Uptake of monosaccharides by guinea-pig cerebral-cortex slices

By the use of 1mm-iodoacetate to inhibit glycolysis in guinea-pig cerebral tissue slices, the kinetics of the uptake of monosaccharides on transfer of tissue from 0° to 37° were studied. d-Ribose, d-galactose, d-mannose, l-sorbose, and d-fructose showed diffusion kinetics, whereas 2-deoxy-d-glucose, d-glucose, d-arabinose and d-xylose showed saturation kinetics.     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

Amino Acid and vitamin requirements of several bacteroides strains

Nutritional studies were performed on nine Bacteroides strains, by use of the methodology and media of anaerobic rumen microbiology. Ristella perfoetens CCI required l-arginine hydrochloride, l-tryptophan, l-leucine, l-histidine hydrochloride, l-cysteine hydrochloride, dl-valine, dl-tyrosine, and the vitamin calcium-d-pantothenate, since scant turbidity developed in media without these nutrients. R. perfoetens was stimulated by glycine, dl-lysine hydrochloride, dl-isoleucine, l-proline, l-glutamic acid, dl-alanine, dl-phenylalanine, dl-methionine, and the vitamins nicotinamide and p-aminobenzoic acid, since maximal turbidity developed more slowly in media without these nutrients than in complete medium. Medium A-23, which was devised for R. perfoetens, contained salts, 0.0002% nicotinamide and calcium d-pantothenate, 0.00001% p-aminobenzoic acid, 0.044% l-tryptophan, 0.09% l-glutamic acid, and 0.1% of the other 13 amino acids listed above. Zuberella clostridiformis and seven strains of R. pseudoinsolita did not require vitamins, and showed no absolute requirement for any one amino acid. Various strains produced maximal turbidity more slowly in media deficient in l-proline, glycine, l-glutamic acid, dl-serine, l-histidine hydrochloride, dl-alanine, or l-cysteine hydrochloride, than in complete medium. These eight strains grew optimally in medium A-23 plus 0.1% dl-serine but without vitamins.     

Regulation of sulfate uptake by amino acids in cultured tobacco cells

The sulfur requirements of tobacco (Nicotiana tabacum L. var. Xanthi) XD cells grown in chemically defined liquid media can be satisfied by sulfate, thiosulfate, l-cyst(e)ine, l-methionine or glutathione, and somewhat less effectively by d-cyst (e) ine, d-methionine or dl-homocyst (e)ine. Sulfate uptake is inhibited after a 2 hr lag by l-cyst (e)ine, l-methionine, l-homocyst(e)ine or l-isoleucine, but not by any of the other protein amino acids, nor by d-cyst(e)ine. l-cyst(e)ine is neither a competitive nor a non-competitive inhibitor of sulfate uptake. Its action most closely resembles apparent uncompetitive inhibition. Inhibition of sulfate uptake by l-cyst(e)ine can be partially prevented by equimolar l-arginine, l-lysine, l-leucine, l-phenylalanine, l-tyrosine or l-tryptophan, but is little affected by any of the other protein amino acids. The effective amino acids are apparent competitive inhibitors of l-cyst(e)ine uptake after a 2 hr lag. Inhibition of sulfate uptake by l-methionine cannot be prevented, nor can uptake of l-methionine be inhibited by any single protein amino acid. The results suggest the occurrence of negative feedback control of sulfate assimilation by the end products, the sulfur amino acids, in cultured tobacco cells.     

Amino Acid Isomerization in the Production of l-Phenylalanine from d-Phenylalanine by Bacteria

To establish an advantageous method for the production of l-amino acids, microbial isomerization of d- and dl-amino acids to l-amino acids was studied. Screening experiments on a number of microorganisms showed that cell suspensions of Pseudomonas fluorescens and P. miyamizu were capable of isomerizing d- and dl-phenylalanines to l-phenylalanine. Various conditions suitable for isomerization by these organisms were investigated. Cells grown in a medium containing d-phenylalanine showed highest isomerization activity, and almost completely converted d- or dl-phenylalanine into l-phenylalanine within 24 to 48 hr of incubation. Enzymatic studies on this isomerizing system suggested that the isomerization of d- or dl-phenylalanine is not catalyzed by a single enzyme, “amino acid isomerase,” but the conversion proceeds by a two step system as follows: d-pheylalanine is oxidized to phenylpyruvic acid by d-amino acid oxidase, and the acid is converted to l-phenylalanine by transamination or reductive amination.     

Structural and Functional Characterization of VanG d-Ala:d-Ser Ligase Associated with Vancomycin Resistance in Enterococcus faecalis

d-Alanyl:d-lactate (d-Ala:d-Lac) and d-alanyl:d-serine ligases are key enzymes in vancomycin resistance of Gram-positive cocci. They catalyze a critical step in the synthesis of modified peptidoglycan precursors that are low binding affinity targets for vancomycin. The structure of the d-Ala:d-Lac ligase VanA led to the understanding of the molecular basis for its specificity, but that of d-Ala:d-Ser ligases had not been determined. We have investigated the enzymatic kinetics of the d-Ala:d-Ser ligase VanG from Enterococcus faecalis and solved its crystal structure in complex with ADP. The overall structure of VanG is similar to that of VanA but has significant differences mainly in the N-terminal and central domains. Based on reported mutagenesis data and comparison of the VanG and VanA structures, we show that residues Asp-243, Phe-252, and Arg-324 are molecular determinants for d-Ser selectivity. These residues are conserved in both enzymes and explain why VanA also displays d-Ala:d-Ser ligase activity, albeit with low catalytic efficiency in comparison with VanG. These observations suggest that d-Ala:d-Lac and d-Ala:d-Ser enzymes have evolved from a common ancestral d-Ala:d-X ligase. The crystal structure of VanG showed an unusual interaction between two dimers involving residues of the omega loop that are deeply anchored in the active site. We constructed an octapeptide mimicking the omega loop and found that it selectively inhibits VanG and VanA but not Staphylococcus aureus d-Ala:d-Ala ligase. This study provides additional insight into the molecular evolution of d-Ala:d-X ligases and could contribute to the development of new structure-based inhibitors of vancomycin resistance enzymes.     

Glutamate, glutamine, aspartate, asparagine, glucose and ketone-body metabolism in chick intestinal brush-border cells

1. Suspensions of isolated chick jejunal columnar absorptive (brush-border) cells respired on endogenous substrates at a rate 40% higher than that shown by rat brush-border cells. 2. Added d-glucose (5 or 10mm), l-glutamine (2.5mm) and l-glutamate (2.5mm) were the only individual substrates which stimulated respiration by chick cells; l-aspartate (2.5 or 6.7mm), glutamate (6.7mm), glutamine (6.7mm), l-alanine (1 or 10mm), pyruvate (1 or 2mm), l-lactate (5 or 10mm), butyrate (10mm) and oleate (1mm) did not stimulate chick cell respiration; l-asparagine (6.7mm) inhibited slightly; glucose (5mm) stimulated more than did 10mm-glucose. 3. Acetoacetate (10mm) and d-3-hydroxybutyrate (10mm) were rapidly consumed but, in contrast to rat brush-border cells, did not stimulate respiration. 4. Glucose (10mm) was consumed more slowly than 5mm-glucose; the dominant product of glucose metabolism during vigorous respiration was lactate; the proportion of glucose converted to lactate was greater with 10mm- than with 5mm-glucose. 5. Glutamate and aspartate consumption rates decreased, and alanine and glutamine consumption rates increased when their initial concentrations were raised from 2.5 to 6.7 or 10mm. 6. The metabolic fate of glucose was little affected by concomitant metabolism of any one of aspartate, glutamate or glutamine except for an increased production of alanine; the glucose-stimulated respiration rate was unaffected by concomitant metabolism of these individual amino acids. 7. Chick cells produced very little alanine from aspartate and, in contrast to rat cells, likewise produced very little alanine from glutamate or glutamine; in chick cells alanine appeared to be predominantly a product of transmination of pyruvate derived from glucose metabolism. 8. In chick cells, glutamate and glutamine were formed from aspartate (2.5 or 6.7mm); aspartate and glutamine were formed from glutamate (2.5mm) but only aspartate from 6.7mm-glutamate; glutamate was the dominant product formed from glutamine (6.7mm) but aspartate only was formed from 2.5mm-glutamine. 9. Chick brush-border cells can thus both catabolize and synthesize glutamine; glutamine synthesis is always diminished by concomitant metabolism of glucose, presumably by allosteric inhibition of glutamine synthetase by alanine. 10. Proline was formed from glutamine (2.5mm) but not from glutamine (2.5mm)+glucose (5mm) and not from 2.5mm-glutamate; ornithine was formed from glutamine (2.5mm)+glucose (5.0mm) but not from glutamine alone; serine was formed from glutamine (2.5mm)+glucose (5mm) and from these two substrates plus aspartate (2.5mm). 11. Total intracellular adenine nucleotides (22μmol/g dry wt.) remained unchanged during incubation of chick cells with glucose. 12. Intracellular glutathione (0.7–0.8mm) was depleted by 40% during incubation of respiring chick cells without added substrates for 75min at 37°C; partial restoration of the lost glutathione was achieved by incubating cells with l-glutamate+l-cysteine+glycine.     

The biological sulphation of l-tyrosyl peptides

1. A rat-liver supernatant preparation can achieve the biological O-sulphation of l-tyrosylglycine and l-tyrosyl-l-alanine at pH7·0. 2. The optimum concentrations of l-tyrosylglycine and l-tyrosyl-l-alanine in this system are 50mm and 60mm respectively. 3. l-Tyrosylglycine yields two sulphated products, whereas l-tyrosyl-l-alanine yields three sulphated products, when used as acceptor for sulphate in the rat-liver system. 4. With both substrates, one of the sulphated products has been identified as the O-sulphate ester of the corresponding parent peptide.     

Accumulation of d-Glucose from Pentoses by Metabolically Engineered Escherichia coli

Escherichia coli that is unable to metabolize d-glucose (with knockouts in ptsG, manZ, and glk) accumulates a small amount of d-glucose (yield of about 0.01 g/g) during growth on the pentoses d-xylose or l-arabinose as a sole carbon source. Additional knockouts in the zwf and pfkA genes, encoding, respectively, d-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E. coli MEC143), increased accumulation to greater than 1 g/liter d-glucose and 100 mg/liter d-mannose from 5 g/liter d-xylose or l-arabinose. Knockouts of other genes associated with interconversions of d-glucose-phosphates demonstrate that d-glucose is formed primarily by the dephosphorylation of d-glucose-6-phosphate. Under controlled batch conditions with 20 g/liter d-xylose, MEC143 generated 4.4 g/liter d-glucose and 0.6 g/liter d-mannose. The results establish a direct link between pentoses and hexoses and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway.     

Sugar transport across the peritubular face of renal cells of the flounder

The transport of some sugars at the antiluminal face of renal cells was studied using teased tubules of flounder (Pseudopleuronectes americanus). The analytical procedure allowed the determination of both free and total (free plus phosphorylated) tissue sugars. The inulin space of the preparation was 0.333 ± 0.017 kg/kg wet wt (7 animals, 33 analyses). The nonmetabolizable α-methyl-D-glucoside entered the cells by a carrier-mediated (phloridzin-sensitive), ouabain-insensitive process. The steady-state tissue/medium ratio was systematically below that for diffusion equilibrium. D-Glucose was a poor inhibitor of α-methyl-glucoside transport, D-galactose was ineffective. The phloridzin-sensitive transport processes of 2-deoxy-D-glucose,D-galactose,and 2-deoxy-D-galactose were associated with considerable phosphorylation. Kinetic evidence suggested that these sugars were transported in free form and subsequently were phosphorylated. 2-Deoxy-D-glucose accumulated in the cells against a slight concentration gradient. This transport was greatly inhibited by D-glucose, whereas α-methyl-glucoside and also D-galactose and its 2-deoxy-derivative were ineffective. D-Galactose and 2-deoxy-D-galactose mutually competed for transport; D-glucose, 2-deoxy-D-glucose, and α-methyl-D-glucoside were ineffective. Studies using various sugars as inhibitors suggest the presence of three carrier-mediated pathways of sugar transport at the antiluminal cell face of the flounder renal tubule: the pathway of α-methyl-D-glucoside (not shared by D-glucose); the pathway commonly shared by 2-deoxy-D-glucose and D-glucose; the pathway shared by D-galactose and 2-deoxy-D-galactose.     

A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge,Identified in Lactobacillus casei Bacteriophage Endolysins

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala⁴→d-Asx-l-Lys³ in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala⁴→l-Ala-(l-Ala/l-Ser)-l-Lys³; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e. d-Ala⁴→d-Asp-l-Lys³. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys³-d-Asn-l-Lys³ bridges replacing the wild-type 4→3 d-Ala⁴-d-Asn-l-Lys³ bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG.     

Alternative Splicing Modulates Ubx Protein Function in Drosophila melanogaster

Polyploidy is an important aspect of the evolution of flowering plants. The potential of gene copies to diverge and evolve new functions is influenced by meiotic behavior of chromosomes leading to segregation as a single locus or duplicated loci. Switchgrass (Panicum virgatum) linkage maps were constructed using a full-sib population of 238 plants and SSR and STS markers to access the degree of preferential pairing and the structure of the tetraploid genome and as a step toward identification of loci underlying biomass feedstock quality and yield. The male and female framework map lengths were 1645 and 1376 cM with 97% of the genome estimated to be within 10 cM of a mapped marker in both maps. Each map coalesced into 18 linkage groups arranged into nine homeologous pairs. Comparative analysis of each homology group to the diploid sorghum genome identified clear syntenic relationships and collinear tracts. The number of markers with PCR amplicons that mapped across subgenomes was significantly fewer than expected, suggesting substantial subgenome divergence, while both the ratio of coupling to repulsion phase linkages and pattern of marker segregation indicated complete or near complete disomic inheritance. The proportion of transmission ratio distorted markers was relatively low, but the male map was more extensively affected by distorted transmission ratios and multilocus interactions, associated with spurious linkages.POLYPLOIDY is common among plants (Masterson 1994; Levin 2002) and is an important aspect of plant evolution. Widespread paleopolyploidy in flowering plant lineages suggests that ancient polyploidization events have contributed to the radiation of angiosperms (Soltis et al. 2009; Van de Peer et al. 2009a). Whole genome duplications are thought to be the sources of evolutionary novelty (Osborn et al. 2003; Freeling and Thomas 2006; Chen 2007; Hegarty and Hiscock 2008; Flagel and Wendel 2009; Leitch and Leitch 2008). Other attributes of polyploids considered to promote evolutionary success include increased vigor, masking of recessive alleles, and reproductive barriers arising from loss of one of the duplicate genes (Soltis and Soltis 2000; Comai 2005; Otto 2007; Van de Peer et al. 2009b). Among crop species, polyploidy likely contributed to trait improvement under artificial selection (Paterson 2005; Udall and Wendell 2006; Dubcovsky and Dvorak 2007; Hovav et al. 2008).Disomic inheritance in polyploids, in contrast to polysomic inheritance, presents opportunities for duplicated genes to diverge and evolve new functions. The relative age of whole genome duplications and the extent of homology between subgenomes greatly influence chromosomal pairing at meiosis (Soltis and Soltis 1995; Wolfe 2001; Ramsey and Schemske 2002). Polysomic inheritance resulting from random chromosome pairing is associated with doubling of a single set of chromosomes. Disomic inheritance resulting from preferential pairing is often associated with polyploidy arising from combinations of divergent genomes. The evolutionary process of diploidization leads to a shift from random to preferential pairing that is not well understood but is genetically defined in systems such as Ph1 of wheat (Triticum aestivum) and PrBn of Brassica napus (Riley and Chapman 1958; Vega and Feldman 1998; Jenczewski et al. 2003). The degree of preferential pairing also affects allelic diversity and the ability to detect linkage. Accurate information about chromosome pairing and whole or partial genome duplications is thus important for both evolutionary studies and in linkage analysis.Such information is extremely limited in the C4 panicoid species Panicum virgatum (switchgrass), which is now viewed as a promising energy crop in the United States and Europe (Lewandowski et al. 2003; McLaughlin and Kszos 2005) and is planted extensively for forage and soil conservation (Vogel and Jung 2001). Little is known about either its genome structure or inheritance. Much current bioenergy feedstock development is focused on tetraploid cytotypes (2n = 4x = 36) due to their higher yield potentials, and an initial segregation study indicated a high degree of preferential pairing in a single F1 mapping population (Missaoui et al. 2005). A once-dominant component of the tallgrass prairie in North America, switchgrass is largely self-incompatible (Martinez-Reyna and Vogel 2002) with predominantly tetraploid or octoploid cytotypes (Hultquist et al. 1997; Lu et al. 1998). Limited gene flow appears possible between different cytotypes suggested by DNA content variation within collection sites and seed lots (Nielsen 1944; Hultquist et al. 1997; Narasimhamoorthy et al. 2008). True diploids appear to be rare (Nielsen 1944; Young et al. 2010). Multivalents in meiosis have not been observed in tetraploids or F1 hybrids between upland and lowland tetraploids, although rare univalents occurred (Barnett and Carver 1967; Martinez-Reyna et al. 2001). However, polysomic inheritance may occur with random bivalent pairing (Howard and Swaminathan 1953).Sustainable production of switchgrass for bioenergy to meet the goal of reducing greenhouse gas emissions will require advances in feedstock production that include improvements in yield (Carroll and Somerville 2009). Switchgrass has extensive genetic diversity and potential for genetic improvements, but each cycle of phenotypic selection can take several years (McLaughlin and Kszos 2005; Parrish and Fike 2005; Bouton 2007). Detailed understanding of genome structure to enable efficient marker-assisted selection (MAS) can speed this process considerably. Complete linkage maps are therefore required to both understand chromosome pairing and allow MAS.We report the construction of the first complete linkage maps of two switchgrass genotypes. The linkage maps provide genetic evidence for disomic inheritance in lowland, tetraploid switchgrass. Gene-derived markers enabled a comparative analysis to sorghum, revealing syntenic relationships between the diploid sorghum genome and the tetraploid switchgrass subgenomes. Transmission ratio distortion and multilocus interactions were analyzed in detail to document their potential influence on map accuracy and map-based studies in switchgrass.     

The Role of a d-Mannosyl-Lipid as an Intermediate in the Synthesis of Polysaccharide in Phaseolus aureus Seedlings

Particulate preparations from Phaseolus aureus produce a d-mannosyl-lipid when treated with GDP-d-mannose. This lipid complex appears to be an active d-mannose donor, and some investigators have proposed that its role might be an obligatory intermediate in mannan synthesis of higher plants. When the partially purified d-mannosyl-lipids, isotopically labeled in the d-mannose moiety, were treated with particulate enzymes under a variety of conditions, a negligible amount of material was produced that behaved as a polysaccharide. Endogenous, particle-bound d-mannosyl-¹⁴C-lipid prepared from P. aureus particles readily transferred d-mannose to GDP to yield GDP-d-mannose and was hydrolyzed to free d-mannose when treated briefly with 0.01 n HCl at 100 C. The d-mannosyl-lipid, therefore, exhibits active d-mannose transfer potential in its endogenous state. When endogenous glycosyl-lipid was incubated in the absence of GDP-d-mannose-¹⁴C, little or no polysaccharide was produced. It was, instead, slowly degraded to d-mannose. Addition of several different unlabeled sugar nucleotides had no effect on the results. Our studies to date, therefore, offer no evidence that the mannosyl-lipid is an obligatory precursor of polysaccharide.     

Substrate Specificity of a Mannose-6-Phosphate Isomerase from Bacillus subtilis and Its Application in the Production of l-Ribose

The uncharacterized gene previously proposed as a mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme was observed at pH 7.5 and 40°C in the presence of 0.5 mM Co²⁺. The isomerization activity was specific for aldose substrates possessing hydroxyl groups oriented in the same direction at the C-2 and C-3 positions, such as the d and l forms of ribose, lyxose, talose, mannose, and allose. The enzyme exhibited the highest activity for l-ribulose among all pentoses and hexoses. Thus, l-ribose, as a potential starting material for many l-nucleoside-based pharmaceutical compounds, was produced at 213 g/liter from 300-g/liter l-ribulose by mannose-6-phosphate isomerase at 40°C for 3 h, with a conversion yield of 71% and a volumetric productivity of 71 g liter⁻¹ h⁻¹.l-Ribose is a potential starting material for the synthesis of many l-nucleoside-based pharmaceutical compounds, and it is not abundant in nature (5, 19). l-Ribose has been produced mainly by chemical synthesis from l-arabinose, l-xylose, d-glucose, d-galactose, d-ribose, or d-mannono-1,4-lactone (2, 17, 23). Biological l-ribose manufacture has been investigated using ribitol or l-ribulose. Recently, l-ribose was produced from ribitol by a recombinant Escherichia coli containing an NAD-dependent mannitol-1-dehydrogenase (MDH) with a 55% conversion yield when 100 g/liter ribitol was used in a 72-h fermentation (18). However, the volumetric productivity of l-ribose in the fermentation is 28-fold lower than that of the chemical method synthesized from l-arabinose (8). l-Ribulose has been biochemically converted from l-ribose using an l-ribose isomerase from an Acinetobacter sp. (9), an l-arabinose isomerase mutant from Escherichia coli (4), a d-xylose isomerase mutant from Actinoplanes missouriensis (14), and a d-lyxose isomerase from Cohnella laeviribosi (3), indicating that l-ribose can be produced from l-ribulose by these enzymes. However, the enzymatic production of l-ribulose is slow, and the enzymatic production of l-ribose from l-ribulose has been not reported.Sugar phosphate isomerases, such as ribose-5-phosphate isomerase, glucose-6-phosphate isomerase, and galactose-6-phosphate isomerase, work as general aldose-ketose isomerases and are useful tools for producing rare sugars, because they convert the substrate sugar phosphates and the substrate sugars without phosphate to have a similar configuration (11, 12, 21, 22). l-Ribose isomerase from an Acinetobacter sp. (9) and d-lyxose isomerase from C. laeviribosi (3) had activity with l-ribose, d-lyxose, and d-mannose. Thus, we can apply mannose-6-phosphate (EC 5.3.1.8) isomerase to the production of l-ribose, because there are no sugar phosphate isomerases relating to l-ribose and d-lyxose. The production of the expensive sugar l-ribose (bulk price, $1,000/kg) from the rare sugar l-ribulose by mannose-6-phosphate isomerase may prove to be a valuable industrial process, because we have produced l-ribulose from the cheap sugar l-arabinose (bulk price, $50/kg) using the l-arabinose isomerase from Geobacillus thermodenitrificans (20) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Schematic representation for the production of l-ribulose from l-arabinose by G. thermodenitrificans l-arabinose isomerase and the production of l-ribose from l-ribulose by B. subtilis mannose-6-phosphate isomerase.In this study, the gene encoding mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in E. coli. The substrate specificity of the recombinant enzyme for various aldoses and ketoses was investigated, and l-ribulose exhibited the highest activity among all pentoses and hexoses. Therefore, mannose-6-phosphate isomerase was applied to the production of l-ribose from l-ribulose.     

Partial Purification and Properties of l-Glutamine d-Fructose 6-Phosphate Amidotransferase from Phaseolus aureus

l-Glutamine d-fructose 6-phosphate amidotransferase (EC 2.6.1.16) was extracted and purified 600-fold by acetone fractionation and diethylaminoethyl cellulose column chromatography from mung bean seeds (Phaseolus aureus). The partially purified enzyme was highly specific for l-glutamine as an amide nitrogen donor, and l-asparagine could not replace it. The enzyme showed a pH optimum in the range of 6.2 to 6.7 in phosphate buffer. Km values of 3.8 mm and 0.5 mm were obtained for d-fructose 6-phosphate and l-glutamine, respectively. The enzyme was competitively inhibited with respect to d-fructose 6-phosphate by uridine diphosphate-N-acetyl-d-glucosamine which had a Ki value of 13 μm. Upon removal of l-glutamine and its replacement by d-fructose 6-phosphate and storage over liquid nitrogen, the enzyme was completely desensitized to inhibition by uridine diphosphate-N-acetyl-d-glucosamine. This indicates that the inhibitor site is distinct from the catalytic site and that uridine diphosphate-N-acetyl-d-glucosamine acts as a feedback inhibitor of the enzyme.     

Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli

Intracellular thiols like L-cystine and L-cystine play a critical role in the regulation of cellular processes. Here we show that Escherichia coli has two L-cystine transporters, the symporter YdjN and the ATP-binding cassette importer FliY-YecSC. These proteins import L-cystine, an oxidized product of L-cystine from the periplasm to the cytoplasm. The symporter YdjN, which is expected to be a new member of the L-cystine regulon, is a low affinity L-cystine transporter (K _m = 1.1 μM) that is mainly involved in L-cystine uptake from outside as a nutrient. E. coli has only two L-cystine importers because ΔydjNΔyecS mutant cells are not capable of growing in the minimal medium containing L-cystine as a sole sulfur source. Another protein YecSC is the FliY-dependent L-cystine transporter that functions cooperatively with the L-cystine transporter YdeD, which exports L-cystine as reducing equivalents from the cytoplasm to the periplasm, to prevent E. coli cells from oxidative stress. The exported L-cystine can reduce the periplasmic hydrogen peroxide to water, and then generated L-cystine is imported back into the cytoplasm via the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (K _m = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein. The double disruption of ydeD and fliY increased cellular levels of lipid peroxides. From these findings, we propose that the hydrogen peroxide-inducible L-cystine/L-cystine shuttle system plays a role of detoxification of hydrogen peroxide before lipid peroxidation occurs, and then might specific prevent damage to membrane lipids.     

Exact Tests for Hardy–Weinberg Proportions

Exact conditional tests are often required to evaluate statistically whether a sample of diploids comes from a population with Hardy–Weinberg proportions or to confirm the accuracy of genotype assignments. This requirement is especially common when the sample includes multiple alleles and sparse data, thus rendering asymptotic methods, such as the common χ²-test, unreliable. Such an exact test can be performed using the likelihood ratio as its test statistic rather than the more commonly used probability test. Conceptual advantages in using the likelihood ratio are discussed. A substantially improved algorithm is described to permit the performance of a full-enumeration exact test on sample sizes that are too large for previous methods. An improved Monte Carlo algorithm is also proposed for samples that preclude full enumeration. These algorithms are about two orders of magnitude faster than those currently in use. Finally, methods are derived to compute the number of possible samples with a given set of allele counts, a useful quantity for evaluating the feasibility of the full enumeration procedure. Software implementing these methods, ExactoHW, is provided.WHEN studying the genetics of a population, one of the first questions to be asked is whether the genotype frequencies fit Hardy–Weinberg (HW) expectations. They will fit HW if the population is behaving like a single randomly mating unit without intense viability selection acting on the sampled loci. In addition, testing for HW proportions is often used for quality control in genotyping, as the test is sensitive to misclassifications or undetected null alleles. Traditionally, geneticists have relied on test statistics with asymptotic χ²-distributions to test for goodness-of-fit with respect to HW proportions. However, as pointed out by several authors (Elston and Forthofer 1977; Emigh 1980; Louis and Dempster 1987; Hernandez and Weir 1989; Guo and Thompson 1992; Chakraborty and Zhong 1994; Rousset and Raymond 1995; Aoki 2003; Maiste and Weir 2004; Wigginton et al. 2005; Kang 2008; Rohlfs and Weir 2008), these asymptotic tests quickly become unreliable when samples are small or when rare alleles are involved. The latter situation is increasingly common as techniques for detecting large numbers of alleles become widely used. Moreover, loci with large numbers of alleles are intentionally selected for use in DNA identification techniques (e.g., Weir 1992). The result is often sparse-matrix data for which the asymptotic methods cannot be trusted.A solution to this problem is to use an exact test (Levene 1949; Haldane 1954) analogous to Fisher''s exact test for independence in a 2 × 2 contingency table and its generalization to rectangular tables (Freeman and Halton 1951). In this approach, one considers only potential outcomes that have the same allele frequencies as observed, thus greatly reducing the number of outcomes that must be analyzed. One then identifies all such outcomes that deviate from the HW null hypothesis by at least as much the observed sample. The total probability of this subset of outcomes, conditioned on HW and the observed allele frequencies, is then the P-value of the test. When it is not possible to enumerate all outcomes, it is still feasible to approximate the P-value by generating a large random sample of tables.The exact HW test has been used extensively and eliminates the uncertainty inherent in the asymptotic methods (Emigh 1980; Hernandez and Weir 1989; Guo and Thompson 1992; Rousset and Raymond 1995). However, there are two difficulties with the application of this method and its interpretation, both of which are addressed in this report.The first issue is the question of how one decides which of the potential outcomes are assigned to the subset that deviates from HW proportions by as much as or more than the observed sample. If the alternative hypothesis is specifically an excess or a dearth of homozygotes, then the tables can be ordered by Rousset and Raymond''s (1995) U-score or, equivalently, by Robertson and Hill''s (1984) minimum-variance estimator of the inbreeding coefficient. However, when no specific direction of deviation from HW is suspected, then there are several possible test statistics that can be used (Emigh 1980). These include the χ²-statistic, the likelihood ratio (LR), and the conditional probability itself. The last option is by far the most widely used (Elston and Forthofer 1977; Louis and Dempster 1987; Chakraborty and Zhong 1994; Weir 1996; Wigginton et al. 2005) and implemented in the GENEPOP software package (Rousset 2008). The idea of using the null-hypothesis probability as the test statistic was originally suggested in the context of rectangular contingency tables (Freeman and Halton 1951), but this idea has been criticized for its lack of discrimination between the null hypothesis and alternatives (Gibbons and Pratt 1975; Radlow and Alf 1975; Cressie and Read 1989). For example, suppose a particular sample was found to have a very low probability under the null hypothesis of HW. Such a result would usually tend to argue against the population being in HW equilibrium. However, if this particular outcome also has a very low probability under even the best-fitting alternative hypothesis, then it merely implies that a rare event has occurred regardless of whether the population is in random-mating proportions. The first part of this report compares the use of probability vs. the likelihood ratio as the test statistic in HW exact tests. Reasons for preferring the likelihood ratio are presented.The second difficulty in performing HW exact tests is the extensive computation needed for large samples when multiple alleles are involved. In this report I present a new algorithm for carrying out these calculations. This method adapts some of the techniques originally developed for rectangular contingency tables in which each possible outcome is represented as a path through a lattice-like network (Mehta and Patel 1983). Unlike the loop-based method currently in use (Louis and Dempster 1987), the new algorithm uses recursion and can be applied to any number of alleles without modification. In addition, it improves the efficiency by about two orders of magnitude, thus allowing the full enumeration procedure to be applied to larger samples and with greater numbers of alleles.The recursion algorithm has been tested successfully on samples with as many as 20 alleles when most of those alleles are rare. However, there are still some samples for which a complete enumeration is not practical. For example, the data from the human Rh locus in Figure 1D would require examining 2 × 10⁵⁶ tables (see below). For such cases a Monte Carlo approach must be used (Guo and Thompson 1992). Several improvements to the method of independent random tables are suggested here to make that approach practical for even the largest of realistic samples, thus eliminating the need for the less-accurate Markov chain approach.Open in a separate windowFigure 1.—Sample data sets: examples that have been used in previous discussions of exact tests for HW proportions. For each data set, a triangular matrix of genotype counts is shown next to the vector of allele counts. (A) From Table 2, bottom row, of Louis and Dempster (1987). (B) From Figure 2 of Guo and Thompson (1992). (C) From the documentation included with the GENEPOP software package (Rousset 2008). (D) From Figure 5 of Guo and Thompson (1992).Finally, I address the problem of determining the number of tables of genotype counts corresponding to a given set of allele counts. This number is needed for determining whether the exact test can be performed by full enumeration. Previously, this number could not be obtained except by actually carrying out the complete enumeration.The methods described are implemented in a software package, ExactoHW, for MacOS X10.5 or later. It is available in compiled form (supporting information, File S1) or as source code for academic use on request from the author.     

A Genetic Analysis of Mortality in Pigs

An analysis of mortality is undertaken in two breeds of pigs: Danish Landrace and Yorkshire. Zero-inflated and standard versions of hierarchical Poisson, binomial, and negative binomial Bayesian models were fitted using Markov chain Monte Carlo (MCMC). The objectives of the study were to investigate whether there is support for genetic variation for mortality and to study the quality of fit and predictive properties of the various models. In both breeds, the model that provided the best fit to the data was the standard binomial hierarchical model. The model that performed best in terms of the ability to predict the distribution of stillbirths was the hierarchical zero-inflated negative binomial model. The best fit of the binomial hierarchical model and of the zero-inflated hierarchical negative binomial model was obtained when genetic variation was included as a parameter. For the hierarchical binomial model, the estimate of the posterior mean of the additive genetic variance (posterior standard deviation in brackets) at the level of the logit of the probability of a stillbirth was 0.173(0.039) in Landrace and 0.202(0.048) in Yorkshire. The implications of these results from a breeding perspective are briefly discussed.LITTER size has been under selection in the Danish pig breeding program since the early 1990s and this resulted in considerable increase in total number born and also in the proportion of stillborn piglets (Sorensen et al. 2000; Su et al. 2007). A number of studies have reported genetic variation for mortality with heritabilities ranging from 0.03 to 0.12. These studies have either assumed normality of the sampling model for mortality (e.g., van Arendonk et al. 1996) or based inferences on a variety of threshold models (e.g., Roehe and Kalm 2000; Arango et al. 2006), and critical investigations of the quality of fit of the models used were not reported.Mortality data, regarded as a trait of the mother, show typically a large proportion of zeros (many litters do not have stillborn piglets). Formal genetic analyses of mortality in pigs accounting for this feature of the data are not available in the literature and this article attempts to fill this gap. The focus here is to study the quality of fit and predictive ability of a number of models and to investigate whether they provide statistical evidence for genetic variation for mortality. The statistical genetic analysis involves fitting various hierarchical models involving three discrete distributions: the Poisson, the binomial, and the negative binomial.The statistical analysis of counts based on discrete parametric distributions has a long and rich history (Johnson and Kotz 1969). In the case of unbounded counts, Poisson regression models are standard, whereas for bounded counts, when the response can be viewed as the number of successes out of a fixed number of trials, regression models based on the binomial distribution are often used (Hall 2000). A restriction of the Poisson model is that it imposes equality of mean and variance. Typically the distribution of counts is overdispersed. In the case of the binomial model the only free parameter is the probability of success, which results in a functional relationship between the mean and the variance. Several possible alternatives have been suggested to obtain more flexible models. For example, the negative binomial distribution has two parameters and allows the mean and variance to be fitted separately (Lawless 1987). An application of the negative binomial model in animal breeding can be found in Tempelman and Gianola (1996, 1999). In the same spirit, a robust alternative to the binomial model is the beta-binomial, which is a mixture of binomials where the unequal probabilities of success vary according to a beta-distribution. In general, hierarchical specifications are needed to explain extra variation that is not accounted for by the sampling model of the data. These involve assigning a distribution to the parameters of the sampling model, directly, as in the case of the negative binomial or beta-binomial models, or indirectly, by embedding these parameters in a linear structure that includes random effects as explanatory variables.There are situations where overdispersion is partly associated with an incidence of zero counts that is greater than expected under the sampling model, as in the present study. Hurdle models (Mullahy 1986; Winkelmann 2000) and zero-inflated models are two instances of finite mixture models commonly used to account for this characteristic of the data. In the present work the excess of zeros is studied using zero-inflated models that are described in Johnson and Kotz (1969) and extended by Lambert (1992). Ridout et al. (1998) provide a review of various zero-inflated models; recent applications of zero-inflated Poisson models in animal breeding are in Rodriguez-Motta et al. (2007) and in Naya et al. (2008). Zero-inflated models assume that the population consists of two sets of observations. In the first set, which may include zeros, observations are realizations from a discrete sampling process indexed by unknown parameters (this set is often referred to as the imperfect state); observations from the second set consist only of zeros and the parameter of interest is the proportion of these individuals. This set is often referred to as the perfect state. Either or both sets of parameters may depend on covariates.This article is organized as follows. material and methods describes the data, details of the models, and their Markov chain Monte Carlo (MCMC) implementation. This is followed by a presentation of the results of the analyses and of MCMC-driven explorative tools for model comparison. The article concludes with a discussion.     

The oxidation of d- and l-glycerate by rat liver

1. The interconversion of hydroxypyruvate and l-glycerate in the presence of NAD and rat-liver l-lactate dehydrogenase has been demonstrated. Michaelis constants for these substrates together with an equilibrium constant have been determined and compared with those for pyruvate and l-lactate. 2. The presence of d-glycerate dehydrogenase in rat liver has been confirmed and the enzyme has been purified 16–20-fold from the supernatant fraction of a homogenate, when it is free of l-lactate dehydrogenase, with a 23–29% recovery. The enzyme catalyses the interconversion of hydroxypyruvate and d-glycerate in the presence of either NAD or NADP with almost equal efficiency. d-Glycerate dehydrogenase also catalyses the reduction of glyoxylate, but is distinct from l-lactate dehydrogenase in that it fails to act on pyruvate, d-lactate or l-lactate. The enzyme is strongly dependent on free thiol groups, as shown by inhibition with p-chloromercuribenzoate, and in the presence of sodium chloride the reduction of hydroxypyruvate is activated. Michaelis constants for these substrates of d-glycerate dehydrogenase and an equilibrium constant for the NAD-catalysed reaction have been calculated. 3. An explanation for the lowered V_max. with d-glycerate as compared with dl-glycerate for the rabbit-kidney d-α-hydroxy acid dehydrogenase has been proposed.