C and the digest was analyzed by fast atom bombardment mass spectrometry. Only one peak in the FAB mass spectrum could correspond to a peptide modified by the episulfonium ion of CEG. These results indicate that although the episulfonium ion of CEG does alkylate human hemoglobin, beta-93 Cys is not the major alkylation target.
Assuntos
Alquilantes/química , Glutationa/análogos & derivados , Hemoglobinas/química , Sequência de Aminoácidos , Biomarcadores/análise , Cromatografia Líquida de Alta Pressão/métodos , Glutationa/química , Hemoglobinas/análise , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
alpha-Tocopherol, a lipid-soluble chain-breaking antioxidant, is important in the protection of biologic membranes and is transported in the body in association with lipids. It has been estimated that the liver contains up to 29% of the alpha-tocopherol stores of the body. Piperonyl butoxide (PIP) is a widely used synthetic methylenedioxyphenyl insecticide synergist. Bile was collected for 1 hr prior to, and 5 hr following, i.p. injection of 1 g/kg PIP. Within 3 hr of PIP administration, biliary alpha-tocopherol output increased to 132% of pretreatment measurements. Hepatic alpha-tocopherol levels of PIP-treated rats decreased to 26% below those of controls at the end of the bile collection period. Biliary bile acid and phospholipid output also increased significantly following PIP treatment. In a second set of experiments, rats were pretreated with either colchicine or one of two doses of vinblastine; both colchicine and the higher dose of vinblastine prevented the PIP-induced decrease in hepatic alpha-tocopherol and the increase in biliary output of alpha-tocopherol, bile acids, and phospholipids. These results suggest that (i) PIP-induced biliary secretion of alpha-tocopherol is associated with altered biliary phospholipid output, (ii) a significant portion of hepatic alpha-tocopherol may function as a highly mobilizable source of antioxidant for possible rapid enterohepatic distribution, and (iii) microtubules may be involved in this process.
Assuntos
Bile/efeitos dos fármacos , Colchicina/farmacologia , Sinergistas de Praguicidas/farmacologia , Butóxido de Piperonila/farmacologia , Vimblastina/farmacologia , Vitamina E/metabolismo , Animais , Bile/metabolismo , Ácidos e Sais Biliares/metabolismo , Colesterol/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Fosfolipídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Vitamina E/antagonistas & inibidoresRESUMO
The effects of host cannibalism on a host-parasitoid system were explored through experiment and modelling. In individual encounters between parasitized and unparasitized Plodia interpunctella larvae, parasitized larvae were more likely to be cannibalized. Inclusion of this differential cannibalism into a simple Lotka-Volterra-type model of host-parasitoid population dynamics generates alternative stable states-including stable coexistence and extinction of the parasitoid - which depend on starting conditions. Possible mechanisms for differential cannibalism, and its implications for studies of host-parasitoid populations and biological control programmes are discussed.