Transient expression of chicken alpha interferon gene in lettuce

We investigated the possibility of producing chicken alpha interferon (ChIFN-α) in transgenic plants.The cDNA encoding ChIFN-a was introduced into lettuce (Lactuca sativa L.) plants by using an agro-infiltration transient expression system.The ChIFN-α gene was correctly transcribed and translated in the lettuce plants according to RT-PCR and ELISA assays.Re-combinant 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 ap-plications 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.     

Transgenic barley with overexpressed PTrx increases aluminum resistance in roots during germination

A transgenic barley line (LSY-11-1-1) with overexpressed Phalaris coerulescens thioredoxin gene (PTrx) was employed to measure the growth, protein oxidation, cell viability, and antioxidase activity in barley roots during germination on the presence of 2 mmol/L AlCl₃ on filter paper. The results show that (1) compared with the non-transgenic barley, LSY-11-1-1 had enhanced root growth, although both were seriously inhibited after AlCl₃ treatment; (2) the degree of protein oxidation and loss of cell viability in roots of LSY-11-1-1 were much less than those in roots of non-transgenic barley, as reflected by lower contents of protein carbonyl and Evans blue uptakes in LSY-11-1-1; (3) activities of catalase (CAT), glutathione peroxidase (GPX), ascorbate peroxidase (APX), and glutathione reductase (GR) in LSY-11-1-1 root tips were generally higher than those in non-transgenic barley root tips, although these antioxidase activities gave a rise to different degrees in both LSY-11-1-1 and non-transgenic barley under aluminum stress. These results indicate that overexpressing PTrx could efficiently protect barley roots from oxidative injury by increasing antioxidase activity, thereby quenching ROS caused by AlCl₃ during germination. These properties raise the possibility that transgenic barley with overexpressed PTrx may be used to reduce the aluminum toxicity in acid soils.     

Influence of fasting on muscle composition and antioxidant defenses of market-size Sparus macrocephalus

The study was conducted to investigate fasting effects on flesh composition and antioxidant defenses of market-size Sparus macrocephalus. Two hundred fish (main initial weight 580 g) were divided into two groups (control and fasted) and reared in 6 cages. After two weeks of adaptation, group I fasted for 28 d; group II was fed normally as a control. In 3, 7, 14, 21 and 28 d, 6 fish per group were sampled for proximate flesh composition, liver antioxidant enzyme activities and malondialdehyde flesh content analyses. In fasted fish, the reduction of lipid content in muscle occurred after day 3, and, compared to controls, the content of protein decreased from day 14, the activities of liver antioxidative enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPX) increased from day 3, and flesh malondialdehyde levels increased from day 21. Flesh fat reduction shows that fasting may be used as a technique to reduce flesh lipid content in Sparus macrocephalus. However, considering flesh protein loss and the subsequent oxidative stress, the fasting technique should be used with precautions. Project (No. 2006C12098) supported by the Science and Technology Department of Zhejiang Province, China     

Expression and purification of bioactive high-purity human midkine in Escherichia coli

Midkine is a heparin-binding growth factor, which plays important roles in the regulation of cell growth and differentiation. The non-tagged recombinant human midkine (rhMK) is therefore required to facilitate its functional studies of this important growth factor. In the present work, rhMK was expressed in Escherichia coli (E. coli) BL21 (DE3). The expression of midkine was efficiently induced by isopropyl-β-D-thiogalactopyranoside (IPTG). After sonication, midkine was recovered in an insoluble form, and was dissolved in guanidine hydrochloride buffer. Renaturation of the denatured protein was carried out in the defined protein refolding buffer, and the refolded protein was purified using S-Sepharose ion-exchange chromatography. The final preparation of the rhMK was greater than 98% pure as measured by sodium dodecylsulfate-polyacrylamid gel electrophoresis (SDS-PAGE) and reverse phase high performance liquid chromatography (RP-HPLC). The purified rhMK enhanced the proliferation of NIH3T3 cells. Project supported in part by the Hi-Tech Research and Development Program (863) of China (No. 2007AA02Z149) and the Science and Technology Commission of Shanghai Municipality (No. 075407071), China     

A CHASE domain containing protein kinaseOsCRL4, represents a newAtCRE1-like gene family in rice

AtCRE1 is known to be a cytokinin receptor inArabidopsis. TheAtCRE1 protein contains CHASE domain at the N-terminal part, followed by a transmitter (histidine kinase) domain and two receiver domains. The N-terminal CHASE domain ofAtCRE1 contains putative recognition sites for cytokinin. Five CHASE domains containing proteins were found in rice,osCRL1a,OsCRL1b,OsCRL2,OsCRL3, andOsCRL4.OsCRL1a,OsCRL1b,OsCRL2 andOsCRL3 contain the four domains existing inCRE1, whereasOsCRL4 only contains the CHASE domain and a putative Ser/Thr protein kinase domain. The authors cloned the encoding geneOsCRL4 and found that it represents a new member of the cytokinin receptor protein in rice. Project supported by the National Natural Science Foundation of China     

Effect of interrupted endogenous BMP/Smad signaling on growth and steroidogenesis of porcine granulosa cells

Bone morphogenetic proteins (BMPs) play a critical role in the growth and steroidogenesis of granulosa cells (GCs). BMP signals act through membrane-bound heteromeric serine/threonine kinase receptors. Upon ligand binding, BMPs activate intracellular Smad proteins and regulate growth and apoptosis in various cell types. The objective of this study was to demonstrate the effects of BMP/Smad signal on growth and steroidogenesis of porcine GCs. A strategy of RNA interference (RNAi)-mediated ‘gene silencing’ of Smad4, a core molecule mediating the intracellular BMP/Smad signal transduction pathways, was used to interrupt endogenous BMP/Smad signaling. Results indicate that Smad4-small interfering RNA (siRNA) caused specific inhibition of Smad4 mRNA and protein expression after transfection. Interrupted endogenous BMP/Smad signaling significantly inhibited growth, and induced apoptosis of porcine GCs, while decreasing estradiol production. In addition, interrupted BMP/Smad signaling significantly (P<0.05) changed the expression of Cyclin D2, CDK4, Bcl-2, and Cyp19a1. These findings provide new insights into how BMP/Smad signaling regulates the growth and steroidogenesis of porcine GCs.     

Survival of the biocontrol agents Brevibacillus brevis ZJY-1 and Bacillus subtilis ZJY-116 on the spikes of barley in the field

Fusarium head blight (FHB) caused byFusarium graminearum is a devastating disease that results in extensive yield losses to wheat and barley. A green fluorescent protein (GFP) expressing plasmid pRP22-GFP was constructed for monitoring the colonization of two biocontrol agents,Brevibacillus brevis ZJY-116, on the spikes of barley and their effect on suppression of FHB. Survival and colonization of theBrevibacillus brevis ZJY-1 andBacillus subtilis ZJY-116 strains on spikes of barley were observed by tracking the bacterial transformants with GFP expression. Our field study revealed that plasmid pRP22-GFP was stably maintained in the bacterial strains without selective pressure. The retrieved GFP-tagged strains showed that the bacterial population fluctuation accorded with that of the rain events. Furthermore, both biocontrol strains gave significant protection against FHB on spikes of barley in fields. The greater suppression of barley FHB disease was resulted from the treatment of barley spikes with biocontrol agents before inoculation withF. graminearum. Project supported by the National Natural Science Foundation of China (No. 30230250) and Science and Technology Committee of Zhejiang Province (No. 2003C22029), China     

Morphological and pathologic changes of experimental chronic atrophic gastritis （CAG） and the regulating mechanism of protein expression in rats

INTRODUCTION In 1998 gastrointestinal pathologists reached aconsensus on the definition of chronic atrophic gas- tritis (CAG), which was described as programmed loss of gastric gland and/or replacement by intestinal glands in gastric mucosa. CAG was recognized to be closely related with development of gastric cancer and listed as precancerous lesions in this meeting (Genta, 1998). According to Correa (1992)’s cascade of gas-tric carcinogenesis, gastric cancer was believed to develop fr…     

MoFLP1, encoding a novel fungal fasciclin-like protein, is involved in conidiation and pathogenicity in Magnaporthe oryzae

Fasciclin family proteins have been identified as cell adhesion molecules in various organisms. In this study, a novel Magnaporthe oryzae fasciclin-like protein encoding gene, named MoFLP1, was isolated from a subtractive suppressive cDNA library and functionally analyzed. Sequence analysis showed that the MoFLP1 gene contains an open reading frame (ORF) of 1050 nucleotides encoding 349 amino acids with a calculated molecular weight of 35.85 kDa and a pI of 7.76. The deduced MoFLP1 protein contains a 17-amino acid secretion signal sequence and an 18-amino acid sequence with the characteristics of a glycosylphosphotidylinositol (GPI) anchor additional signal at its N- and C-terminuses, respectively. Potential N-glycosylation sites and domains involving cell adhesion were also identified in MoFLP1. Sequence analysis and subcellular localization by the expression of MoFLP1-GFP fusion construct in M. oryzae indicated that the MoFLP1 protein is probably localized on the vacuole membrane. Two MoFLP1 null mutants generated by targeted gene disruption exhibited marked reduction of conidiation, conidial adhesion, appressorium turgor, and pathogenicity. Our results indicate that fasciclin proteins play important roles in fungal development and pathogenicity in M. oryzae. Project supported by the National Natural Science Foundation of China (No. 30870101) and the Public Welfare Profession (Agriculture) Research Project (No. 200803008), China     

Expression and significance of cyclin D1, p27kip1 protein in bronchioloalveolar carcinoma

Purpose: To investigate the relationship between expression of cell cycle-related protein cyclin D1, p27kipl and the pathogenesis of bronchioloalveolar carcinoma (BAC) and the value of prediction of prognosis. Methods: Cyclin D 1 and p27kip 1 protein were detected by immunohistochemical En Vision method in 43 BACs. Results: The positivity of cyclin D 1 in BAC was 65.1% (28/43), which was significantly higher than that in normal pulmonary tissue (0/13), P＜0.01. No statistically significant association was found between cyclin D1 expression data and sex, age, tobacco-use history, histologic subtype (mucinous vs nonmucinous), stromal fibrosis, lymph node metastasis, clinical stage or postoperative survival period (P＞0.05), while cyclin D1 expression was found to be negatively correlated with tumor size (P＜0.05). The positivity of p27kipl in BACs was 51.2% (22/43), significantly lower than that in normal pulmonary tissue (12/13), P＜0.01. p27kipl expression level was not associated with sex, age, tobacco-use history, tumor size or histologic subtype (P＞0.05), but was negatively correlated with stromal fibrosis, lymph node metastasis and clinical stage (P＜0.05); and positively associated with postoperative survival period (P＜0.01). The survival rate of p27kipl positive group was significantly higher than that of p27kipl negative group (P＜0.01). No statistically significant correlation was found between cyclin D 1 and p27kipl expression. Conclusions: Increased cyclin D1 expression and decreased p27kip 1 expression are related to the pathogenesis of BAC;decreased p27kipl expression is associated with metastasis progression; immunodetection ofp27kip 1 is useful for assessment of prognosis.     

Availability and toxicity of Fe(II) and Fe(III) in Caco-2 cells

The objective of the present study was to compare the toxicity and availability of Fe(II) and Fe(III) to Caco-2 cells. Cellular damage was studied by measuring cell proliferation and lactate dehydrogenase (LDH) release. The activities of two major antioxidative enzymes [superoxide dismutase (SOD) and glutathione peroxidase (GPx)] and differentiation marker (alkaline phosphatase) were determined after the cells were exposed to different levels of iron salts. The cellular iron concentration was investigated to evaluate iron bioavailability. The results show that iron uptake of the cells treated with Fe(II) is significantly higher than that of the cells treated with Fe(III) (P<0.05). Fe(II) at a concentration >1.5 mmol/L was found to be more effective in reducing cellular viability than Fe(III). LDH release investigation suggests that Fe(II) can reduce stability of the cell membrane. The activities of SOD and GPx of the cells treated with Fe(II) were higher than those of the cells treated with Fe(III), although both of them increased with raising iron supply levels. The results indicate that both Fe(II) and Fe(III) could reduce the cellular antioxidase gene expression at high levels. Project supported by the International Cooperative Project from the Ministry of Science and Technology of China (No. 2006DFA31030), the Bureau of Science and Technology of Zhejiang Province (No. 2006C32019) and HarvestPlus-China (No. 8022), and the Program for Changjiang Scholars and Innovative Research Team in University of China (No. IRT0536)     

Handbook of human performance technology: Principles,practices, potential (3rd ed.) edited by James A. Pershing

James A. Pershing, CPT, PhD, is a professor of education in instructional systems technology and educational inquiry methodology at Indiana University. He is the former editor of the Performance Improvement journal and TechTrends, past director of the International Society for Performance Improvement, and association manager of the Association for Educational Communications and Technology. He is now a consulting reviewer for TechTrends and is editor of the Asia Pacific Cybereducation Journal. The Handbook of Human Performance Technology: Principles, Practices, Potential (3rd ed.; ISBN: 978‐0‐7879‐6530‐0) is published by John Wiley & Sons, Inc. The publisher may be reached online at http://www.pfeiffer.com/WileyCDA/ or contacted at 978–750–8400.     

Hydroxychavicol,a polyphenol from Piper betle leaf extract,induces cell cycle arrest and apoptosis in TP53-resistant HT-29 colon cancer cells

This study aims to elucidate the antiproliferative mechanism of hydroxychavicol(HC).Its effects on cell cycle,apoptosis,and the expression of c-Jun N-terminal kinase(JNK)and P38 mitogen-activated protein kinase(MAPK)in HT-29 colon cancer cells were investigated.HC was isolated from Piper betle leaf(PBL)and verified by high-performance liquid chromatography(HPLC),nuclear magnetic resonance(NMR),and gas chromatography-mass spectrometry(GC-MS).The cytotoxic effects of the standard drug 5-fluorouracil(5-FU),PBL water extract,and HC on HT-29 cells were measured after 24,48,and 72 h of treatment.Cell cycle and apoptosis modulation by 5-FU and HC treatments were investigated up to 30 h.Changes in phosphorylated JNK(pJNK)and P38(pP38)MAPK expression were observed up to 18 h.The half maximal inhibitory concentration(IC₅₀)values of HC(30μg/mL)and PBL water extract(380μg/mL)were achieved at 24 h,whereas the IC₅₀of 5-FU(50μmol/L)was obtained at 72 h.Cell cycle arrest at the G0/G1 phase in HC-treated cells was observed from12 h onwards.Higher apoptotic cell death in HC-treated cells compared to 5-FU-treated cells(P<0.05)was observed.High expression of pJNK and pP38 MAPK was observed at 12 h in HC-treated cells,but not in 5-FU-treated HT-29 cells(P<0.05).It is concluded that HC induces cell cycle arrest and apoptosis of HT-29 cells,with these actions possibly mediated by JNK and P38 MAPK.     

Hepatic glutathione S-transferase activity in mosquitofish (Gambusia affinis) and topmouth gudgeon (Pseudorasobora parva) exposed to fenitrothion

Two common fish species, mosquitofish (Gambusia affinis) and topmouth gudgeon (Pseudorasobora para) were exposed to different concentrations of fenitrothion in static system for 96 h. Hepatic glutathione S-transferase activity was evaluated after 48 and 96 h pesticide exposure, and was also examined in fish pretreated with pepironyl butoxide and triphenyl phosphate and then exposed to fenitrothion. Results indicated presence of intense glutathione S-transferase activity in both species, mosquitofish exhibiting the higher activity. In both species the activity decreased as the concentration of fenitrothion increased, topmouth gudgeon being more susceptible than mosquitofish. In mosquitofish pretreated with pepironyl butoxide, glutathione S-transferase activity was increased (11.8%) compared with the control but in topmouth gudgeon it was decreased (21.6%) at the end of 96 h. Glutathione S-transferase activity was significantly reduced in both species pretreated with triphenyl phosphate at the end of 96 h exposure, topmouth gudgeon being highly susceptible. Ph. D. student, from Southern Ethiopia Health Bureau, Center for Health Research, P. O. Box 317, Awassa, F. R. Ethiopia.     

Alleviative effect of quercetin on germ cells intoxicated by 3-methyl-4-nitrophenol from diesel exhaust particles

As a component of diesel exhaust particles, 3-methyl-4-nitrophenol (4-nitro-m-cresol, PNMC) is also a metabolite of the insecticide fenitrothion and imposes hazardous effects on human health. In the present study, the alleviative effect of a common antioxidant flavonoid quercetin on mouse germ cells intoxicated by PNMC was investigated. Results showed that a single intraperitoneal injection of PNMC at 100 mg/kg induced severe testicular damage after one week. PNMC-treated mice showed a significant loss of germ cells (approximate 40% loss of round germ cells). PNMC caused an increase of hydroxyl radical and hydrogen peroxide production and lipid peroxidation, as well as a decrease in glutathione level, superoxide dismutase and glutathione peroxidase activities. Furthermore, treatment of PNMC increased expression of the pro-apoptotic protein Bax and decreased expression of the anti-apoptotic protein Bcl-XL in germ cells. In addition, testicular caspase-3 activity was significantly up-regulated and germ cell apoptosis was significantly increased in the PNMC-treated mice. In contrast, combined administration of quercetin at 75 mg/kg significantly attenuated PNMC-induced testicular toxicity. These results indicate that the antioxidant quercetin displays a remarkable protective effect on PNMC-induced oxidative damage in mouse testes and may represent an efficient supplement to attenuate reproductive toxicity by environmental toxicants to ensure healthy sperm production.     

Properties of vortex system with random columnar defects at zero temperature

The properties of a two-dimensional vortex system with random columnar defects were studied by cooling the vortex system to zero temperature. The vortex lattice became more and more disordered with increasing pinning strengthf _p. At smallf _p, a triangular vortex, lattice away from the pins was observed. The peak of structure factorS(G₀) of the vortex lattice decreased with pinning strengthf _p, which accorded with the finding that the probability of vortex to be pinned increased withf _p. Some of our results agreed with experimental findings. Project supported by the Ministry of Science and Technology of China (NKBRSF-G19990646) and Zhejiang Province Scientific Foundation (199031).     

Fusion expression of pedA gene to obtain biologically active pediocin PA-1 in Escherichia coli

Two heterologous expression systems using thioredoxin (trxA) as a gene fusion part in Escherichia coli were developed to produce recombinant pediocin PA-1. Pediocin PA-1 structural gene pedA was isolated from Pediococcus acidilactici PA003 by the method of polymerase chain reaction (PCR), then cloned into vector pET32a(+), and expressed as thioredoxin-PedA fusion protein in the host strain E. coli BL21 (DE3). The fusion protein was in the form of inclusion body and was refolded before purification by nickel-iminodiacetic acid (Ni-IDA) agarose resin column. Biological activity of recombinant pediocin PA-1 was analyzed after cleavage of the fusion protein by enterokinase. Agar diffusion test revealed that 512-arbitrary unit (AU) recombinant pediocin PA-1 was obtained from 1 ml culture medium of E. coli (pPA003PED1) using Listeria monocytogenes as the indicator strain. Thioredoxin-PedA fusion gene was further cloned into pET20b(+). Thioredoxin-PedA fusion protein was detected in both the periplasmic and cytoplasmic spaces. The recombinant pediocin PA-1 from the soluble fraction attained 384 AU from 1 ml culture medium of E. coli (pPA003PED2). Therefore, biologically active pediocin PA-1 could be obtained by these two hybrid gene expression methods.     

Effects of α-tocopheryl acetate supplementation in preslaughter diet on antioxidant enzyme activities and fillet quality of commercial-size Sparus macrocephalus

This study examined the effects of dietary α-tocopheryl acetate supplementation on antioxidant enzyme activities and fillet quality in commercial-size Sparus macrocephalus. Three hundred fish [main initial weight （350±12） g] were divided into three groups （E250, E500 and El000） and reared in 9 cages. The fish were fed for 8 weeks with three diets containing different levels of dietary α-tocopheryl acetate （289, 553, 1069 mg/kg）. Over the experimental period, fish were fed to satiation and reached a final mean weight of （465±28） g without significant body weight difference and proximate composition difference. Fillet α-tocopherol was significantly （P〈0.05） different between groups, reaching levels of 14.2, 22.1, 30.9 pg/mg fillet for groups E250, E500 and El000, respectively. Total serum superoxide dismutase （SOD） activity increased significantly （P〈0.05） in fish fed the diets high in α-tocopheryl acetate, but serum glutathione peroxidase （GPX） activity was unaffected. In storage on ice, fillets of fish fed the diets high in α-tocopheryl acetate exhibited significantly lower （P〈0.05） levels of oxidation. These results suggested that increased dietary α-tocopheryl acetate could increase its flesh deposition, increase the activity of SOD and prevent lipid peroxidation ofSparus macrocephalus fillets in retail storage on ice.