Identification and characterization of a putative C. elegans potassium channel gene (Ce-slo-2) distantly related to Ca(2+)-activated K(+) channels

Two putative homologues of large conductance Ca(2+)-activated K(+) channel alpha-subunit gene (slowpoke or slo) were revealed by C. elegans genome sequencing. One of the two genes, F08B12.3 (Ce-slo-2), shows a relatively low amino acid sequence similarity to other Slo sequences and lacks key functional motifs, which are important for calcium and voltage sensing. However, its overall structure and regions of homology, which are conserved in all Slo proteins, suggest that Ce-SLO-2 should belong to the Slo channel family. We have cloned a full-length cDNA of the Ce-slo-2, which encodes a protein containing six putative transmembrane segments with a K(+)-selective pore and a large C-terminal cytosolic domain. Green fluorescent protein (GFP) and whole-mount immunostaining analyses revealed that Ce-slo-2 is specifically expressed in neuronal cells at the nerve ring, at the ventral nerve cord of the mid-body, and at the tail region. We have also identified a putative human counterpart of Ce-slo-2 from a human brain EST database, which shows a stretch of highly conserved amino acid residues. Northern blot and mRNA dot blot analyses revealed a strong and specific expression in brain and skeletal muscle. Taken together, our data suggest that Ce-slo-2 may constitute an evolutionarily conserved gene encoding a potassium channel that has specific functions in neuronal cells.     

Cdc42 and Gsk3 modulate the dynamics of radial glial growth, inter-radial glial interactions and polarity in the developing cerebral cortex

Polarized radial glia are crucial to the formation of the cerebral cortex. They serve as neural progenitors and as guides for neuronal placement in the developing cerebral cortex. The maintenance of polarized morphology is essential for radial glial functions, but the extent to which the polarized radial glial scaffold is static or dynamic during corticogenesis remains an open question. The developmental dynamics of radial glial morphology, inter-radial glial interactions during corticogenesis, and the role of the cell polarity complexes in these activities remain undefined. Here, using real-time imaging of cohorts of mouse radial glia cells, we show that the radial glial scaffold, upon which the cortex is constructed, is highly dynamic. Radial glial cells within the scaffold constantly interact with one another. These interactions are mediated by growth cone-like endfeet and filopodia-like protrusions. Polarized expression of the cell polarity regulator Cdc42 in radial glia regulates glial endfeet activities and inter-radial glial interactions. Furthermore, appropriate regulation of Gsk3 activity is required to maintain the overall polarity of the radial glia scaffold. These findings reveal dynamism and interactions among radial glia that appear to be crucial contributors to the formation of the cerebral cortex. Related cell polarity determinants (Cdc42, Gsk3) differentially influence radial glial activities within the evolving radial glia scaffold to coordinate the formation of cerebral cortex.     

Relationships between structures of hydroxyflavones and their antioxidative effects

Even hydroxyflavones show diverse biological functions, they have two common features such as showing antioxidative effects and containing hydroxyl groups. The authors tested the antioxidative effects of thirty hydroxyflavones using 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay. While the scavenging activity of galangin, 3,5,7-trihydroxyflavone was 52.5%, fisetin, 3,7,3′,4′-tetrahydroxyflavone showed 85.2%. To investigate the relationships between the structures of hydroxyflavones and their antioxidative effects, the three-dimensional quantitative structure–activity relationships were examined.     

Anti-oxidant effect of gold nanoparticles restrains hyperglycemic conditions in diabetic mice

Background  

Oxidative stress is imperative for its morbidity towards diabetic complications, where abnormal metabolic milieu as a result of hyperglycemia, leads to the onset of several complications. A biological antioxidant capable of inhibiting oxidative stress mediated diabetic progressions; during hyperglycemia is still the need of the era. The current study was performed to study the effect of biologically synthesized gold nanoparticles (AuNPs) to control the hyperglycemic conditions in streptozotocin induced diabetic mice.     

Negatively charged amino acids within the intraluminal loop of ryanodine receptor are involved in the interaction with triadin

In mammalian striated muscles, ryanodine receptor (RyR), triadin, junctin, and calsequestrin form a quaternary complex in the lumen of sarcoplasmic reticulum. Such intermolecular interactions contribute not only to the passive buffering of sarcoplasmic reticulum luminal Ca2+, but also to the active Ca2+ release process during excitation-contraction coupling. Here we tested the hypothesis that specific charged amino acids within the luminal portion of RyR mediate its direct interaction with triadin. Using in vitro binding assay and site-directed mutagenesis, we found that the second intraluminal loop of the skeletal muscle RyR1 (amino acids 4860-4917), but not the first intraluminal loop of RyR1 (amino acids 4581-4640) could bind triadin. Specifically, three negatively charged residues Asp4878, Asp4907, and Glu4908 appear to be critical for the association with triadin. Using deletional approaches, we showed that a KEKE motif of triadin (amino acids 200-232) is essential for the binding to RyR1. Because the second intraluminal loop of RyR has been previously shown to contain the ion-conducting pore as well as the selectivity filter of the Ca2+ release channel, and Asp4878, Asp4907, and Glu4908 residues are predicted to locate at the periphery of the pore assembly of the channel, our data suggest that a physical interaction between RyR1 and triadin could play an active role in the overall Ca2+ release process of excitation-contraction coupling in muscle cells.     

Bcl-2 enhances neurite extension via activation of c-Jun N-terminal kinase

Recent studies suggest that Bcl-2 may play an active role in neuronal differentiation. Here, we showed a marked neurite extension in MN9D dopaminergic neuronal cells overexpressing Bcl-2 (MN9D/Bcl-2) or Bcl-X(L) (MN9D/Bcl-X(L)). We found a specific increase in phosphorylation of c-Jun N-terminal kinase (JNK) accompanied by neurite extension in MN9D/Bcl-2 but not in MN9D/Bcl-X(L) cells. Consequently, neurite extension in MN9D/Bcl-2 but not in MN9D/Bcl-X(L) cells was suppressed by treatment with SP600125, a specific inhibitor of JNK. Inhibition of other mitogen-activated protein kinases-including p38 and extracellular signal-regulated kinase-did not affect Bcl-2-mediated neurite extension in MN9D cells. While the expression levels of such protein markers of maturation as SNAP-25, phosphorylated NF-H, and neuron-specific enolase were increased in MN9D/Bcl-2 cells, only upregulation of SNAP-25 was inhibited after treatment with SP600125. Thus, the JNK signal activated by Bcl-2 seems to play an important role during morphological and certain biochemical differentiation in cultured dopaminergic neurons.     

Expression,purification, and crystallization of glutamyl-tRNA(Gln) specific amidotransferase from Bacillus stearothermophilus

Although the genes that encode the glutamyl-tRNA(Gln) (Glu-tRNA(Gln)) specific amidotransferase (Glu-AdTase) from various bacteria and eukaryotic organelles are known, the precise mechanism of the enzyme is still unclear. One of the reasons is that there is no information on the three-dimensional structure of the complex, the Glu-AdTase:Glu-tRNA(Gln):ATP:amino group donor. To obtain the crystals of Glu-AdTase, the Glu-AdTase of Bacillus stearothermophilus was overexpressed and purified after cloning of the gene that encodes the enzyme. The cloned DNA contained the full-length gene cluster that represented the Glu-AdTase of B. stearothermophilus, and was organized as an operon that consisted of three open-reading frames (ORFs). The order of the genes was gatCAB, as shown in Bacillus subtilis. The ORFs showed a high amino-acid homology to those of B. subtilis (A subunit, 73.2%; B subunit, 81.6%; C subunit, 69.5%) and Staphylococcus aureus (A subunit, 61.9%; B subunit, 71.8%; C subunit, 45.9%). The ORFs were re-cloned on the overexpression vector, pTrc99a, and a recombinant pTrcgatCABBST was obtained. The Glu-AdTase that was overexpressed with pTrcgatCABBST in Escherichia coli retained transamidation activity on the mischarged glutamic acid on the tRNA(Gln). It also produced correctly-charged Gln-tRNA(Gln) at 37, 42, and 50 degrees C. Although Glu-AdTases from both B. subtilis and B. stearothermophilus were subjected to crystallization, the micro-crystals were only obtained from the B. stearothermophilus enzyme.     

Fumarate-mediated inhibition of erythrose reductase,a key enzyme for erythritol production by Torula corallina

Torula corallina, a strain presently being used for the industrial production of erythritol, has the highest erythritol yield ever reported for an erythritol-producing microorganism. The increased production of erythritol by Torula corallina with trace elements such as Cu(2+) has been thoroughly reported, but the mechanism by which Cu(2+) increases the production of erythritol has not been studied. This study demonstrated that supplemental Cu(2+) enhanced the production of erythritol, while it significantly decreased the production of a major by-product that accumulates during erythritol fermentation, which was identified as fumarate by instrumental analyses. Erythrose reductase, a key enzyme that converts erythrose to erythritol in T. corallina, was purified to homogeneity by chromatographic methods, including ion-exchange and affinity chromatography. In vitro, purified erythrose reductase was significantly inhibited noncompetitively by increasing the fumarate concentration. In contrast, the enzyme activity remained almost constant regardless of Cu(2+) concentration. This suggests that supplemental Cu(2+) reduced the production of fumarate, a strong inhibitor of erythrose reductase, which led to less inhibition of erythrose reductase and a high yield of erythritol. This is the first report that suggests catabolite repression by a tricarboxylic acid cycle intermediate in T. corallina.     

nor-Oleanene type triterpene glycosides from the leaves of Acanthopanax japonicus

Three new (1-3) and two known (4-5) triterpene glycosides were isolated from the leaves of Acanthopanax japonicus (Araliaceae) and elucidated structurally by mass, 1D, and 2D NMR spectroscopy. All the compounds possessed a nor-oleanene triterpene skeleton as the aglycone. The structures of 1-5 were established as 28-O-alpha-L-rhamno-pyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester of 3beta-hydroxy- 30-nor-olean-12,20(29)-diene-23,28-dioic acid, designated as acanjaposide A, 3beta- hydroxy-23-oxo-30-nor-olean-12,20(29)-diene-28-oic acid, named acanjaposide B, 3beta,20alpha-dihydroxy-23-oxo-30-nor-olean-12-en-28-oic acid, named acanjaposide C, and nipponoside E, a known saponin, respectively.     

Fumarate-Mediated Inhibition of Erythrose Reductase, a Key Enzyme for Erythritol Production by Torula corallina

Torula corallina, a strain presently being used for the industrial production of erythritol, has the highest erythritol yield ever reported for an erythritol-producing microorganism. The increased production of erythritol by Torula corallina with trace elements such as Cu²⁺ has been thoroughly reported, but the mechanism by which Cu²⁺ increases the production of erythritol has not been studied. This study demonstrated that supplemental Cu²⁺ enhanced the production of erythritol, while it significantly decreased the production of a major by-product that accumulates during erythritol fermentation, which was identified as fumarate by instrumental analyses. Erythrose reductase, a key enzyme that converts erythrose to erythritol in T. corallina, was purified to homogeneity by chromatographic methods, including ion-exchange and affinity chromatography. In vitro, purified erythrose reductase was significantly inhibited noncompetitively by increasing the fumarate concentration. In contrast, the enzyme activity remained almost constant regardless of Cu²⁺ concentration. This suggests that supplemental Cu²⁺ reduced the production of fumarate, a strong inhibitor of erythrose reductase, which led to less inhibition of erythrose reductase and a high yield of erythritol. This is the first report that suggests catabolite repression by a tricarboxylic acid cycle intermediate in T. corallina.