Cloning, Expression, and Characterization of a Methionyl Aminopeptidase from a Hyperthermophilic Archaeon Thermococcus sp. NA1

Genomic analysis of a hyperthermophilic archaeon Thermococcus sp. NA1 revealed the presence of an 885-bp open reading frame encoding a protein of 295 amino acids with a calculated molecular mass of 32,981 Da. Analysis of the deduced amino acid sequence showed that amino acid residues important for catalytic activity and the metal binding ligands conserved in all of methionyl aminopeptidases (MetAP) were also conserved and belonged to type IIa MetAP. The protein, designated TNA1_MetAP (Thermococcus sp. NA1 MetAP), was cloned and expressed in Escherichia coli. The recombinant enzyme was a Mn²⁺-, Ni²⁺-, Fe²⁺-, or Co²⁺-dependent metallopeptidase. Optimal MetAP activity against l-methionine p-nitroanilide (Met-pNA) (K _m = 0.68 mM) occurred at pH 7.0 and 80 to 90°C. The MetAP was very unstable compared to Pyrococcus furiosus MetAP, which was completely inactivated by heating at 80°C for 5 min. It seemed likely that the cysteine residue (Cys53) played a critical role in regulating the thermostability of TNA1_MetAP.     

Effect of leptin on insulin resistance of muscle--direct or indirect?

We examined the effect of leptin on the insulin resistance in skeletal muscles by measuring glucose transport. Male Wistar rats were fed rat chow or high-fat diets for 30 days. Before sacrifice, rats fed high-fat diet were subcutaneously injected with leptin (1 mg/kg b.w.) for 3 days. The glucose transport in epitrochlearis and soleus muscles did not differ in the experimental groups under basal conditions, however these values decreased significantly in the rats fed high-fat diet under insulin stimulation (p<0.01). Leptin treatment recovered the decreased glucose transport in epitrochlearis (p<0.05) and soleus muscles (p=0.08). Triglyceride concentrations in soleus muscles were increased significantly in the rats fed high-fat diet as compared to rats fed chow diet (p<0.01), and were decreased significantly by leptin treatment (p<0.01). The glucose transport was measured under basal conditions and after 60 microU/ml of insulin treatment with or without 50 ng/ml of leptin. Leptin had no direct stimulatory effect on glucose transport under both basal and insulin-stimulated conditions in vitro. These results demonstrate that leptin injection to rats fed high-fat diet recovered impaired insulin responsiveness of skeletal muscles and muscle triglyceride concentrations. However, there was no direct stimulatory effect of leptin on insulin sensitivity of skeletal muscles in vitro.     

Hydrogen sulfide inhibits nitric oxide production and nuclear factor-kappaB via heme oxygenase-1 expression in RAW264.7 macrophages stimulated with lipopolysaccharide

Hydrogen sulfide (H(2)S), a regulatory gaseous molecule that is endogenously synthesized by cystathionine gamma-lyase (CSE) and/or cystathionine beta-synthase (CBS) from L-cysteine (L-Cys) metabolism, is a putative vasodilator, and its role in nitric oxide (NO) production is unexplored. Here, we show that at noncytotoxic concentrations, H(2)S was able to inhibit NO production and inducible NO synthase (iNOS) expression via heme oxygenase (HO-1) expression in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS). Both H(2)S solution prepared by bubbling pure H(2)S gas and NaSH, a H(2)S donor, dose dependently induced HO-1 expression through the activation of the extracellular signal-regulated kinase (ERK). Pretreatment with H(2)S or NaHS significantly inhibited LPS-induced iNOS expression and NO production. Moreover, NO production in LPS-stimulated macrophages that are expressing CSE mRNA was significantly reduced by the addition of L-Cys, a substrate for H(2)S, but enhanced by the selective CSE inhibitor beta-cyano-L-alanine but not by the CBS inhibitor aminooxyacetic acid. While either blockage of HO activity by the HO inhibitor, tin protoporphyrin IX, or down-regulation of HO-1 expression by HO-1 small interfering RNA (siRNA) reversed the inhibitory effects of H(2)S on iNOS expression and NO production, HO-1 overexpression produced the same inhibitory effects of H(2)S. In addition, LPS-induced nuclear factor (NF)-kappaB activation was diminished in RAW264.7 macrophages preincubated with H(2)S. Interestingly, the inhibitory effect of H(2)S on NF-kappaB activation was reversed by the transient transfection with HO-1 siRNA, but was mimicked by either HO-1 gene transfection or treatment with carbon monoxide (CO), an end product of HO-1. CO treatment also inhibited LPS-induced NO production and iNOS expression via its inactivation of NF-kappaB. Collectively, our results suggest that H(2)S can inhibit NO production and NF-kappaB activation in LPS-stimulated macrophages through a mechanism that involves the action of HO-1/CO.     

Effect of heat treatment on the antioxidative and antigenotoxic activity of extracts from persimmon (Diospyros kaki L.) peel

Heat treatment of persimmon peel (PP) increased the antioxidative activity of the 70% ethanolic extract (EE) and water extract (WE) from PP. EE and WE both prevented H2O2-induced DNA damage to human peripheral lymphocytes. The antioxidative and antigenotoxic activities of the PP extracts were significantly affected by heating.     

Distribution of AMPA glutamate receptor GluR1 subunit-immunoreactive neurons and their co-localization with calcium-binding proteins and GABA in the mouse visual cortex

The neuronal localization of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor (GluR) subunits is vital as they play key roles in the regulation of calcium permeability. We have examined the distribution of the calcium permeable AMPA glutamate receptor subunit GluR1 in the mouse visual cortex immunocytochemically. We compared this distribution to that of the calcium-binding proteins calbindin D28K, calretinin, and parvalbumin, and of GABA. The highest density of GluR1-immunoreactive (IR) neurons was found in layers II/III. Enucleation appeared to have no effect on the distribution of GluR1-IR neurons. The labeled neurons varied in morphology; the majority were round or oval and no pyramidal cells were labeled by the antibody. Two-color immunofluorescence revealed that 26.27%, 10.65%, and 40.31% of the GluR1-IR cells also contained, respectively, calbindin D28K, calretinin, and parvalbumin. 20.74% of the GluR1-IR neurons also expressed GABA. These results indicate that many neurons that express calcium-permeable GluR1 also express calcium binding proteins. They also demonstrate that one fifth of the GluR1-IR neurons in the mouse visual cortex are GABAergic interneurons.     

AnArabidopsis short root and dwarfism mutant defines a novel locus that mediates both cell division and elongation

Arabidopsis leaf morphology is determined by the coordinated action of cell division and elongation. Of all the hormones that control leaf shape, the brassinosteroids (BRs) are active components in this process. BRs are a group of plant-originated steroidal compounds that induce growth along the long axes of organs. Here, we report the isolation and characterization of a novel mutant,short root and dwarfism (srd). Its dwarf phenotype includes round and curled leaves, reduced fertility, and short hypocotyls in the light and dark. Dwarfism in the aerial portions and a short-root morphology are not rescued by exogenous application of BRs, suggesting thatsrd is not impaired in BR metabolic pathways. Anatomical analysis revealed thatsrd roots are much shorter and thicker than the wild type due to additional layers of cortical cells. A lack of cell elongation but an increase in division results in these short but horizontally swollen roots. A double mutantsrd/bri1-5 also displays the short-root phenotype, implying thatsrd is epistatic tobri1. Cloning and further characterization ofSRD should provide additional information about its role in the determination of leaf shape and root elongation.     

Identification of phytochrome-interacting protein candidates in Arabidopsis thaliana by co-immunoprecipitation coupled with MALDI-TOF MS

Phytochrome-interacting proteins have been extensively studied to elucidate light-signaling pathway in plants. However, most of these proteins have been identified by yeast two-hybrid screening using the C-terminal domain of phytochromes. We used co-immunoprecipitation followed by proteomic analysis in plant cell extracts in an attempt to screen for proteins interacting either directly or indirectly with native holophytochromes including the N-terminal domain as well as C-terminal domain. A total of 16 protein candidates were identified, and were selected from 2-DE experiments. Using MALDI-TOF MS analysis, 7 of these candidates were predicted to be putative phytochrome A-interacting proteins and the remaining ones to be phytochrome B-interacting proteins. Among these putative interacting proteins, protein phosphatase type 2C (PP2C) and a 66-kDa protein were strong candidates as novel phytochrome-interacting proteins, as knockout mutants for the genes encoding these two proteins had impaired light-signaling functions. A transgenic knockout Arabidopsis study showed that a 66-kDa protein candidate regulates hypocotyl elongation in a light-specific manner, and altered cotyledon development under white light during early developmental stages. The PP2C knockout plants also displayed light-specific changes in hypocotyl elongation. These results suggest that co-immunoprecipitation, followed by proteomic analysis, is a useful method for identifying novel interacting proteins and determining real protein-protein interactions in the cell.