Caenorhabditis elegans.The phase II detoxification enzymes execute a major protective role against xenobiotics as well as endogenous toxicants. To understand how xenobiotics regulate phase II enzyme expression, acrylamide was selected as a model xenobiotic chemical, as it induces a large number and a variety of phase II enzymes, including numerous glutathione S-transferases (GSTs) in xenobiotics response pathways (xrep), 24 independent mutants of C. elegans that exhibited abnormal GST expression or regulation against acrylamide were isolated by screening about 3.5×105 genomes of gst::gfp transgenic strains mutagenized with ethyl methanesulfonate (EMS). Complementation testing assigned the mutants to four different genes, named xrep-1, -2, -3, and -4. One of the genes, xrep-1, encodes WDR-23, a nematode homologue of WD repeat-containing protein WDR23. Loss-of-function mutations in xrep-1 mutants resulted in constitutive expression of many GSTs and other phase II enzymes in the absence of acrylamide, and the wild-type xrep-1 allele carried on a DNA construct successfully cured the mutant phenotype of the constitutive enzyme expression.To begin dissecting genetically xrep-1 mutants suggest that a large number of GSTs and other phase II enzymes induced by acrylamide are under negative regulation by XREP-1 (WDR-23), which is likely to be a functional equivalent of mammalian Keap1 and a regulator of SKN-1, a C. elegans analogue of cap-n-collar Nrf2 (nuclear factor erythroid 2-related factor 2).Genetic and cellular characterization of  Here we report on one of four genes defined by these mutants, xrep-1, that encodes a WD repeat-containing protein, a nematode homologue of mammalian WDR23, and provide genetic and cellular evidence that the gene xrep-1 negatively regulates GSTs and some other phase II enzymes.To dissect genetically a gst::gfp transgenic animals, MJCU017 and MJCU047, to screen for mutants abnormally expressing GFP. MJCU017 and MJCU047, which have the chromosomally-integrated gst-4::gfp and gst-30::gfp fusion genes, respectively, emitted no detectable GFP signal in the absence of acrylamide, but emitted a very strong GFP signal from the whole body when treated with acrylamide  I (chromosome I): all of the 16 mutations were recessive, and the affected strains constitutively expressed GST in the whole body without acrylamide. Six, assigned to the same gene on LG II, were also recessive, and their mutants constitutively expressed GST in the body-wall muscle and pharynx only after they had reached the adult stage. Of the remaining two strains, one mutation on LG IV was dominant, with constitutively expressed GST throughout the whole body, whereas the other one, on LG I, was recessive with its mutant expressing no GST even when treated with acrylamide. We called these genes defined by the four complementation groups xrep-1, -2, -3, and -4, in the respective order described above.We mutagenized these transgenic strains with ethyl methanesulfonate (EMS) and screened about 3.5×10xrep-1(k1007) mutation, one of the 16 xrep-1 alleles, we successfully assigned the gene xrep-1 to the middle of LG I between the SNP markers F21C3 and T23G11. Of the total 704 recombinants analyzed, no Hawaiian polymorphism was identified within the genomic region consisting of the cosmid clone D2030, indicating that the mutation site was in or near D2030 (xrep-1(k1007) mutant phenotype (data not shown).In SNP mapping involving the Hawaiian wild-type strain CB4856 crossed with the ar D2030 . We amplar D2030 . FurtherC. elegans GST-4 expression xrep-1 mutations sequenced so far have revealed four missense mutations and one nonsense mutation in the WD repeat domain, suggesting that this domain is important in the regulation of GST expression by keeping it from being induced in the absence of acrylamide  mutant constitutively emits a strong GFP signal from the whole body without acrylamide, and treating it with acrylamide did not further enhance this signal  mutation constitutively expresses GST without acrylamide, we hypothesized that xrep-1 negatively regulated GST-4 expression and that acrylamide triggered GST induction by preventing normal XREP-1 function. To test this hypothesis, we first constructed the xrep-1(+)::rfp fusion gene and transferred it into the xrep-1(k1007) mutant. The xrep-1(+)::rfp completely rescued the xrep-1(k1007) mutant phenotype, but XREP-1::RFP fluorescence was too weak to be useful for the present experiment (data not shown). Therefore, we used a strain, MJCU058 {unc-119(ed3) III, kIs15 IV}. This strain emitted no detectable GST-4::RFP fluorescence signal, with a hardly detectable weak constitutive GST-2::GFP signal from the mouth region (xrep-1(k1007) mutation into the strain MJCU058 to construct MJCU059 {xrep-1(k1007) I; unc-119(ed3) III, kIs15 IV}, which, similarly to the fully induced GST-4::GFP expression pattern of MJCU017, constitutively expressed a strong GST-4::RFP signal from the whole body, without changing the level of the GST-2::GFP signal ::gfp fusion gene ({kEx80; xrep-1(k1007) I; unc-119(ed3) III, kIs15 IV}. The extrachromosomal array kEx80 of MJCU080 rescued the xrep-1(k1007) mutant phenotype and expressed GFP in the cytoplasm and nuclei of neurons, somatic gonads, intestine, and hypodermis over the whole body (xrep-1(+)::gfp transgene. Interestingly, although expectedly, those cells or tissues that retained the xrep-1(+)::gfp transgene, thus keeping the xrep-1(k1007) under rescue, emitted GFP signals. In contrast, cells or tissues without the xrep-1(+)::gfp transgene emitted no GFP signal, thus displaying the xrep-1 mutant phenotype as expected; that is, they emitted the RFP fluorescence signal (xrep-1(+)::gfp that emitted the GFP signal were also induced to emit RFP fluorescence ::gfp transgene. Its repression was eliminated when the animals were treated with xrep-1(RNAi) (xrep-1(k1007) and xrep-1(RNAi) animals show more resistance to aldicarb than do their wild-type counterparts (manuscript in preparation).Notably, when MJCU080 animals were treated with acrylamide for 24 hours, the induction of GST expression was not so strong as would be expected from strains such as MJCU017 . Followi-1(RNAi) . This reIs line MJCU085 {unc-119(ed3) III, kIs84} and confirmed that its expression pattern did not differ from that for the Ex line MJCU080 . We obtained two transcripts from N2 animals, which were then used to synthesize xrep-1a and xrep-1b cDNAs. By sequencing them, we found that xrep-1a cDNA comprises 10 exons  and xrep-1b cDNA consists of 9 exons (exons 3 to 11 with the exclusion of exons 1 and 2). Exons 4 to 11 were identical in both cDNAs except for the 9th exon; the 9th exon in xrep-1b cDNA was 24 bp shorter than that in xrep-1a (xrep-1aProm acDNA::gfp and xrep-1bProm bcDNA::gfp fusion genes ({xrep-1(k1007) I; unc-119(ed3); kIs15 IV}, and obtained at least two lines of transgenic animals for each fusion gene. As observed with the xrep-1(+)::gfp transgene, xrep-1aProm acDNA::gfp expressed GST-4 in a variety of organs, tissues, or cell types in the body, such as pharynx, hypodermis, intestine, and neurons (xrep-1(k1007) mutant phenotype. GST-4 expression in the xrep-1aProm acDNA::gfp transgenic animal was not detected without acrylamide, but was induced with acrylamide (xrep-1bProm bcDNA::gfp transgene did not rescue the xrep-1(k1007) phenotype  phenotype , and 18 of the GSTs were prominently up-regulated when animals were treated with acrylamide xenobiotics response pathway (xrep) in C. elegans by isolating mutants that respond abnormally to acrylamide. We have so far found four xrep genes: three genes  negatively and one gene (xrep-4 I) positively regulate GST expression.Of the phase II enzymes, GSTs are universally found in every organism from bacteria to humans and constitute a large family of enzymes that function as detoxifiers of both endogenous oxidative stress products and exogenous electrophilic chemical compounds xrep-1 encodes XREP-1, a nematode homologue of the WD-repeat containing protein 23 or WDR23. WDR exists in a broad range of organisms from yeasts to mammals as well as plants. It participates in a variety of biochemical, cellular, and organismal processes, such as signal transduction, cytoskeletal dynamics, and RNA processing C. elegans, the bZIP transcription factor SKN-1, which is necessary for mesendodermal differentiation during early embryogenesis xrep-1C. elegans, XREP-1 might control SKN-1, similarly to the mammalian Keap1 for Nfr2 operative in the so-called antioxidant response element (ARE) pathway The negatively regulating gene skn-1 prevented the xrep-1(k1007) mutants and acrylamide-treated animals from expressing GST-4 in most parts of the C. elegans body, except for the pharynx and body-wall muscle  mutants when they were treated with acrylamide (skn-1(RNAi) animals. All five xrep-1 mutation sites so far identified are located in the WDR domain of XREP-1 , and (c) why no or if any mammalian WD40 repeat proteins play a role similar to that for the C. elegans XREP-1.Here we have an emerging picture of a functional similarity between the two pairs of proteins XREP-1/SKN-1 in worms and Keap1/Nrf2 in mammals, as both play a key role in the oxidative and electrophilic stress pathway. At the same time, however, this leaves us with fascinating evolutionary puzzles: (a) how the two counterparts XREP-1 and Keap1, considered as phylogenetically distant relatives (19), have converged to assume essentially an identical role in the pathway critically important in defending organisms against oxidative, xenobiotic, or other life-threatening stresses, (b) why or if really the nematode xrep-1a and xrep-1b, which were PCR products of transcripts from the N2 wild type, and analyzed their function. The xrep-1a cDNA consisting of the exons 4 to 11, which corresponds to a C-terminus of 775 amino acid residues, was sufficient to rescue the mutation xrep-1(k1007) and regulate acrylamide-induced GST expression just as does the entire xrep-1 gene . Thus, we have yet to know what the xrep-1b or any other transcripts of the xrep-1 gene are doing.We obtained two cDNAs, p-1 gene . ContrarC. elegans genetics we have so far identified four xrep genes that regulate the GST expression in the xenobiotics response pathway, and introduced here one of them, xrep-1. The gene xrep-1 that encodes a nematode homologue of WDR23 negatively regulates a large number of phase II enzymes xrep genes while continuing isolation of new Xrep mutants to understand the xenobiotics response pathways and more generally the exogenous/endogenous stress response pathways and their regulation.In summary, with the aid of dpy-5(e61) I, CB1091 unc-13(e1091) I, CB120 unc-4(e120) II, CB364 dpy-18(e364) III, DP38 unc-119(ed3) III, CB138 unc-24(e138) IV, CB270 unc-42(e270) V, CB678 lon-2(e678) X, MJCU017 {unc-119(ed3) III, kIs17 X}{unc-119(ed3) III, kIs41 X}, and MJCU058 {unc-119(ed3) III, kIs15 IV}. NGM plates containing 500 mg/L (about 7 mM) acrylamide were prepared as described by Hasegawa et al. Nematode culturing and handling were carried out at 20°C as described by Brenner C. elegans unc-54 3′UTR region was obtained by cutting the region with EcoRI and SpeI from the gfp vector pPD95.77  and integrated into the equivalent restriction site of pDsRed-Monomer  for rfp (red fluorescence protein) vector pMH06.12-Red. PCR primers for gst-2, gst-4, and gst-30 were designed to amplify predicted promoters for each gene, about 1.2–0.8 kbp upstream from the predicted start sites spanning over full coding regions without the stop codons. PCR-amplified DNA fragments were digested with the appropriate restriction enzymes (gfp vector pPD95.77 (for gst-2::gfp and gst-30::gfp) or the rfp vector pMH06.12-Red (for gst-4::rfp). Each reporter construct (100 µg/mL) so obtained was co-injected with an equal concentration of pDP#MM016B into the gonadal arms of unc-119(ed3) adult hermaphrodites {unc-119(ed3) III; kEx13} and MJCU024 {unc-119(ed3) III; kEx24}. Each transgene's extrachromosomal array was chromosomally integrated by the method of Mitani {unc-119(ed3) III; kIs15 IV} and MJCU047 {unc-119(ed3) III; kIs41 X}. Linkage mapping with the conventional markers (listed in the sub-section Nematode culturing and strains) located the fusion gene gst-4::rfp, gst-2::gfp of MJCU058 integrated in the linkage group IV and, similarly, the fusion gene gst-30::gfp of MJCU047 in X (data not shown). By the standard genetic methods, xrep-1(k1007) was transferred into MJCU058 {unc-119(ed3) III; kIs15 IV} to construct MJCU059 {xrep-1(k1007) I; unc-119(ed3) III; kIs15 IV}. Fluorescence expression patterns were observed with a Nikon SMZ800 dissection microscope equipped with a fluorescence filter.To make reporter constructs, all PCRs were performed with KOD Plus DNA polymerase  on N2 genomic DNA. PCR primer sequences are listed in  enzymes  and ligaxrep-1. The allele xrep-1(k1007) was first mapped to linkage group I by using conventional markers, dpy-5(e61) I, unc-4(e120) II, dpy-18(e364) III, unc-24(e138) IV, unc-42(e270) V, lon-2(e678) X, and their positions were then narrowed by single-nucleotide polymorphism mapping We mutagenized the transgenic strains MJCU017 and MJCU047 with 50 mM ethyl methanesulfonate (EMS), following essentially the method described by Brenner gfp fusion genes, xrep-1(+)::gfp, xrep-1aProm acDNA::gfp, xrep-1bProm bcDNA::gfp, xrep-1aProm bcDNA::gfp, and xrep-1aProm acDNAEx4-11::gfp, were constructed. PCR primer sequences are listed in xrep-1 promoter  plus the deduced coding region was amplified with the primers XREP-1abp_HindIII_For and XREP-1ab_BamHI_Rev (gfp expression vector to obtain the xrep-1(+)::gfp construct. Two isoforms of xrep-1 cDNA, xrep-1a and xrep-1b, were each amplified with the primers XREP-1ap_cDNA_For and XREP-1ab_BamHI_Rev (xrep-1a cDNA) or XREP-1bp_cDNA_For and XREP-1ab_BamHI_Rev (xrep-1b cDNA) (xrep-1a or xrep-1b promoter was amplified with the primers XREP-1abp_HindIII_For and XREP-1ap_cDNA_Rev (xrep-1a promoter) or XREP-1abp_HindIII_For and XREP-1bp_cDNA_Rev (xrep-1b promoter)   by PCR fromoter)  by PCR fonstruct . Next, xDNA::gfp .xrep-1a cDNA containing only exons 4 to 11 was amplified with the primers XREP-1ap_Exon4-11_For and XREP-1ab_BamHI_Rev (xrep-1a transcript. Again the xrep-1a promoter was amplified with primers XREP-1abp_HindIII_For and XREP-1ap_Exon4-11_Rev (xrep-1a cDNA exon 4 to 11 and ligated into the pPD95.77 gfp expression vector to obtain xrep-1aProm acDNAEx4-11::gfp (unc-119(ed3) or MJCU059 {xrep-1(k1007) I; unc-119(ed3) III; kIs15 IV} adult hermaphrodites as described {xrep-1(k1007) I; unc-119(ed3) III; kIs15 IV; kEx80}, MJCU081 {xrep-1(k1007) I; unc-119(ed3) III; kIs15 IV; kEx81}, MJCU085 {unc-119(ed3) III; kIs84}, MJCU086 {xrep-1(k1007) I; unc-119(ed3) III; kIs15 IV; kEx86}, MJCU087 {xrep-1(k1007) I; unc-119(ed3) III; kIs15 IV; kEx87}, MJCU088 {xrep-1(k1007) I; unc-119(ed3) III; kIs15 IV; kEx88}, MJCU089 {xrep-1(k1007) I; unc-119(ed3) III; kIs15 IV; kEx89}, MJCU091 {xrep-1(k1007) I; unc-119(ed3) III; kIs15 IV; kEx91}, MJCU094 {xrep-1(k1007) I; unc-119(ed3) III; kIs15 IV; kEx94}, MJCU097 {xrep-1(k1007) I; unc-119(ed3) III; kIs15 IV; kEx97}, and MJCU098 {xrep-1(k1007) I; unc-119(ed3) III; kIs15 IV; kEx98}.Furthermore, amHI_Rev  from the4-11_Rev  from the-11::gfp . One hunskn-1 cDNA, xrep-1 cDNAEx4-11, or gfp were prepared by PCR amplification of C. elegans N2 cDNA, genomic DNA, or plasmid vector pPD95.77, respectively, with primers . The PCR fragment-ligated plasmid or the blank vector pPD129.36 was used to transform E. coli HT115 Gene fragments of  primers . Each PCE. coli HT115 transformed with each different RNAi plasmid. The animals were then collected and transferred onto NGM plates with or without 500 mg/L acrylamide, seeded with each different E. coli HT115 RNAi bacteria. After 24 to 48 hours of incubation, the animals were observed for GFP expression with both a Nikon SMZ800 dissection microscope equipped with a fluorescence filter and a ZEISS Axiovert 200 microscope equipped with a confocal laser-scanning module.For RNAi experiments, synchronized L1-stage animals were first cultured for 48 hours at 20°C on NGM (containing 50 µg/mL ampicillin and 12.5 µg/mL tetracycline) plates seeded with Figure S1xrep-1). (a) MJCU017 animals treated with blank RNAi. No GST-4::GFP expression is detected . (b) MJCU017 animals treated with xrep-1(RNAi). GST-4::GFP expression is induced. Scale bar, 500 µm.GST-4 expression is induced when the transgenic MJCU017 animals are treated with soaking RNAi of D2020.9 ((1.29 MB TIF)Click here for additional data file.Figure S2xrep-1(k1007) mutation is prevented by skn-1(RNAi) except for that in the pharynx and body-wall muscle. (a-c) GST-4 expression patterns in MJCU017 treated with 500 mg/L acrylamide. (d-f) GST-4 expression patterns in xrep-1(k1007) without acrylamide. (g-i) GST-4 expression patterns of skn-1(RNAi) in xrep-1(k1007) without acrylamide. Scale bars, 50 µm.GST-4 expression induced by acrylamide and (1.33 MB TIF)Click here for additional data file.Figure S3{unc-119(ed3) III; kIs15 IV}. Without acrylamide, no fluorescence signal is detected. (b) Fluorescence image of MJCU058, treated with 500 mg/L of acrylamide at 20°C for 24 hours. RFP fluorescence signal is detected. (c) Fluorescence image of MJCU059 {xrep-1(k1007) I; unc-119(ed3) III; kIs15 IV}. RFP fluorescence signal is detected. (d-f) RFP fluorescence signal in the MJCU058 {unc-119(ed3) III; kIs15 IV} animal, treated with 500 mg/L of acrylamide at 20°C for 24 hours. (d) Head. (e) Vulva. (f) Tail. Scale bars, a-c, 500 µm; d-f, 50 µm.(a) Fluorescence plus bright field images of MJCU058 (2.14 MB TIF)Click here for additional data file.Figure S4xrep-1::gfp fusion genes. (a-c) XREP-1::GFP expression. (d-f) XREP-1aProm acDNA::GFP expression. (g-i) XREP-1bProm bcDNA::GFP expression patterns. (j-l) XREP-1aProm bcDNA::GFP expression. (m-o) XREP-1aProm acDNAEx4-11::GFP expression. Scale bars, 50 µm.Expression patterns of various (1.79 MB TIF)Click here for additional data file.Figure S5{unc-119(ed3) III; kIs17 X}, MJCU028 {unc-119(ed3) III; kEx28} (23), MJCU003 {kEx3} (11), MJCU047 {unc-119(ed3) III; kIs41 X}, and MJCU050 {unc-119(ed3) III; kIs20 III} (21). Control, without xenobiotics; MeHg (500 nM Methylmercury); Paraquat (20 mM Paraquat); tBOOH (1 mM tert-Butyl hydroperoxide); AA (7 mM Acrylamide). (a) GST-4::GFP expression was not detected. Arrows indicate animals. (b) GST-4::GFP expression was induced when animals were treated with MeHg. (c) GST-4::GFP expression was slightly induced (arrows) when animals were treated with paraquat. (d) GST-4::GFP expression was induced when animals were treated with tBOOH. (f) Weak GSTO-2B::CFP expression was detected. (g-i) GSTO-2B::CFP expression was induced when animals were treated with MeHg, paraquat, and tBOOH. (k) Weak GST-7::GFP expression was detected. (l-n) GST-7::GFP expression was induced when animals were treated with MeHg, paraquat, and tBOOH. (p) GST-30::GFP expression was not detected. Arrows indicate animals. (q) GST-30::GFP expression was induced when animals were treated with MeHg (arrows). (r) GST-30::GFP expression was not induced when animals (arrows) were treated with paraquat. (s) GST-30::GFP expression was induced when animals were treated with tBOOH (arrows). (u) Weak UGT-13::GFP expression was detected. (v-x) UGT-13::GFP expression was induced when animals were treated with MeHg, paraquat, and tBOOH.  All of the GST- and UGT-fused GFP expressions were strongly induced when animals were treated with AA (11). Scale bars, 500 µm.GST and UGT responses in transgenic animals against several xenobiotics. Young adult transgenics were transferred into NGM plates containing each xenobiotic and incubated at 25°C for 24 hours. Transgenic animals used in these experiments were MJCU017 (1.64 MB TIF)Click here for additional data file.Table S1gst::reporter fusion genes.Primers for (0.02 MB DOC)Click here for additional data file.Table S2xrep-1::reporter fusion genes.Primers for (0.02 MB DOC)Click here for additional data file.Table S3Primers for RNAi.(0.02 MB DOC)Click here for additional data file.


An outbreak of aseptic meningitis occurred in Tianshui city of Gansu Province, the People's Republic of China, from March to June 2005. A total of 85 patients were clinical confirmed as aseptic meningitis in this outbreak.CVA9 was mainly responsible for this outbreak supported by the clinical manifestations of the patients, epidemiological data of the outbreak, the results of RT-PCR and complete VP1 sequence determination, conventional neutralization assays, IgM serological assays, viral isolation and phylogenetics analysis. Through phylogenetic analysis and homogeneity analysis for partial VP1 gene, the nucleotide and amino acid homologies between Gansu isolates and former Chinese CVA9 strains were 88.2%-96.1% and 97.2%-99.2%, respectively. Multiple transmission chains of CVA9 occurred in different provinces or years in China. Moreover, in order to clarify the genotype of CVA9, Gansu CVA9 strains isolated in this outbreak were compared with other CVA9 isolates based on VP1/2A junction regions (genotyping region) and they might belong to a new genotype of CVA9, which could be assigned for genotype XIII,CVA9 was confirmed as the pathogen responsible for this outbreak. The phylogenetic analysis indicated that the CVA9 strains isolated in this outbreak might belong to a new genotype. Human Enteroviruses (HEV) comprise more than 80 distinct serotypes within the Picornaviridae family, and are currently classified into five species: Poliovirus and HEV-A, HEV-B, HEV-C, and HEV-D . Most inIn China, the repeated outbreaks of HEV-associated diseases occurred in recent years, and these patients were reported to have an aseptic meningitis symptom were usually caused by HEV-B ,8. Here,In this outbreak, the clinical specimens were collected from 22 out of 85 patients during Apirl to June. Like other 63 cases, these 22 patients came from Maiji district of Tianshui city were hospitalized cases and confirmed as aseptic meningitis when these patients were admitted to the hospital. So it clearly showed the epidemiological linkage between 22 patients and 63 other patients.10 cerebrospinal fluids (CSF), 22 stool samples and 22 serum samples were collected from 22 patients with clinical confirmed aseptic meningitis during acute-phase infection after onset; 3 patients were also found to have convalescent sera. A total of 32 etiology specimens and 25 sera obtained during this outbreak were stored at -80°C and -20°C, respectively, for further analysis.Viral RNA was directly extracted from the clinical specimens using a QIAamp Mini Vial RNA Extraction Kit . 10 CSF specimens and 22 stool samples from 22 patients was performed with direct reverse transcription-polymerase chain reaction (RT-PCR) by the pan-enterovirus primer pair including primer PE1 and PE2 as previously described .10 CSF specimens and 22 stool samples were separately inoculated into human rhabdomyosarcoma (RD) cell lines and were cultured in a maintenance medium at 36°C. Cultures that exhibited a characteristic enterovius cytopathic effect (CPE) were identified by RT-PCR and sequencing with the following method. If no CPE was observed in the cultures, these were further cultured for another 7 days. Viral RNA was extracted from culture with CPE by using the QIAamp mini viral RNA extraction kit . The entire VP1 gene was amplified by RT-PCR with the primer pairs including the previous reported primer pair 008 and 011 [Though entire VP1 region were obtained for serotyping of human enterovirus, few sequences were available at GenBank, so it is difficult to analyze the genetical linkage of sequences between China and other countries. In order to clarify the genotype of CVA9, the genotyping region (VP1/2A region) was also amplified with the previous reported primer pair named primer VP+ and 2A- . The RT-The sequence data were aligned, edited, and assembled to obtain the entire VP1 gene and VP1/2A region using Sequencher version 4.0.5 . Sequence alignments were created with ClustalX software 1.81, the pairwise sequence identities of the nucleotide and deduced amino acid sequences were calculated with BioEdit software 7.0. The genetic distances were analyzed with Kimura 2-parameter method of MEGA 4.0 program, and neighbor-joining of phylogenetic analysis were used to provide a more reliable inference of phylogeny with MEGA 4.0 program.22 acute-phase sera collected from the clinical confirmed cases within 2-29 days after onset were detected Coxsackievirus immunoglobulin M (IgM) antibodies using an Serion ELISA classic Coxsackievirus IgM test kits . This kit is quantitative and qualitative tests for detection of human antibodies in serum or plasma against all serotypes of Coxsackievirus.50/50 μl) was calculated according to the Kärber formula [In this study, according to the methods previously described , convent formula .GQ294574 and GQ294575, respectively.The nucleotide sequence of the complete VP1 gene and VP1/2A region for representative strain Gansu05-1/GS/CHN/2005, which was determined in this study, has been deposited in the GenBank nucleotide sequence database under accession number D00627), CVA12(AF081302), CVA16(U05876), CVA19(AF081308), CVA24(AF081311), CVB1(M16560), CVB2(AF081312), CVB3(M16572), CVB4(D00149), CVB5(X67706), CVB6(AF081313), E1(AF081314), E2(AF081315), E3(AF081316), E4(AF081317), E5(AF081320), E6 (AF081321), E7(AF081324), E9(X92886), E11(X80059), E12(X79047), E13(AF081327), E14(AF081328), E15(AF081329), E16(X89545), E17(AF081330), E18(AF081331), E19(AF081332), E20(AF081333), E21(AF081334), E24(AF081335), E25(AF081336), E26(AF081337), E27(AF081338), E29(AF081339), E30(AF081340), E31(AF081344), E32(AF081345), E33(AF081346), EV68(AF081348). GenBank accession numbers for each CVA9 were shown as follows: Griggs(D00627), 06.109.3344(FJ868282), FJ00-127(AY573578), FJ98-90(AY573577), 78-97(AB268124), 49-98(AB268123), 148-00(AB268121), 03-171FCR2(AB167980), 03-144NPC3(AB167979), 03-132NPR2(AB167978), YZ047/SD/CHN/2005/CA9(GQ246517), 04318/SD/CHN/2004/CA9(GQ329731), 01332/SD/CHN/2001/CA9(GQ329730), 00365/SD/CHN/2000/CA9(GQ329729), 97186/SD/CHN/1997/CA9(GQ329728), 97089/SD/CHN/1997/CA9(GQ329727), TR081056-05(AM236967), Can-1-85 (AF166250), Fin-2-95(AF166251), Net-1-79(AF166252), Net-1-64(AF166253), Net-1-81(AF166249), Fin-1-88(AF166248), Den-1-71(AF166247), Net-1-59(AF166246), Net-1-63(AF166245), Net-1-67(AF166244), Net-1-61(AF166243), Mex-1-75(AF166242), Hon-1-77(AF166241), US-CO-1-74(AF166240), US-MD-1-88(AF166239), Can-1-86(AF166238), US-NC-1-83(AF166237), Net-1-71(AF166236), Net-1-78(AF166235), Thai-1-93(AF166234), Net-1-73(AF166233), US-MS-1-78(AF166232), Net-2-79(AF166231), Fin-2-83(AF166230), Fin-1-83(AF166229), Fin-2-97(AF166228), Fin-1-97(AF166227), Fin-1-96(AF166226), Fin-2-96(AF166225), Fin-3-96(AF166224), Fin-2-94(AF166223), Fin-1-94(AF166222), Fin-1-95(AF166221), Fin-2-93(AF166220), Fin-1-93(AF166219).GenBank accession numbers for each human enterovirus were shown as follows: CVA9 , headache (67/85) and vomiting (56/85). The incidence of the relevant cases peaked in late May to early June. All patients were less than 13 years old, and a majority of these patients were 4-5 years old. Numbers of Gregarious children's cases (from kindergarten and primary school) were more than those of scattered children. All patients had good prognosis, and no fatal case was found in this outbreak.The specimens collected from 22 patients were identified by pan-entero RT-PCR, virus isolation/molecular typing, IgM ELISA and NT, and the results were showed in the table 4 CSF sample and 12 stool samples (including 3 CSF samples and 3 stool samples detected from 3 patients) were successfully amplified with pan-enteroviruse primer pair. It indicated the enteroviruse infection responsible for this outbreak.Among them, 8 samples including 2 CSF and 6 stool samples were positive for virus isolation/VP1 RT-PCR, and the other 8 samples were negative and only positive for pan-enteroviruse directly RT-PCR, this maybe due to the lower titer of the virus in the clinical samples. As for the viral isolation, 2 CSF and 6 stool samples out of 32 samples (including one CSF and one stool sample from one patient) were observed with the characteristic enterovirus CPE, which appeared within two passages after inoculation, and the entire VP1 was successfully amplified to obtain the predicted products of 906 bp.Sequence determination revealed that eight viral isolates exhibited 99.9% to 100% homology in their nucleic acid sequences. Therefore, the data showed that all the patients were infected with the similar virus. A viral isolate designated as strain Gansu05-1/GS/CHN/2005 was used for phylogenetic analysis. Furthermore, phylogenetic tree was conducted on the basis of the entire VP1 sequences of human enterovirus prototype strains indicated that the sequences was very close to the prototype strain of CVA9 (strain Griggs)  of former Chinese CVA9 strains was available in the GenBank database. Thus, in order to determine the molecular epidemiology of CVA9 in China, the phylogenetic dendrogram  with 37 CVA9 strains (Figure In this study, the RGD motif within the C-terminal extension of the VP1 capsid protein of CAV9  was alsoEnteroviruses represent the main etiological agents of epidemics of viral meningitis and especially the serotypes related to the HEV-B species, and more than 50% of meningitis cases were due to echovirus 30 . In 2005CVA9 is a member of the A subgroup which exhibits the typical pathogenicity in newborn mice , while tThe genetic heterogeneity of enteroviruses makes it difficult to develop molecular typing that is both reliable and easy to perform. The genomic sequence encoding the VP1 capsid protein gives excellent results because of the high correlation between serotype and genetic information, and the availability of a complete database of reference strains isolated in the 1950's and 1960's. In this study, the pathogen was identified as CVA9 based on the complete VP1 gene. Through phylogenetic analysis and homogeneity analysis for partial VP1 gene, the strain Gansu05-1/GS/CHN/2005 shared highly homology with strains 97186/SD/CHN/1997/CA9 from Shandong province, belonging to different lineages with other 11 Chinese CVA9 strains. It indicated that there were multiple transmissions chains of CVA9 circulation in different provinces in China, and the similar virus were also found in Japan.et al. divided CVA9 strains into 12 genotypes using the criteria designated for PVs that strains sharing >85% identity in the VP1/2A junction region belong to the same genotype [However, it is difficult for systematic genotyping based on VP1 gene because few complete CVA9 VP1 sequences available in the world. Santti genotype . By compIn recent years, CVA9 was also isolated in many other countries including Spain, Finland, Canada, Netherland and United States etc through routine diagnosis for clinical samples from patients suspected viral central nervous system infection ,19. In aIn conclusion, this is the first reporting the outbreak of aseptic meningitis caused by CVA9 in China, and a new genotype of CVA9 was found based on the sequences of VP1/2A region. However, further analysis is needed to investigate the evolution of the CVA9 by obtained more sequences of virus strains and to find out their genetic relationships, and molecular epidemiological surveillance of CVA9 should also be strengthened.An outbreak of aseptic meningitis occurred in Tianshui city of Gansu Province, the People's Republic of China in 2005. In this study, CVA9 was confirmed as the pathogen responsible for this outbreak. The phylogenetic analysis indicated that the CVA9 strains isolated in this outbreak might belong to a new genotype.CVA9: Coxsackievirus A9; RT-PCR: reverse transcription-polymerase chain reaction; CSF: cerebrospinal fluid; HEV: Human Enteroviruses; CPE: cytopathic effect; IgM: immunoglobulin M.The authors declare that they have no competing interests.ALC, ZZ, WBX prepared manuscript. WBX designed the study and organized the coordination. ALC performed data analysis. ALC, DSY, ZZ, LM, HL, JFL, GYL collected specimens and performed RT-PCR, virus isolation, viral identification, Coxsackievirus IgM assays and neutralization test. All authors read and approved the final manuscript.


The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART. RNA silencing (RNAi) is a new gene regulatory mechanism conserved from plants to humans. RNAi mediators are small non-coding RNAs (sncRNAs) that function through sequence specific mRNA targeting to either induce their degradation and/or inhibit translation ,2. In maTo investigate whether RNAi effectors regulate HIV-1 replication, we analyzed virus replication in cells where expression of RNAi effectors was reduced using specific siRNA. HeLa cells were transfected with siRNA specific to RCK/p54, GW182, LSm-1 or XRN1. As controls, HeLa cells were transfected with scrambled siRNA (Scr) or CDK9 specific siRNA and subsequently infected with HIV-1 Figure . KnockdoWe next investigated the physical interaction between RNAi effectors and HIV-1 mRNA. 293 cells were mock transfected or transfected with combinations of pNL4-3, Myc-Ago2, a central component of the RISC complex, or its RNA-binding mutant Myc-Ago2PAZ9 constructs as indicated in figures Emerging evidence suggests the physical and functional interactions between P-bodies and the viral life cycles . Viral mvif gene (pNL4-3Δvif) either alone or with wild-type A3G or A3G mutant lacking antiviral activity (A3Gdm). HIV-1 p24 antigen was measured in culture supernatant 48 hours post-transfection. Interestingly, knock down of RCK/p54 or LSm-1 enhanced HIV-1 production regardless of A3G . APOBEC3G H65R/H257R mutant was previously described . HIV-1 vPBMCs were transfected with siRNA or miRNA using the Nucleofector II Device with the appropriate Nucleofection solution according to the manufacturer's instructions (Amaxa). siRNA corresponding to DGCR8 (5'-CAUCGGACAAGAGUGUGAU(dTdT)-3'), Drosha (5'-CGAGUAGGCUUCGUGACUU(dTdT)-3'), RCK/p54 (5'-GCAGAAACCCUAUGAGAUUUU(dTdT)-3'), LSm-1 (5'-GUGACAUCCUGCCACCUCACUU(dTdT)-3'), GW182 (5'-UAGCGGACCAGACAUUUCU(dTdT)-3'), XRN1 (5'-AGA UGA ACU UAC CGU AGA A(dTdT)-3') and CDK9 (5'-CCAAAGCUUCCCCCUAUAATT(dTdT)-3') were synthesized (MWG). Expression level of knock down proteins was analyzed by Western blotting 48 hours after transfection. Briefly, cell-extracts were resolved on SDS-PAGE gels. Proteins were transferred to PVDF membrane by semi-dry electroblotting and probed overnight at 4°C with the primary antibody , DGCR8 (Proteintech Group), anti-RCK/p54 (Bethyl)or anti-CDK9 (Santa Cruz), washed and incubated with the appropriate secondary antibody (Amersham) for 1 hour. Proteins were visualized by chemiluminescence according to the manufacturer's protocol (Pierce).6 cells/ml) were co-cultured with pre-activated PBMC (106 cells/ml) from the same healthy donor in the presence of 10 U/ml IL-2. The culture medium was collected every 3 or 4 days. Fresh pre-activated healthy PBMCs were added to the culture every 7 days. Viral production was measured by quantifying the amounts of p24 in the culture medium using an ELISA kit (Ingen).Peripheral blood mononuclear cells of HIV-1 infected patients were isolated by lymphocyte separation medium density centrifugation (Lonza). PBMCs from healthy donors were pre-activated using 5 μg/ml PHA /10 U/ml IL-2  for 72 hours. They were then washed once with PBS and once with RPMI medium before co-culture assay. siRNA transfected HIV-infected PBMC (10-Luc+ harboring a luciferase gene (obtained from the NIAID AIDS Reagent Program) was co-transfected with the envelope plasmid pMD.2G encoding the G protein of vesicular stomatitis virus (VSV.G) into human embryonic kidney cells-293T. The virions, named HIV-1VSV-Luc, were collected and filtered using 0.45 μm filters 48 hours post-transfection. HeLa or HeLa CD4+ cells were infected over-night at 37°C, washed and resuspended in DMEM containing 10% FCS. Virus production was monitored in culture supernatant by measuring p24 antigen (Ingen) and by following luciferase activity according to the manufacturer's instructions (Promega).The plasmid pNL4-3-env2, 0.5% NP40 and protease inhibitor). Nuclei were pelleted by centrifugation for 10 minutes at 10,000 rpm. 2 mg of cytoplasmic extracts were loaded on a 7–47% sucrose gradient. Briefly, 5 layers of 7 to 47% sucrose were prepared in sucrose buffer  and diffused at 4°C for 16 hours to obtain a linear sucrose gradient. 2 mg of cytoplasmic extracts were loaded on the top of the column, and centrifuged for 3 hours at 36,000 rpm in a SW41Ti rotor. After ultracentrifugation, 28 fractions were collected and OD at 254 nm was measured in each fraction using a Nanodrop apparatus (Labtech).To isolate cytoplasmic extracts, cells were lysed for 10 minutes in buffer B  containing RNASIN (Promega) and 1 mM DTT, and centrifuged to pellet debris. Supernatants were incubated overnight with mouse anti-Myc mAb 9E10 (Amersham) at 4°C followed by 2 hours incubation with protein G-Sepharose. Immunoprecipitates were washed with RIP buffer, and nucleic acids were extracted with phenol/chloroform/isoamyl alcohol, isopropanol-precipitated, ethanol-washed and resuspended in RNase-free water. Total RNA was DNase I treated and reverse-transcribed using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). RT products were PCR-amplified using either GAPDH , HIV-1 , or HDM2 specific oligonucleotides . PCR products were resolved on 1.5% agarose/TAE gels containing ethidium bromide. In vivo splicing assay and oligonucleotides BSS and SJ4.7A have been described [293 cells were grown in 60 mm dishes and transfected with the indicated plasmids using calcium-phosphate. Cells were harvested 48 hours after transfection, lysed for 15 minutes in RIP buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 2.5 mM MgClescribed The authors declare that they have no competing interest.CBC, OL and LD carried out most experiments. TR initiated and performed the experiments shown in figures


Drosophila has proved useful for the genetic analysis of insulin functions, little is known about the transcription factors and cell lineages involved in insulin production. Within the embryonic central nervous system, the MP2 neuroblast divides once to generate a dMP2 neuron that initially functions as a pioneer, guiding the axons of other later-born embryonic neurons. Later during development, dMP2 neurons in anterior segments undergo apoptosis but their posterior counterparts persist. We show here that surviving posterior dMP2 neurons no longer function in axonal scaffolding but differentiate into neuroendocrine cells that express insulin-like peptide 7 (Ilp7) and innervate the hindgut. We find that the postmitotic transition from pioneer to insulin-producing neuron is a multistep process requiring retrograde bone morphogenetic protein (BMP) signalling and four transcription factors: Abdominal-B, Hb9, Fork Head, and Dimmed. These five inputs contribute in a partially overlapping manner to combinatorial codes for dMP2 apoptosis, survival, and insulinergic differentiation. Ectopic reconstitution of this code is sufficient to activate Ilp7 expression in other postmitotic neurons. These studies reveal striking similarities between the transcription factors regulating insulin expression in insect neurons and mammalian pancreatic β-cells.Insulin and related peptides play important and conserved functions in growth and metabolism. Although  Drosophila have provided many new insights into the functions of insulin and related peptides. It has, however, been more difficult to use Drosophila to study the regulation of insulin, at least in part because the relevant insulinergic cell lineages were not well characterised. Here, we have identified a cell lineage that generates a single Drosophila insulin-producing neuron. This neuron first functions as a pioneer, guiding the axons of other neurons within the central nervous system of the embryo. It then develops long axons that exit the central nervous system to innervate the gut and also begins to express an insulin-like peptide. Genetic analysis identifies four transcription factors and one extrinsic signal that instruct the pioneer neuron to become an insulin-producing neuron. The analysis also reveals similarities between the genetic programmes specifying insulin production by Drosophila neurons and mammalian pancreatic ß-cells. This suggests that Drosophila may, in the future, prove a useful model system for identifying new regulators of human insulin production.Genetic studies using invertebrate model organisms such as  A genetic analysis in the fruit fly reveals similarities between the transcriptional programmes regulating insulin production in mammalian pancreatic β-cells and insect neurons. Insulin, a key regulator of carbohydrate and lipid metabolism, is secreted by the ß-cells of the pancreas . DeficieDrosophila, seven differentially expressed Ilps have been identified as orthologues of mammalian insulins and/or insulin-like growth factors , w, wDrosop mutants B, Mad mu mutants E and S3F mutants , and upoIlp7 RNA A–S3F. Ce mutants , and S6.Drosophila orthologue of vertebrate Mist1, is an important regulator of neuroendocrine cell differentiation . W. Wrpr an isoform D. Ilp7 R isoform K. This iion 87%, K but neiion 87%, L nor Ubxion 87%, M are ablAbd-B, rpr, skl mutants that lacks expression of any Hox genes. dMP2 generation and early specification in A8 occur normally, as revealed by their expression of Odd, Fkh, and Hb9 , indicating that all three Ilp7 regulators contribute in a combinatorial manner to activate Ilp7. Interestingly, combinatorial misexpression of Dimm, Fkh, and Abd-B in motor neurons did not lead to any detectable ectopic expression of Ilp2, an insulin-like peptide known to be expressed in the brain mNSCs H99 mutant embryos, where apoptosis is blocked, ectopic Hb9-positive RP motor neurons are observed in segments A7–A8 [Drosophila and vertebrates [Drosophila, together with the neurotrophic requirement for motor neuron survival in vertebrates, raises the possibility that the same genetic programmes specifying the identities of motor neurons also sensitise them for postmitotic editing via apoptosis.Apoptosis of postmitotic neurons is a widespread feature of normal VNC development ,40, but tebrates ,51. Our rpr/skl but not grim expression. Some support for this idea comes from the observation that loss of hb9 activity blocks rpr/skl-mediated death of dMP2 neurons but not the largely grim-dependent apoptosis of anterior MP1 neurons (unpublished data).Hb9 and Fkh expression in many neurons that do not die suggests a combinatorial mechanism for the control of developmental apoptosis. One possibility is that several transcription factors function in combination to activate the core pro-apoptotic genes. Given the proposed role for Foxa proteins in chromatin accessibility , Fkh exp+ Hb9+ code into an insulinergic Fkh+ Hb9+ Abd-B+ code.An important conclusion from this study is that the combinatorial transcription factor code controlling apoptosis partially overlaps with that regulating insulinergic identity (see next section). Thus, Fkh and Hb9 are both essential components of the codes for anterior apoptosis and also Ilp7 expression, illustrating that these transcription factors play surprising dual roles as pro-apoptotic and pro-differentiation factors within the same neuronal subtype. Importantly, our results also show that the segment-specific Hox protein Abd-B acts as a postmitotic switch, converting the pro-apoptotic FkhDrosophila midline and its vertebrate counterpart, the floor plate [Three Ilp7 regulators  are expressed at least 12 h before Ilp7 is first activated: from the time when the MP2 neuroblast exits the cell cycle. In the case of Hb9, we were able to uncouple two temporally separable functions. Early postmitotic expression of Hb9 is important for its death-activating function, whereas later expression suffices for activating Ilp7. Similarly, the Hox protein Abd-B generates a segment-specific neuropeptide pattern via postmitotic regulation of posterior dMP2 survival and also Ilp7 activation. As vertebrate neuropeptides are also expressed in restricted neuronal populations within specific rostrocaudal domains ,54, theyor plate ,58, Fkh The other two dMP2 regulators identified in this study, Dimm and the BMP pathway, are switched on shortly before the onset of Ilp7 expression. The timing of onset of these two broad neuroendocrine regulators is likely to specify when Ilp7 is first activated, whereas the earlier factors Fkh, Hb9, and Abd-B may contribute more specifically to insulinergic identity. Together, the genetic and expression analyses in this study demonstrate that the combinatorial code of genetic inputs required for Ilp7 expression is assembled in a step-wise manner during postmitotic maturation . ImportaTwo observations from this study indicate that insulinergic combinatorial codes can vary from cell-to-cell and also from one Ilp to another. First, none of the regulators of Ilp7 in dMP2 neurons appear to regulate it in DP neurons. Second, the dMP2 insulinergic code is sufficient to trigger ectopic expression of Ilp7 but not Ilp2 or other neuropeptides such as FMRFa. These findings suggest the existence of additional, as yet unidentified, insulinergic factors in DP neurons and also in the brain mNSCs where Ilp2 is expressed. Recent identification of the neural progenitor for these mNSCs  should fNgn3 to prospective endocrine cells [Pdx-1, following its early input into pancreatic induction [Our finding that an Ilp7-expressing neuron derives from the MP2 lineage reveals that at least some insulinergic regulators are similar in insects and mammals. Three apparent similarities may not be very insulin-specific but reflect more general processes shared by neural and endocrine programmes in many species. First, Notch signalling singles out the MP2 neuroblast and distinguishes its two progeny neurons, while in mammals, it limits pancreatic expression of the “proneural” gene ne cells . Second,ne cells  or via anduction ,60. And nduction , while induction .fkh in dMP2 neurosecretory differentiation described in this study is similar to the functions of HNF3b/Foxa2 in islet maturation and insulin secretion [Drosophila orthologue of Nkx2.2, Vnd, is required for dMP2 formation [Nkx6 (FlyBase name HGTX), is expressed by postmitotic dMP2 neurons [Several more specific regulatory similarities exist between the insulinergic differentiation factors active in postmitotic dMP2 neurons  and pancecretion ,64. In aecretion  and is kormation ,66. Dros neurons , and it  neurons –70. TogedMP2-GAL4, UAS-nls-myc-EGFP (referred to as UAS-nmEGFP), M5Abd-B, UAS-Antennapedia, UAS-Ubx, UAS-abd-A, UAS-Abd-Bm (referred to as UAS-Abd-B), Df(3L)XR38 and Df(3L)H99 deficiencies  [A12wit, B11wit, AUAS-tkv, AUAS-sax, DNUAS-Glued, 1gbb [rev4dimm, P1dimm, UAS-dimm [KK30hb9, JJ154hb9 [UAS-hb9 [6fkh [10Mad, 12Mad [CY27-GAL4 [UAS-fkh [GAL4, UAS-GFPodd [UAS-CD8-GFP [UAS-hid, UAS-rpr [OK371-GAL4 [Ilp2-GAL4 [CyO,Act-GFP or TM3,Ser,Act-GFP balancer chromosomes. y w or 1118w or heterozygous mutant stocks were typically used as control. Crosses were maintained at 25 °C, except for OK371 crosses, which were kept at 21 °C.The following fly stocks were used: ctively) ; witA12,DN, gbb1 ; dimmrevUAS-dimm ; hb9KK30[UAS-hb9 ; fkh6 [7b9 [fkh6 ; Mad10, 0, Mad12 ; CY27-GAY27-GAL4 ; UAS-fkh[UAS-fkh ; oddGAL4 UAS-GFP ; UAS-CD8-CD8-GFP ; UAS-hid UAS-rpr ; OK371-G371-GAL4 ; and Ilplp2-GAL4 . MutantsImmunolabelling was carried out as previously described . AntibodAn antibody to Ilp7 was generated in rabbits against the synthetic peptide RERSQSDWENVWHQET. This sequence is within the predicted B chain and corresponds to amino acids 41–57 of the predicted Ilp7 protein. Peptide synthesis, antibody generation, and affinity purification were done by Eurogentec.Details of a second antibody directed against the predicted A chain are available in dimm, wit, gbb mutants, and rescues  labels the same neuronal types as the anti-Ilp7 antibody, thus confirming its specificity.(C) dMP2 neurons express Ilp7 RNA, as revealed by in situ/immunofluorescence double labelling of first-instar larvae carrying a dMP2 reporter line.(D) Ilp7 peptide sequence, with predicted signal peptide, B, C, and A chains. Optimal predicted cleavage sites, as identified in , are in (E–F′) Detection of mature Ilp7 B chain (E and E′) or A chain (F and F′) in or in close proximity to dMP2 terminals (green) on the visceral muscle of the hindgut (blue). See y w; (C) dMP2-GAL4,UASnGFP/+; (E–F') GAL,,UAS-GFP/UAS-CD8-GFPodd.Genotypes:  (1.7 MB TIF)Click here for additional data file.Figure S2Ilp2-GAL4 reporter line expressed in the insulin-producing mNSCs shows Ilp7-positive projections extending close to the mNSC cell bodies.(A) Expression of nuclear GFP from an Ilp2-Gal4 reveals apparent association between Ilp7- and Ilp2-positive neurites.(B) Expression of a membrane-localised GFP driven by Ilp2-GAL4/UAS-nmEGFP; UAS-nmEGFP/+ and (B) Ilp2-GAL4, UAS-CD8-GFP/+.Genotypes: (A) (723 KB TIF)Click here for additional data file.Figure S3wit (B), gbb (D), and Mad (F) mutants, and reduced in dimm mutants (H). Each of these panels is preceded by a wild-type CNS processed in parallel to illustrate the reduction in levels . Ilp7 is absent from fkh mutant dMP2s (I) and often absent from hb9 mutant dMP2s (J). In Abd-Bm, rpr, skl mutants lacking Abd-B expression in A8 (arrow), no Ilp7 RNA is detected in this segment.The effect on Ilp7 expression observed for all the mutants used in this study occurs at the transcriptional level. Ilp7 RNA is reduced or absent in A12/+wit; (B) A12/witB11wit; (C) 1/+gbb; (D) 1/gbb1gbb; (E) 10/+Mad; (F) 10/Mad12Mad; (G) P1/+dimm; (H) P1/dimmrev4dimm; (I) 6/fkh6fkh; (J) KK30/hb9JJ154hb9; and (K) M5,XR38/AbdBM5,ED225AbdB.Genotypes: (A) (2.2 MB TIF)Click here for additional data file.Figure S4(A) Wild type expression of Ilp7 in a first-instar CNS.sax mutants.(B) Ilp7 peptide is reduced or absent in dMP2 neurons of y w and (B) 4/Df(2R)NCX8sax.Genotypes: (A) (790 KB TIF)Click here for additional data file.Figure S5wit (A), gbb (B), and dimm (C) mutants. Only posterior dMP2 neurons persist, as revealed by their expression of the dMP2 reporter .At the end of embryogenesis, anterior dMP2 neurons have undergone apoptosis in DN in dMP2 and MP1 neurons with GAL4odd does not affect their segmental apoptosis. At the end of embryogenesis, dMP2 neurons are apparent in A6 to A9, whereas MP1 neurons  are present in A5 to A9.(D) Expression of GluedA12/witB11UAS-CD8-GFP/+; dMP2-GAL4,wit; (B) 1/gbb1gbb; UAS-CD8-GFP/dMP2-GAL4; (C) P1/dimmrev4; UAS-CD8-GFP/dMP2-GAL4dimm; and (D) GAL4,UAS-GFP/UAS-GluedDN.oddGenotypes: (A) (324 KB TIF)Click here for additional data file.Figure S6wit mutants, dMP2 neurons express early markers in every segment, such as Odd, Fkh, and Hb9 (A–C).In Posterior dMP2 neurons express Abd-B (D).gbb mutants (H–N).In late embryos, posterior dMP2 neurons express Dimm (E), exit the VNC in the correct nerve (F), and innervate the hindgut (G). The same results were obtained for A12/witB11UAS-CD8-GFP/+; dMP2-GAL4,wit; (H–N) 1/gbb1gbb; UAS-CD8-GFP/dMP2-GAL4.Genotypes: (A–G) (2.9 MB TIF)Click here for additional data file.Figure S7(A) A wild-type first-instar CNS shows Ilp7 expression in three lateral pairs of TV neurons and one single pair of suboesophageal neurons.(B) No ectopic FMRFa is observed when the Ilp7 code is misexpressed in motor neurons.y w and (B) OK371-GAL4/UAS-Abd-B; UAS-dimm,UAS-fkh.Genotypes: (A) (567 KB TIF)Click here for additional data file.Figure S8Cartoon summarizing the dual role of Hb9 as a pro-apoptotic and pro-insulinergic factor, the effects of Fkh on all aspects of the transition from pioneer to neuroendocrine identity, and the anti-apoptotic and pro-Ilp7 functions of Abd-B. See text for details.(1.1 MB TIF)Click here for additional data file.Text S1(33 KB DOC)Click here for additional data file.


C. elegans to the aversive odorant 1-octanol. C. elegans lacking the CAT-2 tyrosine hydroxylase enzyme, which is required for dopamine biosynthesis, are hypersensitive in their behavioral avoidance of dilute concentrations of octanol. Dopamine can also modulate the context-dependent response of C. elegans lacking RGS-3 function, a negative regulator of Gα signaling. rgs-3 mutant animals are defective in their avoidance of 100% octanol when they are assayed in the absence of food , but their response is restored when they are assayed in the presence of food or exogenous dopamine. However, it is not known which receptor might be mediating dopamine's effects on octanol avoidance. Herein we describe a role for the C. elegans D2-like receptor DOP-3 in the regulation of olfactory sensitivity. We show that DOP-3 is required for the ability of food and exogenous dopamine to rescue the octanol avoidance defect of rgs-3 mutant animals. In addition, otherwise wild-type animals lacking DOP-3 function are hypersensitive to dilute octanol, reminiscent of cat-2 mutants. Furthermore, we demonstrate that DOP-3 function in the ASH sensory neurons is sufficient to rescue the hypersensitivity of dop-3 mutant animals, while dop-3 RNAi knockdown in ASH results in octanol hypersensitivity. Taken together, our data suggest that dopaminergic signaling through DOP-3 normally acts to dampen ASH signaling and behavioral sensitivity to octanol.We previously found that dopamine signaling modulates the sensitivity of wild-type  With the possible exception of insects, olfaction is mediated by G protein-coupled signal transduction pathways across species C. elegans genome encodes >500 predicted functional chemosensory GPCRs and, as in other organisms, olfactory signaling in C. elegans is mediated by G protein-coupled signaling cascades The G protein-coupled receptor kinase) and RGS  proteins C. elegans to sensory stimuli that are detected by ASH To allow for appropriate cellular and organismal responses to these environmental stimuli, the level and duration of signaling through GPCRs must be precisely controlled. In the ASH neurons, this is accomplished in part by GRK  biosynthesis Dopamine (DA) and serotonin (5-HT) are believed to signal the presence of food for rgs-3 mutant animals rgs-3 encodes an RGS protein that functions in some C. elegans sensory neurons, including ASH rgs-3 mutants are defective in their responses to strong chemosensory and mechanosensory stimuli in the absence of food . C. elegans lacking RGS-3 function seem to have behavioral defects because increased signaling in the ASH sensory neurons ultimately leads to decreased glutamatergic signaling at the sensory/interneuron synapse 2+ transients in the ASH neurons rgs-3 behavioral defects rgs-3 mutants are significantly improved when assayed in the presence of either food or DA rgs-3 behavioral responses by activating an inhibitory dopaminergic pathway that dampens the increased signaling levels in ASH in the absence of RGS-3 function. Furthermore, as food rescues the rgs-3 behavioral deficit, yet signals the release of both DA and 5-HT, this suggests that endogenous DA signaling may override the effect of 5-HT on the ASH chemosensory signaling circuit. This is consistent with the observation that exogenous DA blocks the 5-HT-dependent increases in the octanol sensitivity of wild-type animals rgs-3 mutants.DA also affects the ASH-mediated responses of sα/Golfα to increase adenylate cyclase activity and cAMP levels in target cells, while stimulation of D2-like receptors couples to Giα/Goα subunits and leads to an inhibition of adenylate cyclase and a decrease in cAMP levels C. elegans, as in vertebrates, dopamine can activate G protein-coupled signaling pathways, and candidate receptors have not only been shown to bind the neurotransmitter, but also to neurotransmitter agonists and antagonists C. elegans behaviors, including food sensing, area restricted search, locomotion, egg-laying, defecation, state-dependent olfactory adaptation and habituation to non-localized mechanical stimulation (tap) Dopaminergic signaling is highly conserved across species. Dopamine receptors are generally grouped into two classes: D1-like receptors signal through GC. elegans genome encodes one D1-like DA receptor (DOP-1), two D2-like receptors (DOP-2 and DOP-3) and one invertebrate specific D1-like receptor (DOP-4) C. elegans DA receptors  resulted in hypersensitivity to dilute octanol rgs-3 animals, suggesting that in well fed animals endogenous DA signals through DOP-3 to modulate octanol behavioral sensitivity. We also show that loss of DOP-3 function in otherwise wild-type animals results in hypersensitivity to dilute octanol, suggesting that endogenous DA normally dampens octanol sensitivity via DOP-3. While DOP-3 transgene reporter expression was not observed in ASH, DOP-3 expression in ASH is sufficient to rescue the octanol hypersensitivity of dop-3 mutant animals, while loss of DOP-3 function in ASH leads to octanol hypersensitivity. Combined, we have uncovered a role for the dopamine receptor DOP-3 in the modulation of octanol sensitivity in C. elegans.The E. coli bacteria). However, rgs-3 animals respond significantly better when they are assayed in the presence of food or exogenous dopamine (DA) C. elegans genome encodes 4 putative DA GPCRs: DOP-1 (D1-like), DOP-2 (D2-like), DOP-3 (D2-like) and DOP-4 (invertebrate specific D1-like). To determine which DA receptor(s) might contribute to the food and DA rescue of rgs-3 octanol avoidance, rgs-3 animals lacking each of the DA receptors were assayed for octanol avoidance in the absence and presence of the bacterial food lawn. Each of the double mutants displayed defective octanol avoidance in the absence of food, taking ∼12 seconds to respond, similar to rgs-3 animals , this is similar to what is seen in tyrosine hydroxylase-deficient mice cat-2 mutant animals are hypersensitive and respond better than wild-type animals to dilute concentrations of octanol dop-2;dop-1dop-3 triple mutants) are hypersensitive to dilute octanol rgs-3 animals suggests that endogenous DA may signal through DOP-3 to regulate octanol sensitivity in wild-type animals. dop-3 single mutants were assayed off food for avoidance of dilute (30% and 10%) octanol. At both concentrations, dop-3 mutant animals responded better than wild-type animals  and isoamyl alcohol (AWC). A range of concentrations was tested for each odorant. In all cases, dop-3 animals were indistinguishable from wild-type animals  were crossed to animals carrying integrated transgenes marking each of the octanol-detecting neurons (osm-10::gfp), AWB (str-1::gfp) or ADL (gpa-15::gfp). Low-level expression was often seen in the ASK sensory neurons that do not detect octanol. Due to their exposed dendritic endings, the head sensory neurons ASH, AWB, ADL, ASJ, ASI and ASK take up lipophilic dyes that mark their cell bodies and projections  neurons . Surprisdop-3 mutant animals. The osm-10 promoter drives expression strongly in ASH and weakly in the ASI head neurons srb-6 promoter drives expression in the ASH, ADL and, to a lesser extent, ADF head neurons osm-10::dop-3 and srb-6::dop-3 transgenes dampened the hypersensitive response of dop-3 mutants to 30% octanol, so that the response of transgeneic animals to dilute octanol was similar to wild-type animals  in both the sense and antisense (sa) orientations in the ASH neurons of otherwise wild-type animals. dop-3 knock-down using either promoter resulted in hypersensitive responses to 30% octanol, similar to dop-3(vs106) animals  faster than wild-type animals C. elegans nervous system consists of just 302 neurons and the physical positions and synaptic connectivity of all of the neurons are known C. elegans an excellent system in which to identify and functionally characterize molecular mechanisms that underlie neuronal signal transduction and regulation.In both vertebrates and invertebrates, biogenic amines contribute to multiple forms of behavioral plasticity ranging from learning and memory to sensitization and tolerance in drug addiction C. elegans navigate their natural soil environment, they encounter sensory signals of varying strengths. In addition, their behavioral responses to these cues are context and experience dependent C. elegans utilize biogenic amines to modulate aversive chemosensory responses as well as olfactory adaptation to attractive chemical cues As C. elegans hermaphrodites have eight dopaminergic neurons that are believed to release DA in response to mechanical stimulation, such as from moving through a bacterial food source C. elegansC. elegans nervous system is known, it does not allow for direct prediction of the site of DA function for a given behavior. Therefore, understanding the contribution of individual receptors and where they are functioning should prove useful in understanding how DA modulates specific behaviors.C. elegans locomotion behaviors rgs-3 mutant animals. In addition, animals lacking DOP-3 function are hypersensitive to dilute octanol, further suggesting that DOP-3 mediates the inhibitory effects of endogenous DA on chemosensory signaling.While DOP-3 has been shown to affect cat-2 and dop-3 mutants are hypersensitive to dilute (30%) octanol, theoretically DA could normally dampen chemosensory response in wild-type animals by acting on either the chemosensory neurons or the interneurons. However, because DA rescues the responses of rgs-3 mutant animals, which have behavioral defects due to enhanced signaling in the chemosensory neurons but decreased glutamatergic signaling at the chemosensory/interneuron synapse dop-3 in ASH (using the same two promoters) results in octanol hypersensitivity similar to dop-3(vs106) loss-of-function animals. Taken together, our data suggest that endogenous DOP-3 likely acts to dampen chemosensory signaling in the ASH sensory neurons that directly detect octanol.As the receptors for biogenic amines sometimes function in cells besides ASH to modulate ASH-mediated behavioral responses iα/Goα C. elegans ASH sensory neurons appear to use polyunsaturated fatty acids (PUFAs) instead of cAMP as second messengers C. elegans.This is consistent with DOP-3 belonging to the D2-like receptor class, which generally leads to decreased adenylate cyclase activity via GC. elegans response to octanol C. elegans locomotion, they have antagonistic effects on egg laying C. elegans should continue to advance our understanding of biogenic amine function and the interaction between modulatory pathways that regulate signaling to ultimately control animal behavior.Biogenic amines can interact in complex ways to ultimately regulate cellular and whole animal responses. For example, although DA, TA and OA all seem to counteract 5-HT in the regulation of E. coli bacteria rgs-3(vs19), LX636 dop-1(vs101), LX702 dop-2(vs105), LX703 dop-3(vs106), FG58 dop-4(tm1392), LX705 dop-1(vs100)dop-3(vs106), FG25 rgs-3(vs19);dop-1(vs101), FG27 rgs-3(vs19);dop-2(vs105), FG29 rgs-3(vs19);dop-3(vs106), FG81 rgs-3(vs19);dop-4(tm1392), FG86 rgs-3(vs19);dop-1(vs100)dop-3(vs106), CB1112 cat-2(e1112), HA1739 rgs-3(vs19);cat-2(e1112), FG83 vsIs33[dop-3::rfp], FG94 vsIs33[dop-3::rfp];rtIs27[osm-10::gfp], FG100 vsIs33[dop-3::rfp];kyIs104[str-1::gfp], FG101 vsIs33[dop-3::rfp];pkIs591[dpy-20(+) + gpa-15::gfp], FG157 dop-3(vs106);udEx7[osm-10::dop-3], FG158 dop-3(vs106);udEx8[osm-10::dop-3], FG161 dop-3(vs106);udEx9[srb-6::dop-3] and FG162 dop-3(vs106);udEx10[srb-6::dop-3], FG196 udEx43[osm-10::dop-3(sense + antisense)], FG197 udEx44[osm-10::dop-3(sense + antisense)], FG194 udEx41[srb-6::dop-3(sense + antisense)], FG195 udEx42[srb-6::dop-3(sense + antisense)].Strains were maintained under standard conditions on NGM agar plates seeded with OP50 osm-10::dop-3(genomic): The unc-47 promoter was removed from pCL35 unc-47::dop-3(genomic) osm-10 upstream promoter region was isolated from CR142 dop-3 genomic clone in the remaining fragment of pCL35.pFG11 srb-6::dop-3(genomic): The ∼1.3 kb srb-6 promoter was first isolated from pHA#355 srb-6 promoter was then removed from pFG10 using SphI and BamHI to be inserted into the SphI/BamHI sites upstream of the dop-3 genomic clone (remaining fragment of pCL35) as described above.pFG12 dop-3 rescue experiments 75 ng/µl of pJM67 elt-2::gfp plasmid osm-10::dop-3(genomic) or pFG12 srb-6::dop-3(genomic). For cell-specific RNAi transgenic experiments 25 ng/µl of pJM67 elt-2::gfp plasmid osm-10::dop-3(sense) and osm-10::dop-3(antisense) or srb-6::dop-3(sense) and srb-6::dop-3(antisense). Exons 6–9 (∼840 bp) of dop-3 were amplified from genomic DNA to generate the sense and antisense fragments; no functional protein should be made from this internal region. Primer sequences are available upon request.Germline transformations were performed as previously described Cell-specific RNAi knock-down experiments were performed as previously described E. coli bacteria (“on food”). As dopamine (DA) is unstable in the presence of salt, 6 mM DA plates were made by spreading 60 µl of a freshly made 1 M stock (dissolved in water) on the surface of a 10 ml NGM plate. The DA was allowed to soak into the plate and equilibrate for 5 minutes prior to the assay, as previously described Well-fed young adult animals were used for analysis, and all behavioral assays were performed on at least two separate days, along with controls. Behavioral assays were performed as previously described vsIs33, which encodes dop-3::rfp, were crossed to animals carrying integrated transgenes marking selected sensory neurons. ASH was marked by rtIs27 (osm-10::gfp), AWB was marked by kyIs104 (str-1::gfp), and ADL and ASK were marked by pkIs591 (gpa-15::gfp). pkIs591 is also expressed in ASH, which is not visible in the focal plane shown in dop-3::rfp (vsIs33) expressing animals were incubated with the lipophilic dye DiO , as previously described Animals carrying the integrated transgene Figure S1vsIs33 were incubated with DiO, shown in green, to mark the cell bodies and projections of these neurons. DOP-3::RFP expression was not seen in ASH, AWB, ADL, ASJ or ASI. Weak DOP-3::RFP expression was often observed in ASK. Scale bar  = 20 µm.DOP-3::RFP is not expressed in the sensory neurons that detect octanol. Six head sensory neurons  take up lipophilic dyes via their exposed sensory endings (10.18 MB TIF)Click here for additional data file.


We determined the prevalence of Shiga toxin-producing Escherichia coli (STEC) in diarrheal stool samples from Nebraska by three methods: cefixime-tellurite sorbitol MacConkey (CT- SMAC) culture, enterohemorrhagic E. coli (EHEC) enzyme immunoassay, and stx1,2 polymerase chain reaction (PCR). Fourteen (4.2%) of 335 specimens were positive by at least one method . Six contained serogroup O157, while non-O157 were as prevalent as O157 serogroups.


We attempted to induce anti-tumour immunity for rejecting pre-B lymphoma derived from E mu/ret transgenic mice (TGM). We established pre-B-lymphoma cell lines of C57BL/6 x Balb/c background (H-2b/d) into which H-2k alloantigen and C3H background were introduced (retL1-6 and retL6-6), and we inoculated BCF1 mice with these immunising tumour cells. After these tumours were rejected by alloantigen (H-2k/C3H background)-specific effector cells, the mice were challenged with the pre-B-lymphoma cell line derived from the original E mu/ret TGM (ret0-2). All non-immunised control mice died within 80 days, whereas half the immunised mice survived for over 300 days. The immunity was also effective against primary pre-B-lymphoma cells from E mu/ret TGM and the ret-driven melanoma cell line (MEL-ret), but not against the pre-B-lymphoma cell line from E mu/myc TGM. This immunity was at least in part mediated by cell-mediated cytotoxicity that was specific to the ret oncogene product or ret-regulated antigen. Next we immunised E mu/ret TGM by inoculating them with retL6-6 cells once every 2 weeks beginning at the age of 1 month. Interestingly, this immunisation enabled the TGM to survive longer than the non-immunised control group (P < 0.05). Moreover, 2 of 11 transgenic mice receiving such immunisation were free from both macroscopic and microscopic tumours at the time when all of the 12 non-immunised control TGM had died from their tumour. This provides a new model for oncogene-linked immunotherapy research.


Aliphatic silicon derivativeswere considerably less active. 8-[(Trimethylsilylmethyl)thio]quinoline (8a) exhibits the highest activityamong quinoline sulfides.Silicon containing pyridine and quinoline sulfides have been prepared using phase transfer catalyticsystem thiol/alkyl halide / solid KOH/18-crown-6 / toluene. The target S-ethers were isolated in yields up to81%. The cytotoxicity of the synthesized compounds was studied. Among pyridine sulfides S-[3-(1-methyl-1-silacyclohexyl)propyl] derivatives


WAF1/CIP1 expression at transcriptional, post-transcriptional as well as translational levels. We previously found that cell growth suppressing retinoid CD437 enhanced expression of p21WAF1/CIP1 and DNA damage inducible GADD45 proteins in part by elevating their mRNA stability.A variety of pathways target CDKI p21WAF1/CIP1 expression. By utilizing MDA-MB-468 HBC cells expressing chimeric rabbit β-globin-p21WAF1/CIP1 transcripts we mapped multiple CD437-responsive sequences located within positions 1195 to 1795 of the 3'-untranslated region of p21WAF1/CIP1 mRNA. Several cytoplasmic proteins present in MDA-MB-468, MCF-7 HBC as well as HL-60R leukemia cells bound specifically, in vitro, with these CD437-responsive sequences. CD437 treatment of cells resulted in elevated binding of ~85 kD and ~55 kD cytoplasmic proteins with putative CD437-responsive sequences. A 12 nt RNA sequence (5'-UGUGGUGGCACA-3') present within CD437-responsive region of p21WAF1/CIP1 mRNA displayed specific and elevated binding with the above noted proteins. Treatment of cells with ActD or CHX prior to CD437 exposure did not abrogate RNA-protein interactions. However, treatment of cytoplasmic protein extracts with proteinase K or alkaline phosphatase resulted in loss of RNA-protein interactions.Here, we investigated molecular mechanisms of CD437-dependent post-transcriptional regulation of p21WAF1/CIP1 mRNA that involves specific RNA-protein interactions that are phosphorylation-dependent, while not requiring nascent transcription or protein synthesis.CD437 regulates cell growth in part by regulating stability of p21 WAF1/CIP1, an important cell-cycle regulatory molecule, was originally identified as a gene regulated by the tumor suppressor protein p53 UTP , [α-32P]CTP  or a combination of both. The 12 nt RNA oligonucleotide (5'-UGUGGUGGCACA-3') was labelled using T4 polynucleotide kinase and [γ32P]ATP  as described [Plasmids pBKS-WAF (581-2110) and pBKS-GADD (10-55) were linearized with NotI and HindIII, respectively, and the T methods . In addiescribed . Radiolaescribed . The rad2, 1.25 mM EDTA, 1.25 mM DTT, 155 mM KCl and 10% glycerol in a total reaction volume of 9 μl. In some cases, proteinase K , calf intestinal alkaline phosphatase , or 2 μl of ~100 ng/μl unlabelled sense strand RNAs were included in the pre-incubation step. The specific competitor RNAs consisted either a160 nt long RNA derived from plasmid pBKS-WAF (1540-1700) or an ~1500 nt RNA derived from plasmid pBKS-WAF (581-2110). The nonspecific competitor RNA consisted of an ~400 nt 3'-UTR subfragment of c-myc mRNA containing an additional 100 nt poly A sequences derived from in vitro transcription of HindIII-digested plasmid pMycSD3, essentially as described [HBC and leukemia cell cytoplasmic as well as nuclear protein extracts were prepared according to procedures described before . HBC cel-WAF 581-10. The nCD437: ; HBC: Human Breast Carcinoma; CDKI: Cyclin-dependent Kinase Inhibitor; ActD: Actinomycin D; CHX: Cycloheximide; ER: Estrogen Receptor; UTR: Untranslated region; FBS: Fetal bovine serum; PCR: Polymerase chain reaction; nt: nucleotides; bp: base pairs; Kb: kilobase pairs; kD: kiloDalton.The authors declare that they have no competing interests.LZ and AKR carried out the majority of experiments including cloning of chimeric RBG constructs, RNA preparations, half-life determinations, northern blots, and analysis of RNA-protein interactions. AW, MID and JAF participated in the design and discussion of various experiments. AKR is the corresponding author responsible for experimental design and coordination, as well as writing of the manuscript and formatting of all the figures. All authors read and approved the final manuscript.


Despite intensive study of the mechanisms of chemotherapeutic drug resistance in human breast cancer, few reports have systematically investigated the mechanisms that underlie resistance to the chemotherapy-sensitizing agent tumor necrosis factor (TNF)-α. Additionally, the relationship between TNF-α resistance mediated by MEK5/Erk5 signaling and epithelial-mesenchymal transition (EMT), a process associated with promotion of invasion, metastasis, and recurrence in breast cancer, has not previously been investigated.To compare differences in the proteome of the TNF-α resistant MCF-7 breast cancer cell line MCF-7-MEK5  and its parental TNF-a sensitive MCF-7 cell line MCF-7-VEC, two-dimensional gel electrophoresis and high performance capillary liquid chromatography coupled with tandem mass spectrometry approaches were used. Differential protein expression was verified at the transcriptional level using RT-PCR assays. An EMT phenotype was confirmed using immunofluorescence staining and gene expression analyses. A short hairpin RNA strategy targeting Erk5 was utilized to investigate the requirement for the MEK/Erk5 pathway in EMT.Proteomic analyses and PCR assays were used to identify and confirm differential expression of proteins. In MCF-7-MEK5 versus MCF-7-VEC cells, vimentin (VIM), glutathione-S-transferase P (GSTP1), and creatine kinase B-type (CKB) were upregulated, and keratin 8 (KRT8), keratin 19 (KRT19) and glutathione-S-transferase Mu 3 (GSTM3) were downregulated. Morphology and immunofluorescence staining for E-cadherin and vimentin revealed an EMT phenotype in the MCF-7-MEK5 cells. Furthermore, EMT regulatory genes SNAI2 (slug), ZEB1 (δ-EF1), and N-cadherin (CDH2) were upregulated, whereas E-cadherin (CDH1) was downregulated in MCF-7-MEK5 cells versus MCF-7-VEC cells. RNA interference targeting of Erk5 reversed MEK5-mediated EMT gene expression.vim, GSTP1/gstp1, and CKB/ckb; and downregulation of KRT8/krt8, KRT19/krt19, and GSTM3/gstm3). We further demonstrate that MEK5-mediated progression to an EMT phenotype is dependent upon intact Erk5 and associated with upregulation of SNAI2 and ZEB1 expression.This study demonstrates that MEK5 over-expression promotes a TNF-α resistance phenotype associated with distinct proteomic changes (upregulation of VIM/ The method of Bradford  was used to detect the protein concentrations, and the samples were stored at -70°C for further analyses.A total of 1 × 10First dimensional electrophoresis was performed using a Protean iso-electronic focus cell unit (BioRad). Precast 11 cm immobilized pH gradient (IPG) strips with pH ranges 5 to 8 were obtained from BioRad. Lysates were thawed and mixed (3:1) with rehydration solution . Two hundred microliters (approximately 180 μg) of protein sample mixture was loaded to each of the IPG strips. The strips were rehydrated with the sample mixture and overlaid with a layer of mineral oil overnight (16 hours). Iso-electric focusing was carried out in three steps under gradient mode: 250 V for 20 minutes, linear ramp; 8,000 V for 2.5 hours, linear ramp; and 8,000 V, rapid ramp to reach 20,000 V-hours.The second dimensional electrophoresis was carried out in a BioRad Criterion electrophoresis cell system. IPG strips were equilibrated in two steps: 20 minutes in 60 mmol/l DTT and 20 minutes in 200 mmol/l of iodoacetamide; both were dissolved in equilibration buffer . IPG strips were sealed on the top of SDS gels (Criterion 8% to 16% Tris-HCl Gel obtained from BioRad) with 0.5% agarose. SDS-PAGE was performed at a constant voltage of 200 V for 55 minutes. Gels were washed with 200 ml de-ionized water three times for 5 minutes each. Bio-safe staining solution  was added to each gel, and the gels were then placed on a shaker for 2 hours. The staining solution was discarded and the stained gels were rinsed with de-ionized water . Stained gels were scanned with a Gel Doc-XR image system (BioRad) and analyzed with the PDQuest software (version 8.01) . The proteins of interest were marked for excision. The spots were excised from the gels by a Quest Spot cutter (BioRad), and digested with trypsin  in 25 mmol/l ammonium bicarbonate overnight at 37°C to release tryptic peptides using a Progest digestion system . Recovered peptides were dried in a Labconco NewCentriVap concentrator .n system , equipped with a Finnigan Micromass AS automatic sampling system. A BioBasic C18 PicoFrit Column  was used for separation. The mobile phases consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile, at a linear gradient from 0% B to 35% B within 35 minutes, along with a gradient from 35% B to 98% B within 8 minutes, kept at 98% B for 2 minutes and then back to 0% B within 2 minutes. The total acquisition time was 60 minutes for each run. The tandem mass spectrometry spectra were analyzed against the ipi.human.v3.27 database using SEQUEST software , and the results were tabulated for each identified protein.The dried protein digests from the 96-well plates were analyzed using an LC-Nanospray-MS® mini kit . The reverse transcription was carried out with a SuperScript first-strand synthesis system (Invitrogen) using Oligo(dT)12–18 primers or with iscript™ cDNA synthesis kit . The primer pairs used to amplify the genes vim (vimentin), krt8 (keratin 8), krt19, hspa4 (HSPA4), gstp1 (glutathione-S-transferase P), gstm3 (glutathione-S-transferase Mu 3) and ckb (creatine kinase B-type), and the genes involved in EMT including cdh1 (E-cadherin), ctnnb1 (β-catenin), snail, slug and δ-ef1 (δ-EF1) were designed using the online tool of Oligo Perfect Designer (Invitrogen), and the endogenous actb (β-actin) was employed as an internal standard. The primer pairs are provided in the additional materials -1-propanesulfonate; CKB: creatine kinase B-type; 2-DE: two-dimensional electrophoresis; DTT: dithiothreitol; EMT: epithelial-mesenchymal transition; Erk: extracellular signal-regulated kinase; GSTM3: glutathione-S-transferase Mu 3; GSTP1: glutathione-S-transferase P; HSP: heat shock protein; IPG: immobilized pH gradient; KRT: keratin; MAPK: mitogen-activated protein kinase; MEK: mitogen-activated protein kinase kinase; PBS: phosphate-buffered saline; RT-PCR: reverse transcription polymerase chain reaction; shRNA: short hairpin RNA; VIM: vimentin; ZEB1: δ-EF1.The authors declare that they have no competing interests.CZ performed 2-DE and RT-PCR, interpreted data, and drafted the manuscript. AMN performed quantitative PCR for EMT gene expression and drafted the manuscript. WX performed RT-PCR. QZ carried out high performance liquid chromatography-tandem mass spectrometry based protein identification and database search. YT performed Western blot analysis. MB conducted RT-PCR analysis. SE generated the MEK5 stable cells and performed colony assays. YZ designed, generated, and validated pSUPER-shRNA-Erk5. LB conducted quantitative PCR analysis of gene expression. DY and CBW conducted RT-PCR and colony assays. RS, JAM, BSB, TEW, and KPN participated in experimental design and interpretation. BS performed immunofluorescence. MEB and GW co-designed the study, directed research efforts, and critically revised the manuscript.A Word file containing a table listing the primer sequences used in all RT-PCR experiments.Click here for fileA Word file containing a table listing all identified proteins that were differentially expressed in MCF-7-MEK5 versus MCF-7-VEC breast cancer cells.Click here for file


However, Bcl-2 overexpression was less effective at preventing cell death that occurred after photoactivation at high levels (50–100 ng ml−1) compared with lower doses (10–25 ng ml−1) of BPD-MA. These results indicate that caspase 3 and 6 activation and their regulation by Bcl-2 may play important roles in photodynamic therapy (PDT)-induced cell killing. © 1999 Cancer Research CampaignTreatment with the photosensitizer benzoporphyrin derivative monoacid ring A  followed by irradiation with visible light induces apoptosis in human acute myelogenous leukaemia HL-60 cells. Photoactivation of BPD-MA induces procaspase 3 (CPP32/Yama/apopain) and procaspase 6 (Mch2) cleavage into their proteolytically active subunits in these cells. The Bcl-2 proto-oncogene product has been shown to protect cells from a number of proapoptotic stimuli. In the present study, the influence of Bcl-2 overexpression on cellular resistance to photoactivation of BPD-MA was studied. Overexpression of Bcl-2 in HL-60 cells prevented apoptosis-related events including caspase 3 and 6 activation, poly(ADP-ribose) polymerase cleavage and the formation of hypodiploid DNA produced by BPD-MA (0–200 ng ml


Tumour necrosis factor-alpha  reduced the interstitial fluid pressure (IFP) to 54-64% (P < 0.05) and the mean arterial blood pressure (MABP) to 70% (P < 0.01) of control values after 5 h in three human melanoma tumour lines transplanted to nude mice.


A square planar copper complex of derivatized NSAID drug (Ketoprofenthiosemicarbazone [3-benzoyl-α-methyl benzene acetic acid thiosemicarbazone]), is characterizedby elemental analysis, spectroscopy, electrochemistry and magnetic susceptibility studies whichexhibits dose-dependent and enhanced antiproliferative effects on human breast cancer cell lineT47D rich in progesterone receptors.


It is concluded that leukotrienes do not mediate either the direct or priming effects of these cytokines and that these classes of anti-inflammatory drugs are therefore unlikely to inhibit the effects of IL-8 and GM-CSF on neutrophil activation.The cytokines interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced the extracellular release of arachidonate metabolites from ionophore-stimulated neutrophils by 145 ± 10%  and 182 ± 11% (n = 16), respectively. To determine whether enhanced leukotriene production mediates the effects of these cytokines on neutrophil activity, two different specific arachidonate 5-lipoxygenase (5-LO) inhibitors, piriprost and MK-886, were used to inhibit leukotriene synthesis. Neither inhibitor affected the upregulation of CD11b β


The crystal structure of complex (1) has been determined by single crystal X-ray diffraction analysis at 173(1) K. Compound (1) contains two triphenyltin moieties linkedby a doubly de-protonated 2,mercaptonicotinic acid (H>2mna). It is an example of a pentacoordinatedPh3SnXY system with an axial-equatorial arrangement of the phenyl groups at Sn(1). Compound (1), exhibitspotent, in vitro, cytotoxicity against sarcoma cancer cells  from the Wistar rat,polycyclic aromatic hydrocarbons  carcinogenesis.A novel triphenyltin(IV) derivative of 2-mercaptonicotinic acid (H


In order to elucidate how Hepatitis C Virus (HCV) interacts with polarized hepatocytes in vivo and how HCV-induced alterations in cellular function contribute to HCV-associated liver disease, a more physiologically relevant hepatocyte culture model is needed. As such, NASA-engineered three-dimensional (3-D) rotating wall vessel (RWV) bioreactors were used in effort to promote differentiation of HCV-permissive Huh7 hepatoma cells.When cultured in the RWV, Huh7 cells became morphologically and transcriptionally distinct from more standard Huh7 two-dimensional (2-D) monolayers. Specifically, RWV-cultured Huh7 cells formed complex, multilayered 3-D aggregates in which Phase I and Phase II xenobiotic drug metabolism genes, as well as hepatocyte-specific transcripts , were upregulated compared to 2-D cultured Huh7 cells. Immunofluorescence analysis revealed that these HCV-permissive 3-D cultured Huh7 cells were more polarized than their 2D counterparts with the expression of HCV receptors, cell adhesion and tight junction markers  significantly increased and exhibiting apical, lateral and/or basolateral localization.These findings show that when cultured in 3-D, Huh7 cells acquire a more differentiated hepatocyte-like phenotype. Importantly, we show that these 3D cultures are highly permissive for HCV infection, thus providing an opportunity to study HCV entry and the effects of HCV infection on host cell function in a more physiologically relevant cell culture system. Hepatitis C virus (HCV), a liver tropic positive-stranded RNA flavivirus, infects ~170 million people worldwide, causing acute and chronic hepatitis and hepatocellular carcinoma -4, allowThe NASA-engineered RWV is a horizontally rotating cylindrical culture vessel which reduces shear and turbulence associated with conventional stirred bioreactors; therefore, it simulates aspects of microgravity similar to the environment encountered during fetal development -14. In cIn the current study we demonstrate that RWV-cultured Huh7 cells formed complex, multilayered, 3-D aggregates that exhibited up-regulation of metabolic and hepatocyte-specific transcripts as well as increased expression and re-localization of tight junction, cell adhesion, and polarity markers. Importantly, these aggregates remained highly permissive for HCV infection suggesting that hepatic polarity does not limit HCV entry in 3-D-cultured Huh7 cells. As such, RWV-cultured Huh7 cells may represent a more appropriate physiologically relevant system for further in vitro studies of HCV entry and infection dynamics.6 Huh7 cells were trypisinized, incubated with 250 mg Cytodex-3 microcarrier beads  for 30 minutes at room temperature in a total volume of 30 ml complete DMEM. Cell-bead complexes were introduced into the RWV bioreactor  at a ratio of 20 cells/bead, transferred to 37°C, and vessel rotation was initiated at 20 rotations per minute. Medium was replenished every 24 h and rotation speed was increased as aggregates developed to maintain cells in free-falling suspension.Huh7 cells . In parallel, Huh7 2-D monolayers were seeded in 8-well chamber slides at 80% confluence and fixed 48 hours post seeding. 3-D aggregates and 2-D monolayer cells were stained with antibodies specific for scavenger receptor class B member 1 (SR-BI) , CD81 , CLDN1 , CD26 , β-Catenin , E-cadherin (Zymed), zona occludens 1 (ZO-1) (Zymed), Occludin (Zymed) or HCV E2 . Alternately, 3-D aggregates were embedded in OCT freezing medium  or paraffin, sectioned and stained with Hoechst dye or Hematoxylin and Eosin (H&E), respectively.Immunofluorescence analysis of aggregates was performed as previously described . BrieflyV E2 (C1 ) overnigHuh7 3-D aggregates were infected with JFH-1 HCVcc at an MOI of 0.01 FFU/cell at day 1, 7 or 14 post RWV seeding by injection of the viral inoculum directly into the RWV. At indicated times post infection (p.i.), medium was harvested for titration analysis and RNA was isolated from ~0.5 ml of aggregates for reverse transcription followed by RTqPCR as described above.Culture supernatants were serially diluted 10-fold and used to infect triplicate Huh7 cultures in 96-well plates. At 24 h p.i., cultures were overlayed with complete DMEM containing 0.4% methylcellulose  to give a final concentration of 0.25% methylcellulose. Seventy-two hours p.i., cells were fixed in 4% paraformaldehyde (Sigma), and immunohistochemically stained for HCV E2 using the anti-HCV E2 antibody C1 . Viral tTo assess the utility of the RWV as a culture method for Huh7 cells, Huh7 cells were cultured on Cytodex-3 microcarrier beads in the RWV for 26 days. Morphological and cytological examination of cultures demonstrated that Huh7 cells efficiently adhered to the collagen-coated microcarrier beads and that these individual beads then assembled to form 3-D tissue-like aggregates containing ~10–20 beads per aggregate Fig. . To deteOne measure of hepatocyte differentiation is up-regulation of expression of transcription factors such as hepatocyte nuclear factors (HNF) ,33, whicWhile the effect of HCV on cell polarity and TJs (and vice-versa) cannot be accurately studied in 2-D monolayer Huh7 cultures , these iAs expected, the cell adhesion molecules E-Cadherin and β-Catenin were membrane localized both in 2-D and 3-D Huh7 cultures; however, there was a profound decrease in the accumulation of nuclear β-Catenin-containing complexes in the 3-D Huh7 aggregates. Because atypical nuclear localization of β-Catenin in transformed cells has been well documented , the losBecause it has been suggested that hepatocyte polarization is inversely related to the permissiveness of the cell for HCVcc infection ,11, we sHere we demonstrate that Huh7 cells cultured in RWV bioreactors form multi-layered tissue-like aggregates that are phenotypically distinct from traditional Huh7 2-D monolayers Fig.  and 2. SRelevant to the study of HCV, expression of the HCV receptors CD81 and SR-B1 were both diffuse and poorly organized in 2-D cultured Huh7s cells, while their expression was increased and localized to apical TJ regions and basolateral-sinusoidal surfaces in 3-D aggregates. Likewise, TJ proteins, which typically localize to the apical surface in polarized hepatocytes , were moTo date, attempts to study HCV in polarized cells have been limited to colorectal adenocarcinoma Caco-2 cells  or HepG2Growing evidence suggests interplay between TJ protein expression, localization and function and HCV infection. Although, the current HCV infectious 2-D Huh7 cell culture system does not amend itself well to elucidating these dynamic relationships, the highly HCV-permissive 3-D Huh7 cell culture system described herein more closely mimics the differentiated and polarized state of hepatocytes. As such the RWV 3-D Huh7 cell culture system should prove useful for understanding the dynamic relationship between HCV and TJ protein expression as well as elucidating how HCV interacts with and disrupts key aspects of hepatocyte physiology.HCV: hepatitis C virus; JFH-1: Japanese Fulminant Hepatitis; RWV: rotating wall vessel; 3-D: three dimensional; 2-D: two-dimensional; HCVcc: hepatitis C virus cell-cultured produced; MOI: multiplicity of infection; FFU: focus forming units; RTqPCR: real-time quantitative PCR; SR-B1: scavenger receptor class B member 1; CLDN1: claudin-1; ZO-1: zona occludens 1; H&E: hematoxylin and eosin; p.i.: post infection; HNF: hepatocyte nuclear factors; α1AT: alpha-1-antitrypisn; TTR: transthyretin; CYP: cytochrome P450s; UGT: UDP-glucuronosyltransferase; TJ: tight junction.The authors declare that they have no competing interests.BS and VT participated in the design of the study, performed the experiments and drafted the manuscript. SLU designed the study and participated in drafting the manuscript. All authors read and approved the final manuscript.


A total of 237 breast carcinomas have been studied with the Courtenay-Mills (C-M) soft agar method. Cell yields and plating efficiencies (PE) were recorded after various enzyme treatments. The highest cell yields and PEs were obtained with the combination of collagenase 0.5%, hyaluronidase 1000 IE ml-1 and DNase 0.1% and an incubation time of 2 h. Eighty percent of the specimens gave greater than 10 colonies, and 60% formed greater than 30 colonies permitting chemosensitivity studies. The C-M method gave significantly higher PEs than the Hamburger-Salmon (H-S) method. Hormone supplements  and also reduced agar concentrations (less than 0.3%) gave marginal stimulation of colony formation. In chemosensitivity studies involving doxorubicin, vincristine and 4-OOH-cyclophosphamide, the C-M method gave dose-response relationships without plateaus.


The effect of idoxifene, a novel anti-oestrogen with less agonist activity than tamoxifen, was compared with that of tamoxifen on the growth of hormone-dependent MCF-7 breast cancer xenografts. Forty tumours were established with oestradiol support in ovariectomized athymic mice, allowed to grow to a median volume of 420 mm3 and then continued with oestradiol, no support, tamoxifen or idoxifene delivered by 1.5-cm silastic capsule. Tumour regression occurred with both anti-oestrogens, although maximum regression was observed following oestradiol withdrawal alone. While prolonged anti-oestrogen therapy was associated with static growth, tumour volumes were significantly lower with idoxifene (P=0.01). After 6 months, 0/10 idoxifene-treated tumours developed acquired resistance compared with 3/10 tumours treated with tamoxifen. In separate experiments, 94 animals were treated initially with oestradiol, tamoxifen, idoxifene or placebo following implantation with 1-mm3 pieces of either wild-type (WT) or tamoxifen-resistant (TR) MCF-7 tumour. After 4 months, only 1/11 WT tumours became established with idoxifene compared with 4/11 with tamoxifen, 8/12 with oestradiol and 0/12 with placebo. Likewise, fewer TR tumours were supported by idoxifene (3/12) than by tamoxifen (8/12) or oestrogen (11/12). These data indicate that, compared with tamoxifen, idoxifene shows reduced growth support of MCF-7 xenografts and may share only partial cross-resistance. Furthermore, the development of acquired anti-oestrogen resistance may be reduced during long-term idoxifene therapy. The drug's reduced agonist activity may, in part, explain these observations and indicate a preferable biochemical profile for breast cancer treatment.


P = 0.002). Three-year overall survival of patients with stable bcl2/IgH-negative conversion was 100% compared to 54% for the remaining patients (P = 0.069); 3-year freedom from progression was respectively 87.5% and 13% (P = 0.005). These results indicate a correlation between bcl-2/IgH expression variations and both clinical response and outcome. Whether this might predict disease outcome early remains to be confirmed. © 1999 Cancer Research CampaignBcl-2, the gene over-expressed in follicular lymphomas (FL), is able to block chemotherapy-induced apoptosis. Consequently, we wondered whether bcl-2/IgH expression variations during treatment of FL could predict the outcome of patients with t-bearing FL. For this purpose, we used a reverse transcription polymerase chain reaction (RT-PCR) assay to analyse 180 serial peripheral blood samples (PBS) during 34 treatment phases in 25 patients with t-bearing FL. In all patients but two, bcl-2/IgH gene expression was demonstrated in pre-treatment samples. During 16 out of the 34 treatment phases (47%), bcl-2/IgH expression became negative: all but one were responders to chemotherapy. This conversion was transient in six cases. In 18 treatment phases, bcl2/IgH expression remained detectable: eight were clinically considered as treatment failures, while eight others achieved PR and two achieved CR. We observed a significant correlation between treatment response and RNA PCR results (


We hypothesized that unexplained increases in nucleoside triphosphates (NTP) observed by 31P magnetic resonance spectroscopy (MRS) after treatment of tumours by DNA-damaging agents were related to chemotherapy-induced up-regulation of the bcl-2 gene and DNA damage prevention and repair processes. To test this hypothesis, we treated HT-29 cells with 10(-4) M nitrogen mustard (HN2) and performed sequential perchloric acid extractions in replicate over 0-18 h. By reference to an internal standard (methylene diphosphonic acid), absolute changes in 31P-detectable high-energy phosphates in these extracts were determined and correlated with changes in bcl-2 protein levels, cell viability, cell cycle, apoptosis and total cellular glutathione (GSH) . After HN2 administration, bcl-2 protein levels in the HT-29 cell line rose at 2 h. Cell viability declined to 25% within 18 h, but apoptosis measured using fluorescence techniques remained in the 1-4% range. Increased cell division was noted at 4 h. Two high-energy interconvertible phosphates, NTP (P < or = 0.006) and phosphocreatine (PCr) (P < or = 0.0002), increased at 2 h concurrently with increased levels of bcl-2 protein and glutathione. This study demonstrates that bcl-2 and glutathione are up-regulated by HN2 and links this to a previously unexplained 31P MRS phenomenon: increased NTP after chemotherapy.