Practitioners of vipassana meditation exhibit enhanced slow wave sleep and REM sleep states across different age groups

Sleep and Biological Rhythms - Intense meditation practices influence brain functions in different ways and at different levels. Earlier studies have shown that meditation practices help to...     

Activation of JAK2-V617F by Components of Heterodimeric Cytokine Receptors

The JAK2-V617F mutation is an important etiologic factor for the development of myeloproliferative neoplasms. The mechanism by which this mutated tyrosine kinase initiates deregulated signals in cells is not completely understood. It is believed that JAK2-V617F requires interactions with homodimeric cytokine receptors to elicit its transforming signal. In this study, we demonstrate that components of heterodimeric cytokine receptors can also activate JAK2-V617F. Expression of IL27Ra, a heterodimeric receptor component, enhanced the activation of JAK2-V617F and subsequent downstream signaling to activation of STAT5 and ERK. In addition, expression of components of the interleukin-3 receptor, IL3Ra and the common β chain, activated JAK2-V617F as well as STAT5 and ERK. Importantly, expression of IL27Ra functionally replaced the requirement of a homodimeric cytokine receptor to promote the activation and transforming activity of JAK2-V617F in BaF3 cells. Tyrosine phosphorylation of IL27Ra was not required to induce activation of JAK2-V617F or STAT5, or to enhance the transforming activity of JAK2-V617F. Expression of IL3Ra or the common β chain in BaF3 cells also enhanced the ability of JAK2-V617F to transform these hematopoietic cells. However, the heterodimeric receptor component IL12RB1 did not enhance the activation or transforming signals of JAK2-V617F in BaF3 cells. IL27Ra also activated the K539L and R683G JAK2 mutants. Together our data demonstrate that in addition to homodimeric receptors, some heterodimeric receptor components can support the activation and transforming signals of JAK2-V617F and other JAK2 mutants. Therefore, heterodimeric receptors may play unappreciated roles in JAK2 activation in the development of hematopoietic diseases including myeloproliferative neoplasms.     

In silico identification of common putative drug targets in <Emphasis Type="Italic">Leptospira interrogans</Emphasis>

Infectious diseases are the leading causes of death worldwide. Hence, there is a need to develop new antimicrobial agents. Traditional method of drug discovery is time consuming and yields a few drug targets with little intracellular information for guiding target selection. Thus, focus in drug development has been shifted to computational comparative genomics for identifying novel drug targets. Leptospirosis is a worldwide zoonosis of global concern caused by Leptospira interrogans. Availability of L. interrogans serovars and human genome sequences facilitated to search for novel drug targets using bioinformatics tools. The genome sequence of L. interrogans serovar Copenhageni has 5,124 genes while that of serovar Lai has 4,727 genes. Through subtractive genomic approach 218 genes in serovar Copenhageni and 158 genes in serovar Lai have been identified as putative drug targets. Comparative genomic approach had revealed that 88 drug targets were common to both the serovars. Pathway analysis using the Kyoto Encyclopaedia of Genes and Genomes revealed that 66 targets are enzymes and 22 are non-enzymes. Sixty two common drug targets were predicted to be localized in cytoplasm and 16 were surface proteins. The identified potential drug targets form a platform for further investigation in discovery of novel therapeutic compounds against Leptospira.     

How does a re-colonizing population of Asian elephants affect the forest habitat?

The Asian elephant Elephas maximus is currently re-colonizing the Bardia National Park in lowland Nepal. We studied their impact on woody vegetation in the nutrient-rich floodplain and in the relatively nutrient-poor sal forest. The types and extent of tree impact were recorded along fixed-width transects (335 km). Species composition, density and size classes ≥8 cm diameter breast height (dbh) were recorded in 15-m radius random plots ( n =95). Impact was higher in the floodplain complex than in the sal-dominated forest. Our hypothesis that elephants were more selective on species in the nutrient-poor sal forest was only partly supported; the niche breadth of impacted trees was slightly higher in the floodplain complex. Pushed-over trees accounted for the highest proportion of impact (55%), followed by killed trees (39%). Of the pushed trees, 10% were not used for food. Among food trees, elephants selectively impacted size class 12–16 cm dbh, whereas non-food trees were impacted independently of size. A large proportion of the freshly browsed trees had been felled previously, indicating that most felled trees survived, enabling elephants to feed on them again. This may reflect an evolutionary adaptation among long-lived species with high site fidelity. Owing to preferential use but low abundance, two species in sal forest, Grewia spp. and Desmodium oojeinense , were found to be particularly vulnerable to local extinction due to elephants. Although the elephants had impacted a large number of species (62, 73% of all), 56.4% of the impacted trees consisted of Mallotus phillippinensis . A recently observed increase in the density of M. phillippinensis and the concurrent reduction of the hardly utilized Shorea robusta indicates that the rapidly growing elephant population may modify the composition of the forest by increasing its preferred food species.     

Substrate specificity and kinetic mechanism of mammalian G9a histone H3 methyltransferase

Lysine-specific murine histone H3 methyltransferase, G9a, was expressed and purified in a baculovirus expression system. The primary structure of the recombinant enzyme is identical to the native enzyme. Enzymatic activity was favorable at alkaline conditions (>pH 8) and low salt concentration and virtually unchanged between 25 and 42 degrees C. Purified G9a was used for substrate specificity and steady-state kinetic analysis with peptides representing un- or dimethylated lysine 9 histone H3 tails with native lysine 4 or with lysine 4 changed to alanine (K4AK9). In vitro methylation of the H3 tail peptide resulted in trimethylation of Lys-9 and the reaction is processive. The turnover number (k(cat)) for methylation was 88 and 32 h(-1) on the wild type and K4AK9 histone H3 tail, respectively. The Michaelis constants for wild type and K4AK9 ((K(m)(pep))) were 0.9 and 1.0 microM and for S-adenosyl-L-methionine (K(m)(AdoMet)) were 1.8 and 0.6 microM, respectively. Comparable kinetic constants were obtained for recombinant histone H3. The conversion of K4AK9 di- to trimethyl-lysine was 7-fold slower than methyl group addition to unmethylated peptide. Preincubation studies showed that G9a-AdoMet and G9a-peptide complexes are catalytically active. Initial velocity data with peptide and S-adenosyl-L-methionine (AdoMet) and product inhibition studies with S-adenosyl-L-homocysteine were performed to assess the kinetic mechanism of the reaction. Double reciprocal plots and preincubation studies revealed S-adenosyl-L-homocysteine as a competitive inhibitor to AdoMet and mixed inhibitor to peptide. Trimethylated peptides acted as a competitive inhibitor to substrate peptide and mixed inhibitor to AdoMet suggesting a random mechanism in a Bi Bi reaction for recombinant G9a where either substrate can bind first to the enzyme, and either product can release first.     

GI side-effects of a possible therapeutic GRF analogue in monkeys are likely due to VIP receptor agonist activity.

Growth hormone (GH) is used or is being evaluated for efficacy in treatment of short stature, aspects of aging, cardiac disorders, Crohn's disease, and short bowel syndrome. Therefore, we synthesized several stable growth hormone-releasing factor (GRF) analogues that could be therapeutically useful. One potent analog, [D-Ala(2),Aib(8, 18,)Ala(9, 15, 16, 22, 24-26,)Gab(27)]hGRF(1-27)NH(2) (GRF-6), with prolonged infusion caused severe diarrhea in monkeys; however, it had no side-effects in rats. Because GRF has similarity to VIP/PACAP and VIPomas cause diarrhea, this study investigated the ability of this and other GRF analogues to interact with the VIP/PACAP receptors. Rat VPAC(1)-R (rVPAC(1)-R), human VPAC(1)-R (hVPAC(1)-R), rVPAC(2)-R and hVPAC(2)-R stably transfected CHO and PANC 1 cells were made and T47D breast cancer cells containing native human VPAC(1)-R and AR4-2J cells containing PAC(1)-R were used. hGRF(1-29)NH(2) had low affinity for both rVPAC(1)-R and rVPAC(2)-R while VIP had a high affinity for both receptors. GRF-6 had a low affinity for both rVPAC(1)-R and rVPAC(2)-R and very low affinity for the rPAC(1)-R. VIP had a high affinity, whereas hGRF(1-29)NH(2) had a low affinity for both hVPAC(1)-R and hVPAC(2)-R. In contrast GRF-6, while having a low affinity for hVPAC(2)-R, had relatively higher affinity for the hVPAC(1)-R. In guinea pig pancreatic acini, all GRF analogues were full agonists at the VPAC(1)-R causing enzyme secretion. These results demonstrate that in contrast to native hGRF(1-29)NH(2,) GRF-6 has a relatively high affinity for the human VPAC(1)-R but not for the human VPAC(2)-R, rat VPAC(1)-R, rat VPAC(2)-R or rat PAC(1)-R. These results suggest that the substituted GRF analog, GRF-6, likely causes the diarrheal side-effects in monkeys by interacting with the VPAC(1)-R. Furthermore, they demonstrate significant species differences can exist for possible therapeutic peptide agonists of the VIP/PACAP/GRF receptor family and that it is essential that receptor affinity assessments be performed in human cells or from a closely related species.     

Efficient plant regeneration from cell suspension-derived callus of East Indian rosewood (Dalbergia latifolia Roxb.)

A procedure is outlined for the establishment of a proliferating cell suspension culture of East Indian rosewood (Dalbergia latifolia Roxb.) and efficient plant regeneration from callus derived from such cultures. Callus was induced from hypocotyl segments derived from 1-week-old axenic seedlings on Murashige and Skoog (1962) medium (MS) containing 10.8 μM naphthaleneacetic acid (NAA) and 2.2 μM benzyladenine (BA). Calli were increased by subculturing on MS supplemented with same growth regulators and 10% coconut water (CW). Friable calli were used to initiate cell suspension cultures. Optimum cell proliferation occurred in MS containing 10.8 μM NAA, 2.2 μM BA and 10% CW, using an initial inoculum cell density of 2%. Cell clumps composed of 20–25 cells harvested from suspension cultures at the exponential growth phase readily formed callus within 3 weeks following plating on the semi-solid MS as above. High-frequency shoot-bud differentiation was induced in these calli on MS containing 2.7 μM NAA and 13.3 μM BA. The regeneration frequency declined at higher BA concentrations. The organogenic potential of the cell suspensions was influenced by the age of the culture. Regenerated shoots were rooted on half-strength MS containing 5.7 μM indole-3-acetic acid, 4.9 μM indole-3-butyric acid and 5.3 μM indole-3-propionic acid. The plantlets were acclimatized and established in soil. Received: 22 August 1997 / Revision received: 21 May 1998 / Accepted: 1 June 1998     

Targeted editing of multiple homologues of GTR1 and GTR2 genes provides the ideal low-seed,high-leaf glucosinolate oilseed mustard with uncompromised defence and yield

Glucosinolate content in the two major oilseed Brassica crops—rapeseed and mustard has been reduced to the globally accepted Canola quality level (<30 μmoles/g of seed dry weight, DW), making the protein-rich seed meal useful as animal feed. However, the overall lower glucosinolate content in seeds as well as in the other parts of such plants renders them vulnerable to biotic challenges. We report CRISPR/Cas9-based editing of glucosinolate transporter (GTR) family genes in mustard (Brassica juncea) to develop ideal lines with the desired low seed glucosinolate content (SGC) while maintaining high glucosinolate levels in the other plant parts for uncompromised plant defence. Use of three gRNAs provided highly efficient and precise editing of four BjuGTR1 and six BjuGTR2 homologues leading to a reduction of SGC from 146.09 μmoles/g DW to as low as 6.21 μmoles/g DW. Detailed analysis of the GTR-edited lines showed higher accumulation and distributional changes of glucosinolates in the foliar parts. However, the changes did not affect the plant defence and yield parameters. When tested against the pathogen Sclerotinia sclerotiorum and generalist pest Spodoptera litura, the GTR-edited lines displayed a defence response at par or better than that of the wild-type line. The GTR-edited lines were equivalent to the wild-type line for various seed yield and seed quality traits. Our results demonstrate that simultaneous editing of multiple GTR1 and GTR2 homologues in mustard can provide the desired low-seed, high-leaf glucosinolate lines with an uncompromised defence and yield.