Role of peroxynitrite in the cardiovascular dysfunction of septic shock.

The intense systemic inflammatory response characterizing septic shock is associated with an increased generation of free radicals by multiple cell types in cardiovascular and non cardiovascular tissues. The oxygen-centered radical superoxide anion (O2 .-) rapidly reacts with the nitrogen-centered radical nitric oxide (NO.) to form the potent oxidant species peroxynitrite. Peroxynitrite oxidizes multiple targets molecules, either directly or via the secondary generation of highly reactive radicals, resulting in significant alterations in lipids, proteins and nucleic acids, with significant cytotoxic consequences. The formation of peroxynitrite is a key pathophysiological mechanism contributing to the cardiovascular collapse of septic shock, promoting vascular contractile failure, endothelial and myocardial dysfunction, and is also implicated in the occurrence of multiple organ dysfunction in this setting. The recent development of various porphyrin-based pharmacological compounds accelerating the degradation of peroxynitrite has allowed to specifically address these pathophysiological roles of peroxynitrite in experimental septic shock. Such agents, including 5,10,15,20-tetrakis(4- sulfonatophenyl)porphyrinato iron III chloride (FeTTPs), manganese tetrakis(4-N-methylpyridyl)porphyrin (MnTMPyP), Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether)pyridyl porphyrin) (FP-15) and WW-85, have been shown to improve the cardiovascular and multiple organ failure in small and large animal models of septic shock. Therefore, these findings support the development of peroxynitrite decomposition catalysts as potentially useful novel therapeutic agents to restore cardiovascular function in sepsis.

Graphitized carbon black in quartz tubes for the sampling of indoor air nicotine and analysis by microwave thermal desorption-capillary gas chromatography

Nicotine in a smoky indoor air environment can be determined using graphitized carbon black as a solid sorbent in quartz tubes. The temperature stability, high purity, and heat absorption characteristics of the sorbent, as well as the permeability of the quartz tubes to microwaves, enable the thermal desorption by means of microwaves after active sampling. Permeation and dynamic dilution procedures for the generation of nicotine in the vapor phase at low and high concentrations are used to evaluate the performances of the sampler. Tube preparation is described and the microwave desorption temperature is measured. Breakthrough volume is determined to allow sampling at 0.1-1 L/min for definite periods of time. The procedure is tested for the determination of gas and paticulate phase nicotine in sidestream smoke produced in an experimental chamber.

Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses.

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.

Different concentrations of Mg++ ions affect nuclear matrix protein distribution during thermal stabilization of isolated nuclei.

The nuclear matrix, a proteinaceous network believed to be a scaffolding structure determining higher-order organization of chromatin, is usually prepared from intact nuclei by a series of extraction steps. In most cell types investigated the nuclear matrix does not spontaneously resist these treatments but must be stabilized before the application of extracting agents. Incubation of isolated nuclei at 37C or 42C in buffers containing Mg++ has been widely employed as stabilizing agent. We have previously demonstrated that heat treatment induces changes in the distribution of three nuclear scaffold proteins in nuclei prepared in the absence of Mg++ ions. We studied whether different concentrations of Mg++ (2.0-5 mM) affect the spatial distribution of nuclear matrix proteins in nuclei isolated from K562 erythroleukemia cells and stabilized by heat at either 37C or 42C. Five proteins were studied, two of which were RNA metabolism-related proteins (a 105-kD component of splicing complexes and an RNP component), one a 126-kD constituent of a class of nuclear bodies, and two were components of the inner matrix network. The localization of proteins was determined by immunofluorescent staining and confocal scanning laser microscope. Mg++ induced significant changes of antigen distribution even at the lowest concentration employed, and these modifications were enhanced in parallel with increase in the concentration of the divalent cation. The different sensitivity to heat stabilization and Mg++ of these nuclear proteins might reflect a different degree of association with the nuclear scaffold and can be closely related to their functional or structural role.

Hsp70 and the Cochaperone StiA (Hop) Orchestrate Hsp90-Mediated Caspofungin Tolerance in Aspergillus fumigatus.

Aspergillus fumigatus is the primary etiologic agent of invasive aspergillosis (IA), a major cause of death among immunosuppressed patients. Echinocandins (e.g., caspofungin) are increasingly used as second-line therapy for IA, but their activity is only fungistatic. Heat shock protein 90 (Hsp90) was previously shown to trigger tolerance to caspofungin and the paradoxical effect (i.e., decreased efficacy of caspofungin at higher concentrations). Here, we demonstrate the key role of another molecular chaperone, Hsp70, in governing the stress response to caspofungin via Hsp90 and their cochaperone Hop/Sti1 (StiA in A. fumigatus). Mutation of the StiA-interacting domain of Hsp70 (C-terminal EELD motif) impaired thermal adaptation and caspofungin tolerance with loss of the caspofungin paradoxical effect. Impaired Hsp90 function and increased susceptibility to caspofungin were also observed following pharmacologic inhibition of the C-terminal domain of Hsp70 by pifithrin-μ or after stiA deletion, further supporting the links among Hsp70, StiA, and Hsp90 in governing caspofungin tolerance. StiA was not required for the physical interaction between Hsp70 and Hsp90 but had distinct roles in the regulation of their function in caspofungin and heat stress responses. In conclusion, this study deciphering the physical and functional interactions of the Hsp70-StiA-Hsp90 complex provided new insights into the mechanisms of tolerance to caspofungin in A. fumigatus and revealed a key C-terminal motif of Hsp70, which can be targeted by specific inhibitors, such as pifithrin-μ, to enhance the antifungal activity of caspofungin against A. fumigatus.

Mutations in the heat-shock protein A9 (HSPA9) gene cause the EVEN-PLUS syndrome of congenital malformations and skeletal dysplasia.

We and others have reported mutations in LONP1, a gene coding for a mitochondrial chaperone and protease, as the cause of the human CODAS (cerebral, ocular, dental, auricular and skeletal) syndrome (MIM 600373). Here, we delineate a similar but distinct condition that shares the epiphyseal, vertebral and ocular changes of CODAS but also included severe microtia, nasal hypoplasia, and other malformations, and for which we propose the name of EVEN-PLUS syndrome for epiphyseal, vertebral, ear, nose, plus associated findings. In three individuals from two families, no mutation in LONP1 was found; instead, we found biallelic mutations in HSPA9, the gene that codes for mHSP70/mortalin, another highly conserved mitochondrial chaperone protein essential in mitochondrial protein import, folding, and degradation. The functional relationship between LONP1 and HSPA9 in mitochondrial protein chaperoning and the overlapping phenotypes of CODAS and EVEN-PLUS delineate a family of "mitochondrial chaperonopathies" and point to an unexplored role of mitochondrial chaperones in human embryonic morphogenesis.