Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli

ABSTRACT: BACKGROUND: Escherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli. RESULTS: Because the redox enzymes can reduce the disulfide that forms on proteins, wefirst tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coli L-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (cysI and cysJ) and the L-cysteine synthase gene (cysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (cysC or cysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coli L-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell . CONCLUSIONS: In this work, we showed that Grx1 and NrdH reduce SSC to L-cysteine, and the generated sulfite is then utilized as the sulfur source to produce additional L-cysteine molecule through the sulfate pathway in E. coli. We also found that co-overexpression of NrdH, CysI, and CysK increases L-cysteine production. Our results propose that the enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from SSC is a novel method for improvement of L-cysteine production.     

Genotyping by allele-specific L-DNA-tagged PCR

We have developed a novel method for typing single-nucleotide polymorphisms (SNPs) that can be applicable to rapid screening. The method involves the fusion of two PCR techniques, allele-specific PCR (AS-PCR) and L-DNA-tagged PCR (LT-PCR), which enables us to label PCR products with sequence-defined tags of mirror-image DNA (L-DNA). PCR products were applied without any purification or denaturation steps to gold surfaces where complementary single-stranded L-DNA was immobilized, and the products were detected with surface plasmon resonance (SPR) imaging. We were able to clearly discriminate 3 genotypes at position 2677 of the MDR1 gene (G/G-homozygote, G/T-heterozygote, and T/T-homozygote) by comparing SPR difference images.     

Migration of mesenchymal cell fated to blastema is necessary for fish fin regeneration

Urodeles and fish have higher regeneration ability in a variety of tissues and organs than do other vertebrate species including mammals. Though many studies have aimed at identifying the cellular and molecular basis for regeneration, relatively little is known about the detailed cellular behaviors and involved molecular basis. In the present study, a small molecule inhibitor was used to analyzed the role of phosphoinositide 3-kinase (PI3K) signaling during regeneration. We showed that the inhibitor disrupted the formation of blastema including the expression of characteristic genes. The failure of blastema formation was due to the impaired migration of mesenchymal cells to the distal prospective blastema region, although it had a little affect on cell cycle activation in mesenchymal cells. Moreover, we found that the epidermal remodeling including cell proliferation, distal cell migration and Akt phosphorylation was also affected by the inhibitor, implying a possible involvement of epidermis for proper formation of blastema. From these data, we propose a model in which distinct signals that direct the cell cycle activation, mesenchymal cell migration and epidermal remodeling coordinate together to accomplish the correct blastema formation and regeneration.     

Affixin activates Rac1 via betaPIX in C2C12 myoblast

Affixin/beta-parvin is an integrin-linked kinase (ILK)-binding focal adhesion protein highly expressed in skeletal muscle and heart. To elucidate the possible role of affixin in skeletal muscle, we established stable C2C12 cell line expressing T7-tagged human affixin (C2C12-affixin cells). Exogenous expression of affixin promotes lamellipodium formation where affixin, ILK alphap21-activated kinase (PAK)-interactive exchange factor (PIX) and betaPIX accumulate. The association of affixin and betaPIX was confirmed by immunoprecipitation and pull down assay. In C2C12-affixin cells, an increased level of activated Rac1 but not Cdc42 was observed, and mutant betaPIX lacking guanine nucleotide exchange factor activity inhibited lamellipodium formation. These results suggest that affixin is involved in reorganization of subsarcolemmal cytoskeletal actin by activation of Rac1 through alpha and betaPIXs in skeletal muscle.     

Direct monitoring of in vivo ER stress during the development of insulin resistance with ER stress-activated indicator transgenic mice

Type 2 diabetes is one of the most prevalent and serious metabolic diseases in the world, and insulin resistance and pancreatic β-cell dysfunction are the hallmarks of the disease. It has been suggested that endoplasmic reticulum (ER) stress is provoked under diabetic conditions and is possibly involved in the development of insulin resistance. In this study, using ER stress-activated indicator (ERAI) transgenic mice which express green fluorescent protein (GFP) under ER stress conditions, we directly monitored in vivo ER stress in various insulin target tissues such as liver, fat, and muscle in diabetic mice with insulin resistance induced by high fat and high sucrose (HF/HS) diet treatment. In the liver of the ERAI transgenic mice, ERAI fluorescence activity was clearly observed as early as after 4 weeks of HF/HS diet treatment, whereas it was not detected at all in the fat and muscle even after 12 weeks of HF/HS diet treatment. These results suggest that induction of ER stress is associated with the development of insulin resistance and that ER stress in the liver may facilitate the development of insulin resistance in the whole body. This is the first report to directly monitor in vivo ER stress in various insulin target tissues during the development of insulin resistance. In addition, our present results suggest that ERAI transgenic mice are very useful for evaluating in vivo ER stress, especially in the liver, during the development of insulin resistance.     

Muscle pump-dependent self-perfusion mechanism in legs in normal subjects and patients with heart failure.

Leg venous pressure markedly falls during upright exercise via a muscle pump effect, creating de novo perfusion pressure. We examined physiological roles of this mechanism in increasing femoral artery blood flow (FABF) and its alterations in chronic heart failure (CHF). In 10 normal subjects and 10 patients with CHF, standard hemodynamic variables, mean ankle vein pressure (MAVP), and FABF with Doppler techniques were obtained during graded upright bicycle exercise. To evaluate a nonspecific blood flow response, normal subjects also performed supine exercise. In normal subjects, MAVP rapidly declined by 45 mmHg and FABF correspondingly increased 5.3-fold without a systemic pressor response during 10 s of light upright exercise at 5 W. Approximately 67% of the blood flow response was attributed to the venous pressure drop-dependent mechanism. In CHF patients, MAVP declined by only 36 mmHg and FABF increased only 1.7-fold during the same upright exercise. The muscle venous pump has an ability to increase FABF at least threefold via the venous pressure drop-dependent mechanism. This mechanism is impaired in CHF patients.     

Occurrence of N-methyl- -aspartate in bivalves and its distribution compared with that of N-methyl- -aspartate and , -aspartate

The presence of N-methyl- -aspartate (NMLA) was demonstrated in bivalves, Corbicula sandai and Tapes japonica. To our knowledge, this is the first report on the occurrence of NMLA in animal tissues. NMLA in bivalve tissues was identified according to the following findings; (a) its derivatives with (+)- and (−)- 1-(9-fluorenyl)ethyl chloroformate (FLEC) behaved identically with those of authentic NMLA, respectively, on high-performance liquid chromatography (HPLC), (b) its derivatives with (+)- and (−)- FLEC behaved identically with (−)- and (+)-FLEC derivatives of authentic N-methyl- -aspartate (NMDA), respectively, on HPLC and (c) its behavior on thin-layer chromatography was the same as those of authentic NMLA. We also describe the distribution of NMDA, and - and -aspartate, to which N-methylaspartate enantiomers are structurally related. NMDA was more widely dirtributed than NMLA in bivalves. These bivalves containing NMLA showed lower -aspartate contents and /( + ) ratios of aspartate, than the bivalves containing NMDA.     

Dynamic mechanism of the self-assembly process of tobacco mosaic virus protein studied by rapid temperature-jump small-angle X-ray scattering using synchrotron radiation

The self-assembly process of tobacco mosaic virus protein (TMVP) was observed by rapid temperature-jump time-resolved solution X-ray small-angle scattering using synchrotron radiation. The temperature-jump device used for the X-ray measurements is rapid enough to cope with even the fastest-assembling process of TMVP, and accumulates data of reasonable signal-to-noise ratios with a minimum total counting time of 7.5 seconds. The measurements suggested that the 20 S disk of TMVP polymerized to stacked disks (short rods). The time to complete stacking varied from approximately 25 seconds to approximately 1200 seconds, depending on the solution condition and magnitude of the temperature gap. Higher protein concentration, ionic strength and temperature favoured faster association. The results were analysed in terms of a set of kinetic equations that describe the two-stage aggregation of TMVP with an equilibrium constant K1, and two rate constants k+2 and k-2 for association and dissociation of disks, respectively. The consistency of the analysis suggests that the TMVP assembly proceeds in two steps of: (1) the aggregation of A-proteins into double-layered disks; and (2) the stacking of double-layered disks. The kinetic analysis indicated that the stacking belongs to the lowest range of protein-protein interaction system.