Identification of Enzymes and Quantification of Metabolic Fluxes in the Wild Type and in a Recombinant Aspergillus oryzae Strain

Two α-amylase-producing strains of Aspergillus oryzae, a wild-type strain and a recombinant containing additional copies of the α-amylase gene, were characterized with respect to enzyme activities, localization of enzymes to the mitochondria or cytosol, macromolecular composition, and metabolic fluxes through the central metabolism during glucose-limited chemostat cultivations. Citrate synthase and isocitrate dehydrogenase (NAD) activities were found only in the mitochondria, glucose-6-phosphate dehydrogenase and glutamate dehydrogenase (NADP) activities were found only in the cytosol, and isocitrate dehydrogenase (NADP), glutamate oxaloacetate transaminase, malate dehydrogenase, and glutamate dehydrogenase (NAD) activities were found in both the mitochondria and the cytosol. The measured biomass components and ash could account for 95% (wt/wt) of the biomass. The protein and RNA contents increased linearly with increasing specific growth rate, but the carbohydrate and chitin contents decreased. A metabolic model consisting of 69 fluxes and 59 intracellular metabolites was used to calculate the metabolic fluxes through the central metabolism at several specific growth rates, with ammonia or nitrate as the nitrogen source. The flux through the pentose phosphate pathway increased with increasing specific growth rate. The fluxes through the pentose phosphate pathway were 15 to 26% higher for the recombinant strain than for the wild-type strain.     

Expression of the Escherichia coli pntA and pntB Genes, Encoding Nicotinamide Nucleotide Transhydrogenase, in Saccharomyces cerevisiae and Its Effect on Product Formation during Anaerobic Glucose Fermentation

We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinant Saccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD⁺ in S. cerevisiae and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD⁺ by NADPH and by NADH in the presence of NADP⁺, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD⁺, NADPH, and NADP⁺ were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD⁺.     

Inhibition of human gastric lipase by intraduodenal fat involves glucagon-like peptide-1 and cholecystokinin

Seven healthy volunteers were intubated with two double lumen nasogastric tubes, one in the stomach, the other in the duodenum. This system allows simultaneous sampling of gastric juice and separate intraduodenal perfusion with a dietary fat (fish oil, 1269 kJ). Gastrin-17 was infused i.v. at a rate of 40 pmol/kg/h throughout the study. Gastric lipase was measured at 15-min intervals as activity (tributyrin) and as immunoreactivity (ELISA). Infusion of gastrin-17 resulted in a stable increase in the plasma concentration from a basal concentration of 8.3±0.8 pmol/l to 41.4±4.2 pmol/l. Perfusion with fat reduced gastric lipase activity from 24.2±5.3 to 7.2±2.5 kU/l (P<0.05), and immunoreactivity from 0.7±0.1 to 0.42±0.1 mg/l (P<0.05). After termination of fat perfusion, gastric lipase secretion increased again, though not reaching preinhibitory concentrations. During the intraduodenal perfusion with fat the plasma concentrations of glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK) increased from 6.9±0.5 to 15.1±1.5 pmol/l (P<0.05) and from 1.2±0.4 to 3.8±0.9 pmol/l (P<0.05). This study reveals a negative effect of fat in the duodenum on gastric lipase secretion. This effect may be mediated by GLP-1 and/or CCK.     

Antisense Silencing of the creA Gene in Aspergillus nidulans

Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in Aspergillus nidulans resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect was approximately one-half of that achieved in a null creA mutant. Unlike results for that mutant, however, growth parameters and colony morphology in the antisense transformants were not affected.     

Identification of Transglutaminase Reactive Residues in Human Osteopontin and Their Role in Polymerization

Osteopontin (OPN) is a highly posttranslationally modified protein present in several tissues where it is implicated in numerous physiological processes. OPN primarily exerts its functions through interaction with integrins via the Arg-Gly-Asp and Ser-Val-Val-Tyr-Gly-Leu-Arg sequences located in the N-terminal part of the protein. OPN can be polymerized by the cross-linking enzyme transglutaminase 2 (TG2), and polymerization has been shown to enhance the biological activity of OPN. However, little is known about the reactivity and location of the glutamine and lysine residues involved in the TG2-mediated modification of OPN. Here we show that TG2 catalyses the incorporation of 5-(Biotinamido)pentylamine at glutamines in both the N- and C-terminal parts of OPN, whereas TG2 primarily incorporated the glutamine-donor peptide biotinyl-TVQQEL-OH into the C-terminal part of OPN. By mass spectrometric analyses we identified Gln34, Gln42, Gln193 and Gln248 as the major TG2 reactive glutamines in OPN. The distribution of reactive Gln and Lys residues in OPN proved to be important, as the full-length protein but not the physiologically highly active integrin-binding N-terminal part of OPN were able to polymerize in a TG2-mediated reaction. Collectively, these data provide important new molecular knowledge about the mechanism of OPN polymerization.     

Re-Meandering of Lowland Streams: Will Disobeying the Laws of Geomorphology Have Ecological Consequences?

We evaluated the restoration of physical habitats and its influence on macroinvertebrate community structure in 18 Danish lowland streams comprising six restored streams, six streams with little physical alteration and six channelized streams. We hypothesized that physical habitats and macroinvertebrate communities of restored streams would resemble those of natural streams, while those of the channelized streams would differ from both restored and near-natural streams. Physical habitats were surveyed for substrate composition, depth, width and current velocity. Macroinvertebrates were sampled along 100 m reaches in each stream, in edge habitats and in riffle/run habitats located in the center of the stream. Restoration significantly altered the physical conditions and affected the interactions between stream habitat heterogeneity and macroinvertebrate diversity. The substrate in the restored streams was dominated by pebble, whereas the substrate in the channelized and natural streams was dominated by sand. In the natural streams a relationship was identified between slope and pebble/gravel coverage, indicating a coupling of energy and substrate characteristics. Such a relationship did not occur in the channelized or in the restored streams where placement of large amounts of pebble/gravel distorted the natural relationship. The analyses revealed, a direct link between substrate heterogeneity and macroinvertebrate diversity in the natural streams. A similar relationship was not found in either the channelized or the restored streams, which we attribute to a de-coupling of the natural relationship between benthic community diversity and physical habitat diversity. Our study results suggest that restoration schemes should aim at restoring the natural physical structural complexity in the streams and at the same time enhance the possibility of re-generating the natural geomorphological processes sustaining the habitats in streams and rivers. Documentation of restoration efforts should be intensified with continuous monitoring of geomorphological and ecological changes including surveys of reference river systems.     

Accessory Gland as a Site for Prothoracicotropic Hormone Controlled Ecdysone Synthesis in Adult Male Insects

Insect steroid hormones (ecdysteroids) are important for female reproduction in many insect species and are required for the initiation and coordination of vital developmental processes. Ecdysteroids are also important for adult male physiology and behavior, but their exact function and site of synthesis remains unclear, although previous studies suggest that the reproductive system may be their source. We have examined expression profiles of the ecdysteroidogenic Halloween genes, during development and in adults of the flour beetle Tribolium castaneum. Genes required for the biosynthesis of ecdysone (E), the precursor of the molting hormone 20-hydroxyecdysone (20E), are expressed in the tubular accessory glands (TAGs) of adult males. In contrast, expression of the gene encoding the enzyme mediating 20E synthesis was detected in the ovaries of females. Further, Spookiest (Spot), an enzyme presumably required for endowing tissues with competence to produce ecdysteroids, is male specific and predominantly expressed in the TAGs. We also show that prothoracicotropic hormone (PTTH), a regulator of E synthesis during larval development, regulates ecdysteroid levels in the adult stage in Drosophila melanogaster and the gene for its receptor Torso seems to be expressed specifically in the accessory glands of males. The composite results suggest strongly that the accessory glands of adult male insects are the main source of E, but not 20E. The finding of a possible male-specific source of E raises the possibility that E and 20E have sex-specific roles analogous to the vertebrate sex steroids, where males produce primarily testosterone, the precursor of estradiol. Furthermore this study provides the first evidence that PTTH regulates ecdysteroid synthesis in the adult stage and could explain the original finding that some adult insects are a rich source of PTTH.     

Modulation of sulfur metabolism enables efficient glucosinolate engineering

Background  

Metabolic engineering in heterologous organisms is an attractive approach to achieve efficient production of valuable natural products. Glucosinolates represent a good example of such compounds as they are thought to be the cancer-preventive agents in cruciferous plants. We have recently demonstrated that it is feasible to engineer benzylglucosinolate (BGLS) in the non-cruciferous plant Nicotiana benthamiana by transient expression of five genes from Arabidopsis thaliana. In the same study, we showed that co-expression of a sixth Arabidopsis gene, γ-glutamyl peptidase 1 (GGP1), resolved a metabolic bottleneck, thereby increasing BGLS accumulation. However, the accumulation did not reach the expected levels, leaving room for further optimization.