iTRAQ-Based Proteomics Investigation of Critical Response Proteins in Embryo and Coleoptile During Rice Anaerobic Germination

Direct-seeding of rice has become popular in recent years due to its low cost and convenience,however,hypoxic condition limits seedling establishment.In this study,weedy rice WR04-6 with high germination ability under anaerobic conditions was used as a gene donor,and we successfully improved the seedling establishment rate of rice cultivar Qishanzhan(QSZ)based on selection of a new rice line R42 from the recombinant inbred line population.R42 inherited high anaerobic germination(AG)ability,and was used for isobaric tags for relative and absolute quantitation(iTRAQ)-based comparative proteomic studies with QSZ to further explore the molecular mechanism of AG.A total of 719 differentially abundant proteins(DAPs)were shared by R42 and QSZ responded to AG,and thus defined as common response DAPs.A total of 300 DAPs that responded to AG were only identified from R42,which were defined as tolerance-specific DAPs.The common response and tolerance-specific DAPs had similar biochemical reaction processes and metabolic pathways in response to anoxic stress,however,they involved different proteins.The tolerance-specific DAPs were involved in amino acid metabolism,starch and sucrose metabolism,tricarboxylic acid cycle pathway,ethylene synthesis pathway,cell wall-associated proteins and activity of active oxygen scavenging enzyme.The in silico protein-protein interactions for the top 60 DAPs indicated that tolerance-specific DAPs had relatively independent protein interaction networks in response to an anoxic environment compared with common response DAPs.The results of physiological indicators showed thatα-amylase and superoxide dismutase activities of R42 were significantly increased under anoxic conditions compared with aerobic conditions.Multiple lines of evidence from western blot,physiological analysis and quantitatDirect seeding of rice has become popular in recent years due to its low cost and convenience,however,hypoxic conditions can limit seedling establishment.In the present study,weedy rice WR04-6 with high germination ability in anaerobic conditions was used as the gene donor and successfully improved the seedling establishment rate of rice cultivar Qishanzhan(QSZ)based on selection of a new rice line R42 from the recombinant inbred line(RIL)population.R42 inherited the had high anaerobic germination(AG)ability,which was used for the isobaric tags for relative and absolute quantitation(iTRAQ)based comparative proteomic studies with QSZ to further explore the molecular mechanism of AG.A total of 719 differentially abundant proteins(DAPs)shared by R42 and QSZ responded to AG and were thus defined as common response DAPs.A total of 300 DAPs that responded to AG were only identified from R42,which were defined as tolerance-specific DAPs.The common response and tolerance-specific DAPs had similar biochemical reaction processes and metabolic pathways in response to anoxic stress,however they involved different proteins.The 300 tolerance-specific DAPs were involved in amino acid metabolism,starch and sucrose metabolism,TCA cycle pathways,ethylene synthesis pathway,cell wall-associated proteins and activity of active oxygen scavenging enzyme.The in silico protein-protein interactions for the top 60 DAPs indicated that tolerance-specific DAPs had relatively independent protein interaction networks in response to an anoxic environment compared with common response DAPs.The results of physiological indicators showed thatα-amylase and SOD activities of R42 were significantly increased under anoxic conditions compared with aerobic conditions.Multiple lines of evidence from western blot,physiological analyses and real-time PCR jointly supported the reliability of proteomics data.In summary,our findings deepened the understanding of the molecular mechanism for the rice response to AG.ive real-time PCR jointly supported the reliability of proteomics data.In summary,our findings deepened the understanding of the molecular mechanism for the rice response to AG.

Protein Bodies in Cotyledon Cells Exhibit Differential Patterns of Legumin-Like Proteins Mobilization during Seedling Germinating States

Olive (Olea europaea L.) tree is one of the most extensive and important agricultural crop in Mediterranean countries due to its beneficial health and nutritional properties and its high economic value. Currently, olive tree constitutes the sixth most important cultivated plant in the world, spreading from the Mediterranean region of origin to new production areas such as Australia, South and North America and South Africa. However, the mobilization processes of storage materials i.e. reserve proteins during seed germination, which are largely involved in essential physiological process including plant growth and development, remain poorly understood. Morphometric and immunohistochemistry analyses of protein bodies contained in olive seed storage tissues, cotyledon and endosperm, were performed by using different microscopy techniques, including light (bright-field and fluorescence) microscopy and transmission electron microscopy. Furthermore, we used legumin-like proteins (11S-type globulins) as a molecular marker to study the mobilization of reserve proteins from PBs of cotyledons at germinating seedling stages by using immunofluorescence assays. Results demonstrated that cotyledon and endosperm are characterized by distinct PBs populations containing legumin-like proteins, distinctly discriminated by the number of PBs per cell and tissue, size, immunofluorescence and histochemical staining. These features reflect differential PBs biogenesis during development and maturation processes in olive seed tissues endosperm and cotyledon, in relation to proteins (polypeptides) final composition, SSPs processing and/or packaging during seed maturation. Three different mobilization patterns of legumin-like proteins were identified for the first time in cotyledon PBs during seedling germinating process. Mature proteins composition and/or processing, cell types and enzyme composition and/or differential activation have been discussed as key features determining how proteins mobilize from PBs for further degradation in the cotyledon.

Altered profiles of nuclear matrix proteins during the differentiation of human gastric mucous adenocarcinoma MGc80-3 cells

AIM： To find and identify specific nuclear matrix proteins associated with proliferation and differentiation of carcinoma cells, which will be potential markers for cancer diagnosis and targets in cancer therapy. METHODS： Nuclear matrix proteins were selectively extracted from MGcS0-3 cells treated with or without hexamethylamine bisacetamide （HMBA）, and subjected to 2-D gel electrophoresis. The resulted protein patterns were analyzed by Melanie software. Spots of nuclear matrix proteins differentially expressed were excised and subjected to in situ digestion with trypsin. Peptide masses were obtained by matrix-assisted laser-desorption/ ionization time of flight mass spectrometry （MALDI-TOFMS） analysis and submitted for database searching using Mascot tool. RESULTS： The MGc80-3 cells were induced into differentiation by HMBA. There were 22 protein spots which changed remarkably in the nuclear matrix, from differentiation of MGcS0-3 cells compared to control. Eleven of which were identified. Seven proteinsactin, prohibitin, porin 31HL, heterogeneous nuclear dbonucleoprotein A2/B1, vimentin, ATP synthase, and heat shock protein 60 were downregulated, whereas three proteins - heat shock protein gp96, heat shock protein 90-beta, and valosin-containing protein were upregulated, and the oxygen-regulated protein was only found in the differentiated MGc80-3 cells. CONCLUSION： The induced differentiation of carcinoma cells is accompanied by the changes of nuclear matrix proteins. Further characterization of those proteins will show the mechanism of cellular proliferation and differentiation, as well as cancer differentiation.

Storage Proteins and Trypsin Inhibitors of an Underutilized Legume, <i>Mucuna</i>: Variability and Their Stability during Germination

The proteins and trypsin inhibitors were isolated from the seeds of different varieties/accessions of an underutilized legume, Mucuna. The crude protein content of all the germplasms of Mucuna is varied from 15% - 26%, showed little variation and contain higher crude protein when compared with other Mucuna species reported earlier and the pulse crops commonly consumed in India. The seeds of all the varieties of Mucuna exhibited trypsin inhibitor activity. The trypsin inhibitor activity varied from 11 - 14 TIA/mg of protein. Not much variation was observed in trypsin inhibitory activities in soaked seeds compared to dry seeds. Germination of Mucuna pruriens has been carried out and the change in the protein content and trypsin inhibitors were monitored. The protein content of the endosperm increased up to 72 hrs of germination and then decreased. The trypsin inhibitory activity decreased with increase in germination time. The trypsin inhibitor activity was decreased from 14.81 TIA/mg to 2.62 TIA/mg (82% reduction in the trypsin inhibitor activity) after 144 hrs germination.

A Cdc2-related protein kinase hPFTAIRE1 from human brain interacting with 14-3-3 proteins

hPFTAIRE1 （PFTK1）, a Cdc2-related protein kinase, is highly expressed in human brain. It exhibits cytoplasmic distribution in Hela cells, although it contains two nuclear localization signals （NLSs） in its N-terminus. To search for its substrates and regulatory components, we screened a two-hybrid library by using the full-length hPFTAIRE1 as a bait. Four 14-3-3 isoforms （β,ε,η,τ） were identified interacting with the hPFTAIRE1. We found a putative 14-3-3 binding consensus motif（RHSSPSS） in the hPFTAIRE 1, which overlapped with its second NLS. Deletion of the RHSSPSS motif or substitution of Ser^119 gwithAla in the conserved binding motif abolished the specific interaction between the hPFTAIRE 1 and the 14-3 -3 proteins. The mutant S 120A hPFTAIRE1 also showed a weak interaction to the 14-3-3 proteins. The results suggested that the Ser^119 is crucial for the interaction between hPFTAIREI and the 14-3-3 proteins. All the hPFTAIRE1 mutants distributed in cytoplasm of Hela cells and human neuroblastoma cells （SH-SY5Y） when fused to the C-terminus of a green fluorescent protein （GFP）, indicating that binding with the 14-3-3 proteins does not contribute to the subcellular localization of the hPFTAIRE1, although the binding may be involved in its signaling regulation.

Genome-wide analysis of OVATE family proteins in cucumber(Cucumis sativus L.)

OVATE family proteins(OFPs)are plant-specific proteins with a conserved OVATE domain that regulate plant growth and development.Although OFPs have been studied in several species,their biological functions remain largely unknown in cucumber(Cucumis sativus L.).This study identified 19 Cs OFPs distributed on seven chromosomes in cucumber.Most Cs OFP genes were expressed in reproductive organs,but with different expression patterns.Ectopic expression of Cs OFP12-16c in Arabidopsis resulted in shorter and blunt siliques.The overall results indicated that Cs OFP12-16c regulates silique development in Arabidopsis and may have a similar function in cucumber.

Effects of natural cerebrolysin on protective proteins and pro-apoptotic molecules in mesenchymal stem cells following beta-amyloid peptide1-40-induced endoplasmic reticulum stress

BACKGROUND： Studies have demonstrated that β-amyloid peptide （Aβ）, a characteristic pathological product of Alzheimer＇s disease （AD）, results in neuronal endoplasmic reticulum stress （ERS）. However, the mechanisms of traditional Chinese medicine against ERS in AD are poorly understood. OBJECTIVE： To measure expression levels of protective proteins （GRP78 and GRP94） of ER molecular partners and pro-apoptotic Caspase-12 ER membrane expression following application of traditional Chinese medicine natural cerebrolysin （NC） to treat Aβ1-40-induced ERS. DESIGN, TIME AND SETTING： A parallel-controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital of Southern Medical University between September 2006 and November 2008. MATERIALS： Sprague Dawley male rats, 6-8 weeks old, were used to harvest tibial and femoral bone marrow. Isolation and purification of mesenchymal stem cells （MSCs） were established from the whole bone marrow by removing non-adherent cells in primary and passage cultures. Aβ1-40 was provided by Sigma, USA. NC was provided by Shenzhen Institute of Integrated Chinese and Western Medicine, China. NC was predominantly composed of Renshen （Radix Ginseng）, Tianma （Rhizoma Gastrodiae）, and Yinxingye （Ginkgo Leaf） in a proportion of 1 ： 2： 2. Following conventional water extraction technology, an extract （1 ： 20） was prepared. Six adult, male, New Zealand rabbits underwent intragastric administration of NC extract （0.976 g/kg per day） for 1 month to prepare NC-positive serum, and the remaining 6 rabbits received intragastric administration of physiological saline to prepare normal blank serum. METHODS： A total of 500 nmol/L Aβ1-40 was used to establish ERS models of primary cultured MSCs. AD cell models were incubated with different doses of NC-positive serum （2.5%, 5%, and 10%）. MSCs treated with normal blank serum served as normal blank controls. MAIN OUTCOME MEASURES： Reverse transcription-polymerase chain reaction and fluorescent immunocytochemistry were respectively used to measure mRNA and protein expression levels of GRP78, GRP94, and Caspase-12 in MSCs. RESULTS： Following Aβ1-40 exposure, mRNA and protein expression levels of GRP78 and GRP94, as well as Caspase-12, significantly increased （P 〈 0.05）, suggesting successful establishment of ERS models. Following NC-positive serum application, mRNA and protein expression levels of GRP78 and GRP94 in MSCs significantly increased （P 〈 0.05 or P 〈 0.01）. However, mRNA and protein expression levels of Caspase-12 significantly decreased （P 〈 0.05, or P 〈 0.01） compared with the ERS model group. These effects were dose-dependent. CONCLUSION： NC downregulated Caspase-12 expression and upregulated GRP78 and GRP94 expression in MSCs in a dose-dependent manner under the state of Aβ1-40-induced ERS.

Cloning and Sequence Analysis of Ma-14-3-3d Encoding a Homologue 14-3-3 Protein from Banana

[Objective] The aim of the study is to clone and analyze the gene encoding 14-3-3 protein from banana. [Method] Combined with PCR amplification, RACE (rapid amplification of cDNA ends) technique was employed to clone 14-3-3 gene from banana; then the amplified sequence was sequenced and homologically analyzed. [Result] A new cDNA homologous with 14-3-3 protein genes were obtained by RT-PCR and RACE ( rapid amplification of cDNA ends ) approaches. The full length of this cDNA was 866 bp encoding 197 amino acids. Alignment of deduced amino acid sequence with those from other plants revealed that the cDNA shared high homology with 14-3-3 protein genes from other plants, and was designated as Musa acuminata 14-3-3 gene (Ma-14-3-3d). Phylogenetic analysis reveals that Ma-14-3-3d has closer genetic relationship with those from monocotyledon species than those from other species. [Conclusion] Ma-14-3-3d belongs to the same lineage of 14-3-3 from monocotyledon.

Changes of Chorophyll protein Complexes and Photosynthetic Activities of Chloroplasts from Lotus (Nelumbo nucifera) Seeds Germinating in Light

The changes of chlorophyll_protein complexes and photosynthetic activities of chloroplast isolated from lotus ( Nelumbo nucifera Gaertn.) seeds germinating under illumination were studied. SDS PAGE analysis of chlorophyll_protein complexes showed that there was only the light harvesting chlorophyll a/b protein complex from PSⅡ (LHCⅡ) precursor in chloroplast from lotus seeds germinated for 2 to 6 days, while LHC Ⅱ 1, and the chlorophyll_protein complex of PSⅠ (CPⅠ) appeared on the 8th day of germination and PSⅡ reaction center complex appeared later. Studies on the polypeptides composition of the chloroplast revealed the following results: 1) Small amount of the 27 kD polypeptide was synthesized in invisible light; 2) The 30 kD polypeptide existed previously in the plumules of the dry seeds; 3) The amount of the 30 kD polypeptide was more than any other polypeptides before germination and decreased gradually throughout germination, while the 27 kD polypeptide changed in the opposite way; 4) In the process of germination, measurement of the electron transport rate and the fluorescence induction kinetics at room temperature showed that PSⅡ activities and efficiency of primary light energy transformation were only experimentally measurable after 7 days of germination and gradually increased afterwards. At the same time, the chl a/b ratio rose from the lower value to normal; 5) The changes of chloroplast membrane components and its functions are concomitant in concert with that of the ultrastructure of chloroplast membranes during germination, as shown in our earlier work . The results have proved again that a different developmental pathway of chloroplast is likely to exist in the lotus plumules, which might provide an important clue for N. nucifera in having an unique position in the phylogeny of the angiosperm.

Overexpression of 14-3-3 protein protects pheochromocytoma cells against 1-methyl-4-phenylpyridinium toxicity

Objective To investigate the effects of 14-3-3 protein overexpression on the 1-methyl-4-phenylpyridinium （MPP^＋） induced pheochromocytoma （PC12） cell death and the potential mechanisms. Methods pcDNA3.1（＋）-14-3-3 plasmids, which could be expressed in mammalian cell, were constructed and transfected into PC 12 cells with Lipofectamine 2000. The expression of 14-3-3 protein, Bcl-2 protein, and BAD protein were determined by western blot. 3-（4,5- dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, microplate reader, and flow cytometric analysis were used to measure cell viability, the caspase activity, and apoptotic ratio respectively. Results （1） The expression of 14-3-3 protein increased significantly three weeks after pcDNA3.1（＋）-14-3-3 plasmids transfected into PC 12 cells. （2） MPP^＋ caused a decrease of cell viability in a dose-dependent manner. At 100μmol/L MPP^＋, cell viability reduced approximately 50%. （3） The caspase activity increased along with the MPP^＋ concentrations rising and reached its maximum value （0.34 μmol/mg protein） at 100 μmol/L MPP＊. However caspase activity decreased significantly when the MPP^＋ concentration exceeded 100 μmol/L. （4） Overexpression of 14-3-3 protein decreased the apoptosis ratio of PC 12 cells treated with 100μmol/L MPP^＋ from 26.5% to 8.6%. （5） Bcl-2 protein tended to decrease but BAD protein tended to increase after treatment of PC 12 cells with 100 μmol/L MPP^＋. Overexpression of 14-3-3 protein significantly increased the cellular level of Bcl-2 protein and decreased that of BAD protein. Conclusion Overexpression of 14-3-3 protein may reduce MPP^＋-induced apoptotic cell death in PC12 cells by up-regulating the Bcl-2 expression and down-regulating the BAD expression. These results may provide a promising target for treatment of Parkinson＇s disease.

Involvement of ERK1/2 and p38 MAPK in up-regulation of 14-3-3 protein induced by hydrogen peroxide preconditioning in PC12 cells

Objective To investigate the protective effects of hydrogen peroxide preconditioning （HPP） on the pheochromocytoma （PC12） cells treated with 1-methyl-4-phenylpyridinium （MPP^＋） and to explore the potential mechanisms. Methods The viability and apoptosis of PC 12 cells were determinded by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and 4′,6′-diamidino-2-phenylindole （DAPI） staining, respectively. The expressions of 14-3-3 protein and phospholylated p38 mitogen-activated protein kinase （MAPK） were determined by Western blot. Enzyme-linked immunosorbent assay （ELISA） was used to measure the activity of extracellular signal-regulated protein kinase 1/2 （ERK1/2）. Results The cell viability decreased and the number of apoptotic cells increased dramatically in MPP^＋ group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP^＋- treated PC 12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK 1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC 12 cells induced by HPP. Conclusion HPP protects PC 12 cells against MPP＋ toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.

Prediction of Protein OmpH in Structure of C47-8 Pasteurella multocida

[Objective] This study aimed to predict the structure of protein OmpH from Pasteurella multocida C47-8 （PmC47-8） strain of yak. [Method] Online BLAST, signal peptide prediction, secondary structure prediction and protein characteristics of sequencing result of gene OmpH from PmC47-8 strain were analyzed. [Result] The similarities of gene OmpH from PmC47-8 with the published 81 OmpH genes were between 84% and 99%; a signal peptide was found with the cleavage sites between 20 and 21 in the polypeptide; secondary structure prediction showed that folding structure accounted for 49.8% and loop structure for 50.2%; it predicted that there were 7 O-glycosylation sites in OmpH protein with the amino acid residual sites of 2, 45, 48, 330, 716, 721, 723, respectively, and 2 N-glycosylation sites with the amino acid residual sites of 15 and 35. [Conclusion] This study lays the foundation for the study on the immunity of OmpH gene from yak.

Effect of Germination on the Nutritional and Protein Profile of Australian Sweet Lupin (<i>Lupinus angustifolius</i>L.)

Australian Sweet Lupin (ASL) has a nutritional profile ideally suited for human consumption with high protein and fibre, but low starch and fat content. The nutritional and protein profile of germinated ASL may be better than ungerminated ASL and these improvements would provide further benefits in its use as an ingredient in food applications. In this study the nutritional components such as protein, crude fibre, fat and protein profile of germinated ASL flour following germination at 25℃ and 90% - 95% relative humidity for 9 days were determined. The changes in the pattern of ASL protein during germination were analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Germination significantly increased crude fibre contents by 450% (db), total protein contents by 38% (db) and decreased the fat content by 70% (db) at day 9 of germination. Electrophoretic analysis of the protein fractions revealed that during germination up to 9 days, some of the high molecular weight proteins disappeared. Germination represents a means to further improve the nutritional profile of the germinated ASL flour with an increased fibre and protein, but lower fat content.

Rice leaf inclination2, a VIN3-1ike protein, regulates leaf angle through modulating cell division of the collar

As an important agronomic trait, inclination of leaves is crucial Ior crop architecture and grain yields. 10 understand the molecular mechanism controlling rice leaf angles, one rice leaf inclination2 （1c2, three alleles） mutant was identified and functionally characterized. Compared to wild-type plants, lc2 mutants have enlarged leaf angles due to increased cell division in the adaxial epidermis of lamina joint. The LC2 gene was isolated through positional cloning, and encodes a vernalization insensitive 3-like protein. Complementary expression of LC2 reversed the enlarged leaf angles of lc2 plants, confirming its role in controlling leaf inclination. LC2 is mainly expressed in the lamina joint during leaf development, and particularly, is induced by the phytohormones abscisic acid, gibberellic acid, auxin, and brassinosteroids. LC2 is localized in the nucleus and defects of LC2 result in altered expression of cell division and hormone-responsive genes, indicating an important role of LC2 in regulating leaf inclination and mediating hormone effects.

Adjusting CA19-9 values to predict malignancy in obstructive jaundice:Influence of bilirubin and C-reactive protein

AIM:To find a possible relationship between inflammation and CA19-9 tumor marker by analyzing data from patients with benign jaundice(BJ) and malignant jaundice(MJ).METHODS:All patients admitted for obstructive jaundice,in the period 2005-2009,were prospectively enrolled in the study,obtaining a total of 102 patients.On admission,all patients underwent complete standard blood test examinations including C-reactive protein(CRP),bilirubin,CA19-9.Patients were considered eligible for the study when they presented obstructive jaundice confirmed by instrumental examinations and increased serum bilirubin levels(total bilirubin > 2.0 mg/dL).The standard cut-off level for CA19-9 was 32 U/mL,whereas for CRP this was 1.5 mg/L.The CA19-9 level was adjusted by dividing it by the value of serum bilirubin or by the CRP value.The patients were divided into 2 groups,MJ and BJ,and after the adjustment a comparison between the 2 groups of patients was performed.Sensitivity,specificity and positive predictive values were calculated before and after the adjustment.RESULTS:Of the 102 patients,51 were affected by BJ and 51 by MJ.Pathologic CA19-9 levels were found in 71.7% of the patients.In the group of 51 BJ patients there were 29(56.9%) males and 22(43.1%) females with a median age of 66 years(range 24-96 years),whereas in the MJ group there were 24(47%) males and 27(53%) females,with a mean age of 70 years(range 30-92 years).Pathologic CA19-9 serum level was found in 82.3% of MJ.CRP levels were pathologic in 66.6% of the patients with BJ and in 49% with MJ.Bilirubin and CA19-9 average levels were significantly higher in MJ compared with BJ(P = 0.000 and P = 0.02),while the CRP level was significantly higher in BJ(P = 0.000).Considering a CA19-9 cut-off level of 32 U/mL,82.3% in the MJ group and 54.9% in the BJ group were positive for CA19-9(P = 0.002).A CA19-9 cut-off of 100 U/mL increases the difference between the two groups:35.3% in BJ and 68.6% in MJ(P = 0.0007).Adjusting the CA19-9 value by dividing it by serum bilirubin level meant that 21.5% in the BJ and 49% in the MJ group remained with a positive CA19-9 value(P = 0.003),while adjusting the CA19-9 value by dividing it by serum CRP value meant that 31.4% in the BJ group and 76.5% in the MJ group still had a positive CA19-9 value(P = 0.000004).Sensitivity,specificity,positive predictive values of CA19-9 > 32 U/mL were 82.3%,45% and 59.1%;when the cutoff was CA19-9 > 100 U/mL they were,respectively,68.6%,64.7% and 66%.When the CA19-9 value was adjusted by dividing it by the bilirubin or CRP values,these became 49%,78.4%,69.4% and 76.5%,68.6%,70.9%,respectively.CONCLUSION:The present study proposes CRP as a new and useful correction factor to improve the diag-nostic value of the CA19-9 tumor marker in patients with cholestatic jaundice.

Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

Effect of Panax notoginseng saponins on the expression of beta-amyloid protein in the cortex of the parietal lobe and hippocampus, and spatial learning and memory in a mouse model of senile dementia

BACKGROUND： The pharmacological actions of Panax notoginseng saponins （PNS） lie in removing free radicals, anti-inflammation and anti-oxygenation. It can also improve memory and behavior in rat models of Alzheimer＇s disease. OBJECTIVE： Using the Morris water maze, immunohistochemistry, real-time PCR and RT-PCR, this study aimed to measure improvement in spatial learning, memory, expression of amyloid precursor protein （App） and β -amyloid （A β ）, to investigate the mechanism of action of PNS in the treatment of AD in the senescence accelerated mouse-prone 8 （SAMP8） and compare the effects with huperzine A. DESIGN, TIME AND SETTING： A completely randomized grouping design, controlled animal experiment was performed in the Center for Research ＆ Development of New Drugs, Guangxi Traditional Chinese Medical University from July 2005 to April 2007. MATERIALS： Sixty male SAMP8 mice, aged 3 months, purchased from Tianjin Chinese Traditional Medical University of China, were divided into four groups： PNS high-dosage group, PNS low-dosage group, huperzine A group and control group. PNS was provided by Weihe Pharmaceutical Co., Ltd. （batch No.： Z53021485, Yuxi, Yunan Province, China）. Huperzine A was provided by Zhenyuan Pharmaceutical Co., Ltd. （batch No.： 20040801, Zhejiang, China）. METHODS： The high-dosage group and low-dosage group were treated with 93.50 and 23.38 mg/kg PNS respectively per day and the huperzine A group was treated with 0.038 6 mg/kg huperzine A per day, all by intragastric administration, for 8 consecutive weeks. The same volume of double distilled water was given to the control group. MAIN OUTCOME MEASURES： After drug administration, learning and memory abilities were assessed by place navigation and spatial probe tests. The recording indices consisted of escape latency （time-to-platform）, and the percentage of swimming time spent in each quadrant. The number of A β 1-40, A β 1-42 and App immunopositive neurons in the brains of SAMP8 mice was analyzed by immunohistochemistry. The mRNA content ofApp, tau, acetylcholinesterase, and synaptophysin （Syp） was tested by real time PCR and RT-PCR. RESULTS： The PCR results show that PNS can downregulate the expression of the App gene and upregulate the expression of the Syp gene in the parietal cortex and hippocampus of SAMP8 mice. The therapeutic effects of the PNS high-dosage group were greater than those of the PNS low-dosage group and the huperzine A group （P 〈 0.05）. The results of the Morris water maze and immunohistochemistry indicated that PNS can improve the capacity for spatial learning and memory in SAMP8 mice, and reduce the content of A β 1-40, A β 1-42 and expression of App in the brains of SAMP8 mice. The therapeutic effects of the PNS high-dosage group were greater than that of the PNS low-dosage group and the huperzine A group （P 〈 0.05）. CONCLUSION： These results support the hypothesis that PNS plays a therapeutic and protective role on the pathological lesions and learning dysfunction of Alzheimer＇s disease. The therapeutic effects of PNS for Alzheimer＇s disease are possibly achieved through downregulating the expression of the App gene and upregulating the expression of the Syp gene. The therapeutic effects of PNS are dose-dependent and are greater than the effect of huperzine A.

Development of a Rapid Multi-residue Assay for Detecting β-lactams Using Penicillin Binding Protein 2x

Objective To develop a rapid multi-residue assay for detecting 16 demanded by the European Union （EU）. Methods A recombinant penicillin-binding protein （PBP） 2x＊ from Streptococcus pneumoniae R6 was expressed in vitro and six β-1actams were conjugated to HRP by four methods. A rapid multi-residue assay for β-1actams was established with PBP2x＊ and HRP-conjugate. Results PBP2x＊ was expressed and purified successfully and the ideal HRP-conjugate was identified. The multi-residue assay was developed. After optimization, penicillin G, ampicillin, amoxicillin, cloxacillin, dicloxacillin, oxacillin, nafcillin, cephalexin, ceftiofur, cefalonium, cefquinome, cefazolin, cefoperazone, cephacetrile, and cephapirin can be detected at levels below MRL in milk with simple pretreatment. Conclusion This assay developed can detect all 16 β-1actams demanded by the European Union （EU）. The whole procedure takes only 45 min and can detect 42 samples and the standards with duplicate analysis.

Accumulation of Carbohydrate and Regulation of 14-3-3 Protein on Sucrose Phosphate Synthase(SPS) Activity in Two Tomato Species

To explore the differences of carbohydrate metabolism in two tomato species and discuss the possible regulation of 14-3-3 proteins on the sucrose phosphate synthase （SPS） activity, we determined the contents of soluble sugar and starch through high performance liquid chromatography （HPLC）. The activities of sugar-metabolizing enzymes were assayed in desalted extract, and the relative expression levels of related genes in sugar metabolism were determined though real-time RT-PCR. The results indicated that glucose and fructose were mainly accumulated during the maturation of the fruit because of the high acid invertase （AI） and neutral invertase （NI） in Micro-Tom （Solanum lycopersicum） fruit, while in Solanum chmielewskii fruit, SPS which went along with the change of sucrose content led to the rapid sucrose increase during the fruit ripening. TFT1 and TFT10, belonging to 14-3-3 protein in tomato, were likely to down-regulated SPS activity during young and intumescence period.

MicroRNA-185-5p mediates regulation of SREBP2 expression by hepatitis C virus core protein

AIM: To investigate the molecular mechanism for regulation of cholesterol metabolism by hepatitis C virus(HCV) core protein in Hep G2 cells.METHODS: HCV genotype 1b core protein was cloned and expressed in Hep G2 cells. The cholesterol content was determined after transfection. The expression of sterol regulatory element binding protein 2(SREBP2) and the rate-limiting enzyme in cholesterol synthesis(HMGCR) was measured by quantitative real-time PCR and immunoblotting after transfection. The effects of core protein on the SREBP2 promoter and 3'-untranslated region were analyzed by luciferase assay. We used different target predictive algorithms, micro RNA(mi RNA) mimics/inhibitors, and site-directed mutation to identify a putative target of a particular mi RNA.RESULTS: HCV core protein expression in Hep G2 cells increased the total intracellular cholesterol level(4.05 ± 0.17 vs 6.47 ± 0.68, P = 0.001), and this increase corresponded to an increase in SREBP2 and HMGCR m RNA levels(P = 0.009 and 0.037, respectively) and protein expression. The molecular mechanism studyrevealed that the HCV core protein increased the expression of SREBP2 by enhancing its promoter activity(P = 0.004). In addition, mi R-185-5p expression was tightly regulated by the HCV core protein(P = 0.041). Moreover, overexpression of mi R-185-5p repressed the SREBP2 m RNA level(P = 0.022) and protein expression. In contrast, inhibition of mi R-185-5p caused upregulation of SREBP2 protein expression. mi R-185-5p was involved in the regulation of SREBP2 expression by HCV core protein. CONCLUSION: HCV core protein disturbs the cholesterol homeostasis in Hep G2 cells via the SREBP2 pathway; mi R-185-5p is involved in the regulation of SREBP2 by the core protein.