Protection of PC12 cells against superoxide-induced damage by isoflavonoids from Astragalus mongholicus / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To further investigate the neuroprotective effects of five isoflavonoids from Astragalus mongholicus on xanthine (XA)/ xanthine oxidase (XO)-induced injury to PC12 cells.</p><p><b>METHODS</b>PC12 cells were damaged by XA/XO. The activities of antioxidant enzymes, MTT, LDH, and GSH assays were used to evaluate the protection of these five isoflavonoids. Contents of Bcl-2 family proteins were determined with flow cytometry.</p><p><b>RESULTS</b>Among the five isoflavonoids including formononetin, ononin, 9, 10-dimethoxypterocarpan-3-O-beta-D-glucoside, calycosin and calycosin-7-O-glucoside, calycosin and calycosin-7-O-glucoside were found to inhibit XA/ XO-induced injury to PC12 cells. Their EC50 values of formononetin and calycosin were 0.05 microg/mL. Moreover, treatment with these three isoflavonoids prevented a decrease in the activities of antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), while formononetin and calycosin could prevent a significant deletion of GSH. In addition, only calycosin and calycosin-7-O-glucoside were shown to inhibit XO activity in cell-free system, with an approximate IC50 value of 10 microg/mL and 50 microg/mL. Formononetin and calycosin had no significant influence on Bcl-2 or Bax protein contents.</p><p><b>CONCLUSION</b>Neuroprotection of formononetin, calycosin and calycosin-7-O-glucoside may be mediated by increasing endogenous antioxidants, rather by inhibiting XO activities or by scavenging free radicals.</p>

Association of SelS mRNA expression in omental adipose tissue with Homa-IR and serum amyloid A in patients with type 2 diabetes mellitus / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Tanis was reported as a putative receptor for serum amyloid A (SAA) involving glucose regulated protein in insulin regulated resistance. It was found to be dysregulated in diabetic rats (Psammomys obesus, Israeli sand rat) and its homologue for humans is SelS/AD-015. The present study analyzed mRNA expression of SelS in omental adipose tissue biopsies from patients with type 2 diabetes mellitus (T2DM), and age- and weight-matched nondiabetic patients, the relationship of SelS mRNA with Homa-IR and serum SAA level.</p><p><b>METHODS</b>Human omental adipose tissues from ten cases of type 2 diabetic patients and twelve cases of nondiabetic individuals were analyzed for the expression level of SelS mRNA by semiquantitative polymerase chain reaction (PCR), Homa-IR estimated by standard formula and SAA level by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>SelS mRNA expression, Homa-IR and serum SAA were higher in T2DM sufferers than in nondiabetic control group. SelS mRNA level was positively correlated with Homa-IR and SAA level in each group.</p><p><b>CONCLUSIONS</b>SelS protein may be involved in insulin resistance in Chinese with T2DM by acting as the SAA receptor, thus playing an important role in the development of T2DM and atherosclerosis.</p>

Expression of caveolin-1 protein in the rat brain and its role in the discrimination learning / 生理学报

Caveolin-1 (Cav-1) is a marker protein for caveolae, and acts as scaffolding protein to regulate the activities of signaling molecules. Previous studies indicate that Cav-1 mainly locates at the base of axonal and dendritic terminals of mouse primary hippocampal neurons and plays an active role in the regulation of injury-induced synaptic and terminal remodeling in central nervous system. The aim of this study was to identify the expression profile of Cav-1 protein in the brains of rats at different ages and to investigate the role of Cav-1 in Y-maze bright-dark discrimination learning (BDL). Firstly, the expressions of Cav-1 in the brains of young (1-month), adult (3-month) and aged (22-month) rats were observed by Western blot. Higher expression in the hippocampus and lower expression in the cortex were shown in the adult rats. It was also found that the score of BDL was related with the expression level of Cav-1. Secondly, using open-field test for spontaneous locomotor activities (SLA) and BDL, the role of Cav-1 in the learning and memory was observed. Compared with that in the control adult group, the Cav-1 protein expression in the hippocampus and prefrontal cortex of Y-maze trained adult rats significantly increased, while no marked changes in the cerebellum. These results suggest that Cav-1 protein is involved in BDL and plays an important role in the plasticity of central nervous system.

Isolation and Transient Expression Assay of PEAMT Gene Promoter from Salicornia europaea / 中国生物工程杂志

Phosphoethanolamine N-methyltransferase (PEAMT) is a key enzyme that catalyzes the synthesis of phosphocholine, which is an important precursor of phosphatidylcholine and glycine betaine. A 1249bp 5'-flanking region of phosphoethanolamine N-methyltransferase gene was isolated by anchored PCR, based on the cDNA sequence of PEAMT from halophyte Salicornia europaea. The transcription start site was identified as A and localized at 301bp upstream of the ATG according to the results of RLM-RACE. In SePEAMT promoter region, many potential cis-acting elements were predicted by PlantCARE and PLACE programs. Aside from the basal transcriptional elements TATA-box and CAAT-box, some stress-responsive motifs such as ABRE, HSE and LTR were found. In addition, some pollen-specific activation-related elements were also present in this region. Binary expression vector was constructed by fusing SePEAMT promoter with GUS gene and designated as pPro. The pPro was transferred into tobacco by Agrobacterium-medicated transformation and transient GUS expression analysis indicated that SePEAMT promoter could drive strong GUS expression.

Study on the Separation and Purification Technology of Catalpol from Rehmannia by Macroporous Adsorption Resins / 中国生物工程杂志

A separation technology of catalpol from Rehmannia with macroporpus adsorbent resins was investigated. The content of catalpol in the extract was determined by high performance liquid chromatography (HPLC). Nine different kinds of macroporous adsorbent resins were studied on the static capacity of adsorption and desorption, and D101 resin was best for the separation of the extraction solution of Rehmannia. The results showed that D101 resin had the highest static adsorption capacity of 69.2mg/g dry resin and its isotherm curve can be well described by Langmuir and Freudlich equation. The 5% ethanol elution on removal of the solvent under reduced pressure provided a brown powder, which was subjected to an open column chromatography on silica gel eluted with a CHCl3–MeOH gradient. The fraction eluted with CHCl3-MeOH (8∶2) was identified as catalpol and the purity of the compound was more than 90% purity by HPLC analysis. The yield of this separation technology was 6%.

pRNA: A Safe,Stable and Large Capacity Molecular Vehicle for Gene Therapy / 中国生物工程杂志

The molecular delivery vectors used in gene therapy need provide the features of safety,stability, efficiency and capacity. The current studies on the structure and action mechanism of pRNA, a packaging RNA of phage?29, showed that pRNA with multiple binding sites can through cell membrane easily and escort exogenous molecules to target cell, without inducing immune reaction. As an ideal nano-scale gene therapy vehicle, pRNA presents a promising application in delivering multiple therapeutic components to detect and treat human diseases.

Site-directed Mutagenesis and Enzymatic Activity Assay of Gln49-Phospholipase A_2 Mutant / 中国生物工程杂志

In order to confirm the role that the 49th amino acid residue plays in enzymatic inactivity of Glutamine 49 phospholipase A2(Gln49-PLA2),site-directed mutagenesis of its 49th amino acid gene codon was conducted using PCR.Aspartic acid 49 phospholipase A2(Asp49-PLA2-Q49D-PLA2),the mutant of Gln49-PLA2 was expressed in E.coli with pET32a+ vector.The fusion protein,expressed as inclusion body,after being denatured,was on-column refolded and purified by immobilized metal affinity chromatography(IMAC),and then cleaved by Factor Xa.The mature Q49D-PLA2 mutant was obtained by Hitrap SP cation exchange and Superdex 75 gel filtration chromatography,with the recovery rate of 1.3%,and the specific activity of the mature Q49D-PLA2 mutant was 72 U/mg.It has been demonstrated that the 49th glutamine amino acid residue is the main reason in enzymatic inactivity of Gln49-PLA2 and the results are helpful for denatured protein refolding,especially in rich disulfide bonds conditions.

Effect of flocculence of a self-flocculating yeast on its tolerance to ethanol and the mechanism / 生物工程学报

Investigation was undertaken for the purpose of examining any possible correlation between flocculence of a self-flocculating fusant of Schizosaccharomyces pombe mutant and Saccharomyces cerevisiae mutant (called fusant SPSC for short) and the tolerance of this strain to ethanol. When exposed to 18% (V/V) ethanol for 7 h at 30 degrees C, 52%, 37% and 9% of viability levels remained for the cells of fusant SPSC and its two parental strains, Sch. pombe mutant and S. cerevisiae mutant respectively. Analysis of phospholipid fatty acid composition of plasma membrane showed that the content of palmitic acid of each flocculating yeast (fusant SPSC or Sch. pombe mutant) was around 2-fold higher than that of free S. cerevisiae mutant, with remarkably lower contents of palmitoleic and oleic acids than the latter. When 0.1 mol/L sodium citrate was initially included in the medium in which cells of each flocculating yeast were grown, free cells rather than aggregates were finally obtained. Furthermore, the content of palmitic acid in the phospholipid fatty acid composition of the plasma membranes of the free cells of each flocculating yeast was found to decrease significantly, with a marked increase in the contents of palmitoleic and oleic acids. As a result, the characteristics of the phospholipid fatty acid composition of the plasma membranes of the free cells of each flocculating yeast were similar to those of S. cerevisiae mutant. Meanwhile, the disappearance of flocculence of each flocculating yeast caused by the action of sodium citrate brought about a steeply decreased tolerance of the free cells to ethanol, thus being equivalent to that of S. cerevisiae mutant. These data suggest that the stronger ethanol tolerance of each flocculating yeast is related to the higher content of palmitic acid in the phospholipid fatty acid composition of the plasma membranes. Thus, the enhancement by flocculence on the tolerance of yeast cells to ethanol as well as its mechanism are first reported in this work.

Studies of chemical constituents and their antioxidant activities from Astragalus mongholicus Bunge / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To evaluate the antioxidant activities of different chemical constituents from Astragalus mongholicus Bunge and their protection against xanthine (XA)/xanthine oxidase (XO)-induced toxicity in PC12 cells.</p><p><b>METHODS</b>The compounds of Astragalus mongholicus Bunge were isolated by chromatography and the structures were elucidated on the basis of spectral data interpretation. Their antioxidant activities were detected by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities in a cell-free system. Meanwhile, the effects against XA/XO-induced toxicity were assessed using MTT assay in PC12 cells.</p><p><b>RESULTS</b>Ten principal constituents were isolated and identified as formononetin (I), ononin (II), calycosin (III), calycosin-7-O-beta-D-glucoside (IV), 9,10-dimethoxypterocarpan-3-O-beta-D-glucoside (V), adenosine (VI), pinitol (VII), daucosterol (VIII), beta-sitoster (IX) and saccharose (X) from Astragalus mongholicus Bunge. The compounds I, III, and IV scavenged DPPH free radicals in vitro. Formononetin and calycosin were found to inhibit XA/XO-induced cell injury significantly, with an estimated EC50 of 50 ng/mL.</p><p><b>CONCLUSION</b>Compound II, VI, and VII are first reported in this plant. Calycosin exhibits the most potent antioxidant activity both in the cell-free system and in the cell system.</p>

Protein amino acid composition of plasma membranes affects membrane fluidity and thereby ethanol tolerance in a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae / 生物工程学报

A combination of three amino acids including 1.0 g/L isoleucine, 0.5 g/L methionine and 2.0 g/L phenylalanine was found to enhance ethanol tolerance of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae. When subjected to 20% (V/V) ethanol for 9 h at 30 degrees C, all cells died whereas 57% remained viable for the cells grown in the presence of the three amino acids. Based on the analysis of protein amino acid composition of plasma membranes and the determination of plasma membrane fluidity by measuring fluorescence anisotropy using diphenylhexatriene as a probe, it was found that the significantly increased ethanol tolerance of cells grown with the three amino acids was due to the incorporation of the supplementary amino acids into the plasma membranes, thus resulting in enhanced ability of the plasma membranes to efficiently counteract the fluidizing effect of ethanol when subjected to ethanol stress. This is the first time to report that plasma membrane fluidity can be influenced by protein amino acid composition of plasma membranes.

Influence of phospholipid fatty acid composition of plasma membrane on sensitivity of plasma membrane ATPase of a self-flocculating yeast to in vivo ethanol activation and its relationship to ethanol tolerance / 生物工程学报

Although alterations in fatty acid composition of phospholipids in plasma membranes had no effect on activities of plasma membrane ATPases of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae cells grown in the absence of ethanol (basal enzymes), they significantly affected the susceptibilities of the enzymes to in vivo activation induced by ethanol: the maximal values for the activated enzymes in cells pregrown with 0.6 mmol/L palmitic, linoleic or linolenic acid respectively were 3.6, 1.5 and 1.2-fold higher than their respective basal levels (in cells grown without ethanol), whereas the corresponding value for cells pregrown in the absence of fatty acid was 2.3-fold, with the concentrations of ethanol for the above maximal in vivo activation of enzymes being 7%, 6%, 6% and 7% (V/V) respectively. The Km values for ATP, the pH profiles, and the sensitivities to orthovanadate of the basal and the activated plasma membrane ATPases were essentially identical; however, the v(max) values of activated enzymes increased significantly. It was found that the characteristics of phospholipid fatty acid composition of plasma membrane leading to the enhanced ethanol tolerance of this strain, were also efficacious to increase the percentage of activation of plasma membrane ATPase per unit of ethanol. These data support a close correlation between the ethanol tolerance of this strain and the sensitivity of its plasma membrane ATPase to the in vivo ethanol-induced activation.

Enhancements in ethanol tolerance of a self-flocculating yeast by calcium ion through decrease in plasmalemma permeability / 生物工程学报

Ca2+ at 1.64 mmol/L markedly increased ethanol tolerance of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae. After 9 h of exposure to 20% (V/V) ethanol at 30 degrees C , no viability remained for the control whereas 50.0% remained for the cells both grown and incubated with ethanol in Ca2+ -added medium. Furthermore, when subjected to 15% (V/V) ethanol at 30 degrees C, the equilibrium nucleotide concentration and plasma membrane permeability coefficient (P' ) of the cells both grown and incubated with ethanol in Ca2+ -added medium accounted for only 50.0% and 29.3% those of the control respectively, indicating that adding Ca2+ can markedly reduce plasma membrane permeability of yeast cells under ethanol stress as compared with the control. Meanwhile, high viability levels acquired by the addition of Ca2+ exactly corresponded to the striking decreases in extracellular nucleotide concentration and P' achieved with identical approach. Therefore, the enhancing effect of Ca2+ on ethanol tolerance of this strain is closely related to its ability to decrease plasma membrane permeability of yeast cells subjected to ethanol stress.

Screening of Phytase-producing Strain and Cloning of Phytase Gene from the Selected Strain / 微生物学通报

A strain with relative higher phytase-producing ability, Aspergillus fumigatus WY-2 was screened from soil. The optimal pH and temperature for activity of the phytase from A.fumigatus WY-2 were 5.5 and 55 ℃, respectively. The gene encoding the phytase was amplified from genomic DNA of the strain by PCR, and a 1.5 kb DNA fragment was obtained and then was cloned into vector pMD18-T. The sequencing analysis revealed that the DNA fragment contained a whole open reading frame (ORF) of phytase gene. The phytase gene was 1459 bp in length included with a 61 bp intron and encoded 465 amino acids. A signal peptide encoding 26 amino acids was found at 5′end of the gene. There were 7 potential glycosylation sites in the phytase. The present phytase showed 91% identity in nucleotide sequence and 91% identity in deduced amino acids sequence to the previously reported A.fumigatus ATCC34625 phytase.