Regionally selective alterations in enzymatic activities and metabolic fluxes during thiamin deficiency

To further elucidate the molecular basis of the selective damage to various brain regions by thiamin deficiency, changes in enzymatic activities were compared to carbohydrate flux through various pathways from vulnerable (mammillary bodies and inferior colliculi) and nonvulnerable (cochlear nuclei) regions after 11 or 14 days of pyrithiamin-induced thiamin deficiency. After 11 days,large decreases (–43 to –59%) in transketolase (TK) occurred in all 3 regions; 2-ketoglutarate dehydrogenase (KGDHC) declined (–45%), but only in mammillary bodies; pyruvate dehydrogenase (PDHC) was unaffected. By day 14, TK remained reduced by 58%–66%; KGDHC was now reduced in all regions (–48 to –55%); PDHC was also reduced (–32%), but only in the mammillary bodies. Thus, the enzyme changes did not parallel the pathological vulnerability of these regions to thiamin deficiency.¹⁴CO₂ production from¹⁴C-glucose labeled in various positions was utilized to assess metabolic flux. After 14 days, CO₂ production in the vulnerable regions declined severely (–46 to 70%) and approximately twice as much as those in the cochlear nucleus. Also by day 14, the ratio of enzymatic activity to metabolic flux increased as much as 56% in the vulnerable regions, but decreased 18 to 30% in the cochlear nuclei. These differences reflect a greater decrease in flux than enzyme activities in the two vulnerable regions. Thus, selective cellular responses to thiamin deficiency can be demonstrated ex vivo, and these changes can be directly related to alterations in metabolic flux. Since they cannot be related to enzymatic alterations in the three regions, factors other than decreases in the activity of these TPP-dependent enzymes must underlie selective vulnerability in this model of thiamin deficiency.Abbreviations KGDHC 2-ketoglutarate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.61, EC 1.6.4.3. - PDHC pyruvate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.12, EC 1.6.4.3 - TK transketolase (EC 2.2.1.1) - TPP thiamin pyrophosphate     

Endothelium specific Weibel-Palade bodies in a continuous human cell line,EA.hy926

Summary Weibel-Palade bodies are ultrastructurally defined organelles found only in vascular endothelial cells. Because endothelium in corpo is very dispersed, isolation and further characterization of this organelle has been dependent on increasing the number of cells in culture. However, primary isolates of endothelial cells have a limited replication potential and tend to senesce in culture. In this report, EA.hy926, a continuously replicating cell line derived from human endothelium, is shown to contain Weibel-Palade bodies. Electron micrographs demonstrate the ultrastructural characteristics of these tissue-specific organelles and their cytoplasmic distribution in EA.hy926 cells. Von Willebrand factor, which has been shown to exist in Weibel Palade bodies, is demonstrated by immunofluorescence in discrete rod-shaped organelles whose size, shape, and distribution are consistent with that of Weibel-Palade bodies in primary endothelial cell cultures. Rapid release of von Willebrand factor can be induced by calcium ionophore, and large multimeric forms of the protein are found in EA.hy926 cells. These two properties are consistent with the function currently ascribed to Weibel Palade bodies: storage of multimerized von Willebrand factor. Thus ultrastructural, immunologic, and functional data establish the existence of this as yet poorly understood tissue-specific organelle in a continuous, vigorously replicating human cell line.     

Statistical analysis for the spatial validity of a model to forecast the daily number of forest fires

In earlier papers a qualitative and quantitative model was developed for predicting the number of forest fires occurring per day. This model permits the forecast at 00.00 hours Universal Time Convention (UTC) of any day (d), the number of forest fires per day for a range of several days (d tod+5) over a particular region. Input data are the number of forest fires in the region during two preceding days (d–2 andd–1) and the type of day (real and evaluated from radiosonde ford–2,d–1,d and predicted from meteorological medium-range forecasts, i.e. of European Centre, ford+1,d+2,d+3,d+4 andd+5. As this model requires data obtained by radiosonde, particularly temperatures and geopotentials at 850 and 700 hPa and dew points (or specific humidity) at 850 hPa, this study investigates the spatial validity of the model in relation to the distance from the radiosonde station (RS). The highest quality forecast is obtained for the region immediately surrounding the RS, and diminishes with increasing distance from it, this being due to the data obtained from the RS not being representative of the atmospheric column over the region. Hence, the derivation of the critical distance for a particular quality level of measurement. Conversely, fixed quality level implies a specific separation between RS and the region for the prediction, with a higher predictive quality implying a shorter distance.     

A spawning aggregation of Nassau grouperEpinephelus striatus (Pisces: Serranidae) in the Mexican Caribbean

Synopsis An annual spawning aggregation of Nassau grouper,Epinephelus striatus, observed off the southern coast of Quintana Roo, Mexico, on full moon days in December and January, was surveyed during the winters from 1991 to 1993. A fish aggregation had been appearing at one traditional site off Mahahual for more than 80 years, but currently it forms at undetermined sites far from the original site. Fish migration was northwards in the fore reef border along the 14 m isobath and isolated groups were observed. Through visual assessment fish aggregations were found to include 200 to 500 individuals. The fishing on Nassau grouper aggregations has been active since 1910 by using hook and line; however, recently fishermen used gill nets. It is necessary to continue the research on this aggregation and establish management guidelines to protect the species.     

Factors affecting transient gene expression in cultured radiata pine cotyledons following particle bombardment

Transfer and expression of the β–glucuronidase gene ( gusA ) in cultured cotyledons of radiata pine ( Pinus radiata D. Don ) were obtained by particle bombardment. Conditions for optimum transient expression were established by using plasmid pB[/12], delivered by gold particles, 1.6 μm in diameter, into 8-day-old cultured cotyledons. Helium pressure of 7.6 MPa, bombardment distance between the stopping screen and the target tissues of 6 cm, and 0.8 μg of plasmid DNA per bombardment proved to be the best parameters for transient expression; using these parameters 79% of bombarded cotyledons showed GUS activity, with 4.3 blue spots per cotyledon. This system was used for studying the expression of several gus-driven promoters the expression of the sunflower ubiquitin gene promoter was higher (99% of positive cotyledons, with 14.2 blue spots per cotyledon) than that of the CaMV 35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower gusA expression, as determined histochemically. These results were confirmed by using the gus fluorometric assay. Use of the sunflower ubiquitin gene promoter resulted in gusA expression up to 20 days after bombardment, with a significant level of gus expressing loci per bombarded cotyledon, whereas with the CaMV 35S promoter gusA expression was lost 12 days after bombardment.     

Cloning of the Canine GALC cDNA and Identification of the Mutation Causing Globoid Cell Leukodystrophy in West Highland White and Cairn Terriers

Globoid cell leukodystrophy, or Krabbe disease, is a severe, autosomal recessive disorder resulting from a deficiency of galactocerebrosidase (GALC) activity. GALC is responsible for the lysosomal catabolism of certain galactolipids, including galactosylceramide and psychosine. In addition to the human patients, there are several naturally occurring animal models for this disease, including the twitcher mouse, West Highland White terriers (WHWT), and Cairn terriers. All species have deficient GALC activity and have the characteristic pathological findings in the nervous system. We now describe the cloning of the canine GALC cDNA and the identification of the disease-causing mutation in both terrier breeds. The 2007-bp open reading frame is 88% identical to that in human, and the deduced amino acid sequence is about 90% identical. However, the 3′-untranslated region is about 1 kb shorter than that in the human. Two nucleotide changes were found in affected dogs, an A to C transversion at cDNA position 473 (Y158S) and a C to T transition at position 1915 (P639S). Expression studies in COS-1 cells demonstrated that the A to C change at 473 is the disease-causing mutation. A rapid test for the identification of the genotype at that position has been developed, and over 100 WHWT and Cairn terriers have been screened. This will allow breeders to mate their dogs selectively and will permit the establishment of a colony of dogs for use in therapy trials.     

Metabolic adaptation of renal carbohydrate metabolism. V.In vivo response of rat renal-tubule gluconeogenesis to different diuretics

We have studied the effects of the diuretics mersalyl, furosemide and ethacrynic acid on renal gluconeogenesis in isolated rat-kidney tubules and on the activities of the most important gluconeogenic and glycolytic enzymes in both fed and fasted rats. Mersalyl (15 mg.kg^–1 animal weight) significantly decreased the rate of gluconeogenesis in well-fed rats (68%) as well as in 24 and 48-h fasted ones (33 and 37% respectively). This inhibition occurred when lactate, pyruvate, glycerol or fructose were used as substrates. Ethacrynic acid at a dose of 50 mg.kg^–1 animal weight provoked a transient inhibition of renal glucose production by almost 20% but only in fed rats with lactate as substrate, whereas the same dose of furosemide did not affect this metabolic pathway.Parallel to these changes, mersalyl caused a significant inhibition in the maximum activity of the most important gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase and glucose 6-phosphatase, in both fed and fasted rats. Neither ethacrynic acid nor furosemide produced any variations in the activities of these enzymes. The activity of the glycolytic enzymes phosphofructokinase and pyruvate kinase was not modified by these diuretics. Nevertheless, the activity of the thiol-enzyme glyceraldehyde 3-phosphate dehydrogenase was severely inhibited by mersalyl and to a lesser extent by the other diuretics. This inhibition was higher in fasted than fed rats. Hence, we conclude that the inhibitory effect of mersalyl on renal gluconeogenesis is due, at least partly, to a decrease in the flux through the gluconeogenic enzymes. Blood glucose was not modified after diuretic treatment in fed animals whereas mersalyl decreased the levels of blood glucose in 24-h fasted rats. Thein vivo effects of diuretics on gluconeogenesis correlate well with the previously observedin vitro effects, although ethacrynic acid was less potent as an inhibitorin vivo, probably because of its rapid clearance.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis (-aminoethylether) N,N,N,N-tetraacetic acid - DTT dithiothreitol - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - TRIS 2-amino-2-hydroxymethyl-1,3-propanediol Publication No. 166 from Drogas, Tóxicos Ambientales y Metabolismo Celular Research Group, Department of Biochemistry and Molecular Biology, University of Granada, Granada, Spain     

Genetics and gibberellin production inGibberella fujikuroi

Gibberella fujikuroi (Fusarium moniliforme) is a complex group of plant pathogens. Some strains produce gibberellic acid and other gibberellins that promote growth and regulate various stages in plant development.The paper describes the research effort directed to development of genetic tools for this species. Furthermore the main features of the gibberellin biosynthetic pathway as established in Gibberella are described.Abbreviations AMO 1618 2-isopropyl-4-(trimethylammonium chloride)-5-methylphenylpiperidine-1-carboxylate - hydroxykaurenoic acid ent-kaur-16-en-7-ol-19-oic acid - kaurenal ent-kaur-16-en-19-al - kaurene ent-kaur-16-ene - kaurenoic acid ent-kaur-16-en-19-oic acid - kaurenol ent-kaur-16-en-19-ol - paclobutrazol 1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-lyl)pentan-3-ol - pefurazoate pent-4-enyl-N-furfuryl-N-imidazol-1-ylcarbonyl-DL-homoa laninate - tetcyclacis 5-(4-chlorophenyl)-3,4,5,9,10-pentaazatetracyclo-5,4,10^2.6,O^8.11-dodeca-3,9-diene - triarimol -(2,4-dichlorophenyl)--phenyl-5-pyrimidine methyl alcohol     

“Cryptic” dinucleotide polymorphism in the 3′ region of the factor IX gene shows substantial variation among different populations

Long tandem dinucleotide repeats composed of alternating purines and pyrimidines [RY(i)] are abundant and highly polymorphic. Simple RY(i) are predominately composed of one tandem repeat of a dinucleotide sequence. In contrast, cryptic RY(i [cRY(i)] are composed of multiple short dinucleotide repeats. Herein, we describe the racial distribution of alleles for a polymorphic cRY(i) in the factor IX gene. Allele I is absent in Asians, whereas allele III is rare or absent in Caucasians or blacks. A polymorphic cRY(i) analyzed previously shows even more dramatic variation among racial groups, hinting that a battery of cRY(i) might have utility in assessing the racial origin of a DNA sample.