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The effect of somatostatin on small intestinal transport in intractable diarrhoea of infancy.

It has been reported that somatostatin may be an effective antisecretory agent in a range of conditions causing severe secretory diarrhoea. In many children, intractable diarrhoeal illnesses result in significant morbidity and mortality. In a group of seven children with secretory diarrhoea, the effect of i.v. infusion of somatostatin (3.5 micrograms/kg stratum plus 3.5 micrograms/kg/h) on the net mucosal flux of salt and water was assessed using an in vivo steady-state perfusion technique. In one of the seven children who had evidence of deranged mucosal secretion and preserved villus function, somatostatin infusion resulted in a moderate reduction in secretion. In the remaining six, it had little or no beneficial effect. Somatostatin did not alter the rate of glucose absorption.

Child↗

Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without stunting growth.

Trehalose plays an important role in stress tolerance in plants. Trehalose-producing, transgenic rice (Oryza sativa) plants were generated by the introduction of a gene encoding a bifunctional fusion (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of Escherichia coli, under the control of the maize (Zea mays) ubiquitin promoter (Ubi1). The high catalytic efficiency (Seo et al., 2000) of the fusion enzyme and the single-gene engineering strategy make this an attractive candidate for high-level production of trehalose; it has the added advantage of reducing the accumulation of potentially deleterious T-6-P. The trehalose levels in leaf and seed extracts from Ubi1::TPSP plants were increased up to 1.076 mg g fresh weight(-1). This level was 200-fold higher than that of transgenic tobacco (Nicotiana tabacum) plants transformed independently with either TPS or TPP expression cassettes. The carbohydrate profiles were significantly altered in the seeds, but not in the leaves, of Ubi1::TPSP plants. It has been reported that transgenic plants with E. coli TPS and/or TPP were severely stunted and root morphology was altered. Interestingly, our Ubi1::TPSP plants showed no growth inhibition or visible phenotypic alterations despite the high-level production of trehalose. Moreover, trehalose accumulation in Ubi1::TPSP plants resulted in increased tolerance to drought, salt, and cold, as shown by chlorophyll fluorescence and growth inhibition analyses. Thus, our results suggest that trehalose acts as a global protectant against abiotic stress, and that rice is more tolerant to trehalose synthesis than dicots.

Adaptation, Physiological↗

PKA and Sch9 control a molecular switch important for the proper adaptation to nutrient availability.

In the yeast Saccharomyces cerevisiae, PKA and Sch9 exert similar physiological roles in response to nutrient availability. However, their functional redundancy complicates to distinguish properly the target genes for both kinases. In this article, we analysed different phenotypic read-outs. The data unequivocally showed that both kinases act through separate signalling cascades. In addition, genome-wide expression analysis under conditions and with strains in which either PKA and/or Sch9 signalling was specifically affected, demonstrated that both kinases synergistically or oppositely regulate given gene targets. Unlike PKA, which negatively regulates stress-responsive element (STRE)- and post-diauxic shift (PDS)-driven gene expression, Sch9 appears to exert additional positive control on the Rim15-effector Gis1 to regulate PDS-driven gene expression. The data presented are consistent with a cyclic AMP (cAMP)-gating phenomenon recognized in higher eukaryotes consisting of a main gatekeeper, the protein kinase PKA, switching on or off the activities and signals transmitted through primary pathways such as, in case of yeast, the Sch9-controlled signalling route. This mechanism allows fine-tuning various nutritional responses in yeast cells, allowing them to adapt metabolism and growth appropriately.

Adaptation, Physiological↗

An unusual pattern of invertase activity development in the thermophilic fungus Thermomyces lanuginosus.

In the thermophilic fungus Thermomyces lanuginosus, invertase displays an unusual pattern of development: the induced activity begins to diminish even before any substantial quantity of sucrose has been utilized or an appreciable amount of biomass has been produced. Despite this pattern of invertase activity, neither the growth rate nor the final mycelial yield is affected adversely. T. lanuginosus invertase is a thiol protein and the enzyme is active when specific sulfhydryl group(s) is in the reduced state. Measurements of reduced coenzyme and glutathione pools in sucrose-growth mycelia excluded oxidative stress as the primary reason for the observed decline in invertase activity. Rather, this unusual pattern of invertase is considered to be due to its localization in the hyphal tips. At the early stage of growth, the number of hyphal tips per unit mass of mycelium is maximum, whereas at later times their numbers do not increase in proportion to the biomass. As a result invertase activity shows an apparent inverse relationship with biomass. The enzyme activity disappears when the inducing carbon source is consumed and growth is completed.

Animals↗

Evidence for the interplay between trehalose metabolism and Hsp104 in yeast.

Disruption of the HSP104 gene in a mutant which cannot accumulate trehalose during heat shock treatment caused trehalose accumulation (H. Iwahashi, K. Obuchi, S. Fujii, and Y. Komatsu, Lett. Appl. Microbiol 25:43-47, 1997). This implies that Hsp104 affects trehalose metabolism. Thus, we measured the activities of enzymes involved in trehalose metabolism. The activities of trehalose-synthesizing and -hydrolyzing enzymes are low in the HSP104 disruption mutant during heat shock. This data is correlated with intracellular trehalose and glucose levels observed in the HSP104 disruption mutant. These results suggest that during heat shock, Hsp104 contributes to the simultaneous increase in both accumulation and degradation of trehalose.

Fungal Proteins↗

Virus particles from conidia of Penicillium species.

Virus particles and their component double-stranded ribonucleic acid (dsRNA) have been isolated from conidia and mycelia of certain Penicillium species. The conidia and mycelia of P. stoloniferum NRRL 5267 contained 75 and 85 mug of dsRNA/g (dry weight), respectively. Of the total dsRNA released from NRRL 5267 conidia, 10% was nonencapsulated. Conidia of P. brevi-compactum NRRL 5260 and P. chrysogenum Q-176 contained 2 and 120 mug of dsRNA/g (dry weight), respectively, whereas mycelium from the two species contained 3 and 95 mug of dsRNA/g (dry weight), respectively. No viruses were isolated from conidia or mycelia of P. stoloniferum NRRL 859. A method is described for disruption of both conidia and mycelia. The technique facilitates the isolation and characterization of fungal viruses and their component dsRNA and also potentiates surveying of fungal isolates for the presence of virus.

Electrophoresis, Polyacrylamide Gel↗

Mutation in Neurospora crasa affecting some of the extracellular enzymes and several growth characteristics.

A mutant of Neurospora crassa, strain T9, which shows reduced activity of glucoamylase, was isolated. The mutant glucoamylase was not altered in pH optimum, or thermostability, but altered in elution profile of Bio-Gel filtration. The T9 mutation led to other distinct phenotypic effects: a poor growth response, resistance to the effect of sorbose, a high activity of extracellular acid phosphatase, and an increased sensitivity to high osmolarity of the growth medium. The discussion was based on the idea that the T9 mutation occurred at a gene governing the synthesis of cell wall precursors and resulted in an alteration in various characteristics related to extracellular enzymes and growth.

Acid Phosphatase↗

Differential amylosaccharide metabolism of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum.

Clostridium thermosulfurogenes displayed faster growth on either glucose, maltose, or starch than Clostridium thermohydrosulfuricum. Both species grew faster on glucose than on starch or maltose. The fermentation end product ratios were altered based on higher ethanol and lactate yields on starch than on glucose. In C. thermohydrosulfuricum, glucoamylase, pullulanase, and maltase were mainly responsible for conversion of starch and maltose into glucose, which was accumulated by a putative glucose permease. In C. thermosulfurogenes, beta-amylase was primarily responsible for degradation of starch to maltose, which was accumulated by a putative maltose permease and then hydrolyzed by glucoamylase. Regardless of the growth substrate, the rates of glucose, maltose, and starch transformation were higher in C. thermosulfurogenes than in C. thermohydrosulfuricum. Both species had a functional Embden-Meyerhof glycolytic pathway and displayed the following catabolic activities: ferredoxin-linked pyruvate dehydrogenase, acetate kinase, NAD(P)-ethanol dehydrogenase, NAD(P)-ferredoxin oxidoreductase, hydrogenase, and fructose-1,6-diphosphate-activated lactate dehydrogenase. Ferredoxin-NAD reductase activity was higher in C. thermohydrosulfuricum than NADH-ferredoxin oxidase activity, but the former activity was not detectable in C. thermosulfurogenes. Both NAD- and NADP-linked ethanol dehydrogenases were unidirectional in C. thermosulfurogenes but reversible in C. thermohydrosulfuricum. The ratio of hydrogen-producing hydrogenase to hydrogen-consuming hydrogenase was higher in C. thermosulfurogenes. Two biochemical models are proposed to explain the differential saccharide metabolism on the basis of species enzyme differences in relation to specific growth substrates.

Clostridium↗

Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli.

It has been shown previously that Escherichia coli accumulates endogenously synthesized trehalose under osmotic stress. We report here that E. coli contained an osmotically regulated trehalose-phosphate synthase which utilized UDP-glucose and glucose 6-phosphate as substrates. In the wild type, the synthase was induced by growth in glucose-mineral medium of elevated osmotic strength and the synthase itself was strongly stimulated by K+ and other monovalent cations. A laboratory strain which expressed the synthase at a high constitutive level was found. GalU mutants, defective in synthesis of UDP-glucose, did not accumulate trehalose. Two genes governing the synthase were identified and named otsA and otsB (osmoregulatory trehalose synthesis). They mapped near 42 min in the flbB-uvrC region. Mutants with an otsA-lacZ or otsB-lacZ operon fusion displayed osmotically inducible beta-galactosidase activity; i.e., the activity was increased fivefold by growth in medium of elevated osmotic strength. Mutants unable to synthesize trehalose (galU, otsA, and otsB) were osmotically sensitive in glucose-mineral medium. But an osmotically tolerant phenotype was restored in the presence of glycine betaine, which also partially repressed the synthesis of synthase in the wild type and of beta-galactosidase in ots-lacZ fusion mutants.

Cloning, Molecular↗

Trehalose transport and metabolism in Escherichia coli.

Trehalose metabolism in Escherichia coli is complicated by the fact that cells grown at high osmolarity synthesize internal trehalose as an osmoprotectant, independent of the carbon source, although trehalose can serve as a carbon source at both high and low osmolarity. The elucidation of the pathway of trehalose metabolism was facilitated by the isolation of mutants defective in the genes encoding transport proteins and degradative enzymes. The analysis of the phenotypes of these mutants and of the reactions catalyzed by the enzymes in vitro allowed the formulation of the degradative pathway at low osmolarity. Thus, trehalose utilization begins with phosphotransferase (IITre/IIIGlc)-mediated uptake delivering trehalose-6-phosphate to the cytoplasm. It continues with hydrolysis to trehalose and proceeds by splitting trehalose, releasing one glucose residue with the simultaneous transfer of the other to a polysaccharide acceptor. The enzyme catalyzing this reaction was named amylotrehalase. Amylotrehalase and EIITre were induced by trehalose in the medium but not at high osmolarity. treC and treB encoding these two enzymes mapped at 96.5 min on the E. coli linkage map but were not located in the same operon. Use of a mutation in trehalose-6-phosphate phosphatase allowed demonstration of the phosphoenolpyruvate- and IITre-dependent in vitro phosphorylation of trehalose. The phenotype of this mutant indicated that trehalose-6-phosphate is the effective in vivo inducer of the system.

Biological Transport↗

Identification and characterization of a novel yeast gene: the YGP1 gene product is a highly glycosylated secreted protein that is synthesized in response to nutrient limitation.

Nutrient starvation in the yeast Saccharomyces cerevisiae leads to a number of physiological changes that accompany entry into stationary phase. The expression of genes whose products play a role in stress adaptation is regulated in a manner that allows the cell to sense and respond to changing environmental conditions. We have identified a novel yeast gene, YGP1, that displays homology to the sporulation-specific SPS100 gene. The expression of YGP1 is regulated by nutrient availability. The gene is expressed at a basal level during "respiro-fermentative" (logarithmic) growth. When the glucose concentration in the medium falls below 1%, the YGP1 gene is derepressed and the gene product, gp37, is synthesized at levels up to 50-fold above the basal level. The glucose-sensing mechanism is independent of the SNF1 pathway and does not operate when cells are directly shifted to a low glucose concentration. The expression of YGP1 also responds to the depletion of nitrogen and phosphate, indicating a general response to nutrient deprivation. These results suggest that the YGP1 gene product may be involved in cellular adaptations prior to stationary phase and may be a useful marker protein for monitoring early events associated with the stress response.

Alkaline Phosphatase↗

Particulate fraction in amniotic fluid at second trimester.

Amniotic fluid at 15-20 weeks contains high concentrations of microvillar enzymes which serve as markers for the prenatal diagnosis of cystic fibrosis. We found that these enzymes were mostly bound to amniotic fluid particulate fraction which eluted in the void volume on Sepharose 6B chromatography and precipitated in ultracentrifugation at 100,000g. Electron microscopy showed that the particulate fraction comprised membrane fragments and small vesicles. It represented less than 1% of the total protein in cell free amniotic fluid. Sorbitol gradient centrifugation showed heterogeneity in the particulate fraction. The enzyme patterns of amniotic fluid and meconium particulate fractions resembled each other. Antiserum against particulate fraction in amniotic fluid strongly reacted with early meconium and fetal small intestine in immunodiffusion, enzyme immunoassay, and staining with immunoperoxidase. The results suggest that most of the enzyme rich particulate fraction in early amniotic fluid originates in the fetal intestine.

Alkaline Phosphatase↗

Production of beta-glucosidase and diauxic usage of sugar mixtures by Candida molischiana.

The fermentation of cellobiose is a rare trait among yeasts. Of the 308 yeast species that utilize cellobiose aerobically, only 12 species ferment it, and only 2 species, Candida molischiana and Candida wickerhamii, also ferment cellodextrins. Candida molischiana produced beta-glucosidase activity on all carbon sources tested, except glucose, mannose, and fructose. When these sugars were added to cultures growing on cellobiose, the synthesis of beta-glucosidase ceased. However, the total amount of enzyme activity remained constant, indicating that the C. molischiana beta-glucosidase is catabolite repressed and not catabolite inactivated. When grown in medium initially containing glucose plus xylose, cellobiose, maltose, mannitol, or glucitol, C. molischiana preferentially utilized glucose and produced little beta-glucosidase activity until glucose was nearly depleted from the medium. When grown in medium containing cellobiose plus either fructose or mannose, the yeast preferentially utilized the monosaccharides and produced little beta-glucosidase activity. Candida molischiana produced beta-glucosidase and co-utilized cellobiose and xylose, maltose, or trehalose. Glucose and fructose, mannose, or trehalose were co-utilized; however, no beta-glucosidase activity was detected. Thus, the order of substrate preference groups appeared to be (glucose, trehalose, fructose, mannose) > (cellobiose, maltose, xylose) > (mannitol, glucitol).

Candida↗

The use of a monoclonal antibody for the rapid purification of kidney neutral endopeptidase ("enkephalinase") solubilized in octyl glucoside.

The neutral endopeptidase ("enkephalinase") of the rabbit brush border membrane has been purified to homogeneity by a rapid immunoaffinity method using a monoclonal antibody. In contrast with other methods used so far, a complete extraction of enkephalinase from the brush border membrane can be achieved with octyl glucoside, without loss of activity. The solubilized enzyme can be selectively separated from the other proteins in a single step using an immunoaffinity column consisting of the monoclonal antibody covalently linked to Sepharose CL-4B. It is demonstrated that enkephalinase can then be recovered in an active form by elution at low pH. The purified enzyme obtained by this method is completely inhibited by thiorphan and appears as a single 94,000 dalton protein after polyacrylamide gel electrophoresis under denaturing and reducing conditions.

Animals↗