Biochemical medicine and metabolic biology最新文献

In 12 out of 32 non-insulin-dependent diabetic subjects, the activity of FAD-linked glycerophosphate dehydrogenase in T lymphocyte homogenates was abnormally low when measured by both a colonimetric and radioisotopic procedure. A comparable situation characterized by a deficient activity of FAD-linked glycerophosphate dehydrogenase in both the colonimetric and radioisotopic assays was only observed once among 26 other subjects including 11 healthy volunteers, 9 non-diabetic patients, 5 type-1 (insulin-dependent) diabetics, and 1 pancreatectomized diabetic. By analogy, it is speculated that an impaired activity of FAD-linked glycerophosphate dehydrogenase in the insulin-producing pancreatic B-cell could represent a far-from-uncommon contributive factor in the pathogenesis of non-insulin-dependent diabetes mellitus.

Deficiency of α₁-antitrypsin (α₁AT), a common hereditary disorder of Caucasians, is associated with an increased risk for early-onset chronic obstructive pulmonary disease and childhood liver dysfunction. The two most common deficiency variants, Pi^S and Pi^Z, are both single base-pair substitutions causing amino acid modifications, although neither mutation creates or destroys a naturally occurring restriction site. Dried blood specimens (DBS) submitted to the New York State Department of Health for mandated newborn screening tests were tested for α₁AT activity using a fluorometric elastase inhibition assay. A second DBS from specimens determined to be α₁AT deficient was phenotyped on an agarose isoelectric focusing gel. Genotypic confirmation was performed by amplifying, directly from a DBS, the regions of the DNA containing the S and Z mutation. The Z mutation was analyzed with a modified primer designed to create an artificial restriction site in the normal allele. TaqI digestion produces two bands, a 157- and a 22-hp fragment. The single base substitution in Pi^Z individuals eliminates this TaqI restriction site, thus showing the same 179-bp fragment before and after digestion. A primer mismatch placed close to the S mutation creates a restriction site in the normal allele, producing a 100-bp product after TaqI digestion. The restriction site is abolished in individuals that carry the S mutation, with a 121-bp product observed before and after digestion. Of 11,081 specimens screened, 3 Pi^Z neonates, all Caucasian, were detected by these methodologies for an estimated incidence of 1:2019 in the Caucasian or 1:3694 in the general population in New York State. Additionally, 5 Pi^Sz, 59 Pi^Mz, 1 Pi^S, and 10 Pi^ms newborns were detected.

We characterized four new mutations in the ornithine transcarbamylase (OTC) gene in three male infants who died from acute neonatal hyperammonemia and one male infant with late onset disease. OTC enzymatic activity was undetectable in the livers of the three neonates, whereas residual enzymatic activity was present in the fourth patient. All 10 exons of the OTC gene were amplified by the polymerase chain reaction (PCR) from genomic DNA of the four patients. The amplified DNA was screened for abnormal gel electrophoretic migration patterns via single-strand conformational polymorphism (SSCP). One patient showed an abnormal SSCP pattern of exon 8 and exon 9, a second patient had an abnormal exon 6, a third had an abnormal exon 9, and the fourth patient revealed an abnormal exon 3. Sequencing of the abnormal exons revealed that the first patient hod a deleterious mutation in exon 9 consisting of a G→T transversion in codon 310 causing a Glu→stop coding change. The abnormal exon 8 of this patient contained a common polymorphism consisting of an A→G transition in codon 270 resulting in Gln→Arg code change. The abnormal exon 6 of the second patient contained an A→G transition in codon 183 causing a Tyr→Cys change. Exon 9 of the third patient contained a deletion of a thymine nucleotide (base 882) resulting in a shift of the reading frame and a code for premature termination 28 codons downstream. The fourth patient with a "milder" clinical presentation had an A→T transversion in exon 3 (codon 88) causing a Lys→Asn change.

Detection of Portuguese carriers for Gaucher disease with urine samples as a source of enzyme was carried out using an immunological procedure employing an anti-glucocerebrosidase monoclonal antibody and by DNA analysis for the presence of the two glucocerebrosidase mutations most frequently found in Portuguese Gaucher patients. Patients, obligate and putative carriers, and individuals unrelated to patients were analyzed. It was found that the vast majority of carriers for the two tested mutations (N370S and L444P), as well as obligate carriers for as yet unidentified mutations, could be distinguished from control subjects with this relatively easy and economic immunological procedure. Furthermore, results obtained for control subjects suggested a high frequency of carriers for the N370S mutation in the Portuguese population. It is concluded that this procedure may be useful in mass screening for carrier detection prior to DNA analysis, particularly in the study of non-Ashkenazi populations in which a significant number of mutations associated with Gaucher disease remain unidentified.

The level of characteristic markers of protein oxidative modification (tryptophan oxidation and sulfhydryl group loss as well as carbonyl and bityrosine formation) and glycation (AGEP formation) have been measured in β_L crystallin purified from the lenses of control, diabetic, and ascorbate-supplemented diabetic animals. These markers were also determined following the application of an in vitro graded oxidative insult. Prior to the application of stress, diabetic lens crystallins, in comparison with control, exhibited a higher content of bityrosine and AGEPs, a lower level of nonoxidized tryptophan, and a loss of sulfhydryl groups. After exposure to the oxidative insult there was a stress-proportional increase of the parameters in all β_L crystallins, irrespective of their source. The effects were most pronounced in the diabetic, in which the already-elevated indicators of oxidative damage were further increased. Dietary supplementation of the diabetic group with ascorbate had a marked effect in preventing β_L crystallin modification in vivo, alleviating the loss of sulfhydryl groups and the oxidation of tryptophan, partially preventing the formation of AGEP and carbonyl groups, but not affecting the formation of bityrosine. Supplementation also inhibited the increase in susceptibility of diabetic β_L crystallin to in vitro oxidative stress, preventing sulfhydryl group loss as well as carbonyl and AGEP group formation. The results are discussed in relation to the proposal that diabetes renders lens crystallins more susceptible to oxidative stress and that this may be a causative factor in cataractogenesis. The possible role of ascorbate in the inhibition, or attenuation, of cataractogenesis is examined.

Spontaneous animal models of inborn errors of metabolism are valuable tools for defining the pathogenesis of these disorders and also the mechanism of various therapeutic approaches. In the present study, we have employed BALB/cByJ mice with an autosomal recessive deficiency of short-chain acyl-CoA dehydrogenase (SCAD). These animals were characterized by a marked urinary excretion of ethylmalonic and methylsuccinic acids along with butyrylglycine. Using adult homozygous mice we have studied the basic cerebral and hepatic profile of carnitine, ammonia, and energy metabolism. The effects of fasting and a short-term supplement of L-carnitine have been evaluated in comparison with control BALB/cJ mice. The mutant mice had low levels of acetyl-CoA and high levels of lactate compared to control mice. Fasting aggravated this condition by further decreasing acetyl-CoA and increasing lactate levels in the mutant mice. Free carnitine levels were significantly decreased in liver with fasting. Long-chain acylcarnitines were significantly lower in the brain of mutant mice. A short-term supplementation of L-carnitine resulted in general increases of carnitine levels in liver and muscle, but they still remained lower in mutant BALB/cByJ mice as compared to control BALB/cJ mice. L-Carnitine treatment increased cerebral CoA-SH levels and both hepatic and cerebral acetyl-CoA levels in mutant mice. Hyperammonemia which has been described frequently in acyl-CoA dehydrogenase deficiencies was not observed in adult BALB/cByJ mice. This could be due to a rapid conjugation of butyryl-CoA with glycine by an increased activity of glycine N-acyltransferase.

The PCR technique was used in a study of the linkage of cystic fibrosis mutations and a polymorphic (GATT)_n repeat in intron 6 of the CFTR gene. Absolute linkage disequilibrium was found between the common ΔF-508 mutation and the (GATT)₆ allele. This allele was also in linkage disequilibrium with other unidentified mutations in the CFTR gene resulting in the pancreatic insufficient form of disease. The frequency of (GATT)_n alleles in the pancreatic sufficient form of CF did not differ significantly from the data obtained in the total population. The significance of the (GATT)_n polymorphic repeat for the diagnosis of CF is discussed.

The uptake and efflux of radioactivity was monitored in rat erythrocytes exposed to or prelabeled with either 3-O-methyl-D-[U-¹⁴C]glucose or D-[U-¹⁴C]glucose. In the case of 3-O-methyl-D-[U-¹⁴C]glucose uptake, the half-life for equilibration of hexose concentration between the extracellular medium and the intracellular 3H₂O space increased from 12.5 to 32.5 min as the concentration of the D-glucose analog was raised from 8.3 to 33.3 mM. In erythrocytes preincubated for 60 min with 3-O-methyl-D-[U-¹⁴C]glucose at the latter two concentrations, the half-life for the fall in cell radioactive content, after correction for the residual radioactivity recovered in the cells after prolonged incubation, amounted to respectively 6.5 and 9.5 min. Thus, whether in terms of uptake or efflux, the equilibration of hexose concentrations across the plasma membrane represented a delayed phenomenon. In this respect, there was no difference between erythrocytes from control or diabetic rats. Comparable conclusions were reached in the study of influent and effluent radioactivity in rat erythrocytes exposed to or prelabeled with D-[U-¹⁴C]glucose. In such a case, the measurement of radioactive acidic metabolites and L-lactic acid indicated that, even after 90 min incubation, the concentration of the hexose in the intracellular 3H₂O space remained lower than its extracellular concentration. It is proposed that the delayed equilibration of hexose concentrations accounts, in part at least, for an artifact of isotopic dilution in the study of radioactive D-glucose metabolism by erythrocytes from control or diabetic rats.

The activity of FAD-linked glycerophosphate dehydrogenase (m-GDH), as well as that of glutamate dehydrogenase and both glutamate-oxalacetate and glutamate-pyruvate transaminases, were measured in islet, liver, and splenocyte homogenates from 6- to 7-week-old female nonobese diabetic mice (NOD) and age- and sex-matched control mice. Despite incipient insulitis and euglycemia, the NOD mice displayed both high islet insulin content and elevated insulinemia. The activity of m-GDH, expressed relative to protein content, was not decreased in islets of NOD mice, despite the fact that such a specific activity is lower in splenic lymphocytes than islet cells. In liver homogenates, the activity of m-GDH was even higher in NOD than control mice. It is proposed, therefore, that in this model of insulin-dependent diabetes no primary decrease in islet m-GDH activity occurs, at variance with the situation recently documented in several animal models of non-insulin-dependent diabetes.