The inositol polyphosphate 5-phosphatases and the apurinic/apyrimidinic base excision repair endonucleases share a common mechanism for catalysis

Inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from the inositol ring of phosphatidylinositol-derived signaling molecules; however, the mechanism of catalysis is only partially characterized. These enzymes play critical roles in regulating cell growth, apoptosis, intracellular calcium oscillations, and post-synaptic vesicular trafficking. The UCLA fold recognition server (threader) predicted that the conserved 300-amino acid catalytic domain, common to all 5-phosphatases, adopts the fold of the apurinic/apyrimidinic (AP) base excision repair endonucleases. PSI-BLAST searches of GENPEPT, using the amino acid sequence of AP endonuclease exonuclease III, identified all members of the 5-phosphatase family with highly significant scores. A sequence alignment between exonuclease III and all known 5-phosphatases revealed six highly conserved motifs containing residues that corresponded to the catalytic residues in the AP endonucleases. Mutation of each of these residues to alanine in the mammalian 43-kDa, or yeast Inp52p 5-phosphatase, resulted in complete loss of enzyme activity. We predict the 5-phosphatase enzymes share a similar mechanism of catalysis to the AP endonucleases, consistent with other common functional similarities such as an absolute requirement for magnesium for activity. Based on this analysis, functional roles have been assigned to conserved residues in all 5-phosphatase enzymes.     

Synchronization and Oscillatory Dynamics in Heterogeneous, Mutually Inhibited Neurons

We study some mechanisms responsible for synchronous oscillations and loss of synchrony at physiologically relevant frequencies (10–200 Hz) in a network of heterogeneous inhibitory neurons. We focus on the factors that determine the level of synchrony and frequency of the network response, as well as the effects of mild heterogeneity on network dynamics. With mild heterogeneity, synchrony is never perfect and is relatively fragile. In addition, the effects of inhibition are more complex in mildly heterogeneous networks than in homogeneous ones. In the former, synchrony is broken in two distinct ways, depending on the ratio of the synaptic decay time to the period of repetitive action potentials (_s/T), where T can be determined either from the network or from a single, self-inhibiting neuron. With _s/T > 2, corresponding to large applied current, small synaptic strength or large synaptic decay time, the effects of inhibition are largely tonic and heterogeneous neurons spike relatively independently. With _s/T < 1, synchrony breaks when faster cells begin to suppress their less excitable neighbors; cells that fire remain nearly synchronous. We show numerically that the behavior of mildly heterogeneous networks can be related to the behavior of single, self-inhibiting cells, which can be studied analytically.     

Multi-Serotype Pneumococcal Nasopharyngeal Carriage Prevalence in Vaccine Na?ve Nepalese Children,Assessed Using Molecular Serotyping

Invasive pneumococcal disease is one of the major causes of death in young children in resource poor countries. Nasopharyngeal carriage studies provide insight into the local prevalence of circulating pneumococcal serotypes. There are very few data on the concurrent carriage of multiple pneumococcal serotypes. This study aimed to identify the prevalence and serotype distribution of pneumococci carried in the nasopharynx of young healthy Nepalese children prior to the introduction of a pneumococcal conjugate vaccine using a microarray-based molecular serotyping method capable of detecting multi-serotype carriage. We conducted a cross-sectional study of healthy children aged 6 weeks to 24 months from the Kathmandu Valley, Nepal between May and October 2012. Nasopharyngeal swabs were frozen and subsequently plated on selective culture media. DNA extracts of plate sweeps of pneumococcal colonies from these cultures were analysed using a molecular serotyping microarray capable of detecting relative abundance of multiple pneumococcal serotypes. 600 children were enrolled into the study: 199 aged 6 weeks to <6 months, 202 aged 6 months to < 12 months, and 199 aged 12 month to 24 months. Typeable pneumococci were identified in 297/600 (49·5%) of samples with more than one serotype being found in 67/297 (20·2%) of these samples. The serotypes covered by the thirteen-valent pneumococcal conjugate vaccine were identified in 44·4% of samples containing typeable pneumococci. Application of a molecular serotyping approach to identification of multiple pneumococcal carriage demonstrates a substantial prevalence of co-colonisation. Continued surveillance utilising this approach following the introduction of routine use of pneumococcal conjugate vaccinates in infants will provide a more accurate understanding of vaccine efficacy against carriage and a better understanding of the dynamics of subsequent serotype and genotype replacement.     

Tolerance of Ornamental Succulent Plant Crown of Thorns (Euphorbia milli) to Chromium and its Remediation

The potential of an ornamental shrub Crown of thorns (Euphorbia milli) was evaluated for remediation of soil contaminated with Cr. The plant is one of the rare succulent ornamental shrubs with a slow to moderate growth rate and is capable of blooming almost year-round. The plant could tolerate well up to 75 mg of applied Cr and beyond that there was mortality of plants. Though the plant could not be classified as a hyperaccumulator, the plant was still very efficient in translocating Cr from roots to shoots as evident from the data on uptake and translocation efficiency values. The translocation efficiency of over 80% in our study demonstrates that a large proportion of Cr has been translocated to the harvestable biomass of the plant and therefore, this plant could be effectively recommended for the remediation of soils contaminated with low to medium level of contamination i.e., up to 50 mg/kg soil.     

Peptide segments in protein-protein interfaces

An important component of functional genomics involves the understanding of protein association. The interfaces resulting from protein-protein interactions - (i) specific, as represented by the homodimeric quaternary structures and the complexes formed by two independently occurring protein components, and (ii) non-specific, as observed in the crystal lattice of monomeric proteins - have been analysed on the basis of the length and the number of peptide segments. In 1000 A2 of the interface area, contributed by a polypeptide chain, there would be 3.4 segments in homodimers, 5.6 in complexes and 6.3 in crystal contacts. Concomitantly, the segments are the longest (with 8.7 interface residues) in homodimers. Core segments (likely to contribute more towards binding) are more in number in homodimers (1.7) than in crystal contacts (0.5), and this number can be used as one of the parameters to distinguish between the two types of interfaces. Dominant segments involved in specific interactions, along with their secondary structural features, are enumerated.     

RLIP76, a glutathione-conjugate transporter, plays a major role in the pathogenesis of metabolic syndrome

Purpose

Characteristic hypoglycemia, hypotriglyceridemia, hypocholesterolemia, lower body mass, and fat as well as pronounced insulin-sensitivity of RLIP76^−/− mice suggested to us the possibility that elevation of RLIP76 in response to stress could itself elicit metabolic syndrome (MSy). Indeed, if it were required for MSy, drugs used to treat MSy should have no effect on RLIP76^−/− mice.

Research Design and Methods

Blood glucose (BG) and lipid measurements were performed in RLIP76^+/+ and RLIP76^−/− mice, using Ascensia Elite Glucometer® for glucose and ID Labs kits for cholesterol and triglycerides assays. The ultimate effectors of gluconeogenesis are the three enzymes: PEPCK, F-1,6-BPase, and G6Pase, and their expression is regulated by PPARγ and AMPK. The activity of these enzymes was tested by protocols standardized by us. Expressions of RLIP76, PPARα, PPARγ, HMGCR, pJNK, pAkt, and AMPK were performed by Western-blot and tissue staining.

Results

The concomitant activation of AMPK and PPARγ by inhibiting transport activity of RLIP76, despite inhibited activity of key glucocorticoid-regulated hepatic gluconeogenic enzymes like PEPCK, G6Pase and F-1,6-BP in RLIP76^−/− mice, is a salient finding of our studies. The decrease in RLIP76 protein expression by rosiglitazone and metformin is associated with an up-regulation of PPARγ and AMPK.

Conclusions/Significance

All four drugs, rosiglitazone, metformin, gemfibrozil and atorvastatin failed to affect glucose and lipid metabolism in RLIP76^−/− mice. Studies confirmed a model in which RLIP76 plays a central role in the pathogenesis of MSy and RLIP76 loss causes profound and global alterations of MSy signaling functions. RLIP76 is a novel target for single-molecule therapeutics for metabolic syndrome.     

Molecular diversity analysis of eggplant (Solanum melongena) genetic resources

Eggplant (Solanum melongena), a vegetable that is cultivated worldwide, is of considerable importance to agriculture in China. We analyzed the diversity of this plant using inter-simple sequence repeat (ISSR) and RAPD procedures to subdivide 143 Chinese-cultivated eggplants based on coefficient of parentage, genetic diversity index (GDI) and canonical discriminant analysis. ISSR markers were more effective than RAPD markers for detecting genetic diversity, which ranged from 0.10-0.51, slightly lower than what is known from other crops. Our ISSR/RAPD data provide molecular evidence that coincides with morphological-based classification into three varieties and further subdivision into eight groups, except for two groups. Intensive use of elite parents and extensive crossing within groups have resulted in increased coefficient of parentage and proportional contribution but decreased GDI during the past decades. The mean coefficient of parentage and proportional contribution increased from 0.05 to 0.10% and from 3.22 to 6.46% during 1980-1991 and 1992-2003, respectively. The GDI of landraces was 0.21, higher than the 0.09 and 0.08 calculated for the hybrid cultivars released during the two periods. The recent introduction of alien genotypes into eggplant breeding programs may broaden the genetic base.     

The SH2 domain containing inositol polyphosphate 5-phosphatase-2: SHIP2

Phosphoinositides are membrane-bound signaling molecules that recruit, activate and localize target effectors to intracellular membranes regulating apoptosis, cell proliferation, insulin signaling and membrane trafficking. The SH2 domain containing inositol polyphosphate 5-phosphatase-2 (SHIP2) hydrolyzes phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) generating phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). Overexpression of SHIP2 inhibits insulin-stimulated phosphoinositide 3-kinase (PI3K) dependent signaling events. Analysis of diabetic human subjects has revealed an association between SHIP2 gene polymorphisms and type 2 diabetes mellitus. Genetic ablation of SHIP2 in mice has generated conflicting results. SHIP2 knockout mice were originally reported to show lethal neonatal hypoglycemia resulting from insulin hypersensitivity, but in addition to inactivating the SHIP2 gene, the Phox2a gene was also inadvertently deleted. Another SHIP2 knockout mouse has now been generated which inactivates the SHIP2 gene but leaves Phox2a intact. These animals show normal insulin and glucose tolerance but are highly resistant to weight gain on high fat diets, exhibiting an obesity-resistant phenotype. Therefore, SHIP2 remains a significant therapeutic target for the treatment of both obesity and type 2 diabetes.     

Oncogene-induced telomere dysfunction enforces cellular senescence in human cancer precursor lesions

In normal human somatic cells, telomere dysfunction causes cellular senescence, a stable proliferative arrest with tumour suppressing properties. Whether telomere dysfunction-induced senescence (TDIS) suppresses cancer growth in humans, however, is unknown. Here, we demonstrate that multiple and distinct human cancer precursor lesions, but not corresponding malignant cancers, are comprised of cells that display hallmarks of TDIS. Furthermore, we demonstrate that oncogenic signalling, frequently associated with initiating cancer growth in humans, dramatically affected telomere structure and function by causing telomeric replication stress, rapid and stochastic telomere attrition, and consequently telomere dysfunction in cells that lack hTERT activity. DNA replication stress induced by drugs also resulted in telomere dysfunction and cellular senescence in normal human cells, demonstrating that telomeric repeats indeed are hypersensitive to DNA replication stress. Our data reveal that TDIS, accelerated by oncogene-induced DNA replication stress, is a biological response of cells in human cancer precursor lesions and provide strong evidence that TDIS is a critical tumour suppressing mechanism in humans.