SummaryWe investigated whether termination of transcripts with a self-cleaving ribozyme can enhance nuclear retention and serve as a tool to decrease speci®c plant gene expression. Nuclear retention was ®rst monitored in tobacco using the b-glucuronidase gene terminated with either the 35S CaMV 3¢ untranslated sequence (UTR) or a cis-acting ribozyme. Northern blot analysis of nuclear RNA and total RNA, and in situ hybridizations showed that the ribozyme-terminated transcripts were preferentially retained in the nucleus of transgenic tobacco. Ribozyme-terminated transcripts were subsequently tested as a gene down-regulation strategy in soybean. The embryo-speci®c D-12 fatty acid desaturase FAD2-1 gene was targeted because its down-regulation elevates oleic acid content of seed storage lipids. Both ribozyme-terminated antisense and standard antisense constructs were capable of gene downregulation, producing over 57% oleic acid compared with less than 18% in wild-type seed. Ribozyme termination cassettes were also constructed to evaluate sense transcripts for single gene downregulation and the simultaneous down-regulation of two embryo-speci®c genes in soybean using a single promoter. Eight independent soybean transformants were screened that harboured standard plus sense or ribozyme terminated FAD2-1 cassette. Two of the eight ribozyme terminated transformants displayed oleic acids levels in the seed storage lipids of over 75%, while none of the standard plus sense FAD2-1 lines showed elevated oleic acid phenotypes. The dual constructs targeted FAD2-1 and the FatB gene encoding a palmitoyl-thioesterase. Five transgenic soybean lines harbouring the dual constructs had oleic acid levels, greater than 85%, and saturated fatty acids levels, less than 6%. Thus, ribozyme termination of transcripts can be utilized to speci®cally down-regulate endogenous gene expression in soybean.

Abstract. Light microscopic and ultrastructural changes were observed in chicks challenged with North American Serpulina pilosicoli, a weakly 6-hemolytic intestinal spirochete (WBHIS) associated with human and canine intestinal spirochetosis. Chicks in control groups received trypticase soy broth or canine Serpulina innocens. The birds were necropsied at weekly intervals, and the ceca were processed for bacteriologic and pathologic examinations. No WBHIS were isolated from the ceca of chicks in the control groups, but WBHIS with genotypes similar to the parent isolates were isolated from the ceca of chicks inoculated with human and canine S. pilosicoli. Gross examination revealed no significant changes in the ceca of chicks at any time postinoculation. Light microscopic examination revealed no spirochetal attachment in the ceca of chicks in control groups. In contrast, focal to diffuse thickening of the brush border of the surface epithelium along with dilation of the crypt lumina and mild focal lamina propria heterophil infiltration were present in the ceca of chicks inoculated with human and canine S. pilosicoli. Scanning electron microscopic examination revealed focal to confluent spirochetal attachment mainly in the furrow region at the periphery of the crypt units. Transmission electron microscopic examination revealed spirochetes attached to the brush border of the cecal epithelium, causing effacement of the microvilli and disruption of the terminal web microfilaments. The cecal epithelium of chicks inoculated with the canine S. pilosicoli also had caplike elevations of the apical membrane at the point of attachment of the spirochetes together with large numbers of vesicles in the cytoplasm immediately beneath the terminal web and evidence of spirochetal invasion beyond the mucosal barrier. The changes observed suggested that the mechanism of attachment of human and canine S. pilosicoli to the cecal epithelium of chicks was analogous to but different from that described previously for other attaching and effacing gastroenteric bacterial pathogens of human beings and animals.

Colorectal cancer (CRC) is the second largest cause of cancer deaths in the United States. A key barrier that prevents better outcomes for this type of cancer as well as other solid tumors is the lack of effective therapies against the metastatic disease. Thus there is an urgent need to fill this gap in cancer therapy. We utilized a 2D-DIGE proteomics approach to identify and characterize proteins that are differentially regulated between primary colon tumor and liver metastatic deposits of the IGF1R-dependent GEO human CRC xenograft, orthotopically implanted in athymic nude mice that may serve as potential therapeutic targets against CRC metastasis. We observed increased expression of ezrin in liver metastasis in comparison to the primary colonic tumor. Increased ezrin expression was further confirmed by western blot and microarray analyses. Ezrin, a cytoskeletal protein belonging to Ezrin-Radixin-Moesin (ERM) family plays important roles in cell motility, invasion and metastasis. However, its exact function in colorectal cancer is not well characterized. Establishment of advanced GEO cell lines with enhanced liver-metastasizing ability showed a significant increase in ezrin expression in liver metastasis. Increased phosphorylation of ezrin at the T567 site (termed here as p-ezrin T567) was observed in liver metastasis. IHC studies of human CRC patient specimens showed an increased expression of p-ezrin T567 in liver metastasis compared to the primary tumors of the same patient. Ezrin modulation by siRNA, inhibitors and T567A/D point mutations significantly downregulated inhibitors of apoptosis (IAP) proteins XIAP and survivin that have been linked to increased aberrant cell survival and metastasis and increased cell death. Inhibition of the IGF1R signaling pathway by humanized recombinant IGF1R monoclonal antibody MK-0646 in athymic mouse subcutaneous xenografts resulted in inhibition of p-ezrin T567 indicating ezrin signaling is downstream of the IGF1R signaling pathway. We identified increased expression of p-ezrin T567 in CRC liver metastasis in both orthotopically implanted GEO tumors as well as human patient specimens. We report for the first time that p-ezrin T567 is downstream of the IGF1R signaling and demonstrate that ezrin regulates cell survival through survivin/XIAP modulation.

Shearman, L.; Mathiesen, M.; Zhou, Y.J.; Arredondo-Peter, R.; Sarath, Gautam; and Klucas, R. V., "Nonsymbiotic hemoglobins in rice are synthesized during germination and in differentiating cell types" (2001 Abstract: Nonsymbiotic hemoglobins (ns-Hbs) previously have been found in monocots and dicots; however, very little is known about the tissue and cell type localization as well as the physiological function(s) of these oxygen-binding proteins. We report the immunodetection and immunolocalization of ns-Hbs in rice (Oryza sativa L.) by Western blotting and in situ confocal laser scanning techniques. Ns-Hbs were detected in soluble extracts of different tissues from the developing rice seedling by immunoblotting. Levels of ns-Hbs increased in the germinating seed for the fi rst six days following imbibition and remained relatively constant thereafter. In contrast, ns-Hb levels decreased during leaf maturation. Roots and mesocotyls contained detectable, but low levels of ns-Hbs. Split-seed experiments revealed that ns-Hbs are synthesized de novo during seed germination and are expressed in the absence of any signal originating from the embryo. Immunolocalization of ns-Hbs by con-focal microscopy indicated the presence of ns-Hbs primarily in differentiated and differentiating cell types of the developing seedling, such as the aleurone, scutellum, root cap cells, sclerenchyma, and tracheary elements. To our knowledge, this is the fi rst report of the specifi c cellular localization of these proteins during seedling development.

The development and characterization of effective anticancer drugs against colorectal cancer (CRC) is of urgent need since it is the second most common cause of cancer death. The study was designed to evaluate the effects of two IGF-1R antagonists, MK-0646, a recombinant fully humanized monoclonal antibody and OSI-906, a small molecule tyrosine kinase inhibitor on CRC cells. Xenograft study was performed on IGF-1R-dependent CRC cell lines for analyzing the antitumor activity of MK-0646 and OSI-906. Tumor proliferation and apoptosis were assessed using Ki67 and TUNEL assays, respectively. We also performed in vitro characterization of MK-0646 and OSI-906 treatment on CRC cells to identify mechanisms associated with drug-induced cell death. Exposure of the GEO and CBS tumor xenografts to MK-0646 or OSI-906 led to a decrease in tumor growth. TUNEL analysis showed an increase of approximately 45–55% in apoptotic cells in both MK-0646 and OSI-906 treated tumor samples. We report the novel finding that treatment with IGF-1R antagonists led to downregulation of X-linked inhibitor of apoptosis (XIAP) protein involved in cell survival and inhibition of cell death. In conclusion, IGF-1R antagonists (MK-0646 and OSI-906) demonstrated single agent inhibition of subcutaneous CRC xenograft growth. This was coupled to pro-apoptotic effects resulting in downregulation of XIAP and inhibition of cell survival. We report a novel mechanism by which MK-0646 and OSI-906 elicits cell death in vivo and in vitro. Moreover, these results indicate that MK-0646 and OSI-906 may be potential anticancer candidates for the treatment of patients with IGF-1R-dependent CRC.

Abstract. Porcine colonic spirochetosis is a nonfatal diarrheal disease that affects pigs during the growing and finishing stages of production. The disease is caused by Serpulina pilosicoli, a newly recognized species of pathogenic intestinal spirochete. Antimicrobial therapy aimed at reducing the infection may be helpful in controlling spirochetal diarrhea. In this study, the in vitro antimicrobial susceptibilities of the reference isolate S. pilosicoli P43/6/78 from the United Kingdom and 19 field isolates obtained from pigs in Canada (n ϭ 5) and the United States (n ϭ 14) were determined against the antimicrobial agents carbadox, gentamicin, lincomycin, and tiamulin, all of which are commonly used for control of the related pathogenic intestinal spirochete S. hyodysenteriae. Additionally, the susceptibility or resistance of each isolate against each antimicrobial agent was estimated on the basis of available data on the in vitro antimicrobial susceptibility breakpoints of S. hyodysenteriae. Each isolate was identified on the basis of phenotypic and genotypic markers, and the minimum inhibitory concentration of each antimicrobial agent was determined by the agar-dilution method. All the isolates were susceptible to carbadox and tiamulin. The percentages of isolates susceptible, intermediate, and resistant to lincomycin were 42.1%, 42.1%, and 15.8%, respectively. Slightly less than half of the isolates (47.4%) were susceptible to gentamicin, and the remainder (52.6%) were resistant. Implementation of rational control measures to reduce infection by S. pilosicoli should improve overall health and productivity in swine herds.Until recently, porcine hemolytic intestinal spirochetes consisted of 2 taxonomically recognized species in the genus Serpulina: the strongly ␤-hemolytic spirochete Serpulina hyodysenteriae, the agent of swine dysentery, and the weakly ␤-hemolytic intestinal spirochete (WBHIS) S. innocens, a nonpathogenic spirochete of the swine colon. 7,13,31 However, an association between a WBHIS, recently classified as S. pilosicoli, and a nonfatal diarrheal disease of growing pigs, called porcine intestinal spirochetosis or porcine colonic spirochetosis (PCS), has been recognized. 1,[5][6][7][8][9][10][11][12]19,[24][25][26][27][28][32][33][34][35][36][37] With more intensive swine production, where the efficiency of weight gain is closely monitored, PCS has been recognized as a major contributing factor to reduced performance in growing and finishing pigs. 5,6,8,9,10,12,34 Koch's postulates for S. pilosicoli have been fulfilled using gnotobiotic pigs 25 and conventional pigs. 3,5,[33][34][35] Conventional pigs challenge exposed with S. pilosicoli have diarrhea and reduced performance as a result of colonization and inflammation of the cecum and colon. 3,5,[33][34][35] Clinical signs of S. pilosicoli infection consist of diarrhea, which is transient to persistent. The feces from diarrheic pigs are watery to mucoid in consistency,

Abstract. Pathogenic intestinal spirochetes of swine include Serpulina hyodysenteriae, a strongly ß-hemolytic spirochete that causes swine dysentery, and S. pilosicoli, a weakly ß-hemolytic intestinal spirochete (WBHIS) that causes porcine colonic spirochetosis. Because of the existence of nonpathogenic WBHIS in the normal swine colon, it is important to develop laboratory procedures for accurate identification of S. pilosicoli. The purpose of the present study was to assess hippurate hydrolysis and polymerase chain reaction (PCR) amplification of specific 16S ribosomal RNA (rRNA) sequences for identification of porcine S. pilosicoli. Additionally, the enteropathogenicity of 8 field isolates of porcine S. pilosicoli was determined by challenge exposure of 1-dayold chicks and sequential histologic examination of the cecal mucosa. The field isolates of porcine S. pilosicoli hydrolyzed hippurate and yielded S. pilosicoli-specific products by PCR amplification of 16S rRNA sequences. Although all of the field isolates of porcine S. pilosicoli attached to the cecal epithelium of challenge-exposed chicks by day 21 postinoculation, some isolates had locally invasive phenotypes. We concluded that identification of porcine S. pilosicoli could be made on the basis of results of hippurate hydrolysis and 16S rRNA PCR amplification. Challenge inoculation of 1-day-old chicks followed by histologic examination of the cecal mucosa demonstrated the enteropathogenicity of porcine S. pilosicoli.Previously, all weakly ß-hemolytic intestinal spiro-Europe, and Australia. Additionally, S. pilosicoli has chetes (WBHIS) inhabiting the large intestine of swine a broad host range; spirochetes with a similar morwere assigned to the nonpathogenic spirochete species, phology have been seen in the cecum and colon of Serpulina innocens. 1,13,16,17,28,29 The porcine WBHIS now human beings, 5,6,30 nonhuman primate, 5,30 dogs, 6 guinhave been shown to consist of a diverse group in ad-ea pigs, 22 and opossums 36 often with clinical signs or dition to S. innocens, based on differences in the num-lesions of colonic spirochetosis. ber and arrangement of periplasmic flagella, 4,16,21,26,32,35 Because S. pilosicoli is weakly ß-hemolytic, it is important to differentiate it from S. innocens, using rouhippurate hydrolysis, 4,8,27,35 multilocus enzyme electro-tine laboratory procedures. A 16S rRNA PCR assay phoretic patterns, 21 DNA hybridization using whole-specific for S. pilosicoli is available; 25 however, there genome 26 and gene-specific 18,23 probes, DNA finger-is evidence indicating that some human S. pilosicoli printing by arbitrarily primed polymerase chain re-isolates yield negative results with this method. 5 The action (PCR), 4,23 ribosomal RNA gene restriction frag-purpose of the present study was to assess hippurate ment length polymorphisms or ribotyping, 31 sequence hydrolysis and PCR amplification of S. pilosicoli-speanalyses of 16S ribosomal RNA and DNA, 9,29 PCR cific 16S rRNA sequences for identification of porcine amplification of a 1...

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