RESUMEN
AIMS/HYPOTHESIS: The aim of the study was to investigate the use of hyperglycaemic clamp tests to identify individuals who will develop diabetes among insulinoma-associated protein-2 antibody (IA-2A)-positive first-degree relatives (IA-2A(+) FDRs) of type 1 diabetic patients. METHODS: Hyperglycaemic clamps were performed in 17 non-diabetic IA-2A(+) FDRs aged 14 to 33 years and in 21 matched healthy volunteers (HVs). Insulin and C-peptide responses were measured during the first (5-10 min) and second (120-150 min) release phase, and after glucagon injection (150-160 min). Clamp-induced C-peptide release was compared with C-peptide release during OGTT. RESULTS: Seven (41%) FDRs developed diabetes 3-63 months after their initial clamp test. In all phases they had lower C-peptide responses than non-progressors (p < 0.05) and HVs (p < 0.002). All five FDRs with low first-phase release also had low second-phase release and developed diabetes 3-21 months later. Two of seven FDRs with normal first-phase but low second-phase release developed diabetes after 34 and 63 months, respectively. None of the five FDRs with normal C-peptide responses in all test phases has developed diabetes so far (follow-up 56 to 99 months). OGTT-induced C-peptide release also tended to be lower in progressors than in non-progressors or HVs, but there was less overlap in results between progressors and the other groups using the clamp. CONCLUSIONS/INTERPRETATION: Clamp-derived functional variables stratify risk of diabetes in IA-2A(+) FDRs and may more consistently identify progressors than OGTT-derived variables. A low first-phase C-peptide response specifically predicts impending diabetes while a low second-phase response may reflect an earlier disease stage. TRIAL REGISTRATION: ClinicalTrials.gov NCT00654121 FUNDING: The insulin trial was financially supported by Novo Nordisk Pharma nv.
Asunto(s)
Autoanticuerpos/sangre , Diabetes Mellitus/epidemiología , Adolescente , Adulto , Péptido C/sangre , Diabetes Mellitus/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Familia , Técnica de Clampeo de la Glucosa , Antígenos HLA-DQ/genética , Humanos , Hiperglucemia , Insulina/sangre , Anamnesis , Valores de Referencia , Medición de Riesgo , Adulto JovenRESUMEN
Purification to homogeneity of the N-acetylmuramoyl-L-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) from human serum has been achieved with a high yield. By molecular sieving chromatography, a molecular weight of 120,000-130,000 has been found for the native enzyme. Polyacrylamide gel electrophoresis under native conditions gave a unique band of Mr = 125,000. The same technique performed under denaturing conditions revealed that the protein is a dimer composed of one subunit of Mr = 57,000 and another of Mr = 70,000. In isoelectrofocalization assays, the amidase behaved as an acidic protein. Ethylenediaminetetraacetate inhibited the enzyme activity; the Mg2+ requirement was confirmed. The simultaneous presence of sulfhydryl groups and disulfide bonds in the protein was evidenced by the inhibitions produced by different thiol-blocking reagents and by several thiol-bearing substances. Direct measurements established the presence of two accessible thiol groups and the occurrence of nine disulfide bonds per protein molecule. Studies of substrate hydrolyzing capacities showed a marked preference for the muramoyl tripeptide derived from the Escherichia coli or Bacillus cereus mureins, the disaccharide tetrapeptide and the bis disaccharide tetra-tetrapeptide from E. coli were also good substrates. Activities on small muropeptides of other composition are also reported. Whole (insoluble) peptidoglycans representing the main bacterial chemotypes were submitted to the enzyme action; although with weak specific activities, the human amidase was nevertheless able to release soluble peptides from some of them. A bacteriolytic capacity on some microorganisms cannot be excluded. Results are discussed and the human enzyme is compared to presently known microbial muramoyl amidases.
Asunto(s)
Amidohidrolasas/sangre , N-Acetil Muramoil-L-Alanina Amidasa/sangre , Secuencia de Aminoácidos , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Humanos , Focalización Isoeléctrica , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , N-Acetil Muramoil-L-Alanina Amidasa/aislamiento & purificación , Oligopéptidos , Especificidad por SustratoRESUMEN
Heat-shock protein 70 (HSP70)-related proteins are classified in two main subfamilies: the DnaK subfamily and the HSP110/SSE1 subfamily. We have characterized the first plant member of the HSP110/SSE1 subfamily, HSP91. At least two, tightly linked genes encoding HSP91 are present per haploid Arabidopsis genome. HSP91 is constitutively expressed in non-stressed Arabidopsis plants and is transiently induced by heat shock.
Asunto(s)
Arabidopsis/metabolismo , Proteínas de Escherichia coli , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/genética , Familia de Multigenes , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Proteínas de Arabidopsis , Células CHO , Bovinos , Cricetinae , ADN Complementario , Genes de Plantas , Proteínas del Choque Térmico HSP110 , Proteínas HSP70 de Choque Térmico/química , Humanos , Ratones , Datos de Secuencia Molecular , Filogenia , Técnica del ADN Polimorfo Amplificado Aleatorio , Homología de Secuencia de Aminoácido , Programas InformáticosRESUMEN
We applied total error profiling to evaluate the conversion of a known proinsulin (PI) enzyme-linked immunosorbent assay (ELISA) into a time-resolved fluorescence immunoassay (TRFIA). The formula and acceptance criteria proposed by the Ligand Binding Assay Bioanalytical Focus Group (LBABFG) of the American Association of Pharmaceutical Scientists (AAPS) were applied. We found that the expected dynamic range enlargement with TRFIA compared to ELISA ([0.5-240] versus resp. [0.7-98] pmol/L) is limited by an interference of C-peptide when present in the sample at high concentrations (>7000 pmol/L).
Asunto(s)
Fluoroinmunoensayo/métodos , Proinsulina/sangre , Diabetes Mellitus Tipo 1/sangre , HumanosRESUMEN
AIMS/HYPOTHESIS: Insulin resistance has been proposed as a risk factor for type 1 diabetes. We investigated whether adiponectin, an insulin sensitiser, can serve as an additional predictive marker for type 1 diabetes in first-degree relatives of known patients. METHODS: Adiponectin was followed in 211 persistently islet antibody-positive (Ab+) first-degree relatives of type 1 diabetic patients and in 211 age- and sex-matched persistently antibody-negative relatives, and correlated with antibody status, random proinsulin:C-peptide ratio and HLA-DQ genotype. During follow-up, 37 Ab+ relatives developed type 1 diabetes. RESULTS: In the group of 422 relatives, baseline adiponectin correlated inversely with age and BMI and was lower in male than in female participants, especially after 15 years of age (p < 0.001). There was no correlation with antibody status or later development of diabetes. In 24 Ab+ relatives sampled fasted, adiponectin levels correlated significantly with homeostasis model assessment of insulin sensitivity (p = 0.006). In Ab+ relatives (n = 211), adiponectin levels could not predict type 1 diabetes nor complement risk assessment based on islet antibodies, HLA-DQ genotype and pancreatic hormones in Cox regression analysis. CONCLUSIONS/INTERPRETATION: Adiponectin levels do not contribute to the prediction of type 1 diabetes in Ab+ relatives.
Asunto(s)
Adiponectina/genética , Diabetes Mellitus Tipo 1/epidemiología , Adiponectina/sangre , Autoanticuerpos/sangre , Biomarcadores/sangre , Estudios de Cohortes , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Femenino , Antígenos HLA-DQ/genética , Humanos , Resistencia a la Insulina , Masculino , Núcleo Familiar , Polimorfismo Genético , Valor Predictivo de las PruebasRESUMEN
AIMS/HYPOTHESIS: We investigated whether random proinsulin levels and proinsulin:C-peptide ratio (PI:C) complement immune and genetic markers for identifying relatives at high risk of type 1 diabetes. MATERIALS AND METHODS: During an initial sampling, random glycaemia, proinsulin, PI:C and HLA DQ genotype were determined in 561 non-diabetic first-degree relatives who had been positive for islet autoantibodies on one or more occasions and in 561 age- and sex-matched persistently antibody-negative relatives. RESULTS: During follow-up (median 62 months), 46 relatives with antibodies at entry developed type 1 diabetes. At baseline, antibody-positive relatives (n=338) had higher PI:C values (p<0.001) than antibody-negative subjects with (n=223) or subjects without (n=561) later seroconversion. Proinsulin and PI:C were graded according to risk of diabetes as expressed by positivity for (multiple) antibodies or IA-2 antibodies, especially in persons carrying the high-risk HLA DQ2/DQ8 genotype and in prediabetic relatives. In the presence of multiple or IA-2 antibodies, a PI:C ratio exceeding percentile 66 of all antibody-negative relatives at entry (n=784) conferred a 5-year diabetes risk of 50% and 68%, respectively (p<0.001 vs 13% for same antibody status with PI:CAsunto(s)
Autoanticuerpos/sangre
, Péptido C/sangre
, Diabetes Mellitus Tipo 1/diagnóstico
, Proinsulina/sangre
, Adulto
, Niño
, Preescolar
, Diabetes Mellitus Tipo 1/sangre
, Femenino
, Antígenos HLA-DQ/genética
, Hormonas/sangre
, Humanos
, Lactante
, Recién Nacido
, Islotes Pancreáticos/inmunología
, Masculino
, Persona de Mediana Edad
, Linaje
, Estado Prediabético/diagnóstico
, Estado Prediabético/metabolismo
, Valor Predictivo de las Pruebas
RESUMEN
N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28) specifically hydrolyzes the bacterial cell wall peptidoglycans (or mureins) and the muropeptides. The enzyme splits these molecules into two parts: the peptide subunits and the glycan strands or moieties. The bacterial peptidoglycans and their derived muropeptides display a number of biological properties. Removal of the glycosidic part of these molecules abolishes their beneficial as well as their detrimental properties. We report the high level of enzymatic activity found in all mammalian (including human) sera tested. The enzyme also occurred in human saliva, milk, cerebrospinal fluid, and synovial liquid. Mucosal tissue from different parts of the mammalian digestive tract exhibited enzymatic activity, but the enzyme was not detectable in the lumen content. The range of substrate specificity of the human enzyme was evaluated by measuring its action on the peptidoglycans extracted from several bacterial strains and representing different chemotypes and structures. Time course of the muramylalanine amidase and of the lysozyme (both of human origin) activities on some of these peptidoglycans are also reported, with the enzymes acting separately or together. From these data, we would speculate that a probable physiological role of the muramylalanine amidase is the maintenance of adequate ratios between the biologically active muropeptides and their inactive derivatives in the organism, the amidase activity antagonizing the production of biologically active molecules by lysozyme.
Asunto(s)
Muramidasa/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Peptidoglicano/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Carbohidratos , Bovinos , Humanos , Mucosa Intestinal/enzimología , Datos de Secuencia Molecular , N-Acetil Muramoil-L-Alanina Amidasa/sangre , N-Acetil Muramoil-L-Alanina Amidasa/líquido cefalorraquídeo , Conejos , Ratas , Ovinos , Especificidad por SustratoRESUMEN
Human serum N-acetylmuramyl-L-alanine amidase was purified to homogeneity by a relatively short procedure including affinity chromatography. For this purpose, a specific adsorbent was prepared by coupling the main substrate of the enzyme, N-acetyl-muramyl-L-alanine-gamma-D-glutamyl-L-meso-2,6[3,4,5-3H] diaminopimelic acid, to a divinylsulfone agarose gel. The enzyme is unable to hydrolyze this muramylpeptide when it is attached as a ligand to the gel, whereas a high affinity is conserved. In addition to affinity chromatography, the presented purification scheme includes ion exchange chromatography on DEAE-Sepharose and molecular sieving on Superdex 200. The enzyme was purified 739-fold with a yield of 22.5%. One single band at 135 kDa was obtained on native gradient PAGE. Gradient PAGE in denaturing conditions gave one single band at 74 kDa, which was lowered to 64 kDa when the enzyme was denatured in nonreducing conditions. This suggests that the native enzyme is a dimer consisting of two subunits of identical molecular weight with only intramolecular disulfide bonds. Isoelectric focusing gave one single band at pI 5.0. Glycan detection before and after treatment with N-glycosidase F showed that the enzyme is a glycoprotein. Further analysis by lectin immuno detection on dot blots confirmed that the enzyme is an N-glycosylated protein of complex type with sialic acid, terminally linked alpha (2-->6) to galactose or N-acetylgalactosamine. The 15 amino acid N-terminal sequence was determined by microsequence analysis.
Asunto(s)
Cromatografía de Afinidad/métodos , Glicoproteínas/sangre , N-Acetil Muramoil-L-Alanina Amidasa/sangre , Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Secuencia de Aminoácidos , Western Blotting , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/aislamiento & purificación , Humanos , Focalización Isoeléctrica , Datos de Secuencia Molecular , N-Acetil Muramoil-L-Alanina Amidasa/aislamiento & purificación , Unión Proteica , Conformación Proteica , Análisis de SecuenciaRESUMEN
Ascorbate peroxidases are important enzymes that detoxify hydrogen peroxide within the cytosol and chloroplasts of plant cells. To better understand their role in oxidative stress tolerance, the transcriptional regulation of the apx1 gene from Arabidopsis was studied. The apx1 gene was expressed in all tested organs of Arabidopsis; mRNA levels were low in roots, leaves, and stems and high in flowers. Steady-state mRNA levels in leaves or cell suspensions increased after treatment with methyl viologen, ethephon, high temperature, and illumination of etiolated seedlings. A putative heat-shock cis element found in the apx1 promoter was shown to be recognized by the tomato (Lycopersicon esculentum) heat-shock factor in vitro and to be responsible for the in vivo heat-shock induction of the gene. The heat-shock cis element also contributed partially to the induction of the gene by oxidative stress. By using in vivo dimethyl sulfate footprinting, we showed that proteins interacted with a G/C-rich element found in the apx1 promoter.