Purification of phosphate-dependent glutaminase from isolated mitochondria of Ehrlich ascites-tumour cells.

Phosphate-dependent glutaminase was purified to homogeneity from isolated mitochondria of Ehrlich ascites-tumour cells. The enzyme had an Mr of 135,000 as judged by chromatography on Sephacryl S-300. SDS/polyacrylamide-gel electrophoresis displayed two protein bands, with Mr values of 64,000 and 56,000. Two major immunoreactive peptides of Mr values of 65,000 and 57,000 were found by immunoblot analysis using anti-(rat kidney glutaminase) antibodies. The concentration-dependences for both glutamine and phosphate were sigmoidal, with S0.5 values of 7.6 mM and 48 mM, and Hill coefficients of 1.5 and 1.6, respectively. The glutaminase pH optimum was 9. The activation energy of the enzymic reaction was 58 kJ/mol. The enzyme showed a high specificity towards glutamine. A possible explanation for the different kinetic behaviour found for purified enzyme and for isolated mitochondria [Kovacević (1974) Cancer Res. 34, 3403-3407] should be that a conformational change occurs when the enzyme is extracted from the mitochondrial inner membrane.     

Metabolic adaptation of the renal carbohydrate metabolism. I. Effects of starvation on the gluconeogenic and glycolytic fluxes in the proximal and distal renal tubules

Summary The influence of starvation on renal carbohydrate metabolism was studied in the proximal and distal fragments of the nephron. Starvation induced a double and opposite adaptation mechanism in both fractions of the renal tubule. In renal proximal tubules, the gluconeogenic flux was stimulated progressively during a period of 48 hours of starvation (2.15 fold), due, in part, to a significant increase in the fructose 1,6-bisphosphatase and phosphoenolpyruvate carboxykinase activities although with different characteristics. Fructose 1,6-bisphosphatase activity from this tubular fragment increased only at subsaturating subtrate concentration (68%) which involved a significant decrease in the Km (35%) for fructose 1,6-bisphosphate while there was no change in Vmax. This behaviour clearly indicates that it is related to modifications in the activity of the preexistent enzyme in the cell. Proximal phosphoenolpyruvate carboxykinase activity increased proportionally at both substrate concentrations (86 and 89% respectively) which brought about changes in Vmax without changes in Kin, all of which are in accordance with variations in the cellular levels of the enzyme. In the renal distal tubules, the glycolytic capacity drastically decreased throughout the starvation time. At 48 hours 65% of inhibition was shown. We have found a short term regulation of phosphofructokinase activity by starvation which involves an increase in Km (2.2 fold) without changes in Vmax, as a result of these kinetic changes, an inactivation of phosphofructokinase was detected at subsaturating concentration of fructose 6-phosphate. On the contrary, this nutritional state did not modify the kinetic behaviour of renal pyruvate kinase. Finally, neither proximal glycolytic nor distal gluconeogenic capacities and related enzymes activities were changed during starvation.     

Degradation of diarylethane structures by Pseudomonas fluorescens biovar I

Pseudomonas fluorescens biovar I was isolated from a pulp mill effluent based on its ability to grow on synthetic media containing 1,2-diarylethane structures as the sole carbon and energy source. Analysis of samples taken from cultures of this strain in benzoin or 4,4-dimethoxybenzoin (anisoin), showed that cleavage between the two aliphatic carbons takes place prior to ring fission. Intermonomeric cleavage was also obtained with crude extracts. Substrates of this reaction were only those 1,2-diarylethane compounds that supported growth of the bacterium. The purification and partial characterization of an enzyme that catalyzes the NADH-dependent reduction of the carbonyl group of benzoin and anisoin is also reported.     

Regional brain GABA metabolism and release during hepatic coma produced in rats chronically treated with carbon tetrachloride

Hepatic coma was induced in rats chronically treated with CCl₄, by means of a single injection of ammonium acetate. The activities of glutamate decarboxylase (GAD) and GABA transaminase (GABA-T), as well as the synaptosomal uptake and release of [³H]GABA, were measured in the following brain areas of the comatose rats: cortex, striatum, hypothalamus, hippocampus, midbrain and cerebellum. Hepatic coma was associated with a general decrease of GAD activity, whereas GABA-T activity was diminished only in the hypothalamus, striatum and midbrain. During hepatic coma, the K⁺-stimulated [³H]GABA release was notably diminished in the striatum and cerebellum, whereas a significant increase was observed in the hippocampus. [³H]GABA uptake increased in most regions after CCl₄ treatment, independently of the presence of coma. The results indicate that GABAergic transmission seems to be decreased in most cerebral regions during hepatic coma.     

Intestinal parasites in the Camiri, Gutierrez and Boyuibe areas, Santa Cruz Department, Bolivia

A parasitological study was carried out on 381 apparently healthy subjects from Camiri, Boyuibe, Gutierrez. Intestinal parasites and non-pathogenic protozoa were present in 78.7% of the population sampled; multiple infections were observed in 67.7% of the parasitized individuals. The protozoon most commonly found was Entamoeba coli (in 40.7% of specimens), followed by Giardia intestinalis (30.7%), Iodamoeba bütschlii (10%), Chilomastix mesnili (8.7%). Other protozoon parasites also present were Enteromonas hominis (3.4%), Retortamonas intestinalis (2.4%), Cryptosporidium (2.1%), Endolimax nana (2.1%), Balantidium coli (1.8%) and Pentatrichomonas hominis (0.8%). The helminths observed were hookworms (28.6%), Trichuris trichiura (19.7%), Ascaris lumbricoides (9.7%), Hymenolepis nana (8.7%), Trichostrongylus (5.5%), Strongyloides stercoralis (1.8%), Taenia (5 cases) and Enterobius (6 cases). Prevalence for nematodes is probably underestimated in the 3-9 years age group because of a mebendazole treatment given 5 weeks before the survey, under a Program of P D C of the Ministry of Health. The sample from Camiri was found to be the most parasitized (84.1%). An extraordinarily high infection rate was found in two urban institutions, as well as in Itanambicua, a rural community close to Camiri. No significant differences were observed in parasitic prevalence between rural and urban environments. Exposure to contamination with human and animal faeces, overcrowding and poor sanitation habits are some of the factors responsible for the parasitic situation evidenced.     

Epidemiology of tegumentary leishmaniasis in Mérida, Venezuela. I. Diversity and dispersion of phlebotomine species at 3 altitude levels and its possible role in the transmission of the disease

As part of an epidemiological study on leishmaniasis in Merida, Venezuela, the diversity and dispersion of sandflies species found in 15 localities between 175 m and 1,960 m.a.s.l., are presented. From 7,126 collected sandflies (5,132 female and 1,994 male), 24 species were identified, 10 of them recognized as anthropophilic. The relation species-altitude is presented, and the species composition found in human dwellings, periodomestic and sylvatic areas, are recorded. The possible role of the identified species on the transmission of leishmaniasis in the andean region, is discussed.     

Molecular analysis of HLA-A2.4 functional variant KLO: close structural and evolutionary relatedness to the HLA-A2.2 subtype

The structure of an HLA-A2.4 functional variant (A2.4c) expressed on donor KLO has been examined by comparative peptide mapping with other HLA-A2 antigens of known structure and radiochemical sequencing. All the peptide differences between A2.4c and A2.1 could be accounted for by five amino acid changes at positions 9, 43, 66, 95, and 156. The nature of residues 9, 43, and 95 in A2.4c was determined by sequencing to be identical to those in A2.2Y. The nature of residue 156 in A2.4c was also assigned as identical to that in A2.2Y on the basis of the identity of the corresponding peptide in its chromatographic comparison with A2.2Y. Position 66 was unique to A2.4c. It was determined to be an Asn residue instead of the Lys present in all other HLA-A2 antigens of known structure. This was the only detected amino acid difference between A2.4c and A2.2Y. The results indicate that, from a structural point of view, A2.4c is most closely related to the A2.2 subtype antigens and not to other A2.4 antigens. The data are compatible with the assumption that A2.4c was derived from A2.2Y by a single point mutation event.     

A quantitative study of enterotoxin production by sheep milk staphylococci

Of 124 staphylococcal strains isolated from sheep milk, 78 produced enterotoxin A, B, C, or D when evaluated by an enzyme-linked immunosorbent assay. Enterotoxins A and D, elaborated by 44 and 43 strains, respectively, showed the highest incidence. Enterotoxin production by coagulase-negative strains (one Staphylococcus cohnii, three S. epidermidis, five S. haemolyticus, and four S. xylosus) was detected. Linear and logarithmic-logarithmic regressions of optical density on enterotoxin concentration yielded the best-fitting equations for enterotoxin quantitation. A significantly higher incidence of enterotoxin producers and significantly higher levels of enterotoxins produced were recorded for coagulase-positive, thermostable nuclease-positive, hemolysis-positive, or mannitol-positive strains. Mannitol utilization was the best test for discriminating between enterotoxigenic and nonenterotoxigenic staphylococci.     

An HLA-A2 population variant with structural polymorphism in the α3 region

The HLA-A2 antigen expressed by donor OZB can be distinguished from the main HLA-A2.1 subtype by isoelectric focusing - it is one charge unit more acidic — and by some alloreactive T-cell clones but not by cytolytic T lymphocyte lines. The structure of variant OZB has been examined by comparative peptide mapping with A2.1 and radiochemical sequence analysis. The two molecules were found to differ in a single tryptic peptide from the 0 region, spanning residues 220–243. The amino acid sequence of this peptide from variant OZB revealed that there was only one amino acid change of Glu instead of Ala at position 236, a hitherto invariant residue in class I HLA antigens. All previously characterized HLA or H-2 natural variants have structural changes restricted to the 1 and/or 2 domains. Thus, variant OZB is unique in that (1) it has one amino acid change in 3 and (2) it has no changes in l and 2. The only detected substitution of this variant may be accounted for by a single base change at the DNA level, suggesting that it might have resulted from a point mutation in the A2.1 gene. The structural features of variant OZB open a novel way to examine the influence of polymorphism in 3 on cytolytic T-cell recognition of naturally occurring class I antigens.Abbreviations CTL cytolytic T lymphocytes - HPLC high performance liquid chromatography - IEF isoelectric focusing - MHC major histocompatibility complex