Down-regulation of Rac activity during beta 2 integrin-mediated adhesion of human neutrophils

In human neutrophils, beta2 integrin engagement mediated a decrease in GTP-bound Rac1 and Rac2. Pretreatment of neutrophils with LY294002 or PP1 (inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases, respectively) partly reversed the beta2 integrin-induced down-regulation of Rac activities. In contrast, beta2 integrins induced stimulation of Cdc42 that was independent of Src family members. The PI 3-kinase dependence of the beta2 integrin-mediated decrease in GTP-bound Rac could be explained by an enhanced Rac-GAP activity, since this activity was blocked by LY204002, whereas PP1 only had a minor effect. The fact that only Rac1 but not Rac2 (the dominating Rac) redistributed to the detergent-insoluble fraction and that it was independent of GTP loading excludes the possibility that down-regulation of Rac activities was due to depletion of GTP-bound Rac from the detergent-soluble fraction. The beta2 integrin-triggered relocalization of Rac1 to the cytoskeleton was enabled by a PI 3-kinase-induced dissociation of Rac1 from LyGDI. The dissociations of Rac1 and Rac2 from LyGDI also explained the PI 3-kinase-dependent translocations of Rac GTPases to the plasma membrane. However, these accumulations of Rac in the membrane, as well as that of p47phox and p67phox, were also regulated by Src tyrosine kinases. Inasmuch as Rac GTPases are part of the NADPH oxidase and the respiratory burst is elicited in neutrophils adherent by beta2 integrins, our results indicate that activation of the NADPH oxidase does not depend on the levels of Rac-GTP but instead requires a beta2 integrin-induced targeting of the Rac GTPases as well as p47phox and p67phox to the plasma membrane.     

Hypoxia inducible factor activates the transforming growth factor-alpha/epidermal growth factor receptor growth stimulatory pathway in VHL(-/-) renal cell carcinoma cells

Bi-allelic-inactivating mutations of the VHL tumor suppressor gene are found in the majority of clear cell renal cell carcinomas (VHL(-/-) RCC). VHL(-/-) RCC cells overproduce hypoxia-inducible genes as a consequence of constitutive, oxygen-independent activation of hypoxia inducible factor (HIF). While HIF activation explains the highly vascularized nature of VHL loss lesions, the relative role of HIF in oncogenesis and loss of growth control remains unknown. Here, we report that HIF plays a central role in promoting unregulated growth of VHL(-/-) RCC cells by activating the transforming growth factor-alpha (TGF-alpha)/epidermal growth factor receptor (EGF-R) pathway. Dominant-negative HIF and enzymatic inhibition of EGF-R were equally efficient at abolishing EGF-R activation and serum-independent growth of VHL(-/-) RCC cells. TGF-alpha is the only known EGF-R ligand that has a VHL-dependent expression profile and its overexpression by VHL(-/-) RCC cells is a direct consequence of HIF activation. In contrast to TGF-alpha, other HIF targets, including vascular endothelial growth factor (VEGF), were unable to stimulate serum-independent growth of VHL(-/-) RCC cells. VHL(-/-) RCC cells expressing reintroduced type 2C mutants of VHL, and which retain the ability to degrade HIF, fail to overproduce TGF-alpha and proliferate in serum-free media. These data link HIF with the overproduction of a bona fide renal cell mitogen leading to activation of a pathway involved in growth of renal cancer cells. Moreover, our results suggest that HIF might be involved in oncogenesis to a much higher extent than previously appreciated.     

Food effects on the absorption and pharmacokinetics of cocoa flavanols

Macronutrients in food and gastric acid are known to have a pronounced effect on the metabolism of many xenobiotics, an effect that impacts their efficacy as bioactive agents. In this investigation we assessed the impact of select food treatments and the histamine H(2)-receptor antagonist Famotidine (Pepcid-AC) on flavanol absorption and metabolism. Four crossover intervention studies were conducted with 6 subjects each. Volunteers consumed sugar-free, flavanol-rich cocoa (0.125 g/kg body wt) alone, with macronutrient-rich foods (8.75 or 17.5 kJ/kg subject body wt) or Famotidine (Pepcid-AC). Blood samples were drawn at 5 time points including baseline. Plasma samples were analyzed for epicatechin and catechin flavanols by HPLC. Pharmacokinetic parameters were assessed using non-compartmental methodology. When provided at 17.5 kJ/kg subject body weight (approximately 4 kcal/kg), sugar and bread test meals increased flavanol area under the curve (AUC) values to 140% of control values (P < 0.05). A corresponding tendency for plasma antioxidant capacity to increase was observed for the cocoa treatment at 1.5 and 2.5 h (P < 0.17, P < 0.06, respectively). The ability of treatment meals to affect AUC values was positively correlated with treatment carbohydrate content (r = 0.83; P< 0.02). In contrast to carbohydrate rich meals, lipid and protein rich meals and Famotidine treatment had minimal effects on flavanol absorption. Based on C(max) and AUC values, this data suggests that the uptake of flavanols can be increased significantly by concurrent carbohydrate consumption.     

Stability of the I-motif structure is related to the interactions between phosphodiester backbones

The i-motif DNA tetrameric structure is formed of two parallel duplexes intercalated in a head-to-tail orientation, and held together by hemiprotonated cytosine pairs. The four phosphodiester backbones forming the structure define two narrow and wide grooves. The short interphosphate distances across the narrow groove induce a strong repulsion which should destabilize the tetramer. To investigate this point, molecular dynamics simulations were run on the [d(C2)]4 and [d(C4)]4 tetramers in 3'E and 5'E topologies, for which the interaction of the phosphodiester backbones through the narrow groove is different. The analysis of the simulations, using the Molecular Mechanics Generalized Born Solvation Area and Molecular Mechanics Poisson-Boltzmann Solvation Area approaches, shows that it is the van der Waals energy contribution which displays the largest relative difference between the two topologies. The comparison of the solvent-accessible area of each topology reveals that the sugar-sugar interactions account for the greater stability of the 3'E topology. This stresses the importance of the sugar-sugar contacts across the narrow groove which, enforcing the optimal backbone twisting, are essential to the base stacking and the i-motif stability. Tighter interactions between the sugars are observed in the case of N-type sugar puckers.     

Increased resistance of neutrophils to deformation upon cooling and rate of recovery on rewarming

Increase in the resistance to deformation of neutrophils upon exposure to the cold may impair their passage through microvessels. However, the potential for such rheological changes to cause prolonged microvascular obstruction in cooled tissue will depend on whether and at what rate the neutrophils recover on rewarming. We tested the ability of neutrophils to pass through micropore filters, and found that neutrophils cooled to 10 degrees C for 10-20 minutes could block either 5 microm or 8 microm pore filters. On return to 37 degrees C, flow resistance remained impaired briefly but recovered over about 5 minutes. The kinetics of changes in flow resistance in the cold and on rewarming were linked to kinetics of actin polymerisation during these periods. However, they were not closely linked to distortion of cell shape in the cold, which recovered only slowly with rewarming. The results suggest that while rigid neutrophils might occlude capillaries in cold tissue, mechanical obstruction should not be long-lived on rewarming. Moreover, rigid neutrophils washed out of cold tissue should experience only temporary mechanical trapping in remote tissues.     

Characterization of extradiol dioxygenases from a polychlorinated biphenyl-degrading strain that possess higher specificities for chlorinated metabolites

Recent studies demonstrated that 2,3-dihydroxybiphenyl 1,2-dioxygenase from Burkholderia sp. strain LB400 (DHBDLB400; EC 1.13.11.39) cleaves chlorinated 2,3-dihydroxybiphenyls (DHBs) less specifically than unchlorinated DHB and is competitively inhibited by 2',6'-dichloro-2,3-dihydroxybiphenyl (2',6'-diCl DHB). To determine whether these are general characteristics of DHBDs, we characterized DHBDP6-I and DHBDP6-III, two evolutionarily divergent isozymes from Rhodococcus globerulus strain P6, another good polychlorinated biphenyl (PCB) degrader. In contrast to DHBDLB400, both rhodococcal enzymes had higher specificities for some chlorinated DHBs in air-saturated buffer. Thus, DHBDP6-I cleaved the DHBs in the following order of specificity: 6-Cl DHB > 3'-Cl DHB approximately DHB approximately 4'-Cl DHB > 2'-Cl DHB > 4-Cl DHB > 5-Cl DHB. It also cleaved its preferred substrate, 6-Cl DHB, three times more specifically than DHB. Interestingly, some of the worst substrates for DHBDP6-I were among the best for DHBDP6-III (4-Cl DHB > 5-Cl DHB approximately 6-Cl DHB approximately 3'-Cl DHB > DHB > 2'-Cl DHB approximately 4'-Cl DHB; DHBDP6-III cleaved 4-Cl DHB two times more specifically than DHB). Generally, each of the monochlorinated DHBs inactivated the enzymes more rapidly than DHB. The exceptions were 4-Cl DHB for DHBDP6-I and 2'-Cl DHB for DHBDP6-III. As observed in DHBDLB400, chloro substituents influenced the reactivity of the dioxygenases with O2. For example, the apparent specificities of DHBDP6-I and DHBDP6-III for O2 in the presence of 2'-Cl DHB were lower than those in the presence of DHB by factors of >60 and 4, respectively. DHBDP6-I and DHBDP6-III shared the relative inability of DHBDLB400 to cleave 2',6'-diCl DHB (apparent catalytic constants of 0.088 +/- 0.004 and 0.069 +/- 0.002 s(-1), respectively). However, these isozymes had remarkably different apparent K(m) values for this compound (0.007 +/- 0.001, 0.14 +/- 0.01, and 3.9 +/- 0.4 micro M for DHBDLB400, DHBDP6-I, and DHBDP6-III, respectively). The markedly different reactivities of DHBDP6-I and DHBDP6-III with chlorinated DHBs undoubtedly contribute to the PCB-degrading activity of R. globerulus P6.     

Membrane topology of a metabotropic glutamate receptor

The metabotropic glutamate receptors (mGluRs) have been predicted to have a classical seven transmembrane domain structure similar to that seen for members of the G-protein-coupled receptor (GPCR) superfamily. However, the mGluRs (and other members of the family C GPCRs) show no sequence homology to the rhodopsin-like GPCRs, for which this seven transmembrane domain structure has been experimentally confirmed. Furthermore, several transmembrane domain prediction algorithms suggest that the mGluRs have a topology that is distinct from these receptors. In the present study, we set out to test whether mGluR5 has seven true transmembrane domains. Using a variety of approaches in both prokaryotic and eukaryotic systems, our data provide strong support for the proposed seven transmembrane domain model of mGluR5. We propose that this membrane topology can be extended to all members of the family C GPCRs.     

PPAR-gamma ligands modulate effects of LPS in stimulated rat synovial fibroblasts

This work demonstrated the constitutive expressionof peroxisome proliferator-activated receptor (PPAR)- and PPAR-in rat synovial fibroblasts at both mRNA and protein levels. A decrease in PPAR- expression induced by 10 µg/ml lipopolysaccharide (LPS) was observed, whereas PPAR- mRNA expression was not modified. 15-Deoxy-^12,14-prostaglandin J₂(15d-PGJ₂) dose-dependently decreased LPS-induced cyclooxygenase (COX)-2 (80%) and inducible nitric oxide synthase (iNOS) mRNA expression (80%), whereas troglitazone (10 µM) only inhibited iNOS mRNA expression (50%). 15d-PGJ₂ decreasedLPS-induced interleukin (IL)-1 (25%) and tumor necrosis factor(TNF)- (40%) expression. Interestingly, troglitazone stronglydecreased TNF- expression (50%) but had no significant effect onIL-1 expression. 15d-PGJ₂ was able to inhibitDNA-binding activity of both nuclear factor (NF)-B and AP-1.Troglitazone had no effect on NF-B activation and was shown toincrease LPS-induced AP-1 activation. 15d-PGJ₂ andtroglitazone modulated the expression of LPS-induced iNOS, COX-2, andproinflammatory cytokines differently. Indeed, troglitazone seems tospecifically target TNF- and iNOS pathways. These results offer newinsights in regard to the anti-inflammatory potential of the PPAR-ligands and underline different mechanisms of action of15d-PGJ₂ and troglitazone in synovial fibroblasts.

     

Protease-activated receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway

Trypsin and mast cell tryptase can signal to epithelial cells, myocytes, and nerve fibers of the respiratory tract by cleaving proteinase-activated receptor 2 (PAR2). Since tryptase inhibitors are under development to treat asthma, a precise understanding of the contribution of PAR2 to airway inflammation is required. We examined the role of PAR2 in allergic inflammation of the airway by comparing OVA-sensitized and -challenged mice lacking or overexpressing PAR2. In wild-type mice, immunoreactive PAR2 was detected in airway epithelial cells and myocytes, and intranasal administration of a PAR2 agonist stimulated macrophage infiltration into bronchoalveolar lavage fluid. OVA challenge of immunized wild-type mice stimulated infiltration of leukocytes into bronchoalveolar lavage and induced airway hyperreactivity to inhaled methacholine. Compared with wild-type animals, eosinophil infiltration was inhibited by 73% in mice lacking PAR2 and increased by 88% in mice overexpressing PAR2. Similarly, compared with wild-type animals, airway hyperreactivity to inhaled methacholine (40 micro g/ml) was diminished 38% in mice lacking PAR2 and increased by 52% in mice overexpressing PAR2. PAR2 deletion also reduced IgE levels to OVA sensitization by 4-fold compared with those of wild-type animals. Thus, PAR2 contributes to the development of immunity and to allergic inflammation of the airway. Our results support the proposal that tryptase inhibitors and PAR2 antagonists may be useful therapies for inflammatory airway disease.