RESUMEN
Few studies have investigated the effects of exercise training on sleep physiology in well-trained athletes. We investigated changes in sleep markers, mood state and exercise performance in well-trained cyclists undergoing short-term intensified training and carbohydrate nutritional intervention. Thirteen highly-trained male cyclists (age: 25 ± 6y, [Formula: see text]O2max: 72 ± 5 ml/kg/min) participated in two 9-day periods of intensified training while undergoing a high (HCHO) or moderate (CON) carbohydrate nutritional intervention before, during and after training sessions. Sleep was measured each night via wristwatch actigraphy. Mood state questionnaires were completed daily. Performance was assessed with maximal oxygen uptake ([Formula: see text]. Percentage sleep time fell during intensified training (87.9 ± 1.5 to 82.5 ± 2.3%; p < 0.05) despite an increase in time in bed (456 ± 50 to 509 ± 48 min; p = 0.02). Sleep efficiency decreased during intensified training (83.1 ± 5.3 to 77.8 ± 8.6%; p < 0.05). Actual sleep time was significantly higher in CON than HCHO throughout intensified training. Mood disturbance increased during intensified training and was higher in CON than HCHO (p < 0.05). Performance in the [Formula: see text] exercise protocol fell significantly with intensified training. The main findings of this study were that 9-days of intensified training in highly-trained cyclists resulted in significant and progressive declines in sleep quality, mood state and maximal exercise performance.
Asunto(s)
Afecto/fisiología , Ciclismo/fisiología , Ciclismo/psicología , Carbohidratos de la Dieta/administración & dosificación , Educación y Entrenamiento Físico/métodos , Sueño/fisiología , Actigrafía , Adulto , Rendimiento Atlético/fisiología , Estudios Cruzados , Método Doble Ciego , Humanos , Masculino , Consumo de Oxígeno/fisiologíaRESUMEN
The aim of this study was to determine whether participating in a cross-country skiing stage race (Tour de Ski; TDS) affects subsequent illness incidence, training, and race performance. Self-reported training and illness data from 44 male and female elite cross-country skiers were included. In total, 127 years of data were collected (2-3 seasons per athlete). Illness incidence, training load, and performance in international competitions were calculated for athletes who did and did not participate in TDS. Forty-eight percent of athletes reported becoming ill during or in the days immediately after taking part in TDS vs 16% of athletes who did not participate. In both groups, illness incidence was somewhat lower for female athletes. For male athletes, race performance was significantly worse for 6 weeks following TDS vs 6 weeks before TDS. Furthermore, while female athletes who participated in TDS performed relatively better than controls in Olympics/World Championships, male athletes who participated in TDS typically performed worse in subsequent major championships. Participating in TDS appears to result in â¼ 3-fold increase in risk of illness in this period. Male athletes appear more prone to illness and also see a drop in race performance following TDS, possibly linked to differences in training load before and after the event.
Asunto(s)
Rendimiento Atlético/fisiología , Enfermedades Gastrointestinales/epidemiología , Acondicionamiento Físico Humano/fisiología , Infecciones del Sistema Respiratorio/epidemiología , Esquí/fisiología , Adulto , Rendimiento Atlético/estadística & datos numéricos , Femenino , Enfermedades Gastrointestinales/microbiología , Frecuencia Cardíaca , Humanos , Incidencia , Ácido Láctico/sangre , Masculino , Acondicionamiento Físico Humano/estadística & datos numéricos , Autoinforme , Factores Sexuales , Esquí/estadística & datos numéricos , Adulto JovenRESUMEN
This study utilized two-dimensional gel electrophoresis (2DE) to illustrate the compositional differences between in vitro salivary conditioning films (denoted pellicles) formed on human enamel as well as on the dental materials titanium and poly(methyl methacrylate). The salivary pellicles were formed by immersing each surface in individual tubes containing small volumes of freshly collected whole saliva. Saliva remaining in the tubes after the pellicle formation for 2 h was visualized by means of 2DE and silver staining. The results showed that the protein patterns in 2DE of the liquid phase of saliva left after the exposure to the respective surfaces, regarding proteins <100 kDa in size, were different depending on the surface used. Several protein groups and/or individual proteins were shown to be distinct for each surface used.
Asunto(s)
Esmalte Dental/metabolismo , Materiales Dentales , Saliva/química , Saliva/metabolismo , Peso MolecularRESUMEN
Hereditary transthyretin (TTR) amyloidosis is a rare often fatal form of systemic amyloidosis, that until recently was considered intractable, with the patients dying from the disease 5-15 years after onset. The phenotype of the disease varies according to the type of mutation, but generally the heart and/or the nervous system is affected. Liver and in some cases heart transplantation has now been shown to stop the progress of the disease, but the outcome depends on the patients' status at the time of operation, as no substantial improvement of the patients' symptoms has been noted after the procedure. Thus an early diagnosis is of importance for the outcome. In the following, we summarize our knowledge of the amyloidogenic TTR mutations found in the Scandinavian countries, their symptoms, how to settle the diagnosis and the outcome of transplantation. Besides, the problems arising from our capability to genetically test asymptomatic members of affected families for the trait will be discussed.
Asunto(s)
Amiloidosis Familiar/genética , Mutación/genética , Prealbúmina/genética , Edad de Inicio , Neuropatías Amiloides/etiología , Amiloidosis Familiar/diagnóstico , Amiloidosis Familiar/terapia , Enfermedades Gastrointestinales/etiología , Asesoramiento Genético , Cardiopatías/etiología , Trasplante de Corazón , Humanos , Enfermedades Renales/etiología , Trasplante de Hígado , Complicaciones Posoperatorias/etiología , Países Escandinavos y NórdicosRESUMEN
In a previous article (Yallop and Svendsen 2001), recombinant CHO and BHK cell lines, expressing the human glucagon receptor and the gastric inhibitory peptide receptor, respectively, showed reduced growth rates and altered nutrient utilisation when grown with increasing concentrations of G418. This response was associated with an increased expression of the neo (r) protein, while expression of the recombinant membrane receptors remained unaltered. The metabolic response was characterised in both cell lines by an increase in the specific rate of glutamine utilisation and in CHO cells by a decrease in the yield of lactate from glucose, suggesting a change in the flux of glucose through central metabolism. The aim of this study was to further elucidate these metabolic changes by determining the activity and relative expression of key enzymes involved in glucose and glutamine metabolism. For both CHO and BHK cells, there was an increase in the activity of glutaminase, glutamate dehydrogenase and glutamine synthetase, suggesting an increased flux through the glutaminolysis pathway. The activity of glucose-6-phosphate dehydrogenase and pyruvate carboxylase in CHO cells was also increased whilst lactate dehydrogenase activity remained unaltered, suggesting an increased flux to the pentose phosphate pathway and TCA cycle, respectively. The activity of these enzymes in BHK cells was unchanged. Quantitative RT-PCR showed that expression levels of glutaminase and pyruvate carboxylase were the same with and without G418, indicating that the differences in activities were likely due to post-translational modifications.
RESUMEN
BACKGROUND: The voltage-gated, rapid-delayed rectifier current (I(Kr)) is important for repolarization of the heart, and mutations in the genes coding for the K+-ion channel conducting this current, i.e., KCNH2 for the alpha-subunit HERG and KCNE2 for the beta-subunit MiRP1, cause acquired and congenital long Q-T syndrome (LQTS) and other cardiac arrhythmias. METHODS: We developed a robust single-strand conformation polymorphism-heteroduplex screening analysis, with identical thermocycling conditions for all PCR reactions, covering all of the coding exons in KCNH2 and KCNE2. The method was used to screen 40 unrelated LQTS patients. RESULTS: Eleven mutations, of which six were novel, were found in KCNH2. Interestingly, six mutations were found in the region of the gene coding for the Per-Arnt-Sim (PAS) and PAS-S1 regions of the HERG protein, stressing the need to examine the entire gene when screening for mutations. No mutations were found in KCNE2, suggesting that direct involvement of MiRP1 in LQTS is rare. Furthermore, four novel single-nucleotide polymorphisms (SNPs) and one amino acid polymorphism (R1047L) were identified in KCNH2, and one novel SNP and one previously known amino acid polymorphism (T8A) were found in KCNE2. CONCLUSIONS: The potential role of rare polymorphisms in the HERG/MiRP1 K+-channel should be clarified with respect to drug interactions and susceptibility to arrhythmia and sudden death.
Asunto(s)
Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Síndrome de QT Prolongado/genética , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/genética , Transactivadores , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go , Análisis Heterodúplex , Humanos , Síndrome de QT Prolongado/congénito , Mutación , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Canales de Potasio/sangre , Regulador Transcripcional ERGRESUMEN
Polygalacturonases represent the most abundant carbohydrate hydrolase family in the Arabidopsis thaliana genome, and they are thought to be involved in nearly all of the developmental processes requiring cell wall modifications during the life cycle of the plant. By phylogenetic analysis, plant polygalacturonases fall into at least three groups, one of which is distinguished from the others by the presence of an additional N-terminal domain. We have used RDPG1, the polygalacturonase involved in pod dehiscence in oilseed rape (Brassica napus), as a model to investigate the function of this domain. We have confirmed that this domain is absent in the mature protein by determination of the N-terminal sequence of mature RDPG1 purified from oilseed rape pod. We have furthermore investigated the accumulation and subcellular localization of the precursor containing the N-terminal domain and of the mature protein throughout the development and maturation of the pod. Using recombinant expression in Pichia pastoris, we have produced the RDPG1 precursor, and we present evidence that the N-terminal domain of plant polygalacturonases is not involved in folding or inactivation of the precursor but may play a role in the intracellular transport of this protein family via a novel regulated secretion pathway.
Asunto(s)
Brassica/enzimología , Poligalacturonasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Hidrólisis , Microscopía Electrónica , Datos de Secuencia Molecular , Pichia/genética , Poligalacturonasa/química , Poligalacturonasa/genética , Poligalacturonasa/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Alkalophilic Bacillus alpha-amylase (ABA) was produced in the yeast Pichia pastoris with a yield of 50 mg L(-1) of culture supernatant. The recombinant protein, rABA, was glycosylated at seven of the nine sites for potential N-glycosylation as identified by automated peptide sequencing and MALDI-TOF MS of tryptic fragments. The number of hexose units within each glycan chain was found to vary from 8 to 18 as calculated from the masses of glycosylated peptide fragments. Temperature stability measurements in the absence of substrate showed that the T(50) of glycosylated rABA and its endoglycosidase H-deglycosylated form was 76 degrees C while that of ABA purified from Bacillus was 89 degrees C thus demonstrating that the original temperature stability of ABA was not retained by rABA. The relative thermoperformance, i.e., the activity at 80 degrees C relative to that at 37 degrees C was 0.9 +/- 0.3 for rABA. Removal of all seven N-linked glycans by endoglycosidase H increased the relative thermoperformance to 2.4 +/- 0.6, compared to the value of 3.5 +/- 1.1 for ABA. Thus, removal of the N-linked glycans did not improve the thermostability of rABA but modified its thermoperformance to approach that of the original Bacillus enzyme. rABA had the highest activity around pH 6. Treatment of rABA with endoglycosidase H shifted the pH activity profile in a more alkaline direction approaching the pH activity profile of ABA.
Asunto(s)
Bacillus/enzimología , alfa-Amilasas/química , alfa-Amilasas/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Estabilidad de Enzimas , Glicopéptidos/química , Glicosilación , Calor , Cinética , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/química , Pichia , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tripsina , alfa-Amilasas/aislamiento & purificaciónRESUMEN
It is widely reported that the growth of recombinant bacteria and yeast is adversely affected by increased metabolic load caused by the maintenance of plasmid copy number and recombinant protein expression. Reports suggest that recombinant mammalian systems are similarly affected by increased metabolic load. However, in comparison to bacterial systems relatively little information exists. It was the aim of this study to test the effects of recombinant gene expression on the growth and metabolism of two industrially important cell lines. A BHK and CHO cell line were stably transfected with the human gastric inhibitory peptide (h-GIP)and glucagon receptor respectively. Selection was by way of the neomycin resistance (neo (r)) gene using G418.The growth and metabolism of both cell lines was affected by the presence of G418 in a manner indicative of increased metabolic load and which appeared to be caused by over-expression of the neomycin resistance protein. The two cell lines differed in their metabolic response to G418, which suggested that some cell lines or clones may be better able to tolerate a metabolic load than others. Growth under increased metabolic load was affected by medium composition with serum, insulin and glutamine concentration as influencing factors. Implications for the use of G418 are discussed.
RESUMEN
The proteomes of exponentially growing and stationary cells of Lactobacillus delbrueckii ssp. bulgaricus grown in rich medium (MRS) were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE) and quantified after Coomassie staining. Stationary cells grown in MRS were inoculated in reconstituted skim milk, and "early" protein synthesis during the first 30 min of fermentation in milk was monitored by [35S]methionine labeling and 2-DE. In contrast to exponentially growing or stationary cells, the predominant "early" proteins were small (< 15 kDa) and of low pI (< 5.3). Quantification of the proteome of the "early" lag phase based on 47 "spots" revealed that only three "early" proteins accounted for more than 80% of the total label. They were identified as pI 4.7 and 4.9 isoforms of the heat-stable phosphoryl carrier protein (HPr) with 45.2 and 9.4% of total label, respectively, and an unknown protein called EPr1 ("early" protein 1) with 26.6% of total label. Although an N-terminal sequence of 19 amino acids was obtained, no homologs to EPr1 could be found. De novo synthesis of the 10 and 60 kDa heat shock proteins (GroES and GroEL) was considerably lower (0.04 and 0.9% of total label, respectively), indicating only low levels of stress. Synthesis of triosephosphate isomerase (Tpi) as marker for glycolytic enzymes reached only 0.08% of total label. Our results demonstrate that inoculation in milk, resulting in a change from glucose to lactose as carbon source, imposes only little need for synthesis of stress or glycolytic enzymes, as sufficient proteins are present in the stationary, MRS-grown cells. The high level of expression of the pI 4.7 isoform of HPr suggests a regulatory function of the presumed Ser-46 phosphorylated form of HPr.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Electroforesis en Gel Bidimensional , Regulación Bacteriana de la Expresión Génica , Lactobacillus/metabolismo , Leche/microbiología , Triosa-Fosfato Isomerasa/análisis , Triosa-Fosfato Isomerasa/biosíntesis , Triosa-Fosfato Isomerasa/genética , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Bovinos , Chaperonina 10/análisis , Chaperonina 10/biosíntesis , Chaperonina 10/genética , Chaperonina 60/análisis , Chaperonina 60/biosíntesis , Chaperonina 60/genética , Colorantes , Medios de Cultivo/farmacología , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Procesamiento de Imagen Asistido por Computador , Lactobacillus/efectos de los fármacos , Lactobacillus/ultraestructura , Espectrometría de Masas , Datos de Secuencia Molecular , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/análisis , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/biosíntesis , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Isoformas de Proteínas/análisis , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Proteoma , ARN Bacteriano/biosíntesis , ARN Mensajero/biosíntesis , Colorantes de Rosanilina , Alineación de Secuencia , Análisis de Secuencia de Proteína , Tinción con Nitrato de Plata , Coloración y Etiquetado/métodos , Factores de TiempoRESUMEN
The sulfatase from the snail Heli pomatia is widely used for analytical applications. We have investigated the content of sulfatases in H. pomatia, using a biochemical and a molecular approach. A 112-kDa protein from the intestinal juice of H. pomatia comigrated with sulfatase activity when chromatographed on Sephacryl S300 and concanavalin A-Sepharose. The N-terminal amino acid sequence of the protein was similar to one of three sulfatase motifs defined by sequence alignment of known sulfatases. Degenerate primers designed from the motifs and the N-terminal amino acid sequence obtained were used to generate PCR fragments and to isolate both a full-length and a 3'-truncated cDNA encoding H. pomatia sulfatases, designated SULF1 and SULF2. SULF1 consists of 503 amino acids and shows 53-55% identity to the mammalian arylsulfatase B. The amino acid sequence deduced from the 878-bp SULF2 cDNA fragment is 55% identical with SULF1. Both SULF1 and SULF2 contain the cysteine residue conserved in the active site of many sulfatases, which is known to be posttranslationally modified into formylglycine in eukaryotic sulfatases. However, the SULF1 and SULF2 cDNAs do not code for the protein purified. This indicates the presence of at least three sulfatase genes in H. pomatia.
Asunto(s)
ADN Complementario/metabolismo , Caracoles Helix/enzimología , Caracoles Helix/genética , Sulfatasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía en Gel , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Plásmidos , Homología de Secuencia de Aminoácido , Sulfatasas/químicaRESUMEN
The monoclonal antibody hGR-2 F6 has been raised against the human glucagon receptor and shown to act as a competitive antagonist. As a first step in the structural characterization of the receptor, the crystal structure of the Fab fragment from this antibody is reported at 2.1 A resolution. The hGR-2 F6 Fab crystallizes in the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 76.14, b = 133.74, c = 37.46 A. A model generated by homology modelling was used as an aid in the chain-tracing and the Fab fragment structure was subsequently refined (final R factor = 21.7%). The structure obtained exhibits the typical immunoglobulin fold. Complementarity-determining regions (CDRs) L1, L2, L3, H1 and H2 could be superposed onto standard canonical CDR loops. The H3 loop could be classified according to recently published rules regarding loop length, sequence and conformation. This loop is 14 residues long, with an approximate beta-hairpin geometry, which is distorted somewhat by the presence of two trans proline residues at the beginning of the loop. It is expected that this H3 loop will facilitate the design of synthetic probes for the glucagon receptor that may be used to investigate receptor activity.
Asunto(s)
Anticuerpos Monoclonales/química , Fragmentos Fab de Inmunoglobulinas/química , Receptores de Glucagón/antagonistas & inhibidores , Receptores de Glucagón/inmunología , Secuencia de Aminoácidos , Cristalografía por Rayos X , Receptor del Péptido 1 Similar al Glucagón , Humanos , Fragmentos Fab de Inmunoglobulinas/farmacología , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/química , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Estructura Secundaria de Proteína , Receptores de Glucagón/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Alineación de SecuenciaRESUMEN
Investigation of proteins extracted from wheat bran lead to the isolation of a 37 kDa polypeptide extracted from a polyacrylamide gel. Extensive internal peptide sequence information of this protein identified it as a glyoxalase I. Glyoxalase I activity in crude wheat bran extract was measured to 1 U/mg protein (1U=1 µmol S-lactoyl glutathione formed/min). Degenerate primers were designed and used for PCR-RACE-based cloning of the corresponding composite cDNA sequence (AJ243528). The wheat bran glyoxalase I amino acid sequence is very similar to the translated sequence of a RNA transcript induced by desiccation of the resurrection grass Sporobulus stapfianus, suggesting a role for glyoxalase in de- or rehydration of plant tissue. The 37 kDa wheat enzyme belongs to a group of monomeric glyoxalases and is composed of two similar halves each representing the full-length human glyoxalase I enzyme. A survey of glyoxalase I sequences, including one (not previously reported) from Drosophila melanogaster, is presented and alignments of these sequences show that amino acid residues involved in co-ordinating zinc or interaction with the substrate are conserved. The alignments indicate a non-linear evolution of glyoxalase I enzymes.
RESUMEN
Barley (Hordeum vulgare L.) storage proteins, which have a high content of proline (Pro) and glutamine, are cleaved by cysteine endoproteases to yield peptides with a Pro next to the N-terminal and/or C-terminal amino acid residues. A peptidase cleaving after Xaa-Pro- at the N terminus of peptides was purified from green barley malt. It was identified as a serine-type dipeptidyl peptidase (DPP), based on inhibitor studies, and the nature of the cleavage product. It is a monomeric glycoprotein with an apparent molecular mass of 105 kD (85 kD after deglycosylation), with a pI of 3.55 and a pH optimum at 7.2. Substrate specificity was determined with a series of fluorogenic peptide substrates with the general formula Xaa-Pro-AMC, where Xaa is an unspecified amino acid and AMC is 7-amino-4-methylcoumarin. The best substrates were Xaa = lysine and arginine, while the poorest were Xaa = aspartic acid, phenylalanine, and glutamic acid. The K(m) values ranged from 0.071 to 8.9 microM, compared with values of 9 to 130 microM reported for mammalian DPP IVs. We discuss the possible role of DPP IV in the degradation of small Pro-containing peptides transported from the endosperm to the embryo of the germinating barley grain.
Asunto(s)
Dipeptidil Peptidasa 4/aislamiento & purificación , Hordeum/enzimología , Secuencia de Aminoácidos , Cromatografía por Intercambio Iónico , Cumarinas/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Péptidos/química , Péptidos/metabolismo , Especificidad por SustratoRESUMEN
In a four-generation family with long QT syndrome, syncopes and torsades de pointes ventricular tachycardia (TdP) were elicited by abrupt awakening in the early morning hours. The syndrome was associated with a novel KCNH2 missense mutation, G572R, causing the substitution of a glycine residue at position 572, at the end of the S5 transmembrane segment of the HERG K(+)-channel, with an arginine residue. This segment is involved in the channel pore and the mutation may cause a reduction in the rapidly activating delayed rectifier K+ current (Ikr), or changed gating properties of the ion channel, leading to prolonged cardiac repolarization. The electrocardiograms of affected persons showed prolonged QT interval and notched T waves. Despite treatment with atenolol, 200 mg twice daily, the proband still experienced TdP episodes. Three untreated relatives of the proband died suddenly, and unexpectedly, at 18, 32, and 57 years of age. The G572R mutation is thus associated with a high mortality rate, and the clinical presentation illustrates that some mutations may not be controllable by just beta-blockade.
Asunto(s)
Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Síndrome de QT Prolongado/genética , Mutación Missense , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/genética , Transactivadores , Adolescente , Adulto , Secuencia de Aminoácidos , Análisis Mutacional de ADN , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go , Resultado Fatal , Femenino , Humanos , Síndrome de QT Prolongado/diagnóstico , Síndrome de QT Prolongado/terapia , Masculino , Datos de Secuencia Molecular , Linaje , Fenotipo , Homología de Secuencia de Aminoácido , Regulador Transcripcional ERGRESUMEN
2-Butyne-1,4-diamine (DABI) is a mechanism-based inhibitor of copper-containing plant amine oxidases; the number of turnovers that leads to enzyme inactivation is approximately 20. The product of DABI oxidation is a very reactive aminoallene that reacts with an essential nucleophilic group at the enzyme active site, forming a covalently bound pyrrole and producing an inactive enzyme. The inactivated enzyme shows a new absorption maximum at 295 nm and gives coloured derivatives with p-dimethylaminobenzaldehyde and p-dimethylaminocinnamaldehyde that are spectrally similar to the products of pyrrole treated with the above reagents. Resonance Raman spectra of the p-dimethylaminobenzaldehyde adduct of pyrrole and the inactivated enzyme show very high degree of similarity, supporting the idea that the product of inactivation is indeed a bound pyrrole. The bound pyrrole is formed already in the anaerobic step of the reaction, while the topa semiquinone radical is not affected, as shown by the EPR and stopped-flow absorption measurements. Peptides containing the DABI binding site were obtained by proteolysis of inactivated enzyme, isolated by HPLC and analysed by amino acid sequencing and MS. The crystal structure of the amine oxidase from pea has been determined; inhibition is caused mainly by the highly reactive DABI product, 4-amino-2-butynal, binding to a nucleophilic residue at the entrance to the substrate channel. As other DABI labelled peptides were also found and no free DABI product was detected by MS after complete inhibition of the enzyme, it is likely that the DABI product binds also to other solvent exposed nucleophilic residues on the enzyme surface.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/antagonistas & inhibidores , Diaminas/farmacología , Inhibidores Enzimáticos/farmacología , Plantas/enzimología , Amina Oxidasa (conteniendo Cobre)/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Espectrometría de Masas , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
The first committed steps in the biosynthesis of the two cyanogenic glucosides linamarin and lotaustralin in cassava are the conversion of L-valine and L-isoleucine, respectively, to the corresponding oximes. Two full-length cDNA clones that encode cytochromes P-450 catalyzing these reactions have been isolated. The two cassava cytochromes P-450 are 85% identical, share 54% sequence identity to CYP79A1 from sorghum, and have been assigned CYP79D1 and CYP79D2. Functional expression has been achieved using the methylotrophic yeast, Pichia pastoris. The amount of CYP79D1 isolated from 1 liter of P. pastoris culture exceeds the amounts that putatively could be isolated from 22,000 grown-up cassava plants. Each cytochrome P-450 metabolizes L-valine as well as L-isoleucine consistent with the co-occurrence of linamarin and lotaustralin in cassava. CYP79D1 was isolated from P. pastoris. Reconstitution in lipid micelles showed that CYP79D1 has a higher k(c) value with L-valine as substrate than with L-isoleucine, which is consistent with linamarin being the major cyanogenic glucoside in cassava. Both CYP79D1 and CYP79D2 are present in the genome of cassava cultivar MCol22 in agreement with cassava being allotetraploid. CYP79D1 and CYP79D2 are actively transcribed, and production of acyanogenic cassava plants would therefore require down-regulation of both genes.
Asunto(s)
Sistema Enzimático del Citocromo P-450/fisiología , Glucósidos/biosíntesis , Glucósidos/metabolismo , Manihot/enzimología , Nitrilos/metabolismo , Secuencia de Aminoácidos , Southern Blotting , Catálisis , Células Cultivadas , Clonación Molecular , Sistema Enzimático del Citocromo P-450/genética , Isoleucina/metabolismo , Manihot/genética , Modelos Químicos , Datos de Secuencia Molecular , Pichia/metabolismo , Plantas Modificadas Genéticamente/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Valina/metabolismoRESUMEN
Hordeins, the natural substrates of barley (Hordeum vulgare) cysteine endoproteases (EPs), were isolated as protein bodies and degraded by purified EP-B from green barley malt. Cleavage specificity was determined by synthesizing internally quenched, fluorogenic tetrapeptide substrates of the general formula 2-aminobenzoyl-P(2)-P(1)-P(1)'-P(2)' 1-tyrosine(NO(2))-aspartate. The barley EPs preferred neutral amino acids with large aliphatic and nonpolar (leucine, valine, isoleucine, and methionine) or aromatic (phenylalanine, tyrosine, and tryptophan) side chains at P(2), and showed less specificity at P(1), although asparagine, aspartate, valine, and isoleucine were particularly unfavorable. Peptides with proline at P(1) or P(1)' were extremely poor substrates. Cleavage sites with EP-A and EP-B preferred substrate sequences are found in hordeins, their natural substrates. The substrate specificity of EP-B with synthetic peptides was used successfully to predict the cleavage sites in the C-terminal extension of barley beta-amylase. When all of the primary cleavage sites in C hordein, which occur mainly in the N- and C-terminal domains, were removed by site-directed mutagenesis, the resulting protein was degraded 112 times more slowly than wild-type C hordein. We suggest that removal of the C hordein terminal domains is necessary for unfolding of the beta-reverse turn helix of the central repeat domain, which then becomes more susceptible to proteolytic attack by EP-B.
Asunto(s)
Cisteína Endopeptidasas/química , Hordeum/química , Metaloendopeptidasas/química , Proteínas de Plantas/química , Secuencia de Aminoácidos , Aminoácidos/química , Glútenes , Hidrólisis , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , beta-Amilasa/químicaRESUMEN
Variant forms and post-translational modifications of transthyretin (TTR) can be identified by electrospray ionisation mass spectrometry (ESI-MS). The aim of the present study was to investigate thiol conjugation of transthyretin and it's relation to age and symptomatic amyloid disease in different populations of variant TTR carriers. Plasma samples from 70 individuals from Denmark, Argentina, Sweden and Japan, with 2 different TTR mutations were analysed. The percentage cysteine (Cys) conjugated wild and variant TTR were calculated from the corresponding peaks of the spectra, and multiple regression analysis was employed to disclose relationships between age, symptomatic amyloid disease and origin. Age, origin and presence of symptomatic disease, were found to be independent factors related to transthyretin conjugation. A higher percentage of conjugated to unconjugated TTR was disclosed in symptomatic, but not in asymptomatic carriers. In summary: Thiol conjugation of TTR is dependent on age and presence of symptomatic amyloid disease. Furthermore, it varies between different populations. Variant TTR is more susceptible to thiol conjugation than the wild type. Post-translational factors may be related to amyloid formation and/or toxicity.
Asunto(s)
Envejecimiento/metabolismo , Amiloidosis/metabolismo , Prealbúmina/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Adolescente , Adulto , Anciano , Amiloidosis/genética , Amiloidosis/fisiopatología , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , MutaciónRESUMEN
Amyloidosis is a group of diseases characterized by amyloid deposition in various tissues. The diseases can roughly be divided into hereditary and non-hereditary forms. The hereditary forms are related to a mutation in the serum protein transthyretin which is produced mainly in the liver. The inheritance is autosomal dominant. A family in Denmark has earlier been described as having inherited cardiac amyloidosis with a mutation at amino acid number 111 in the transthyretin protein. The family now has been re-examined because of new diagnostic and therapeutic possibilities. The aims of the study were to identify carriers and non-carriers of the mutant transthyretin methionine 111 linked familial amyloid disease, to detect early signs of the restrictive cardiomyopathy and other clinical manifestations of this disease. Clinical, echocardiographic and genetic examination was carried out. Out of 125 living family members, 99 were available for examination. Twenty-five persons were heterozygous carriers of the mutant transthyretin methionine 111 genotype, while 74 were non-carriers. Eight carriers, all above the age of 35, showed echocardiographic abnormalities suggestive of developing or manifest restrictive cardiomyopathy. Nine carriers had carpal tunnel syndrome as opposed to none of the non-carriers. It is concluded that for early detection of familial amyloid cardiomyopathy, echocardiography is the investigation of choice. The first sign is diastolic dysfunction detected as an abnormal relaxation pattern. Carpal tunnel syndrome appears to be the earliest presenting clinical symptom. Early liver transplantation seems to be curative.