A soluble diacylglycerol acyltransferase is involved in triacylglycerol biosynthesis in the oleaginous yeast Rhodotorula glutinis

The biosynthesis of triacylglycerol (TAG) occurs in the microsomal membranes of eukaryotes. Here, we report the identification and functional characterization of diacylglycerol acyltransferase (DGAT), a member of the 10 S cytosolic TAG biosynthetic complex (TBC) in Rhodotorula glutinis. Both a full-length and an N-terminally truncated cDNA clone of a single gene were isolated from R. glutinis. The DGAT activity of the protein encoded by RgDGAT was confirmed in vivo by the heterologous expression of cDNA in a Saccharomyces cerevisiae quadruple mutant (H1246) that is defective in TAG synthesis. RgDGAT overexpression in yeast was found to be capable of acylating diacylglycerol (DAG) in an acyl-CoA-dependent manner. Quadruple mutant yeast cells exhibit growth defects in the presence of oleic acid, but wild-type yeast cells do not. In an in vivo fatty acid supplementation experiment, RgDGAT expression rescued quadruple mutant growth in an oleate-containing medium. We describe a soluble acyl-CoA-dependent DAG acyltransferase from R. glutinis that belongs to the DGAT3 class of enzymes. The study highlights the importance of an alternative TAG biosynthetic pathway in oleaginous yeasts.

MPK-09, a small molecule inspired from bioactive styryllactone restores the wild-type function of mutant p53

In the search for more efficacious and less toxic cancer drugs, the tumor suppressor p53 protein has long been a desirable therapeutic target. In the recent past, few independent studies have demonstrated that the antitumor activity of wild-type p53 can be restored in cancer cells harboring mutant form of p53 using small molecule activators. In this study, we describe a novel small molecule MPK-09, which is selective and highly potent against allele specific p53 mutations mainly, R175H, R249S, R273H, R273C, and E285K. Except E285K, all other mutations tested are among the six ``hot spot'' p53 mutations reported in majority of human cancer. Furthermore, our study conclusively demonstrates that the apoptotic activity of the small molecule MPK-09 against cancer cells harboring R273C and E285K mutations is due to restoration of the wild-type conformation to the corresponding mutant form of p53.

Malaria Parasite-Synthesized Heme Is Essential in the Mosquito and Liver Stages and Complements Host Heme in the Blood Stages of Infection

Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, d-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of Plasmodium berghei (Pb). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using 4-(14) C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.

Identification and characterization of starvation induced msdgc-1 promoter involved in the c-di-GMP turnover

C-di-GMP Bis-(3'-5')-cyclic-dimeric-guanosine monophosphate], a second messenger is involved in intracellular communication in the bacterial species. As a result several multi-cellular behaviors in both Gram-positive and Gram-negative bacteria are directly linked to the intracellular level of c-di-GMP. The cellular concentration of c-di-GMP is maintained by two opposing activities, diguanylate cyclase (DGC) and phosphodiesterase (PDE-A). In Mycobacterium smegmatis, a single bifunctional protein MSDGC-1 is responsible for the cellular concentration of c-di-GMP. A better understanding of the regulation of c-di-GMP at the genetic level is necessary to control the function of above two activities. In this work, we have characterized the promoter element present in msdgc-1 along with the + 1 transcription start site and identified the sigma factors that regulate the transcription of msdgc-1. Interestingly, msdgc-1 utilizes SigA during the initial phase of growth, whereas near the stationary phase SigB containing RNA polymerase takes over the expression of msdgc-1. We report here that the promoter activity of msdgc-1 increases during starvation or depletion of carbon source like glucose or glycerol. When msdgc-1 is deleted, the numbers of viable cells are similar to 10 times higher in the stationary phase in comparison to that of the wild type. We propose here that msdgc-1 is involved in the regulation of cell population density. (C) 2013 Elsevier B.V. All rights reserved.

Viscoelastic Behavior of Human Lamin A Proteins in the Context of Dilated Cardiomyopathy

Lamins are intermediate filament proteins of type V constituting a nuclear lamina or filamentous meshwork which lines the nucleoplasmic side of the inner nuclear membrane. This protein mesh provides a supporting scaffold for the nuclear envelope and tethers interphase chromosome to the nuclear periphery. Mutations of mainly A-type lamins are found to be causative for at least 11 human diseases collectively termed as laminopathies majority of which are characterised by aberrant nuclei with altered structural rigidity, deformability and poor mechanotransduction behaviour. But the investigation of viscoelastic behavior of lamin A continues to elude the field. In order to address this problem, we hereby present the very first report on viscoelastic properties of wild type human lamin A and some of its mutants linked with Dilated cardiomyopathy (DCM) using quantitative rheological measurements. We observed a dramatic strain-softening effect on lamin A network as an outcome of the strain amplitude sweep measurements which could arise from the large compliance of the quasi-cross-links in the network or that of the lamin A rods. In addition, the drastic stiffening of the differential elastic moduli on superposition of rotational and oscillatory shear stress reflect the increase in the stiffness of the laterally associated lamin A rods. These findings present a preliminary insight into distinct biomechanical properties of wild type lamin A protein and its mutants which in turn revealed interesting differences.

Distribution, Demography, and Conservation of Lion-tailed Macaques (Macaca silenus) in the Anamalai Hills Landscape, Western Ghats, India

The status of the endemic and endangered lion-tailed macaque (Macaca silenus) has not been properly assessed in several regions of the Western Ghats of southern India. We conducted a study in Parambikulam Forest Reserve in the state of Kerala to determine the distribution, demography, and status of lion-tailed macaques. We laid 5km(2) grid cells on the map of the study area (644km(2)) and made four replicated walks in each grid cell using GPS. We gathered data on lion-tailed macaque group locations, demography, and site covariates including trail length, duration of walk, proportion of evergreen forest, height of tallest trees, and human disturbance index. We also performed occupancy modeling using PRESENCE ver. 3.0. We estimated a minimum of 17 groups of macaques in these hills. Low detection and occupancy probabilities indicated a low density of lion-tailed macaques in the study area. Height of the tallest trees correlated positively whereas human disturbance and proportion of evergreen forest correlated negatively with occupancy in grid cells. We also used data from earlier studies carried out in the surrounding Anamalai Tiger Reserve and Nelliyampathy Hills to discuss the conservation status in the large Anamalai Hills Landscape. This landscape harbors an estimated population of 1108 individuals of lion-tailed macaques, which is about one third of the entire estimated wild population of this species. A conservation plan for this landscape could be used as a model for conservation in other regions of the Western Ghats.

Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, Groundnut bud necrosis virus

The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 50 a phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.

In the elephant's seed shadow: the prospects of domestic bovids as replacement dispersers of three tropical Asian trees

As populations of the world's largest animal species decline, it is unclear how ecosystems will react to their local extirpation. Due to the unique ecological characteristics of megaherbivores such as elephants, seed dispersal is one ecosystem process that may be affected as populations of large animals are decimated. In typically disturbed South Asian ecosystems, domestic bovids (cattle, Bos primigenius, and buffalo, Bubalus bubalis) may often be the species most available to replace Asian elephants (Elephas maximus) as endozoochorous dispersers of large-fruited mammal-dispersed species. We use feeding trials, germination trials, and movement data from the tropical moist forests of Buxa Tiger Reserve (India) to examine whether domestic bovids are viable replacements for elephants in the dispersal of three largefruited species: Dillenia indica, Artocarpus chaplasha, and Careya arborea. We find that (1) once consumed, seeds are between 2.5 (C. arborea) and 26.5 (D. indica) times more likely to pass undigested into elephant dung than domestic bovid dung; and (2) seeds from elephant dung germinated as well as or better than seeds taken from bovid dung for all plant species, with D. indica seeds from elephant dung 1.5 times more likely to germinate. Furthermore, since wild elephants have less constrained movements than even free-roaming domestic bovids, we calculate that maximum dispersal by elephants is between 9.5 and 11.2 times farther than that of domestic bovids, with about 20% of elephant-dispersed seeds being moved farther than the maximum distance seeds are moved by bovids. Our findings suggest that, while bovids are able to disperse substantial numbers of seeds over moderate distances for two of the three study species, domestic bovids will be unable to routinely emulate the reliable, long-distance dispersal of seeds executed by elephants in this tropical moist forest. Thus while domestic bovids can attenuate the effects of losing elephants as dispersers, they may not be able to prevent the decline of various mammal-dispersed fruiting species in the face of overhunting, habitat fragmentation, and climate change.

Probing the role of highly conserved residues in triosephosphate isomerase-analysis of site specific mutants at positions 64 and 75 in the Plasmodial enzyme

Highly conserved residues in enzymes are often found to be clustered close to active sites, suggesting that functional constraints dictate the nature of amino acid residues accommodated at these sites. Using the Plasmodiumfalciparum triosephosphate isomerase (PfTIM) enzyme () as a template, we have examined the effects of mutations at positions 64 and 75, which are not directly involved in the proton transfer cycle. Thr (T) occurring at position 75 is completely conserved, whereas only Gln (Q) and Glu (E) are accommodated at position 64. Biophysical and kinetic data are reported for four T75 (T75S/V/C/N) and two Q64 (Q64N/E) mutants. The dimeric structure is weakened in the Q64E and Q64N mutants, whereas dimer integrity is unimpaired in all four T75 mutants. Measurement of the concentration dependence of enzyme activity permits an estimate of K-d values for dimer dissociation (Q64N=73.79.2nm and Q64E=44.6 +/- 8.4nm). The T75S/V/C mutants have activities comparable to the wild-type enzyme, whereas a fourfold drop is observed for T75N. All four T75 mutants show a dramatic fall in activity between 35 degrees C and 45 degrees C. Crystal structure determination of the T75S/V/N mutants provides insights into the variations in local interactions, with the T75N mutant showing the largest changes. Hydrogen-bond interactions determine dimer stability restricting the choice of residues at position 64 to Gln (Q) and Glu (E). At position 75, the overwhelming preference for Thr (T) may be dictated by the imperative of maintaining temperature stability of enzyme activity.

Ischaemic concentrations of lactate increase TREK1 channel activity by interacting with a single histidine residue in the carboxy terminal domain

Key points The physiological metabolite, lactate and the two-pore domain leak potassium channel, TREK1 are known neuroprotectants against cerebral ischaemia. However, it is not known whether lactate interacts with TREK1 channel to provide neuroprotection. In this study we show that lactate increases TREK1 channel activity and hyperpolarizes CA1 stratum radiatum astrocytes in hippocampal slices. Lactate increases open probability and decreases longer close time of the human (h)TREK1 channel in a concentration dependent manner. Lactate interacts with histidine 328 (H328) in the carboxy terminal domain of hTREK1 channel to decrease its dwell time in the longer closed state. This interaction was dependent on the charge on H328. Lactate-insensitive mutant H328A hTREK1 showed pH sensitivity similar to wild-type hTREK1, indicating that the effect of lactate on hTREK1 is independent of pH change. AbstractA rise in lactate concentration and the leak potassium channel TREK1 have been independently associated with cerebral ischaemia. Recent literature suggests lactate to be neuroprotective and TREK1 knockout mice show an increased sensitivity to brain and spinal cord ischaemia; however, the connecting link between the two is missing. Therefore we hypothesized that lactate might interact with TREK1 channels. In the present study, we show that lactate at ischaemic concentrations (15-30mm) at pH7.4 increases TREK1 current in CA1 stratum radiatum astrocytes and causes membrane hyperpolarization. We confirm the intracellular action of lactate on TREK1 in hippocampal slices using monocarboxylate transporter blockers and at single channel level in cell-free inside-out membrane patches. The intracellular effect of lactate on TREK1 is specific since other monocarboxylates such as pyruvate and acetate at pH7.4 failed to increase TREK1 current. Deletion and point mutation experiments suggest that lactate decreases the longer close dwell time incrementally with increase in lactate concentration by interacting with the histidine residue at position 328 (H328) in the carboxy terminal domain of the TREK1 channel. The interaction of lactate with H328 is dependent on the charge on the histidine residue since isosteric mutation of H328 to glutamine did not show an increase in TREK1 channel activity with lactate. This is the first demonstration of a direct effect of lactate on ion channel activity. The action of lactate on the TREK1 channel signifies a separate neuroprotective mechanism in ischaemia since it was found to be independent of the effect of acidic pH on channel activity. Key points The physiological metabolite, lactate and the two-pore domain leak potassium channel, TREK1 are known neuroprotectants against cerebral ischaemia. However, it is not known whether lactate interacts with TREK1 channel to provide neuroprotection. In this study we show that lactate increases TREK1 channel activity and hyperpolarizes CA1 stratum radiatum astrocytes in hippocampal slices. Lactate increases open probability and decreases longer close time of the human (h)TREK1 channel in a concentration dependent manner. Lactate interacts with histidine 328 (H328) in the carboxy terminal domain of hTREK1 channel to decrease its dwell time in the longer closed state. This interaction was dependent on the charge on H328. Lactate-insensitive mutant H328A hTREK1 showed pH sensitivity similar to wild-type hTREK1, indicating that the effect of lactate on hTREK1 is independent of pH change.

Mutations in the nucleotide binding and hydrolysis domains of Helicobacter pylori MutS2 lead to altered biochemical activities and inactivation of its in vivo function

Background: Helicobacter pylori MutS2 (HpMutS2), an inhibitor of recombination during transformation is a non-specific nuclease with two catalytic sites, both of which are essential for its anti-recombinase activity. Although HpMutS2 belongs to a highly conserved family of ABC transporter ATPases, the role of its ATP binding and hydrolysis activities remains elusive. Results: To explore the putative role of ATP binding and hydrolysis activities of HpMutS2 we specifically generated point mutations in the nucleotide-binding Walker-A (HpMutS2-G338R) and hydrolysis Walker-B (HpMutS2-E413A) domains of the protein. Compared to wild-type protein, HpMutS2-G338R exhibited similar to 2.5-fold lower affinity for both ATP and ADP while ATP hydrolysis was reduced by similar to 3-fold. Nucleotide binding efficiencies of HpMutS2-E413A were not significantly altered; however the ATP hydrolysis was reduced by similar to 10-fold. Although mutations in the Walker-A and Walker-B motifs of HpMutS2 only partially reduced its ability to bind and hydrolyze ATP, we demonstrate that these mutants not only exhibited alterations in the conformation, DNA binding and nuclease activities of the protein but failed to complement the hyper-recombinant phenotype displayed by mutS2-disrupted strain of H. pylori. In addition, we show that the nucleotide cofactor modulates the conformation, DNA binding and nuclease activities of HpMutS2. Conclusions: These data describe a strong crosstalk between the ATPase, DNA binding, and nuclease activities of HpMutS2. Furthermore these data show that both, ATP binding and hydrolysis activities of HpMutS2 are essential for the in vivo anti-recombinase function of the protein.