Protein-tyrosine phosphatase SHP-1 is expressed at high levels in hematopoietic cells and at moderate levels in many other cell types including epithelial cells. Although SHP-1 has been shown to be a negative regulator of multiple signaling pathways in hematopoietic cells, very little is known about the biological role of SHP-1 in epithelial cells. In order to elucidate the mechanism(s) responsible for the loss of proliferative potential once committed intestinal epithelial cells begin to differentiate, the role and regulation of SHP-1 were analyzed in both intact epithelium as well as in well established intestinal cell models recapitulating the crypt-villus axis in vitro. Results show that SHP-1 was expressed in the nuclei of all intestinal epithelial cell models as well as in epithelial cells of intact human fetal jejunum and colon. Expression and phosphatase activity levels of SHP-1 were much more elevated in confluent growth-arrested intestinal epithelial cells and in differentiated enterocytes as well. Overexpression of SHP-1 in intestinal epithelial crypt cells significantly inhibited dhfr, c-myc, and cyclin D1 gene expression but did not interfere with c-fos gene expression. In contrast, a mutated inactive form of SHP-1 had no effect on these genes. SHP-1 expression significantly decreased beta-catenin/TCF-dependent transcription in intestinal epithelial crypt cells. Immunoprecipitation experiments revealed that beta-catenin is one of the main binding partners and a substrate for SHP-1. Taken together, our results indicate that SHP-1 may be involved in the regulation of beta-catenin transcriptional function and in the negative control of intestinal epithelial cell proliferation.

In the past, the use of delta ribozyme as a therapeutic tool was limited because substrate specificity was thought to be determined by only 8 nucleotides. Recently, we have accumulated evidence suggesting that the substrate sequence upstream of the cleavage site, which is not involved in the binding with the delta ribozyme, appears to be essential in the selection of an appropriate cleavage site. To understand the role of this region in efficient cleavage, we synthesized a collection of small substrates that possessed single and multiple mutations in positions -1 to -4 and determined the kinetic parameters of their cleavage using a model antigenomic delta ribozyme. Some substrates were found to be uncleavage, whereas others showed >60-fold difference in relative specificity between the least and most efficiently cleaved substrates. The base at each position from -1 to -4 contributes differently to the ability of a substrate to be cleaved. An optimal sequence for positions -1 to -4 was determined to be -1HRHY(-4) (H = U, C, or A). These results shed light on new features that contribute to the substrate requirement of delta ribozyme cleavage and should increase interest in the use of this unique ribozyme.

Abstract-To␣ Me)Phe analogues showed high antagonistic potencies (pA 2 ) at both the human (8.8, 7.7, and 8.7, respectively) and rabbit (8.6, 7.8, and 8.6, respectively) B 1 receptors. No antagonistic effects (pA 2 Ͻ5) were observed on the B 2 receptors that mediate the contractile effects of BK on the human umbilical vein, the rabbit jugular vein, and the guinea pig ileum. Moreover, these new B 1 antagonists were found to be resistant to in vitro degradation by purified angiotensin-converting enzyme from rabbit lung. The N ␣ -acetylated forms, R 892 and R 914, were resistant to aminopeptidases from human plasma. In vivo antagonistic potencies (ID 50 ) of B 1 receptor antagonists were evaluated in anesthetized lipopolysaccharide-treated (for B 1 receptor) and nontreated (for B 2 receptor) rabbits against the hypotensive effects of exogenous desArg 9 BK and BK. R 892 efficiently inhibited (ID 50 2.8 nmol/kg IV) hypotension induced by desArg 9 BK without affecting that evoked by BK (ID 50 Ͼ600 nmol/kg IV

Somatostatin, or its structural analog SMS 201-995 (SMS), is recognized to exert a growth-inhibitory action in rat pancreas, but the cellular mechanisms are not completely understood. This study was undertaken to evaluate the effect of SMS on p42/p44 MAP kinases and phosphatidylinositol 3-kinase activation and to analyze expression of some cell cycle regulatory proteins in relation to pancreatic acinar cell proliferation in vivo (rat pancreas), as well as in the well-established tumoral cell line AR4-2J. We herein report that: 1) SMS inhibits caerulein-induced pancreatic weight and DNA content and abolishes epidermal growth factor (EGF)-stimulated AR4-2J proliferation; 2) SMS only moderately reduces the stimulatory effect of caerulein on p42/p44 MAP kinase activities in pancreas and has no effect on EGF-stimulated MAP kinase activities in AR4-2J cells; 3) SMS repressed caerulein-induced Akt activity in normal pancreas; 4) SMS has a strong inhibitory action on cyclin E expression induced by caerulein in pancreas and EGF in AR4-2J cells and as expected, the resulting cyclin E-associated cyclin-dependent kinase (cdk)2 activity, as well as pRb phosphorylation, are blunted by SMS treatment in both models; and 5) SMS suppresses mitogen-induced p27(Kip1) down-regulation, as well as marginally induces p21(Cip) expression. Thus, our data suggest that somatostatin-induced growth arrest is mediated by inhibition of phosphatidylinositol 3-kinase pathway and by enhanced expression of p21(Cip) and p27(Kip1), leading to repression of pRb phosphorylation and cyclin E-cdk2 complex activity.

Somatostatin, or its structural analog SMS 201-995 (SMS), is recognized to exert a growth-inhibitory action in rat pancreas, but the cellular mechanisms are not completely understood. This study was undertaken to evaluate the effect of SMS on p42/p44 MAP kinases and phosphatidylinositol 3-kinase activation and to analyze expression of some cell cycle regulatory proteins in relation to pancreatic acinar cell proliferation in vivo (rat pancreas), as well as in the well-established tumoral cell line AR4-2J. We herein report that: 1) SMS inhibits caerulein-induced pancreatic weight and DNA content and abolishes epidermal growth factor (EGF)-stimulated AR4-2J proliferation; 2) SMS only moderately reduces the stimulatory effect of caerulein on p42/p44 MAP kinase activities in pancreas and has no effect on EGF-stimulated MAP kinase activities in AR4-2J cells; 3) SMS repressed caerulein-induced Akt activity in normal pancreas; 4) SMS has a strong inhibitory action on cyclin E expression induced by caerulein in pancreas and EGF in AR4-2J cells and as expected, the resulting cyclin E-associated cyclin-dependent kinase (cdk)2 activity, as well as pRb phosphorylation, are blunted by SMS treatment in both models; and 5) SMS suppresses mitogen-induced p27(Kip1) down-regulation, as well as marginally induces p21(Cip) expression. Thus, our data suggest that somatostatin-induced growth arrest is mediated by inhibition of phosphatidylinositol 3-kinase pathway and by enhanced expression of p21(Cip) and p27(Kip1), leading to repression of pRb phosphorylation and cyclin E-cdk2 complex activity.