Biochemical medicine and metabolic biology最新文献

The incorporation of radioactivity from [1-¹⁴C]galactose into TCA-precipitable material was determined in skin fibroblasts derived from 11 galactosemic patients deficient in galactose 1-phosphate uridyl transferase (GALT-). "R" ratios (designated the R phenotype) were defined as the ratio between [¹⁴C]galactose incorporation and [³H]leucine incorporation. Results were expressed as a percentage of the controls. In the GALT-strains this ratio varied from strain to strain, presumably depending on the efficiency of the secondary route via the UDP-galactose pyrophosphorylase pathway. In 10 GALT-patients without late serious clinical manifestations, the R phenotype varied hom 37 to 57% of the control value. In the 11th patient, the R phenotype was only 20% of the control. Thus, we obtained a significantly lower R phenotype in one patient who was distinguished from the others by having very severe delayed neurological complications, although compliance to galactose-free diet was good. We suggest that, in this patient, the development of the UDP-galactose pyrophosphorylase pathway was not sufficient to ensure the availability of enough galactose for the necessary synthesis of glycoproteins and glycolipids. Thus the R phenotype may be an indicator of the risk of late neurological complications. The determination of the R phenotype of GALT-patients may therefore be valuable. However, further investigations of galactosemic patients with neurological complications are required to confirm this relationship.

The effects of growth hormone treatment and dietary alanine supplementation, individually and in combination, were studied in five patients with organic acidemias. Three patients had propionic acidemia, one had 3-hydroxyisobutyric acidemia, and one had a defect in isoleucine metabolism. Two patients with propionic acidemia had decreased growth hormone secretion in response to provocative stimuli (intravenous L-arginine and oral levodopa or clonidine); the remaining subjects had sufficient growth hormone secretion. Three of four subjects in whom IGF₁ was measured showed subnormal concentrations at baseline (including two with normal growth hormone secretory responses). All patients showed an increase in linear growth with growth hormone. In the four patients studied, all had a significant increase in nitrogen retention over baseline with alanine or growth hormone alone, or with the combination of growth hormone and alanine, with a much greater effect of growth hormone. Lean body mass and body fat composition tended to become normal with treatment. Protein tolerance increased, and when the patients′ dietary protein intakes were increased between 20 and 60% they maintained positive nitrogen balance, without a significant increase in metabolite excretion. One patient with propionic acidemia expired during the time of the study, following a course of recurrent pancreatitis and an episode of acute basal ganglia infarction. All of the other subjects showed clinical improvement (decreased incidence of ketoacidotic episodes and decreased frequency of hospital admission and school absence) during treatment, and even the patient who expired remained metabolically stable up to and through the terminal event. We conclude that growth hormone may be of value in the management of patients with organic acidemia.

Differences in Ahd-2 at the DNA sequence level were characterized in mouse strains with variable ethanol preferences. The 5′ region and the region surrounding the active site of Ahd-2 were compared to detect differences which could affect ethanol sensitivity. Only minor differences were found among the strains in the two regions. These differences cannot explain their variable ethanol preference and the implications of sequence identities among the divergent strains in these regions has yet to be determined.

The Friedreich ataxia (FRDA) locus is localized on chromosome 9q13 in an interval less than 1 Mb between markers D9S202/FR1 and FR5. We cloned the FRDA candidate region in YACs, and we started a systematic search for transcripts in this region using the cDNA selection approach. Several overlapping cDNA clones mapping near the telomeric end of the FRDA minimum genetic region were isolated. Zoo blot analysis demonstrated that these cDNAs are well conserved among different species. A transcript of 4.8 kb was identified by hybridization to a Northern blot containing human brain poly(A)⁺ RNA. Partial sequence of these clones showed 100% homology with a previously described anonymous brain cDNA (EST01251). A search for mutations of this gene in FRDA patients and carriers is in progress. No mutations have been found to date, but we have identified a DNA polymorphysm. This polymorphism was nonrecombinant with the disease in a previously described FRDA pedigree in which a recombination had occurred with more telomeric markers.

In the present study, we investigated the effects of different diacylglycerols in comparison with phorbol 12-myristate 13-acetate (PMA) on eicosanoid-independent phospholipase A₂ (PLA₂) activation in human platelets and neutrophils. Eicosanoid-independent PLA₂ activation was measured under conditions where both cyclooxygenase and lipoxygenases were blocked by BW755C. In the presence of PMA (50 nM), the amount of mass arachidonic acid (AA) released represented 400 and 257% of control (without PMA) in A23187-stimulated platelets and neutrophils, respectively, while 1,2-dioctanoylglycerol (1,2-DiC₈) and 1-oleoyl-2-acetyl-sn-glycerol (OAG) had increased the eicosanoid-independent AA release by 150 and 117-134% of control, in platelets and neutrophils, respectively. Our results further demonstrate that 1,3-dioctanoylglycerol (1,3-DiC₈), a poor activator of protein kinase C (PKC), is nearly as effective as diacylglycerols, such as OAG and 1,2-DiC₈ (activators of PKC) in priming PLA₂ activation, but is less effective than PMA as a priming agent. However, all three diacylglycerols were less effective than PMA as priming agents. Furthermore, diacylglycerols including 1,3-DiC₈ exerted a much greater effect on PLA₂ activation in platelets than in neutrophils. Neither 1,3-DiC₈ nor 1,2-DiC₈ and OAG had any significant priming effect on the accumulation of palmitic and stearic acids, while PMA caused a substantial accumulation of these fatty acids in platelets, but not in neutrophils. We also found that exogenously added OAG underwent significant hydrolysis even in unstimulated platelets, but not in neutrophils, suggesting that exogenously added OAG may be readily accessible for diacylglycerol (DAG) lipase/PLA₁ in platelets. It is possible that the priming of PLA₂ by diacylglycerols in both cell types may involve a PKC-independent mechanism, whereas that by PMA may involve both PKC-dependent and PKC-independent mechanisms. The differential effects of PMA and diacylglycerols on PLA₂/DAG lipase activation observed between platelets and neutrophils may stem from the differences in the predominance of certain enzyme isoforms, requiring specific factors such as cytosolic/exogenous Ca²⁺, receptor-agonist interaction, enzyme-diacylglycerol interactions, and PKC and tyrosine kinase mediated phosphorylations.

Eight mouse monoclonal antibodies directed against the acetylated N-terminal part of the type 1 human VDAC Porin 31HL clearly discriminate type 1 and type 2 mammalian porin channels. This is shown by comparing synthetic N-terminal peptides of either channel type in Western dot blots or by ELISA. The data support the specificity of the anti-Porin 31HL antibodies and thus give further support to our recent observations on extramitochondrial expression of VDAC. In the plasmalemma of different mammalian cells VDAC forms part of an ubiquitous chloride channel complex, which in patch clamp measurements may figure as the outwardly rectifying depolarization-induced chloride channel that is affected in cystic fibrosis.

Tay-Sachs disease (TSD) is caused by mutations in the gene encoding the α-subunit of β-hexosaminidase A (HexA). This disease is more prevalent in certain ethnic groups such as Jews of Ashkenazi origin. Three mutations are most commonly found among the latter population: a 4-nucleotide insertion in exon 11, a transversion at the splice site in intron 12, and the adult onset mutation in exon 7. The frequency and distribution of these mutations among Ashkenazi and non-Ashkenazi Jews were examined: 96% of the Ashkenazi carriers bore one of these mutations, while in only 30% of the non-Ashkenazi Jewish carriers were the mutations identified. The percentage distribution of the exon 11:intron 12:exon 7 and unidentified mutant allele(s) was 82:10:4:4 among 152 Ashkenazi carriers, and 16:12:2:70 among non-Ashkenazi Jewish carriers. When the non-Ashkenazi Jewish population was divided into two groups according to the geographical distance from Eastern Europe, it was obvious that the ancestral origin of the subjects bearing the exon 11 allele was predominantly from countries bordering Eastern and Central Europe, such as Turkey, Bulgaria, and Georgia. In carriers from other geographical areas of North Africa and the Middle East, this allele was about fivefold less frequent. The result is compatible with the assumption that this gene, of which the tested individuals were unaware, originates from interethnic marriage in neighboring populations. However, regardless of the ancestral origin, the intron 12 allele was quite evenly distributed throughout the Jewish population. It is our hypothesis that this allele existed in Jews even before the European diaspora was formed following the grand exile of the Jews by the Romans (70 AD).

The relationship between growth rate and various parameters of the heme biosynthetic pathway was studied in two cell lines of rat fibroblasts (REabl-1 and REabl-3) transfected with v-abl oncogene, coded by the Abelson murine leukemia virus, and subjected to glucocorticoid dependent transformation. In the REabl-1 cell line, whose growth rate was only slightly affected by dexamethasone (DX), almost no change was noticed either in heme content or in the enzymatic activities of aminolevulinate synthase (ALAS), porphobilinogen deaminase (PBGD), and ferrochelatase (FC) in the presence of various concentrations of DX. In the REabl-3 cell line, exhibiting a growth rate highly sensitive to DX, a significant reduction in intracellular heme concomitantly with decreases in ALAS and FC activities and a threefold increase in PBGD were noted. The fact that incubation with 10⁻⁵M hemin did not result in a decrease in ALAS activity raised the possibility that REabl cells lack a negative feedback control mechanism. The relationships between transformation, growth rate, and heme biosynthetic pathway are discussed.

To better understand the effects of lipid-lowering drugs on the transfer of esterified cholesterol (EC) between lipoproteins, we investigated the changes induced by gemfibrozil administration on the unidirectional transfer of radiolabeled EC from high density lipoproteins (HDL) to very low (VLDL) and low density lipoproteins (LDL) in 10 normolipidemic subjects. HDL, VLDL/LDL, and the d > 1.21 g/ml fraction containing cholesterol ester transfer protein (CETP) were isolated from plasma before and after 8 weeks of gemfibrozil administration. The same fractionation procedure was applied to aliquots of a control plasma pool to permit different recombination experiments. When the CETP fractions of the subjects studied were incubated in the presence of control HDL and VLDL/LDL, no effect of gemfibrozil was observed on the rate of EC transfer, indicating that the drug did not induce any change in the plasma transfer activity. When HDL of the subjects studied were recombined with the CETP fraction and VLDL/LDL isolated from the control plasma pool, the rate of EC transfer was decreased by 43% after gemfibrozil administration. Thus, the drug induced a decrease in the HDL-dependent transfer of EC. This effect was accompanied by a decrease of the triglyceride (TG)/EC ratio in HDL, a decrease of the Stokes radius of large HDL determined after gradient gel electrophoresis, and an increase of the HDL viscosity. Since both HDL size and viscosity are in part dependent upon their TG/EC ratio, further investigations will be necessary to answer the question as to whether one of these structural criteria is predominant for the regulation of the HDL dependent transfer of EC.