Pathogenicity in Francisella tularensis subspecies

.Sequencing of the non-pathogenic Francisella tularensis sub-species novicida U112, and comparison with two pathogenic sub-species, provides insights into the evolution of pathogenicity in these species.

Endothelial cells release nitric oxide (NO) acutely in response to increased laminar fluid shear stress, and the increase is correlated with enhanced phosphorylation of endothelial nitric-oxide synthase (eNOS). Phosphoamino acid analysis of eNOS from bovine aortic endothelial cells labeled with [ 32 P]orthophosphate demonstrated that only phosphoserine was present in eNOS under both static and flow conditions. Fluid shear stress induced phosphate incorporation into two specific eNOS tryptic peptides as early as 30 s after initiation of flow. The flow-induced tryptic phosphopeptides were enriched, separated by capillary electrophoresis with intermittent voltage drops, also known as "peak parking," and analyzed by collision-induced dissociation in a tandem mass spectrometer. Two phosphopeptide sequences determined by tandem mass spectrometry, TQpSFSLQER and KLQTRPpSPGPPPAEQLLSQAR, were confirmed as the two flow-dependent phosphopeptides by co-migration with synthetic phosphopeptides. Because the sequence (RIR)TQpSFSLQER contains a consensus substrate site for protein kinase B (PKB or Akt), we demonstrated that LY294002, an inhibitor of the upstream activator of PKB, phosphatidylinositol 3-kinase, inhibited flow-induced eNOS phosphorylation by 97% and NO production by 68%. Finally, PKB phosphorylated eNOS in vitro at the same site phosphorylated in the cell and increased eNOS enzymatic activity by 15-20-fold.Endothelial nitric-oxide synthase (eNOS 1 or type III NOS) is one of three isoenzymes that converts L-arginine to L-citrulline and nitric oxide (NO). Endothelial cells synthesize NO tonically and increase NO production in response to agonists and increased fluid shear stress (FSS). Endothelial NO contributes to blood vessel homeostasis by regulating vessel tone (1), cell growth (2), platelet aggregation (3), and leukocyte binding to endothelium (4). In vivo eNOS is both myristoylated and palmitoylated. These modifications increase eNOS compartmentalization to plasmalemmal caveolae and facilitate release of NO from cells (5-7). In caveolae, which are small plasmalemmal invaginations that sequester signaling proteins (8), eNOS specifically interacts with the scaffolding protein caveolin-1 through a caveolin (9, 10) binding motif (11), located near the domain that binds Ca 2ϩ /calmodulin. Recent studies suggest that the activity of eNOS is regulated in a reciprocal manner through caveolin-1 inhibition and Ca 2ϩ /calmodulin stimulation (12-14).Increased FSS stimulates an increase in free intracellular calcium [Ca 2ϩ ] i from intracellular stores (15, 16) leading to a Ca 2ϩ /calmodulin-dependent increase in eNOS activity. However, recent investigations show that increases in [Ca 2ϩ ] i do not fully explain the rapid rise in NO production in response to FSS (17). Exposure of bovine aortic endothelial cells (BAEC) to 25 dynes/cm 2 FSS for 30 s caused a 7-fold rise in NO production and a corresponding 2-fold increase in eNOS phosphorylation, whereas the calcium ionophore A23187 neither caused rapid NO production...

No abstract

Objective-Free fatty acids (FFA) are commonly elevated in diabetes and obesity and have been shown to impair nitric oxide (NO) production by endothelial cells. However, the signaling pathways responsible for FFA impairment of NO production in endothelial cells have not been characterized. Insulin receptor substrate-1 (IRS-1) regulation is critical for activation of endothelial nitric oxide synthase (eNOS) in response to stimulation by insulin or fluid shear stress. Methods and Results-We demonstrate that insulin-mediated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, eNOS, and NO production are significantly inhibited by treatment of bovine aortic endothelial cells with 100 mol/L FFA composed of palmitic acid for 3 hours before stimulation with 100 nM insulin. This FFA preparation also increases, in a dose-dependent manner, IKK␤ activity, which regulates activation of NF-B, a transcriptional factor associated with inflammation. Similarly, elevation of other common FFA such as oleic and linoleic acid also induce IKK␤ activation and inhibit insulin-mediated eNOS activation. Overexpression of a kinase inactive form of IKK␤ blocks the ability of FFA to inhibit insulin-dependent NO production, whereas overexpression of wild-type IKK␤ recapitulates the effect of FFA on insulin-dependent NO production. Conclusions-Elevated levels of common FFA found in human serum activate IKK␤ in endothelial cells leading to reduced NO production, and thus may serve to link pathways involved in inflammation and endothelial dysfunction. Key Words: diabetes Ⅲ endothelial dysfunction Ⅲ endothelial nitric oxide synthase Ⅲ free fatty acids Ⅲ IKK␤ Ⅲ nitric oxide E ndothelial dysfunction is a hallmark of diabetic vascular disease and can be described as impairment in the generation and function of nitric oxide (NO) as a vasodilator and vascular homeostatic agent. Insulin's physiological action in the vasculature promotes vasodilation through increased NO production and resultant enhanced blood flow may couple metabolic and hemodynamic homeostasis. Insulin increases NO production in endothelial cells through an IRS-1 and phosphatidylinositol 3-kinase (PI3-kinase)-dependent pathway that results in phosphorylation of endothelial nitric oxide synthase (eNOS) by Akt in a calcium-independent manner. 1,2 Similarities have been demonstrated between insulin signaling in endothelial cells and in classic insulin-responsive cells such as skeletal muscle cells, hepatocytes, and adipocytes. Mechanisms of impaired insulin signaling in these better-characterized insulin responsive cells are likely to be relevant to endothelial dysfunction in diabetes that is not well understood. See page 889Metabolic abnormalities found in diabetes and obesity include increases in the circulating levels of cytokines such as tumor necrosis factor-␣ (TNF-␣) and metabolites such as free fatty acids (FFAs), diacylglycerol, and fatty acyl-coenzyme A. Resistance of target tissues to the effects of insulin has been attributed to alteration(s) in cellular resp...