Withaferin A: a proteasomal inhibitor promotes healing after injury and exerts anabolic effect on osteoporotic bone.

Withania somnifera or Ashwagandha is a medicinal herb of Ayurveda. Though the extract and purified molecules, withanolides, from this plant have been shown to have different pharmacological activities, their effect on bone formation has not been studied. Here, we show that one of the withanolide, withaferin A (WFA) acts as a proteasomal inhibitor (PI) and binds to specific catalytic ß subunit of the 20S proteasome. It exerts positive effect on osteoblast by increasing osteoblast proliferation and differentiation. WFA increased expression of osteoblast-specific transcription factor and mineralizing genes, promoted osteoblast survival and suppressed inflammatory cytokines. In osteoclast, WFA treatment decreased osteoclast number directly by decreasing expression of tartarate-resistant acid phosphatase and receptor activator of nuclear factor kappa-B (RANK) and indirectly by decreasing osteoprotegrin/RANK ligand ratio. Our data show that in vitro treatment of WFA to calvarial osteoblast cells decreased expression of E3 ubiquitin ligase, Smad ubiquitin regulatory factor 2 (Smurf2), preventing degradation of Runt-related transcription factor 2 (RunX2) and relevant Smad proteins, which are phosphorylated by bone morphogenetic protein 2. Increased Smurf2 expression due to exogenous treatment of tumor necrosis factor α (TNFα) to primary osteoblast cells was decreased by WFA treatment. This was corroborated by using small interfering RNA against Smurf2. Further, WFA also blocked nuclear factor kappa-B (NF-kB) signaling as assessed by tumor necrosis factor stimulated nuclear translocation of p65-subunit of NF-kB. Overall data show that in vitro proteasome inhibition by WFA simultaneously promoted osteoblastogenesis by stabilizing RunX2 and suppressed osteoclast differentiation, by inhibiting osteoclastogenesis. Oral administration of WFA to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased expression of osteogenic genes. WFA supplementation improved trabecular micro-architecture of the long bones, increased biomechanical strength parameters of the vertebra and femur, decreased bone turnover markers (osteocalcin and TNFα) and expression of skeletal osteoclastogenic genes. It also increased new bone formation and expression of osteogenic genes in the femur bone as compared with vehicle groups (Sham) and ovariectomy (OVx), Bortezomib (known PI), injectible parathyroid hormone and alendronate (FDA approved drugs). WFA promoted the process of cortical bone regeneration at drill-holes site in the femur mid-diaphysis region and cortical gap was bridged with woven bone within 11 days of both estrogen sufficient and deficient (ovariectomized, Ovx) mice. Together our data suggest that WFA stimulates bone formation by abrogating proteasomal machinery and provides knowledge base for its clinical evaluation as a bone anabolic agent.

Arsenate exposure affects amino acids, mineral nutrient status and antioxidants in rice (Oryza sativa L.) genotypes.

Simulated pot experiments were conducted on four rice (Oryza sativa L.) genotypes (Triguna, IR-36, PNR-519, and IET-4786) to examine the effects of As(V) on amino acids and mineral nutrient status in grain along with antioxidant response to arsenic exposure. Rice genotypes responded differentially to As(V) exposure in terms of amino acids and antioxidant profiles. Total amino acid content in grains of all rice genotypes was positively correlated with arsenic accumulation. While, most of the essential amino acids increased in all cultivars except IR-36, glutamic acid and glycine increased in IET-4786 and PNR-519. The level of nonprotein thiols (NPTs) and the activities of superoxide dismutase (SOD; EC 1.15.1.1), glutathione reductase (GR; EC 1.6.4.2) and ascorbate peroxidase (APX; EC 1.11.1.11) increased in all rice cultivars except IET-4786. A significant genotypic variation was also observed in specific arsenic uptake (SAU; mg kg(-1)dw), which was in the order of Triguna (134) > IR-36 (71) > PNR-519 (53) > IET-4786 (29). Further, application of As(V) at lower doses (4 and 8 mg L(-1) As) enhanced the accumulation of selenium (Se) and other nutrients (Fe, P, Zn, and S), however, higher dose (12 mg L(-1) As) limits the nutrient uptake in rice. In conclusion, low As accumulating genotype, IET-4786, which also had significantly induced level of essential amino acids, seems suitable for cultivation in moderately As contaminated soil and would be safe for human consumption.

Differential transcriptional expression following thidiazuron-induced callus differentiation developmental shifts in rice.

Very little is known about molecular events associated with callus differentiation in indica rice. The genes expressed differentially during shoot meristem initiation were identified on genomic arrays applied to efficiently regenerating rice calli. A thidiazuron (TDZ; N-phenyl-N-thiadiazol-1,2,3-5,ylurea)-dependent regeneration protocol was developed for efficient embryogenesis in indica rice. The regenerating embryogenic calli induced by TDZ for 10 days showed transcriptional modulation of a number of genes associated with photosynthesis, hormone metabolism, plant development, signal transduction, light response, and plant defense. Eighteen candidate miRNAs were predicted to target the genes expressed differentially in the embryogenic calli grown in TDZ-containing medium. The majority of the photosynthesis-related genes up-regulated in differentiating calli were not expressed or were down-regulated in developing seeds and inflorescences. Most of the genes down-regulated in differentiating calli were up-regulated in developing seeds. The transcriptome of differentiating callus most closely resembled that of the germinating whole seed.

Cadmium-induced biochemical responses of Vallisneria spiralis.

The following study was carried out to investigate the cadmium (Cd) accumulating potential of Vallisneria. After subjecting plants to different concentrations of Cd, it was observed that plants are able to accumulate ample amount of metal in their roots (5,542 microg g(-1) dw) and leaves (4,368 microg g(-1) dw) in a concentration- and duration-dependent manner. Thus, it is evident that the accumulation in roots was 1.3 times higher than the shoots. It was also noted that with increasing Cd accumulation, roots of the plant appeared darker in color and harder in texture. In response to metal exposure, amount of low molecular weight antioxidants such as cysteine and nonprotein thiols (NP-SH) and activity of enzymes such as APX and GPX were significantly enhanced at lower concentrations of Cd, followed by decline at higher doses. It was also observed that in exposed plants, activity of APX enzyme was higher in roots (ca. 3 times) as compared to leaves. However, chlorophyll and protein content was found to decline significantly in a dose-dependent manner. Results suggested that due to its high accumulation potential, Vallisneria may be effectively grown in water bodies moderately contaminated with Cd.

Arsenic affects mineral nutrients in grains of various Indian rice (Oryza sativa L.) genotypes grown on arsenic-contaminated soils of West Bengal.

The exposure of paddy fields to arsenic (As) through groundwater irrigation is a serious concern that may not only lead to As accumulation to unacceptable levels but also interfere with mineral nutrients in rice grains. In the present field study, profiling of the mineral nutrients (iron (Fe), phosphorous, zinc, and selenium (Se)) was done in various rice genotypes with respect to As accumulation. A significant genotypic variation was observed in elemental retention on root Fe plaque and their accumulation in various plant parts including grains, specific As uptake (29-167 mg kg(-1) dw), as well as As transfer factor (4-45%). Grains retained the least level of As (0.7-3%) with inorganic As species being the dominant forms, while organic As species, viz., dimethylarsinic acid and monomethylarsonic acid, were non-detectable. In all tested varieties, the level of Se was low (0.05-0.12 mg kg(-1) dw), whereas that of As was high (0.4-1.68 mg kg(-1) dw), considering their safe/recommended daily intake limits, which may not warrant their human consumption. Hence, their utilization may increase the risk of arsenicosis, when grown in As-contaminated areas.

Lead bioaccumulation potential of an aquatic macrophyte Najas indica are related to antioxidant system.

Plants of Najas indica bioaccumulated significantly higher amounts of Pb (3554 microg g(-1) dw) when, exposed to varying concentrations of Pb(NO(3))(2).This also led to increased malondialdehyde (MDA), electrical conductivity (EC) and H(2)O(2) content. In response to this, the activities of antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT) and glutathione reductase (GR) were elevated along with the induction of various molecular antioxidants including GSH, cysteine, ascorbic acid and proline. Further, Pb exposed plants showed significantly increased cysteine synthase and glutathione-S-transferase activity. Visible symptoms of toxicity were evident at 50 microM after 4d showing chlorosis and fragmentation of leaves with mucilaginous discharge. It seems that bioaccumulated Pb is efficiently tolerated by Najas plants through activation of antioxidant system and thiolic pathways which was evident by the increased biomass up to 10 microM Pb. Therefore, it appears that due to metal tolerance characteristics with high concentration factor these plants can find use in phytoremediation of aquatic system highly contaminated by Pb.

Characterization of native microalgal strains for their chromium bioaccumulation potential: phytoplankton response in polluted habitats.

Due to its various uses, Cr contamination has become widespread in a diverse array of environments. The present study was carried out during 2007-2008 to investigate the accumulation potential of metals (Cr, Cu, Fe, Mn, Ni and Zn) and metalloid (As) by green (GA) and blue green (BGA) microalgae growing naturally in selected Cr-contaminated sites in districts Unnao and Kanpur (Uttar Pradesh, India). This investigation is a preliminary work to identify suitable native microalgae for biomonitoring and phytoremediation purposes. A total of 22 GA and 11 BGA were encountered in three seasons (summer, rainy and winter). Among these, the accumulation potential was evaluated in high biomass producing strains of BGA (three) and GA (nine). The maximum accumulation of Cr was shown by Phormedium bohneri (8550 microg g(-1)dw) followed by Oscillatoria tenuis (7354 microg g(-1)dw), Chlamydomonas angulosa (5325 microg g(-1)dw), Ulothrix tenuissima (4564 microg g(-1)dw), and Oscillatoria nigra (1862 microg g(-1)dw); all of which demonstrated a transfer factor of >10% for Cr. The results also indicate that the phytoplankton diversity was modified by Cr pollution. BGA represented the dominant community where Cr concentration was higher (11.84 and 2.27 mg L(-1)) (r=0.695), whereas GA showed negative correlation with respect to Cr concentration (r=-0.567). In conclusion, different algal species were able to grow in Cr-contaminated sites and to accumulate significant amounts of Cr with a high transfer factor.

Phytochelatins and antioxidant systems respond differentially during arsenite and arsenate stress in Hydrilla verticillata (L.f.) Royle.

Serious contamination of aquatic systems by arsenic (As) in different parts of the world calls for the development of an in situ cost-effective phytoremediation technology. In the present investigation, plants of Hydrilla verticillata (L.f.) Royle were exposed to various concentrations of arsenate (As(V)) (0-250 microM) and arsenite (AsIII) (0-25 microM) and analyzed for accumulation responses vis-à-vis biochemical changes. Total As accumulation was found to be higher in plants exposed to AsIII (315 microg g(-1) dw at 25 microM) compared to As(V) (205 microg g(-1) dw at 250 microM) after 7 d of treatment. Plants tolerated low concentrations of As(III) and As(V) by detoxifying the metalloid through augmented synthesis of thiols such as phytochelatins and through increased activity of antioxidant enzymes. While As(V) predominantly stimulated antioxidant enzyme activity, As(III) primarily caused enhanced levels of thiols. The maximum amount of As chelated by PCs was found to be about 39% in plants exposed to As(III) (at 10 microM) and 35% in As(V) exposed plants (at 50 microM) after 4 d. Only the respective highest concentrations of As(III) (25 microM) and As(V) (250 microM) proved toxic for normal plant growth after prolonged treatment. Thus, H. verticillata forms a promising candidate for the phytoremediation of As contaminated water.

Equity in Swedish health care reconsidered: new results based on the finite mixture model.

This paper reconsiders the equity issue in Swedish health care utilization previously analysed by Gerdtham (Health Econ 1997; 6: 303-319) within the framework of the standard two-part model. Departing from the user/non-user distinction, we use the more flexible framework of the finite mixture model that distinguishes between frequent/infrequent users. Our results indicate that the support for the inequity hypothesis reported by Gerdtham is sensitive to model specification and the way standard errors of coefficients are estimated. The new framework offers an alternative perspective on the magnitude of the income-related difference in health care utilization.

Organization and post-transcriptional processing of the psb B operon from chloroplasts of Populus deltoides.

Chloroplast genes are typically organized into polycistronic transcription units that give rise to complex sets of mono- and oligo-cistronic overlapping RNAs through a series of processing steps. The psbB operon contains genes for the PSII (psbB, psbT, psbH) and cytochrome b(6)f (petB and petD) complexes which are needed in different amounts during chloroplast biogenesis. The functional significance of gene organization in this polycistronic unit, containing information for two different complexes, is not known and is of interest. To determine the organization and expression of these complexes, studies have been carried out on crop plants by different groups, but not much information is known about trees. We present the nucleotide sequences of PSII genes and RNA profiles of the genes located in the psbB operon from Populus deltoides, a tree species. Although the gene organization of this operon in P. deltoides is similar to that in other species, a few variations have been observed in the processing scheme.

Characterization of the orf31-petG gene cluster from the plastid genome of Populus deltoides.

The orf31-petG gene cluster is located approximately 1.2 kb away from the psbEFLJ operon in the chloroplast genome of Populus deltoides. The orf31 (ycf7) encodes an unidentified polypeptide while the petG gene encodes subunit V of an important component, cytochrome b6/f complex, involved in photosynthetic electron transport. We have determined the nucleotide sequence of the orf31-petG gene cluster from the plastid genome of a tree, Populus deltoides. Our sequence analysis suggests that these genes possess high homology with the published sequences of these genes from other plants. Northern analysis suggests development dependent transcription of the orf31-petG cluster in leaves.

Expression of chloroplastic genes during autumnal senescence in a deciduous tree Populus deltiodes.

In Populus deltoides, a deciduous tree, the development on new leaves starts in the month of March, the leaves reach maturity by October and fall by December. Changes in the composition and function of the photosynthetic apparatus were analysed during autumnal senescence. With the progress of senescence, there was an initial increase followed by a decrease in the steady state levels of psbA, psbD/C and psaA/B gene transcripts. Decrease in the steady state level of D1 protein was faster than that of Cytochrome f. The decline in LHCP level was seen only during late senescence. Although the leaves continue to look green and healthy till late November, the electron transport driven by individual photosystems started declining by October end suggesting the onset of senescence.