Human Resistome Study with Metagenomic Sequencing Data / 한양의대학술지

With the introduction of synthetic antibiotics, many lives including humans and animals have been saved against bacterial infection. An increasing level of antibiotics use, however, raises serious problems of multi-drug resistance and transferring of resistance genes across different environments and countries. Advances in high-throughput sequencing technology and efficient bioinformatics methods allow us to perform a large-scale screening and analysis of resistomes in the human and environmental microbiomes. Recent studies on human microbiomes have revealed a diverse distribution of resistance genes and their transferring activities in the communities. This review discusses recent progresses in metagenomic approaches to identify resistance genes in the human microbiome, including genomic sequence search and functional metagenomics methods. Using Rifampicin ADP-ribosyltransferase as an example, an integrative approach that analyzes the sequences and three-dimensional structures of the proteins derived from resistance genes is also introduced.

Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

BACKGROUND: The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. METHODS: The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. RESULTS: A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. CONCLUSIONS: The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run.

Correlation Between Virulence Genotype and Fluoroquinolone Resistance in Carbapenem-Resistant Pseudomonas aeruginosa

BACKGROUND: Pseudomonas aeruginosa is a clinically important pathogen that causes opportunistic infections and nosocomial outbreaks. Recently, the type III secretion system (TTSS) has been shown to play an important role in the virulence of P. aeruginosa. ExoU, in particular, has the greatest impact on disease severity. We examined the relationship among the TTSS effector genotype (exoS and exoU), fluoroquinolone resistance, and target site mutations in 66 carbapenem-resistant P. aeruginosa strains. METHODS: Sixty-six carbapenem-resistant P. aeruginosa strains were collected from patients in a university hospital in Daejeon, Korea, from January 2008 to May 2012. Minimum inhibitory concentrations (MICs) of fluoroquinolones (ciprofloxacin and levofloxacin) were determined by using the agar dilution method. We used PCR and sequencing to determine the TTSS effector genotype and quinolone resistance-determining regions (QRDRs) of the respective target genes gyrA, gyrB, parC, and parE. RESULTS: A higher proportion of exoU+ strains were fluoroquinolone-resistant than exoS+ strains (93.2%, 41/44 vs. 45.0%, 9/20; P< or =0.0001). Additionally, exoU+ strains were more likely to carry combined mutations than exoS+ strains (97.6%, 40/41 vs. 70%, 7/10; P=0.021), and MIC increased as the number of active mutations increased. CONCLUSIONS: The recent overuse of fluoroquinolone has led to both increased resistance and enhanced virulence of carbapenem-resistant P. aeruginosa. These data indicate a specific relationship among exoU genotype, fluoroquinolone resistance, and resistance-conferring mutations.

Effects of Pseudomonas quinolone signal on the virulence of Pseudomonas aeruginosa / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of Pseudomonas quinolone signal (PQS) on the virulence of Pseudomonas aeruginosa.</p><p><b>METHODS</b>Pseudomonas aeruginosa strain PAO1 was treated with PQS alone, PQS plus farnesol, or farnesol alone. The transcriptional levels of the regulator gene ExsA and virulence protein gene ExoS of type III secretion system were examined using quantitative real-time PCR, and spectrophotometry was employed to detect pyocyanin production in the bacteria. The adhesion and invasiveness of the treated PAO1 in cultured alveolar epithelial cells A549 were assessed on plate count agar, and their effects on the survival of a mouse model of peritonitis was compared.</p><p><b>RESULTS</b>The increase or decrease of PQS did not affect the growth of PAO1. Compared with the untreated bacteria, PQS-treated PAO1 showed obviously increased transcription levels of ExsA and ExoS (P<0.01) and pyocyanin production, which was significantly lowered by farnesol (P<0.01). In A549 cell cultures, farnesol-treated PAO1 exhibited significantly lowered adhesion and invasiveness, while PQS-treated PAO1 caused a significantly decreased survival time of mice with peritonitis (P<0.01). Farnesol treatment did not obviously affected ExsA transcription (P>0.05) but caused a significant reduction in the transcriptional level of Exos (P<0.05) in PAO1. PQS showed no significant effect on the adhesion and invasiveness of PAO1 (P<0.05).</p><p><b>CONCLUSION</b>PQS can maintain the adhesion and invasiveness of Pseudomonas aeruginosa, and in the hosts of the bacteria, PQS concentration is positively correlated with pyocyanin production and hence negatively with the survival time of the hosts.</p>

Carriage of the Pseudomonas aeruginosa virulence factors and prognosis after infection / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate drug-resistance and carriage of virulence factors of Pseudomonas aeruginosa (Pa) isolated from children.</p><p><b>METHOD</b>Thirty-eight strains of Pa were collected and isolated in pediatric clinic during 2006-2009, and tests were undertaken to identify bacteria and susceptibility test was performed using VITEK-2 COMPACT GNI and AST-GN13 cards. The virulence factors were confirmed by using polymerase chain reaction (PCR) and sequencing.</p><p><b>RESULT</b>All the 38 strains of Pa were resistant to ampicillin, ampicillin/sulbactam, cefazolin, nitrofurantoin, trimethoprim/sulfamethoxazole, resistance rates were 100%. Except for ceftriaxone (60.53%), the resistance rates to other antibiotics were all below 16%. PCR test showed that all the 38 strains of Pa carried exotoxin A(toxA) and nitric oxide reductase A (norA), however, detective ratio of the other virulence factors, exoenzyme Y (exoY) was 84.21% (32/38), exoenzyme S (exoS) 57.89% (22/38), pyocyanin (pyp) 42.11% (16/38), exoenzyme U (exoU) 34.21% (13/38), and 38 strains of Pa did not carry exoenzyme T (exoT) and elastase B (lasB) without exception. By analyzing tests, we discovered that 3 pan-drug resistant strains of Pa were all combination of exo U+/pyp+, there were 4 strains of Pa which were moderately-resistant to imipenem, including exoU+/pyp+/exoY+ (2 isolates), exo U+/pyp+ (1 isolate), and exoY+/exoS+ (1 isolates). It indicated that the drug-resistance rate of exoU+/pyp+ is much higher, compared with exoS+ and exoY+. Molecular epidemiological detection revealed that 2 of 3 extensive-resistance strains of Pa were the same clone, but another one had 96.3% of homology with them.</p><p><b>CONCLUSION</b>The above mentioned 34.21% of Pa isolated from children carried virulence factors toxA, norA, exoS, exoY, pyp and exoU. The strains with exoU/pyp had rather high resistance. The strains with pyp had strong toxicity, they easily cause generalized infection, the patients with them had very high mortality.</p>

Comparison of antibiotic resistance and virulence factors in pigmented and Non-pigmented Pseudomonas aeruginosa / Comparación de la resistencia antibiótica y los factores de virulencia en las Pseudomonas aeruginosa pigmentadas y no pigmentadas

OBJECTIVES: Pseudomonas aeruginosa produces multiple virulence factors that have been implicated in pathogenesis and quorum sensing. The aim of this study was to determine differences in the virulence factors of pigmented and non-pigmented P aeruginosa isolates. METHODS: Associations were assessed between pigment production (pyocyanin and pyoverdin) and production of DNase, elastase, lipase, protease, siderophore, twitching motility, antibiotic resistance patterns and virulence-associated genes in 57 non-duplicate P aeruginosa isolates from wounds, sputum, urine, high vaginal swab (HVS), ear, eye and respiratory tract swabs and aspirates of peritoneum and ulcers. RESULTS: Most (82.5%) of the isolates produced either pigment. Pigmented isolates produced more frequently and significant more (p < 0.05) DNase, elastase, lipase protease, and siderophore. Imipenem was the only antibiotic to which all isolates were susceptible (p < 0.05), while 93% and 32% were resistant to tetracycline and norfloxacin, respectively. There was however no significant difference between pigmented and non-pigmented isolates when antibiotic resistance was compared. While isolates had multiple virulence-associated genes, exoS (51%), rhlA (37%) and rhlB (46%) were the predominant genes detected. Except for exoY, genes were present in pigmented isolates more frequently than in non-pigmented isolates. CONCLUSION: The results of this study suggest that antibiotic resistance per se might not be associated with the pigment production in P aeruginosa. However, pigment production appeared to be more significantly associated with multi-drug resistance, presence ofvirulence-associated genes, and expression ofcertain virulence factors, most notably elastase, protease, siderophore and DNase activity. Since pigment production is easy to determine, this might to be a good starting point to identify the virulence status ofan isolate.

OBJETIVO: Pseudomonas aeruginosa produce múltiples factores de virulencia que han estado implicados en patogénesis y detección de quórum (quorum sensing). El objetivo de este estudio fue determinar las diferencias en los factores de virulencia de aislados de P aeruginosa pigmentada y no pigmentada. MÉTODO: Se evaluaron las asociaciones entre la producción de pigmentos (piocianina y pioverdina) y la producción de Dnasa, elastasa, lipasa, proteasa, sideróforos, motilidad asociada a superficies (twitching), patrones de resistencia antibiótica, y genes asociados con virulencia en 57 aislados de P aeruginosa no duplicados, de heridas, esputo, orina, exudado vaginal, exudados de oídos, ojos, y vías respiratorias, y aspirados de peritoneo y úlceras. RESULTADOS: La mayor parte (82.5%) de los aislados produjeron uno de los pigmentos. Los aislados pigmentados produjeron con mayor frecuencia y más significativamente (p < 0.05). Dnasa, elastasa, lipasa, proteasa, y siderósforos. Imipenem fue el único antibiótico al que todos los aislados eran susceptibles (p < 0.05), mientras que el 93% y el 32% fueron resistentes a la tetraciclina y a la norfloxacina, respectivamente. Sin embargo, no hubo diferencia significativa entre los aislados pigmentados y los no pigmentados cuando se comparaba la resistencia antibiótica. Si bien los aislados tenían múltiples genes asociados con la virulencia, exoS (51%), rhlA (37%) y rhlB (46%) fueron los genes predominantes detectados. Con excepción de exoY, los genes estuvieron presentes en aislados pigmentados con mayor frecuencia que en los aislados no pigmentados. CONCLUSIÓN: Los resultados de este estudio sugieren que la resistencia antibiótica per se podría no estar asociada con la producción de pigmentos en P aeruginosa. Sin embargo, la producción de pigmentos parecía estar asociada más significativamente con la resistencia a las multidrogas, la presencia de genes asociados con la virulencia, y la expresión de ciertos factores de virulencia, en particular la actividad de la elastasa, la proteasa, los sideróforos, y la Dnasa. Puesto que la producción de pigmentos es fácil de determinar, esto podría ser un buen punto de partida para identificar el estado de virulencia de un aislado.

Clinical significance of virulence-related genes of type III secretion system of Pseudomonas aeruginosa / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the clinical significance of virulence genes exo U and exo S of type III secretion system (TTSS) of Pseudomonas aeruginosa (PA).</p><p><b>METHODS</b>One hundred and eighty-nine clinical isolates of PA were collected from five hospitals. The incidence of virulence genes exo U and exo S in PA were determined with PCR. Minimum inhibitory concentration of anti-bacterial drug for PA was determined with microdilution method. The clinical features and outcomes of 60 hospitalized patients colonized or infected with exo U+/exo S- positive or exo U-/exo S+ positive PA isolated from sputum were analyzed retrospectively. Data were processed with chi-square test.</p><p><b>RESULTS</b>Among the 189 PA isolates, 85.2% (161/189) harbored TTSS genes, including exo U-/exo S+ type (120 isolates), exo U+/exo S- type (31 isolates), exo U-/exo S- type (7 isolates), and exo U+/exo S+ type (3 isolates). 72.0% (72/100) isolates from sputum and 81.5% (44/54) isolates from blood belonged to exo U-/exo S+ genotype. Compared with those of TTSS-negative isolates, the antimicrobial resistance of TTSS-positive isolates to cefoperazone/sulbactam, ceftazidime, amikacin, and cefepime were lower (with χ² value respectively 10.1, 16.1, 9.3, 33.8, P values all below 0.01). The antimicrobial resistance to all examined drug between exo U-/exo S+ type and exo U+/exo S- type isolates was close (with χ² values from 0.08 to 2.04, P values all above 0.05). Patients detected with exo U+/exo S- positive PA isolated from sputum were significantly associated with PA infection, and they usually had history of tracheal intubation, ICU hospitalization, and combined use of drugs for anti-infection treatment. Patients detected with exo U-/exo S+ positive PA isolated from sputum were significantly associated with PA colonization, which had basic lung disease and better outcome than the former infection type.</p><p><b>CONCLUSIONS</b>The TTSS exists in most clinical isolates of PA. Detection of exo U or exo S of PA isolated from sputum is helpful for the analysis of clinical features and outcome of patients.</p>

Target-specific cytotoxic activity of recombinant fusion toxin C-CPE-ETA' against CLDN-3,4-overexpressing ovarian cancer cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>The aim of this study was to explore the possibility of creating a toxin, C-CPE-ETA', by fusing C-terminal high affinity binding domain of CPE (C-CPE) with a truncated form of Pseudomonas aeruginosa exotoxin A (ETA') and to examine whether C-CPE-ETA' could specifically target CLDN-3, 4 molecule and the targeted toxin was cytotoxic against CLDN-3,4-overexpressing ovarian cancer.</p><p><b>METHODS</b>CLDN-3 and CLDN-4 expressions were analyzed at the mRNA level in three ovarian cancer cell lines and epithelial ovarian cancer tissues from 20 patients. After transforming an expression plasmid of C-CPE-ETA' into E. coli BL21 (DE3) plysS strain, the recombinant protein was purified using His-Bind resin chromatography column and analyzed by Western blot and Coomassie blue staining. The specific binding, proapoptotic and cytolytic activities were evaluated by flow cytometry, fluorescence microscopy with the JC-1 probe and MTT assay in CLDN-3,4-overexpressing ovarian cancer cells.</p><p><b>RESULTS</b>Quantitive RT-PCR results showed there existed high levels of CLDN-3 and CLDN-4 in ovarian cancer cells, CAOV3, OVCAR3 and SKOV3. Moreover, high expressions of CLDN-3 and CLDN-4 were observed in 90.0% (18/20) and 60.0% (12/20) of ovarian cancer tissues, with an expression level 10-fold higher than that in the normal ovarian tissue. A 58 000 recombinant protein C-CPE-ETA' was demonstrated by Western blot and Coomassie blue staining. Purified and recombinant C-CPE-ETA' was bound with high affinity to CLDN-3,4-overexpressing ovarian cancer cells, CAOV3, OVCAR3 and SKOV3 cells. C-CPE-ETA' was strongly proapoptotic and cytotoxic towards the CLDN-3,4-overexpressing ovarian cancer cells. The concentration of IC(50) was 7.364 ng/ml for CAOV3 cells, 8.110 ng/ml for OVCAR3 cells and 22.340 ng/ml for SKOV3 cells, respectively. However, control CLDN-3,4-deficient cell line HUVEC was not susceptible to the recombinant C-CPE-ETA' at a concentration up to 10 µg/ml.</p><p><b>CONCLUSIONS</b>The C-CPE-ETA' protein exhibits remarkably specific cytotoxicity for CLDN-3,4-overexpressing ovarian cancer cells. Its therapeutic potential warrants further development for ovarian cancer molecular targeted therapy.</p>

Construction, expression and immunogenicity analysis of a fusion protein containing M2e of influenza A virus fused to a modified Pseudomonas aeruginosa exotoxin A / 病毒学报

M2 protein of type A influenza virus is a good candidate for universal influenza vaccine, exotoxin A of Pseudomonas aeruginosa may facilitate the immunogenicity of M2 protein. We constructed and expressed a prokaryotic expression plasmid containing a chimeric gene of M2 extracellular coding region and a partial PEA gene, and observed the immunoprotection in BALB/c mice vaccinated with the fusion protein. The fusion protein (ntPE-M2e) was generated by inserting the coding sequence of the M2e in place of Ib loop in PEA. This fusion protein was used to immunize BALB/c mice by subcutaneously injection with incomplete Freund's adjuvant and boost at weeks 3 and 7. The immunized mice were challenged with influenza virus strain A/PR/34/8. The fusion protein (ntPE-M2e) immunization protected mice against lethal viral challenge. ELISA and ELISPOT results demonstrated that the fusion protein could induce a strong systemic immune response against synthetic M2e peptide, and virus replication in the lungs of mice was inhibited in comparison with the control. This study provides foundation for developing broad-spectrum vaccines against type A influenza viruses.

Pro-apoptotic effect on osteosarcoma SOSP-9607 cells by human recombinant caspase-6 fusion protein / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the pro-apoptotic effect of Her-2 targeted recombinant caspase-6 fusion protein on osteosarcoma SOSP-9607 cells.</p><p><b>METHODS</b>Recombinant immunocasp-6 was generated by sequential fusion of the genes of a signal peptide, a single-chain Her-2 antibody (e23sFv), a PEA translocation domain (PEA aa253-364) and an active caspase-6. The immunocasp-6 gene was cloned into pCMV plasmid to construct a kind of eukaryotic expression vector, i.e. pCMV-e23sfv-PE II-caspase-6 (abbr. pCMV-6) and transfected into SOSP-9607 cells. Murine xenograft models were randomly divided into two groups that received i.m. injections of liposome encapsulated pCMV-6 or pCMV alone. The tumor volume and weight of the nude mice and the tumor weight of the cured mice were observed and statistically analyzed. The morphological changes of the tumors were examined with HE staining, apoptotic morphology of the tumor was observed by TUNEL staining and the gene expression was analyzed by immunohistochemical staining.</p><p><b>RESULTS</b>The tumor growth of the mice in the treatment group was significantly slower than that of the control group (P = 0.001). The weight of the nude mice in the treatment group was significantly higher than that of the control group (P = 0.0002). The tumor weight of the mice in the treatment group was significantly lower than that of the control group (P = 0.0006). HE and TUNEL staining of the tumor of nude mice in the treatment groups showed typical characteristics of apoptosis, while normal structure was found in the control group. Furthermore, caspase-6 was not found in the tumor and muscle tissues in the control group, but only in the treatment group by immunohistochemistry.</p><p><b>CONCLUSION</b>Immunocasp-6 can selectively recognize and bind to and kill HER-2 positive osteosarcoma cells, therefore, to offer some foundation for the clinical treatment of osteosarcoma.</p>

Pseudomonas aeruginosa Exotoxin A Reduces Chemoresistance of Oral Squamous Carcinoma Cell via Inhibition of Heat Shock Proteins 70 (HSP70)

PURPOSE: Oral squamous carcinoma (OSCC) cells exhibit resistance to chemotherapeutic agent-mediated apoptosis in the late stage of malignancy. Increased levels of heat shock proteins 70 (HSP70) in cancer cells are known to confer resistance to apoptosis. Since recent advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers, we investigated the effect of Pseudomonas aeruginosa exotoxin A (PEA) on HSP70 expression and induction of apoptosis in chemoresistant OSCC cell line (YD-9). MATERIALS AND METHODS: The apoptotic effect of PEA on chemoresistant YD-9 cells was confirmed by MTT, Hoechst and TUNEL stains, DNA electrophoresis, and Western blot analysis. RESULTS: While YD-9 cells showed high resistance to chemotherapeutic agents such as etoposide and 5-fluorouraci (5-FU), HSP70 antisense oligonucelotides sensitized chemoresistant YD-9 cells to etoposide and 5-FU. On the other hand, PEA significantly decreased the viability of YD-9 cells by deteriorating the HSP70-relating protecting system through inhibition of HSP70 expression and inducing apoptosis in YD-9 cells. Apoptotic manifestations were evidenced by changes in nuclear morphology, generation of DNA fragmentation, and activation of caspases. While p53, p21, and E2F-1 were upregulated, cdk2 and cyclin B were downregulated by PEA treatment, suggesting that PEA caused cell cycle arrest at the G2/M checkpoint. CONCLUSION: Therefore, these results indicate that PEA reduced the chemoresistance through inhibition of HSP70 expression and also induced apoptosis in chemoresistant YD-9 cells.

Orf1/SpcS chaperones ExoS for type three secretion by Pseudomonas aeruginosa / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Pseudomonas aeruginosa is a ubiquitous and opportunistic pathogen that uses the type III secretion system (TTSS) to inject effector proteins directly into the cytosol of target cells to subvert the host cell's functions. Specialized bacterial chaperones are required for effective secretion of some effectors. To identify the chaperone of ExoS, the representative effector secreted by the TTSS of P. aeruginosa, we analyzed the role of a postulated chaperone termed Orf1.</p><p><b>METHODS</b>By allelic exchange, we constructed the mutant with the deletion of gene Orf1. Analysis of secreted and cell-associated fractions was performed by SDS-PAGE and Western blotting. Using strain expressing in trans Orf1, tagged by V5 polypeptide and histidine, protein-protein interaction was determined by affinity resin pull-down assay in combination with MALDI-TOF. The role of Orf1 in the expression of exoS was evaluated by gene reporter analysis.</p><p><b>RESULTS</b>Pull-down assay showed that Orf1 binds to ExoS and ExoT. Secretion profile analysis showed that Orf1 was necessary for the optimal secretion of ExoS and ExoT. However, Orf1 had no effect on the expression of exoS.</p><p><b>CONCLUSION</b>Orf1 is important for the secretion of ExoS probably by maintaining ExoS in a secretion-competent conformation. We propose to name Orf1 as SpcS for "specific Pseudomonas chaperone for ExoS".</p>

Rapid detection of Pseudomonas aernginosa by the fluorescence quantitative TaqMan PCR assay targetting ETA gene / 生物工程学报

Pseudomonas aernginosa (PA) is one of the most universal pathogens in clinical diagnosis, and conventional detection assay has many disadvantages. In this research, a pair of specific primers and a TaqMan fluorescent probe were designed in the conservative region of ETA gene by the method of bioinformatics analysis, the detection method for PA was successfully developed. Different gradient concentrations of PA DNA and various pathogen DNA were amplified by fluorescence quantitative PCR (FQ-PCR) to confirm the specificity and sensitivity of the developed method. Results showed that the developed detection assay is more sensible and specific by comparison to the conventional FQ-PCR method, and it is valuable for research and application prospects.

Endogenous ADP-ribosylation of eukaryotic elongation factor 2 and its 32 kDa tryptic fragment

Eukaryotic elongation factor 2 (eEF-2) can undergo ADP-ribosylation in the absence of diphtheria toxin. The binding of free ADP-ribose and endogenous transferase-dependent ADP-ribosylation were distinct reactions for eEF-2, as indicated by different findings. Incubation of eEF-2 tryptic fragment 32/33 kDa (32F) with NAD was ADP-ribosylated and gave rise to the covalent binding of ADP-ribose to eEF-2. 32F was revealed to be at the C-terminal by Edman degradation sequence analysis. In our study, the elution of 32F from SDS-PAGE was ADP-ribosylated both in the presence and absence of diphtheria toxin. These results suggest that endogenous ADP-ribosylation of 32F might be related to protein synthesis. This modification appears to be important for the cell function.

Type III secretion study of popN- mutant of Pseudomonas aeruginosa and proteases degradation / 生物工程学报

Pseudomonas aeruginosa is an important opportunistic human pathogen. It encodes many virulence factors and one of them is type III secretion system (TTSS). Effectors proteins can be delivered into host cells directly by this system, causing necrosis or apoptosis. popN gene is the first gene in the popN operon of TTSS gene cluster. To investigate its function, popN gene deletion mutant was generated in this study, and we found this mutant can secrete effectors proteins constitutively under non-inducting condition in DMEM medium containing serum. The results indicated that PopN is a negative regulator of the TTSS expression. However, no secreted effector proteins were detectable when the popN- mutant was grown in LB medium under non-inducting condition. To investigate the possible reasons, effects of growth status and protease (s) inhibitors on the TTSS were investigated. We present evidences that indicate protease mediated degradation of secreted effector proteins played a key role in the phenotypic inconsistency of popN- mutant.

Relationship of genetic polymorphism in APE1 and ADPRT to risks of chronic benzene poisoning / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To explore the relationship between genetic polymorphisms in apurinic/apyrimidinic endonuclease (APE1) and ADP ribosyltransferase (ADPRT) and individuals' susceptibility to chronic benzene poison ing (BP).</p><p><b>METHODS</b>A case-control study was conducted. One hundred and fifty-two B P patients and 152 workers occupationally exposed to benzene without poisoning manifestations were investigated. The mismatched bases combined to create restriction site with restrained fragment length polymorphism technique (CRS-RFLP) was used for detecting the single nucleotide polymorphisms (SNPs) at Asp148Glu of APE1 gene and Val762Ala of ADPRT gene.</p><p><b>RESULTS</b>There was no significant difference in the distribution of genotypes of APE1Asp148Glu and ADPRTVal762Ala between the patients and the control groups. Compared with individuals having genotype of APE1Asp148Glu T/T without habit of alcohol consumption, there was a 4.13 times increased risk of BP for the alcohol user with genotype of APE1Asp148Glu T/T (OR = 4.13, 95% CI: 1.07 - 15.85, P = 0.03). The analysis of Logistic regression showed that smoking may play some role in modifying the risk of cironic benzene poisoning (OR = 0.33, 95% CI: 0.14 - 0.75, P = 0.01).</p><p><b>CONCLUSION</b>The genetic polymorphisms in APE1Asp148Glu, ADPRTVal762Ala are not related to the risk of BP. Potential interaction is found between alcohol consumption and polymorphism of APE1Asp148Glu. Further study is needed to elucidate this interaction.</p>

Expression of toxin-related human mono-ADP-ribosyltransferase 3 in human testes / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To investigate wether the corresponding protein of mono-ADP-ribosyltransferase 3 (ART3) mRNA is expressed in human testes and, if so, whether the expression is cell type-specific.</p><p><b>METHODS</b>ART3 mRNA was determined in human testes and sperm by reverse transcription-polymerase chain reaction (RT-PCR). The glycosyl-phosphatidylinositol linkage of ART3 was shown by treating ART3-transfected HEK-293-T cells with phospholipase C. Fluorescent activated cell sorter (FACS)-analyses were used to detect ART3 on mature spermatozoa and immunohistological studies to detect the protein in testes.</p><p><b>RESULTS</b>ART3 protein was shown to be present in testes. It was found on spermatocytes only. It was absent from spermatogonia, spermatids and spermatozoa. The absence of ART3 from spermatozoa was confirmed by FACS-analysis. ART3 protein was detected neither within a seminoma nor on Leydig cells.</p><p><b>CONCLUSION</b>Here we show for the first time that ART3 protein is expressed in testes in particular on spermatocytes, indicating that ART3 exerts a specific function only required at a particular stage of spermatogenesis.</p>

High level expression, purification and cytotoxicity of IL-10(18-57)-PE40 / 生物工程学报

The objective of the experiment is to explore the purification and production of immunotoxin. The chimeric toxin, which is composed of 40 peptides of interleukin 10 (from amino acids 18 to 57) fused to a mutant form of Pseudomonas extoxin (PE) devoid of its native cell recognition domain. Two kinds of prokaryotic expression vector containing the chimeric toxin IL-10(18-57)-PE40 were constructed respectively. After induction of IPTG for 3 hours, IL-10(18-57)-PE40 was expressed highly in cytoplasmic fraction in Rosettablue(DE3), and was directed to periplasmic space as soluble form in E. coli BL21(DE3)pLysS . Western -blotting showed that the expressed protein could react with the specific rabbit sera against LHRH-PE40. With the application of salting out of (NH4)2SO4, hydrophobic interaction chromatography, Cu-affinity chromatography and anion exchange chromatography, the purity of IL-10(18-57)-PE40 was about 96%. The cytotoxicity assay, Cell-ELISA and fluorescent antibody test support the hypothesis that IL-10(18-57) based ligand-mediated cytotoxicity can serve to target cytotoxic agents in vitro.

Studies of the expression, purification, renaturation and biologic activity of an anti-CEA immunotoxin / 生物工程学报

A recombinant immunotoxin named CEA/PE38/KDEL was constructed, which was composed of anti-CEA single-chain Fv and the truncated and modified form of Pseudomonas exotoxin (PE38/KDEL). The CEA/PE38/KDEL immunotoxin was expressed in the E. coli strain BL21 (DE3)-star as inclusion bodies. The denatured inclusion bodies were purified with Ni-NTA chelate agarose, then the constant gradient dialysis was used to perform the refolding of the CEA/PE38/KDEL immunotoxin. Results of FACS and MTT assay indicate that the refolded immunotoxins keep potent and specific cytotoxicity to tumor cells bearing CEA antigens.

Growth inhibitory effects of recombinant granzyme B containing different N-terminal translocating peptides / 生物工程学报

Translocating protein and translocating peptides have therapeutic potential against tumors by exposing the cytotoxic domains of toxic proteins to the cell cytosol. The aim of this study is to investigate the effect of N-terminally fused PE translocating peptides on granzyme B (GrBa) activity. PE II-GrBa fusion protein genes were constructed by replacing N-terminal signal and acidic dipeptide sequence of human granzyme B gene with two truncated translocating sequences of Pseudomonas exotoxin A (PE II aa 280-364/358) by recombinant PCR, and then cloned into pIND inducible expression vector. The resulting pIND-PE II-GrBa expression vectors were co-transfected with assistant plasmid pVgRXR into HeLa cells through lipofectamine, followed by selection on G418 and zeocin. The resistant cells were collected and induced with ponasterone A. Western blot analysis demonstrated that ponasterone A induction caused the expression of PE II-GrBa fusion proteins, and indirect immunofluorescence detected giant sized multinucleated cells, suggesting cytoskeletal and mitotic abnormalities as reported in our previous studies. Western blot, enzymatic activity assay and cell counting analysis indicated that two types of PE II-GrBa fusion proteins were capable of cleaving both endogenous and exogenous substrates of granzyme B, and inhibiting the growth of cells. The PE II (aa 280-358)-GrBa was shown to have higher serine protease activity and stronger growth inhibitory effect. Such inhibition was presumably associated with G2 arrest as determined by cell cycle analysis. These data prove that PE II-GrBa fusion proteins have cell inhibitory effect similar to GrBa, and that the shorter PE-derived peptide exerts less influence on GrBa activity. This study helps to optimize the construction of recombinant protein comprising translocating peptides and cytotoxic molecules for tumor cell killing.