Population Structure With Localized Haplotype Clusters

We propose a multilocus version of F_ST and a measure of haplotype diversity using localized haplotype clusters. Specifically, we use haplotype clusters identified with BEAGLE, which is a program implementing a hidden Markov model for localized haplotype clustering and performing several functions including inference of haplotype phase. We apply this methodology to HapMap phase 3 data. With this haplotype-cluster approach, African populations have highest diversity and lowest divergence from the ancestral population, East Asian populations have lowest diversity and highest divergence, and other populations (European, Indian, and Mexican) have intermediate levels of diversity and divergence. These relationships accord with expectation based on other studies and accepted models of human history. In contrast, the population-specific F_ST estimates obtained directly from single-nucleotide polymorphisms (SNPs) do not reflect such expected relationships. We show that ascertainment bias of SNPs has less impact on the proposed haplotype-cluster-based F_ST than on the SNP-based version, which provides a potential explanation for these results. Thus, these new measures of F_ST and haplotype-cluster diversity provide an important new tool for population genetic analysis of high-density SNP data.GENOME-WIDE data sets from worldwide panels of individuals provide an outstanding opportunity to investigate the genetic structure of human populations (Conrad et al. 2006; International Hapmap Consortium 2007; Jakobsson et al. 2008; Auton et al. 2009). Populations around the globe form a continuum rather than discrete units (Serre and Paabo 2004; Weiss and Long 2009). However, notions of discrete populations can be appropriate when, for example, ancestral populations were separated by geographic distance or barriers such that little gene flow occurred.F_ST (Wright 1951; Weir and Cockerham 1984; Holsinger and Weir 2009) is a measure of population divergence. It measures variation between populations vs. within populations. One can calculate a global measure, assuming that all populations are equally diverged from an ancestral population, or one can calculate F_ST for specific populations or for pairs of populations while utilizing data from all populations (Weir and Hill 2002). One use of F_ST is to test for signatures of selection (reviewed in Oleksyk et al. 2010).F_ST may be calculated for single genetic markers. For multiallelic markers, such as microsatellites, this is useful, but single-nucleotide polymorphisms (SNPs) contain much less information when taken one at a time, and thus it is advantageous to calculate averages over windows of markers (Weir et al. 2005) or even over the whole genome. The advantage of windowed F_ST is that it can be used to find regions of the genome that show different patterns of divergence, indicative of selective forces at work during human history.Another measure of human evolutionary history is haplotype diversity. Haplotype diversity may be measured using a count of the number of observed haplotypes in a region or by the expected haplotype heterozygosity based on haplotype frequencies in a region. Application of this regional measure to chromosomal data can be achieved by a haplotype block strategy (Patil et al. 2001) or by windowing (Conrad et al. 2006; Auton et al. 2009).One problem with the analysis of population structure based on genome-wide panels of SNPs is that a large proportion of the SNPs were ascertained in Caucasians, potentially biasing the results of the analyses. Analysis based on haplotypes is less susceptible to such bias (Conrad et al. 2006). This is because haplotypes can be represented by multiple patterns of SNPs; thus lack of ascertainment of a particular SNP does not usually prevent observation of the haplotype. On a chromosome-wide scale, one cannot directly use entire haplotypes, because all the haplotypes in the sample will almost certainly be unique, thus providing no information on population structure. Instead one can use haplotypes on a local basis, either by using windows of adjacent markers or by using localized haplotype clusters, for example those obtained from fastPHASE (Scheet and Stephens 2006) or BEAGLE (Browning 2006; Browning and Browning 2007a).Localized haplotype clusters are a clustering of haplotypes on a localized basis. At the position of each genetic marker, haplotypes are clustered according to their similarity in the vicinity of the position. Both fastPHASE and BEAGLE use hidden Markov modeling to perform the clustering, although the specific models used by the two programs differ.Localized haplotype clusters derived from fastPHASE have been used to investigate haplotype diversity, to create neighbor-joining trees of populations, and to create multidimensional scaling (MDS) plots (Jakobsson et al. 2008). It was found that haplotype clusters showed different patterns of diversity to SNPs, while the neighbor-joining and MDS plots were similar between haplotype clusters and SNPs.In this work, we apply windowed F_ST methods to localized haplotype clusters derived from the BEAGLE program (Browning and Browning 2007a,b, 2009). We consider population-average, population-specific, and pairwise F_ST estimates (Weir and Hill 2002). Population-average F_ST''s either assume that all the populations are equally diverged from a common ancestor, which is not realistic, or represent the average of a set of population-specific values. This can be convenient in that the results are summarized by a single statistic; however, information is lost. A common procedure is to calculate F_ST for each pair of populations, and these values reflect the degree of divergence between the two populations. Different levels of divergence are allowed for each pair of populations but each estimate uses data from only that pair of populations. On the other hand, population-specific F_ST''s allow unequal levels of divergence in a single analysis that makes use of all the data.We compare results from the localized haplotype clusters to those using SNPs directly. The results of applying localized haplotype clusters to population-specific F_ST estimation are very striking, showing better separation of populations and a more realistic pattern of divergence than for population-specific F_ST estimation using SNPs directly. We also use BEAGLE''s haplotype clusters in a haplotype diversity measure and investigate the relationship between this measure of haplotype-cluster diversity and the recombination rate.     

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.     

Recharacterization of the mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase as 4-oxo-l-proline reductase (EC 1.1.1.104)

Early studies revealed that chicken embryos incubated with a rare analog of l-proline, 4-oxo-l-proline, showed increased levels of the metabolite 4-hydroxy-l-proline. In 1962, 4-oxo-l-proline reductase, an enzyme responsible for the reduction of 4-oxo-l-proline, was partially purified from rabbit kidneys and characterized biochemically. However, only recently was the molecular identity of this enzyme solved. Here, we report the purification from rat kidneys, identification, and biochemical characterization of 4-oxo-l-proline reductase. Following mass spectrometry analysis of the purified protein preparation, the previously annotated mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase (BDH2) emerged as the only candidate for the reductase. We subsequently expressed rat and human BDH2 in Escherichia coli, then purified it, and showed that it catalyzed the reversible reduction of 4-oxo-l-proline to cis-4-hydroxy-l-proline via chromatographic and tandem mass spectrometry analysis. Specificity studies with an array of compounds carried out on both enzymes showed that 4-oxo-l-proline was the best substrate, and the human enzyme acted with 12,500-fold higher catalytic efficiency on 4-oxo-l-proline than on (R)-β-hydroxybutyrate. In addition, human embryonic kidney 293T (HEK293T) cells efficiently metabolized 4-oxo-l-proline to cis-4-hydroxy-l-proline, whereas HEK293T BDH2 KO cells were incapable of producing cis-4-hydroxy-l-proline. Both WT and KO HEK293T cells also produced trans-4-hydroxy-l-proline in the presence of 4-oxo-l-proline, suggesting that the latter compound might interfere with the trans-4-hydroxy-l-proline breakdown in human cells. We conclude that BDH2 is a mammalian 4-oxo-l-proline reductase that converts 4-oxo-l-proline to cis-4-hydroxy-l-proline and not to trans-4-hydroxy-l-proline, as originally thought. We also hypothesize that this enzyme may be a potential source of cis-4-hydroxy-l-proline in mammalian tissues.     

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.     

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.     

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.     

Depletion of L-arginine induces autophagy as a cytoprotective response to endoplasmic reticulum stress in human T lymphocytes

L-arginine (L-Arg) deficiency results in decreased T-cell proliferation and impaired T-cell function. Here we have found that L-Arg depletion inhibited expression of different membrane antigens, including CD247 (CD3ζ), and led to an ER stress response, as well as cell cycle arrest at G₀/G₁ in both human Jurkat and peripheral blood mitogen-activated T cells, without undergoing apoptosis. By genetic and biochemical approaches, we found that L-Arg depletion also induced autophagy. Deprivation of L-Arg induced EIF2S1 (eIF2α), MAPK8 (JNK), BCL2 (Bcl-2) phosphorylation, and displacement of BECN1 (Beclin 1) binding to BCL2, leading to autophagosome formation. Silencing of ERN1 (IRE1α) prevented the induction of autophagy as well as MAPK8 activation, BCL2 phosphorylation and XBP1 splicing, whereas led T lymphocytes to apoptosis under L-Arg starvation, suggesting that the ERN1-MAPK8 pathway plays a major role in the activation of autophagy following L-Arg depletion. Autophagy was required for survival of T lymphocytes in the absence of L-Arg, and resulted in a reversible process. Replenishment of L-Arg made T lymphocytes to regain the normal cell cycle profile and proliferate, whereas autophagy was inhibited. Inhibition of autophagy by ERN1, BECN1 and ATG7 silencing, or by pharmacological inhibitors, promoted cell death of T lymphocytes incubated in the absence of L-Arg. Our data indicate for the first time that depletion of L-Arg in T lymphocytes leads to a reversible response that preserves T lymphocytes through ER stress and autophagy, while remaining arrested at G₀/G₁. Our data also show that the L-Arg depletion-induced ER stress response could lead to apoptosis when autophagy is blocked.     

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.     

Negative Regulation of AMP-activated Protein Kinase (AMPK) Activity by Macrophage Migration Inhibitory Factor (MIF) Family Members in Non-small Cell Lung Carcinomas

AMP-activated protein kinase (AMPK) is a nutrient- and metabolic stress-sensing enzyme activated by the tumor suppressor kinase, LKB1. Because macrophage migration inhibitory factor (MIF) and its functional homolog, d-dopachrome tautomerase (d-DT), have protumorigenic functions in non-small cell lung carcinomas (NSCLCs) but have AMPK-activating properties in nonmalignant cell types, we set out to investigate this apparent paradox. Our data now suggest that, in contrast to MIF and d-DTs AMPK-activating properties in nontransformed cells, MIF and d-DT act cooperatively to inhibit steady-state phosphorylation and activation of AMPK in LKB1 wild type and LKB1 mutant human NSCLC cell lines. Our data further indicate that MIF and d-DT, acting through their shared cell surface receptor, CD74, antagonize NSCLC AMPK activation by maintaining glucose uptake, ATP production, and redox balance, resulting in reduced Ca²⁺/calmodulin-dependent kinase kinase β-dependent AMPK activation. Combined, these studies indicate that MIF and d-DT cooperate to inhibit AMPK activation in an LKB1-independent manner.     

Stable changes to calcium fluxes in mitochondria isolated from rat livers perfused with α-adrenergic agonists and with glucagon

1. Human uterine cervical stroma was found to contain a Ca²⁺-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein–Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4–8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4×10⁵ by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10μm) or leupeptin (10μm) was also found to be inhibitory, but chymostatin (40μg/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.     

The Cell Shape-determining Csd6 Protein from Helicobacter pylori Constitutes a New Family of l,d-Carboxypeptidase

Helicobacter pylori causes gastrointestinal diseases, including gastric cancer. Its high motility in the viscous gastric mucosa facilitates colonization of the human stomach and depends on the helical cell shape and the flagella. In H. pylori, Csd6 is one of the cell shape-determining proteins that play key roles in alteration of cross-linking or by trimming of peptidoglycan muropeptides. Csd6 is also involved in deglycosylation of the flagellar protein FlaA. To better understand its function, biochemical, biophysical, and structural characterizations were carried out. We show that Csd6 has a three-domain architecture and exists as a dimer in solution. The N-terminal domain plays a key role in dimerization. The middle catalytic domain resembles those of l,d-transpeptidases, but its pocket-shaped active site is uniquely defined by the four loops I to IV, among which loops I and III show the most distinct variations from the known l,d-transpeptidases. Mass analyses confirm that Csd6 functions only as an l,d-carboxypeptidase and not as an l,d-transpeptidase. The d-Ala-complexed structure suggests possible binding modes of both the substrate and product to the catalytic domain. The C-terminal nuclear transport factor 2-like domain possesses a deep pocket for possible binding of pseudaminic acid, and in silico docking supports its role in deglycosylation of flagellin. On the basis of these findings, it is proposed that H. pylori Csd6 and its homologs constitute a new family of l,d-carboxypeptidase. This work provides insights into the function of Csd6 in regulating the helical cell shape and motility of H. pylori.     

l-Glutamate-Dependent Medium Alkalinization by Asparagus Mesophyll Cells : Cotransport or Metabolism?

Mechanically isolated Asparagus sprengeri Regel mesophyll cells cause alkalinization of the suspension medium on the addition of l-glutamate or its analog l-methionine-d,l-sulfoximine. Using a radiolabeled pH probe, it was found that both compounds caused internal acidification whereas l-aspartate did not. Fusicoccin stimulated H⁺ efflux from the cells by 111% and the uptake of l-[U-¹⁴C]glutamate by 55%. Manometric experiments demonstrated that, unlike l-methionine-d,l-sulfoximine, l-glutamate stimulated CO₂ evolution from nonilluminated cells. Simultaneous measurements of medium alkalinization and ¹⁴CO₂ evolution upon the addition of labeled l-glutamate showed that alkalinization was immediate and reached a maximum value after 45 minutes whereas ¹⁴CO₂ evolution exhibited a lag before its appearance and continued in a linear manner for at least 100 minutes. Rates of alkalinization and uptake of l-[U-¹⁴C]glutamate were higher in the light while rates of ¹⁴CO₂ evolution were higher in the dark. The major labeled product of glutamate decarboxylation, γ-aminobutyric acid, was found in the cells and the suspension medium. Its addition to the cell suspension did not result in medium alkalinization and evidence indicates that it is lost from the cell to the medium. The data suggest that the origin of medium alkalinization is co-transport not metabolism, and that the loss of labeled CO₂ and γ-aminobutyric acid from the cell result in an overestimation of the stoichiometry of the H⁺/l-glutamate uptake process.     

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.     

Chirality Matters: Synthesis and Consumption of the d-Enantiomer of Lactic Acid by Synechocystis sp. Strain PCC6803

Both enantiomers of lactic acid, l-lactic acid and d-lactic acid, can be produced in a sustainable way by a photosynthetic microbial cell factory and thus from CO₂, sunlight, and water. Several properties of polylactic acid (a polyester of polymerized lactic acid) depend on the controlled blend of these two enantiomers. Recently, cyanobacterium Synechocystis sp. strain PCC6803 was genetically modified to allow formation of either of these two enantiomers. This report elaborates on the d-lactic acid production achieved by the introduction of a d-specific lactate dehydrogenase from the lactic acid bacterium Leuconostoc mesenteroides into Synechocystis. A typical batch culture of this recombinant strain initially shows lactic acid production, followed by a phase of lactic acid consumption, until production “outcompetes” consumption at later growth stages. We show that Synechocystis is able to use d-lactic acid, but not l-lactic acid, as a carbon source for growth. Deletion of the organism''s putative d-lactate dehydrogenase (encoded by slr1556), however, does not eliminate this ability with respect to d-lactic acid consumption. In contrast, d-lactic acid consumption does depend on the presence of glycolate dehydrogenase GlcD1 (encoded by sll0404). Accordingly, this report highlights the need to match a product of interest of a cyanobacterial cell factory with the metabolic network present in the host used for its synthesis and emphasizes the need to understand the physiology of the production host in detail.     

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 and Sugar Transport in Rabbit Ileum

L-Alanine and 3-O-methyl-D-glucose accumulation by mucosal strips from rabbit ileum has been investigated with particular emphasis on the interaction between Na and these transport processes. L-Alanine is rapidly accumulated by mucosal tissue and intracellular concentrations of approximately 50 mM are reached within 30 min when extracellular L-alanine concentration is 5 mM. Evidence is presented that intracellular alanine exists in an unbound, osmotically active form and that accumulation is an active transport process. In the absence of extracellular Na, the final ratio of intracellular to extracellular L-alanine does not differ significantly from unity and the rate of net uptake is markedly inhibited. Amino acid accumulation is also inhibited by 5 x 10^-5 M ouabain. 3-O-methyl-D-glucose accumulation by this preparation is similarly affected by ouabain and by incubation in a Na-free medium. The effects of amino acid accumulation, of ouabain, and of incubation in a Na-free medium on cell water content and intracellular Na and K concentrations have also been investigated. These results are discussed with reference to the two hypotheses which have been suggested to explain the interaction between Na and intestinal nonelectrolyte transport.     

Physiology and Substrate Specificity of Two Closely Related Amino Acid Transporters,SerP1 and SerP2, of Lactococcus lactis

The serP1 and serP2 genes found adjacently on the chromosome of Lactococcus lactis strains encode two members of the amino acid-polyamine-organocation (APC) superfamily of secondary transporters that share 61% sequence identity. SerP1 transports l-serine, l-threonine, and l-cysteine with high affinity. Affinity constants (K_m) are in the 20 to 40 μM range. SerP2 is a dl-alanine/dl-serine/glycine transporter. The preferred substrate appears to be dl-alanine for which the affinities were found to be 38 and 20 μM for the d and l isomers, respectively. The common substrate l-serine is a high-affinity substrate of SerP1 and a low-affinity substrate of SerP2 with affinity constants of 18 and 356 μM, respectively. Growth experiments demonstrate that SerP1 is the main l-serine transporter responsible for optimal growth in media containing free amino acids as the sole source of amino acids. SerP2 is able to replace SerP1 in this role only in medium lacking the high-affinity substrates l-alanine and glycine. SerP2 plays an adverse role for the cell by being solely responsible for the uptake of toxic d-serine. The main function of SerP2 is in cell wall biosynthesis through the uptake of d-alanine, an essential precursor in peptidoglycan synthesis. SerP2 has overlapping substrate specificity and shares 42% sequence identity with CycA of Escherichia coli, a transporter whose involvement in peptidoglycan synthesis is well established. No evidence was obtained for a role of SerP1 and SerP2 in the excretion of excess amino acids during growth of L. lactis on protein/peptide-rich media.     

Inositol Metabolism in Plants. V. Conversion of Myo-inositol to Uronic Acid and Pentose Units of Acidic Polysaccharides in Root-tips of Zea mays

The metabolism of myo-inositol-2-¹⁴C, d-glucuronate-1-¹⁴C, d-glucuronate-6-¹⁴C, and l-methionine-methyl-¹⁴C to cell wall polysaccharides was investigated in excised root-tips of 3 day old Zea mays seedlings. From myo-inositol, about one-half of incorporated label was recovered in ethanol insoluble residues. Of this label, about 90% was solubilized by treatment, first with a preparation of pectinase-EDTA, then with dilute hydrochloric acid. The only labeled constituents in these hydrolyzates were d-galacturonic acid, d-glucuronic acid, 4-O-methyl-d-glucuronic acid, d-xylose, and l-arabinose, or larger oligosaccharide fragments containing these units. Medium external to excised root-tips grown under sterile conditions in myo-inositol-2-¹⁴C contained labeled polysaccharide.     

The Conserved miR-51 microRNA Family Is Redundantly Required for Embryonic Development and Pharynx Attachment in Caenorhabditis elegans

A major question about cytokinesis concerns the role of the septin proteins, which localize to the division site in all animal and fungal cells but are essential for cytokinesis only in some cell types. For example, in Schizosaccharomyces pombe, four septins localize to the division site, but deletion of the four genes produces only a modest delay in cell separation. To ask if the S. pombe septins function redundantly in cytokinesis, we conducted a synthetic-lethal screen in a septin-deficient strain and identified seven mutations. One mutation affects Cdc4, a myosin light chain that is an essential component of the cytokinetic actomyosin ring. Five others cause frequent cell lysis during cell separation and map to two loci. These mutations and their dosage suppressors define a signaling pathway (including Rho1 and a novel arrestin) for repairing cell-wall damage. The seventh mutation affects the poorly understood RNA-binding protein Scw1 and severely delays cell separation when combined either with a septin mutation or with a mutation affecting the septin-interacting, anillin-like protein Mid2, suggesting that Scw1 functions in a pathway parallel to that of the septins. Taken together, our results suggest that the S. pombe septins participate redundantly in one or more pathways that cooperate with the actomyosin ring during cytokinesis and that a septin defect causes septum defects that can be repaired effectively only when the cell-integrity pathway is intact.THE fission yeast Schizosaccharomyces pombe provides an outstanding model system for studies of cytokinesis (McCollum and Gould 2001; Balasubramanian et al. 2004; Pollard and Wu 2010). As in most animal cells, successful cytokinesis in S. pombe requires an actomyosin ring (AMR). The AMR begins to assemble at the G₂/M transition and involves the type II myosin heavy chains Myo2 and Myp2 and the light chains Cdc4 and Rlc1 (Wu et al. 2003). Myo2 and Cdc4 are essential for cytokinesis under all known conditions, Rlc1 is important at all temperatures but essential only at low temperatures, and Myp2 is essential only under stress conditions. As the AMR constricts, a septum of cell wall is formed between the daughter cells. The primary septum is sandwiched by secondary septa and subsequently digested to allow cell separation (Humbel et al. 2001; Sipiczki 2007). Because of the internal turgor pressure of the cells, the proper assembly and structural integrity of the septal layers are essential for cell survival.Septum formation involves the β-glucan synthases Bgs1/Cps1/Drc1, Bgs3, and Bgs4 (Ishiguro et al. 1997; Le Goff et al. 1999; Liu et al. 1999, 2002; Martín et al. 2003; Cortés et al. 2005) and the α-glucan synthase Ags1/Mok1 (Hochstenbach et al. 1998; Katayama et al. 1999). These synthases are regulated by the Rho GTPases Rho1 and Rho2 and the protein kinase C isoforms Pck1 and Pck2 (Arellano et al. 1996, 1997, 1999; Nakano et al. 1997; Hirata et al. 1998; Calonge et al. 2000; Sayers et al. 2000; Ma et al. 2006; Barba et al. 2008; García et al. 2009b). The Rho GTPases themselves appear to be regulated by both GTPase-activating proteins (GAPs) and guanine-nucleotide-exchange factors (GEFs) (Nakano et al. 2001; Calonge et al. 2003; Iwaki et al. 2003; Tajadura et al. 2004; Morrell-Falvey et al. 2005; Mutoh et al. 2005; García et al. 2006, 2009a,b). In addition, septum formation and AMR function appear to be interdependent. In the absence of a normal AMR, cells form aberrant septa and/or deposit septal materials at random locations, whereas a mutant defective in septum formation (bgs1) is also defective in AMR constriction (Gould and Simanis 1997; Le Goff et al. 1999; Liu et al. 1999, 2000). Both AMR constriction and septum formation also depend on the septation initiation network involving the small GTPase Spg1 (McCollum and Gould 2001; Krapp and Simanis 2008). Despite this considerable progress, many questions remain about the mechanisms and regulation of septum formation and its relationships to the function of the AMR.One major question concerns the role(s) of the septins. Proteins of this family are ubiquitous in fungal and animal cells and typically localize to the cell cortex, where they appear to serve as scaffolds and diffusion barriers for other proteins that participate in a wide variety of cellular processes (Longtine et al. 1996; Gladfelter et al. 2001; Hall et al. 2008; Caudron and Barral 2009). Despite the recent progress in elucidating the mechanisms of septin assembly (John et al. 2007; Sirajuddin et al. 2007; Bertin et al. 2008; McMurray and Thorner 2008), the details of septin function remain obscure. However, one prominent role of the septins and associated proteins is in cytokinesis. Septins concentrate at the division site in every cell type that has been examined, and in Saccharomyces cerevisiae (Hartwell 1971; Longtine et al. 1996; Lippincott et al. 2001; Dobbelaere and Barral 2004) and at least some Drosophila (Neufeld and Rubin 1994; Adam et al. 2000) and mammalian (Kinoshita et al. 1997; Surka et al. 2002) cell types, the septins are essential for cytokinesis. In S. cerevisiae, the septins are required for formation of the AMR (Bi et al. 1998; Lippincott and Li 1998). However, this cannot be their only role, because the AMR itself is not essential for cytokinesis in this organism (Bi et al. 1998; Korinek et al. 2000; Schmidt et al. 2002). Moreover, there is no evidence that the septins are necessary for AMR formation or function in any other organism. A further complication is that in some cell types, including most Caenorhabditis elegans cells (Nguyen et al. 2000; Maddox et al. 2007) and some Drosophila cells (Adam et al. 2000; Field et al. 2008), the septins do not appear to be essential for cytokinesis even though they localize to the division site.S. pombe has seven septins, four of which (Spn1, Spn2, Spn3, and Spn4) are expressed in vegetative cells and localize to the division site shortly before AMR constriction and septum formation (Longtine et al. 1996; Berlin et al. 2003; Tasto et al. 2003; Wu et al. 2003; An et al. 2004; Petit et al. 2005; Pan et al. 2007; Onishi et al. 2010). Spn1 and Spn4 appear to be the core members of the septin complex (An et al. 2004; McMurray and Thorner 2008), and mutants lacking either of these proteins do not assemble the others at the division site. Assembly of a normal septin ring also depends on the anillin-like protein Mid2, which colocalizes with the septins (Berlin et al. 2003; Tasto et al. 2003). Surprisingly, mutants lacking the septins are viable and form seemingly complete septa with approximately normal timing. These mutants do, however, display a variable delay in separation of the daughter cells, suggesting that the septins play some role(s) in the proper completion of the septum or in subsequent processes necessary for cell separation (Longtine et al. 1996; An et al. 2004; Martín-Cuadrado et al. 2005).It is possible that the septins localize to the division site and yet are nonessential for division in some cell types because their role is redundant with that of some other protein(s) or pathway(s). To explore this possibility in S. pombe, we screened for mutations that were lethal in combination with a lack of septins. The results suggest that the septins cooperate with the AMR during cytokinesis and that, in the absence of septin function, the septum is not formed properly, so that an intact system for recognizing and repairing cell-wall damage becomes critical for cell survival.