The f luidity of Synechocystis membranes was adjusted in vivo by temperature acclimation, addition of f luidizer agent benzyl alcohol, or catalytic lipid hydrogenation specific to plasma membranes. The reduced membrane physical order in thylakoids obtained by either downshifting growth temperature or administration of benzyl alcohol was paralleled with enhanced thermosensitivity of the photosynthetic membrane. Simultaneously, the stress-sensing system leading to the cellular heat shock (HS) response also has been altered. There was a close correlation between thylakoid f luidity levels, monitored by steady-state 1,6-diphenyl-1,3,5-hexatriene anisotropy, and threshold temperatures required for maximal activation of all of the HS-inducible genes investigated, including dnaK, groESL, cpn60, and hsp17. The causal relationship between the pre-existing thylakoid physical order and temperature set point of both the transcriptional activation and the de novo protein synthesis was the most striking for the 17-kDa HS protein (HSP17) associated mostly with the thylakoid membranes. These findings together with the fact that the in vivo modulation of lipid saturation within cytoplasmic membrane had no effect on HS response suggest that thylakoid acts as a cellular thermometer where thermal stress is sensed and transduced into a cellular signal leading to the activation of HS genes.

Background-The role of NO in the mechanism of preconditioning is not understood. Therefore, we studied the effect of preconditioning and subsequent ischemia/reperfusion on myocardial NO content in the presence of an NO synthase (NOS) inhibitor. Methods and Results-Isolated working rat hearts were subjected to preconditioning protocols of 3 intermittent periods of rapid pacing or no-flow ischemia of 5 minutes' duration each followed by a test 30 minutes of global no-flow ischemia and 15 minutes of reperfusion. Test ischemia/reperfusion resulted in a deterioration of myocardial function and a considerable increase in cardiac NO content as assessed by electron spin resonance. Preconditioning improved postischemic myocardial function and markedly decreased test ischemia/reperfusion-induced NO accumulation. In the presence of 4.6 mol/L N G -nitro-L-arginine (LNA), basal cardiac NO content decreased significantly, although test ischemia/reperfusion-induced functional deterioration and NO accumulation were not affected in nonpreconditioned hearts. However, the protective effects of preconditioning on both test ischemia/reperfusion-induced functional depression and NO accumulation were abolished. When 4.6 mol/L LNA was administered after preconditioning, it failed to block the effect of preconditioning. In the presence of 46 mol/L LNA, ischemia/reperfusion-induced NO accumulation was significantly decreased and postischemic myocardial function was improved in nonpreconditioned hearts. Conclusions-Our results show that (1) although NO synthesis by the heart is necessary to trigger classic preconditioning, preconditioning in turn attenuates the accumulation of NO during ischemia/reperfusion, and (2)

The mitochondrial protein horse heart cytochrome c was specifically spin-labeled with succinimidyl-2,2,5, 5-tetramethyl-3-pyrroline-1-oxyl-carboxylate on different lysine residues at positions 86, 87, 72, 8, or 25, respectively. Site-specifically labeled species were separated chromatographically and identified by peptide sequencing of tryptic digests. The monolabeled protein was bound to negatively charged phospholipid membranes composed of dioleoylphosphatidylglycerol, and the accessibility of the spin-labeled lysine residues to lipid-soluble molecular oxygen and to lipid-impermeant chromium maltolate was determined from the saturation properties of the ESR spectra. The accessibilities of the spin-labeled proteins relative to those obtained for phospholipids spin-labeled in the headgroup region, in the presence of unlabeled protein, identify the position of the spin-labeled lysine residues relative to the phospholipid bilayer surface. We have found that cytochrome c does not penetrate into the membrane interior and that the active side of cytochrome c in the protein-membrane interaction is the side on which lys86, lys87, and lys72 are located.

Electron microscopic analysis of gap-junction-like structures isolated from an arthropod (Nephrops nowegicus) and composed of a 16-kDa polypeptide, show the functionaI unit to be a star-shaped hexamer of protein arranged around a central channel which runs perpendicular to the plane of the membrane. Estimations of the molecular volume carried out on an averaged projection are consistent with a subunit mass of 16-18 kDa. Fourier transform infrared spectroscopy indicates a high a-helical content for the protein, supporting secondary-structure predictions of four transmembrane a helices/monomer. The averaged projection shows a close resemblance to a hexamer of the 16-kDa protein built on the basis of a four a-helical bundle [Finbow,

Although the physiological regulatory function of the gasotransmitter NO (a diatomic free radical) was discovered decades ago, NO is still in the frontline research in biomedicine. NO has been implicated in a variety of physiological and pathological processes; therefore, pharmacological modulation of NO levels in various tissues may have significant therapeutic value. NO is generated by NOS in most of cell types and by non-enzymatic reactions. Measurement of NO is technically difficult due to its rapid chemical reactions with a wide range of molecules, such as, for example, free radicals, metals, thiols, etc. Therefore, there are still several contradictory findings on the role of NO in different biological processes. In this review, we briefly discuss the major techniques suitable for measurement of NO (electron paramagnetic resonance, electrochemistry, fluorometry) and its derivatives in biological samples (nitrite/nitrate, NOS, cGMP, nitrosothiols) and discuss the advantages and disadvantages of each method. We conclude that to obtain a meaningful insight into the role of NO and NO modulator compounds in physiological or pathological processes, concomitant assessment of NO synthesis, NO content, as well as molecular targets and reaction products of NO is recommended.