In a previous report (1978, Proc. Nat. Acad. Sci. USA 75, 3479-3483), we showed that recipient strains of Streptococcusfaecalis excrete a heat-stable substance (sex pheromone) which induces donor cells carrying certain conjugative plasmids to become adherent, generating the cell-to-cell contact necessary for plasmid transfer. Since donors themselves could be induced to aggregate or "clump" by recipient filtrates, the substance was referred to as "clumping-inducing agent" (CIA). In this report, we present a simplified assay for CIA and determine the level of activity in filtrates prepared at various stages of growth. We also present evidence that recipient cells produce multiple pheromones, each specific for donors harboring a particular class of plasmids. Whereas a recipient that acquires a conjugative plasmid no longer produces the corresponding CIA, it still produces CIAs specific for donors with different conjugative plasmids. In addition, an analysis of 100 clinical isolates of S. faecalis showed that drug-resistant strains are significantly more likely to respond to and produce CIA activities than drug-sensitive strains. A model is discussed describing the relationships of sex pheromones to the mating process.

Background The hedgehog (HH) signaling pathway is a key regulator in tumorigenesis of pancreatic adenocarcinoma (PDA) and is up-regulated in PDA cancer stem cells (CSCs). GDC-0449 is an oral small-molecule inhibitor of HH pathway. This study assessed the effect of GDC-0449-mediated HH inhibition in paired biopsies, followed by combined treatment with gemcitabine, in patients with metastatic PDA. Methods Twenty-five patients were enrolled of which 23 underwent core biopsies at baseline and following 3 weeks of GDC-0449. On day 29, 23 patients started weekly gemcitabine while continuing GDC-0449. We evaluated GLI1 and PTCH1 inhibition, change in CSCs, Ki-67, fibrosis, and assessed tumor response, survival and toxicity. Results On pre-treatment biopsy, 75% of patients had elevated sonic hedgehog (SHH) expression. On post-treatment biopsy, GLI1 and PTCH1 decreased in 95.6% and 82.6% of 23 patients, fibrosis decreased in 45.4% of 22 and Ki-67 in 52.9% of 17 evaluable patients. No significant changes were detected in CSCs pre- and post-biopsy. The median progression-free and overall survival for all treated patients was 2.8 and 5.3 months. The response and disease control rate was 21.7% and 65.2%. No significant correlation was noted between CSCs, fibrosis, SHH, Ki-67, GLI1, PTCH1 (baseline values, or relative change on post-treatment biopsy) and survival. Grade >3 adverse events were noted in 56% of patients. Conclusion We show that GDC-0449 for 3 weeks leads to down-modulation of GLI1 and PTCH1, without significant changes in CSCs compared to baseline. GDC-0449 and gemcitabine was not superior to gemcitabine alone in the treatment of metastatic pancreatic cancer.

Abstract. Selectin-ligands on T cells contribute to the recruitment of circulating cells into chronic inflammatory lesions in the skin and elsewhere. This report provides the first evidence that a single fucosyltransferase, termed FucT-VII, controls the synthesis of E-selectin ligands in human T-lymphoblasts. The FucT-IV transferase (the ELFT enzyme), in contrast, constructs lower avidity E-selectin ligands and requires enzyme levels found only in myeloid cells. Treatment of Jurkat cells with phorbol myristate acetate increased the expression of sialylated LewisX-related (sLeX-related) epitopes and induced the synthesis of E-selectin ligands functional at physiologic levels of linear shear-stress. Northern analysis revealed a parallel increase in the steady-state level of FucT-VII mRNA, but there were no increases in the two other leukocyte-associated fucosyltransferases (FucT-IV and VI). The stable transfection of the FucT-VII gene into Jurkat cells induced high levels of the sLeX-related epitopes and the synthesis of E-selectin ligands which equaled or exceeded the avidity of those on circulating lymphocytes. The growth of T-lymphoblasts under conditions which induced expression of the sLe x,a epitopes increased the level of FucT-VII mRNA, the synthesis of sialylated-Lewis x structures by cell-free extracts and the synthesis of E-selectin ligands equal in avidity to those on FucT-VII transfectants. In contrast, neither the mRNA levels nor activities of the FucT-IV and VI enzymes increased in association with E-selectin ligand synthesis in T-lymphoblasts. Myeloid cell lines, unlike lymphoblasts, expressed high levels of both the FucT-VII and IV enzymes in conjunction with E-selectin ligands raising the possibility that both enzymes contributed to ligand synthesis. FucT-IV transfected Jurkat cells synthesized low avidity ligands for E-selectin but only in association with the CDw65 (VIM-2) carbohydrate epitope. Only blood neutrophils and myeloid cell lines expressed this epitope at the levels associated with E-ligand synthesis in the transfectants. In contrast, native Jurkat cells, blood monocytes, blood lymphocytes, and cultured T-lymphoblasts expressed low levels or none. We conclude that FucT-VII is a principal regulator of E-selectin ligand synthesis in human T-lymphoblasts while both FucT-VII and FucT-IV may direct ligand synthesis in some myeloid cells. Correlative studies in several species suggest that these molecules participate in recruitment. T-lymphocytes expressing carbohydrate epitopes associated with ligands for

Recipient strains of Streptococcus faecalis excrete multiple, peptide sex pheromones that induce mating responses in donors harboring certain conjugative plasmids. Acquisition of plasmid DNA leads to a "shutting off" of pheromone excretion, and such cells become responsive to exogenous pheromone. Data are presented showing that donors excrete low levels of a modified, inactive form of the pheromone. This substance, when mixed in excess with active pheromone, inhibits pheromone activity (probably by competition for a receptor site on the donor). Modified forms of both cPDI and cADI were revealed, and each appeared to have a mass about 350-400 daltons larger than the active pheromone. In both cases, pheromone activity could be regenerated by treatment with phosphodiesterase II.Recipient strains of Streptococcusfaecalis excrete multiple, heatstable, peptide sex pheromones specific for donor strains harboring certain conjugative plasmids (1-4). Donors respond by synthesizing an adherent surface structure, which facilitates the formation of mating aggregates upon contact (random collisions) with recipients. The response involves the appearance of a unique antigen which uniformly coats the donor surface (5, 6). In addition, an undefined "preparation for plasmid transfer" takes place (3). If exposed to a culture filtrate of recipients, donors are induced to self clump; for this reason pheromone is also referred to as "clumping inducing agent." The clumping response provides a basis for quantitating (by dilution) pheromone activity (2).When plasmid DNA is acquired by the recipient bacterium, production of the related pheromone ceases and the strain becomes responsive to exogenous pheromone. However, the cell continues to produce other pheromones specific for donors harboring different conjugative plasmids (2, 4). The pheromones are designated by their relationship to the plasmid originally used to detect them. Thus, cADi refers to the pheromone to which strains harboring pADI respond, whereas cPD1 refers to the pheromone related to pPD1.In this paper we address the mechanism by which plasmid acquisition leads to the "shutting off" of endogenous pheromone production. We present evidence that donor cells harboring pADi [a 37.8-megadalton Table 1. The construction of plasmid-bearing strains was by mixing overnight cultures of appropriate donors and recipients (0.05 ml of donors with 0.5 ml of recipients with 4.5 ml of fresh broth) and allowing the mixture to incubate for 2-4 hr prior to plating on an appropriate selective medium. Insertions of Tn916 (determines resistance to tetracycline) and Tn917 (determines resistance to erythromycin) into plasmid DNA were derived as described (8,9,11,12). Drug concentrations in selective media were as described (2). Unless otherwise noted, the medium used throughout this study was N2GT (Oxoid Nutrient Broth no. 2 supplemented with 0.2% glucose and 0.1 M Tris-HCI, pH 7.7). Turbidity was monitored by using a KlettSummerson colorimeter with a no. 54 (green) filter.The mutant d...

Multiorgan apoptosis occurs during sepsis. Following cecal ligation and puncture (CLP) in rats, thymocytes underwent apoptosis in a time-dependent manner. C5a blockade dramatically reduced thymocyte apoptosis as measured by thymic weight, binding of annexin V to thymocytes, and laddering of thymocyte DNA. When C5a was generated in vivo by infusion of purified cobra venom factor (CVF), thymocyte apoptosis was significantly increased. Similar results were found when CVF was injected in vivo during the early stages of CLP. In animals 12 hours after induction of CLP, there was an increase in the activities of caspase-3, -6, and -9, but not caspase-1 and -8. Cytosolic cytochrome c levels increased by twofold, whereas mitochondrial levels showed a 50% decrease. Western blot analysis revealed that the content of Bcl-X L (but not of Bcl-2, BAX, Bad, and Bim) significantly decreased in thymocytes after CLP. C5a blockade in the sepsis model almost completely inhibited caspase-3, -6, and -9 activation, significantly preserved cytochrome c in the mitochondrial fraction, and restored Bcl-X L expression. These data suggest that systemic activation of complement induces C5a-dependent apoptosis of thymocytes and that the blockade of C5a during sepsis rescues thymocytes from apoptosis.