Mechanism of glucose-6-phosphate dehydrogenase-mediated regulation of coronary artery contractility

We previously identified glucose-6-phosphate dehydrogenase (G6PD) as a regulator of vascular smooth muscle contraction. In this study, we tested our hypothesis that G6PD activated by KCl via a phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-protein kinase C (PKC) pathway increases vascular smooth muscle contraction and that inhibition of G6PD relaxes smooth muscle by decreasing intracellular Ca(2+) ([Ca(2+)](i)) and Ca(2+) sensitivity to the myofilament. Here we show that G6PD is activated by membrane depolarization via PKC and PTEN pathway and that G6PD inhibition decreases intracellular free calcium ([Ca(2+)](i)) in vascular smooth muscle cells and thus arterial contractility. In bovine coronary artery (CA), KCl (30 mmol/l) increased PKC activity and doubled G6PD V(max) without affecting K(m). KCl-induced PKC and G6PD activation was inhibited by bisperoxo(pyridine-2-carboxyl)oxovanadate (Bpv; 10 μmol/l), a PTEN inhibitor, which also inhibited (P < 0.05) KCl-induced CA contraction. The G6PD blockers 6-aminonicotinamide (6AN; 1 mmol/l) and epiandrosterone (EPI; 100 μmol/l) inhibited KCl-induced increases in G6PD activity, [Ca(2+)](i), Ca(2+)-dependent myosin light chain (MLC) phosphorylation, and contraction. Relaxation of precontracted CA by 6AN and EPI was not blocked by calnoxin (10 μmol/l), a plasma membrane Ca(2+) ATPase inhibitor or by lowering extracellular Na(+), which inhibits the Na(+)/Ca(2+) exchanger (NCX), but cyclopiazonic acid (200 μmol/l), a sarcoplasmic reticulum Ca(2+) ATPase inhibitor, reduced (P < 0.05) 6AN- and EPI-induced relaxation. 6AN also attenuated phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Ser855, a site phosphorylated by Rho kinase, inhibition of which reduced (P < 0.05) KCl-induced CA contraction and 6AN-induced relaxation. By contrast, 6AN increased (P < 0.05) vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser239, indicating that inhibition of G6PD increases PKA or PKG activity. Inhibition of PKG by RT-8-Br-PET-cGMPs (100 nmol/l) diminished 6AN-evoked VASP phosphorylation (P < 0.05), but RT-8-Br-PET-cGMPs increased 6AN-induced relaxation. These findings suggest G6PD inhibition relaxes CA by decreasing Ca(2+) influx, increasing Ca(2+) sequestration, and inhibiting Rho kinase but not by increasing Ca(2+) extrusion or activating PKG.     

Locking and unlocking of ribosomal motions

During the ribosomal translocation, the binding of elongation factor G (EF-G) to the pretranslocational ribosome leads to a ratchet-like rotation of the 30S subunit relative to the 50S subunit in the direction of the mRNA movement. By means of cryo-electron microscopy we observe that this rotation is accompanied by a 20 A movement of the L1 stalk of the 50S subunit, implying that this region is involved in the translocation of deacylated tRNAs from the P to the E site. These ribosomal motions can occur only when the P-site tRNA is deacylated. Prior to peptidyl-transfer to the A-site tRNA or peptide removal, the presence of the charged P-site tRNA locks the ribosome and prohibits both of these motions.     

Picrorhiza kurrooa: current status and tissue culture mediated biotechnological interventions

Picrorhiza kurrooa, one of the important plant species among the various medicinal plants, is endemic to Himalaya. As the plant is useful in the treatment of various diseases, e.g., hepatic disorders, gastric troubles, anemia, asthma, etc., illegal collection from the wild is increasing and now this plant is banned for export in any form and listed as ‘endangered’. Ecological studies carried out on this species in last few decades suggested that the availability of this species in its specific habitats is comparatively lower than other associate species. Possible factors responsible for this depletion are increasing demand in the pharmaceutical industries, habitat specificity, heavy exploitation from the wild, unorganized cultivation practices etc. Biotechnology is playing a crucial role to conserve this important plant species. The past 23 years have witnessed a progressive biotechnological advances made in P. kurrooa. People have published various reports on establishments of in vitro culture techniques including micropropagation, synthetic seed production, plant regeneration via callus-mediated shoot organogenesis, adventitious shoot regeneration, genetic transformation through Agrobacterium rhizogenes, secondary metabolite analysis etc. This review attempts to focus on present ecological status and provide a comprehensive account on the tissue culture-mediated biotechnological interventions made in P. kurrooa for improvement and conservation of this medicinally important plant.     

Shoot growth phenology of co-existing evergreen and deciduous species in an oak forest

Shoot growth phenology was compared for the saplings of evergreen and deciduous woody species sharing the same microsite. Growth initiation occurred earlier in evergreens (among co-stratal species) while deciduous species completed their growth earlier. Shoot growth rate was significantly greater (P<0.01) for deciduous trees than evergreen trees. The amount of shoot elongations and shoot diameter was also significantly greater (P<0.01) for deciduous trees than evergreens. On the other hand, among shrubs the amount of shoot elongation and shoot diameter was greater for evergreens but the rate of elongation and diameter was more or less similar for both. The duration of shoot elongation and shoot diameter was significantly longer in evergreens than the deciduous species. Leaf packing (number of leaves per shoot) was significantly more dense in evergreen trees (P<0.01) than in deciduous tree species. Leaf packing was more dense in evergreen than deciduous shrubs but the difference was not significant. Leaf area (per individual leaf) at full expansion was significantly greater (P<0.01) in deciduous species. Leaf dry mass and specific leaf mass in the initial stage was significantly greater for evergreen species than for deciduous species. The number of buds/10 cm of shoot was higher in evergreens. However, the per cent mortality was also higher in them.     

Non-timber forest products (NTFPs): a viable option for biodiversity conservation and livelihood enhancement in central Himalaya

The present study aims to document detail information of some of the selected wild edible having enormous potential for livelihood enhancement and socio-economic development by making a variety of value added products. To this end, some of the wild edibles of central Himalaya were selected and prioritized for harnessing their economic potential along with their detail information in terms of distribution, ethnobiology, phenophases and appropriate time of harvesting so as to make communities well aware about the resource availability and their harvesting period round the year. The cost-benefit analysis of each value added product prepared from selected wild edibles was worked out in detail and these analyses revealed that total monetary output, as well as the net return, is very high for all value added products prepared. Since wild edible fruits or other edible parts can be collected from wild free of cost except labour is involved in collection of these wild edibles bio-resources. In addition, information on a participatory action research framework & approaches for promoting participatory conservation of these wild edible species were also highlighted for appropriate management of these resources. The present attempt provides a practical example of sustainable utilization of wild edibles, their potential in livelihood improvement of local people, distribution and phenophases and availability in natural conditions, participatory conservation of these wild edibles may help policy planners at the regional and national levels to link livelihood/socio-economic development with conservation.     

Identification and characterization of the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana

Despite the importance of riboflavin as the direct precursor of the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the physiologically relevant catalyst dephosphorylating the riboflavin biosynthesis pathway intermediate 5‐amino‐6‐ribitylamino‐2,4(1H,3H) pyrimidinedione 5′‐phosphate (ARPP) has not been characterized from any organism. By using as the query sequence a previously identified plastidial FMN hydrolase AtcpFHy1 (At1g79790), belonging to the haloacid dehalogenase (HAD) superfamily, seven candidates for the missing ARPP phosphatase were found, cloned, recombinantly expressed, and purified. Activity screening showed that the enzymes encoded by AtcpFHy1, At4g11570, and At4g25840 catalyze dephosphorylation of ARPP. AtcpFHy1 was renamed AtcpFHy/PyrP1, At4g11570 and At4g25840 were named AtPyrP2 and AtGpp1/PyrP3, respectively. Subcellular localization in planta indicated that AtPyrP2 was localized in plastids and AtGpp1/PyrP3 in mitochondria. Biochemical characterization of AtcpFHy/PyrP1 and AtPyrP2 showed that they have similar K_m values for the substrate ARPP, with AtcpFHy/PyrP1 having higher catalytic efficiency. Screening of 21 phosphorylated substrates showed that AtPyrP2 is specific for ARPP. Molecular weights of AtcpFHy/PyrP1 and AtPyrP2 were estimated at 46 and 72 kDa, suggesting dimers. pH and temperature optima for AtcpFHy/PyrP1 and AtPyrP2 were ~7.0–8.5 and 40–50°C. T‐DNA knockout of AtcpFHy/PyrP1 did not affect the flavin profile of the transgenic plants, whereas silencing of AtPyrP2 decreased accumulation of riboflavin, FMN, and FAD. Our results strongly support AtPyrP2 as the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana. The identification of this enzyme closes a long‐standing gap in understanding of the riboflavin biosynthesis in plants.     

Contribution of “substrate shuttles” in the transport of extramitochondrial reducing equivalents by hepatic mitochondria from chronic alcohol-fed mice

Effects of chronic alcohol treatment have been investigated on the rates of extramitochondrial NADH utilization by hepatic mitochondria in the presence or absence of “malate-aspartate shuttle,” oxidation of ethanol, α-glycerophosphate, and the activity of succinic dehydrogenase, along with the changes in the intrahepatic distribution of aspartate aminotransferase. The rates of blood alcohol clearance, hepatic alcohol dehydrogenase activity, and NADPH-dependent microsomal ethanol oxidation were also studied after different time intervals of alcohol withdrawal from chronically alcohol-fed animals. Hepatic mitochondria from chronically ethanol-fed mice (ethanol withheld 20 hr before sacrifice) utilized extramitochondrial NADH at rates 25–40% higher than the corresponding pair-fed controls. Addition of malateaspartate shuttle components to mitochondria from control and ethanol-fed groups resulted in 70 and 90% stimulation of NADH utilization, respectively. Mitochondria from both groups showed respiratory control upon ADP addition (state 3). Preincubation with amino-oxyacetate or hydrazine, which inhibit aspartate aminotransferase activity, prevented the stimulatory effect of malate-aspartate shuttle on NADH utilization. Mitochondria from livers of chronic ethanol-fed mice in the presence of reconstituted malate-aspartate shuttle showed 30–40% higher utilization of ethanol than the corresponding pair-fed control animals. The rate of mitochondrial α-glycerophosphate utilization by alcohol-fed animals was significantly higher than the control group. Succinic dehydrogenase activity measured as an index of mitochondrial permeability in the absence of Ca²⁺ showed 85% higher activity in alcoholtreated group than the control animals. Chronic ethanol feeding for 4 weeks resulted in an increase in the activity of hepatic aspartate aminotransferase in the cytoplasmic fraction and a corresponding decrease in the mitochondrial fraction. Alcohol withdrawal from chronic alcohol-fed animals resulted in a decrease in the blood alcohol clearance rate after 10 days. Furthermore, a lack of correlation was observed between the rates of blood alcohol clearance and the activity of hepatic alcohol dehydrogenase on one hand, and between the rates of blood alcohol clearance and the microsomal ethanol-oxidizing activity on the other.     

Floral ontogeny inMazus pumilus (Scrophulariaceae)

InMazus pumilus, all the floral appendages are initiated in acropetal sequence in the second cell layer (except stamens) of the floral primordium by periclinal divisions. The actinomorphic calyx tube is formed due to zonal growth. The zygomorphy in corolla is evident from the inception of petal primordia which arise sequentially as independent units in order of one anterior, a pair of anterio-lateral followed by a pair of posterio-lateral. Later these primordia exhibit differential growth because of which zygomorphy becomes more pronounced. The upper corolla tube is formed by interprimordial growth and lower corolla tube by zonal growth. Stamens are initiated in the third layer of the floral apex. Unlike sepals and petals, in the development of stamens (4) underlying cells of corpus also contribute. Posterior stamen is absent. The stamens become epipetalous because of interprimordial and zonal growth in the common region below the bases of petals as well as stamens. The two carpel primordia arise as crescent shaped structures which become continuous due to interprimordial growth. The ovary is formed by a ring of zonal meristem. The style develops later between stigma and ovary because of intercalary growth. The residual apex grows vertically along with the ovary and forms the septum of the ovary. All the floral appendages exhibit similar pattern of histogenesis and early growth suggesting thereby the appendicular nature of these appendages.