Particle Simulation of Oxidation Induced Band 3 Clustering in Human Erythrocytes

Oxidative stress mediated clustering of membrane protein band 3 plays an essential role in the clearance of damaged and aged red blood cells (RBCs) from the circulation. While a number of previous experimental studies have observed changes in band 3 distribution after oxidative treatment, the details of how these clusters are formed and how their properties change under different conditions have remained poorly understood. To address these issues, a framework that enables the simultaneous monitoring of the temporal and spatial changes following oxidation is needed. In this study, we established a novel simulation strategy that incorporates deterministic and stochastic reactions with particle reaction-diffusion processes, to model band 3 cluster formation at single molecule resolution. By integrating a kinetic model of RBC antioxidant metabolism with a model of band 3 diffusion, we developed a model that reproduces the time-dependent changes of glutathione and clustered band 3 levels, as well as band 3 distribution during oxidative treatment, observed in prior studies. We predicted that cluster formation is largely dependent on fast reverse reaction rates, strong affinity between clustering molecules, and irreversible hemichrome binding. We further predicted that under repeated oxidative perturbations, clusters tended to progressively grow and shift towards an irreversible state. Application of our model to simulate oxidation in RBCs with cytoskeletal deficiency also suggested that oxidation leads to more enhanced clustering compared to healthy RBCs. Taken together, our model enables the prediction of band 3 spatio-temporal profiles under various situations, thus providing valuable insights to potentially aid understanding mechanisms for removing senescent and premature RBCs.     

The effect of the different components of the inhibitor β complex in peelings of resting potato tubers on the respiration and uptake of inorganic phosphate

The four different inhibitors or groups of inhibitors, called I, II, IV and V, which could be separated from the inhibitor β complex from peelings of resting potatoes ( Solanum tuberosum L., variety Majestic) by thin layer chromatography in chloroform: 96% acetic acid (95:5 v/v), stimulated the oxygen uptake of discs of potato tubers during the first 4 to 5 h after addition of the substances. Number I was less active, the other three more active. Furthermore II, IV, and V significantly inhibited the uptake of inorganic phosphate by potato discs measured 24 and 5 h after addition of the substances, so that they seem to be uncouplers of oxidative phosphorylation.     

Short-Term,Intermittent Fasting Induces Long-Lasting Gut Health and TOR-Independent Lifespan Extension

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RNAi-based transgene conferred extreme resistance to the geminivirus causing apical leaf curl disease in potato

Potato apical leaf curl disease is an emerging geminiviral disease in tropics and subtropics. It was reported for the first time in the year 1999 in northern plains of India but quickly spread to almost all potato growing regions of the country largely due to prevalence of warmer weather during early crop growth, thereby favoring whitefly vector. The problem of apical leaf curl disease in India became more severe due to lack of seed indexing for this virus in conventional seed production scheme. Although it accounts for major yield loss, there is no conventional source of resistance available in potato against Tomato Leaf Curl New Delhi Virus-Potato (ToLCNDV-Potato) that causes this disease in potato. In the present study, we have investigated the potential use of RNAi for obtaining resistance against this DNA virus in potato. The replication-associated protein gene (AC1) of the virus was used to obtain pathogen-derived resistance. The AC1 gene was PCR amplified from field-infected potato leaves, cloned and sequenced (JN393309). It showed 93% sequence similarity with the AC1 gene of Tomato Leaf Curl Virus-New Delhi (TOLCV-NDe; DQ169056) virus. Transgenic plants encoding the AC1 gene in three different orientations, viz. sense, antisense and hairpin loop, were raised. Transgenic lines when challenge inoculated with ToLCNDV-Potato showed different levels of resistance for all three constructs. Transgene integration and copy number in selected transgenic lines were determined by qPCR and further confirmed by Southern blot analysis. Though a reduction in viral titer was observed in transgenic lines encoding either antisense or hairpin loop constructs of AC1 gene, the latter transgenics showed most significant results as shown by reduction in the level of symptom expression in glasshouse screening as well as real-time data of in vivo virus concentration. In fact, we obtained a few totally asymptomatic transgenic lines with hairpin loop strategy.     

Multiple roles of mobile active center loops in the E1 component of the Escherichia coli pyruvate dehydrogenase complex—Linkage of protein dynamics to catalysis

The region encompassing residues 401–413 on the E1 component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli comprises a loop (the inner loop) which was not seen in the X-ray structure in the presence of thiamin diphosphate, the required cofactor for the enzyme. This loop is seen in the presence of a stable analogue of the pre-decarboxylation intermediate, the covalent adduct between the substrate analogue methyl acetylphosphonate and thiamin diphosphate, C2α-phosphonolactylthiamin diphosphate. It has been shown that the residue H407 and several other residues on this loop are required to reduce the mobility of the loop so electron density corresponding to it can be seen once the pre-decarboxylation intermediate is formed. Concomitantly, the loop encompassing residues 541–557 (the outer loop) appears to work in tandem with the inner loop and there is a hydrogen bond between the two loops ensuring their correlated motion. The inner loop was shown to: (a) sequester the active center from carboligase side reactions; (b) assist the interaction between the E1 and the E2 components, thereby affecting the overall reaction rate of the entire multienzyme complex; (c) control substrate access to the active center. Using viscosity effects on kinetics it was shown that formation of the pre-decarboxylation intermediate is specifically affected by loop movement. A cysteine-less variant was created for the E1 component, onto which cysteines were substituted at selected loop positions. Introducing an electron spin resonance spin label and an ¹⁹F NMR label onto these engineered cysteines, the loop mobility was examined: (a) both methods suggested that in the absence of ligand, the loop exists in two conformations; (b) line-shape analysis of the NMR signal at different temperatures, enabled estimation of the rate constant for loop movement, and this rate constant was found to be of the same order of magnitude as the turnover number for the enzyme under the same conditions. Furthermore, this analysis gave important insights into rate-limiting thermal loop dynamics. Overall, the results suggest that the dynamic properties correlate with catalytic events on the E1 component of the pyruvate dehydrogenase complex.     

VEGF-B: A survival,or an angiogenic factor?

Despite its early discovery and high sequence homology to the other VEGF family members, the biological function of VEGF-B remained debatable for a long time, and VEGF-B has received little attention from the field thus far. Recently, we and others have found that (1) VEGF-B is a potent survival factor for different types of cells by inhibiting apoptosis via suppressing the expression of BH3-only protein and other apoptotic/cell death-related genes. (2) VEGF-B has a negligible role in inducing blood vessel growth in most organs. Instead, it is critically required for blood vessel survival. VEGF-B targeting inhibited pathological angiogenesis by abolishing blood vessel survival in different animal models. (3) Using different types of neuro-injury and neurodegenerative disease models, VEGF-B treatment protected endangered neurons from apoptosis without inducing undesired blood vessel growth or permeability. Thus, VEGF-B is the first member of the VEGF family that has a potent survival/anti-apoptotic effect, while lacking a general angiogenic activity. Our work thus advocates that the major function of VEGF-B is to act as a “survival,” rather than an “angiogenic” factor and implicates a therapeutic potential of VEGF-B in treating different types of vascular and neurodegenerative diseases.Key words: VEGF-B, survival factor, angiogenesis, apoptosis, vascular biology