Transport of l(-)malic acid and other dicarboxylic acids in the yeast Candida sphaerica

Summary dl-Malic acid grown cells of Candida sphaerica (anamorph of Kluyveromyces marxianus) formed a saturable transport system that mediated accumulative transport of l(-)malic acid with the following kinetic parameters at pH 5.0: V _max, 0.44 nmol l(-)malate·s^-1 per milligram dry weight; K _m ,0.1 mM l(-)malate. Initial uptake of the acid was accompanied by disappearance of extracellular protons, the rates of which followed Michaelis-Menten kinetics as a function of the acid concentration. Variation with extracellular pH of the K _m values, calculated either as the concentrations of anions or of undissociated acid, pointed to anions as the transported form. Furthermore, accumulated free acid suffered rapid efflux after the addition of the protonophore carbonylcyanide-M-chlorophenyl-hydrazone (CCCP). These results suggested that the transport system was a dicarboxylate-proton symporter. The system was inducible and was subject to glucose repression. Succinic, fumaric, -ketoglutaric, oxaloacetic and d-malic acid, but not maleic, malonic, oxalic nor l(+)-tartaric acid, apparently used the same transport system since they acted as competitive inhibitors of l(-)malic acid transport and induced proton movements that followed Michaelis-Menten kinetics. Experiments with glucose-repressed cells showed that undissociated dicarboxylic acid (measured with labelled succinic acid) entered the cells slowly by simple diffusion. The permeability of the cells for undissociated acid increased exponentially with pH, the diffusion constant increasing 100-fold between pH 3.5 and 6.0.     

Intracellular free [Ca2+] in human skeletal muscle with myopathic carnitine deficiency

Carnitine is required for the transport of activated long chain fatty acids through the mitochondrial inner membrane. We measured the intracellular free calcium concentration [( Ca2+]i) by means of a calcium selective microelectrode in skeletal muscle biopsies obtained from nine patients in which myopathic carnitine deficiency (MCD) was diagnosed, and from six subjects with no evidence of neuromuscular disease. Intact intercostal muscle bundles were dissected and then split for electron microscopic studies and electrophysiological measurements. The [Ca2+]i in muscle fibers from MCD patients was 0.46 +/- 0.02 mumol.l-1 (mean +/- SEM) and 0.10 +/- 0.01 mumol.l-1 in control subjects. At the electron microscopic level, the predominant abnormality was the presence of lipid vacuoles between the myofibrils. These results show that in patients with myopathic carnitine deficiency there is a significant increase in the resting myoplasmic calcium concentration which might be related to a malfunction of some mechanisms responsible for the homeostasis of intracellular calcium.     

Regulation of reduced nitrogen assimilation in Rhodobacter capsulatus E1F1

The phototrophic bacterium Rhodobacter capsulatus E1F1 assimilates ammonia and other forms of reduced nitrogen either through the GS/GOGAT pathway or by the concerted action of l-alanine dehydrogenase and aminotransferases. These routes are light-independent and very responsive to the carbon and nitrogen sources used for cell growth. GS was most active in cells grown on nitrate or l-glutamate as nitrogen sources, whereas it was heavily adenylylated and siginificantly repressed by ammonium, glycine, l-alanine, l-aspartate, l-asparagine and l-glutamine, under which conditions specific aminotransferases were induced. GOGAT activity was kept at constitutive levels in cells grown on l-amino acids as nitrogen sources except on l-glutamine where it was significantly induced during the early phase of growth. In vitro, GOGAT activity was strongly inhibited by l-tyrosine and NADPH. In cells using l-asparagine or l-aspartate as nitrogen source, a concerted induction of l-aspartate aminotransferase and l-asparaginase was observed. Enzyme level enhancements in response to nitrogen source variation involved de novo protein synthesis and strongly correlated with the cell growth phase.Abbreviations ADH l-alanine dehydrogenase - AOAT l-alanine:2-oxoglutarate aminotransferase - Asnase l-asparaginase - GOAT Glycine: oxaloacetate aminotransferase - GOGAT Glutamate synthase - GOT l-aspartate: 2-oxoglutarate aminotransferase - GS Glutamine synthetase - HPLC High-Pressure Liquid Chromatography - MOPS 2-(N-morpholino)propanesulfonic acid - MSX l-methionine-d,l-sulfoximine     

Sequence divergence and open regions of RNA secondary structures in the envelope regions of the 17 human immunodeficiency virus isolates.

Genetic variation during the course of infection of an individual is a remarkable feature of the acquired immune deficiency syndrome (AIDS) disease. This variation has been studied for the envelope protein encoding regions of seventeen different sequences from various isolates of human immunodeficiency virus (HIV) using multiple sequence comparison and calculation of variability. The open regions with little intramolecular base pairing in these envelope sequences are predicted by a recently developed statistical method. The minimum length L for a run of hypervariable sites, conserved sites, or open regions that gives significance at the 1% (or 0.1%) level is then determined by a scan statistical method. The results show that significant clusters of open regions predicted at the RNA levels correlate with significant clusters of hypervariable sites in the HIV envelope gene. Those significant genomic variations in HIVs seem to be manifested mainly in the extracellular portion of the envelope protein. Twelve potential antigenic determinants are predicted using an antigenic index method. Interestingly, the majority of the significant hypervariable regions in the exterior envelope protein (gp120) were predicted potential epitopes.     

Purification and properties of the DNA-binding protein HPB12 from Bacillus subtilis nucleoid

We report the purification to homogeneity of a 12 KDa protein (HPB12) present in the nucleoids of Bacillus subtilis. From the purification data the abundance of the protein was estimated to about 20,000 monomers per cell. The HPB12 protein is heat-stable and acid-soluble and binds preferentially to supercoiled and linearized double-stranded DNAs. The binding of the protein to the supercoiled DNA occurs very rapidly and appears to be cooperative. Moreover, the complexes are extremely stable and do not dissociate after 90 min. These properties are consistent with a role of the HPB12 protein in the structure of the B. subtilis chromosome.     

Permeability of human granulocytes to dimethyl sulfoxide

The permeability of the membrane of human granulocytes to the permeating solute dimethyl sulfoxide (DMSO) was studied using the Onsager form of the phenomenological equations derived from the theory of irreversible thermodynamics. Changes in cellular volume were monitored with an electronic particle counter as samples of that population were introduced into hypertonic osmotica. Temperature and concentration sensitivity analyses of the permeability coefficients were carried out. It is shown that the introduction of the Onsager formalism allows further insight into the observed transport phenomena. It was found that DMSO may affect the water permeability properties of the membrane for that population of cells.     

Pathophysiological variations in the rat liver plasma membrane serine proteinase activity

The effects of fasting, diabetes, cholestasis, two-third hepatectomy and adrenalectomy on the rat liver plasma membrane serine proteinase activity were studied. Our results show a significant decrease of the enzyme activity during fasting (-50%), during experimental diabetes (-50%), in regenerating liver after partial hepatectomy (-70%) and after extrahepatic cholestasis (-70%). No modifications are noted when the rats are bilaterally adrenalectomized. These findings suggest that the enzyme activity may be linked to the level of circulating insulin, and may be regulated in physiological cellular proliferation so as to prevent undesirable protein degradation.     

Effects of acetylene on hydrogenases from the sulfate reducing and methanogenic bacteria

The effect of acetylene on the activity of the three types of hydrogenase from the anaerobic sulfate reducing bacteria has been investigated. The (Fe) hydrogenase is resistant to inhibition by acetylene while the nickel-containing hydrogenases are inhibited by acetylene with the (NiFe) hydrogenase being 10-50 fold more sensitive than the (NiFeSe) hydrogenase. In addition the Ni(III) EPR signal (g approximately 2.3) of the "as isolated" (NiFe) hydrogenase was significantly decreased in intensity upon exposure to acetylene.     

Prenatal diagnosis of familial amyloidotic polyneuropathy: evidence for an early expression of the associated transthyretin methionine 30

Summary Transthyretin methionine 30 (TTR Met 30), which is associated with familial amyloidotic polyneuropathy, originates in a single base substitution (A for G) in the second exon of the TTR gene. This autosomal dominant disease can be diagnosed by RFLP analysis of NsiI-digested DNA. The amplification of DNA by PCR improves the diagnosis method, making it suitable for prenatal diagnosis. Using PCR-amplified DNA, prenatal diagnosis of two at-risk fetuses was performed. Control Met 30 and normal DNA (either genomic or produced by site directed mutagenesis) were processed in parallel. The diagnosis was made by hybridization with allele-specific oligonucleotide probes, and later confirmed by screening of the mutant protein in the amniotic fluid and, when possible, in the sera from the newborns. TTR Met 30 was detected in the amniotic fluid of a positive fetus whose father was the carrier of the mutation. This indicates that the mutant protein is expressed very early in development.