Pathogenicty and immune prophylaxis of cag pathogenicity island gene knockout homogenic mutants

AIM: To clarify the role of cag pathogenicity island (cagPAI) of Helicobacter pylori (H pylori) in the pathogenicity and immune prophylaxis of H pylori infection. METHODS: Three pairs of H pylori including 3 strains of cagPAI positive wildtype bacteria and their cagPAI knockout homogenic mutants were utilized. H pylori binding to the gastric epithelial cells was analyzed by flow cytometry assays. Apoptosis of gastric epithelial cells induced by H pylori was determined by ELISA assay. Prophylaxis effect of the wildtype and mutant strains was compared by immunization with the sonicate of the bacteria into mice model. RESULTS: No difference was found in the apoptasis between cagPAI positive and knockout H pylori strains in respective of the ability in the binding to gastric epithelial cells as well as the induction of apoptosis. Both types of the bacteria were able to protect the mice from the infection of H pylori after immunization, with no difference between them regarding to the protection rate as well as the stimulation of the proliferation of splenocytes of the mice. CONCLUSION: The role of cagPAI in the pathogenicity and prophylaxis of Hpyloriinfection remains to be cleared.

Replication and gene expression of mutant hepatitis B virus in a transgenic mouse containing the complete viral genome with mutant sgene

AIM:To establish the transgenic mouse line harbouring complete hepatitis B virus (HBV) genome with mutant s gene (adr subtype).METHODS: Transgenic mice were generated by microinjecting HBV genome into fertilized eggs. Integration, expression, replication of HBV gene and histological changes in transgenic mice were estimated by genomic DNA PCR, serum DNA PCR, Southern blot, ELISA, HE staining, immunohistochemistry and transmission electron microscopy. Transgenic mice with HBsAg positive in serum were bred and analyzed.RESULTS: A total of 288 eggs survived from microinjections were transplanted into the oviducts of 13 pseudopregnant mice and 49 pups were produced. Twenty-six mice were identified to have the integrated HBV gene. Serum HBsAg and HBeAg were detected in 2 of 43 mice. HBsAg and HBcAg in cytoplasm or nuclei of hepatocytes were detected in 10 mice. Founders with HBsAg in serum were named lineages G145R-15 and G145R-18. Of the 16 F1 offsprings generated by G145R-15 founder, 12 were positive for HBV genome with PCR, 10 were positive for HBsAg and HBcAg with immunohistochemistry and 7 were positive for HBsAg and HBeAg with ELISA. Only 1 of 8 F1 offsprings generated by G145R-18 founder was survived and it was detected positive for HBV genome, HBsAg, HBcAg and HBeAg. Both of the two lineages had some pathological characteristics of mild chronic hepatitis B in the liver, such as swelling of hepatocytes and focal hepatocellular necrosis and parenchymal lymphomononuclear cell infiltrate.CONCLUSION: Transgenic mice harbouring HBV with mutant s gene can be generated. The HBV genes are integrated in the transgenic mice genome and can be expressed, replicated, packaged and excreted. HBV DNA can be stably transmitted in the transgenic mice.

Structure prediction and activity analysis of human heme oxygenase-1 and its mutant

AIM: To predict wild human heme oxygenase-1 (whHO-1) and hHO-1 His25Ala mutant (△hHO-1) structures, to done and express them and analyze their activities.METHODS: Swiss-PdbViewer and Antheprot 5.0 were used for the prediction of structure diversity and physicalchemical changes between wild and mutant hHO-1, hHO-1 His25Ala mutant cDNA was constructed by site-directed mutagenesis in two plasmids of E.COli DH5α Expression products were purified by ammonium sulphate precipitation and Q-Sepharose Fast Flow column chromatography, and their activities were measured,RESULTS: rHO-1 had the structure of a helical fold with the heme sandwiched between heme-heme oxygenase-1 helices. Bond angle, dihedral angle and chemical bond in the active pocket changed after Ala25 was replaced by His25, but Ala25 was still contacting the surface and the electrostatic potential of the active pocket was negative. The mutated enzyme kept binding activity to heme. Two vectors pBHO-1 and pBHO-1(M) were constructed and expressed. Ammonium sulphate precipitation and column chromatography yielded 3.6-fold and 30-fold higher purities of whHO-1, respectively. The activity of △hHO-1 was reduced 91.21% after mutation comparedwith whHO-1.CONCLUSION: Proximal His25 ligand is crucial for normal hHO-1 catalytic activity. △hHO-1 is deactivated by mutation but keeps the same binding site as whHO-1. △hHO-1 might be a potential inhibitor of whHO-1 for preventing neonatal hyperbilirubinemia.

Heterotrimeric G protein α subunit is involved in rice brassinosteroid response

Heterotrimeric G proteins are known to function as messengers in numerous signal transduction pathways.The nullmutation of RGA(rice heterotrimeric G protein α subunit),which encodes the α subunit of heterotrimeric G proteinin rice,causes severe dwarfism and reduced responsiveness to gibberellic acid in rice.However,less is known aboutheterotrimeric G protein in brassinosteroid(BR)signaling,one of the well-understood phytohormone pathways.In thepresent study,we used root elongation inhibition assay,lamina inclination assay and coleoptile elongation analysis todemonstrated reduced sensitivity of dl mutant plants(caused by the null mutation of RGA)to 24-epibrassinolide(24-epiBL),which belongs to brassinosteroids and plays a wide variety of roles in plant growth and development.Moreover,RGA transcript level was decreased in 24-epiBL-treated seedlings in a dose-dependent manner.Our results show thatRGA is involved in rice brassinosteroid response,which may be beneficial to elucidate the molecular mechanisms of Gprotein signaling and provide a novel perspective to understand BR signaling in higher plants.

Efflux pump gene hefA of Helicobacter pylori plays an important role in multidrug resistance

AIM: To determine whether efflux systems contribute to multidrug resistance of H pylori. METHODS: A chloramphenicol-induced multidrug resistance model of six susceptible H pylori strains (5 isolates and H pylori NCTC11637) was developed. Multidrug-resistant (MDR) strains were selected and the minimal inhibitory concentration (MIC) of eryth-romycin, metronidazole, penicillin G, tetracycline, and ciprofloxacin in multidrug resistant strains and their parent strains was determined by agar dilution tests. The level of mRNA expression of hefA was assessed by fluorescence real-time quantitative PCR. A H pylori LZ1026 knockout mutant (ΔH pylori LZ1026) for (puta-tive) efflux protein was constructed by inserting the kanamycin resistance cassette from pEGFP-N2 into hefA, and its susceptibility profiles to 10 antibiotics were evaluated. RESULTS: The MIC of six multidrug-resistant strains (including 5 clinical isolates and H pylori NCTC11637) increased signifi cantly (≥ 4-fold) compared with their parent strains. The expression level of hefA gene was significantly higher in the MDR strains than in their parent strains (P = 0.033). A H pylori LZ1026 mutant was successfully constructed and the ΔH pylori LZ1026 was more susceptible to four of the 10 antibiotics. All the 20 strains displayed transcripts for hefA that con-fi rmed the in vitro expression of these genes.CONCLUSION: The efflux pump gene hefA plays an important role in multidrug resistance of H pylori.

IL-17RD （Sef or IL-17RLM） interacts with IL-17 receptor and mediates IL-17 signaling

Interleukin-17 （IL-17 or IL-17A） production is a hallmark of TH17 cells, a new unique lineage of CD4^＋ T lymphocytes contributing to the pathogenesis of multiple autoimmune and inflammatory diseases. IL-17 receptor （IL-17R or IL-17RA） is essential for IL-17 biological activity. Emerging data suggest that the formation of a heteromeric and/or homomeric receptor complex is required for IL-17 signaling. Here we show that the orphan receptor IL-17RD （Sef, similar expression to FGF genes or IL-17RLM） is associated and colocalized with IL-17R. Importantly, IL-17RD mediates IL-17 signaling, as evaluated using a luciferase reporter driven by the native promoter of 24p3, an IL-17 target gene. In addition, an IL-17RD mutant lacking the intraeellnlar domain dominant-negatively suppresses IL-17R- mediated IL-17 signaling. Moreover, IL-17RD as well as IL-17R is associated with TRAF6, an IL-17R downstream molecule. These results indicate that IL-17RD is a part of the IL-17 receptor signaling complex, therefore providing novel evidence for IL-17 signaling through a heteromeric and/or homomeric receptor complex.

Feasibility of herpes simplex virus type 1 mutants labeled with radionuclides for tumor treatment

For over one hundred years, viruses have been recognized as capable of killing tumor cells. At present, people are still researching and constructing more suitable oncolytic viruses for treating different malignant tumors. Although extensive studies have demonstrated that herpes simplex virus type 1 (HSV-1) is the most potential oncolytic virus, therapies based on herpes simplex virus type 1 vectors still arouse bio-safety and risk management issues. Researchers have therefore introduced the new idea of treating cancer with HSV-1 mutants labeled with radionuclides, combining radionuclide and oncolytic virus therapies. This overview briefly summarizes the status and mechanisms by which oncolytic viruses kill tumor cells, discusses the application of HSV-1 and HSV-1 derived vectors for tumor therapy, and demonstrates the feasibility and prospect of HSV-1 mutants labeled with radionuclides for treating tumors.

Improved production of spiramycin by mutant Streptomyces ambofaciens

Strain improvement and medium optimization to increase the productivity of spiramycin were carried out. Of oil tolerant mutant strains screened, one mutant, Streptomyces ambofaciens XC 2-37, produced 9% more spiramycin than the parent strain S. ambofaciens XC 1-29. The effects of soybean oil and propyl alcohol on spiramycin production with S. ambofaciens XC 2-37 were studied. The potency of S. ambofaciens XC 2-37 was improved by 61.8% with addition of 2% soybean oil in the fermentation medium and 0.4% propyl alcohol at 24 hours after incubation. The suitable time for feeding propyl alcohol is at 24 hours after incubation in flask fermentation and at 20 hours after incubation in fermentor fermentation. The new process with S. ambofaciens XC 2-37 was scaled up for industrial scale production of spiramycin in a 60 m3 fermentor in Xinchang Pharmaceutical Factory, Zhejiang Medicine Company, Ltd., China, and the potency and productivity of fermentation were improved by 42.9%.

Preliminary study on a gravity－insensitive rice mutant

A gravity-insensitive mutant was isolated from rice (Oryza sativa L. cv. Zhonghua 11) transformed by Agrobacterium tumefaciens. The mutant's shoot growth (prostrate growth) was insensitive to gravity; whereas root growth displayed a normal positive gravitropism. Histological observation of root caps and leaf sheaths indicated that there was no significant difference in the number and size of amyloplasts in cells of the mutant and cells of the wild type.

Differential effects of p63 mutants on transactivation of p53 and/or p63 responsive genes

p63, known to play a role in development, has more recently also been implicated in cancer progression. Mutations in p63 have been shown to be responsible for several human developmental diseases. Differential splicing of the p63 gene gives rise to p63 isoforms, which can act either as tumor suppressors or as oncogene. In this report, we studied the effects of naturally occurring TAp637 mutants on the regulation of p53/p63 and p63 specific target genes. We observed significant differences among p63 mutants to regulate the p53/p63 and p63 specific target genes. Additionally, we observed a differential effect of p63 mutants on wildtype-p63-mediated induction ofp53/p63 and p63 specific target genes. We also demonstrated that these mutants differentially regulate the binding of wildtype p63 to the promoter of target genes. Furthermore, the effects of these mutants on cell death and survival were consistent with their ability to regulate the downstream targets when compared to wildtype TAp63T. In summary, our data demonstrate that p63 mutants exhibit differential effects on p63 and p53/p63 specific target genes and on the induction of apoptosis, and provide further insight into the function of p63.

Comparison of the Immunogenicities of HIV-1 Mutants Based on Structural Modification of env

Eleven env mutants were designed and generated by site-directed mutagenesis of the regions around NAb epitopes and deletions of variable regions in env. The immunogenicities of the generated mutants were evaluated using single-cycle infection neutralization assays with two pseudoviruses and IFN-γ ELISPOT. Overall, five mutants (dWt, M2, M5-2, M5-1 and dM7) induced higher neutralization activities for both pseudoviruses than plasmid Wt, while only two of the mutants (dWt and M5-2) showed significant differences (P<0.05). Two mutants (M2 and dM2) induced more Env-specific T cells than plasmid Wt. Statistically however, significance was only reached for mutant M2. Thus, properly modified HIV-1 Env may have the potential to induce potent cellular and humoral immune responses.

Germline MLH1 and MSH2 mutational spectrum including frequent large genomic aberrations in Hungarian hereditary non-polyposis colorectal cancer families:Implications for genetic testing

AIM: To analyze the prevalence of germline MLH1 and MSH2 gene mutations and evaluate the clinical characteristics of Hungarian hereditary non-polyposis colorectal cancer (HNPCC) families. METHODS: Thirty-six kindreds were tested for mutations using conformation sensitive gel electrophoreses, direct sequencing and also screening for genomic rearrangements applying multiplex ligation-dependent probe amplifi cation (MLPA). RESULTS: Eighteen germline mutations (50%) were identifi ed, 9 in MLH1 and 9 in MSH2. Sixteen of these sequence alterations were considered pathogenic, the remaining two were non-conservative missense alterations occurring at highly conserved functional motifs. The majority of the defi nite pathogenic mutations (81%, 13/16) were found in families fulfilling the stringent Amsterdam Ⅰ/Ⅱ criteria, including three rearrangements revealed by MLPA (two in MSH2 and one in MLH1). However, in three out of sixteen HNPCC-suspected families (19%), a disease-causing alteration could be revealed. Furthermore, nine mutations described here are novel, and none of the sequence changes were found in more than one family.CONCLUSION: Our study describes for the f irst time the prevalence and spectrum of germline mismatch repair gene mutations in Hungarian HNPCC and suspected-HNPCC families. The results presented here suggest that clinical selection criteria should be relaxed and detection of genomic rearrangements should be included in genetic screening in this population.

NEW ANTIMICROBIAL SENSITIVITY TESTS OF BIOFILM OF STREPTOCOCCUS MUTANS IN ARTIFICIAL MOUTH MODEL

Objective To develop a new antimicrobial sensitivity test model for oral products in vitro.Methods A biofilm artificial mouth model for antimicrobial sensitivity tests was established by modifying the LKB chromatography chamber. Using sodium fluoride and Tea polyphenol as antimicrobial agent and Streptococcus mu-tans as target, sensitivity tests were studied. Results The modeling biofilm assay resulted in a MIC of 1. 28mg/ ml for fluoride against S. mutans, which was 32 times the MIC for broth maco-dilution method. The differential resistance of bacteria bioflim to antimicrobial agent relative to planktonic cells was also demonstrated. Conclusion The biofilm artificial mouth model may be useful in oral products test.

Construction of DNA damage response gene pprI function-deficient and function-complementary mutants in Deinococcus radiodurans

PprI, a DNA damage response factor from the extraordinary radioresistant bacterium Deinococcus radi- odurans, plays a central regulatory role in multiple DNA damage repair. In this study, a fusion DNA fragment carry- ing kanamycin resistance gene with the D. radiodurans groEL promoter was cloned by PCR amplification and reversely inserted into the pprI locus in the genome of the wild-type strain R1. The resulting pprI-deficient strain, designated YR1, was very sensitive to ionizing radiation. Meanwhile, the re- combinant DNA fragment was cloned into the shuttle vector pRADZ3, and resulted in plasmid pRADK with kanamycin resistance in D. radiodurans. The fragments containing com- plete pprI gene and 3′-terminal deletion pprI△ were cloned into plasmid pRADK. The resulted plasmids designated pRADKpprI and pRADKpprI△ were then transformed to YR1. Results show that YR1 carrying pRADKpprI was able to fully restore the extreme radioresistance to the same level as the wild-type D. raiodurans R1, whereas YR1 pRADKpprI△ failed to do so. Construction of DNA repair switch PprI function-deficient and function-complementary mutants in D. radiodurans is not only useful to elucidating the relationship between domains and functions of PprI pro- tein, but also opens the door to the further studies of the bio- logical functions of PprI protein in vivo.

Genetic analysis and mapping of rice(Oryza sativa L.)male-sterile(OsMS-L)mutant

A rice male-sterile mutant OsMS-L of japonica cultivar 9522 background, was obtained in M4 population treated with Co γ-Ray. Genetic analysis indicated that the 60 male-sterile phenotype was controlled by a single recessive gene. Results of tissue section showed that at microspore stage, OsMS-L tapetum was retarded. Then tapetal cells ex- panded and microspores degenerated. No matured pollens were observed in OsMS-L anther locus. To map OsMS-L lo- cus, an F2 population was constructed from the cross be- tween the OsMS-L (japonica) and LongTeFu B(indica). Firstly, the OsMS-L locus was roughly mapped between two SSR markers, RM109 and RM7562 on chromosome 2. And then eleven polymorphic markers were developed for further fine fine-mapping. At last the OsMS-L locus was mapped between the two InDel markers, Lhs10 and Lhs6 with genetic distance of 0.4 cM, respectively. The region was delimited to 133 kb. All these results were useful for further cloning and functional analysis of OsMS-L.

Genetic analysis and identification of a large leaf angles (lla) mutant in rice

Mutants are essential genetic materials to elucidation of biological functions of genes involved.

Construction and characteri-zation of double mutants in nitrogenase of Klebsiella pneumoniae

Two mutants in nitrogenase of Klebsiella pneu-moniae are constructed by site-directed mutagenesis and gene replacement procedure, which express the nitrogenases with Lysine and Glutamine substituting for a-Glutamine 190 and a-Histidine 194 respectively (Kp-Q a190 K and Kp-H a194 Q). The above two substitutions are respectively intro-duced into a nifV mutant (expressing a citrate-containing nitrogenase) and sequentially two double mutants are ob-tained (Kp-Q a190 K-nifV- and Kp-H a194 Q-nifV-). All four mutants exhibit strict Nif- phenotype under the N2-fixation condition and fail to grow diazotrophically. Altered ni-trogneases are effectively depressed and the C2H2 reduction analysis shows that the double substitutions in Kp-Q a190 K-nifV abolish cell C2H2 reduction activity, but Kp-H a194 Q-nifV- cells maintain a C2H2 reduction activity at 10% of that of wild type. Whole cell C2D2 reduction by all four mu-tants in comparison to the wild type and nifV mutant is also detected. The results show that only single a-Gln194 substitu-tion does not perturb the stereospecificity of protonation of C2D2. These results indicate that the a- Glutamine 190 and its combination with homocitrate are essential to the catalytic activity of nitrogenase and it is proposed that a-Glutamine 190 and its combination with homocitrate are involved in the proton and/or electron transfer to FeMoco. The nitrogenases from these double mutants will be useful in further analysis of the entry of the proton and/or electron to FeMoco and the substrate binding sites.

The secretion of lecithinase of Pseudomonas alcaligenes S2 was via type II secretion pathway

Strain S2 is a lecithin (or phosphatidylcholine)- solubilizing bacterium, which was isolated from the rice rhizosphere in rural areas of Beijing, China. On the basis of a polyphasic study involving phenotypic tests, physiological and biochemical tests, 16S rDNA sequence analysis, G+C content determination and DNA-DNA hybridizations analy-sis, strain S2 was identified as Pseudomonas alcaligenes. P. alcaligenes S2 was mutagenized with Tn5 and four mutants showing decreased or increased solubilizing ability of lecithin were isolated based on the halo size around colonies on the solid plate supplemented with egg yolk. To characterize the genes of P. alcaligenes S2 involved in solubilization of lecithin, the EcoR I fragments of the chromosomes from the four mutant strains carrying a single transposon were cloned, and the DNA sequences flanking the Tn5 were determined. The Tn5 insertion sites in the mutants M808, M1329 and M1400, showing decreased solubilizing ability of lecithin, were found to be located in the xcpS, xcpX and xcpW , respectively, whose products XcpS, XcpX and XcpW were the components of type II secretion pathway. Complementation of xcpS, xcpX and xcpW could restore the corresponding mutants M808, M1329 and M1400 to solubilize lecithin. The data suggested that mutation in one of these xcp genes would lead to the absence of mature lecithinase secretion into the extracellular medium. The data also indicated that the secretion of leci-thin-hydrolyzing enzyme of P. alcaligenes was via type II secretion pathway. In the mutant M20 showing increasing lecithin-hydrolyzing activity, the interrupted gene showed 86% identity with chpA of Pseudomonas aeruginosa PAO1, whose product plays an important role in controlling twitch-ing motility of the bacterial cells.

Observation of fiber ultr-astructure of Ligon lintless mutant in upland cotton during fiber elongation

Lintless mutant is a super-short fiber mutant in upland cotton only 4—8 mm in fiber length and also named Ligon cotton controlled by one dominant gene Li1. Fiber ul- trastructure of the mutant (Li1) and its wild type (li1) in situ and in vitro was observed under an electron microscope to understand its cytological characteristics during the fiber cell elongation. The results showed that the mutant fiber in situ had thinner cytoplasm, more small vacuoles, less mitochon- dria, Golgi apparatus and endoplasmic reticula, and there were more starch granules which were free or packed in the amyloplast beside the cell wall than that of wild type. It was indicated that scarcity of functional organelles and disability of transformation from starch to sugar might be associated with the fact that the mutant fiber cell was aborted too early to elongate into normal length. Mutant ovule in some media containing GA3 could produce a kind of huge callus that grew faster than normal ovules. The callus was covered with many white, loose, and semitransparent fiber-like cells that apt to get off from ovule. These fiber-like cells were multi- cellular fibers generated by cell division and had black dots just like pigment glands in the stem and leaf of cotton. There were lots of micro-tubes beside cytoplasm membrane of the multicellular fiber, which were thought to be primary preparation for second wall deposition of multicellular fiber. It was indicated that GA3 might induce the expression of gene(s) that kept inactive in the field condition and then stimulate the original fiber cell in vitro to undergo division again.