Krü ppel-like factor 4 (KLF4) is an epithelial cell-enriched, zinc finger-containing transcription factor, the expression of which is associated with growth arrest. Previous studies show that constitutive expression of KLF4 inhibits DNA synthesis but the manner by which KLF4 exerts this effect is unclear. In the present study, we developed a system in which expression of KLF4 is controlled by a promoter that is induced upon treatment of cells containing the receptors for the insect hormone, ecdysone, with ponasterone A, an ecdysone analogue. The rate of proliferation of a stably transfected colon cancer cell line, RKO, was significantly decreased following addition of ponasterone A when compared with untreated cells. Flow cytometric analyses indicated that the inducible expression of KLF4 caused a block in the G 1 /S phase of the cell cycle. A similar block was observed when ecdysone receptor-containing RKO cells were infected with a replication-defective recombinant adenovirus containing an inducible KLF4 and treated with ponasterone A. Results of these studies provide evidence that the inhibitory effect of KLF4 on cell proliferation is mainly exerted at the G 1 /S boundary of the cell cycle.

Intestinal-enriched Krü ppel-like factor (IKLF or KLF5) belongs to the family of mammalian Krü ppel-like transcription factors. Previous studies indicate that expression of IKLF is enriched in the proliferating crypt epithelial cells of the intestinal tract. However, the biological function of IKLF is unknown. In the current study, we have shown that the level of IKLF mRNA was nearly undetectable in serum-deprived NIH3T3 fibroblasts but became acutely and significantly increased upon the addition of fetal bovine serum or the phorbol ester, PMA. This induction required protein synthesis because it was prevented by cycloheximide. Transfection of IKLF into NIH3T3 cells resulted in the formation of foci in a manner similar to that caused by the activated Ha-ras oncogene. Constitutive expression of IKLF in transfected NIH3T3 cells significantly increased the rate of proliferation when compared with cells transfected with an empty vector. The growth of IKLF-transfected cells was no longer inhibited by cell-cell contact or by low serum content. Moreover, these cells proliferated in an anchorage-independent fashion. We conclude that IKLF encodes a delayed early response gene product that positively regulates cellular proliferation and may give rise to a transformed phenotype when overexpressed.

SummaryBacterial alternative RNA polymerase sigma factors are key global adaptive response regulators with a likely role in Mycobacterium tuberculosis pathogenesis. We constructed a mutant lacking the sigma factor gene, sigC , by allelic exchange, in the virulent CDC1551 strain of M. tuberculosis and compared the resulting mutant with the isogenic wild-type strain and complemented mutant strain. In vitro , compared to the wild-type and complemented strains, the mutant was found to have similar ability to survive in both murine bone marrow-derived macrophages and activated J774 macrophages.

No abstract

No abstract

Strains of enterotoxigenic Escherichia coli that express K88 fimbriae are among the most common causes of diarrhea in young pigs. Adhesion of bacteria to receptors on intestinal epithelial cells, mediated by K88 fimbriae, is the initial step in the establishment of infection. Three antigenic variants of K88 fimbriae exist in nature: K88ab, K88ac, and K88ad. K88ac is the most prevalent and may be the only variant of significance in swine disease. Each K88 fimbrial variant is composed of multiple antigenic determinants. Some of these determinants are shared among the three variants and may be referred to as conserved epitopes, whereas others are unique to a specific variant and may be referred to as variable epitopes. In this study, monoclonal antibodies (MAbs) specific to either variable or conserved epitopes of K88ac fimbriae were produced. The specificity of each MAb was tested by enzyme-linked immunosorbent and immunoblot assays. Fab fragments were prepared from these MAbs and were tested for their ability to block the binding of K88-positive bacteria and purified fimbriae to porcine enterocyte brush border vesicles and purified K88 receptors, respectively. The purified receptors were intestinal mucin-type sialoglycoproteins (

Three antigenic variants of the K88 fimbrial adhesin exist in nature, K88ab, K88ac, and K88ad. Enterotoxigenic Escherichia coli (ETEC) strains that produce these fimbriae cause life-threatening diarrhea in some but not all young pigs. The susceptibility of pigs to these organisms has been correlated with the adherence of bacteria to isolated enterocyte brush borders. Whether that correlation holds for multiple K88 variants and over a broad genetic base of pigs is unknown and was the impetus for this study. We also desired to examine the correlation of the expression of a porcine intestinal brush border mucin-type glycoprotein (IMTGP) which binds K88ab and K88ac with the susceptibility of piglets to K88 ؉ ETEC. Of 31 neonatal gnotobiotic pigs inoculated with K88ab ؉ or K88ac ؉ ETEC, 13 developed severe diarrhea, became dehydrated, and died or became moribund. Another pig became severely lethargic but not dehydrated. In vitro brush border adherence analysis was not possible for 10 of the severely ill pigs due to colonization by challenge strains. However, of the 17 pigs that did not become severely ill, 8 (47%) had brush borders that supported the adherence of K88ab ؉ and K88ac ؉ bacteria in vitro, suggesting a poor correlation between in vitro brush border adherence and piglet susceptibility to K88 ؉ ETEC. By contrast, the expression of IMTGP was highly correlated with susceptibility to K88 ؉ ETEC. Of the 12 pigs that produced IMTGP, 11 developed severe diarrhea. The other pig that produced IMTGP became lethargic but not severely diarrheic. Only 2 of 18 pigs that did not produce IMTGP became severely diarrheic. Colonizing bacteria were observed in histologic sections of intestines from all pigs that expressed IMTGP except for the one that did not develop severe diarrhea. However, colonizing bacteria were observed in histologic sections from only one pig that did not produce IMTGP. The bacterial concentration in the jejuna and ilea of pigs expressing IMTGP was significantly greater (P < 0.005) than that in pigs not expressing IMTGP. These observations suggest the IMTGP is a biologically relevant receptor for K88ab ؉ and K88ac ؉ E. coli or a correlate for expression for such a receptor.

Three antigenic variants of the K88 fimbrial adhesin exist in nature, K88ab, K88ac, and K88ad. Enterotoxigenic Escherichia coli (ETEC) strains that produce these fimbriae cause life-threatening diarrhea in some but not all young pigs. The susceptibility of pigs to these organisms has been correlated with the adherence of bacteria to isolated enterocyte brush borders. Whether that correlation holds for multiple K88 variants and over a broad genetic base of pigs is unknown and was the impetus for this study. We also desired to examine the correlation of the expression of a porcine intestinal brush border mucin-type glycoprotein (IMTGP) which binds K88ab and K88ac with the susceptibility of piglets to K88 ؉ ETEC. Of 31 neonatal gnotobiotic pigs inoculated with K88ab ؉ or K88ac ؉ ETEC, 13 developed severe diarrhea, became dehydrated, and died or became moribund. Another pig became severely lethargic but not dehydrated. In vitro brush border adherence analysis was not possible for 10 of the severely ill pigs due to colonization by challenge strains. However, of the 17 pigs that did not become severely ill, 8 (47%) had brush borders that supported the adherence of K88ab ؉ and K88ac ؉ bacteria in vitro, suggesting a poor correlation between in vitro brush border adherence and piglet susceptibility to K88 ؉ ETEC. By contrast, the expression of IMTGP was highly correlated with susceptibility to K88 ؉ ETEC. Of the 12 pigs that produced IMTGP, 11 developed severe diarrhea. The other pig that produced IMTGP became lethargic but not severely diarrheic. Only 2 of 18 pigs that did not produce IMTGP became severely diarrheic. Colonizing bacteria were observed in histologic sections of intestines from all pigs that expressed IMTGP except for the one that did not develop severe diarrhea. However, colonizing bacteria were observed in histologic sections from only one pig that did not produce IMTGP. The bacterial concentration in the jejuna and ilea of pigs expressing IMTGP was significantly greater (P < 0.005) than that in pigs not expressing IMTGP. These observations suggest the IMTGP is a biologically relevant receptor for K88ab ؉ and K88ac ؉ E. coli or a correlate for expression for such a receptor.