Anion effects on the structural organization of spinach thylakoid membranes

Changes in the conformation of spinach thylakoid membranes were monitored in 5-doxyl stearic acid (SAL)-treated thylakoid membranes in the presence of various anions (Cl⁻, Br⁻, I⁻, NO₂ ⁻, SO₄ ²⁻, PO₄ ³⁻). The presence of anions made the thylakoid membrane more fluid. The extent of change in membrane fluidity differed with different anion and was reversible.     

55 kDa outer-membrane protein from short-chain fatty acids exposed Salmonella enterica serovar Typhi induces apoptosis in macrophages

Various pathogens including Salmonella species are known to induce apoptosis in host cell types during their infection processes. However, the bacterial components capable of inducing apoptosis have not been fully understood. It is now known that in vivo expression of virulence determinants differ from the expression under in vitro conditions. Therefore, in the present study, attempts were made to evaluate the apoptotic potential of outer-membrane protein (OMP) from short-chain fatty acids (SCFA) exposed Salmonella enterica serovar Typhi. Short-chain fatty acids exposure is one of the in vivo stresses encountered by the pathogen in the intestine. Therefore, to simulate the in vivo condition, S. enterica serovar Typhi was grown in the presence of SCFA and its OMP profile was analyzed. The apoptotic potential of 55 kDa protein expressed with enhanced intensity under the SCFA stress was evaluated. Murine peritoneal macrophages interacted with 55 kDa protein showed DNA fragmentation, changes in fluorescence and exposure of phosphatidylserine on their outer leaflets. Levels of nitrite and citrulline were found to be increased in the supernatant of macrophages after interacting them with 55 kDa protein. However, the enzymatic activity of superoxide dismutase was found to be decreased as compared to that of the control (uninteracted) macrophages. These observations indicate that increased levels of nitrite and decreased levels of superoxide dismutase may be one of the mechanisms to induce apoptosis in macrophages by SCFA induced 55 kDa OMP. These findings may help us better understand the pathophysiology of the disease during the host pathogen interactions.     

Stability of 100 homo and heterotypic coiled-coil a-a' pairs for ten amino acids (A, L, I, V, N, K, S, T, E, and R)

We present the thermal stability monitored by circular dichroism (CD) spectroscopy at 222 nm of 100 heterodimers that contain all possible coiled-coil a-a' pairs for 10 amino acids (I, V, L, N, A, K S, T, E, and R). This includes the stability of 36 heterodimers for 6 amino acids (I, V, L, N, A, and K) previously described and 64 new heterodimers including the 4 amino acids (S, T, E, and R). We have calculated a double mutant alanine thermodynamic cycle to determine a-a' pair coupling energies to evaluate which a-a' pairs encourage specific dimerization partners. The four new homotypic a-a' pairs (T-T, S-S, R-R, E-E) are repulsive relative to A-A and have destabilizing coupling energies. Among the 90 heterotypic a-a' pairs, the stabilizing coupling energies contain lysine or arginine paired with either an aliphatic or a polar amino acid. The range in coupling energies for each amino acid reveals its potential to regulate dimerization specificity. The a-a' pairs containing isoleucine and asparagine have the greatest range in coupling energies and thus contribute dramatically to dimerization specificity, which is to encourage homodimerization. In contrast, the a-a' pairs containing charged amino acids (K, R, and E) show the least range in coupling energies and promiscuously encourage heterodimerization.     

Structure, folding, and misfolding of Cu,Zn superoxide dismutase in amyotrophic lateral sclerosis

Fourteen years after the discovery that mutations in Cu, Zn superoxide dismutase (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS), the mechanism by which mutant SOD1 exerts toxicity remains unknown. The two principle hypotheses are (a) oxidative damage stemming from aberrant SOD1 redox chemistry, and (b) misfolding of the mutant protein. Here we review the structure and function of wild-type SOD1, as well as the changes to the structure and function in mutant SOD1. The relative merits of the two hypotheses are compared and a common unifying principle is outlined. Lastly, the potential for therapies targeting SOD1 misfolding is discussed.     

Tumour necrosis factor alpha mediated apoptosis in murine macrophages by Salmonella enterica serovar Typhi under oxidative stress

Invasive Salmonella has been reported to induce apoptosis of macrophages as part of its infection process, which may allow it to avoid detection by the innate immune system. However, the induction of apoptosis under the different host environments remains to be examined, including the oxidative stress experienced by pathogens in the macrophage milieu. To simulate in vivo oxidative conditions, Salmonella enterica serovar Typhi was grown in the presence of hydrogen peroxide and its ability to induce apoptosis of murine macrophages was assessed. Analysis of data revealed that oxidative stressed S. Typhi caused apoptotic cell death in 51% of macrophages, whereas S. Typhi grown under normal conditions accounted for apoptotic cell death in only 32% of macrophages. A significant increase in the levels of oxidants and decrease in the antioxidant was also observed which correlated with the increased generation of tumour necrosis factor alpha, interleukin-1alpha and interleukin-6. These results suggest that tumour necrosis factor alpha in conjunction with other cytokines may induce apoptotic cell death through the up-regulation of lipid peroxidation and down-regulation of superoxide dismutase. This finding may help us to understand better the host-pathogen interactions and may be of clinical importance in the development of preventive intervention against infection.     

C-tail mediated modulation of somatostatin receptor type-4 homo- and heterodimerizations and signaling

Somatostatin receptors show great diversity in response to agonist mediated receptor-specific homo- and heterodimerizations. Here, using photobleaching-fluorescence resonance energy transfer, immunocytochemistry, western blot and co-immunoprecipitation, we investigated dimerization, trafficking, coupling to adenylyl cyclase and signaling of human somatostatin receptor-4 (hSSTR4) in HEK-293 cells. We also determined the role of the C-tail of hSSTR4 on physiological responses of the cells. wt-hSSTR4 exogenously expressed in HEK-293 cells exhibits constitutive dimerization, inhibits forskolin-stimulated cAMP, and displays agonist dependent changes in pERK1/2 and pERK5 expressions. Upon C-tail deletion, the receptor loses membrane expression and ability to dimerize and inhibition of cAMP and pERK5 however, displays several-fold increases in the expression of pERK1/2. Chimeric hSSTR4 with the C-tail of hSSTR5 functions like wt-hSSTR4, in contrast, with the C-tail of hSSTR1 functions like C-tail deleted hSSTR4. hSSTR4 dimerization and signaling are associated with increased cyclin-dependent-kinase p27^kip1 expression and inhibition of the cell proliferation. We also report heterodimerization between hSSTR4/hSSTR5, but not between hSSTR4/hSSTR1, with significant changes in receptor functions. Taken together, these data define a novel mechanism for the role of hSSTR4 in cell proliferation and modulation of signaling pathways.     

Ratio of the vitreous vascular endothelial growth factor and pigment epithelial-derived factor in Eales disease

Eales disease (ED) is an idiopathic inflammatory venous occlusion of the peripheral retina. As neovascularization is prominent in ED, this study attempts to look at the ratio of VEGF, the angiogenic factor, and PEDF, an anti-angiogenic factor in the vitreous of ED patients in comparison with the macular hole (MH) and Proliferative Diabetic Retinopathy (PDR). Vitreous levels of VEGF and PEDF were determined in the undiluted vitreous specimen obtained from 26 ED cases, 17 PDR, and seven patients with MH. The vitreous levels of VEGF and PEDF were estimated by ELISA. The immunohistochemistry (IHC) for VEGF and PEDF were done in the epiretinal membrane of ED and PDR case. The VEGF/PEDF ratio was found to be significantly increased in ED (p = 0.014) and PDR (p = 0.000) compared to MH. However the ratio was 3.5-fold higher in PDR than ED (p = 0.009). The IHC data on the ERM specimen from ED showed the presence of VEGF and PEDF similar to PDR. The high angiogenic potential seen as the ratio of VEGF/PEDF correlates with the peak clinical onset of the disease in the age group 21–30 years and the diseases usually self-resolves above the age of 40, which is reflected by the low ratio of VEGF/PEDF. The study shows that the VEGF/PEDF ratio is significantly increased in ED though the angiogenic potential is higher in PDR than in ED. Clinically Eales Disease is known as a self-limiting disease, while PDR is a progressive disease.     

Induction of an osteocyte-like phenotype by fibroblast growth factor-2

Cycloheximide (CHX) is one of the most interesting protein synthesis inhibitors. For this reason, fluorescent derivatives of CHX could find useful applications in cell biology. We report the successful synthesis of a set of novel fluorescent derivatives of CHX. The effect of different functional groups on the biological activity of CHX was studied upon their modification through suitable strategies, i.e., acetylation of the hydroxyl group and reductive amination of the ketone group. The first route induced a complete loss of biological activity, while the second approach allowed a retained inhibition of protein synthesis, as demonstrated by in vitro translation assays. Various fluorescent dyes for reductive amination were tested (i.e., ANTS, APTS, and Rhodamine-123), and the success of the syntheses was demonstrated by diverse analytical techniques. Cycloheximide labeling with fluorescent dyes is a promising approach for developing fluorescence reporters for various applications, both in vitro (fluorescence spectroscopy) and in vivo (live imaging).     

Characterization of photosystem II heterogeneity in response to high salt stress in wheat leaves (<Emphasis Type="Italic">Triticum aestivum</Emphasis>)

The effect of high salt stress on PS II heterogeneity was investigated in wheat (Triticum aestivum) leaves. On the basis of antenna size, PS II has been classified into three forms, i.e., α, β, and γ centers while on the basis of electron transport properties of the reducing side of the reaction centers, two distinct forms of PS II have been suggested, i.e., Q_B reducing centers and Q_B non-reducing centers. The chlorophyll a (Chl a) fluorescence transients, which can quantify PS II behavior, were recorded using PEA to derive OJIP in vivo with high time resolution and further analyzed according to JIP test. Our results showed that with an increase in the salt concentration during growth, the number of Q_B non-reducing centers increased. In antenna size heterogeneity the number of β and γ centers increased while the number of α centers decreased. A change in the energetic connectivity between the PS II units was also observed. Recovery studies showed that antenna heterogeneity was completely recovered from damage at 0.5 M NaCl concentration and partially recovered at 1 M NaCl concentration while reducing side heterogeneity showed no recovery at all after 0.5 M onwards.