The Influence of pH and Temperature on Cellulolytic and Pectolytic Activity of Cylindrocarpon destructans (Zins.) Scholt.

In studies on the effect of pH and temperature on cellulolytic and pectolytic activity of C. destructans, it was found that the isolates used produced only endoglucanases. The temperature and pH affected the synthesis of these enzymes. Fungi cultured at 26°C produced more of these enzymes than those grown at the two other temperatures. At 10°C, only one isolate produced minute amounts of endoglucanases. None of fungi studied exhibited cellulolytic activity in cultures grown at 20°C. Cellulolytic activity was found only in acidic media (pH 5.0). The fungi studied exhibited higher pectolytic than cellulolytic activity. In the post culture liquids of these organisms, both types of pectolytic enzymes (exo- and endo-PMG) were detected. Different temperature and pH values affected the production of these enzymes differently in various isolates.     

2-Bromoethyl glycosides in glycoside synthesis: preparation of glycoproteins containing alpha-L-Fuc-(1----2)-D-Gal and beta-D-Gal-(1----4)-D-GlcNAc

The applicability of 2-bromoethyl glycosides in carbohydrate synthesis is demonstrated by the synthesis of glycosides of alpha-L-Fuc-(1----2)-D-Gal and beta-D-Gal-(1----4)-D-GlcNAc. The bromoethyl aglycon was transformed into the methoxycarbonylethylthioethyl spacer, which allowed coupling of the sugars to proteins (BSA and KLH).     

(Beta alpha)8-barrel proteins of tryptophan biosynthesis in the hyperthermophile Thermotoga maritima.

To better understand the evolution of a key metabolic pathway, we have sequenced the trpCFBA gene cluster of the hyperthermophilic bacterium Thermotoga maritima. The genes were cloned by complementation in vivo of trp deletion strains of Escherichia coli. The new sequences, together with earlier findings, establish that the trp operon of T.maritima has the order trpE(G.D)CFBA, which might represent the ancestral organization of the tryptophan operon. Heterologous expression of the trp(G.D) and trpC genes in E.coli and N-terminal sequencing of their polypeptide products showed that their translation is initiated at the rate start codons TTG and ATC, respectively. Consequently, the N-terminus of the trp(G.D) fusion protein is 43 residues shorter than previously postulated. Amino acid composition and sequence analyses of the protein products of T.maritima trpC (indoleglycerol phosphate synthase), trpF (phosphoribosyl anthranilate isomerase) and trpA (alpha-subunit of tryptophan synthase) suggest that these thermostable (beta alpha)8-barrel proteins may be stabilized by additional salt bridges, compared with the mesostable forms. Another notable feature is the predicted lack of the N-terminal helix alpha 0 in the alpha-subunit of tryptophan synthase.     

Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production

Metabolic engineering in microbes could be used to produce large amounts of valuable metabolites that are difficult to extract from their natural sources and too expensive or complex to produce by chemical synthesis. As a step towards the production of Taxol in the yeast Saccharomyces cerevisiae, we introduced heterologous genes encoding biosynthetic enzymes from the early part of the taxoid biosynthetic pathway, isoprenoid pathway, as well as a regulatory factor to inhibit competitive pathways, and studied their impact on taxadiene synthesis. Expression of Taxus chinensis taxadiene synthase alone did not increase taxadiene levels because of insufficient levels of the universal diterpenoid precursor geranylgeranyl diphosphate. Coexpression of T. chinensis taxadiene synthase and geranylgeranyl diphosphate synthase failed to increase levels, probably due to steroid-based negative feedback, so we also expressed a truncated version of 3-hydroxyl-3-methylglutaryl-CoA reductase (HMG-CoA reductase) isoenzyme 1 that is not subject to feedback inhibition and a mutant regulatory protein, UPC2-1, to allow steroid uptake under aerobic conditions, resulting in a 50% increase in taxadiene. Finally, we replaced the T. chinensis geranylgeranyl diphosphate synthase with its counterpart from Sulfolobus acidocaldarius, which does not compete with steroid synthesis, and codon optimized the T. chinensis taxadiene synthase gene to ensure high-level expression, resulting in a 40-fold increase in taxadiene to 8.7±0.85 mg/l as well as significant amounts of geranylgeraniol (33.1±5.6 mg/l), suggesting taxadiene levels could be increased even further. This is the first demonstration of such enhanced taxadiene levels in yeast and offers the prospect for Taxol production in recombinant microbes.     

Sexual Dimorphism in Juvenile Hormone Synthesis by Corpora Allata and in Juvenile Hormone Acid Methyltransferase Activity in Corpora Allata and Accessory Sex Glands of Some Lepidoptera

Summary

The kinetic profiles of vitellin accumulation in the oyster ovary during oocyte growth and the effects in vivo and in vitro of estradiol-17β (E₂) on vitellin formation were examined in this study. The relative vitellin content measured by an enzyme-linked immunosorbent assay (ELISA) shows an apparent increase as the oocyte develops. Immunoblotting of the vitellin using anti-vitellin indicated that two main bands (179 and 110 kD), which begin to accumulate at an early stage of maturation, become pronounced during oocyte growth. Meanwhile, the major peak of the intact form of vitellin (530 kD) in gel filtration also enlarges with oocyte growth, supporting the results of immunoblot analysis and vitellin determination. E₂ treatment in vivo causes significant increases in oocyte diameter and vitellin content in the female oyster. A similar trend was observed in ovarian tissues cultured in the presence of E₂. It is concluded that E₂ is one of the major factors which control the vitellogenesis in the oyster and that the ovary is undoubtedly the site of synthesis of vitellin.     

Coupling Nutrient Uptake and Energy Flow in Headwater Streams

Nutrient cycling and energy flow in ecosystems are tightly linked through the metabolic processes of organisms. Greater uptake of inorganic nutrients is expected to be associated with higher rates of metabolism [gross primary production (GPP) and respiration (R)], due to assimilatory demand of both autotrophs and heterotrophs. However, relationships between uptake and metabolism should vary with the relative contribution of autochthonous and allochthonous sources of organic matter. To investigate the relationship between metabolism and nutrient uptake, we used whole-stream and benthic chamber methods to measure rates of nitrate–nitrogen (NO₃–N) uptake and metabolism in four headwater streams chosen to span a range of light availability and therefore differing rates of GPP and contributions of autochthonous carbon. We coupled whole-stream metabolism with measures of NO₃–N uptake conducted repeatedly over the same stream reach during both day and night, as well as incubating benthic sediments under both light and dark conditions. NO₃–N uptake was generally greater in daylight compared to dark conditions, and although day-night differences in whole-stream uptake were not significant, light–dark differences in benthic chambers were significant at three of the four sites. Estimates of N demand indicated that assimilation by photoautotrophs could account for the majority of NO₃–N uptake at the two sites with relatively open canopies. Contrary to expectations, photoautotrophs contributed substantially to NO₃–N uptake even at the two closed-canopy sites, which had low values of GPP/R and relied heavily on allochthonous carbon to fuel R.     

An Ecosystem View of the Restoration of the Kissimmee River

Restoration of the Kissimmee River and floodplain ultimately will involve restoring 70 km of river channel and riparian zone and 11,000 ha of wetland over a period of two decades. Restoring ecosystem integrity is a crucial goal of the project, and the evaluation program is designed to assess the success of this endeavor. Major components of the riverine and floodplain ecosystem will be evaluated, guided by conceptual models of their structure and function. These studies will be referenced to historic conditions of the past and to present-day conditions in the channelized system. Enhanced connectivity and interactions between the river and floodplain, the interplay of abiotic and biotic variables, and interactions between trophic levels will restructure the channelized river and the largely drained floodplain that now exist. The key to evaluating the success of this ambitious project will be selecting measurements of the structure and function of the river and floodplain ecosystems that are responsive to this large-scale manipulation. The timing and duration of floodplain inundation, improved dissolved oxygen conditions, germination and establishment of wetland vegetation, and enhancement and expansion of rheophilic benthic invertebrate populations are critical initial elements of restoration. Further expected outcomes are an increase in the primary productivity of the ecosystem, expansion of the fish community into the reopened channels and onto the reflooded floodplain, and improved visitation and use by waterbirds in the restored regions. We highlight predictions of some of these key linkages and primary structural and functional attributes of the restored river and floodplain that should be measured.     

Effect of DHEA Glutamate Release from Synaptosomes of Rats at Different Ages

Dehydroepiandrosterone (DHEA) exerts multiple effects in the central nervous system. Most of them seem to be mediated through their nongenomic actions on neurotransmitter receptors, and these actions occur within seconds or milliseconds. DHEA increases neuronal excitability, enhances neuronal plasticity, and has neuroprotective properties. By investigating glutamate release from synaptosomes of rats at different ages (from 17 days to 12 months), we observed that (i) there is an increase in basal and K(+)-stimulated L-[3H] glutamate release in rats at 12 months old, when compared to other ages; and (ii) there is an inhibitory effect of DHEA on basal L-[3H] glutamate release in 12 months old. This inhibitory effect of DHEA could be related to its reported protective role against excitotoxicity caused by overstimulation of the glutamatergic system and ageing.