RESUMEN
INTRODUCTION: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) booster vaccination campaign and the emergence of SARS-CoV-2 Omicron variants impact the prevalence and levels of SARS-CoV-2 antibodies in the Netherlands. In this study we determined antibody levels across age groups, the impact of Omicron variant infections, and the effect of booster vaccinations on antibody levels. METHODS: In September and December 2021 and in February 2022, over 2000 Dutch blood donors were tested for presence of SARS-CoV-2 antibodies. Donations were selected based on age, sex, and region of residence, to provide an optimal coverage and representation of the Dutch population. RESULTS: Levels of vaccination-induced spike antibodies decreased over time in all age groups. Donors vaccinated with Janssen or AstraZeneca had significantly lower antibody levels than donors vaccinated with Pfizer or Moderna vaccine. Boostering with an mRNA vaccine elevated antibody levels in all age-groups irrespective of the initial vaccine. In donors aged < 56 years, the proportion of infected donors almost doubled between December 2021 and February 2022. CONCLUSION: The booster vaccination campaign increased antibody levels in all age-groups. After a booster vaccination, donors initially vaccinated with AstraZeneca or Janssen vaccine showed antibody levels similar to donors initially vaccinated with an mRNA vaccine. The emergence of the SARS-CoV-2 Omicron variant in the Netherlands caused a substantial increase in donors with infection-induced antibodies, especially among younger donors.
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Donantes de Sangre , COVID-19 , Humanos , SARS-CoV-2 , COVID-19/epidemiología , COVID-19/prevención & control , Anticuerpos Antivirales , VacunaciónAsunto(s)
Donantes de Sangre/estadística & datos numéricos , COVID-19/epidemiología , Infecciones por Parvoviridae/epidemiología , Parvovirus B19 Humano/aislamiento & purificación , Infecciones Asintomáticas/epidemiología , Transfusión Sanguínea , Portador Sano/virología , Humanos , Países Bajos/epidemiología , Infecciones por Parvoviridae/transmisión , SARS-CoV-2/aislamiento & purificaciónRESUMEN
BACKGROUND AND OBJECTIVES: Hepatitis E virus (HEV) is a known transfusion-transmissible agent. HEV infection has increased in prevalence in many developed nations with RNA detection in donors as high as 1 in 600. A high proportion of HEV infections are asymptomatic and therefore not interdicted by donor exclusion criteria. To manage the HEV transfusion-transmission (TT) risk some developed nations have implemented HEV RNA screening. In Australia, HEV is rarely notified; although locally acquired infections have been reported, and the burden of disease is unknown. The purpose of this study was to determine the frequency of HEV infection in Australian donors and associated TT risk. MATERIALS AND METHODS: Plasma samples (n = 74 131) were collected from whole blood donors during 2016 and screened for HEV RNA by transcription-mediated amplification (TMA) in pools of six. Individual TMA reactive samples were confirmed by RT-PCR and, if positive, viral load determined. Prevalence data from the study were used to model the HEV-TT risk. RESULTS: One sample in 74 131 (95% CI: 1 in 1 481 781 to 1 in 15 031) was confirmed positive for HEV RNA, with an estimated viral load of 180 IU/ml, which is below that typically associated with TT. Using a transmission-risk model, we estimated the risk of an adverse outcome associated with TT-HEV of approximately 1 in 3·5 million components transfused. CONCLUSION: Hepatitis E virus viremia is rare in Australia and lower than the published RNA prevalence estimates of other developed countries. The risk of TT-HEV adverse outcomes is negligible, and HEV RNA donor screening is not currently indicated.
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Donantes de Sangre , Virus de la Hepatitis E/genética , Hepatitis E/epidemiología , ARN Viral/sangre , Australia , Hepatitis E/sangre , Virus de la Hepatitis E/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , Prevalencia , Medición de RiesgoRESUMEN
BACKGROUND AND OBJECTIVE: In several Western countries, silent endemic hepatitis E virus (HEV) infection is common among blood donors. Immunocompromised persons may develop chronic hepatitis E, but the relevance of endemic HEV for immunocompetent persons remains largely unknown. We investigated the immune status and travel history in cases of hepatitis E in the Netherlands. STUDY DESIGN: Between January 2009 and May 2014, physicians throughout the Netherlands submitted samples from 4067 hepatitis patients to Sanquin Diagnostic Services for HEV antibody testing. For the 144 patients testing positive for HEV IgM and HEV RNA, travel behavior and immune status were assessed. Complete information was obtained for 81 patients. RESULTS: Surprisingly, the majority of patients (52/81, 64%) were immunocompetent and did not travel outside Europe. HEV genotyping was obtained for 47 non-traveling patients, all concerned HEV genotype 3. DISCUSSION: Our findings suggest that currently in Western countries the impact of hepatitis E for non-traveling, immunocompetent persons is underestimated. Historically cases of hepatitis A, B and C, but not cases of hepatitis E, are notifiable and warrant preventive measures. However, in parts of Western Europe HEV may have become the most important source of viral hepatitis, in immunocompetent and in immunosuppressed persons. Pending measures against the ongoing transmission of HEV genotype 3 in parts of Europe, physicians should consider hepatitis E in dealing with new hepatitis patients.
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Virus de la Hepatitis E , Hepatitis E/epidemiología , Hepatitis E/etiología , Terapia de Inmunosupresión , Viaje , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Virus de la Hepatitis E/clasificación , Virus de la Hepatitis E/genética , Virus de la Hepatitis E/inmunología , Humanos , Huésped Inmunocomprometido , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Países Bajos/epidemiología , Filogenia , ARN Viral , Adulto JovenRESUMEN
In Europe, the dynamics of endemic hepatitis E virus (HEV) infection remain enigmatic. We studied the presence of silent HEV infection among Dutch blood donors. Using donations collected throughout the Netherlands in 2011 and 2012, 40,176 donations were tested for HEV RNA in 459 pools of 48 or 480 donations. Deconstruction of the reactive pools identified 13 viraemic donors. In addition, 5,239 donors were tested for presence of anti-HEV IgG and IgM and for HEV RNA when IgM-positive. Of the 5,239 donations, 1,401 (27%) tested repeat-positive for HEV IgG, of which 49 (3.5%) also tested positive for anti-HEV IgM. Four of the HEV IgM-positive donors tested positive for HEV RNA. HEV IgG seroprevalence ranged from 13% among donors younger than 30 years to 43% in donors older than 60 years. The finding of 17 HEV RNA-positive donations among 45,415 donations corresponds to one HEV-positive blood donation per day in the Netherlands. For 16 of the 17 HEV RNA-positive donors, genotyping succeeded, revealing HEV genotype 3, which is circulating among Dutch pigs. Apparently, silent HEV infection is common in the Netherlands, which possibly applies to larger parts of Europe.
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Donantes de Sangre , Anticuerpos Antihepatitis/sangre , Virus de la Hepatitis E/aislamiento & purificación , Hepatitis E/diagnóstico , Adulto , Anciano , Femenino , Anticuerpos Antihepatitis/genética , Hepatitis E/epidemiología , Hepatitis E/virología , Virus de la Hepatitis E/genética , Virus de la Hepatitis E/inmunología , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Masculino , Persona de Mediana Edad , Países Bajos/epidemiología , Reacción en Cadena de la Polimerasa , ARN Viral/genética , ARN Viral/aislamiento & purificación , Estudios SeroepidemiológicosRESUMEN
Large outbreaks of Q fever in the Netherlands from 2007 to 2009 were monitored using notification data of acute clinical Q fever. However, the notification system provides no information on infections that remain subclinical or for which no medical attention is sought. The present study was carried out immediately after the peak of the 2009 outbreak to estimate the ratio between Coxiella burnetii infections and Q fever notifications. In 23 postcode areas in the high-incidence area, notification rates were compared with seroconversion rates in blood donors from whom serial samples were available. This resulted in a ratio of one Q fever notification to 12.6 incident infections of C. burnetii. This ratio is time and place specific and is based on a small number of seroconversions, but is the best available factor for estimating the total number of infections. In addition, as subclinical C. burnetii infection may lead to chronic Q fever, the ratio can be used to estimate the expected number of chronic Q fever patients in the coming years and as input for costbenefit analyses of screening options.
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Coxiella burnetii/aislamiento & purificación , Brotes de Enfermedades , Fiebre Q/epidemiología , Adulto , Anciano , Donantes de Sangre/estadística & datos numéricos , Notificación de Enfermedades/métodos , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Países Bajos/epidemiología , Fiebre Q/sangre , Fiebre Q/diagnósticoRESUMEN
To identify additional potential functions for the multi-PDZ domain containing protein Na+/H+ exchanger regulatory factor 2 (NHERF2), which is present in the apical domain of intestinal epithelial cells, proteomic studies of mouse jejunal villus epithelial cell brush border membrane vesicles compared wild-type to homozygous NHERF2 knockout FVB mice by a two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-iTRAQ approach. Jejunal architecture appeared normal in NHERF2 null in terms of villus length and crypt depth, Paneth cell number, and microvillus structure by electron microscopy. There was also no change in proliferative activity based on BrdU labeling. Four brush border membrane vesicles (BBMV) preparations from wild-type mouse jejunum were compared with four preparations from NHERF2 knockout mice. LC-MS/MS identified 450 proteins in both matched wild-type and NHERF2 null BBMV; 13 proteins were changed in two or more separate BBMV preparations (9 increased and 4 decreased in NHERF2 null mice), while an additional 92 proteins were changed in a single BBMV preparation (68 increased and 24 decreased in NHERF2 null mice). These proteins were categorized as 1) transport proteins (one increased and two decreased in NHERF2 null); 2) signaling molecules (2 increased in NHERF2 null); 3) cytoskeleton/junctional proteins (4 upregulated and 1 downregulated in NHERF2 null); and 4) metabolic proteins/intrinsic BB proteins) (2 upregulated and 1 downregulated in NHERF2 null). Immunoblotting of BBMV was used to validate or extend the findings, demonstrating increase in BBMV of NHERF2 null of MCT1, coronin 3, and ezrin. The proteome of the NHERF2 null mouse small intestinal BB demonstrates up- and downregulation of multiple transport proteins, signaling molecules, cytoskeletal proteins, tight junctional and adherens junction proteins, and proteins involved in metabolism, suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.
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Yeyuno/metabolismo , Fosfoproteínas/genética , Proteoma/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Animales , Cadherinas/metabolismo , Proliferación Celular , Cromatografía Liquida , Citoesqueleto/metabolismo , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente , Masculino , Ratones , Ratones Noqueados , Microvellosidades/genética , Microvellosidades/metabolismo , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , beta Catenina/metabolismoRESUMEN
Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCß3, E-cadherin, p120, ß-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.
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Yeyuno/metabolismo , Fosfoproteínas/genética , Proteoma/análisis , Intercambiadores de Sodio-Hidrógeno/genética , Vesículas Transportadoras/metabolismo , Animales , Cadherinas/análisis , Cromatografía por Intercambio Iónico , Femenino , Immunoblotting , Inmunohistoquímica , Yeyuno/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Microvellosidades/metabolismo , Microvellosidades/ultraestructura , Fosfoproteínas/metabolismo , Proteómica/métodos , Intercambiadores de Sodio-Hidrógeno/metabolismo , Espectrometría de Masas en Tándem , beta Catenina/análisisRESUMEN
We investigated the role of the Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na(+) absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.
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Colon/metabolismo , Absorción Intestinal/fisiología , Yeyuno/metabolismo , Fosfoproteínas/deficiencia , Cloruro de Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Inmunohistoquímica , Mucosa Intestinal/metabolismo , Ratones , Microvellosidades/ultraestructura , Intercambiador 3 de Sodio-HidrógenoRESUMEN
Metabolite profiling in succinate semialdehyde dehydrogenase (SSADH; Aldh5a1-/-) deficient mice previously revealed elevated gamma-hydroxybutyrate (GHB) and total GABA in urine and total brain and liver extracts. In this study, we extend our metabolic characterization of these mutant mice by documenting elevated GHB and total GABA in homogenates of mutant kidney, pancreas and heart. We quantified beta-alanine (a GABA homolog and putative neurotransmitter) to address its potential role in pathophysiology. We found normal levels of beta-alanine in urine and total homogenates of mutant brain, heart and pancreas, but elevated concentrations in mutant kidney and liver extracts. Amino acid analysis in mutant total brain homogenates revealed no abnormalities except for significantly decreased glutamine, which was normal in mutant liver and kidney extracts. Regional amino acid analysis (frontal cortex, parietal cortex, hippocampus and cerebellum) in mutant mice confirmed glutamine results. Glutamine synthetase protein and mRNA levels in homogenates of mutant mouse brain were normal. We profiled organic acid patterns in mutant brain homogenates to assess brain oxidative metabolism and found normal concentrations of Kreb's cycle intermediates but increased 4,5-dihydroxyhexanoic acid (a postulated derivative of succinic semialdehyde) levels. We conclude that SSADH-deficient mice represent a valid metabolic model of human SSADH deficiency, manifesting focal neurometabolic abnormalities which could provide key insights into pathophysiologic mechanisms.
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Aldehído Oxidorreductasas/deficiencia , Encéfalo/metabolismo , Animales , Western Blotting , Ácidos Carboxílicos/metabolismo , Modelos Animales de Enfermedad , Femenino , Glutamato-Amoníaco Ligasa/metabolismo , Glutamina/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Miocardio/metabolismo , Especificidad de Órganos , Oxidación-Reducción , Páncreas/metabolismo , ARN Mensajero/metabolismo , Oxibato de Sodio/metabolismo , Succionato-Semialdehído Deshidrogenasa , beta-Alanina/metabolismo , beta-Alanina/orina , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Succinate semialdehyde dehydrogenase (ALDH5A1, encoding SSADH deficiency is a defect of 4-aminobutyric acid (GABA) degradation that manifests in humans as 4-hydroxybutyric (gamma-hydroxybutyric, GHB) aciduria. It is characterized by a non-specific neurological disorder including psychomotor retardation, language delay, seizures, hypotonia and ataxia. The current therapy, vigabatrin (VGB), is not uniformly successful. Here we report the development of Aldh5a1-deficient mice. At postnatal day 16-22 Aldh5a1-/- mice display ataxia and develop generalized seizures leading to rapid death. We observed increased amounts of GHB and total GABA in urine, brain and liver homogenates and detected significant gliosis in the hippocampus of Aldh5a1-/- mice. We found therapeutic intervention with phenobarbital or phenytoin ineffective, whereas intervention with vigabatrin or the GABAB receptor antagonist CGP 35348 (ref. 2) prevented tonic-clonic convulsions and significantly enhanced survival of the mutant mice. Because neurologic deterioration coincided with weaning, we hypothesized the presence of a protective compound in breast milk. Indeed, treatment of mutant mice with the amino acid taurine rescued Aldh5a1-/- mice. These findings provide insight into pathomechanisms and may have therapeutic relevance for the human SSADH deficiency disease and GHB overdose and toxicity.
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Aldehído Oxidorreductasas/genética , Anticonvulsivantes/uso terapéutico , Convulsiones/tratamiento farmacológico , Convulsiones/genética , Animales , Secuencia de Bases , Encéfalo/metabolismo , Cartilla de ADN , Genotipo , Proteína Ácida Fibrilar de la Glía/metabolismo , Hidroxibutiratos/metabolismo , Inmunohistoquímica , Ratones , Ratones Noqueados , Fenobarbital/uso terapéutico , Fenitoína/uso terapéutico , Receptores de GABA-B/metabolismo , Convulsiones/enzimología , Succionato-Semialdehído DeshidrogenasaRESUMEN
Inherited succinic semialdehyde dehydrogenase (SSADH; EC1.2.1.24; McKusick 271980) deficiency is a defect of GABA degradation which leads to accumulation of 4-hydroxybutyric acid (gamma-hydroxybutyric acid; GHB) in physiologic fluids of patients. Prenatal diagnosis (PND) was performed in three at-risk pregnancies employing combinations of: (1) reverse-transcription-polymerase chain reaction (RT-PCR) and genomic DNA amplification followed by sequencing using isolated leukocytes or cultured human lymphoblasts; (2) GHB quantitation in amniotic fluid; or (3) SSADH enzyme assay in chorionic villus (CV) and/or amniocytes. In two pregnancies, all analyses were concordant for prediction of disease status in the fetus. In the third case, enzyme activity in CV (deficient) and metabolite analysis in amniotic fluid (normal) were discordant. For clarification, mutation analysis was undertaken in CV, confirming heterozygosity for the mutation previously identified in the proband. We hypothesize that delayed transit time for shipment of CV between Greece and the United States (8 days) led to enhanced degradation of heterozygous SSADH enzyme activity. Our data demonstrate the importance of combined metabolite, enzyme, and DNA analysis for increased accuracy in the PND of SSADH deficiency.
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Aldehído Oxidorreductasas/deficiencia , Diagnóstico Prenatal , Aldehído Oxidorreductasas/genética , Femenino , Heterocigoto , Humanos , Mutación , Reacción en Cadena de la Polimerasa , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Succionato-Semialdehído DeshidrogenasaRESUMEN
BACKGROUND: Several methods have been published for measuring gamma-aminobutyric acid transaminase (GABA-T) activity, but these methods are either impracticable because of the use of radioisotopes or insufficiently sensitive to determine small enzyme activities in leukocyte extracts. We developed a direct and sensitive enzyme method. METHODS: We developed a stable-isotope dilution method for the measurement of [15N]glutamic acid derived from [15N]GABA and alpha-ketoglutaric acid, catalyzed by GABA-T. The method for analysis of [15N]glutamic acid comprised a solid-phase extraction procedure to isolate this analyte from incubation samples. After derivatization, [15N]glutamic acid was quantified by gas chromatography-mass spectrometry relative to its 2H5-labeled internal standard. In addition to [15N]GABA, [15N]beta-alanine was a cosubstrate. RESULTS: GABA-T-deficient lymphoblasts showed diminished enzyme activity, with both [15N]GABA and [15N]beta-alanine as substrate. Vigabatrin inhibited the enzyme activity for both substrates. CONCLUSIONS: The activity of GABA-T can be accurately determined by our procedure using 15N-labeled substrate, measuring the formation of [15N]glutamic acid. Our results with [15N]beta-alanine indicate that GABA and beta-alanine transaminases are identical.
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4-Aminobutirato Transaminasa/metabolismo , Alanina Transaminasa/metabolismo , Linfocitos/enzimología , 4-Aminobutirato Transaminasa/química , Alanina Transaminasa/química , Humanos , Técnicas In Vitro , Técnicas de Dilución del Indicador , Isótopos de Nitrógeno , Espectrometría de Masa por Ionización de Electrospray , beta-Alanina/metabolismoRESUMEN
An 4-mo-old male was found to have an isolated increase in 2-methylbutyrylglycine (2-MBG) and 2-methylbutyrylcamitine (2-MBC) in physiologic fluids. In vitro oxidation studies in cultured fibroblasts using 13C- and 14C-labeled branched chain amino acids indicated an isolated block in 2-methylbutyryl-CoA dehydrogenase (2-MBCDase). Western blotting revealed absence of 2-MBCDase protein in fibroblast extracts; DNA sequencing identified a single 778 C>T substitution in the 2-MBCDase coding region (778 C>T), substituting phenylalanine for leucine at amino acid 222 (L222F) and absence of enzyme activity for the 2-MBCDase protein expressed in Escherichia coli. Prenatal diagnosis in a subsequent pregnancy suggested an affected female fetus, supporting an autosomal recessive mode of inheritance. These data confirm the first documented case of isolated 2-MBCDase deficiency in humans.
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Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Isoleucina/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/sangre , Errores Innatos del Metabolismo de los Aminoácidos/sangre , Secuencia de Bases , Carnitina/análogos & derivados , Carnitina/sangre , Cartilla de ADN , ADN Complementario , Femenino , Humanos , Lactante , Masculino , Oxidorreductasas/genética , Embarazo , Diagnóstico PrenatalRESUMEN
N-myristoylation is a covalent protein modification that can promote the association of proteins with membranes. De Jonge, Hogema, and Tilly discuss how N-myristoylation may be involved in triggering Fas ligand-induced apoptosis in mammals, and in adapting to conditions of high salt in plants. The pro-apoptotic protein BID is unique in that its proteolytic cleavage product, tBID, is posttranslationally myristoylated. In contrast, the plant accessory protein SOS3 undergoes "classical" cotranslational N-myristoylation. N-myristoylation is essential for the proper functioning of these proteins in regulating the signaling pathways (apoptosis and adaptation to salt stress, respectively) in which they are involved.
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Apoptosis/fisiología , Ácido Mirístico/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Cloruro de Sodio/farmacología , Animales , Apoptosis/efectos de los fármacos , Humanos , Cloruro de Sodio/metabolismoRESUMEN
Bacterial growth on one or more carbon sources requires careful control of the uptake and metabolism of these carbon sources. In Escherichia coli, the phosphorylation state of enzyme IIAGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is involved in this control in two ways. The unphosphorylated form of IIAGlc causes 'inducer exclusion', the inhibition of uptake of a number of non-PTS carbon sources, including lactose uptake by the lactose permease. The phosphorylated form of enzyme IIAGlc probably activates adenylate cyclase. In cells growing on lactose, enzyme IIAGlc was approximately 50% dephosphorylated, suggesting that lactose could inhibit its own uptake. This inhibition could be demonstrated by comparing lactose uptake rates in the wild-type strain and in a mutant in which the lactose carrier was insensitive to inducer exclusion. In this deregulated mutant strain, lactose was consumed much faster, and large amounts of glucose were excreted. It was shown that enzyme IIAGlc was dephosphorylated more strongly and that the cAMP level was lower in the mutant, most probably causing the observed decrease in lac expression level. When the lac expression level in the mutant strain was increased to that of the parent strain by adding exogenous cAMP, growth on lactose was slower, suggesting that enzyme IIAGlc-mediated inhibition of lactose uptake and downregulation of the lac expression level protected the cells against excessive lactose influx. An even stronger increase in the lac expression level in a mutant lacking enzyme IIAGlc caused complete growth arrest. We conclude that the autoregulatory mechanism that controls lactose uptake is an important mechanism for the cells in adjusting the uptake rate to their metabolic capacity.
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Proteínas de Escherichia coli , Escherichia coli/metabolismo , Homeostasis/fisiología , Lactosa/metabolismo , Proteínas de Transporte de Membrana/fisiología , Proteínas de Transporte de Monosacáridos , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/fisiología , Simportadores , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Glucosa/metabolismo , Glucosa-6-Fosfato/metabolismo , Isopropil Tiogalactósido/metabolismo , Fosforilación , Factores de Tiempo , beta-Galactosidasa/metabolismoRESUMEN
The main mechanism causing catabolite repression in Escherichia coli is the dephosphorylation of enzyme IIAGlc, one of the enzymes of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The PTS is involved in the uptake of a large number of carbohydrates that are phosphorylated during transport, phosphoenolpyruvate (PEP) being the phosphoryl donor. Dephosphorylation of enzyme IIAGlc causes inhibition of uptake of a number of non-PTS carbon sources, a process called inducer exclusion. In this paper, we show that dephosphorylation of enzyme IIAGlc is not only caused by the transport of PTS carbohydrates, as has always been thought, and that an additional mechanism causing dephosphorylation exists. Direct monitoring of the phosphorylation state of enzyme IIAGlc also showed that many carbohydrates that are not transported by the PTS caused dephosphorylation during growth. In the case of glucose 6-phosphate, it was shown that transport and the first metabolic step are not involved in the dephosphorylation of enzyme IIAGlc, but that later steps in the glycolysis are essential. Evidence is provided that the [PEP]-[pyruvate] ratio, the driving force for the phosphorylation of the PTS proteins, determines the phosphorylation state of enzyme IIAGlc. The implications of these new findings for our view on catabolite repression and inducer exclusion are discussed.
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Inducción Enzimática/fisiología , Escherichia coli/metabolismo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Proteínas Bacterianas/metabolismo , Transporte Biológico/fisiología , Metabolismo de los Hidratos de Carbono , Carbohidratos/farmacología , Escherichia coli/enzimología , Glucosa-6-Fosfato/farmacología , Metilgalactósidos/metabolismo , Mutación/genética , Fosfoenolpiruvato/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Ácido Pirúvico/metabolismo , Tiogalactósidos/metabolismoRESUMEN
The main mechanism causing catabolite repression by glucose and other carbon sources transported by the phosphotransferase system (PTS) in Escherichia coli involves dephosphorylation of enzyme IIA(Glc) as a result of transport and phosphorylation of PTS carbohydrates. Dephosphorylation of enzyme IIA(Glc) leads to 'inducer exclusion': inhibition of transport of a number of non-PTS carbon sources (e.g. lactose, glycerol), and reduced adenylate cyclase activity. In this paper, we show that the non-PTS carbon source glucose 6-phosphate can also cause inducer exclusion. Glucose 6-phosphate was shown to cause inhibition of transport of lactose and the non-metabolizable lactose analogue methyl-beta-D-thiogalactoside (TMG). Inhibition was absent in mutants that lacked enzyme IIA(Glc) or were insensitive to inducer exclusion because enzyme IIA(Glc) could not bind to the lactose carrier. Furthermore, we showed that glucose 6-phosphate caused dephosphorylation of enzyme IIA(Glc). In a mutant insensitive to enzyme IIA(Glc)-mediated inducer exclusion, catabolite repression by glucose 6-phosphate in lactose-induced cells was much weaker than that in the wild-type strain, showing that inducer exclusion is the most important mechanism contributing to catabolite repression in lactose-induced cells. We discuss an expanded model of enzyme IIA(Glc)-mediated catabolite repression which embodies repression by non-PTS carbon sources.
Asunto(s)
Escherichia coli/metabolismo , Glucosa-6-Fosfato/metabolismo , Lactosa/metabolismo , Transporte Biológico , Proteínas de Escherichia coli , Gluconatos/metabolismo , Metilgalactósidos/metabolismo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Fosforilación , Tiogalactósidos/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
While catabolite repression by glucose has been studied extensively and is understood in large detail in Enterobacteriaceae, catabolite repression by carbohydrates that are not transported by the phosphotransferase system (PTS) has always remained an enigma. Examples of non-PTS carbohydrates that cause catabolite repression in Escherichia coli are gluconate, lactose and glucose 6-phosphate. In this article it is shown that enzyme IIA(Glc) of the PTS is not involved in catabolite repression by these carbon sources. Carbon sources that caused strong catabolite repression of beta-galactosidase lowered the concentration of both cAMP and the cAMP receptor protein (CRP). A strong correlation was found between the amounts of cAMP and CRP and the strength of the repression. The levels of cAMP and CRP were modulated in various ways. Neither overproduction of CRP nor an increased cAMP concentration could completely relieve the repression by glucose 6-phosphate, lactose and gluconate. Simultaneously increasing the cAMP and the CRP levels was lethal for the cells. In a mutant expressing a constant amount of cAMP-independent CRP* protein, catabolite repression was absent. The same was found in a mutant in which lac transcription is independent of cAMP/CRP. These results, combined with the fact that both the cAMP and the CRP levels are lowered by glucose 6-phosphate, lactose and gluconate, lead to the conclusion that the decreased cAMP and CRP levels are the cause of catabolite repression by these non-PTS carbon sources.