Parthenolide inhibits proliferation of vascular smooth muscle cells through induction of G0/G1 phase cell cycle arrest

Objective: This study is to determine the effect of the natural product parthenolide, a sesquiterpene lactone isolated from extracts of the herb Tanacetum parthenium, on the proliferation of vascular smooth muscle cells (VSMCs). Methods: Rat aortic VSMCs were isolated and cultured in vitro, and treated with different concentrations of parthenolide (10, 20 and 30 μmol/L). [³H]thymidine incorporation was used as an index of cell proliferation. Cell cycle progression and distribution were determined by flow cytometric analysis. Furthermore, the expression of several regulatory proteins relevant to VSMC proliferation including IκBα, cyclooxygenase-2 (Cox-2), p21, and p27 was examined to investigate the potential molecular mechanism. Results: Treatment with parthenolide significantly decreased the [³H]thymidine incorporation into DNA by 30%~56% relative to control values in a dose-dependent manner (P<0.05). Addition of parthenolide also increased cell population at G₀/G₁ phase by 19.2%~65.7% (P<0.05) and decreased cell population at S phase by 50.7%~84.8% (P<0.05), which is consistent with its stimulatory effects on p21 and p27. In addition, parthenolide also increased IκBα expression and reduced Cox-2 expression in a time-dependent manner. Conclusion: Our results show that parthenolide significantly inhibits the VSMC proliferation by inducing G₀/G₁ cell cycle arrest. IκBα and Cox-2 are likely involved in such inhibitory effect of parthenolide on VSMC proliferation. These findings warrant further investigation on potential therapeutic implications of parthenolide on VSMC proliferation in vivo.     

Anti-allergic effects of ethanol extracts from brown seaweeds

Ethanol extracts of brown seaweeds from Pakistan and China were isolated and compared for their antiallergenic activities. They included Sargassum tennerimum (ST) and Sargassum cervicorne (SC) from Pakistan, and Sargassum graminifolium turn (SG), Sargassum thunbergii (STH), and Laminaria japonica (LJ) from China. The ethanol extracts of these brown seaweeds were optimized at 85% (v/v) ethanol for the maximum yield of phlorotannin, an inhibitor against hyaluronidase. Total phlorotannins contained in the crude extracts were measured as 1.71% (SG), 0.74% (STH), 0.97% (LJ), 3.30% (SC), and 5.06% (ST). The 50% inhibitory concentrations (IC₅₀) of Pakistani SC and ST were 109.5 and 21 μg/ml, respectively, lower than those of Chinese SG, STH, and LJ (134, 269, and 148 μg/ml, respectively). An antiallergic drug, disodium cromoglycate (DSCG), had an IC₅₀=39 μg/ml, and a natural inhibitor of hyaluronidase, catechin, had an IC₅₀=20 μg/ml. The IC₅₀ of ST extract was found similar to that of catechin (21 vs 20 μg/ml) and lower than that of DSCG (21 vs 39 μg/ml). This suggests that ST is a potent inhibitor of hyaluronidase, indicating a promising future development of natural antiallergic medicines or functional foods.     

Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtil＆ was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone a-factor （MFals）, and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction （PCR）-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae （SC-βG） was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A （PrA） activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/（h·ml） after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.     

Astragalus mongholicus ameliorates renal fibrosis by modulating HGF and TGF-β in rats with unilateral ureteral obstruction

Astragalus mongholicus (AM) derived from the dry root of Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao is a widely used traditional Chinese medicine. The present study investigated the potential role of AM on renal fibrosis on a rat model of unilateral ureteral obstruction (UUO). We divided 48 Sprague-Dawley rats randomly into 4 groups: sham-operated group (Sham), untreated UUO group, AM-treated (10 g/(kg·d)) UUO group, and losartan-treated (20 mg/(kg·d)) UUO group as positive control. Haematoxylin & eosin (HE) and Masson staining were used to study the dynamic histological changes of the kidneys 7 and 14 d after operation. The expressions of fibronectin (FN), type I collagen (colI), hepatocyte growth factor (HGF), transforming growth factor-β1 (TGF-β1), and α-smooth muscle actin (α-SMA) were analyzed by real-time polymerase chain reaction (PCR), immunohistochemistry staining, and Western blot. Results show that, similar to losartan, AM alleviated the renal damage and decreased the deposition of FN and colI from UUO by reducing the expressions of TGF-β1 and α-SMA (P<0.05), whereas HGF increased greatly with AM treatment (P<0.05). Our findings reveal that AM could retard the progression of renal fibrosis. The renoprotective effect of AM might be related to inhibition of myofibroblast activation, inducing of HGF and reducing of TGF-β1 expression.     

Expressions of cardiac sympathetic norepinephrine transporter and β1-adrenergic receptor decreased in aged rats

Evidence suggests that the deterioration of communication between the sympathetic nervous system and cardiovascular system always accompanies the aging of human and animals. Cardiac sympathetic norepinephrine (NE) transporter (NET) on presynaptic membrane is a predominant component to eliminate released NE in the synaptic cleft and maintains the sensitivity of the β-adrenergic receptor (β-AR). In the present study, we investigated NET and β₁-AR mRNA levels and sympathetic nerve density in cardiac sympathetic ganglion and left ventricular myocardium in 2- and 16-month-old rats with Northern blot analysis and immunohistochemistry. The expression levels of NET mRNA, NET protein and β₁-AR mRNA in the ganglia or myocardia of 16-month-old rats were markedly reduced by 67%, 26%, and 43%, respectively, in comparison with those in 2-month-old rats. Our results also show that aging induces a strong decrease of the catecholaminergic nerve fiber density.     

茵陈等六种中药对肝葡萄糖醛酸转移酶的体外诱导作用

为了解中药治疗新生儿黄疸是否通过酶的诱导作用,用洋地黄皂甙作为体外肝葡萄糖醛酸转移酶(UGT)诱导的阳性对照物。用Black法测定茵陈(Artemisia capillaris)、黄连(Coptis chinensis)、大黄(Rheum officinale)、黄芩(Scutellaria baicalensis)、栀子(Fructus gardeniae)及甘草(Glycyrrhiza glabra)的水提物对Wistar大鼠肝匀浆在体外UGT酶诱导作用。结果表明,在体外实验,这6种中药水提取物不能增加大鼠肝细胞中UGT的活性。     

Detection of plasmid-mediated IMP-1 metallo-β-lactamase and quinolone resistance determinants in an ertapenem-resistant Enterobacter cloacae isolate

Objective: To investigate the mechanism of carbapenem resistance and the occurrence of plasmid-mediated quinolone resistance determinants qnr and aac(6′)-Ib-cr in a clinical isolate of Enterobacter cloacae. Methods: An ertapenem-resistant E. cloacae ZY106, which was isolated from liquor puris of a female gastric cancer patient in a Chinese hospital, was investigated. Antibiotic susceptibilities were determined by agar dilution method. Conjugation experiments, isoelectric focusing, polymerase chain reaction (PCR), and DNA sequence analyses of plasmid-mediated carbapenemases and quinolone resistance determinants were preformed to confirm the genotype. Outer membrane proteins (OMPs) were examined by urea-sodium dodecyl sulfatepolyacrylamide gel electrophoresis (Urea-SDS-PAGE). Results: Minimum inhibitory concentrations (MICs) of imipenem, meropenem, and ertapenem for ZY106 were 2, 4, and 16 μg/ml, respectively. Conjugation studies with Escherichia coli resulted in the transfer of significantly reduced carbapenem susceptibility. ZY106 produced IMP-1 metallo-β-lactamase and CTX-M-3 extended-spectrum β-lactamase, and E. coli transconjugant produced IMP-1. Plasmid-mediated quinolone resistance determinant qnrS1 was detected in ZY106. Transfer of the qnrS1-encoding-plasmid into E. coli by conjugation resulted in intermediate resistance to ciprofloxacin in E. coli transconjugant. Urea-SDS-PAGE analysis of OMPs showed that ZY106 lacked an OMP of approximately 38 kDa. Conclusion: It is the first IMP-1-producing Enterobacteriaceae in China and the first report of a clinical isolate that harbors both blaIMP and qnrS genes as well. The blaIMP-1, blaCTX-M-3, and qnrS1 are encoded at three different plasmids. IMP-1 combined with the loss of an OMP possibly resulted in ertapenem resistance and reduced imipenem and meropenem susceptibility in E. cloacae.     

HPLC法研究三种皮类药材不同贮藏期药效成分的含量变化

目的 利用高效液相色谱法探究杜仲、厚朴和黄柏三种皮类药材不同贮藏期药效成分的含量变化。方法 分别采用回流、超声和闪式三种方式对同批贮存一年以及贮存五年的杜仲、厚朴和黄柏三种皮类药材进行提取,并利用高效液相色谱法对提取液中有效成分进行定量分析。结果 与贮藏期一年的药材相比,经过5年的贮存,三种皮类药材的药效物质含量均随着贮藏期延长或多或少的降低,其中杜仲皮中桃叶珊瑚苷的含量减少70%以上;厚朴皮中厚朴酚及和厚朴酚的含量减少1%以下;黄柏皮中盐酸小檗碱的含量减少10%以上。结论 三种皮类药材的药效物质在贮藏期变化总体有相似的规律,贮藏时间对杜仲皮皮中桃叶珊瑚苷的含量影响较大,对厚朴皮中厚朴酚与和厚朴酚的含量影响最小,而对黄柏皮中盐酸小檗碱的含量影响较小。研究结果为三种皮类药材的质量控制提供了科学依据。     

Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29

An antifungal protein was isolated from a culture of Bacillus subtilis strain B29. The isolation procedure comprised ion exchange chromatography on diethylaminoethyl (DEAE)-52 cellulose and gel filtration chromatography on Bio-Gel® P-100. The protein was absorbed on DEAE-cellulose and Bio-Gel® P-100. The purified antifungal fraction was designated as B29I, with a molecular mass of 42.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pI value 5.69 by isoelectric focusing (IEF)-PAGE, and 97.81% purity by high performance liquid chromatography (HPLC). B29I exhibited inhibitory activity on mycelial growth in Fusarium oxysporum, Rhizoctonia solani, Fusarium moniliforme, and Sclerotinia sclerotiorum. The 50% inhibitory concentrations (IC₅₀) of its antifungal activity toward Fusarium oxysporum and Rhizoctonia solani were 45 and 112 μmol/L, respectively. B29I also demonstrated an inhibitory effect on conidial spore germination of Fusarium oxysporum and suppression of germ-tube elongation, and induced distortion, tumescence, and rupture of a portion of the germinated spores.     

酸浆对炎症及花生四烯酸代谢途径的影响

探讨酸浆的抗炎镇痛作用及机制. 抗炎实验采用角叉菜胶致大鼠足肿胀和大鼠棉球肉芽肿模型;镇痛实验采用小鼠扭体法. 各模型均随机分为4组:模型组、阳性药吲哚美辛组、酸浆宿萼提取物高剂量组、低剂量组. 测定大鼠足肿胀度,胸腺、脾脏指数,肉芽肿干重,观察小鼠扭体情况,检测足肿胀模型血清中磷脂酶A₂(phospholipaseA₂, PLA₂)、环氧合酶-2(cyclooxygenase 2, COX-2)、前列腺素E₂(prostaglandin E₂, PGE₂)、5-脂氧合酶(5-lipoxygenase, 5-LOX)、白三烯B₄(leukotriene B₄, LTB₄)水平. 实验表明:酸浆宿萼提取物对急、慢性炎症均有显著抑制作用且对机体免疫器官具有一定的保护作用,对急性炎症血清中PLA₂、COX-2、PGE₂、5-LOX、LTB₄均具有抑制作用. 酸浆宿萼提取物可能通过抑制COX-2、5-LOX活性从而双重抑制花生四烯酸(arachidonic acid, AA)代谢途径发挥抗炎作用.      

A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1 × 106 ml^-1, the percentages of conidium germination and appressorium formation were （97.98±0.67）% and （97.88±0.45）%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 lag total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA （complementary DNA） library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.     

Assessment of volumetric bone mineral density of the femoral neck in postmenopausal women with and without vertebral fractures using quantitative multi-slice CT

Objective: To demonstrate the validity and reliability of volumetric quantitative computed tomography (vQCT) with multi-slice computed tomography (MSCT) and dual energy X-ray absorptiometry (DXA) for hip bone mineral density (BMD) measurements, and to compare the differences between the two techniques in discriminating postmenopausal women with osteoporosis-related vertebral fractures from those without. Methods: Ninety subjects were enrolled and divided into three groups based on the BMD values of the lumbar spine and/or the femoral neck by DXA. Groups 1 and 2 consisted of postmenopausal women with BMD changes <−2SD, with and without radiographically confirmed vertebral fracture (n=11 and 33, respectively). Group 3 comprised normal controls with BMD changes ≥−1SD (n=46). Post-MSCT (GE, LightSpeed16) scan reconstructed images of the abdominal-pelvic region, 1.25 mm thick per slice, were processed by OsteoCAD software to calculate the following parameters: volumetric BMD values of trabecular bone (TRAB), cortical bone (CORT), and integral bone (INTGL) of the left femoral neck, femoral neck axis length (NAL), and minimum cross-section area (mCSA). DXA BMD measurements of the lumbar spine (AP-SPINE) and the left femoral neck (NECK) also were performed for each subject. Results: The values of all seven parameters were significantly lower in subjects of Groups 1 and 2 than in normal postmenopausal women (P<0.05, respectively). Comparing Groups 1 and 2, 3D-TRAB and 3D-INTGL were significantly lower in postmenopausal women with vertebral fracture(s) [(109.8±9.61) and (243.3±33.0) mg/cm³, respectively] than in those without [(148.9±7.47) and (285.4±17.8) mg/cm³, respectively] (P<0.05, respectively), but no significant differences were evident in AP-SPINE or NECK BMD. Conclusion: the femoral neck-derived volumetric BMD parameters using vQCT appeared better than the DXA-derived ones in discriminating osteoporotic postmenopausal women with vertebral fractures from those without. vQCT might be useful to evaluate the effect of osteoporotic vertebral fracture status on changes in bone mass in the femoral neck.     

Preliminary studies on the chemical characterization and antioxidant properties of acidic polysaccharides from Sargassum fusiforme

In order to investigate the antioxidant properties of the polysaccharides from the brown alga Sargassum fusiforme, the crude polysaccharides from S. fusiforme (SFPS) were extracted in hot water, and the lipid peroxidation inhibition assay exhibited that SFPS possessed a potential antioxidant activity. Hence, two purely polymeric fractions, SFPS-1 and SFPS-2 were isolated by the column of DEAE (2-diethylaminoethanol)-Sepharose Fast Flow, with their molecular weights of 51.4 and 30.3 kDa determined by high performance gel permeation chromatography (HPGPC). They were preliminarily characterized using chemical analysis in combination of infrared (IR) and nuclear magnetic resonance (NMR) spectroscopies and found to contain large amounts of uronic acids and β-glycosidical linkages. The antioxidant activities of these two SFPS fractions were evaluated using superoxide and hydroxyl radical-scavenging assays. The results show that the antioxidant ability of SFPS-2 was higher than that of SFPS-1, probably correlating with the molecular weight and uronic acid content.     

Science Letters: Transient expression of chicken alpha interferon gene in lettuce

We investigated the possibility of producing chicken alpha interferon （ChlFN-α） in transgenic plants. The cDNA encoding ChlFN-α was introduced into lettuce （Lactuca sativa L.） plants by using an agro-infiltration transient expression system. The ChlFN-a gene was correctly transcribed and translated in the lettuce plants according to RT-PCR and ELISA assays. Recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus （VSV） replication on chicken embryonic fibroblast （CEF）. The results demonstrate that biologically active avian cytokine with potential pharmaceutical applications could be expressed in transgenic lettuce plants and that it is possible to generate interferon protein in forage plants for preventing infectious diseases of poultry.     

Expression, purification and characterization of a phyAm-phyCs fusion phytase

The phyA^m gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichiapastoris in order to expand the pH profile ofphytase and decrease the cost of production. The fusion phytase phyA^m-phyCs gene was successfully overexpressed in P. pastoris as an active and extracellular phytase. The yield of total extracellular fusion phytase activity is （25.4±0.53） U/ml at the flask scale and （159.1±2.92） U/ml for high cell-density fermentation, respectively. Purified fusion phytase exhibits an optimal temperature at 55 ℃ and an optimal pH at 5.5-6.0 and its relative activity remains at a relatively high level of above 70% in the range ofpH 2.0 to 7.0. About 51% to 63% of its original activity remains after incubation at 75 ℃ to 95 ℃ for 10 min. Due to heavy glycosylation, the expressed fusion phytase shows a broad and diffuse band in SDS-PAGE （sodium dodecyl sulfate-polyacrylamide gel electrophoresis）. After deglycosylation by endoglycosidase H （EndoHf）, the enzyme has an apparent molecular size of 95 kDa. The characterization of the fusion phytase was compared with those ofphyCs andphyA^m.     

Ginsenosides stimulated the proliferation of mouse spermatogonia involving activation of protein kinase C

The effect of ginsenosides on proliferation of type A spermatogonia was investigated in 7-day-old mice. Spermatogonia were characterized by c-kit expression and cell proliferation was assessed by immunocytochemical demonstration of proliferating cell nuclear antigen (PCNA). After 72-h culture, Sertoli cells formed a confluent monolayer to which numerous spermatogonial colonies attached. Spermatogonia were positive for c-kit staining and showed high proliferating activity by PCNA expression. Ginsenosides (1.0~10 μg/ml) significantly stimulated proliferation of spermatogonia. Activation of protein kinase C (PKC) elicited proliferation of spermatogonia at 10⁻⁸ to 10⁻⁷ mol/L and the PKC inhibitor H₇ inhibited this effect. Likewise, ginsenosides-stimulated spermatogonial proliferation was suppressed by combined treatment of H₇. These results indicate that the proliferating effect of ginsenosides on mouse type A spermatogonia might be mediated by a mechanism involving the PKC signal transduction pathway.     

Synthesis and electrochemical properties of LiNi0.8Al0.2−xTixO2 cathode materials by an ultrasonic-assisted co-precipitation method

A new co-precipitation route was proposed to synthesize LiNi_0.8Al_0.2−xTi_xO₂ (x=0.0-0.20) cathode materials for lithium ion batteries, with Ni(NO₃)₂, Al(NO₃)₃, LiOH·H₂O, and TiO₂ as the starting materials. Ultrasonic vibration was used during preparing the precursors, and the precursors were protected by absolute ethanol before calcination in the air. The influences of doped-Ti content, calcination temperature and time, additional Li content, and ultrasonic vibration on the structure and properties of LiNi_0.8Al_0.2−xTi_xO₂ were investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM), and charge-discharge tests, respectively. The results show that the optimal molar fraction of Ti, calcination temperature and time, and additional molar fraction of Li for LiNi_0.8Al_0.2−xTi_xO₂ cathode materials are 0.1, 700°C, 20 h, and 0.05, respectively. Ti doping facilitates the formation of the α-NaFeO₂ layered structure, and ultrasonic vibration improves the electrochemical performance of LiNi_0.8Al_0.2−xTi_xO₂.     

Preparation,crystal structure and quantum chemical investigation of [Li(NTO) (H2O)]

[Li(NTO)(H₂O)₂] was prepared by mixing the aqueous solution of 3-nitro-1,2,4-triazol-5-one (NTO) and lithium hydroxide. The crystal structure of [Li(NTO)(H₂O)₂] was determined by single crystal diffraction analysis. The crystal is monoclinic, space group P2₁/n with crystal parameters of a = 0.742 0(2) nm, b = 0.344 9(1) nm,c = 2.490 6(3) nm, β= 94.89(1)°, Z = 4,D _c , = 1.799 g cm⁻³,V = 0.635 nm³, μ = 1.591 cm⁻¹, F(000) = 392. The finalR is 0.051. The MNDO MO calculation shows that the coordinate bonds of title compound possess certain extent of covalent character. O₂ atom of NTO anion is bonded to Li atom; the nitro group will be lost first when NTO is decomposed.     

Effect of CO2 enrichment on the glucosinolate contents under different nitrogen levels in bolting stem of Chinese kale (Brassica alboglabra L.)

The effects of CO₂ enrichment on the growth and glucosinolate (GS) concentrations in the bolting stem of Chinese kale (Brassica alboglabra L.) treated with three nitrogen (N) concentrations (5, 10, and 20 mmol/L) were investigated. Height, stem thickness, and dry weights of the total aerial parts, bolting stems, and roots, as well as the root to shoot ratio, significantly increased as CO₂ concentration was elevated from 350 to 800 μl/L at each N concentration. In the edible part of the bolting stem, 11 individual GSs were identified, including 7 aliphatic and 4 indolyl GSs. GS concentration was affected by the elevated CO₂ concentration, N concentration, and CO₂×N interaction. At 5 and 10 mmol N/L, the concentrations of aliphatic GSs and total GSs significantly increased, whereas those of indolyl GSs were not affected, by elevated atmospheric CO₂. However, at 20 mmol N/L, elevated CO₂ had no significant effects on the concentrations of total GSs and total indolyl GSs, but the concentrations of total aliphatic GSs significantly increased. Moreover, the bolting stem carbon (C) content increased, whereas the N and sulfur (S) contents decreased under elevated CO₂ concentration in the three N treatments, resulting in changes in the C/N and N/S ratios. Also the C/N ratio is not a reliable predictor of change of GS concentration, while the changes in N and S contents and the N/S ratio at the elevated CO₂ concentration may influence the GS concentration in Chinese kale bolting stems. The results demonstrate that high nitrogen supply is beneficial for the growth of Chinese kale, but not for the GS concentration in bolting stems, under elevated CO₂ condition.