During B-Z Transition There is No Large Scale Breakage of Watson-Crick Base Pairs A Direct Demonstration Using 500 MHz 1H NMR Spectroscopy

Abstract

Monitoring of the Watson-Crick GNH1 proton in poly(dG-dC)-poly(dG-dC) at 500 MHz in 90% H₂0:10% D₂o at 30° C as a function of NaCl concentration (1.5 to 3.6 M), demonstrates that the bases retain Watson-Crick pairing throughout the transition. This observation unequivocally demonstrates that during the B-Z transition there is no large scale and detectable base pair opening and that macroscopically the phenomenon can be described as a direct helix to helix transition. We present frame by frame, an energetically sound stereodynamical trajectory for this transfiguration from right-handed B-DNA to left-handed Z-DNA.     

Modulation of hypercholesterolemia-induced alterations in apolipoprotein B and HMG-CoA reductase expression by selenium supplementation

Various studies demonstrated a significant association between the trace element selenium (Se), hypercholesterolemia and the risk of cardiovascular disorders. Present study was aimed to reveal the role of Se supplementation in modulation of hypercholesterolemia-induced changes in apolipoprotein B (apoB) and 3-hydroxy 3-methylglutaryl co-enzyme A (HMG-CoA) reductase expression during experimental hypercholesterolemia in Sprague-Dawley male rats. Animals were fed 0.2 and 1 ppm Se-supplemented control diet as well as 2% cholesterol-supplemented diet for 3 months. Apolipoprotein B levels were measured by ELISA and Western blot. HMG-CoA reductase mRNA expression was studied by RT-PCR. ApoB levels increased significantly on 2% cholesterol-supplemented diet feeding. On 1 ppm Se supplementation apoB levels decreased significantly. HMG-CoA reductase mRNA expression decreased significantly on cholesterol-supplemented diet feeding and on 1 ppm Se supplementation the mRNA expression further decreased. So the present results demonstrate that 1 ppm Se supplementation is responsible for down regulation of apoB and HMG-CoA reductase expression during hypercholesterolemia. These findings highlight the therapeutic potential of selenium supplementation in lipid metabolism.     

Resolving an ancient, rapid radiation in Saxifragales

Despite the prior use of approximately 9000 bp, deep-level relationships within the angiosperm clade, Saxifragales remain enigmatic, due to an ancient, rapid radiation (89.5 to 110 Ma based on the fossil record). To resolve these deep relationships, we constructed several new data sets: (1) 16 genes representing the three genomic compartments within plant cells (2 nuclear, 10 plastid, 4 mitochondrial; aligned, analyzed length = 21,460 bp) for 28 taxa; (2) the entire plastid inverted repeat (IR; 26,625 bp) for 17 taxa; (3) "total evidence" (50,845 bp) for both 17 and 28 taxa (the latter missing the IR). Bayesian and ML methods yielded identical topologies across partitions with most clades receiving high posterior probability (pp = 1.0) and bootstrap (95% to 100%) values, suggesting that with sufficient data, rapid radiations can be resolved. In contrast, parsimony analyses of different partitions yielded conflicting topologies, particularly with respect to the placement of Paeoniaceae, a clade characterized by a long branch. In agreement with published simulations, the addition of characters increased bootstrap support for the putatively erroneous placement of Paeoniaceae. Although having far fewer parsimony-informative sites, slowly evolving plastid genes provided higher resolution and support for deep-level relationships than rapidly evolving plastid genes, yielding a topology close to the Bayesian and ML total evidence tree. The plastid IR region may be an ideal source of slowly evolving genes for resolution of deep-level angiosperm divergences that date to 90 My or more. Rapidly evolving genes provided support for tip relationships not recovered with slowly evolving genes, indicating some complementarity. Age estimates using penalized likelihood with and without age constraints for the 28-taxon, total evidence data set are comparable to fossil dates, whereas estimates based on the 17-taxon data are much older than implied by the fossil record. Hence, sufficient taxon density, and not simply numerous base pairs, is important in reliably estimating ages. Age estimates indicate that the early diversification of Saxifragales occurred rapidly, over a time span as short as 6 million years. Between 25,000 and 50,000 bp were needed to resolve this radiation with high support values. Extrapolating from Saxifragales, a similar number of base pairs may be needed to resolve the many other deep-level radiations of comparable age in angiosperms.     

A novel mTOR inhibitor is efficacious in a murine model of colitis

Ulcerative colitis is an autoimmune-inflammatory disease characterized by increased proliferation of colonic epithelial cells, dysregulation of signal transduction pathways, elevated mucosal T cell activation, increased production of proinflammatory cytokines, and enhanced leukocyte infiltration into colonic interstitium. Several compounds that possess antiproliferative properties and/or inhibit cytokine production exhibit a therapeutic effect in murine models of colitis. Mammalian target of rapamycin (mTOR), a protein kinase regulating cell proliferation, is implicated in colon carcinogenesis. In this study, we report that a novel haloacyl aminopyridine-based molecule (P2281) is a mTOR inhibitor and is efficacious in a murine model of human colitis. In vitro studies using Western blot analysis and cell-based ELISA assays showed that P2281 inhibits mTOR activity in colon cancer cells. In vitro and in vivo assays of proinflammatory cytokine production revealed that P2281 diminishes induced IFN-gamma production but not TNF-alpha production, indicating preferential inhibitory effects of P2281 on T cell function. In the dextran sulfate sodium (DSS) model of colitis, 1) macroscopic colon observations demonstrated that P2281 significantly inhibited DSS-induced weight loss, improved rectal bleeding index, decreased disease activity index, and reversed DSS-induced shortening of the colon; 2) histological analyses of colonic tissues revealed that P2281 distinctly attenuated DSS-induced edema, prominently diminished the leukocyte infiltration in the colonic mucosa, and resulted in protection against DSS-induced crypt damage; and 3) Western blot analysis showed that P2281 blocks DSS-induced activation of mTOR. Collectively, these results provide direct evidence that P2281, a novel mTOR inhibitor, suppresses DSS-induced colitis by inhibiting T cell function and is a potential therapeutic for colitis. Given that compounds with anticancer activity show promising anti-inflammatory efficacy, our findings reinforce the cross-therapeutic functionality of potential drugs.     

Effect of phyllotactic position and cultural treatments toward successful direct shoot organogenesis in dwarf ‘Pixie’ grapevine (Vitis vinifera L.)

In Vitis spp. where somatic embryogenesis-based regeneration predominates, an efficient, reproducible and robust method of direct shoot organogenesis from leaf explant material has been established in the dwarf wine grape ‘Pixie’ (Vitis vinifera). This regeneration system was achieved by testing the response of leaf material in two stages of development, and pre-conditioning the explant material in dark conditions and/or in liquid media prior to excising from the plant and placing it on solidified media. The pre-excision treatments included (1) a dark period of 24 h, with no regeneration medium; (2) soaking in regeneration medium followed by a dark period of 24 h; (3) a dark period of 24 h followed by soaking in liquid VRM (Vitis Regeneration Medium); (4) vacuum infiltration in liquid VRM followed by a dark period of 24 h; and (5) a control of no pre-conditioning treatment. Excised leaves from pre-treated intact plants in vitro significantly increased the frequency of shoot organogenesis. The most responsive explant material consisted of young semi-translucent apical leaves varying in size from 3 to 8 mm in length. The most successful combinations of factors contributing to shoot organogenesis involved the solely dark-exposed apical leaves or the soaking in VRM followed by a dark period. These results are expected to facilitate Vitis-related research in genetics, functional genomics, physiology, and other fields.     

Rootstock scion somatogenetic interactions in perennial composite plants

The ancient plant production practice of grafting which instantly imparts new physiological properties to the desirable scion still remains shrouded in mystery. Yet, grafting remains a widely used technique in the production of several horticultural species. In a composite grafted plant, rootstocks control many aspects of scion growth and physiology including yield and quality attributes as well as biotic and abiotic stress tolerance. Broadly, physical, physiological, biochemical and molecular mechanisms have been reviewed to develop an integrated understanding of this enigmatic process that challenges existing genetic paradigms. This review summarizes the reported mechanisms underlying some of the economically important traits and identifies several key points to consider when conducting rootstock scion interaction experiments. Study of the somatogenetic interactions between rootstock and scion is a field that is ripe for discovery and vast improvements in the coming decade. Further, utilization of rootstocks based on a better understanding of the somatogenetic interactions is highly relevant in the current agricultural environment where there is a need for sustainable production practices. Rootstocks may offer a non-transgenic approach to rapidly respond to the changing environment and expand agricultural production of annual and perennial crops where grafting is feasible in order to meet the global food, fiber and fuel demands of the future.     

Poly(dA-dT)•Poly(dA-dT) in Low Salt Appears to be a Left-Handed B-Helix Combined Use of Chemical Theory,Fiber Diffraction and NMR Spectroscopy

Abstract

Poly(dA-dT)?poly(dA-dT) can adopt the B- and D- forms in the fibrous state. Theoretical energy calculations and fiber diffraction analyses suggest that there can be three structural models of poly(dA-dT)?poly(dA-dT) in each of these two forms viz right and left-handed Watson Crick models and left-handed Hoogsteen—a total of six possible models. Fiber data for the polymer in the B- or the D-form or energy calculations cannot distinguish any one model from the other. However, a comparison of observed proton chemical shifts with the theoretically computed ones and the NOE studies on exchangeable and nonexchangeable protons suggest that poly(dA-dT)?poly(dA-dT) in low salt solution exists predominantly in the left-handed B-conformation.     

Metabotropic glutamate receptor 6 signaling enhances TRPM1 calcium channel function and increases melanin content in human melanocytes

Mutations in TRPM1, a calcium channel expressed in retinal bipolar cells and epidermal melanocytes, cause complete congenital stationary night blindness with no discernible skin phenotype. In the retina, TRPM1 activity is negatively coupled to metabotropic glutamate receptor 6 (mGluR6) signaling through Gα_o and TRPM1 mutations result in the loss of responsiveness of TRPM1 to mGluR6 signaling. Here, we show that human melanocytes express mGluR6, and treatment of melanocytes with L‐AP4, a type III mGluR‐selective agonist, enhances Ca²⁺ uptake. Knockdown of TRPM1 or mGluR6 by shRNA abolished L‐AP4‐induced Ca²⁺ influx and TRPM1 currents, showing that TRPM1 activity in melanocytes is positively coupled to mGluR6 signaling. Gα_o protein is absent in melanocytes. However, forced expression of Gα_o restored negative coupling of TRPM1 to mGluR6 signaling, but treatment with pertussis toxin, an inhibitor of G_i/G_o proteins, did not affect basal or mGluR6‐induced Ca²⁺ uptake. Additionally, chronic stimulation of mGluR6 altered melanocyte morphology and increased melanin content. These data suggest differences in coupling of TRPM1 function to mGluR6 signaling explain different cellular responses to glutamate in the retina and the skin.