A developmental biological study of aldolase gene expression in Xenopus laevis

We cloned cDNAs for Xenopus aldolases A, B and C. These three aldolase genes are localized on different chromosomes as a single copy gene. In the adult, the aldolase A gene is expressed extensively in muscle tissues, whereas the aldolase B gene is expressed strongly in kidney, liver, stomach and intestine, while the aldolase C gene is expressed in brain, heart and ovary. In oocytes aldolase A and C mRNAs, but not aldolase B mRNA, are extensively transcribed. Thus, aldolase A and C mRNAs, but not B mRNA, occur abundantly in eggs as maternal mRNAs, and strong expression of aldolase B mRNA is seen only after the late neurula stage. We conclude that aldolase A and C mRNAs are major aldolase mRNAs in early stages of Xenopus embryogenesis which proceeds utilizing yolk as the only energy source, aldolase B mRNA, on the other hand, is expressed only later in development in tissues which are required for dietary fructose metabolism.We also isolated the Xenopus aldolase C genomic gene (ca. 12 kb) and found that its promoter (ca. 2 kb)contains regions necessary for tissue-specific expression and also a GC rich region which is essential for basal transcriptional activity.

The expression of c-src gene in the carcinogenesis process of human cardia adenocarcinoma

AIM To investigate the activation, expression of c src gene and its role in the carcinogenetic process of human cardia adenocarcinoma (CA). METHODS Fifty six cases of CA, 34 cases of normal, 36 cases of protiferative epithelia adjacent to carcinoma, and 20 cases of lymph node metastases of CA were studied for PP60 c src , the expression product of c src gene immunohistochemically by using the specific monoclonal antibody, Mab327. RESULTS The positive rates of PP60 c src in the normal epithelia, protiferative epithelia, CA and lymph node metastases were 29 4% (10/*!34), 94 4% (34/*!36), 71 4% (40/*!56) and 60 0% (12/*!20) , respectively, among them, the differences of the positive rates were statistically significant ( P <0 01) . The expression levels of PP60 c src in CA and proliferative epithelia were significantly higher than that in the normal epithelia ( P <0 01) . The PP60 c src positive rates in the papillary, tubular, poorly differentiated and mucous adenocarcinoma were 75 0% (6/*!8) , 81 8% (18/*!22) , 50 0% (10/*!20) and 100 0% (6/*!6) , respectively, whereas those of tubular and mucous adenocarcinomas were significantly higher than those of papillary and poorly differentiated adenocarcinomas ( P <0 05) , and the PP60 c src expression levels of tubular and mucous adenocarcinomas were also significantly higher than those of papillary and poorly differentiated adenocarcinomas ( P <0 01) . CONCLUSION The activation and expression of c src gene are associated with the initiation and development of human CA; the protein amount of PP60 c src increased during the process of carcinogenesis; and PP60 c src expression is also related to lymph node metastases.

Expression Profiles of <i>psbA, ALS, EPSPS</i>, and Other Chloroplastic Genes in Response to PSII-, ALS-, and EPSPS-Inhibitor Treatments in <i>Kochia scoparia</i>

Kochia (Kochia scoparia L. Schrad.), also known as tumbleweed, is an economically important annual C4 broadleaf weed found throughout the US Great Plains. Several herbicides with different modes of action are used in the management of kochia. The effect of commonly used herbicides on the expression of their target site(s) and photosynthetic/chloroplastic genes is poorly understood in weed species, including kochia. The objective of this research was to characterize the expression profiles of herbicide target-site genes, KspsbA, KsALS, and KsEPSPS upon treatment with PSII- (e.g. atrazine), ALS- (e.g. chlorsulfuron), and EPSPS- (e.g. glyphosate)-inhibitors, respectively, in kochia. Furthermore, the expression of genes involved in photosynthesis (e.g. KsRubisco, KsCAB, and KsPPDK) was also determined in response to these herbicide treatments. KspsbA was strongly upregulated (>200-fold) 24 h after atrazine treatment. Transcript levels of the KsALS or KsEPSPS genes were 7 and 3-fold higher 24 h after chlorsulfuron or glyphosate treatment, respectively. KsRubisco, a Calvin cycle gene important for CO2 fixation, was upregulated 7 and 2.6-fold 8 and 24 h after glyphosate and chlorsulfuron treatments, whereas it downregulated 8 and 24 h after atrazine treatment. The transcript levels of KsPPDK remained unchanged after glyphosate treatment but increased 1.8-fold and decreased 2-fold at 24 h after chlorsulfuron and atrazine treatments, respectively. KsCAB remained unchanged after chlorsulfuron treatment, but was downregulated after glyphosate and atrazine treatments. The results show that herbicide treatments not only affect the respective target-site gene expression, but also influence the genes involved in the critical photosynthetic pathway.

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.

Retrovirus-mediated herpes simplex virus thymidine kinase gene therapy approach for hepatocellular carcinoma

The therapeutic effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system on hepatocellular carcinoma was studied in this experiment. The tk-containing retroviral recombinants were used to infect hepatoma cells (BEL-7402) and the cells were treated with ganciclovir (0-1000 microg/ml). The results showed that HSV-tk gene could be efficiently transferred in vitro into hepatoma cells and stably expressed. The growth potential of the tk-containing cells was significantly inhibited by GCV (P

Expression of core gene cDNA of Chinese hepatitis C virus in HepG2 cell line

Objective: To clone core gene cDNA of Chinese hepatitis C virus (HCV) into eukaryotic expression vector pTM3 and to express HCV core antigen in HepG2 cells. Methods: Core gene cDNA of HCV was introduced into eukaryotic expression vector pTM3. Using vaccinia virus/bacteriophage T7 hybrid expression system, HepG2cells were trans feeted with the recombinant plasmid pTM3-Q534 by lipofectin. Results: From the transfected bacteria Top10F, 2pTM3-Q534 clones containing the recombinant plasmid were identified from randomly selected 10ampicillin-resistant colonies. By indirect immunofluorescence technique HCV core protein was identified in HepG2cells trans feeted with the recombinant plasmid. Conclusion: The construction of a recombinant plasmid and the expression of core gene cDNA of HCV in HepG2 were successful.

Cloning and expression of core gene cDNA of Chinese hepatitis C virus in cosmid pTM3

AIM To clone core gene cDNA of Chinesehepatitis C virus(HCV)into eukaryoticexpression vector cosmid pTM3 and to expressHCV core antigen in HepG2 cells.METHODS Core gene cDNA of HCV wasintroduced into eukaryotic expression vectorcosmid pTM3.Using vaccinia virus/bacteriophage T7 hybrid expression system,HepG2 cells were transfected with therecombinant plasmid pTM3-Q534 by lipofectin.RESULTS From the transfected bacteriaTop10F’,2 pTM3-Q534 clones containing therecombinant plasmid were identified fromrandomly selected 10 ampicillin-resistantcolonies.By reverse transcription PCR andindirect immunofluorescence technique,HCVRNA and core protein was identified in HepG2cells transfected with the recombinant plasmid.CONCLUSION The construction of arecombinant plasmid and the expression of coregene cDNA of HCV in HepG2 was successful.

Characterization and Expression Analysis of Protein Kinase C Gene from Dunaliella salina

Protein kinase C (PKC) has a crucial role in signal transduction for a variety of biologically active substances which activate cellular functions and proliferation. We previously isolated the full-length PKC gene from Dunaliella salina (DsPKC) using rapid amplification of cDNA ends (RACE) and RT-PCR methods. And we submitted the mRNA sequence of DsPKC gene to NCBI (Genbank No. JN625213). In the present paper, the DsPKC gene open reading frame obtained by PCR was cloned into pGS-21a vector and transformed into Escherichia coli to generate the fusion protein. Bioinformatics analysis revealed that DsPKC gene was a member of serine/threonine kinase with two conserved domains and highly conserved motifs. The DsPKC was highly expressed upon induction with isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0.2 mmol L 1 at 37℃. Under salt stress, the fu- sion protein Green Fluorescent Protein (GFP)-DsPKC was transferred from the cytoplasm to the cell membrane. The expression pat- tern of DsPKC gene was analyzed using real-time quantitative PCR, and indicated that DsPKC gene was up-regulated by 3.0 mol L 1 NaCl at 12 h, which was significantly higher than in control values (P < 0.05). These results suggest that the DsPKC gene plays an important role in response to salt stress in D. salina.

Cloning and Expression of PKR Gene of Ctenopharyngodon idellus Induced by PolyI:C in vitro

[Objective] The study aimed at cloning PKR gene from Ctenopharyngodon idellus induced by PolyI：C in vitro,so as to provide foundation for study on the anti-virus genes of C.idellus.[Method] By referring to the PKR gene sequences of zebra fish（AJ852023.1） and Carassius auratus（AY293929.1） in Genbank,three pairs of degenerate primers were designed with Primer Premier 5.0 software;in vitro C.idellus kidney cells（CIK） were treated with 100 μg/ml of Poly I：C for 12,36 and 48 h,and then total RNA of the cells treated was extracted for amplifying the PKR gene by RT-PCR.[Result] The PKR gene was amplified from the cells treated with Poly I：C for 36 and 48 h,but not from the cells treated for 12 h;in addition,the expression level increased with the processing time.Part of the amplified sequence of C.idellus shared the homology of 100% and 81.48% with the sequences of carp and zebra fish separately.[Conclusion] Part of the PKR gene sequence was cloned successfully from C.idellus.Moreover,we have proved that PolyI：C induction is effective for PKR protein expression,which will provide reference for treating viral diseases of C.idellus.

Serial changes in expression of functionally clustered genes in progression of liver fibrosis in hepatitis C patients

AIM: To investigate the relationship of changes in expression of marker genes in functional categories or molecular networks comprising one functional category or multiple categories in progression of hepatic fibrosis in hepatitis C (HCV) patients. METHODS: Marker genes were initially identified using DNA microarray data from a rat liver fibrosis model. The expression level of each fibrosis associated marker gene was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) in clinical biopsy specimens from HCV-positive patients (n = 61). Analysis of changes in expression patterns and interactions of marker genes in functional categories was used to assess the biological mechanism of fibrosis. RESULTS: The profile data showed several biological changes associated with progression of hepatic fibrosis. Clustered genes in functional categories showed sequential changes in expression. Several sets of clustered genes, including those related to the extracellular matrix (ECM), inflammation, lipid metabolism, steroid metabolism, and some transcription factors important for hepatic biology showed expression changes in the immediate early phase (F1/F2) of fibrosis. Genes associated with aromatic amino acid (AA) metabolism, sulfur-containing AA metabolism and insulin/ Wnt signaling showed expression changes in the middle phase (F2/F3), and some genes related to glucose metabolism showed altered expression in the late phase of fibrosis (F3/F4). Therefore, molecular networks showing serial changes in gene expression are present in liver fibrosis progression in hepatitis C patients. CONCLUSION: Analysis of gene expression profiles from a perspective of functional categories or molecular networks provides an understanding of disease and suggests new diagnostic methods. Selected marker genes have potential utility for biological identification of advanced fibrosis.

Design,delivery and efficacy testing of therapeutic nucleic acids used to inhibit hepatitis C virus gene expression in vitro and in vivo

Despite major achievements in the treatment ofchronic hepatitis C with the combination ofinterferons and the nucleoside analog ribavirin themajority of patients with chronic hepatitis C virus(HCV) infection cannot be treated effectively.Toimprove this response rate we used antisensetechnologies to inhibit HCV translation as possibleadditional option for experimental treatment.Antisense oligodeoxynucleotides(ODN) are

The Special Expression and Comparison of the c-kit Protein in Spermatogenesis of Three Species of Locusts of Arcypteridae

The aim of this study was to elucidate the expression and regulation of the c.kit protein in spermatogenesis of locusts. Immunohistochemistry and biological statistics were used to investigate the expression of the c-kit protein in four representative phases of spermatogenesis of three dominant species of locusts of Arcypteridae （Orthoptera： Acridoidea）, namely, Omocestus viridulus （Linnaeus）, Euchorthippus unicolor （Ikonn.）, and Euchorthippus vittatus Zheng, and so on, in Siping area of Jilin Province, China. The results revealed the following： （1） There was weak positive expression of the c-kit protein in spermatogonia and the positive granules were thinner; （2） there was a strong positive expression of the c-kit protein in primary spermatocyte and the positive granules became the largest than in all developmental stages; （3） the c-kit protein positive expression became stronger in secondary spermatocyte, while the positive granules became thinner; （4） there was strong positive expression of the c-kit protein and the positive granules were thinner in mature sperm, which were distributed on its head and tail; （5） there were strong positive protein granules massing at the end of spermary; （6） the positive intensity of the c-kit protein in spermatogenesis was significantly different among different species of locusts. The data suggested that the c-kit protein may play a crucial role in spermatogenesis as well as maintain the physiological action of sperms and fertilization, regulate the developmental speed of spermatogenesis, and/or maintain species isolation, etc.

Down-Regulated Expression of RACK1 Gene by RNA Interference Enhances Drought Tolerance in Rice

The receptor for activated C-kinase 1 （RACK1） is a highly conserved scaffold protein with versatile functions, and plays important roles in the regulation of plant growth and development. Transgenic rice plants, in which the expression of RACK1 gene was inhibited by RNA interference （RNAi）, were studied to elucidate the possible functions of RACK1 in responses to drought stress in rice. Real-time PCR analysis showed that the expression of RACK1 in transgenic rice plants was inhibited by more than 50%. The tolerance to drought stress of the transgenic rice plants was higher as compared with the non-transgenic rice plants. The peroxidation of membrane and the production of malondialdehyde were significantly lower and the superoxide dismutase activity in transgenic rice plants was significantly higher than those in non-trangenic rice plants It is suggested that RACK1 negatively regulated the redox system-related tolerance to drought stress of rice plants.

Expression,purification and immunocharacteristics of recombination UreB protein of H.pylori

INTRODUCTIONHelicobacter pylori (H . pylori) is associated with the development of chronic gastritis ,peptic ulcer and gastric cancer and gastric MALT lymphoma[1-9],H .pylori has many antigens ,including urease ,heat shock protein and vacuolating cytotoxin and so on ,and urease is an important factor in the colinization of the gastric mucosa and suspected to cause damage to the gastric mucosa[10-14].At the same time ,urdase is also one of the important protective antigens .

Cloning of the non-structural gene 3 of hepatitis C virus and its inducible expression in cultured cells

AIM To study the inducible expression of hepatitis C virus ns3 gene (HCV ns3) in eukaryotic cells.METHODS The ns3 gene was obtained from plasmid pBns3 by polymerase chain reaction and inserted into the cloning vector pGEM-T. Then, the ns3 was subcloned into the vector pMSG to generate dexamethasone (DM)-inducible expression plasmid pMSG-ns3. CHO cells were transfected by pMSG-ns3 using calcium phosphate precipitation method and cultivated for 12 h-24 h. The transfected cells were induced with DM and the transient expression of NS3 protein was analyzed by ELISA and Western-blot methods.RESULTS After treated with 3×10-8mol/ L DM, the expression of NS3 was observed in the transfected CHO cells. A slightly higher level of NS3 was shown along with the time of DM treatment.CONCLUSION The inducible expressing vector pMSG-ns3 might be helpful for further studies of the characteristics of the ns3 gene in vivo.

cDNA Cloning, Bioinformatic and Tissue-specific Expression Analysis of Porcine JARID1C Gene

Jumonji, AT-rich interactive domain 1C （JARID1C） protein belongs to the highly conserved ARID protein family, which is involved in chromatin remodeling and transcriptional regulation during cell growth, differentiation, and development. In humans, this gene plays a vital role in normal brain development and function. Using an in silico approach in combination with 5＇ rapid amplification of cDNA ends （5＇ RACE）, the full-length cDNA of JARIDIC （GenBank accession No. EF139241） from porcine ovary, which contains 5,908 bp nucleotides, with an open reading frame （ORF） of 4,548 bp, has been cloned. The putative porcine JARID 1C protein, which is located in the nucleus, encodes 1,516 amino acids with a molecular weight of 170 kDa and a pI of 5.44. Bioinformatic prediction indicates that the protein contains several conserved domains： a JmjN domain, an ARID domain, a JmjC domain, a C5HC2 zinc finger domain, and a PHD zinc finger domain. Similarity comparisons for nucleic and amino acid sequences reveal that the porcine JARID1C protein shares a high identity with its dog, mouse, rat, and human counterparts. The phylogenetic tree of the JARID1 subfamily proteins has been constructed to reveal the evolutionary relationship of various species. Real-time PCR analysis shows that the JARIDIC gene is expressed in various tissues, but at different levels. The expression levels of this gene are higher in the brain and gonad than in other tissues, suggesting that the JARID1C protein plays a role in porcine brain and gonad functions.

Cloning and expression of NS3 cDNA fragment of HCV genome of Hebei isolate in E.coli

AIM To obtain greater antigenicity of HCV NS3 protein. METHODS The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients. The DNA sequence was determined by dideoxy mediated chain termination method using T7 polymerase. HCV NS3 protein was expressed in E. coli . RESULTS Sequence analysis indicated that the HCV isolate of this study belongs to HCV Ⅱ; SDS PAGE demonstrated an M r 23800 and an M r 22000 recombinant protein band which amount to 14% and 11% of the total bacterial proteins separately. Western blotting and ELISA showed NS3 protein possessed greater antigenicity. CONCLUSION Recombinant HCV NS3 protein was expressed successfully, which provided the basis for developing HCV diagnostic reagents.

HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro

AIM： TO establish a cell culture system with longterm replication of hepatitis C virus （HCV） genome and expression of viral antigens in vitro. METHODS： HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect na＇ive cells. The presence of minus （antisense） RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques （flow cytometry and Western blot） respectively. RESULTS： The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies （anti-core, and anti-E1）. Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION： HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.

Cloning and Expression of a Chitinase Gene from Serratia marcescens Strain C8-8

A 1 692 bp long chitinase-encoding ch/A gene was cloned from the genomic DNA of Serrat/a marcescens strain C8-8 by PCR, which was speculated to en- code a 563 aa long polypeptide chain with molecular weight of about 60.9 kD. Homolog analysis showed that the chiA gene sequence cloned from C8-8 shared the highest similarity with cMA sequences from Serrat/a maresscens strains 141 （ DQ 990373.1 ） and 14041 （ DQ 493896. 1 ）, which reached 99%. Domain analysis showed that N-termlnal （23 aa） of the chiA gene cloned from C8-8 harbored typical signal peptide sequence, while C-telminal harbored the other two domains, in- eluding the PKD region （73 aa） and chitinase catalytic region （387 aa）. The PCR fragment was digested with restriction endonucleases and cloned into plasmid pET28a. The recombinant plasmid pET＇28a-ch/A was firstly transformed into Escherichia coli DI-I5 , and then transformed into expression host E. coli DH3 to express ch/A gene. The recombinant strain DH3 chiA could produce transparent hydrolysis circles on the colloidal chitin plate induced by isopropyl-l-thiogalactopyranoside （IFrG）. SDS-PAGE electrophoresis analysis showed that, a protein with relative molecular weight of about 60 kD was expressed by the recombinant strain DH3 chiA, which was consistent with the except molecular weight. After initial purification, biological activity test showed that the recombinant expression product could hydrolyze chitin, which produced transparent hydrolysis circles on the colloidal chitin plates. Results indicated that chiA gene from Serrat/a marcescens strain C8-8 had biological functions and could be utilized as a potential biological control factor.

THE EFFECTS OF RETINOIC ACID ON EXPRESSION OF C-MYC, C-FOS IN LEUKEMIC PROMYELOCYTES

The expression of c-myc, c-fos of leukemic promyelocytes (HL-60 and acute promyelocytic leukemia cells) from 18 acute promyelocytic leukemia (APL) patients treated with all-trans retinoic acid (RA) in vitro was studied. There was no expression of c-fos in HL-60 cells and APL cells from 17 patients. But in one case, a slight expression of c-fos in leukemic cells was observed, and the alteration of expression level was found during the treatment of the cells with RA in vitro. The expression of c-myc in HL-60 cells induced by RA was altered, decrease in the early, increase in the middle, and decline in the later stage were found. The c-myc expression in leukemic cells of eighteen APL patients was variable. There was c-myc expression in eleven APL cells, but no expression in the others. The APL cells with c-myc expression were treated with RA in vitro to observe the kinetic changes of c-myc RNA level. The results showed that the expression of c-myc was gradually decreased except in few cases. Using in situ hybridization technique for detecting the alteration of c-myc expression in leukemic cells of two APL patients. the high level of c-myc before RA treatment and low level of c-myc expression after obtaining complete remission induced by RA were found. The possibility of different proto-oncogenes implicated differentiation was discussed.