Escherichia coli flavohemoglobin is an efficient alkylhydroperoxide reductase

Escherichia coli flavohemoglobin (HMP) is shown to be capable of catalyzing the reduction of several alkylhydroperoxide substrates into their corresponding alcohols using NADH as an electron donor. In particular, HMP possesses a high catalytic activity and a low Km toward cumyl, linoleic acid, and tert-butyl hydroperoxides, whereas it is a less efficient hydrogen peroxide scavenger. An analysis of UV-visible spectra during the stationary state reveals that at variance with classical peroxidases, HMP turns over in the ferrous state. In particular, an iron oxygen adduct intermediate whose spectrum is similar to that reported for the oxo-ferryl derivative in peroxidases (Compound II), has been identified during the catalysis of hydrogen peroxide reduction. This finding suggests that hydroperoxide cleavage occurs upon direct binding of a peroxide oxygen atom to the ferrous heme iron. Competitive inhibition of the alkylhydroperoxide reductase activity by carbon monoxide has also been observed, thus confirming that heme iron is directly involved in the catalytic mechanism of hydroperoxide reduction. The alkylhydroperoxide reductase activity taken together with the unique lipid binding properties of HMP suggests that this protein is most likely involved in the repair of the lipid membrane oxidative damage generated during oxidative/nitrosative stress.     

Muscarinic subtypes profile modulation within a series of new antagonists,bridged bicyclic derivatives of 2,2-diphenyl-[1,3]-dioxolan-4-ylmethyl-dimethylamine

A set of new muscarinic antagonists, bridged bicyclic derivatives of 2,2-diphenyl-[1,3]-dioxolan-4-ylmethyl-dimethylamine (1), was synthesized and tested to evaluate their affinity and selectivity for M(1), M(2), M(3) and M(4) receptor subtypes. The conformational constraint of 1 in a bicyclic structure, and the variation in distance and stereochemistry of the active functions allowed us to modulate the selectivity of interaction with the M(1)-M(3) receptor subtypes. The most interesting compound was (cis,trans)-2-(2,2-diphenylethyl)-5-methyl-tetrahydro-[1,3]dioxolo[4,5-c]pyrrole oxalate (6), which is equipotent with Pirenzepine on rabbit vas deferens (M(1)-putative) but shows a better selectivity profile.     

CD80+Gr-1+ myeloid cells inhibit development of antifungal Th1 immunity in mice with candidiasis

To find out whether polymorphonuclear neutrophils (PMN), abundantly recruited in disseminated Candida albicans infection, could directly affect the activation of Th cells we addressed the issues as to whether murine PMN, like their human counterparts, express costimulatory molecules and the functional consequence of this expression in terms of antifungal immune resistance. To this purpose, we assessed 1) the expression of CD80 (B7-1) and CD86 (B7-2) molecules on peripheral, splenic, and inflammatory murine Gr-1+ PMN; 2) its modulation upon interaction with C. albicans in vitro, in vivo, and in human PMN; 3) the effect of Candida exposure on the ability of murine PMN to affect CD4+ Th1 cell proliferation and cytokine production; and 4) the mechanism responsible for this effect. Murine PMN constitutively expressed CD80 molecules on both the surface and intracellularly; however, in both murine and human PMN, CD80 expression was differentially modulated upon interaction with Candida yeasts or hyphae in vitro as well as in infected mice. The expression of the CD86 molecule was neither constitutive nor inducible upon exposure to the fungus. In vitro, Gr-1+ PMN were found to inhibit the activation of IFN-gamma-producing CD4+ T cells and to induce apoptosis through a CD80/CD28-dependent mechanism. A population of CD80+Gr-1+ myeloid cells was found to be expanded in conventional as well as in bone marrow-transplanted mice with disseminated candidiasis, but its depletion increased the IFN-gamma-mediated antifungal resistance. These data indicate that alternatively activated PMN expressing CD80 may adversely affect Th1-dependent resistance in fungal infections.     

Expression of the Extracellular Fatty Acid Binding Protein (Ex-FABP) during Muscle Fiber Formationin Vivoandin Vitro

We report that Ex-FABP, an extracellular protein belonging to the lipocalin family and involved in the extracellular transport of long-chain fatty acids, is expressed in the forming myotubes bothin vivoandin vitro.The presence of the protein and of the mRNA was observed in newly formed myotubes at early stages of chick embryo development by immunohistochemistry and byin situhybridization. At later stages of development myofibers still expressed both the mRNA and the protein. Ex-FABP expression was observed also in the developing myocardium and the muscular layer of large blood vessels. In agreement with these findings, an initial expression of the mRNA and protein secretion by cultured chicken myoblasts were observed only after the onset of myoblast fusion. Double-immunofluorescence staining of these cultured cells revealed that multinucleate myotubes were stained by antibodies directed against both the Ex-FABP and the sarcomeric myosin, whereas immature myotubes and single myoblasts were not. When added to cultured myoblasts, antibodies against the Ex-FABP induced a strong enhancement of the production of the same protein. In all experiments some cell sufferance and a transient impairment of myotube formation were also observed. The finding that the continuous removal of the Ex-FABP from the culture medium of myoblasts, due to the formation of immune complexes, resulted in an overproduction of the protein suggests a feedback (autocrine) control during myotube differentiation and maturation. We propose that the requirement for increased transport and metabolism of free fatty acid released from the membrane phospholipids and storage lipids, mediated by Ex-FABP, may be essential during differentiation of multinucleated myotubes or that an increased local demand of fatty acids and metabolites may act as a local hormone in tissues differentiating and undergoing morphogenesis.     

Effect of caffeic acid on tert-butyl hydroperoxide-induced oxidative stress in U937

Nonvitamin phenolic compounds are ubiquitous in food plants and therefore potentially present in human plasma in a diet-dependent concentration. The aim of this study was to evaluate the ability of caffeic acid, a phenolic acid with antioxidant activity, to affect cellular response in U937 human monocytic cells to t-butyl hydroperoxide-induced oxidative stress. In our experimental conditions caffeic acid was incorporated into cells without any cytotoxic effect. Caffeic acid-treated cells showed an increased resistance to oxidative challenge, as revealed by an higher percent of survival and the maintenance of an higher proliferative capacity in respect to control cells. This effect seems to be due to the ability of caffeic acid to reduce glutathione depletion and to inhibit lipid peroxidation during tBOOH treatment. It can be concluded that caffeic acid exerts an antioxidant action inside the cell, responsible for the observed modulation of the cellular response to oxidative challenge. Due to its presence in the diet, therefore, caffeic acid may play a role in the modulation of oxidative processes in vivo.     

Susceptibility to Heavy Metals and Cadmium Accumulation in Aerobic and Anaerobic Thermophilic Microorganisms Isolated from Deep-Sea Hydrothermal Vents

Thirty thermophilic strains isolated from heavy metal-rich hydrothermal vent sites at Lau Basin were tested for their susceptibility to cadmium, zinc, cobalt, and nickel. The 14 aerobic spore formers belonging to the genus Bacillus, 6 anaerobic fermenters from the order Thermotogales, and 10 anaerobic sulfur reducers from the order Thermococcales could be clearly distinguished according to their metal susceptibilities. The Thermococcales were found to exhibit the highest resistance to cadmium and zinc, whereas Thermotogales were highly sensitive to these metals. In contrast, the Thermotogales displayed the highest resistance to cobalt ions. No clear distinction could be established between the metal susceptibilities of these strains and seven reference organisms used for comparative studies. Cadmium resistance, slightly inducible in some cadmium-resistant bacilli, was not plasmid mediated. The amount of cadmium immobilized by the Thermotogales was related to their level of resistance to this metal. Received: 24 February 2000 / Accepted: 28 March 2000     

Structure-activity relationship at alpha-adrenergic receptors within a series of imidazoline analogues of cirazoline

Several analogues of cirazoline (2), a selective alpha1-adrenoreceptor agonist, were prepared and their pharmacological profiles studied. Although at the alpha1-adrenoreceptor all the compounds displayed a significant agonist activity, at the alpha2-adrenoreceptor they showed either agonist or antagonist activity depending on the nature of the phenyl substituent. The qualitative structure-activity relationship led us to the conclusion that the oxygen atom in the side-chain is essential for alpha1-agonist activity, while the cyclopropyl ring is not, and may be replaced by several groups. Of the groups studied, isopropoxy appears to be the best. Instead, the same substitution (i.e., isopropoxy for the cyclopropyl ring) at alpha2-adrenoreceptors causes a reversal of activity. On the other hand, the cyclopropyl ring seems to be important for alpha1-selectivity. Compound 20 is the most potent alpha1-agonist of the series, being equiactive with cirazoline on rat vas deferens and in pithed rat.     

Serum and glucocorticoid-regulated kinase Sgk1 inhibits insulin-dependent activation of phosphomannomutase 2 in transfected COS-7 cells

Serum- and glucocorticoid-regulated kinase (Sgk1) is considered to be an essential convergence point for peptide and steroid regulation of ENaC-mediated sodium transport. We tried to identify molecular partners of Sgk1 by yeast two-hybrid screening. Yeast two-hybrid screening showed a specific interaction between Sgk1 and phosphomannomutase (PMM)2, the latter of which is an enzyme involved in the regulation of glycoprotein biosynthesis. The interaction was confirmed in intact cells by coimmunoprecipitation and colocalization detected using confocal microscopy. We were then able to demonstrate that Sgk1 phosphorylated PMM2 in an in vitro assay. In addition, we found that the enzymatic activity of PMM2 is upregulated by insulin treatment and that Sgk1 completely inhibits PMM2 activity both in the absence and in the presence of insulin stimulation. These data provide evidence suggesting that Sgk1 may modulate insulin action on the cotranslational glycosylation of glycoproteins. Sgk; protein glycosylation; CDGIa     

Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector

Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses, to generate multicistronic retroviral vectors with efficient translation of four cistrons. Using the T-cell receptor:CD3 complex as a test system, we show that a single 2A peptide-linked retroviral vector can be used to generate all four CD3 proteins (CD3epsilon, gamma, delta, zeta), and restore T-cell development and function in CD3-deficient mice. We also show complete 2A peptide-mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer-based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.