Crystal structure of alpha-hordothionin at 1.9 Angstrom resolution

Crystal structure of ubiquitous toxin from barley alpha-hordothionin (alpha-HT) has been determined at 1.9A resolution by X-ray crystallography. The primary sequence as well as the NMR solution structure of alpha-HT firmly established that alpha-HT belongs to a family of membrane active plant toxins-thionins. Since alpha-HT crystallized in a space group (P4(1)2(1)2) that is different from the space group (I422) of previously determined alpha(1)- and beta-purothionins, and visocotoxin A3, therefore, it provided independent information on protein-protein interactions that may be relevant to the toxin mechanism. The structure of alpha-HT not only confirms overall architectural features (crambin fold) but also provides an additional confirmation of the role for crucial solute molecules, that were postulated to be directly involved in the mechanism of toxicity for thionins.     

Detection of luciferase gene sequences in nonluminescent bacteria from the Chesapeake Bay

Washed excised roots of rice (Oryza sativa) produced H(2), CH(4) and fatty acids (millimolar concentrations of acetate, propionate, butyrate; micromolar concentrations of isovalerate, valerate) when incubated under anoxic conditions. Surface sterilization of the root material resulted in the inactivation of the production of CH(4), a strong reduction of the production of fatty acids and a transient (75 h) but complete inhibition of the production of H(2). Radioactive bicarbonate was incorporated into CH(4), acetate, propionate and butyrate. About 20-40% of the fatty acid carbon originated from CO(2) reduction. In the presence of phosphate, CH(4) was exclusively produced from H(2)/CO(2), since phosphate selectively inhibited acetoclastic methanogenesis. Acetoclastic methanogenesis was also selectively inhibited by methyl fluoride, while chloroform or 2-bromoethane sulfonate inhibited CH(4) production completely. Production of CH(4), acetate, propionate and butyrate from H(2)/CO(2) was always exergonic with Gibbs free energies <-20 kJ mol(-1) product. Chloroform inhibited the production of acetate and the incorporation of radioactive CO(2) into acetate. Simultaneously, H(2) was no longer consumed and accumulated, indicating that acetate was produced from H(2)/CO(2). Chloroform also resulted in increased production of propionate and butyrate whose formation from CO(2) became more exergonic upon addition of chloroform. Nevertheless, the incorporation of radioactive CO(2) into propionate and butyrate was inhibited by chloroform. The accumulation of propionate and butyrate in the presence of chloroform probably occurred by fermentation of organic matter, rather than by reduction of acetate and CO(2). [U-(14)C]Glucose was indeed converted to acetate, propionate, butyrate, CO(2) and CH(4). Radioactive acetate, CO(2) and CH(4) were also products of the degradation of [U-(14)C]cellulose and [U-(14)C]xylose. Addition of chloroform and methyl fluoride did not affect the product spectrum of [U-(14)C]glucose degradation. The application of combinations of selective inhibitors may be useful to elucidate anaerobic metabolic pathways in mixed microbial cultures and natural microbial communities.     

Downregulation of MDM2 stabilizes p53 by inhibiting p53 ubiquitination in response to specific alkylating agents

p53 is stabilized in response to DNA damaging stress. This stabilization is thought to result from phosphorylation in the N-terminus of p53, which inhibits p53:MDM2 binding, and prevents MDM2 from promoting p53 ubiquitination. In this report, the DNA alkylating agents mitomycin C (MMC) and methylmethane sulfonate (MMS), as well as UV radiation, stabilized p53 in a manner independent of phosphorylation in p53 N-terminus. This stabilization coincided with decreased levels of MDM2 mRNA and protein, and a corresponding decrease in p53 ubiquitination. Importantly, MDM2 overexpression inhibited the stabilization of p53 and decrease in ubiquitination following MMC, MMS, and UV treatment. This indicates that downregulation of MDM2 contributes to the stabilization of p53 in response to these agents.     

Subcellular glutathione contents in isolated hepatocytes treated with L-buthionine sulfoximine

The glutathione contents of the mitochondrial and cytosolic fractions and extracellular space of isolated hepatocytes decrease when glutathione synthesis is inhibited with L-buthionine sulfoximine. Mitochondrial glutathione is depleted to 50% of its initial value whereas the cytosolic pool is completely emptied after 2 h incubation in the presence of inhibitor. The mitochondrial glutathione content was only fully depleted when L-buthionine sulfoximine was added together with phorone (2,6-dimethyl-2,5-heptadiene-4-one), a substrate of the glutathione S-transferases (E.C. 2.5.1.18).     

Influence of growth hormone and thyroxine on thermotropic effects of respiration and 1-anilino-8-naphthalene sulfonate fluorescence and on lipid composition of cardiac membranes

The effects of hypophysectomy and subsequent administration of growth hormone and/or L-thyroxine on thermotropic properties of State 3 respiration (ADP-induced), cholesterol, phospholipid and fatty acid composition of phospholipid fraction were examined in myocardial mitochondria of rats. Temperature-dependence of 1-anilino-8-naphthalene sulfonate fluorescence was determined in vesicles prepared from lipids of heart mitochondria. Transition temperature obtained from the Arrhenius plots of respiration occurred at 21 and 24°C for heart mitochondria of normal and hypophysectomized rats, respectively. Most notably, after hypophysectomy the rate of respiration was lower below 24°C, but was progressively higher above that temperature when compared to normal rats. The energy of activation was 148 and 36% larger below and above the transition temperature, respectively. Growth hormone restored almost completely the energy of activation and respiratory rates to normal levels. Administration of L-thyroxine, with or without growth hormone, did not significantly change the rate of respiration but decreased the transition temperature to 17.7–17.0°C. Lipid and phospholipid content, as well as percent distribution of phospholipids and their fatty acid composition were not statistically different among the different groups of rats. Only cholesterol content was increased after hypophysectomy. Administration of growth hormone and thyroxine did not significantly change the total unsaturation index of fatty acids, but growth hormone increased the content of arachidonic acid (20 : 4) by 70% but decreased the docosahexaenoic acid (22 : 6) three times which may have a beneficial effect on mitochondrial membranes. These and other results suggest that hormones exert different effects on subcellular organelles in different tissues, like heart and liver.     

Bimutation breeding of Aspergillus niger strain for enhancing β-mannanase production by solid-state fermentation

A parent strain Aspergillus niger LW-1 was mutated by the compound mutagenesis of vacuum microwave (VMW) and ethyl methane sulfonate (EMS). A mutant strain, designated as A. niger E-30, with high- and stable-yield β-mannanase was obtained through a series of screening. The β-mannanase activity of the mutant strain E-30, cultivated on the basic fermentation medium at 32 °C for 96 h, reached 36,675 U/g dried koji, being 1.98-fold higher than that (18,501 U/g dried koji) of the parent strain LW-1. The purified E-30 β-mannanase, a glycoprotein with a carbohydrate content of 19.6%, had an apparent molecular weight of about 42.0 kDa by SDS–PAGE. Its optimal pH and temperature were 3.5 and 65 °C, respectively. It was highly stable at a pH range of 3.5–7.0 and at a temperature of 60 °C and below. The kinetic parameters K_m and V_max, toward locust bean gum and at pH 4.8 and 50 °C, were 3.68 mg/mL and 1067.5 U/mg, respectively. The β-mannanase activity was not significantly affected by an array of metal ions and EDTA, but strongly inhibited by Ag⁺ and Hg²⁺. In addition, the hydrolytic conditions of konjak glucomannan using the purified E-30 β-mannanase were optimized as follows: konjak gum solution 240 g/L (dissolved in deionized water), hydrolytic temperature 50 °C, β-mannanase dosage 120 U/g konjak gum, and hydrolytic time 8 h.     

Sodium tanshinone IIA sulfonate protects against Aβ-induced cell toxicity through regulating Aβ process

Sodium tanshinone IIA sulfonate (STS) has been reported to prevent Alzheimer's disease (AD). However, the mechanism is still unknown. In this study, two in vitro models, Aβ-treated SH-SY5Y cells and SH-SY5Y human neuroblastoma cells transfected with APPsw (SH-SY5Y-APPsw cells), were employed to investigate the neuroprotective of STS. The results revealed that pretreatment with STS (1, 10 and 100 µmol/L) for 24 hours could protect against Aβ (10 µmol/L)-induced cell toxicity in a dose-dependent manner in the SH-SY5Y cells. Sodium tanshinone IIA sulfonate decreased the concentrations of reactive oxygen species, malondialdehyde, NO and iNOS, while increased the activities of superoxide dismutase and glutathione peroxidase in the SH-SY5Y cells. Sodium tanshinone IIA sulfonate decreased the levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in the SH-SY5Y cells. In addition, Western blot results revealed that the expressions of neprilysin and insulin-degrading enzyme were up-regulated in the SH-SY5Y cells after STS treatment. Furthermore, ELISA and Western blot results showed that STS could decrease the levels of Aβ. ELISA and qPCR results indicated that STS could increase α-secretase (ADAM10) activity and decrease β-secretase (BACE1) activity. In conclusion, STS could protect against Aβ-induced cell damage by modulating Aβ degration and generation. Sodium tanshinone IIA sulfonate could be a promising candidate for AD treatment.     

Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: the biological relevance of the excluded volume effect

The excluded volume effect (EVE) rules all life processes. It is created by macromolecules that occupy a given volume thereby confining other molecules to the remaining space with large consequences on reaction kinetics and molecular assembly. Implementing EVE in fibroblast culture accelerated conversion of procollagen to collagen by procollagen C-proteinase (PCP/BMP-1) and proteolytic modification of its allosteric regulator, PCOLCE1. This led to a 20-30- and 3-6-fold increased collagen deposition in two- and three-dimensional cultures, respectively, and creation of crosslinked collagen footprints beneath cells. Important parameters correlating with accelerated deposition were hydrodynamic radius of macromolecules and their negative charge density.     

Kinetic and equilibrium studies of copper biosorption onto <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> base using direct determination of copper by a voltammetric method

This paper provided information on the use of linear sweep anodic stripping voltammetry for evaluating the process of copper biosorption onto Pseudomonas aeruginosa. This technique was suited to determine the concentration of free copper ion on site on the mercaptoethane sulfonate modified gold electrode surface without any pretreatment. It was in favor of the study of kinetic process as the fast changing kinetic data characteristic just after the beginning of biosorption could be accurately depicted. Based on the electrochemical results, the kinetics and equilibrium of biosorption were systematically examined. The pseudo-second-order kinetic model was used to correlate the kinetic experimental data and the kinetic parameters were evaluated. The Langmuir and Freundlich models were applied to describe the biosorption equilibrium. It was found that the Langmuir isotherm fitted the experimental data better than the Freundlich isotherm. Maximum adsorption capacity of copper ion onto Pseudomonas aeruginosa was 0.9355 μmol mg⁻¹ (about 59.4417 mg g⁻¹).