Biorefining of wood: combined production of ethanol and xylanase from waste fiber sludge

The possibility to utilize fiber sludge, waste fibers from pulp mills and lignocellulose-based biorefineries, for combined production of liquid biofuel and biocatalysts was investigated. Without pretreatment, fiber sludge was hydrolyzed enzymatically to monosaccharides, mainly glucose and xylose. In the first of two sequential fermentation steps, the fiber sludge hydrolysate was fermented to cellulosic ethanol with the yeast Saccharomyces cerevisiae. Although the final ethanol yields were similar, the ethanol productivity after 9.5?h was 3.3?g/l/h for the fiber sludge hydrolysate compared with only 2.2?g/l/h for a reference fermentation with similar sugar content. In the second fermentation step, the spent fiber sludge hydrolysate (the stillage obtained after distillation) was used as growth medium for recombinant Aspergillus niger expressing the xylanase-encoding Trichoderma reesei (Hypocrea jecorina) xyn2 gene. The xylanase activity obtained with the spent fiber sludge hydrolysate (8,500?nkat/ml) was higher than that obtained in a standard medium with similar monosaccharide content (1,400?nkat/ml). Analyses based on deglycosylation with N-glycosidase?F suggest that the main part of the recombinant xylanase was unglycosylated and had molecular mass of 20.7?kDa, while a minor part had N-linked glycosylation and molecular mass of 23.6?kDa. Chemical analyses of the growth medium showed that important carbon sources in the spent fiber sludge hydrolysate included xylose, small aliphatic acids, and oligosaccharides. The results show the potential of converting waste fiber sludge to liquid biofuel and enzymes as coproducts in lignocellulose-based biorefineries.     

A variable selection index for the compensation of correlated genetic change

Summary A selection index for two traits has been constructed which allows partial restriction for one of the traits. The index is used in a situation where correlated response to selection in one sex is compenstated for by selection for other traits in the opposite sex. A numerical example is given.     

Juniper fields in Sogn, Western Norway, a man-made vegetation type

Juniperus communis has been used e.g. as poles (posts), haysticks and wall-coating. In some districts it has even been an important export article. The fields of columnar juniper are the result of a long history of cultivation and grazing. In this article the main types of juniper fields in Sogn, Western Norway are presented, and their relationships to soil conditions and traditional use are discussed. According to structure, utilization and development four main types of juniper fields are recognized: I. Traditional juniper fields, II. Semi-cultural juniper fields, III. Secondary juniper fields and IV. Semi-natural juniper fields. Due to changes in agricultural methods, juniper fields are becoming rare.     

Acute Effect of Ammonia on Branched-Chain Amino Acid Oxidation and Incorporation into Proteins in Astrocytes and in Neurons in Primary Cultures

14CO2 production and incorporation of label into proteins from the labeled branched-chain amino acids, leucine, valine, and isoleucine, were determined in primary cultures of neurons and of undifferentiated and differentiated astrocytes from mouse cerebral cortex in the absence and presence of 3 mM ammonium chloride. Production of 14CO2 from [1-14C]leucine and [1-14C]valine was larger than 14CO2 production from [U-14C]leucine and [U-14C]valine in both astrocytes and neurons. In most cases more 14CO2 was produced in astrocytes than in neurons. Incorporation of labeled branched-chain amino acids into proteins varied with the cell type and with the amino acid. Addition of 3 mM ammonium chloride greatly suppressed 14CO2 production from [1-14C]-labeled branched chain amino acids but had little effect on 14CO2 production from [U-14C]-labeled branched-chain amino acids in astrocytes. Ammonium ion, at this concentration, suppressed the incorporation of label from all three branched-chain amino acids into proteins of astrocytes. In contrast, ammonium ion had very little effect on the metabolism (oxidation and incorporation into proteins) of these amino acids in neurons. The possible implications of these findings are discussed, especially regarding whether they signify variations in metabolic fluxes and/or in magnitudes of precursor pools.     

Role of the polar head group stereoconfiguration in the cation-induced aggregation of dimyristoylphosphatidylglycerol vesicles

Cation-induced aggregation of small unilamellar vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphatidyl-sn-1'-glycerol (1'-DMPG), the corresponding 3' stereoisomer (3'-DMPG), and their 1:1 mixture was studied as a function of the concentration of different mono- and divalent cations. The order of efficiency, Na+ greater than Li+ greater than K+ greater than Cs+, of the monovalent cations to induce the aggregation of DMPG vesicles is the same for both stereoisomers and their mixture. However, significant differences in the Na+-induced aggregation of 1'-DMPG and 3'-DMPG were evident. The threshold concentration of aggregation by Na+ was 0.35 M for 3'-DMPG, 0.55 M for 1'-DMPG, and 0.50 M for the mixed liposomes. Such difference in the aggregation of DMPG stereoisomers was not observed for the other mono- and divalent cations. The higher affinity of 3'-DMPG for Na+ is suggested to be due to a slightly different favored conformation of the head group glycerol moiety. Aggregation of the stereoisomers by 1 M NaCl was identical, indicating that the differences in the affinity of 1'-DMPG and 3'-DMPG for sodium can be overcome by very high ionic strength. Inclusion of 20 mol % cholesterol in vesicles enhanced the aggregation of 1'-DMPG and decreased the aggregation of 3'-DMPG by Na+ and thus abolished the difference between the two stereoisomers.     

Computer-graphics interpretations of residue exchanges between the alpha, beta and gamma subunits of human-liver alcohol dehydrogenase class I isozymes

Three-dimensional models of human alcohol dehydrogenase subunits have been constructed, based on the homologous horse enzyme, with computer graphics. All types of class I subunits (alpha, beta, and gamma) and the major allelic variants (beta 1/beta 2 and gamma 1/gamma 2) have been studied. Residue differences between the E-type subunit of the horse enzyme and any of the subunits of the human isozymes occur at 64 positions, about half of which are isozyme-specific. About two thirds of the substitutions are at the surface and all differences can be accommodated in highly conserved three-dimensional structures. The model of the gamma isozyme is most similar to the crystallographically analyzed horse liver E-type alcohol dehydrogenase, and has all the functional residues identical to those of the E subunit except for one which is slightly smaller: Val-141 in the substrate pocket. The residues involved in coenzyme binding are generally conserved between the horse enzyme and the alpha, beta, and gamma types of the human enzyme. In contrast, single exchanges of these residues are the ones involved in the major allelic differences (beta 1 versus beta 2 and gamma 1 versus gamma 2), which affects the overall rate of alcohol oxidation since NADH dissociation is the rate-determining step. Residue 47 is His in beta 2 and Arg in the beta 1, gamma 1, and gamma 2 subunits, and in horse liver alcohol dehydrogenase. Both His and Arg can make a hydrogen bond to a phosphate oxygen atom of NAD; hence the lower turnover rate of beta 1 apparently derives from a charge effect. The substitution to Gly in the alpha subunit results in one less hydrogen bond in NAD binding, and consequently in rapid dissociation. This may explain why the overall rate is an order of magnitude faster than that of beta 1. The important difference between gamma 1 and gamma 2 is an exchange at position 271 from Arg to Gln which can give a hydrogen bond from Gln in gamma 2 to the adenine of NAD. The tighter binding to gamma 2 can account for the slower overall catalytic rate in this isozyme. The kinetics and interactions of cyclohexanol and benzyl alcohol with the isozymes were judged by docking experiments using an interactive fitting program.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pyruvate decarboxylation in astrocytes and in neurons in primary cultures in the presence and the absence of ammonia

Oxidative decarboxylation of [1-¹⁴C]pyruvate was studied in primary cultures of neurons and of astrocytes. The rate of this process, which is a measure of carbon flow into the tricarboxylic acid (TCA) cycle and which is inhibited by its end product, acetyl CoA, was determined under conditions which would either elevate or reduce the components of the malate-aspartate shuttle (MAS). Addition of aspartate (1 mM) was found to stimulate pyruvate decarboxylation in astrocytes whereas addition of glutamate (or glutamine) had no effect. Since aspartate is a precursor for extramitochondrial malate, and thus intramitochondrial oxaloacetate, whereas glutamate and glutamine are not, this suggests that an increase in oxaloacetate level stimulates TCA cycle activity. Conversely, a reduction of the glutamate content by 3 mM ammonia, which might reduce exchange between glutamate and aspartate across the mitochondrial membrane, suppressed pyruvate decarboxylation. This effect was abolished by addition of glutamate or glutamine or exposure to methionine sulfoximine (MSO). These findings suggest that impairment of MAS activity by removal of MAS constituents decreases TCA cycle activity whereas replenishment of these compounds restores the activity of the TCA cycle. No corresponding effects were observed in neurons.     

Energy metabolism in glutamatergic neurons,GABAergic neurons and astrocytes in primary cultures

Several aspects of energy metabolism (glucose utilization, lactate production,¹⁴CO₂ production from labeled glucose, glutamate or pyruvate, oxygen consumption and contents of ATP and phosphocreatine) were measured in cerebellar granule cells (glutamatergic) in primary cultures and compared with corresponding data for cerebral cortical neurons (mainly GABA-ergic) and astrocytes. Cerebellar granule cells and astrocytes were metabolically more active than cerebral cortical neurons. Glutamate which is utilized as a major metabolic fuel as astrocytes and, to a lesser extent, in cerebral cortical neurons, was virtually not oxidized in cerebellar granule cells.Special Issue dedicated to Prof. Holger Hydén.     

Linkage relationships in the bovine MHC region. High recombination frequency between class II subregions

Class II genes of the bovine major histocompatibility complex (MHC) have been investigated by Southern blot analysis using human DNA probes. Previous studies revealed the presence of bovine DO , DQ , DQ , DR and DR genes, and restriction fragment length polymorphisms for each of these genes were documented. In the present study, the presence of three additional class II genes, designated DZ , DY , and DY , are reported. DZ was assumed to correspond to the human DZ gene while the other two were designated DY because their relationship to human class II genes could not be firmly established. The linkage relationships among bovine class II genes and two additional loci, TCP1B and C4, were investigated by family segregation analysis and analysis of linkage disequilibrium. The results clearly indicated that all these loci belong to the same linkage group. This linkage group is divided into two subregions separated by a fairly high recombination frequency. One region includes the C4, DQ , DQ , DR and DR loci and the other one is composed of the DO DY , DY , and TCPIB loci. No recombinant was observed within any of these subregions and there was a strong or fairly strong linkage disequilibrium between loci within groups. In contrast, as many as five recombinants among three different families were detected in the interval between these subregions giving a recombination frequency estimate of 0.17 ± 0.07. The fairly high recombination frequency observed between class 11 genes in cattle is strikingly different from the corresponding recombination estimates in man and mouse. The finding implies either a much larger molecular distance between some of the bovine class II genes or alternatively the presence of a recombinational hot spot in the bovine class II region.     

Ethylene effects on cambial activity and cell wall formation in hypocotyls of Picea abies seedlings

Ethylene regulation of cell division in the vascular cambium and cell wall formation was studied in hypocotyls of Norway spruce ( Picea abies [L.] Karst.) seedlings. Cuttings from 6-week-old seedlings were placed in water culture to which compounds affecting the synthesis and action of ethylene were added. After a 3-week treatment period, growth, ethylene production, morphology and cell wall composition of the hypocotyls were determined. Addition of high concentrations of the potent ethylene releasing agent 2-chloroethylphosphonic acid (ethrel), which increased ethylene emission by more than twice compared to control plants, inhibited the expansion of xylem cells while stimulating the incorporation of cell wall material, especially cellulose. Addition of small amounts of ethrel, which slightly stimulated ethylene emission, led to increases in the size of xylem cells, the amount of phloem tissue and the number of intercellular spaces in the cortex, and thus to increased hypocotyl diameter. However, no significant change in cell wall composition was detected. When ethylene production was decreased by adding Co²⁺ to the nutrient solution, differentiation of new xylem was disturbed, but the rate of cell division was not affected. Although the incorporation of cell wall material was inhibited, the proportions of lignin and cellulose in the wall appeared to remain unchanged. Silver ions stimulated the expansion of both xylem and cortex cells, but had no significant effect on cell wall formation. We conclude that ethylene has a role in regulating the incorporation of wall carbohydrates.