Sirtuin 1 and 7 mediate resveratrol-induced recovery from hyper-anxiety in high-fructose-fed prediabetic rats

Hyperglycaemia in diabetes is either caused by reduced availability of insulin (type 1 diabetes, T1D) or insulin resistance to the cells (type 2 diabetes, T2D). In recent years, the prevalence of T2D has increased to an alarming proportion, encompassing 95% of the total diabetic burden, probably due to economy-driven changes in lifestyle. Recent epidemiological studies show comorbid depression, anxiety and related mental illness. To explore the molecular mechanisms underlying this comorbid conditions, we used Sprague–Dawley rats on high-fructose diet for 8 weeks to induce prediabetic condition. Rats with this metabolic syndrome also showed hyper-anxiety when they were subjected to anxiety-related behavioural assays. Rats were administered with resveratrol, an activator of sirtuins, and metformin, a standard antidiabetic drug, simultaneously with fructose. We observed that resveratrol was more effective in protecting from both the metabolic (prediabetic) and affective (anxiety) disorders than metformin. Molecular studies showed that recovery was associated with the upregulation of few nuclear sirtuins that act epigenetically – Sirt 1 and 7, which were significantly attenuated in the striatum of prediabetic rats. In conclusion, our study showed that hyper-anxiety associated with prediabetic condition is ameliorated by resveratrol through modulation of sirtuins, which is more or less similar to metformin.     

De novo sphingolipid synthesis is essential for viability, but not for transport of glycosylphosphatidylinositol-anchored proteins, in African trypanosomes

De novo sphingolipid synthesis is required for the exit of glycosylphosphatidylinositol (GPI)-anchored membrane proteins from the endoplasmic reticulum in yeast. Using a pharmacological approach, we test the generality of this phenomenon by analyzing the transport of GPI-anchored cargo in widely divergent eukaryotic systems represented by African trypanosomes and HeLa cells. Myriocin, which blocks the first step of sphingolipid synthesis (serine + palmitate --> 3-ketodihydrosphingosine), inhibited the growth of cultured bloodstream parasites, and growth was rescued with exogenous 3-ketodihydrosphingosine. Myriocin also blocked metabolic incorporation of [3H]serine into base-resistant sphingolipids. Biochemical analyses indicate that the radiolabeled lipids are not sphingomyelin or inositol phosphorylceramide, suggesting that bloodstream trypanosomes synthesize novel sphingolipids. Inhibition of de novo sphingolipid synthesis with myriocin had no adverse effect on either general secretory trafficking or GPI-dependent trafficking in trypanosomes, and similar results were obtained with HeLa cells. A mild effect on endocytosis was seen for bloodstream trypanosomes after prolonged incubation with myriocin. These results indicate that de novo synthesis of sphingolipids is not a general requirement for secretory trafficking in eukaryotic cells. However, in contrast to the closely related kinetoplastid Leishmania major, de novo sphingolipid synthesis is essential for the viability of bloodstream-stage African trypanosomes.     

Relative Reactivity of Ribosyl 2′-OH vs. 3′-OH in Concentrated Aqueous Solutions of Phosphoimidazolide Activated Nucleotides

Phosphoimidazolide activated ribomononucleotides (*pN, see structure) are useful substrates for the non-enzymatic synthesis of oligonucleotides. In the presence of metal ions, aqueous solutions of *pN yield primarily the two internucleotide-linked (pN^2'pN and pN^3'pN) and the pyrophosphate-linked (N^5'ppN) dimers. Small amounts of cyclic dimers and higher oligomers are also produced. In this study the relative reactivity of 2-OH vs. 3-OH was determined from the ratio of the yields of pN^2'pN vs. pN^3'pN. Experiments were performed at 23 °C in the range 7.2 pH 8.4 with substrates that differ in nucleobase (guanosine (G), cytidine (C), uridine (U), and adenosine (A)) and leaving group (imidazole (Im), 2-methylimidazole (2-MeIm) and 2,4-dimethylimidazole (2,4-diMeIm)). Two metal ions (Mg²⁺ or Mn²⁺) were employed as catalysts. The conditions used here, i.e. a substrate concentration in the range 0.1 M to 1.0 M and metal ion concentration in the range 0.05 M to 0.2 M, favor base-stacking interactions. The ratio pN^2'pN: pN^3'pN = 2-5: 3-5 was found independent of nucleobase and typically varied between 2 to 3 indicating that the 2-OH is about 2 to 3 times more reactive than the 3-OH. *pN with Im, compared to 2-MeIm and 2,4-diMeIm leaving group, produce lower yields of internucleotide linked dimers, and a higher pN^2'pN: pN^3'pN ratio. Trends in the data, observed with all three leaving groups, suggest an increase in pN^2'pN: pN^3'pN ratio with decreasing substrate concentration (up to 5.47 with 0.051 M ImpG). The observations are in accord with earlier studies reporting a relative reactivity 2'-5': 3'-5'= 6 to 9 obtained with Im as the leaving group, in dilute nucleotide solutions and under conditions that disfavor stacking. It is speculated that the concentration induced change in the relative reactivity is the result of self-association via base-stacking that enhances selectively the proximity of the 3-OH of one molecule to the reactive P-N bond of an other molecule. The implication of these conclusions for oligomerization/ligation reactions is discussed.     

Drosophila perlecan modulates FGF and hedgehog signals to activate neural stem cell division

Mutations in the Drosophila trol gene cause cell cycle arrest of neuroblasts in the larval brain. Here, we show that trol encodes the Drosophila homolog of Perlecan and regulates neuroblast division by modulating both FGF and Hh signaling. Addition of human FGF-2 to trol mutant brains in culture rescues the trol proliferation phenotype, while addition of a MAPK inhibitor causes cell cycle arrest of the regulated neuroblasts in wildtype brains. Like FGF, Hh activates stem cell division in the larval brain in a Trol-dependent fashion. Coimmunoprecipitation studies are consistent with interactions between Trol and Hh and between mammalian Perlecan and Shh that are not competed with heparin sulfate. Finally, analyses of mutations in trol, hh, and ttv suggest that Trol affects Hh movement. These results indicate that Trol can mediate signaling through both of the FGF and Hedgehog pathways to control the onset of stem cell proliferation in the developing nervous system.     

Regulation (alteration) of activity and conformation of sucrase by coaggregation with cellobiase in culture medium of Termitomyces clypeatus

Extracellular sucrase (S) of Termitomyces clypeatus was aggregated with cellobiase (C) in culture filtrate and coaggregates of sucrase to cellobiase with different activity ratios (S/C) were obtained during purification. Specific activity of the enzyme decreased significantly, after purification of sucrase free from cellobiase. Purified sucrase was characterized as a glycoprotein of molar mass around 55kDa as indicated by SDS-PAGE and HPGPLC. K(m) and V(max) of the purified enzyme were determined as 34.48 mM and 13.3 U/mg, respectively, at optimum temperature (45 degrees C) and pH (5.0). Substrate affinity and reaction velocity of the purified enzyme, free from cellobiase, was lowered by approximately 3.5 and 55 times, respectively, than that of the enzyme obtained from culture filtrate. The instant regain of sucrase activity up to the extent of 41% was obtained on in vitro addition of cellobiase (free from sucrase) to the enzyme in incubation mixture. Conformation of the enzyme free from cellobiase appeared to be significantly different from that of the coaggregate, as analyzed by circular dichroic and light scattering spectroscopy. It was concluded that activity and conformation of sucrase is regulated (altered) by heteroaggregation with cellobiase in the fungus.     

A nonradioactive assay method for determination of enzymatic activity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)

A sensitive and nonradioactive assay method for activity determination of Rubisco is described. The method is based on thin-layer chromatographic separation of 3-phosphoglycerate (3-PGA) and D-ribulose-1,5-bisphosphate (RuBP). This assay method allows the quantitative determination of Rubisco activity. Rates of carbon dioxide fixation on RuBP determined by this method were comparable to those obtained independently by other methods. This assay method is reproducible and relatively free from interference.     

Climate Drivers on Malaria Transmission in Arunachal Pradesh,India

The present study was conducted during the years 2006 to 2012 and provides information on prevalence of malaria and its regulation with effect to various climatic factors in East Siang district of Arunachal Pradesh, India. Correlation analysis, Principal Component Analysis and Hotelling’s T² statistics models are adopted to understand the effect of weather variables on malaria transmission. The epidemiological study shows that the prevalence of malaria is mostly caused by the parasite Plasmodium vivax followed by Plasmodium falciparum. It is noted that, the intensity of malaria cases declined gradually from the year 2006 to 2012. The transmission of malaria observed was more during the rainy season, as compared to summer and winter seasons. Further, the data analysis study with Principal Component Analysis and Hotelling’s T² statistic has revealed that the climatic variables such as temperature and rainfall are the most influencing factors for the high rate of malaria transmission in East Siang district of Arunachal Pradesh.