Effect of 3-5 monocyclizations of angiotensin II and 4-aminoPhe6-Ang II on AT2 receptor affinity

The endogenous angiotensin II (Ang II) and the synthetic AT(2) selective agonist 4-aminoPhe(6)-Ang II respond very differently to identical cyclizations. Cyclizations of Ang II by thioacetalization, involving the 3 and 5 amino acid residue side chains, provided ligands with almost equipotent binding affinities to Ang II at the AT(2) receptor. In contrast, the same cyclization procedures applied on the AT(2) selective 4-aminoPhe(6)-Ang II delivered significantly less potent AT(2) receptor ligands, although the AT(2)/AT(1) selectivity was still very high. The fact that different structure-activity relationships are observed after imposing conformational restrictions on Ang II and 4-aminoPhe(6)-Ang II, respectively, suggests that the peptides, despite large similarities might adopt quite different backbone conformations when binding to the AT(2) receptor.     

HLM1, an essential signaling component in the hypersensitive response,is a member of the cyclic nucleotide-gated channel ion channel family

The hypersensitive response (HR) in plants is a programmed cell death that is commonly associated with disease resistance. A novel mutation in Arabidopsis, hlm1, which causes aberrant regulation of cell death, manifested by a lesion-mimic phenotype and an altered HR, segregated as a single recessive allele. Broad-spectrum defense mechanisms remained functional or were constitutive in the mutant plants, which also exhibited increased resistance to a virulent strain of Pseudomonas syringae pv tomato. In response to avirulent strains of the same pathogen, the hlm1 mutant showed differential abilities to restrict bacterial growth, depending on the avirulence gene expressed by the pathogen. The HLM1 gene encodes a cyclic nucleotide-gated channel, CNGC4. Preliminary study of the HLM1/CNGC4 gene pro-duct in Xenopus oocytes (inside-out patch-clamp technique) showed that CNGC4 is permeable to both K(+) and Na(+) and is activated by both cGMP and cAMP. HLM1 gene expression is induced in response to pathogen infection and some pathogen-related signals. Thus, HLM1 might constitute a common downstream component of the signaling pathways leading to HR/resistance.     

Superantigen-induced regulatory T cells display different suppressive functions in the presence or absence of natural CD4+CD25+ regulatory T cells in vivo

Repeated exposures to both microbial and innocuous Ags in vivo have been reported to both eliminate and tolerize T cells after their initial activation and expansion. The remaining tolerant T cells have been shown to suppress the response of naive T cells in vitro. This feature is reminiscent of natural CD4(+)CD25(+) regulatory T cells. However, it is not known whether the regulatory function of in vivo-tolerized T cells is similar to the function of natural CD4(+)CD25(+) regulatory T cells. In this study, we demonstrate that CD4(+)CD25(+) as well as CD4(+)CD25(-) T cells isolated from mice treated with superantigen three consecutive times to induce tolerance were functionally comparable to natural CD4(+)CD25(+) regulatory T cells, albeit more potent. The different subpopulations of in vivo-tolerized CD4(+) T cells efficiently down-modulated costimulatory molecules on dendritic cells, and their suppressive functions were strictly cell contact dependent. Importantly, we demonstrate that conventional CD4(+)CD25(-) T cells could also be induced to acquire regulatory functions by the same regimen in the absence of natural regulatory T cells in vivo, but that such regulatory cells were functionally different.     

Keratinocyte growth factor stimulates migration and hyaluronan synthesis in the epidermis by activation of keratinocyte hyaluronan synthases 2 and 3

Keratinocyte growth factor (KGF) activates keratinocyte migration and stimulates wound healing. Hyaluronan, an extracellular matrix glycosaminoglycan that accumulates in wounded epidermis, is known to promote cell migration, suggesting that increased synthesis of hyaluronan might be associated with the KGF response in keratinocytes. Treatment of monolayer cultures of rat epidermal keratinocytes led to an elongated and lifted cell shape, increased filopodial protrusions, enhanced cell migration, accumulation of intermediate size hyaluronan in the culture medium and within keratinocytes, and a rapid increase of hyaluronan synthase 2 (Has2) mRNA, suggesting a direct influence on this gene. In stratified, organotypic cultures of the same cell line, both Has2 and Has3 with the hyaluronan receptor CD44 were up-regulated and hyaluronan accumulated in the epidermis, the spinous cell layer in particular. At the same time the expression of the early differentiation marker keratin 10 was inhibited, whereas filaggrin expression and epidermal permeability were less affected. The data indicate that Has2 and Has3 belong to the targets of KGF in keratinocytes, and support the idea that enhanced hyaluronan synthesis acts an effector for the migratory response of keratinocytes in wound healing, whereas it may delay keratinocyte terminal differentiation.     

Control of endothelial cell proliferation by calcium influx and arachidonic acid metabolism: a pharmacological approach

In physiological conditions, endothelial cell proliferation is strictly controlled by several growth factors, among which bFGF and VEGF are the most effective. Both bind to specific tyrosine kinase receptors and trigger intracellular signal cascades. In particular, bFGF stimulates the release of arachidonic acid (AA) and its metabolites in many types of endothelial cells in culture. In bovine aortic endothelial cells, it has been suggested that AA is released by the recruitment of cytosolic phospholipase A2 (cPLA2). AA metabolites are involved in the control of both endothelial cell motility (mostly via the cyclooxygenase pathway) and proliferation (via the lipoxygenase (LOX) cascade). On the other hand, evidence has been provided for a proliferative role of AA-induced calcium influx. By using a pharmacological approach, we have tried to elucidate the contribution to bovine aortic endothelial proliferation of the different pathways leading to production of AA and its metabolites. Two main informations were obtained by our experiments: first, AA release is not entirely due to cPLA2 involvement, but also to DAG lipase recruitment; second, cyclooxygenase derivatives play a role in the control of cell proliferation, and not only of motility. Moreover, by combining proliferation assays and single cell calcium measurements, we show that the blocking effect of carboxyamido-triazole (CAI), an inhibitor of tumor growth and angiogenesis acting on calcium influx-dependent pathways, including AA metabolism, is at least in part due to a direct effect on AA-induced calcium influx.     

Mutagenic and genotoxic evaluation of bisphenol F diglycidyl ether (BFDGE) in prokaryotic and eukaryotic systems

The epoxy resin bisphenol F diglycidyl ether (BFDGE), was examined for its mutagenicity in prokaryotic assays (Salmonella typhimurium His(-) and Escherichia coli Trp(-) tests) and its genotoxicity in eukaryotic systems (sister chromatid exchange (SCE) and micronucleus tests in human lymphocytes), in the presence or absence of an exogenous metabolizing system (S9 from rat liver). In the prokaryotic tests, the concentrations of BFDGE ranged between 100 and 5000 micro g per plate, and in the eukaryotic assays from 12.5 to 62.5 micro g/ml. The compound is able to induce mutagenic effects in bacterial strains TA100, TA1535, WP2uvrA and IC3327, as revealed by the increase observed in the number of induced revertants. With respect to the genotoxicity assays, BFDGE induces an increase in the frequency of sister chromatid exchanges and micronuclei in human peripheral blood lymphocytes.     

Metmyoglobin and methemoglobin catalyze the isomerization of peroxynitrite to nitrate

Hemoproteins, in particular, myoglobin and hemoglobin, are among the major targets of peroxynitrite in vivo. The oxygenated forms of these proteins are oxidized by peroxynitrite to their corresponding iron(iii) forms (metMb and metHb). This reaction has previously been shown to proceed via the corresponding oxoiron(iv) forms of the proteins. In this paper, we have conclusively shown that metMb and metHb catalyze the isomerization of peroxynitrite to nitrate. The catalytic rate constants were determined by stopped-flow spectroscopy in the presence and absence of 1.2 mM CO(2) at 20 and 37 degrees C. The values obtained for metMb and metHb, with no added CO(2) at pH 7.0 and 20 degrees C, are (7.7 +/- 0.1) x 10(4) and (3.9 +/- 0.2) x 10(4) M(-1) s(-1), respectively. The pH-dependence of the catalytic rate constants indicates that HOONO is the species that reacts with the iron(iii) center of the proteins. In the presence of 1.2 mM CO(2), metMb and metHb also accelerate the decay of peroxynitrite in a concentration-dependent way. However, experiments carried out at pH 8.3 in the presence of 10 mM CO(2) suggest that ONOOCO(2)(-), the species generated from the reaction of ONOO(-) with CO(2), does not react with the iron(iii) center of Mb and Hb. Finally, we showed that different forms of Mb and Hb protect free tyrosine from peroxynitrite-mediated nitration. The order of efficiency is metMbCN < apoMb < metHb < metMb < ferrylMb < oxyHb < deoxyHb < oxyMb. Taken together, our data show that myoglobin is always a better scavenger than hemoglobin. Moreover, the globin offers very little protection, as the heme-free (apoMb) and heme-blocked (metMbCN) forms only partly prevent nitration of free tyrosine.     

Metabolic efficiency of liver mitochondria in rats with decreased thermogenesis

We have studied changes in hepatic mitochondrial efficiency induced by 24-h fasting or acclimation at 29 degrees C, two conditions of reduced thermogenesis. Basal and palmitate-induced proton leak, which contribute to mitochondrial efficiency, are not affected after 24-h fasting, when serum free triiodothyronine decreases significantly and serum free fatty acids increase significantly. In rats at 29 degrees C, in which serum free triiodothyronine and fatty acids decrease significantly, basal proton leak increases significantly, while no variation is found in palmitate-induced proton leak. The present results indicate that mitochondrial efficiency in the liver is not related to a physiological decrease in whole body thermogenesis.     

Vitamin C prevents hyperoxia-mediated vasoconstriction and impairment of endothelium-dependent vasodilation

High arterial blood oxygen tension increases vascular resistance, possibly related to an interaction between reactive oxygen species and endothelium-derived vasoactive factors. Vitamin C is a potent antioxidant capable of reversing endothelial dysfunction due to increased oxidant stress. We tested the hypotheses that hyperoxic vasoconstriction would be prevented by vitamin C, and that acetylcholine-mediated vasodilation would be blunted by hyperoxia and restored by vitamin C. Venous occlusion strain gauge plethysmography was used to measure forearm blood flow (FBF) in 11 healthy subjects and 15 congestive heart failure (CHF) patients, a population characterized by endothelial dysfunction and oxidative stress. The effect of hyperoxia on FBF and derived forearm vascular resistance (FVR) at rest and in response to intra-arterial acetylcholine was recorded. In both healthy subjects and CHF patients, hyperoxia-mediated increases in basal FVR were prevented by the coinfusion of vitamin C. In healthy subjects, hyperoxia impaired the acetylcholine-mediated increase in FBF, an effect also prevented by vitamin C. In contrast, hyperoxia had no effect on verapamil-mediated increases in FBF. In CHF patients, hyperoxia did not affect FBF responses to acetylcholine or verapamil. The addition of vitamin C during hyperoxia augmented FBF responses to acetylcholine. These results suggest that hyperoxic vasoconstriction is mediated by oxidative stress. Moreover, hyperoxia impairs acetylcholine-mediated vasodilation in the setting of intact endothelial function. These effects of hyperoxia are prevented by vitamin C, providing evidence that hyperoxia-derived free radicals impair the activity of endothelium-derived vasoactive factors.