Ribosome assembly factor URB1 contributes to colorectal cancer proliferation through transcriptional activation of ATF4.

Ribosome assembly factor URB1 is essential for ribosome biogenesis. However, its latent role in cancer remains unclear. Analysis of The Cancer Genome Atlas database and clinical tissue microarray staining showed that URB1 expression was upregulated in colorectal cancer (CRC) and prominently related to clinicopathological characteristics. Silencing of URB1 hampered human CRC cell proliferation and growth in vitro and in vivo. Microarray screening, ingenuity pathway analysis, and JASPAR assessment indicated that activating transcription factor 4 (ATF4) and X-box binding protein 1 (XBP1) are potential downstream targets of URB1 and could transcriptionally interact through direct binding. Silencing of URB1 significantly decreased ATF4 and cyclin A2 (CCNA2) expression in vivo and in vitro. Restoration of ATF4 effectively reversed the malignant proliferation phenotype of URB1-silenced CRC cells. Dual-luciferase reporter and ChIP assays indicated that XBP1 transcriptionally activated ATF4 by binding with its promoter region. X-box binding protein 1 colocalized with ATF4 in the nuclei of RKO cells, and ATF4 mRNA expression was positively regulated by XBP1. This study shows that URB1 contributes to oncogenesis and CRC growth through XBP1-mediated transcriptional activation of ATF4. Therefore, URB1 could be a potential therapeutic target for CRC.

An overview of type E botulism in China.

The geographical distribution of C. botulinum type E and its associated disease, type E botulism in China, is different from that in other areas of the world. Cases of type E botulism generally arise in costal regions. In China, however, type E botulism is found primarily in the Qinghai-Tibet plateau of northwest China far from the ocean, at an altitude of approximately 4-5 km. The foods most commonly associated with the disease are fermented grain and beans as well as raw meat. A suspected outbreak of type E botulism poisoning in the central costal region of China in the 1990s prompted the collection and analysis of samples of mud, sand, and fish from the region. The toxin produced by type E botulinum was found in these samples. Surprisingly, though, upon further analysis, the strain isolated from the samples was identified not as type E C. botulinum, but as the neurotoxigenic bacterium Clostridium butyricum.

[Effects of Tcd A on Rho GTPases and the Cytoskeleton of Leukemia Cell Line K562].

OBJECTIVE: To investigate the effect of clostridium difficile toxin A(TcdA) on the Rho GTPases and the cytoskeleton in K562 cells. METHODS: K562 cells were cultured in vitro with different concentration of TcdA.The effect of TcdA proliferation of cells was detected by MTT method after the K562 cells were stimulated with TcdA for 24,48 and 72h; the expression of cdc42, RhoA, Rac1 mRNA was assessed by RT-PCR; the changes of the microtubule, the microfilament were observed by confocal laser scanning microscopy. RESULTS: The proliferation of K562 cells was inhibited after exposure to TcdA for 24, 48 and 72h, and the inhibitory rate was 47.67% in the treatment for 48 h. the cdc42，RhoA and Rac1 mRNA expressions in the experimental groups decreased after treated with TcdA(P<0.05), which positively correlated with concentration of TcdA. Also, the microfilament decreased ,which was observed by confocal laser scanning microscopy. CONCLUSION: TcdA inhibites K562 cell proliferation and induces apoptosis, TcdA can change the cytoskeleton structure through the cytoskeletal protein genes cdc42 and RhoA, Rac1 mRNA expression,. It is related with cell microfilament content decreasing.

Development of an ELISA kit using monoclonal antibody to Clostridium difficile toxin A.

AIM: To establish an ELISA kit using monoclonal antibodies against Clostridium difficile (C. difficile) toxin A. METHODS: An indirect sandwich ELISA was described using the purified rabbit monospecific antiserum as capturing antibody. After the polystyrene microtitre plates with 96 flat-bottomed wells were coated with rabbit antiserum, the wells were blocked with 100 g/L BSA in PBS-T. C. difficile toxin A or culture filtrates were added to each well and then monoclonal antibodies IgG-horseradish peroxidase conjugate was added as detecting antibody, tetramethylbenzidine was used as substrate and A450 of the stopped reacting product was recorded in an automated plate reader. RESULTS: The tested specimens included culture filtrates of 2 strains of toxigenic C. difficile, 2 strains of non-toxigenic C. difficile, 26 strains of E. coli, 2 strains of S. dysenteriae, 1 strain of Bif. infantis, 5 strains of V. cholera, 2 strains of S. typhi, 7 strains of C. botulinum, 1 strain of toxigenic C. sordllii, and 1 strain of C. butyricum. A total of 47 strains of culture filtrates were all negative except for 2 strains of toxigenic C. difficile. The detective limitation of toxin A was 0.1 ng/mL. CONCLUSION: An ELISA kit with high specificity and excellent sensitivity for the rapid detection of C. difficile toxin A was established. It will be a useful tool for diagnostic test of C. difficile toxin A.

Simplified purification method for Clostridium difficile toxin A.

AIM: To establish the purification method for Clostridium difficile (C. difficile) toxin A. METHODS: C. difficile VPI 10463 filtrate was cultured anaerobically by the dialysis bag methods. And then the toxin A was purified by precipitation with 500 g/L (NH4)2SO4 and acid precipitation at pH 5.5, followed by ion-exchange chromatography on DEAE-Toyopearl. RESULTS: Purified toxin A exhibited only one band on native polyacrylamide gel electrophoresis (native-PAGE) and Ouchterlony double immunodiffusion. The molecular weight of toxin A was estimated to be 550,000. The purified toxin A had a protein concentration of 0.881 mg/mL. The minimum lethal dose was 1X10(6) MLD/mL (i.p.mice). The cytotoxic titer was 10(7) CU/mg. The haemagglutinate activity was at a concentration of 1.72 microg/mL. The ratio of fluid volume (mL) accumulated to the length (cm) of the loop was 2.46. CONCLUSION: The modified method for purification of toxin A of C. difficile was simple and convenient. It may be even more suitable for purification of toxin A on large scales.

[Study of dental caries and correlated factors of 12-year-old children in Dongxiang, Baoan and Yugu races].

OBJECTIVE: To investigate the epidemiology of dental caries and its correlated factors of 12-year-old children in Dongxiang, Baoan and Yugu races. METHODS: According to the method of third national oral health epidemiologic investigation, 448 12-year-old children in Dongxiang, Baoan and Yugu races were randomly collected and the epidemiological investigation of dental caries, oral bacteriological detection and oral hygiene behavior were carried out. RESULTS: 1) The caries prevalence rate of Dongxiang, Baoan and Yugu races were 40.52%, 44.29%, 46.45%, respectively. The average caries of Dongxiang, Baoan and Yugu races were 0.92, 0.90, 1.13, respectively. 2) The main ranks of Streptococcus mutans in saliva were class 2 and class 3 in Dongxiang and Baoan races. However, it was class 0 or class 1 in Yugu race. The level of Streptococcus mutans in dental plaque was higher in Dongxiang and Baoan races than in Yugu race. 3) The children's everyday brushing rate was higher in Yugu race than in Dongxiang and Baoan races (P<0.01). But there were no difference between Dongxiang and Baoan races. CONCLUSION: The caries prevalence rates of 12-year-old children in Dongxiang, Baoan and Yugu races are high. The main factors of high caries prevalence rate were low brushing rate and dental plaque couldn't be removed effectively. Oral health education should be strengthened in the three race areas.

[Preparation and characterization of monoclonal antibody against Clostridium difficile toxin A].

AIM: To prepare monoclonal antibodies (mAbs) against Clostridium difficile toxin A and identify their properties. METHODS: BALB/c mice were immunized with C.difficile toxin A. The splenocytes from immunized mice were fused with myeloma cells Sp2/0. The hybridoma cells were screened by indirect ELISA and limiting dilution method. The titer and relative affinity of ascitic mAbs were determined by ELISA. Specificity of mAbs was analyzed by Western blot. RESULTS: Six hybridoma cell lines (2H7, 3E9, 4B5, 5C10, 6G8 and 8A1) secreting mAbs against C.difficile toxin A were obtained. The Ig classes and subclasses of mAbs 2H7, 3E9 and 6G8 were IgM, mAbs 4B5 and 8A1 were IgG1, and mAb 5C10 was IgG2a. All 6 mAbs had no neutralization activity. Epitope recognized by 5 mAbs(2H7, 4B5, 5C10, 6G8 and 8A1) differed from that by mAb 3E9. Relative affinities of mAbs 8A1 and 4B5 were all above 10(5), and those of other 4 mAbs were 10(4). Western blot analysis no-denatured PAGE showed that all 6 mAbs reacted to C.difficile toxin A with M(r) being 55 x 10(4), and under the condition of denatured SDS-PAGE, Western blot analysis showed that all 6 mAbs reacted to subunits of C.difficile toxin A with M(r) being 5 x 10(4)-24 x 10(4). CONCLUSION: Six mAbs against C.difficile toxin A with high titers were obtained successfully with satisfactory specificity and relative affinity, which will be useful for detection of C.difficile toxin A.