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Mycobacterium tuberculosis, the primary agent of tuberculosis, must acquire iron from the host to cause infection. To do so, it releases high-affinity iron-binding siderophores Mycobacterium tuberculosis, the primary causative agent of tuberculosis, infects one-third of humanity and is one of the world's most important infectious agents. M. tuberculosis is a facultative intracellular pathogen. In the host, it multiplies intracellularly in mononuclear phagocytes, and it also appears to multiply extracellularly at least in lung cavities. Its capacity to infect the host is closely linked to its ability to acquire iron. Serum containing poorly saturated transferrin, such as human serum, is tuberculostatic, and its tuberculostatic effect is neutralized by the addition of iron (1, 2).Free iron is very limited in the host, particularly in extracellular sites, owing to the high affinity with which it is held by host iron-binding proteins, chiefly transferrin and lactoferrin. To obtain iron at sites where it is limited, many pathogens have developed high-affinity iron-binding molecules of their own called siderophores, which can remove iron from host ironbinding molecules. Mycobacteria have been shown by Macham, Ratledge, Barclay, and colleagues (3-5) to produce small water-soluble siderophores called exochelins. This group of investigators has proposed that exochelins bind iron in the extracellular aqueous environment and transport the metal to another high-affinity iron-binding molecule located in the cell wall ofM. tuberculosis-mycobactin (3). Mycobactin is a highly lipophilic molecule thought to facilitate the transport of iron across the cell wall to the interior of the bacterium (6).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Both exochelins and mycobactins are induced by low concentrations of iron in broth medium (4, 7). There are two general types of exochelins, classified according to their extractability in organic solvents (8). The chloroform-insoluble exochelins, produced by saprophytic mycobacteria, are not extractable into any organic solvent. The chloroform-soluble exochelins, produced by slow-growing pathogenic mycobacteria, including M. tuberculosis, are extractable into chloroform (5).Mycobactins have been extensively studied and their structures delineated (7). In contrast, individual exochelins have not been purified previously and neither their structure nor composition has been described. In this paper, we describe the purification of exochelins of M. tuberculosis from both a virulent (Erdman) and an avirulent (H37Ra) strain and their characterization by MS. MATERIALS AND METHODSMedium and Reagents. Modified iron-deficient Sauton's broth medium (9) was prepared with 1-10 ,uM Fe3+ and without Tween. Mycobactin J was purchased from Allied Monitor (Fayette, MO).Bacteria. M. tuberculosis Erdman (ATCC catalog no. 35801) and H37Ra (A...

We previously demonstrated the expression of bitter taste receptors of the type 2 family (T2R) and the ␣-subunits of the G protein gustducin (G␣ gust) in the rodent gastrointestinal (GI) tract and in GI endocrine cells. In this study, we characterized mechanisms of Ca 2ϩ fluxes induced by two distinct T2R ligands: denatonium benzoate (DB) and phenylthiocarbamide (PTC), in mouse enteroendocrine cell line STC-1. Both DB and PTC induced a marked increase in intracellular [Ca 2ϩ (3,13,19,60). Molecular sensing by GI cells plays a critical role in the control of multiple fundamental functions, including digestion, food intake, and metabolic regulation. Although these fundamental control systems have been known for a considerable time, the initial molecular recognition events that sense the chemical composition of the luminal contents have remained poorly understood. The gustatory system has been selected during evolution to detect nonvolatile nutritive and beneficial (sweet) compounds as well as potentially harmful (bitter) substances (24, 34). In particular, bitter taste has evolved as a central warning signal against the ingestion of potentially toxic substances, including plant alkaloids and other environmental toxins (21, 65). Specialized neuroepithelial taste receptor cells, organized within taste buds in human and rodent lingual epithelium, expressed a family of bitter taste receptors (referred as T2Rs) (1, 6, 46). These putative taste receptors belong to the guanine nucleotide-binding regulatory protein (G protein)-coupled receptor (GPCR) superfamily (1), which are characterized by seven transmembrane ␣-helices (32). Extensive genetic and biochemical evidence indicate that specific G proteins, gustducin and transducin, mediate bitter and sweet gustatory signals in the taste buds of the lingual epithelium (47,48,62,63,73). More recently, phospholipase C␤ 2 (PLC␤ 2 ) and TRPM5, a member (melastatin subtype 5) of the transient receptor potential (TRP) family (49), have been linked to bitter and sweet signal transduction (55, 56, 81). There is evidence for the activation of multiple second messenger pathways and ion channels in individual taste cells (1, 82). Clearly, taste signal transduction is complex and multifactorial and there is still much that is unknown about individual taste cell regulation.Outside the tongue, expression of the ␣-subunit of gustducin (G␣ gust ) has been also localized to gastric (28, 75) and pancreatic (27) cells, suggesting that a taste-sensing mechanism may also exist in the digestive system. Indeed, we demonstrated the expression of members of the bitter taste receptors of the T2R family in the mouse and rat GI tract and in mouse and rat enteroendocrine cells in culture (74,75). More recently, these results have been confirmed (45) and extended to the expression of the sweet taste receptors of the T1R family (15). Collectively, these findings demonstrated the expression of taste signal transduction pathways in cells of the GI tract of mice and rats.The intracellular signal transd...

Although NF-kappaB plays an important role in pancreatitis, mechanisms underlying its activation remain unclear. We investigated the signaling pathways mediating NF-kappaB activation in pancreatic acinar cells induced by high-dose cholecystokinin-8 (CCK-8), which causes pancreatitis in rodent models, and TNF-alpha, which contributes to inflammatory responses of pancreatitis, especially the role of PKC isoforms. We determined subcellular distribution and kinase activities of PKC isoforms and NF-kappaB activation in dispersed rat pancreatic acini. We applied isoform-specific, cell-permeable peptide inhibitors to assess the role of individual PKC isoforms in NF-kappaB activation. Both CCK-8 and TNF-alpha activated the novel isoforms PKC-delta and -epsilon and the atypical isoform PKC-zeta but not the conventional isoform PKC-alpha. Inhibition of the novel PKC isoforms but not the conventional or the atypical isoform resulted in the prevention of NF-kappaB activation induced by CCK-8 and TNF-alpha. NF-kappaB activation by CCK-8 and TNF-alpha required translocation but not tyrosine phosphorylation of PKC-delta. Activation of PKC-delta, PKC-epsilon, and NF-kappaB with CCK-8 involved both phosphatidylinositol-specific PLC and phosphatidylcholine (PC)-specific PLC, whereas with TNF-alpha they only required PC-specific PLC for activation. Results indicate that CCK-8 and TNF-alpha initiate NF-kappaB activation by different PLC pathways that converge at the novel PKCs (delta and epsilon) to mediate NF-kappaB activation in pancreatic acinar cells. These findings suggest a key role for the novel PKCs in pancreatitis.

Background The unpredictable nature of peptide binding to surfaces requires optimization of experimental containers to be utilized. Objective To demonstrate the variable recoveries of peptides from multiple surfaces commonly employed in peptide research by testing the recovery of radiolabeled 125I-endocrine peptides under different conditions and provide guidelines for determining the surfaces to use for other peptides. Methods 125I-labeled peptides (ghrelin, sulfated cholecystokinin-8, corticotropin releasing factor, glucagon-like peptide-1 (GLP-1), insulin, leptin, nesfatin-1, peptide YY) representing a wide spectrum in net charge, size, end groups and modifications were incubated for 48h in glass and plastic tubes untreated or coated with siliconizing fluid. Best surfaces were chosen and peptides incubated with bovine serum albumin (BSA, 1%) with or without subsequent lyophilization. Recovery of 125I-peptides was determined by γ-counting. Results Important differences in 125I-peptide binding capacities to various types of surfaces exist. Siliconization decreased while addition of BSA improved recovery from surfaces tested. Lyophilizing solutions containing 125I-peptides and BSA in the tubes best suited for individual peptides rendered >89% recovery for all peptides. Ghrelin specifically displaced 125I-ghrelin from borosilicate glass while GLP-1 and Fmoc-arginine did not. Conclusion Choosing the appropriate experimental container avoids unpredictable peptide loss resulting in inaccurate measurements and false conclusions.