Microbial indicators for sawdust and bark compost stability and humification processes

To what extent some microbial index ratios are suitable for use as early criteria for the level of compost stability during aerobic composting of coniferous sawdust and bark at mesophilic conditions was studied. Evolution of the specific respiration activity (CO₂-C/biomass C) and the ratios between some groups of microorganisms were followed as a function of composting time. The specific respiration activity was found to be an early and most reliable indicator of compost stability. The peroxidase and polyphenoloxidase enzyme activity during composting, as well as the composition of newly-formed humus substances were studied. The duration of composting increased the quality of newly-formed humus substances (C_h.a.:C_f.a ratio; Ca-complexed humic acid and resistance of organo-mineral complexes). The quality of humus substances could be used to assess compost stability. However, the results can be applied only under defined conditions.     

Digestive enzymes in midgut cells,endo-and ectoperitrophic contents,and peritrophic membranes of Spodoptera frugiperda (lepidoptera) larvae

In the midgut of Spodoptera frugiperda larvae, subcellular fractionation data suggest that aminopeptidase and part of amylase, carboxypeptidase A, dipeptidase, and trypsin are bound to the microvillar membranes; that major amounts of soluble dipeptidase, cellobiase, and maltase are trapped in the cell glycocalyx; and finally that soluble carboxypeptidase, amylase, and trypsin occur in intracellular vesicles. Most luminal acetylglucosaminidase is soluble and restricted to the ectoperitrophic contents. Aminopeptidase occurs in minor amounts bound to membranes both in the ectoperitrophic contents and incorporated in the peritrophic membrane. Amylase, carboxypeptidase A, and trypsin are found in minor amounts in the ectoperitrophic contents (both soluble and membrane-bound) and in major amounts in the peritrophic membrane with contents. Part of the activities recovered in the last mentioned contents corresponds to enzyme molecules incorporated in the peritrophic membrane. The results suggest that initial digestion is carried out in major amounts by enzymes in the endoperitrophic space and, in minor amounts, by enzymes immobilized in the peritrophic membrane. Intermediate and final digestion occur at the ectoperitrophic space or at the surface of midgut cells. The results also lend support to the hypothesis that amylase and trypsin are derived from membrane-bound forms, are released in soluble form by a microapocrine mechanism, and are partly incorporated into the peritrophic membrane. © 1994 Wiley-Liss, Inc.     

1H and 15N resonance assignments and secondary structure of the carbon monoxide complex of sperm whale myoglobin

Summary Sequence-specific backbone ¹H and ¹⁵N resonance assignments have been made for 95% of the amino acids in sperm whale myoglobin, complexed with carbon monoxide (MbCO). Many assignments for side-chain resonances have also been obtained. Assignments were made by analysis of an extensive series of homonuclear 2D spectra, measured with unlabeled protein, and both 2D and 3D ¹H-¹⁵N-correlated spectra obtained from uniformly ¹⁵N-labeled myoglobin. Patterns of medium-range NOE connectivities indicate the presence of eight helices in positions that are very similar to those found in the crystal structures of sperm whale myoglobin. The resonance assignments of MbCO form the basis for determination of the solution structure and for hydrogen-exchange measurements to probe the stability and folding pathways of myoglobin. They will also form a basis for assignment of the spectra of single-site mutants with altered ligand-binding properties.     

Protein dynamics: conformational disorder,vibrational coupling and anharmonicity in deoxy-hemoglobin and myoglobin

In this work we study the temperature dependence of the Soret band lineshape of deoxymyoglobin and deoxyhemoglobin, in the range 300–20 K. To fit the measured spectra we use an approach originally proposed by Champion and coworkers (Srajer et al. 1986; Srajer and Champion 1991). The band profile is modelled as a Voigt function that accounts for the coupling with low frequency vibrational modes, whereas the coupling with high frequency modes is responsible for the vibronic structure of the spectra. Moreover, owing to the position of the iron atom out of the mean heme plane, inhomogeneous broadening brings about a non-Gaussian distribution of 0–0 electronic transition frequencies. The reported analysis enables us to isolate the various contributions to the overall bandwidth, and their temperature dependence points out the relevance of low frequency vibrations and of large scale anharmonic motions starting at temperatures higher than 170 K. Information on the mean iron-heme plane distance and on its temperature dependence, as well as on the heme pocket conformational disorder, is also obtained.Abbreviations Cc Carbon monoxide - Hb Human deoxyhemoglobin A - HbCO human carbonmonoxyhemoglobin A - SWMb spermwhale deoxymyoglobin - SWMbCO spermwhale carbonmonoxymyoglobin - HbO₂ human oxyhemoglobin A - SWMb³⁺-H₂O spermwhale aquometmyoglobin     

Concerning the mechanism of increased thermogenesis in rats treated with dehydroepiandrosterone

Dehydroepiandrosterone (DHEA) treatment of rats decreases gain of body weight without affecting food intake; simultaneously, the activities of liver malic enzyme and cytosolic glycerol-3-P dehydrogenase are increased. In the present study experiments were conducted to test the possibility that DHEA enhances thermogenesis and decreases metabolic efficiency via trans-hydrogenation of cytosolic NADPH into mitochondrial FADH₂ with a consequent loss of energy as heat. The following results provide evidence which supports the proposed hypothesis: (a) the activities of cytosolic enzymes involved in NADPH production (malic enzyme, cytosolic isocitrate dehydrogenase, and aconitase) are increased after DHEA treatment; (b) cytosolic glycerol-3-P dehydrogenase may use both NAD⁺ and NADP⁺ as coenzymes; (c) activities of both cytosolic and mitochondrial forms of glycerol-3-P dehydrogenase are increased by DHEA treatment; (d) cytosol obtained from DHEA-treated rats synthesizes more glycerol-3-P during incubation with fructose-1,6-P₂ (used as source of dihydroxyacetone phosphate) and NADP⁺; the addition of citratein vitro further increases this difference; (e) mitochondria prepared from DHEA-treated rats more rapidly consume glycerol-3-P added exogenously or formed endogenously in the cytosol in the presence of fructose-1,6-P₂ and NADP⁺.     

Colonization ofRetama raetam seeds by fungi and their significance in seed germination

Examination by scanning electron microscopy and incubation on potato-dextrose agar medium showed that dry seeds ofRetama raetam were externally free of fungi. When planted in sandy loam soil, the seeds become colonized with eleven soil-borne fungal species. The fungi were isolated on cellulose agar, pectin agar and lignin agar media.Aspergillus flavus, A. niger, A. fumigatus, Penicillium capsolatum andFusarium oxysporum had broad occurrence and were recovered on all the three media. The production of hydrolytic enzymes by the isolated fungi depends on the substrate and species.Penicillium capsolatum, P. spinulosum andA. niger had wide enzymatic amplitude and they were able to produce cellulolytic, pectolytic and lignolytic activities on corresponding substrates as well as on seed-coat-containing media. The lignolytic activities of the isolated species exceptChaetomium bostrychodes andTrichoderma viride were enhanced by applying the seed-coat materials as C- source rather than lignin. SoakingR. raetam seeds in culture filtrates of most of the fungi grown on seed-coat-supplemented media induced a pronounced and distinct stimulating effect on seed germination. The most effective filtrates were those ofP. capsolatum, P. spinulosum andSporotrichum pulverulentum.     

Temperature-dependent sex determination in reptiles: Proximate mechanisms,ultimate outcomes,and practical applications

In many egg-laying reptiles, the incubation temperature of the egg determines the sex of the offspring, a process known as temperature-dependent sex determination (TSD). In TSD sex determination is an “all or none” process and intersexes are rarely formed. How is the external signal of temperature transduced into a genetic signal that determines gonadal sex and channels sexual development? Studies with the red-eared slider turtle have focused on the physiological, biochemical, and molecular cascades initiated by the temperature signal. Both male and female development are active processes—rather than the crganized/default system characteristic of vertebrates with genotypic sex determination—that require simultaneous activation and suppression of testis- and ovary-determining cascades for normal sex determination. It appears that temperature accomplishes this end by acting on genes encoaing for steroidogenic enzymes and steroid hormone receptors and modifying the endocrine microenvironment in the embryo. The temperature experienced in development also has long-term functional outcomes in addition to sex determination. Research with the leopard gecko indicates that incubation temperature as well as steroid hormones serve as organizers in shaping the adult phenotype, with temperature modulating sex hormone action in sexual differentiation. Finally, practical applications of this research have emerged for the conservation and restoration of endangered egg-laying reptiles as well as the embryonic development of reptiles as biomarkers to monitor the estrogenic effects of common environmental contaminants. © 1994 Wiley-Liss, Inc.     

Thermostable β-Glycosidase from Sulfolobus Solfataricus

The Sulfolobus solfataricus β-glycosidase (Sβgly) is a thermostable and thermophilic glycosyl-hydrolase with broad substrate specificity. The enzyme hydrolizes β-D-gluco-, fuco-, and galactosides, and a large number of /Winked glycoside dimers and oligomers, linked β1-3, β1-4, and β1-6, It is able to hydrolize oligosaccharides with up to 5 glucose residues. Furthermore, it is also able to promote transglycosylation reactions. The corresponding gene has been cloned and overexpressed both in yeast and Escherichia coli. Based on sequence and functional data, the Sβgly has been assigned to the so-called BGA family of glycosyl-hydrolases, including β-glycosidases, β-galactosidases and phosho-β-galactosidases from mesophilic and thermophilic organisms of the three domains. The Sβgly has been crystallized and the resolution of its structure is in progress. Because of its special properties, the enzymes has considerable biotechnological potential.     

Seasonal determinations of extracellular hydrolytic activities in heterotrophic and mixed heterotrophic/autotrophic biofilms from two contrasting rivers

The temporal changes in extracellular enzyme activities in freshwater microbial biofilms were examined in two contrasting river sites in North Wales over a 12 month period. Sites were a first order, unshaded oligotrophic upland stream (Nant Waen) and a fourth order, mildly eutrophic river with riparian tree cover (River Clywedog). When algal populations were low, biofilms of the more eutrophic site supported greater enzyme activities and higher population densities than the oligotrophic site. Composition, concentration and origin of substrates available to the respective biofilm communities influenced extracellular processing patterns. Reduction in algal populations depressed total and extracellular activities in biofilms from the first order site, suggesting that biofilm communities here were maintained by in situ primary production. Biofilms from Nant Waen were often found to contain higher extracellular activities per cell than the more eutrophic River Clywedog biofilms, which might represent the enhanced ability of an oligotrophic biofilm to accumulate extracellular enzymes. In contrast, light and darkgrown River Clywedog biofilms were not enzymatically distinct, inferring a less important role for biofilm phototrophs. Some evidence was found for increased reliance on allochthonous substrates in the River Clywedog for biofilm maintenance.     

Gluco-oligosaccharide synthesis by free and immobilized beta-glucosidase

Gluco-oligosaccharides were synthesized through the enzymatic condensation of D-glucose at high concentration using a commercial almond beta-glucosidase. The synthesis reactions were carried out with both free and immobilized enzyme, with or without sorbitol, an efficient depressor of water activity (a(w)) in the presence of different glucose concentrations. The yield and the composition of the gluco-oligosaccharides produced changed with the reaction mixture and the form of the enzyme used (free or immobilized). The use of 5 M glucose solution permitted only disaccharides to be obtained, whereas with a glucose concentration of 7.5 M glucose, di-, tri-, and tetrasaccharides were produced. A 7.5 M glucose solution used with 4.4 M sorbitol gave three times more disaccharides than the same solution without sorbitol. Moreover, the immobilized enzyme was much more active in synthesis. The synthesis yield (oligomers mg/mL . mg of enzyme) after immobilization was 573% compared to that of the free enzyme, when a 7.5 M glucose solution was tested. The effects of substrate concentration, sorbitol addition and enzyme immobilization were investigated. (c) 1993 John Wiley & Sons, Inc.