The fluidity of Synechocystis membranes was adjusted in vivo by temperature acclimation, addition of fluidizer 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 fluidity 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 μmol/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 μmol/L LNA was administered after preconditioning, it failed to block the effect of preconditioning. In the presence of 46 μmol/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) blockade of ischemia/reperfusion-induced accumulation of cardiac NO by preconditioning or by an appropriate concentration of NOS inhibitor alleviates ischemia/reperfusion injury as demonstrated by enhanced postischemic function.

Electron microscopic analysis of gap‐junction‐like structures isolated from an arthropod (Nephrops norvegicus) and composed of a 16‐kDa polypeptide, show the functional 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 α‐helical content for the protein, supporting secondary‐structure predictions of four transmembrane α helices/monomer. The averaged projection shows a close resemblance to a hexamer of the 16‐kDa protein built on the basis of a four α‐helical bundle [Finbow, M. E., Eliopoulos, E. E., Jackson, P. J., Keen, J. N., Meagher, L., Thompson, P., Jones, P. C. & Findlay, J. B. C. (1992) Protein Eng. 5, 7–15]. The reconstructed image is also similar to that obtained for gap‐junction‐like channels isolated from a related arthropod [Homarus americanus; Sikerwar, S. S., Downing, K. H. & Glaeser, R. M. (1991) J. Struct. Biol. 106, 255–263] whose protein content was unknown but which we demonstrate may be composed of a related 16‐kDa protein.Previous studies have shown a high sequence identity of the Nephrops 16‐kDa protein with the 16‐kDa proteolipid subunit c of the vacuolar H⁺‐ATPase, both of which in turn bear similarity to the 8‐kDa proteolipid subunit of the F₁F₀‐ATP synthase. Expression of cDNA coding for the Nephrops 16‐kDa protein in Saccharomyces cerevisiae, in which the endogenous gene coding for the V‐ATPase proteolipid has been inactivated, restores V‐ATPase activity and cell growth.