羊蹄甲属植物种子的裂解色谱鉴定法研究

本文探索了裂解气相色谱法作为植物种子分类表征方法的可能性。对豆科植物中不同属、不同亚属及相同亚属的共10种种子的裂解色谱图,用分区编码法进行区别,分析结果表明,不同种子最少有一个编码不同,方法简易快速,可为高等植物种子的区分提供一种佐证手段。     

13C-NMR spectroscopy of human serum high density lipoprotein enriched with labelled phospholipids.

Native human serum high density lipoprotein (HDL) (d = 1.063--1.21g x cm-3) was enriched with phosphatidylcholines labelled with 13C in the polar head group ([N-13CH3]choline) and in the fatty acyl chains ([26-13C]cholesterol) and its linoleic acid ester using the previously described exchange method (Stoffel et al. 1978). The properties of the HDL particles with the exchanged lipid classes were the same as those of the native particles (Mr, CD, fluorescence, lipid and apoprotein stoichiometry, electrophoretic mobility). The T1-times were very similar to those obtained previously with recombined apolipoprotein-[13C]lipid complexes and further support our proposals concerning lipid and apoprotein interactions in the HDL particle.     

Activation of the Endoplasmic Reticulum Calcium Sensor STIM1 and Store-Operated Calcium Entry by Rotavirus Requires NSP4 Viroporin Activity

Rotavirus nonstructural protein 4 (NSP4) induces dramatic changes in cellular calcium homeostasis. These include increased endoplasmic reticulum (ER) permeability, resulting in decreased ER calcium stores and activation of plasma membrane (PM) calcium influx channels, ultimately causing a 2- to 4-fold elevation in cytoplasmic calcium. Elevated cytoplasmic calcium is absolutely required for virus replication, but the underlying mechanisms responsible for calcium influx remain poorly understood. NSP4 is an ER-localized viroporin, whose activity depletes ER calcium, which ultimately leads to calcium influx. We hypothesized that NSP4-mediated depletion of ER calcium activates store-operated calcium entry (SOCE) through activation of the ER calcium sensor stromal interaction molecule 1 (STIM1). We established and used a stable yellow fluorescent protein-expressing STIM1 cell line (YFP-STIM1) as a biosensor to assess STIM1 activation (puncta formation) by rotavirus infection and NSP4 expression. We found that STIM1 is constitutively active in rotavirus-infected cells and that STIM1 puncta colocalize with the PM-localized Orai1 SOCE calcium channel. Expression of wild-type NSP4 activated STIM1, resulting in PM calcium influx, but an NSP4 viroporin mutant failed to induce STIM1 activation and did not activate the PM calcium entry pathway. Finally, knockdown of STIM1 significantly reduced rotavirus yield, indicating STIM1 plays a critical role in virus replication. These data demonstrate that while rotavirus may ultimately activate multiple calcium channels in the PM, calcium influx is predicated on NSP4 viroporin-mediated activation of STIM1 in the ER. This is the first report of viroporin-mediated activation of SOCE, reinforcing NSP4 as a robust model to understand dysregulation of calcium homeostasis during virus infections.     

Effect of external stresses on protein conformation: a computer modelling study

The increasing use of digital technologies such as mobile phones has led to major health concerns about the effects of non-ionizing pulsed radiation exposure. We believe that the health implications of exposure to radiation cannot be fully understood without establishing the molecular mechanisms of biological effects of pulsed microwaves. We aim to establish methods for studying the molecular mechanisms of protein structural and energetic changes occurring due to external stresses related to non-ionizing radiation by using a combination of experimental and theoretical approaches. In this paper, we present the results from our fully atomistic simulation study of chemical and thermal stress response of a prototype protein, insulin. We performed a series of molecular dynamics simulations of insulin in solution under equilibrium conditions, under chemical stress (imitated by reducing the disulfide bonds in the protein molecule), and under short-lived thermal stress (imitated by increasing simulation temperature for up to 2 ns). The resultant protein conformational behaviour was analysed for various properties with the aim of establishing analysis routines for classification of protein unfolding pathways and associated molecular mechanisms.Submitted as a record of the 2002 Australian Biophysical Society meeting     

Genotype-dependent expression of endothelial nitric oxide synthase (eNOS) and its regulatory proteins in cultured endothelial cells

DNA polymorphisms in endothelial nitric oxide synthase (eNOS) gene have been shown to be associated with constitutive eNOS expression and coronary artery disease (CAD). In the present study we explored the hypothesis whether genotype-dependent effects can be maintained in vitro during replication, or the effect is conditional on in vivo biological environments. Human umbilical vein endothelial cells (HUVEC) were collected and cultured from 89 normal deliveries of Mexican Americans. The cells were treated with or without cigarette smoking extracts (CSE) and genotypes of eNOS polymorphisms were determined by PCR. We measured the levels of eNOS by ELISA and its binding proteins including heat-shock protein 90 (Hsp-90) and caveolin-1 by Western blotting. The rare C allele for the promoter T786C polymorphism (0.2), and the rare 4 x 27-bp repeat allele in the intron 4 (0.30) were different from those reported in other populations. Yet, the rare T allele in the exon 7 (G894T polymorphism) was similar as others. After four passages in vitro, both the intron 4 and promoter polymorphisms maintained significant effects on eNOS mRNA levels in HUVECs (P < 0.05). However, the effects on eNOS protein and enzyme activity were less consistent. Although primary smokers had significantly lower eNOS protein levels (P < 0.05), the in vitro CSE treatment on cultured HUVECs only resulted in a significant reduction in NO levels as measured by the stable metabolites of nitrite/nitrate (P < 0.001). Neither Hsp-90 nor caveolin-1--important eNOS regulators--appears to mediate the genotypesmoking effects on eNOS expression although HUVECs did produce more Hsp-90 when exposed to CSE. Our study demonstrates that endothelial cells maintain genotype-dependent expression even after the deprivation of in vivo environment. However, the cigarette smoking-genotype interaction may require such in vivo conditions to be manifested.     

Endogenous nitric oxide activation protects against cigarette smoking induced apoptosis in endothelial cells

Cigarette-induced endothelial dysfunction could be an early mediator of atherosclerosis. In this study, we explored the mechanisms of cigarette smoke extract (CSE)-induced human aortic endothelial cells (HAEC) apoptosis. We found that 10-65% of HAECs underwent apoptotic changes when HAECs were exposed to 0.001-0.02 cigarette equivalent unit of CSE for 4 h. CSE activated the caspases-3 and 8, the p38 MAP kinase and stress activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK). Specific inhibitors of p38 MAP or SAPK/JNK reduced CSE-induced caspase activation. We further showed that eNOS pre-activation by L-arginine reduced endothelial apoptosis from 65% to 5%; and eNOS inhibition by N-omega-nitro-L-arginine methyl ester accentuated CSE-induced endothelial apoptosis. We suggest that appropriate endogenous NO production may be an important protective mechanism against smoking-induced endothelial damage.     

Enhancement of carbon dioxide fixation by alteration of illumination duringChlorella vulgaris-Buitenzorg's growth

Alteration of illumination with optimum carbon dioxide fixation-based curve in this research successfully enhanced the CO₂-fixation (q_co2) capability ofChlorella vulgaris Buitenzorg cultivated in a bubble column photo bioreactor. The level of CO₂ fixation was up to 1.91 times that observed from cultivation with intensification of illumination on an optimum growth-based curve. During 144 h of cultivation, alteration of light intensity on an optimum CO₂-fixation-based curve produced a q_CO2 of 6.68 h^?1. Increases in light intensity based on a curve of optimum CO₂-fixation produced a final cell concentration of about 5.78 g/L. Both cultivation methods were carried out under ambient pressure at a temperature of 29°C with a superficial gas velocity of 2.4 m/h (U_G). Cells were grown on Beneck medium in a 1.0 L Bubble Column Photo bioreactor illuminated by aPhillips Halogen Lamp (20 W/12 V/50 Hz). The inlet gas had a carbon dioxide content of 10%.     

Protein flexibility: multiple molecular dynamics simulations of insulin chain B

Multiple molecular dynamics simulations totaling more than 100 ns were performed on chain B of insulin in explicit solvent at 300 K and 400 K. Despite some individual variations, a comparison of the protein dynamics of each simulation showed similar trends and most structures were consistent with NMR experimental values, even at the elevated temperature. The importance of packing interactions in determining the conformational transitions of the protein was observed, sometimes resulting in conformations induced by localized hydrophobic interactions. The high temperature simulation generated a more diverse range of structures with similar elements of secondary structure and populated conformations to the simulations at room temperature. A broad sampling of the conformational space of insulin chain B illustrated a wide range of conformational states with many transitions at room temperature in addition to the conformational states observed experimentally. The T-state conformation associated with insulin activity was consistently present and a possible mechanism of behavior was suggested.