RESUMEN
How anaerobic bacteria protect themselves against nitric oxide-induced stress is controversial, not least because far higher levels of stress were used in the experiments on which most of the literature is based than bacteria experience in their natural environments. This results in chemical damage to enzymes that inactivates their physiological function. This review illustrates how transcription control mechanisms reveal physiological roles of the encoded gene products. Evidence that the hybrid cluster protein, Hcp, is a major high affinity NO reductase in anaerobic bacteria is reviewed: if so, its trans-nitrosation activity is a nonspecific secondary consequence of chemical inactivation. Whether the flavorubredoxin, NorV, is equally effective at such low [NO] is unknown. YtfE is proposed to be an enzyme rather than a source of iron for the repair of iron-sulfur proteins damaged by nitrosative stress. Any reaction catalyzed by YtfE needs to be revealed. The concentration of NO that accumulates in the cytoplasm of anaerobic bacteria is unknown, but indirect evidence indicates that it is in the pM to low nM range. Also unknown are the functions of the NO-inducible cytoplasmic proteins YgbA, YeaR, or YoaG. Experiments to resolve some of these questions are proposed.
Asunto(s)
Bacterias Anaerobias/metabolismo , Óxido Nítrico/metabolismo , Estrés Nitrosativo/fisiología , Oxidorreductasas/metabolismo , Anaerobiosis/fisiología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Hierro-Azufre/metabolismo , Transcripción Genética/genéticaRESUMEN
In a dispersal-limited species that has evolved reproductive character displacement at a contact zone, a cline in mating behaviour may result if gene flow diffuses alleles out of the contact zone into allopatric populations. Prior work has found such a clinal pattern in the shield-back katydid Aglaothorax morsei, in which the male calling songs in a sympatric population have a displaced, short interpulse interval that increases in length with increasing distance from the contact zone. In this study, molecular phylogenetic and female preference data show that (1) sympatric populations result from secondary contact, (2) hybridization in sympatry has resulted in unidirectional mitochondrial introgression and (3) female preferences are consistent with reproductive character displacement and could generate a cline in mating behaviour. These data together suggest a history of reinforcement, generally considered rare in acoustically communicating insects; thus, Aglaothorax represents an important example of a rarely documented evolutionary process.
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Hibridación Genética , Ortópteros , Simpatría , Animales , Evolución Biológica , Femenino , Masculino , FilogeniaRESUMEN
After growth of gonococci in the presence of cytidine monophospho-N-acetyl-neuraminic acid (CMP-NANA), their 4.5-kD lipooligosaccharide (LOS) component was increased by approximately 400 daltons, whereas the LOS of strains lacking the 4.5-kD component were unaffected. Expression of mAb-defined epitopes on the 4.5-kD component was decreased on LOS of strains grown in CMP-NANA, and treatment of the LOS with neuraminidase reversed this affect. Gonococci incubated with human PMNs also had decreased expression of the 4.5-kD+ epitopes. A detergent extract of gonococci incorporated radiolabeled NANA in the LOS, suggesting the presence of a sialyltransferase in gonococci. Exogenous sialyltransferases also could use LOS as an acceptor.
Asunto(s)
Antígenos Bacterianos/inmunología , Ácido N-Acetilneuramínico Citidina Monofosfato/metabolismo , Epítopos/inmunología , Lipopolisacáridos/inmunología , Neisseria gonorrhoeae/inmunología , Ácidos Siálicos/metabolismo , Animales , Anticuerpos Monoclonales , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Lipopolisacáridos/aislamiento & purificación , Microscopía Electrónica , Neisseria gonorrhoeae/ultraestructura , Neuraminidasa , Neutrófilos/microbiología , Radioinmunoensayo , Glándula Submandibular/enzimología , PorcinosRESUMEN
OBJECTIVES: The Shape of Training report recommended that full registration is aligned with medical school graduation. As part of a General Medical Council-funded study about the preparedness for practice of UK medical graduates, we explored UK stakeholders' views about this proposal using qualitative interviews (30 group and 87 individual interviews) and Framework Analysis. SETTING: Four UK study sites, one in each country. PARTICIPANTS: 185 individuals from eight stakeholder groups: (1) foundation year 1 (F1) doctors (n=34); (2) fully registered trainee doctors (n=33); (3) clinical educators (n=32); (4) undergraduate/postgraduate Deans, and Foundation Programme Directors (n=30); (5) other healthcare professionals (n=13); (6) employers (n=7); (7) policy and government (n=11); (8) patient and public representatives (n=25). RESULTS: We identified four main themes: (1) The F1 year as a safety net: patients were protected by close trainee supervision and 'sign off' to prevent errors; trainees were provided with a safe environment for learning on the job; (2) Implications for undergraduate medical education: if the proposal was accepted, a 'radical review' of undergraduate curricula would be needed; undergraduate education might need to be longer; (3) Implications for F1 work practice: steps to protect healthcare team integration and ensure that F1 doctors stay within competency limits would be required; (4) Financial, structural and political implications: there would be cost implications for trainees; clarification of responsibilities between undergraduate and postgraduate medical education would be needed. Typically, each theme comprised arguments for and against the proposal. CONCLUSIONS: A policy change to align the timing of full registration with graduation would require considerable planning and preliminary work. These findings will inform policymakers' decision-making. Regardless of the decision, medical students should take on greater responsibility for patient care as undergraduates, assessment methods in clinical practice and professionalism domains need development, and good practice in postgraduate supervision and support must be shared.
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Competencia Clínica/estadística & datos numéricos , Competencia Clínica/normas , Educación de Pregrado en Medicina , Médicos/estadística & datos numéricos , Médicos/normas , Investigación Cualitativa , Adulto , Estudios Transversales , Femenino , Humanos , Entrevistas como Asunto , Masculino , Persona de Mediana Edad , Reino Unido , Adulto JovenRESUMEN
BACKGROUND/OBJECTIVES: Enteral feeding will induce remission in as many as 80-90% of compliant patients with active Crohn's disease (CD), but its method of action remains uncertain. This study was designed to examine its effects on the colonic microbiome. METHODS/SUBJECTS: Healthy volunteers and patients with CD followed a regimen confined to enteral feeds alone for 1 or 2 weeks, respectively. Chemicals excreted on breath or in faeces were characterised at the start and at the end of the feeding period by gas chromatography/mass spectrometry. RESULTS: One week of feeding in healthy volunteers caused significant changes in stool colour and deterioration in breath odour, together with increased excretion of phenol and indoles on the breath. Feeding for 2 weeks in patients with CD produced significant improvements in symptoms and a decrease in the concentration of C-reactive protein. The faecal concentrations of microbial products, including short-chain fatty acids (SCFAs), and potentially toxic substances, including 1-propanol, 1-butanol and the methyl and ethyl esters of SCFAs, showed significant falls. CONCLUSIONS: A significant change occurs in the production of microbial metabolites after enteral feeding in both healthy volunteers and patients with CD. Many of those detected in CD are toxic and may feasibly lead to the immunological attack on the gut microbiota, which is characteristic of inflammatory bowel disease. The reduction in the production of such metabolites after enteral feeding may be the reason for its effectiveness in CD.
Asunto(s)
Colon , Enfermedad de Crohn/terapia , Nutrición Enteral , Microbioma Gastrointestinal , 1-Butanol/metabolismo , 1-Propanol/metabolismo , Adolescente , Adulto , Anciano , Bacterias/metabolismo , Toxinas Bacterianas/metabolismo , Proteína C-Reactiva/metabolismo , Colon/metabolismo , Colon/microbiología , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/microbiología , Ésteres/metabolismo , Ácidos Grasos Volátiles/metabolismo , Heces/química , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
In many tissues, hyperglycemia alters the activities of the Na(+)-dependent myo-inositol (Na/MI) transporter, Na(+)-K(+)-ATPase, and protein kinase C (PKC). However, little is known concerning adaptive changes in renal proximal tubular function after acute or chronic hyperglycemia. We examined hyperglycemia-induced changes in Na/MI transport, Na(+)-K(+)-ATPase activity, and PKC activity using three proximal tubule-like cell lines (JTC12, LLC-PK1, and OK/E cells) and primary cultures of human proximal tubular epithelium (HK cells) cultured for varying periods in low- or high-glucose media, myo-Inositol (MI) transport was mediated by a high-affinity (Km approximately 50 mumol/l) Na(+)-dependent saturable process in the four cell lines. Hyperglycemia produced a time-dependent and persistent increase in Na/MI transport in all cell lines. Chronic hyperglycemia increased the Km for MI transport in LLC-PK1 cells and increased the Vmax in both LLC-PK1 and JTC12 cells. Glucose competitively inhibited Na/MI transport in all low-glucose cells and in high-glucose HK, JTC12, and OK/E cells but had no effect on transport in high-glucose LLC-PK1 cells. Acute hyperglycemia also produced time-dependent increases in Na(+)-K(+)-ATPase activity in all cell lines, a change that persisted only in HK cells. A 24-h exposure to high glucose had no effect on PKC activity in any of the cell lines but increased Ca/phospholipid-dependent PKC activity in membrane fractions from chronically high-glucose LLC-PK1 and OK/E cells. These data suggest that hyperglycemia causes acute changes in proximal tubule function and long-lived adaptive responses in Na/MI transport and the PKC signaling pathway.
Asunto(s)
Hiperglucemia/metabolismo , Inositol/metabolismo , Túbulos Renales Proximales/metabolismo , Proteína Quinasa C/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Transporte Biológico , Membrana Celular/enzimología , Células Cultivadas , Citosol/enzimología , Humanos , Técnicas In Vitro , Factores de TiempoRESUMEN
The DNA sequence containing the start of the Escherichia coli nirB gene is reported. The N-terminal amino acid sequence of purified NADH-dependent nitrite reductase coincided with that predicted from the DNA sequence, confirming that nirB is the structural gene for nitrite reductase apoprotein and identifying the translation start point. Using nuclease S1 mapping, the sole transcription startpoint for the nirB gene was found 23 or 24 base-pairs upstream from the ATG initiation codon. By subcloning successively smaller DNA fragments into a beta-galactosidase expression vector plasmid, we located the promoter within a sequence bounded by a TaqI site at +14 with respect to the transcription startpoint and a HpaII site at -208. Measurements in vivo of beta-galactosidase expression and RNA levels due to nirB promoter activity showed that this promoter was activated during anaerobic growth. Optimal activity was found only after anaerobic growth in the presence of nitrite. The sequence of the nirB promoter is compared with sequences found at other anaerobically activated promoters.
Asunto(s)
ADN Bacteriano/genética , Escherichia coli/genética , Genes Bacterianos , NADH NADPH Oxidorreductasas/genética , Nitrito Reductasas/genética , Regiones Promotoras Genéticas , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Escherichia coli/enzimología , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Nitrito-Reductasa (NAD(P)H) , Nitrito Reductasas/metabolismo , Nitritos/metabolismo , Oxígeno/metabolismo , ARN Bacteriano/biosíntesis , ARN Mensajero/biosíntesis , Transcripción GenéticaRESUMEN
We examined the effects of prolonged exposure to parathyroid hormone (PTH) and the protein kinase C (PKC) activator mezerein (MEZ) on cyclic adenosine monophosphate (cAMP) production, PKC activity, and Na(+)-dependent phosphate (Na/Pi) transport in an opossum kidney cell line (OK/E). A 5 minute exposure to PTH stimulated, while a 6 h incubation reduced, cAMP production, Na/Pi transport was maximally inhibited under desensitizing conditions and was not affected by reintroduction of the hormone. MEZ pretreatment (6 h) enhanced PTH-, cholera toxin (CTX)-, and forskolin (FSK)-stimulated cAMP production, suggesting enhanced Gs alpha coupling and increased adenylyl cyclase activity. However, PKA- and PKC-dependent regulation of Na/Pi were blocked in MEZ-treated cells. The PTH-induced decrease in cAMP production was associated with a reduction in membrane-associated PKC activity while MEZ-induced increases in cAMP production were accompanied by decreases in membrane and cytosolic PKC activity. Enhanced cAMP production was not accompanied by significant changes in PTH/PTH related peptide (PTHrP) receptor affinity or number, nor was the loss of Na/Pi transport regulation associated with changes in PKA activity. The results indicate that down-regulation of PKC by PTH or MEZ differentially modulates cAMP production and regulation of Na/Pi transport. The distinct effects of PTH and MEZ on PKC activity suggest that agonist-specific activation and/or down-regulation of PKC isozyme(s) may be involved in the observed changes in cAMP production and Na/Pi transport.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , AMP Cíclico/biosíntesis , Diterpenos , Riñón/efectos de los fármacos , Hormona Paratiroidea/toxicidad , Fosfatos/metabolismo , Proteína Quinasa C/metabolismo , Terpenos/toxicidad , Animales , Sitios de Unión , Transporte Biológico Activo , División Celular/efectos de los fármacos , Células Cultivadas , Toxina del Cólera/toxicidad , Colforsina/toxicidad , Regulación hacia Abajo , Activación Enzimática/efectos de los fármacos , Riñón/citología , Riñón/metabolismo , Zarigüeyas , Isótopos de Fósforo , Sodio/farmacologíaRESUMEN
Structural alterations in the parathyroid hormone (PTH) molecule produce marked changes in biologic activity. We examined the relative sensitivity of PTH-stimulated cAMP formation and PTH-inhibitable Na+-dependent phosphate transport responses to bovine PTH analogs [bPTH-(1-34), bPTH-(1-84), 8,18-norleucine-34-tyrosinamide bPTH-(1-34), bPTH-(7-34)-amide, 8,18-norleucine-34-tyrosinamide bPTH-(3-34), transaminated bPTH-(1-34)] and the human PTH-related peptide of malignancy (1-34) in cultured opossum kidney cells. The rank order of potency for stimulation of cAMP formation was bPTH-(1-34) = hPTHrP-(1-34) greater than nle bPTH-(1-34) greater than bPTH-(1-84) much greater than TAbPTH-(1-34). Nle bPTH-(3-34) and bPTH-(7-34) did not affect cAMP formation in intact cells at concentrations up to 10 microM. The rank order of potency for the inhibition of phosphate transport was bPTH-(1-34) = hPTHrP-(1-34) greater than nle bPTH-(1-34) greater than bPTH-(1-84) = TAbPTH-(1-34) greater than nle bPTH-(3-34). TAbPTH-(1-34) was a full agonist and inhibited phosphate transport at concentrations that did not increase cAMP formation, but nle bPTH-(3-34) was a partial agonist in spite of its inability to stimulate cAMP formation. Bovine PTH-(7-34) had no effect on phosphate transport. This study indicates that changes in the PTH molecule produce analogs that apparently discriminate between the cAMP-stimulating activity and phosphate transport-inhibiting activities of the native hormone.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
AMP Cíclico/biosíntesis , Riñón/metabolismo , Hormona Paratiroidea/farmacología , Aminación , Animales , Transporte Biológico Activo/efectos de los fármacos , Línea Celular , Riñón/citología , Riñón/efectos de los fármacos , Zarigüeyas , Hormona Paratiroidea/antagonistas & inhibidores , Hormona Paratiroidea/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Fosfatos/farmacocinética , Sodio/fisiología , Relación Estructura-ActividadRESUMEN
Many G protein-coupled receptor agonists activate p42/p44 mitogen-activated protein kinase (MAPK), using signaling pathways that are a function of receptor, G protein-coupled, and effector complement. In opossum kidney (OK) cells, activation of endogenous PTH receptors caused a time- (peak within 15-30 min, sustained for approximately 2 h) and dose-dependent (EC50 approximately 3 x 10(-10) M) activation of MAPK. Immunoblot analysis with an activation- specific MAPK antibody indicated that PTH activated both p42 and p44 MAPK. Epidermal growth factor (EGF) also activated p42 and p44MAPK in a time- (peak at 5 min, return to basal within 2 h) and dose-dependent (EC50 approximately 3 ng/ml) fashion. PTH-dependent MAPK activation was mimicked by the protein kinase C activator (PKC) phorbol myristate acetate (PMA), and the protein kinase A activators 8 bromo-cAMP (8-Br-cAMP) and forskolin but was not affected by pertussis toxin pretreatment. PMA or 8-Br-cAMP pretreatment blocked MAPK activation by reexposure to each kinase activator but caused no significant reduction in MAPK activation by PTH. MAPK activation by PTH, EGF, and 8-Br-cAMP was inhibited by the MAPK kinase inhibitor PD98059 and an EGF receptor (EGFR)-selective inhibitor tyrphostin AG1478. AG1478 also blocked MAPK activation by insulin-like growth factor-1 and platelet-derived growth factor. EGF and PTH caused time- and AG1478-sensitive phosphorylation of the EGFR, but EGFR desensitization did not affect MAPK activation by PTH. EGF, PMA, and low doses of PTH (10(12) to 10(-9) M) stimulated while 8-Br-cAMP and high doses of PTH (10(-8) to 10(-6) M) inhibited [3H]thymidine uptake. These data demonstrate that PTH activates MAPK and suggest that PKC, protein kinase A, and the EGFR play roles in PTH signaling. The biphasic effect of PTH on DNA synthesis suggests that MAPK activation by the hormone leads to distinct cellular responses.
Asunto(s)
Riñón/enzimología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Hormona Paratiroidea/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Células Cultivadas , ADN/biosíntesis , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Zarigüeyas , Fosforilación , Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The hormonal regulation of Na+-dependent phosphate transport was studied in opossum kidney (OK) cells. PTH caused time- and concentration-dependent decreases in Na+-dependent phosphate transport, with 10 pM PTH-(1-34) producing a 19% decline in phosphate transport. The EC50 for PTH inhibition of phosphate transport was 50 pM. Kinetic analyses of phosphate transport indicated that PTH decreased the maximum velocity without affecting the Km for phosphate. PTH increased cAMP formation with an EC50 of 10 nM. 8-Bromo-cAMP and (Bu)2cAMP also inhibited phosphate transport. Forskolin increased cAMP formation and decreased phosphate transport, whereas the cyclase-inactive forskolin analog 1,9-dideoxyforskolin also inhibited phosphate transport. The PTH analog [8,18-norleucine,34-tyrosinamide]PTH-(3-34) reduced phosphate transport at concentrations from 10 nM to 30 microM, but did not increase cAMP formation at concentrations up to 10 microM. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine produced concentration-dependent decreases in PTH-stimulated cAMP formation, but did not influence PTH inhibition of Na+-dependent phosphate transport. Vasoactive intestinal polypeptide and prostaglandin E1 increased cAMP formation in OK cells, but were weak inhibitors of phosphate transport. This study suggests that cAMP may not be the only transmembrane signaling mechanism involved in the regulation of Na+-dependent phosphate transport by PTH-(1-34) in OK cells.
Asunto(s)
AMP Cíclico/farmacología , Didesoxiadenosina/análogos & derivados , Riñón/metabolismo , Zarigüeyas/metabolismo , Hormona Paratiroidea/farmacología , Fosfatos/metabolismo , Sodio/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Alprostadil/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Bucladesina/farmacología , Línea Celular , Colforsina/farmacología , AMP Cíclico/biosíntesis , Desoxiadenosinas/análogos & derivados , Desoxiadenosinas/farmacología , Cinética , Fragmentos de Péptidos/farmacología , Teriparatido , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
The occurrence and properties of PTH-related peptide (PTH-RP) in milk was investigated. PTH-RP was purified to homogeneity from human and bovine milk using heat and acid to precipitate milk proteins followed by ion exchange chromatography and reverse-phase HPLC. The peak of PTH-RP from HPLC was detected using a sensitive bone cell bioassay. A single band of peptide was detected on silver-stained polyacrylamide gels, which migrated as a 20-21-kDa macromolecule. PTH-RP isolated from either human or bovine milk had similar electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The partially purified bovine PTH-RP stimulated cAMP production in UMR106-01 and OK cell lines and elicited a concentration-dependent inhibition of sodium-dependent phosphate transport in OK cells. Incubation of milk extracts with an anti-PTH antiserum did not affect their bioactivity, whereas an antihuman PTH-RP 1-34 antiserum markedly reduced the cAMP response of UMR106-01 cells to the immunoabsorbed milk extracts. A PTH antagonist, norleu PTH 3-34, blocked the stimulation of cAMP production in UMR106-01 cells treated with milk extracts. PTH-RP immunoreactivity and bioactivity occurred in milk extracts of diverse animals from both eutherian and metatherian (marsupial) species. Porcine colostrum also had immunoreactive PTH-RP, although the levels were lower than the immunoreactive PTH-RP concentrations observed in milk samples collected at 7 and 14 days of lactation. Thus, a 20-21-KDa PTH-RP is secreted into milk where it could play a role in the development of suckling, newborn animals.
Asunto(s)
Leche Humana/análisis , Leche/análisis , Proteínas/aislamiento & purificación , Adenilil Ciclasas/metabolismo , Animales , Bioensayo , Western Blotting , Bovinos , Cromatografía Líquida de Alta Presión , Calostro/análisis , AMP Cíclico/biosíntesis , Activación Enzimática/efectos de los fármacos , Femenino , Cabras , Humanos , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Proteína Relacionada con la Hormona Paratiroidea , Fosfatos/metabolismo , Proteínas/farmacología , Porcinos , Células Tumorales CultivadasRESUMEN
A mutant of Escherichia coli, JCB606, shown to be pleiotropically deficient in the formation of endogenous membrane and periplasmic c-type cytochromes, synthesised the apo form of the exogenous cytochrome c550 from Paracoccus denitrificans, but not the holo form. In contrast, a cytoplasmically located holo form of Hydrogenobacter thermophilus cytochrome c552 was found in E. coli JCB606. These findings support the proposition that the formation of the cytoplasmic H. thermophilus cytochrome c552 in E. coli does not involve the physiological pathway of c-type cytochrome biosynthesis in E. coli and that the haem insertion may be catalysed.
Asunto(s)
Grupo Citocromo c/biosíntesis , Escherichia coli/genética , Bacterias Gramnegativas Quimiolitotróficas/genética , Mutación , Grupo Citocromo c/análisis , Grupo Citocromo c/genética , Citoplasma/química , Citoplasma/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Immunoblotting , Peso Molecular , Paracoccus denitrificans/genética , Plásmidos , Precursores de Proteínas/análisis , Transformación BacterianaRESUMEN
Ten patients were fed by nasogastric tube for 5 days after major surgery of the head and neck. Five were fed by continuous infusion 24 h/day using an enteral nutrition pump and five were fed comparable quantities by 2-h bolus administration between 0600 and 2200 h. Those fed by bolus had lower resting oxygen consumption on the 4th and 5th postoperative days and better cumulative nitrogen balance over the 5 days than the continuously fed group. It appears that metabolically it may be better to use an intermittent feeding regimen than a continuous one when feeding patients postoperatively via a nasogastric tube.
Asunto(s)
Nutrición Enteral , Nitrógeno/metabolismo , Consumo de Oxígeno , Nutrición Parenteral , Cabeza/cirugía , Humanos , Pruebas de Función Hepática , Masculino , Persona de Mediana Edad , Cuello/cirugía , Periodo PosoperatorioRESUMEN
Gonococci examined directly from urethral exudates are resistant to killing by human serum, but most strains become susceptible on subculture. Previous work with gonococci grown in vitro indicates that resistance in vivo is due to sialylation of gonococcal lipopolysaccharide (LPS) by a host factor, cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) or a related compound present in urogenital secretions and blood cells including phagocytes, which exude during inflammation. This sialylation inhibits the reaction between bactericidal IgM in serum and its target LPS sites. Here, we confirm the indication by using gonococci grown in vivo. Crucial to the above conclusions was the marked reduction of CMP-NANA-conferred serum resistance when gonococci were treated with neuraminidase to remove sialyl groups from their LPS. We now show that the serum resistance of gonococci in urethral exudates was reduced by treatment with neuraminidase from more than 95% (calculated in relation to controls incubated with heated serum) to 2-11% according to sample and incubation time. Subculture of the gonococci also reduced resistance to 9-11% but resistance was restored to more than 95% by incubation with CMP-NANA. This work is the culmination of an investigation that underlines the need to identify specific host factors and the virulence determinants they induce in vivo in future studies of pathogenicity.
Asunto(s)
Actividad Bactericida de la Sangre , Ácido N-Acetilneuramínico Citidina Monofosfato/farmacología , Exudados y Transudados/microbiología , Neisseria gonorrhoeae/efectos de los fármacos , Neuraminidasa/farmacología , Uretra/microbiología , Clostridium perfringens/enzimología , Exudados y Transudados/efectos de los fármacos , Humanos , Neisseria gonorrhoeae/crecimiento & desarrolloRESUMEN
The resistance of gonococci in most patients to complement mediated killing by human serum is due to sialylation of their lipopolysaccharide (LPS) which prevents bactericidal antibody from reacting with target sites. Two of the host factors responsible are: cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA), a well-known sialylating agent, and another factor which enhances the transfer of sialyl groups from CMP-NANA to LPS catalysed by a gonococcal sialyltransferase. The bacterial determinant of resistance is a conserved LPS component of about 4.5 kDa which is sialylated at a terminal Gal beta 1-4GlcNAc site on its side chain. The sialylated LPS forms a surface coat which is stainable by ruthenium red and connected with previously described 'capsules'. These observations sparked off an explosion of research. Recent publications show that sialylation of LPS by CMP-NANA affects additional important aspects of gonococcal pathogenicity, notably interactions with antibodies and phagocytes, and rendering the gonococcal surface more 'host-like'. Also, the observations have prompted an examination of LPS from some other pathogens for the presence of sialyl groups with positive results for Neisseria meningitidis and Haemophilus influenzae.
Asunto(s)
Lipopolisacáridos/química , Neisseria gonorrhoeae/química , Neisseria gonorrhoeae/patogenicidad , Anticuerpos Antibacterianos , Proteínas del Sistema Complemento , Citotoxicidad Inmunológica , Humanos , Técnicas In Vitro , Lipopolisacáridos/inmunología , Neisseria gonorrhoeae/inmunología , Ácidos Siálicos/químicaRESUMEN
The periplasmic nitrate reductase, NapA, from Escherichia coli was identified as a 90 kDa molybdoprotein which comigrated during polyacrylamide gel electrophoresis with the di-haem c-type cytochrome, NapB. The DNA sequence of the 5' end of the napA gene and the N-terminal amino acid sequences of both NapA and NapB were determined. The 36 residue leader peptide for NapA includes the double-arginine motif typical of proteins to which complex redox cofactors are attached in the cytoplasm prior to targeting to the periplasm. The pre-NapA leader sequence is both unexpectedly long and, unless two successive proteolysis steps are involved, is cleaved at the unprecedented sequence G-Q-Q-. Nap activity was suppressed during growth in the presence of tungstate and was absent from a mutant unable to synthesise the molybdopterin cofactor.
Asunto(s)
Coenzimas , Grupo Citocromo c/aislamiento & purificación , Escherichia coli/enzimología , Molibdeno , Nitrato Reductasas/aislamiento & purificación , Periplasma/enzimología , Secuencia de Aminoácidos , Arginina , Secuencia de Bases , Dimerización , Escherichia coli/genética , Metaloproteínas/metabolismo , Datos de Secuencia Molecular , Cofactores de Molibdeno , Nitrato Reductasas/efectos de los fármacos , Nitrato Reductasas/genética , Periplasma/genética , Precursores de Proteínas/genética , Señales de Clasificación de Proteína/genética , Pteridinas/metabolismo , Compuestos de Tungsteno/farmacologíaRESUMEN
We previously reported that a 17.5-kDa haem-binding polypeptide accumulates in Escherichia coli K-12 mutants defective in an essential gene for cytochrome c assembly, ccmF, and speculated that this polypeptide is either CcmE or CcmG. The haem-containing polypeptide, which is associated with the cytoplasmic membrane, has now been identified by N-terminal sequencing to be CcmE. The haem-dependent peroxidase activity of CcmE is clearly visible not only in a ccmF mutant, but also in ccmG and ccmH mutants, implying that CcmE functions either before or in the same step as CcmF, CcmG and CcmH in cytochrome c maturation. A trxA mutant, like the dipZ mutant, was unable to assemble c-type cytochromes or catalyse formate-dependent nitrite reduction: both activities were restored in the trxA and dipZ, but not ccmG, mutants by the reducing agent, 2-mercaptoethanesulphonic acid. Our data suggest that haem transferred across the cytoplasmic membrane by the CcmABCD complex becomes associated with CcmE, possibly by a labile covalent bond, before it is transferred to the cytochrome c apoproteins by the periplasmic haem lyase encoded by ccmF and ccmH. We further propose that CcmG is essential to reduce the disulphide bonds formed in cytochrome c apoproteins by DsbA, before haem is attached by the haem lyase. Electrons for disulphide bond reduction are supplied from thioredoxin in the cytoplasm via DipZ in the membrane, but can be replaced by the chemical reductant, 2-mercaptoethanesulphonic acid. According to this model, CcmG is the last protein in the reducing pathway which interacts stereospecifically with the apoprotein.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Hemo/metabolismo , Hemoproteínas/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Grupo Citocromo c/biosíntesis , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Unión al Hemo , Hemoproteínas/química , Hemoproteínas/genética , Nitritos/metabolismo , Oxidación-Reducción , Plásmidos/genética , Fracciones SubcelularesRESUMEN
Two derivatives of plasmid pAT153 which arose spontaneously in phosphate-limited continuous cultures of Escherichia coli HB101[pAT153] have been characterised. The smaller plasmid, pLAB-446, resulted from a deletion of 446 base pairs of DNA covering the translation start and codon sequence of the tetracycline resistance gene. The other plasmid, pLAB135, differed from pAT153 only by an A:T to G:C transition in the transcribed but untranslated leader region of the tet gene which decreased tet transcription by 84%, possibly due to the formation of a more stable stem-loop structure in the mRNA.
Asunto(s)
Escherichia coli/genética , Plásmidos , Resistencia a la Tetraciclina , Secuencia de Bases , Medios de Cultivo , ADN Bacteriano/genética , Escherichia coli/efectos de los fármacos , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Fosfatos/metabolismo , Regiones Promotoras Genéticas , Recombinación Genética , Transcripción GenéticaRESUMEN
Examined before subculture, gonococci in 18 urethral exudates collected from different patients were serum-resistant. For 15 exudates, the resistance was drastically reduced by treatment with neuraminidase and by one subculture on laboratory media. It was restored by incubation with cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA). Electron microscopic examination of gonococci in eight exudates showed a surface structure stained by Ruthenium red which disappeared in most samples when they were treated with neuraminidase. These results were identical with those of previous studies on in vitro grown gonococci which had shown that serum resistance is due to sialylation of a 4.5-kDa conserved component of gonococcal lipopolysaccharide (LPS) by host CMP-NANA, which masks the target site for bactericidal IgM and renders surface LPS stainable by Ruthenium red. The serum resistance of gonococci in the remaining three exudates was not reduced by neuraminidase nor by subculture. The mechanism of this stable resistance is unknown.