S100A8 mediates the activation of P65/HLA-B/S100A8/BCL-2/Caspase-9 (-3) pathway in laryngeal carcinogenesis

S100 calcium binding protein A8 (S100A8),a possible novel member of NF-kappa B signal pathway in laryngeal squamous cell carcinoma (LSCC),interacts with human leukocyte antigen B (HLA-B) which carries an NF-kappa B binding site within the enhancer A. The objective of this study was to explore the molecular mechanism of S100A8 in laryngeal carcinogenesis. RT-PCR,Western blotting and immuno-histochemistry staining were applied to evaluate the expression levels of IKKα,P65,REL-B,S100A8,APAF-1 and BCL-2 genes. The signal transduction passway in which S100A8 might participate was explored by RNA interference. Flow cytometry,TUNEL assay and cell invasion in vitro were used to detect the biological behavior of Hep2 cells induced by S100A8 gene. Our results showed that high expression of S100A8 was related to tumorigenesis in LSCC and negatively correlated with the degree of differentiation,indicating that S100A8 gene could inhibit apoptosis and promote metastasis in LSCC. Additionally,the suppression of S100A8 by RNA interference down-regulated BCL-2 but not APAF-1,P65 and IKKα,while,the suppression of P65 could significantly down-regulate the expression of S100A8 gene. In conclusion,S100A8 plays an important role in P65/HLA-B/S100A8/BCL-2/Caspase-9 (-3) pathway in laryngeal carcinoma.     

High frequency of p53 intronic point mutations in laryngeal squamous cell carcinoma

Intronic point mutations are rare and totally unknown for human laryngeal squamous cell carcinoma (LSCC). To explore the relationship of p53 gene intronic mutation to the development of human LSCC, DNA was extracted from both tumor tissues and matched normal tissues of 55 patients with LSCC in northeast of China. Polymerase chain reaction amplification-single strand conformational polymorphism (PCR-SSCP) combined with silver staining and DNA direct sequencing were used to detect mutations in exons 7～8 (p53E7 and p53E8) and introns 7～8 (p53I7 and p53I8) of p53 gene. The p53E7 mutation was detected in 17 out of 55 patients, and the p53I7 mutation in 21 patients. No mutation was found at p53E8 or p53I8 site. The difference between tumor group and paired normal group on the rates of both p53E7 and p53I7 mutations was statistically significant. The rate of p53I7 mutations in tumor tissue was higher than that of normal tissue, and so was that of p53E7. Sequence analysis revealed that most p53I7 mutations were at the nucleotides in the branch point sequence or the polypyrimidine tract in the 3′-splice acceptor site of the intron 7. The high incidence of p53 gene intronic mutation in LSCC indicates that genetic changes within the noncoding region of the p53 gene may serve as an alternative mechanism of activating the pathogenesis of human laryngeal squamous cell carcinoma. Mutations in the noncoding region of this gene should be further studied.     

Partial inhibition of CDK4 gene expression leads to the extension of G1 phase of V79-8 cells

V79-8 is an abnormal cell line which does not have detectable G1 and G2 phases in its cell cycle. This cell line is derived from V79 cell line which has Gl phase but lacks G2 phase. By using an anti-sense approach, CDK4 gene expression was partially inhibited to find whether CDK4 might contribute to the lack of Gl phase in V79-8 cells. Anti-CDK4 anti-sense plasmid was constructed and used to transfect V79-8 cells. Clones of transfected cells (V79-8-asCDK4) were examined, in comparison with V79-8 cells, to determine its growth curve, cell doubling-time (GT), the level of CDK4 gene expression and the levels of expression of some other growth related genes. V79-8-asCDK4 cells showed a slower growth rate with a doubling time 2.5-h longer than that of V79-8 cells. A flow cytometry (FCM) analysis demonstrated that the 2.5 h increase of the doubling time of V79-8-asCDK4 cells was mainly due to the appearance of Gl phase because its G2 + M phase was not significantly different from that of V79-8 cells. The decrease of CDK4 gene expression in V79-8-asCDK4 cells was shown by Northern-blot. Changes in the expression levels of the growth-related genes TGF-β, cyclin D1 and Rb were also detected in V79-8-asCDK4 cells. CDK4 functions mainly in G1 and at the transition between G1 and S phases. Expression of an anti-sense CDK4 gene fragment reduces the levels of endogenous CDK4, CDK4/cyclinD kinase activity and the phosphorylation of Rb. These events may postpone the inactivation of the check-point leading to the delay of entry into S phase and the reappearance of G1 phase in V79-8-asCDK4 cells.     

Involvement of Jasmonate-signaling pathway in the herbivore-induced rice plant defense

The expression patterns of eight defense- related genes in the herbivore-infested and jasmonate- treated (jasmonic acid, JA and its derivative MeJA) rice leaves were analyzed using RT-PCR. The results showed that Spodoptera litura Fabricius (Lepidoptera: Noctuidae) herbi-vory induced the expression of lipoxygenase (LOX) and al-lene oxide synthase (AOS) genes that are involved in the jasmonate-signaling pathway. Moreover, S. litura damage resulted in the expression of farnesyl pyrophosphate syn-thase (FPS), Bowman-birk proteinase inhibitor (BBPI), phenylalanine ammonia-lyase (PAL) and other rice defense- related genes that were also induced by aqueous JA treat-ment or gaseous MeJA treatment. These indicated that in rice leaves, the JA-related signaling pathway was involved in the S. litura-induced chemical defense. Mechanical damage and brown planthopper (BPH), Nilaparvata lugens (St錶) (Homoptera: Delphacidae) damage induced the expression of LOX gene, but both treatments did not induce the expression of AOS gene. However, BPH damage induced the expression of acidic pathogen-related protein 1 (PR-1a), Chitinase (PR-3), and PAL genes, which is involved in the salicylate- signaling pathway. It was suggested that salicylate-related signaling pathway or other pathways, rather than jas-monate-signaling pathway was involved in the BPH-induced rice plant defense.     

Construction and characterization of partially ntrC-deleted mutants in Alcaligenes faecalis

To study the effect of ntrC gene product on the expression and regulation of other important nitrogen-fixing genes in Alcaligenes faecalis, partially ntrC-deleted mutants of A. faecalis have been generated. To start with, the ntrC gene of A. faecalis was cloned into a suicide plasmid pSUP202 to create a recombinant plasmid pSUM1. The ntrC gene in pSUM1 was then replaced by a lacZ-Kmr fragment resulted in the generation of a plasmid pSUM2. The lacZ fragment in pSUM2 was further removed and a plasmid pSUM3 produced. As a second step, the plasmid pSUM2 or pSUM3 was introduced into the wild type of A. faecalis A1501 by conjugation and two partially ntrC-deleted mutants A15CM1 (ntrC∷lacZ) and A15CM2 (ntrC-) were obtained. To understand the regulatory effect of the NtrC on the expression of nifH and nifA, a nifH-lacZ gene or a nifA-lacZ gene was introduced into the ntrC- mutant by conjugation. The results indicated that: (ⅰ) although the ntrC- mutant was nif + , its nitrogen fixation activity was only 20% that of the wild type; (ⅱ) the ntrC- mutant failed to grow on the medium containing nitrate as a sole nitrogen source; (ⅲ) the regulation of ntrC gene expression did not require its own product; (ⅳ) the expression of nifH in A . faecalis was positively regulated by the ntrC. Deletion of the ntrC resulted in the reduction of nifH expression or even totally inactivated nitrogen fixation; (ⅴ) there was no obvious influence on the expression of nifA in A. faecalis if the ntrC gene was deleted.     

Activity of the truncated promoter of pollen-specific G9 gene of cotton and the transgenic recessive nuclei-sterile tobacco

A 557 bp fragment from the translation initiation site of the G9 gene expressed in maturing pollens of cotton was isolated from genomic DNA of upland cotton (Gossypium hirsutum L.) cv. “Zhongkang 17”, and two expression vectors for plant transformation were constructed via fusing this fragment with β-glucuronidase gene (Gus) and cytotoxin gene Barnase. The promoter activity of this fragment was demonstrated via transient expression of Gus gene in cotton and by the integrated expression of Barnase gene in tobacco. This promoter can initiate the expression of exogenous gene specifically and efficiently in plant pollen. The transgenic tobacco plant containing G9-Barnase fusion gene showed the characteristics of recessive nuclei-sterility.     

Knockdown of <Emphasis Type="Italic">MRP4</Emphasis> by lentivirus-mediated siRNA improves sensitivity to adriamycin in adriamycin-resistant acute myeloid leukemia cells

Chemotherapy remains the standard treatment for acute myeloid leukemia;however,the emergence of drug resistance is a major hurdle in the successful treatment of leukemia.The expression of multidrug resistance-associated protein 4(MRP4)induces re- sistance in the adriamycin-resistant acute myeloid leukemia cell line,K562/ADR.The aim of this study was to investigate whether knockdown of MRP4 by lentivirus-mediated siRNA could improve the sensitivity of K562/ADR cells to adriamycin.Five lenti- virus-mediated short hairpin RNAs(lv-shRNAs-MRP4)were designed to trigger the gene silencing RNA interference(RNAi) pathway.The efficiency of lentivirus-mediated siRNA infection into K562/ADR cells was determined using fluorescence mi- croscopy to observe lentivirus-mediated GFP expression.MRP4 expression in infected K562/ADR cells was evaluated by real- time PCR and Western blot analysis.The MTS assay was used to measure cell viability and flow cytometry was used to measure apoptosis.The transfection efficiency of K562/ADR cells was over 80 percent.The gene silencing efficacy of lv-shRNA1-MRP4 was superior to the other constructs.Infection of K562/ADR cells with lv-shRNA1-MRP4 led to strong inhibition of MRP4 mRNA and protein expression.Combined treatment with lv-shRNA1-MRP4 and adriamycin decreased cell growth and increased apoptosis compared to treatment with lv-shRNA1-MRP4 or adriamycin alone.These data indicate that in K562/ADR cells MRP4 is involved in drug resistance mechanisms and that lentivirus-mediated knockdown of MRP4 may enhance sensitivity to adriamycin.     

Identification of seed-specific promoter nap300 and its comparison with 7S promoter

By fusing seed-specific promoter nap300 with β-glucuronidase gene, it was found that this about 300bp DNA fragment was sufficient to direct seed-specific gene expression. The substitution mutation in both distB and proxB elements had a little effect on the expression efficiency and almost no effect on the organ-specific expression pattern. In the experiment designed to compare nap300 with 7S promoter, the result showed that tissue specificity for nap300 was higher than that for 7S, and its expression level was lower than 7S's. There was no big difference in their expression pattern, and the maximal activity stage for the two promoters was identical, which indicated they could be used simultaneously for expressing different foreign genes in seeds.     

Non-viral gene carrier mediated short hairpin RNA interference for inhibition of tumor cells growth

A tumor-targeting gene vector G250mAb-PEI-PEG has been prepared by modification of polyethylenimine （PEI） with polyethyleneglycol （PEG） and G250, a monoclonal antibody against the G250 antigen on tumor cell surface. The transfection efficiency was as high as 70% in G250 positive HeLa cells, whereas the transfection efficiency was relatively low （30%） in normal NIH3T3 cells. A plasmid encoding the short hairpin RNA （shRNA） specific for nucleostemin gene （NS） was efficiently transfected into the HeLa cells with this nonviral gene vector. RNA interference down-regulated the expression of NS gene in HeLa cells, inhibited cells proliferation and induced apoptosis. However, the growth and activity of the NIH3T3 cells were not affected under the same treatment. These results indicate that the reported nonviral gene vector, G250mAb-PEI-PEG, can target and efficiently deliver genes into HeLa cells, and has the potential for the cervical cancer treatment.     

Overexpression and export of Vibrio anguillarum metalloprotease in Escherichia coil

Vibrio anguillarum metalloprotease, an extracellular zinc metalloprotease involved in the virulence mechanism of Vibrio anguillarum, is synthesized from the empA gene as a 611-residue precursor and naturally secreted via Sec secretion pathway in Vibrio anguillarum. In this study, hetemlogous expression of the empA gene encoding metallopmtease and export of the recombinant metallopmtease in Escherichia coli were examined. The empA gene was subcloned into pBAD24 with arabinose promoter and sequenced. The sequence encoded a polypeptide （611 amino acids） consisting of four domains： a signal peptide, an Nterminal pmpoptide, a mature region and a C-terminal pmpoptide. The empA gene inserted in plasmid pBAD24 was overexpressed in TOP10 strain of E. coli after arabinose induction. The 36kDa polypeptide of the recombinant metallopmtease as the mature pmtease was further confirmed by SDS-PAGE and im- munoblotting. It was found that recombinant metallopmtease with the EmpA activity and antigenicity was exported into the poriplasm of Escherichia coli cells via Sec translocation pathway, whereas it was secreted into extracellular environments in V. anguillarum. The results imply that the expression, export and processing mechanism of the protein in E. coli are similar to those in V. anguillarum.     

Suitable internal control genes for qRT-PCR normalization in cotton fiber development and somatic embryogenesis

The mechanisms of cotton fiber development and somatic embryogenesis have been explored sys-tematically with microarray and suppression subtractive hybridization. Real-time RT-PCR provides the simultaneous measurement of gene expression in many different samples,with which the data from microarray or others can be confirmed in detail. To achieve accurate and reliable gene expression re-sults,normalization of real-time PCR data against one or several internal control genes is required,which should not fluctuate in different tissues during various stages of development. We assessed the gene expression of 7 frequently used housekeeping genes,including 18S rRNA,Histone3,UBQ7,Actin,Cyclophilin,Gbpolyubiquitin-1 and Gbpolyubiquitin-2,in a diverse set of 21 cotton samples. For fiber developmental series the expression of all housekeeping genes had the same down tendency after 17 DPA. But the expression of the AGP gene(arabinogalactan protein) that has high expression level at the later fiber development stage was up-regulated from 15 to 27 DPA. So the relative absolute quanti-fication should be an efficient and convenient method for the fiber developmental series. The expres-sion of nonfiber tissues series varied not so much against the fiber developmental series. And three best control genes Histone3,UBQ7 and Gbpolyubiquitin-1 have to be used in a combinated way to get better normalization.     

Overexpression and export of Vibrio anguillarum metalloprotease in Escherichia coli

Vibrio anguillarum metalloprotease, an extracellular zinc metalloprotease involved in the virulence mechanism of Vibrio anguillarum, is synthesized from the empA gene as a 611-residue precursor and naturally secreted via Sec secretion pathway in Vibrio anguillarum. In this study, heterologous expression of the empA gene encoding metalloprotease and export of the recombinant metalloprotease in Escherichia coli were examined. The empA gene was subcloned into pBAD24 with arabinose promoter and sequenced. The sequence encoded a polypeptide (611 amino acids) consisting of four domains: a signal peptide, an N-terminal propeptide, a mature region and a C-terminal propeptide. The empA gene inserted in plasmid pBAD24 was overexpressed in TOP10 strain of E. coli after arabinose induction. The 36kDa polypeptide of the recombinant metalloprotease as the mature protease was further confirmed by SDS-PAGE and immunoblotting. It was found that recombinant metalloprotease with the EmpA activity and antigenicity was exported into the periplasm of Escherichia coli cells via Sec translocation pathway, whereas it was secreted into extracellular environments in V. anguillarum. The results imply that the expression, export and processing mechanism of the protein in E. coli are similar to those in V. anguillarum.     

Polycistronic strategy for cyanobacterial expression vector construction: Co-transcription of a human gene and a selective marker gene

A polycistronic expression vector, pKGA-NTF1, was constructed for the cyanobacterium. Within this vector, the spectinomycin/streptomycin resistance gene (aadA) facilitated the selection of transformants when co-transcribed with favorite genes. A natural glnA gene was selected as the platform to introduce the plasmid into a neutral site of the Synechococcus sp. PCC 7002 chromosome. Function of the vector was demonstrated by the insertion of a modified human Trefoil factor 3 gene (NTF1) to upstream of the aadA gene and by the analyses of the transformed strains. Antibiotics resistance assays showed that the dicistronic expression cassette conferred high spectinomycin resistance to both the E. coli cells and the Synechococcus cells. PCR analysis and Western-blot analysis were carried out to confirm the integration and expression of the NTF1 gene, respectively. Through simple molecular manipulations, the artificial polycistronic structure described here can be conveniently used to express other favorable genes or operons in cyanobacteria, and to study the cyanobacterial gene expression as well.     

Robust and regulatory expression of defensin A gene driven by vitellogenin promoter in transgenic Anopheles stephensi

The use of genetically modified mosquitoes to reduce or replace field populations is a new strategy to control mosquito-borne diseases. The precondition of the implementation of this strategy is the ability to manipulate the genome of mosquitoes and to induce specific expression of the effector molecules driven by a suitable promoter. The objective of this study is to evaluate the expression of defensin A gene of Anopheles sinensis under the control of a vitellogenin promoter in transgenic Anopheles ste- phensi. The regulatory region of Anopheles gambiae vitellogenin was cloned and subcloned into transfer vector pSLFa consisting of an expression cassette with defensin A coding sequence. Then, the expression cassette was transferred into transformation vector pBac[3xP3-DsRedafm] using Asc I di- gestion. The recombinant plasmid DNA of pBac[3xP3DsRed-AgVgT2-DefA] and helper plasmid DNA of phsp-pBac were micro-injected into embryos of An. stephensi. The positive transgenic mosquitoes were screened by observing specific red fluorescence in the eyes of G1 larvae. Southern blot analysis showed that a single-copy transgene integrated into the genome of An. stephensi. RT-PCR analysis showed that the defensin A gene expressed specifically in fat bodies of female mosquitoes after a blood meal. Interestingly, the mRNA of defensin A is more stable compared with that of the endogenous vitellogenin gene. After multiple blood meals, the expression of defensin A appeared as a reducible and non-cycling type, a crucial feature for its anti-pathogen effect. From data above, we concluded that the regulatory function of the Vg promoter and the expression of defensin A gene were relatively con- served in different species of anopheles mosquitoes. These molecules could be used as candidates in the development of genetically modified mosquitoes.     

Construction and characterization of partiallyntrC-deleted mutants inAlcaligenes faecalis

To study the effect of ntrC gene product on the expression and regulation of other important nitrogen-fixing genes in Alcaligenes faecalis, partially ntrC-deleted mutants of A. faecalis have been generated. To start with, the ntrC gene of A. faecalis was cloned into a suicide plasmid pSUP202 to create a recombinant plasmid pSUM1. The ntrC gene in pSUM1 was then replaced by a lacZ-Km^r fragment resulted in the generation of a plasmid pSUM2. The lacZ fragment in pSUM2 was further removed and a plasmid pSUM3 produced. As a second step, the plasmid pSUM2 or pSUM3 was introduced into the wild type of A. faecalis A1501 by conjugation and two partially ntrC-deleted mutants A15CM1 (ntrC∷lacZ) and A15CM2 (ntrC ^- ) were obtained. To understand the regulatory effect of the NtrC on the expression of nifH and nifA, a nifH-lacZ gene or a nifH-lacZ gene was introduced into the ntrC^- mutant by conjugation. The results indicated that: (ⅰ) although the ntrC^-mutant was nif ⁺ , its nitrogen fixation activity was only 20% that of the wild type; (ⅱ) the ntrC^- mutant failed to grow on the medium containing nitrate as a sole nitrogen source; (ⅲ) the regulation of ntrC gene expression did not require its own product; (ⅳ) the expression of nifH in A . faecalis was positively regulated by the ntrC. Deletion of the ntrC resulted in the reduction of nifH expression or even totally inactivated nitrogen fixation; (ⅴ) there was no obvious influence on the expression of nifA in A. faecalis if the ntrC gene was deleted.     

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.