Cell-autonomous enhancement of glutamate-uptake by female astrocytes

Since gonadal female hormones act on and protect neurons, it is well known that the female brain is less vulnerable to stroke or other brain insults than the male brain. Although glial functions have been shown to affect the vulnerability of the brain, little is known if such a sex difference exists in glia, much less the mechanism that might cause gender-dependent differences in glial functions. In this study, we show that in vitro astrocytes obtained from either female or male pups show a gonadal hormone-independent phenotype that could explain the gender-dependent vulnerability of the brain. Female spinal astrocytes cleared more glutamate by GLAST than male ones. In addition, motoneurons seeded on female spinal astrocytes were less vulnerable to glutamate than those seeded on male ones. It is suggested that female astrocytes uptake more glutamate and reveal a stronger neuroprotective effect against glutamate than male ones. It should be noted that such an effect was independent of gonadal female hormones, suggesting that astrocytes have cell-autonomous regulatory mechanisms by which they transform themselves into appropriate phenotypes.     

A novel xylanase with tolerance to ethanol, salt, protease, SDS, heat, and alkali from actinomycete Lechevalieria sp. HJ3

A xylanase-coding gene (xynAHJ3, 1,104 bp) was cloned from Lechevalieria sp. HJ3 harbored in a saline soil sampled from Heijing town, aka the "town of salt", on the famous "Silk Route of the South". The gene encodes a 367-residue polypeptide (XynAHJ3) with the highest identity of 74.0 % with the endoxylanase from Streptomyces thermocarboxydus HY-15. The coding sequence of the mature protein (without the predicted signal peptide from M1 to S22) of xynAHJ3 was expressed in Escherichia coli BL21 (DE3). The activity of the purified recombinant XynAHJ3 (rXynAHJ3) was apparently optimal at 70 °C and pH 6.0, retained greater than 55 % xylanase activity at a concentration of 0.2-2.0 M Na(+) and 26 % at 4.0 M Na(+) (pH 7.5 20 °C), and showed 110.2 and 44.2 % xylanase activities in the presence of 100 mM SDS (pH 6.0 37 °C) and 10 % ethanol (pH 5.0 37 °C), respectively. rXynAHJ3 activity was stable at 50 °C and pH 4.0-11.0 for more than 60 min, in trypsin or proteinase K at 20 °C for 24 h (pH 7.5), in 10 % ethanol (v/v) (pH 5.0) at 30 or 37 °C for 72 h, in 80 % ethanol (v/v) for 1 h, and in 0.6 or 3 M NaCl (20 °C, pH 7.5) for 72 h. Compared with the majority of xylanases with tolerance to ethanol, salt, SDS, or protease (K (m) values of 1.42-15.1 mg ml(-1)), rXynAHJ3 showed a low K (m) value (0.8 mg ml(-1)) and showed only limited amino acid sequence identity with those other xylanases (less than 47 %).     

On the relationship between mitochondrial ATPase and the inner membrane

Digestion of the submitochondrial particle (ETP_H) with a proteolytic enzyme, Nagarse, selectively and completely removed the headpieces from the membrane without damaging the electron transfer chain. By determining the amount of protein released by the Nagarse treatment, it was calculated that the headpieces represent 16±0.5% of the total protein of the submitochondrial particles.In respiring ETP_H, membrane-bound AMP was found to be an acceptor of inorganic phosphate, and this esterification led to the formation of membrane-bound ADP. About 70% of the membranebound adenine nucleotides were found to be tightly bound to the intrinsic proteins of the membrane. A transphosphorylation reaction was observed between external and membrane-bound ADP.Abbreviations F₁ coupling factor one - OSCP oligomycin-sensitivity conferring protein - TRU tripartite repeating unit - ETP_H phosphorylating electron transfer particle     

Reversible Activation of Halophilic β-lactamase from Methanol-Induced Inactive Form: Contrast to Irreversible Inactivation of Non-Halophilic Counterpart

Effects of a water-miscible organic solvent, methanol, on the structure and activity of halophilic β-lactamase derived from Chromohalobacter sp.560 (HaBla), were investigated by means of circular dichroism (CD) measurement and enzymatic activity determination. Beta-lactamase activity was enhanced about 1.2-fold in the presence of 10–20% methanol. CD measurement of HaBla revealed different structures depending on the methanol concentration: native-like active form (Form I) in 10–20% methanol and methanol-induced inactive form at higher concentration (Form II in 40–60% and Form III in 75–80% methanol). Incubation of HaBla with 40% methanol led to the complete loss of activity within ~80 min accompanied by the formation of Form II, whose activity was recovered promptly up to ~80% of full activity upon dilution of the methanol concentration to 10%. In addition, when the protein concentration was sufficiently high (e.g., 0.7 mg/ml), HaBla activity of Form III in 75% methanol could be recovered in the same way (with slightly slower recovery rate), upon dilution of the methanol concentration. In contrast, non-halophilic β-lactamase from Escherichia coli K12 strain MG1655 (EcBla) was irreversibly denatured in the presence of 40% methanol. HaBla showed remarkable ability to renature from the methanol-induced inactive states.     

RNA and a cell wall component of Enterococcus faecalis IC-1 are required for phagocytosis and interleukin 12 production by the mouse macrophage cell line J774.1

Enterococcus faecalis is a resident lactic acid bacterium in the human intestine. Its immunostimulatory action was reported to be enhanced by heat sterilization. To investigate its beneficial actions, we evaluated the ability of 10 E. faecalis strains to induce interleukin-12 (IL-12) production in a mouse macrophage cell line, J774.1 and found that the strain, E. faecalis IC-1, had a potent IL-12-inducing ability. Furthermore, we investigated the underlying mechanism by treating IC-1 cells with RNase or lysozyme. Its activity almost disappeared and an antagonist of Toll-like receptor (TLR) 7 inhibited this activity. Moreover, lysozyme-treated IC-1 bacteria were not phagocytized by J774.1 cells, and did not induce IL-12 production. Based on our results, we propose that macrophages recognize the cell wall components of IC-1, leading to phagocytosis. The IC-1 RNA is then recognized by TLR7, which induces the production of IL-12.     

Stereoselective synthesis of 2,3,7-trimethylcyclooctanone and related compounds and determination of their relative and absolute configurations by the MalphaNP acid method

The copper/chiral phosphoramidite (L(1))-catalyzed conjugate addition of dimethylzinc to cycloocta-2,7-dienone 4, followed by the methylation of the intermediate enolate, yielded a single isomer of 7,8-dimethylcyclooct-2-enone (+)-5. Compound (+)-5 was subjected to the second conjugate addition with ent-L(1) giving only one stereoisomer of 2,3,7-trimethylcyclooctanone (+)-6, which was converted to 2,3,7-trimethylcyclooctanol 7. To determine the relative and absolute configurations of these compounds, the (1)H NMR anisotropy method using (S)-(+)-2-methoxy-2-(1-naphthyl)propionic acid {(S)-(+)-MalphaNP acid} 1 was applied. Racemic alcohol (+/-)-7 was esterified with (S)-(+)-MalphaNP acid 1 yielding diastereomeric esters, which were efficiently separated by HPLC on silica gel affording the first-eluted MalphaNP ester (-)-10a and the second-eluted one (-)-10b. The relative and absolute configurations of ester (-)-10a were determined to be (S;1R,2S,3R,7S) by analyzing the (1)H and (13)C NMR spectra of (-)-10a and (-)-10b, especially their HSQC-TOCSY and NOESY spectra, and by applying the MalphaNP anisotropy method. The alcohol 7 formed from (+)-6 was similarly esterified with (S)-(+)-MalphaNP acid 1 yielding an MalphaNP ester, which was identical with (-)-10a, and the relative and absolute configurations of 2,3,7-trimethylcyclooctanone (+)-6 were determined to be (2S,3R,7S).     

Discovery of indenopyrazoles as EGFR and VEGFR-2 tyrosine kinase inhibitors by in silico high-throughput screening

A series of indenopyrazoles 8 and 9 were designed and synthesized as EGFR tyrosine kinase inhibitors by in silico high-throughput screening. Compounds 8b and 8d showed significant inhibition of A431 cell growth (GI50 = 0.062 and 0.057 microM, respectively). Compounds 8b and 9a showed inhibitory activity toward both EGFR and VEGFR-2 (KDR) tyrosine kinases, whereas 8d inhibited VEGFR-2 tyrosine kinase, exclusively.