Determinants of redox sensitivity in RsrA, a zinc-containing anti-sigma factor for regulating thiol oxidative stress response

Various environmental oxidative stresses are sensed by redox-sensitive regulators through cysteine thiol oxidation or modification. A few zinc-containing anti-sigma (ZAS) factors in actinomycetes have been reported to respond sensitively to thiol oxidation, among which RsrA from Streptomyces coelicolor is best characterized. It forms disulfide bonds upon oxidation and releases bound SigR to activate thiol oxidative stress response genes. Even though numerous ZAS proteins exist in bacteria, features that confer redox sensitivity to a subset of these have been uncharacterized. In this study, we identified seven additional redox-sensitive ZAS factors from actinomycetes. Comparison with redox-insensitive ZAS revealed characteristic sequence patterns. Domain swapping demonstrated the significance of the region K(33)FEHH(37)FEEC(41)SPC(44)LEK(47) that encompass the conserved HX(3)CX(2)C (HCC) motif. Mutational effect of each residue on diamide responsive induction of SigR target genes in vivo demonstrated that several residues, especially those that flank two cysteines (E39, E40, L45, E46), contribute to redox sensitivity. These residues are well conserved among redox-sensitive ZAS factors, and hence are proposed as redox-determinants in sensitive ZAS. H37A, C41A, C44A and F38A mutations, in contrast, compromised SigR-binding activity significantly, apparently affecting structural integrity of RsrA. The residue pattern around HCC motif could therefore serve as an indicator to predict redox-sensitive ZAS factors from sequence information.     

Effects of Adrenalectomy and Replacement Therapy of Corticosterone on Cell Proliferation and Neuroblast Differentiation in the Rat Dentate Gyrus

Newly generated neurons in the dentate gyrus differentiate into mature granule cells. In the present study, we observed the effects of adrenalectomy (ADX) and corticosterone replacement therapy (CRT) on cell death, cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus (SZDG). For this, the animals received vehicle or CRT after ADX, and were sacrificed 5 or 42 days later. Plasma corticosterone levels were very low in the adrenalectomized groups, whereas CRT after ADX significant increased serum corticosterone levels at 42 days, not 5 days, after ADX. ADX induced some neuronal damage in the dentate gyrus at 5 days post-ADX. CRT did not significantly reduce the neuronal damage at 5 days post-ADX; however, neuronal damage was not shown at 42 post-ADX with CRT. Ki67 (a marker for cell proliferation) and doublecortin (DCX, a marker for neuronal differentiation) immunoreaction was detected in the SZDG. ADX transiently increased cell proliferation and neuroblast differentiation 5 days after ADX, not 42 days, after ADX, and the CRT 42 days after ADX prominently decreased cell proliferation and neuroblast differentiation in the dentate gyrus. These results suggest that adrenal corticosteroid hormone is not essential for cell proliferation and neuroblast differentiation in long-term period after ADX.     

Differential effects of treadmill exercise in early and chronic diabetic stages on parvalbumin immunoreactivity in the hippocampus of a rat model of type 2 diabetes

In the present study, we investigated the effects of treadmill exercise in early and chronic diabetic stages on parvalbumin (PV) immunoreactivity in the subgranular zone of the dentate gyrus of Zucker diabetic fatty (ZDF) and its lean control rats (ZLC). To investigate the effects, ZLC and ZDF rats at 6 or 23 weeks of age were put on a treadmill with or without running for 1 h/day/5 consecutive days at 16–22 m/min for 5 weeks or 12–16 m/min for 7 weeks, respectively. Physical exercise in pre-diabetic rats prevented onset of diabetes, while exercise in rats at chronic diabetic stage significantly reduced blood glucose levels. In addition, physical exercise in the pre-diabetic rats significantly increased PV immunoreactive fibers in the strata oriens and radiatum of the CA1-3 region and in the polymorphic and molecular layers of the dentate gyrus compared to that in sedentary controls. However, in rats at chronic stages, PV immunoreactivity was slightly increased in the CA1-3 region as well as in the dentate gyrus compared to that in the sedentary controls. These results suggest that physical exercise has differential effects on blood glucose levels and PV immunoreactivity according to diabetic stages. Early exercise improves diabetic phenotype and PV immunoreactive fibers in the rat hippocampus.     

Comparison of GAD65 and 67 Immunoreactivity in the Lumbar Spinal Cord Between Young Adult and Aged Dogs

We investigated distribution and age-related changes in two isoforms of GABA synthesizing enzymes, glutamic acid decarboxylase (GAD) 65 and 67, in the lumbar levels (L(5)-L(6)) of the dog spinal cord. Male German shepherds were used at 1-2 years (young adult dogs) and 10-12 years (aged dogs) of age. GAD65 immunoreaction was observed in neuropil, not in cell bodies, in all laminae of the adult lumbar spinal cord: Many punctate GAD65-immunoreactive structures were shown in all laminae. The density of GAD65 immunoreactive structures was highest in laminae I-III, and lowest in lamina VII. In the aged dog, the distribution pattern of GAD65 immunoreactivity was similar to that in the adult dog; however the density of GAD65-immunoreactive structures and its protein levels were significantly increased in the aged lumbar spinal cord. GAD67 immunoreaction in the adult dog was also distributed in all laminae of the lumbar spinal cord like GAD65; however, we found that small GAD67-immunoreactive cell bodies were observed in laminae II, III and VIII. In the aged dogs, GAD67 immunoreactivity and its protein levels were also increased compared to those in the adult group. In conclusion, our results indicate that the distribution of GAD65-immunoreactive structures is different from GAD67-immunoreactive structures and that their immunoreactivity in the aged dogs is much higher than the adult dogs.     

Comparison of Glucocorticoid Receptor and Ionized Calcium-Binding Adapter Molecule 1 Immunoreactivity in the Adult and Aged Gerbil Hippocampus Following Repeated Restraint Stress

Stress leads to changes in homeostasis and internal balance of the body and is known to be one of important factors in the development of several diseases. In the present study, we investigated changes in glucocorticoid receptor (GR) and ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactivity in the adult and aged gerbil hippocampus after chronic restraint stress. Serum corticosterone level was much higher in both the stress-groups than the control groups. No neuronal death was found in all hippocampal subregions of the adult and aged gerbil after chronic restraint stress. GR immunoreactivity was decreased in both the adult and aged groups after repeated restraint stress; however, GR immunoreactivity in the adult-stress-group was decreased much more than that in the aged-stress-group. Iba-1 immunoreactive microglia were hypertrophied and activated in the adult group after repeated restraint stress; in the aged-stress-group, there was no any significant change in Iba-1 immunoreactive microglia. In brief, level of GR, not Iba-1, in the hippocampus was much decreased in the adult gerbil compared to the aged gerbil following chronic restraint stress.     

Regulation of intestinal stem cell proliferation by human methyl-CpG-binding protein-2 in Drosophila

Recent studies have suggested the involvement of epigenetic factors such as methyl-CpG-binding protein-2 (MeCP2) in tumorigenesis. In addition, cancer may represent a stem cell-based disease, suggesting that understanding of stem cell regulation could provide valuable insights into the mechanisms of tumorigenesis. However, the function of epigenetic factors in stem cell regulation in adult tissues remains poorly understood. In the present study, we investigated the role of human MeCP2 (hMeCP2), a bridge factor linked to DNA modification and histone modification, in stem cell proliferation using adult Drosophila midgut, which appears to be an excellent model system to study stem cell biology. Results show that enterocyte (EC)-specific expression of hMeCP2 in adult midgut using an exogenous GAL4/UAS expression system induced intestinal stem cell (ISC) proliferation marked by staining with anti-phospho-histone H3 antibody and BrdU incorporation assays. In addition, hMeCP2 expression in ECs activated extracellular stress-response kinase signals in ISCs. Furthermore, expression of hMeCP2 modulated the distribution of heterochromatin protein-1 in ECs. Our data suggests the hypothesis that the expression of hMeCP2 in differentiated ECs stimulates ISC proliferation, implying a role of MeCP2 as a stem cell regulator.     

Novel enzymatic production of trehalose from sucrose using amylosucrase and maltooligosyltrehalose synthase-trehalohydrolase

Trehalose (α-d-glucopyranosyl α-d-glucopyranoside) is an important non-reducing disaccharide used in the food industry due to its mild sweetness (45% that of sucrose), low cariogenicity, high glass transition temperature, low hygroscopicity, and protein protection properties. In this study, we accomplished the production of trehalose from sucrose as a sole substrate using a novel dual-enzyme system, in which amylosucrase (ASase) and maltooligosyltrehalose synthase-trehalohydrolase (MTSH) fusion enzyme were employed. The biotransformation of sucrose to trehalose was confirmed by high-performance anion-exchange chromatography (HPAEC) analysis. Trehalose was successfully produced by both simultaneous and sequential methods by using ASase and MTSH. A higher trehalose production yield (3.15 ± 0.83 mM trehalose/20 mM sucrose) was observed in the sequential method than the simultaneous method (1.43 ± 0.14 mM trehalose/20 mM sucrose), indicating that the production of maltooligosaccharides from sucrose by ASase was an important step in the biosynthesis of trehalose.     

A novel alkaline lipase from Ralstonia with potential application in biodiesel production

With the aim of isolating a biocatalyst able to catalyze biodiesel production from microbial source, Ralstonia sp. CS274 was isolated and a lipase from the strain (RL74) was purified. Molecular weight of RL74 was estimated to be 28,000 Da by SDS-PAGE. The activity was highest at 50-55 °C and pH 8.0-9.5 and was stable at pH 7.0-12.0 and up to 45 °C. It was resistant to oxidizing and reducing agents and the activity was enhanced by detergents. RL74 was 1,3 specific and K_m and V_max for p-nitrophenyl palmitate were 2.73 ± 0.6 mM and 101.4 ± 1.9 mM/min mg, respectively. N-terminal amino acid sequence showed partial homology with that of Penicillium lipases. RL74 produced biodiesel more efficiently in palm oil than in soybean oil; and the production was highest at pH 8.0, at 5% methanol and at 20% water content.     

Characterization of 2-octenoyl-CoA carboxylase/reductase utilizing pteB from Streptomyce avermitilis

The filipin biosynthetic gene cluster of Streptomyces avermitilis contains pteB, a homolog of crotonyl-CoA carboxylase/reductase. PteB was predicted to be 2-octenoyl-CoA carboxylase/reductase, supplying hexylmalonyl-CoA to filipin biosynthesis. Recombinant PteB displayed selective reductase activity toward 2-octenoyl-CoA while generating a broad range of alkylmalonyl-CoAs in the presence of bicarbonate.