Effect of salinity and different nitrogen sources on the activity of antioxidant enzymes and indole alkaloid content in Catharanthus roseus seedlings

The activities of antioxidant enzymes viz. glutathione reductase, GR; superoxide dismutase, SOD; peroxidase, POD; catalase, CAT and glutathione-S-transferase, GST and alkaloid accumulation were investigated in leaf pairs (apical, middle, basal) and in roots of Catharanthus roseus seedlings under the conditions of different nitrogen sources (20 mM KNO(3) and 2 mM NH(4)Cl) and salinity, in the absence (non-saline control) and in the presence of 100 mM NaCl in the nutrient solution. Salinity caused a reduction in plant biomass. The biomass production of ammonium-fed plants was lower than that of nitrate-fed plants. The antioxidant enzymes exhibited higher activity in saline-treated plants. Changes in antioxidant enzyme activity caused by different nitrogen sources differed in all leaf pairs, as well as in roots of C. roseus. Ammonium-fed plants showed higher CAT, GR and GST activity in leaf pairs as well as in roots, while POD and SOD activity were higher in nitrate-fed plants. Higher peroxidase activity concomitant with the increased accumulation of alkaloid was found in all leaf pairs, as well as in roots of C. roseus of NO(3)(-) fed plants as compared to NH(4)(+) fed plants.     

Salt Stress-induced Responses in Growth and Metabolism in Callus Cultures and Differentiating In Vitro Shoots of Indian Ginseng (Withania somnifera Dunal)

In vitro-grown shoots and calli of Withania somnifera, an important medicinal plant, were exposed to various types of salts under in vitro culture conditions. Membrane permeability, lipid peroxidation, and the antioxidant system increased in shoots as well as in unorganized callus tissues under all the three concentrations of KCl, NaCl, KNO₃, NaNO₃, and CaCl₂. The growth responses of shoots and callus cultures under various salt treatments revealed that the tissue could grow better under NaCl and KNO₃ compared to other salts and the in vitro shoots appeared healthy at 50?mM concentration of NaCl and KNO_3. The activity of antioxidant enzymes such as catalase (CAT), ascorbate peroxidase, guaiacol peroxidase, lipoxygenase, polyphenol oxidase, and glutathione reductase increased under salt treatments, especially at higher concentrations. The greatest activity increase was recorded for peroxidases, whereas CAT was the least responsive. Only two isoforms, Mn-superoxide dismutase (Mn-SOD) and Fe-SOD, could be visualized in callus tissue while Cu/Zn-SOD was absent. Diaphorase 4 was totally missing in callus tissue and was detected only in shoots. Phenolics accumulated at all the concentrations of the salts tested as an induced protective response. The higher concentration of CaCl₂ produced maximum increases in antioxidants and enzymatic activities compared to other salts. Thus, for W. somnifera the presence of excess calcium in the growing medium is most deleterious compared to other salts. Results also suggest that the nonenzymatic and enzymatic antioxidant systems of both the tissues played a primary role in combating the imposed salt stress.     

Multifunctional role of Bcl-2 in malignant transformation and tumorigenesis of Cr(VI)-transformed lung cells

B-cell lymphoma-2 (Bcl-2) is an antiapoptotic protein known to be important in the regulation of apoptosis in various cell types. However, its role in malignant transformation and tumorigenesis of human lung cells is not well understood. We previously reported that chronic exposure of human lung epithelial cells to the carcinogenic hexavalent chromium Cr(VI) caused malignant transformation and Bcl-2 upregulation; however, the role of Bcl-2 in the transformation is unclear. Using a gene silencing approach, we showed that Bcl-2 plays an important role in the malignant properties of Cr(VI)-transformed cells. Downregulation of Bcl-2 inhibited the invasive and proliferative properties of the cells as well as their colony forming and angiogenic activities, which are upregulated in the transformed cells as compared to control cells. Furthermore, animal studies showed the inhibitory effect of Bcl-2 knockdown on the tumorigenesis of Cr(VI)-transformed cells. The role of Bcl-2 in malignant transformation and tumorigenesis was confirmed by gene silencing experiments using human lung carcinoma NCI-H460 cells. These cells exhibited aggressive malignant phenotypes similar to those of Cr(VI)-transformed cells. Knockdown of Bcl-2 in the H460 cells inhibited malignant and tumorigenic properties of the cells, indicating the general role of Bcl-2 in human lung tumorigenesis. Ingenuity Pathways Analysis (IPA) revealed potential effectors of Bcl-2 in tumorigenesis regulation. Additionally, using IPA together with ectopic expression of p53, we show p53 as an upstream regulator of Bcl-2 in Cr(VI)-transformed cells. Together, our results indicate the novel and multifunctional role of Bcl-2 in malignant transformation and tumorigenesis of human lung epithelial cells chronically exposed to Cr(VI).     

Kaposi's sarcoma-associated herpesvirus forms a multimolecular complex of integrins (alphaVbeta5, alphaVbeta3, and alpha3beta1) and CD98-xCT during infection of human dermal microvascular endothelial cells, and CD98-xCT is essential for the postentry stage of infection

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface heparan sulfate (HS) and α3β1 integrin during the early stages of infection of human dermal microvascular endothelial cells (HMVEC-d) and human foreskin fibroblasts (HFF), and these interactions are followed by virus entry overlapping with the induction of preexisting host cell signal pathways. KSHV also utilizes the amino acid transporter protein xCT for infection of adherent cells, and the xCT molecule is part of the cell surface heterodimeric membrane glycoprotein CD98 (4F2 antigen) complex known to interact with α3β1 and αVβ3 integrins. KSHV gB mediates adhesion of HMVEC-d, CV-1, and HT-1080 cells and HFF via its RGD sequence. Anti-αV and -β1 integrin antibodies inhibited the cell adhesion mediated by KSHV-gB. Variable levels of neutralization of HMVEC-d and HFF infection were observed with antibodies against αVβ3 and αVβ5 integrins. Similarly, variable levels of inhibition of virus entry into adherent HMVEC-d, 293 and Vero cells, and HFF was observed by preincubating virus with soluble α3β1, αVβ3, and αVβ5 integrins, and cumulative inhibition was observed with a combination of integrins. We were unable to infect HT1080 cells. Virus binding and DNA internalization studies suggest that αVβ3 and αVβ5 integrins also play roles in KSHV entry. We observed time-dependent temporal KSHV interactions with HMVEC-d integrins and CD98/xCT with three different patterns of association and dissociation. Integrin αVβ5 interaction with CD98/xCT predominantly occurred by 1 min postinfection (p.i.) and dissociated at 10 min p.i., whereas α3β1-CD98/xCT interaction was maximal at 10 min p.i. and dissociated at 30 min p.i., and αVβ3-CD98/xCT interaction was maximal at 10 min p.i. and remained at the observed 30 min p.i. Fluorescence microscopy also showed a similar time-dependent interaction of αVβ5-CD98. Confocal-microscopy studies confirmed the association of CD98/xCT with α3β1 and KSHV. Preincubation of KSHV with soluble heparin and α3β1 significantly inhibited this association, suggesting that the first contact with HS and integrin is an essential element in subsequent CD98-xCT interactions. Anti-CD98 and xCT antibodies did not block virus binding and entry and nuclear delivery of viral DNA; however, viral-gene expression was significantly inhibited, suggesting that CD98-xCT play roles in the post-entry stage of infection, possibly in mediating signal cascades essential for viral-gene expression. Together, these studies suggest that KSHV interacts with functionally related integrins (αVβ3, α3β1, and αVβ5) and CD98/xCT molecules in a temporal fashion to form a multimolecular complex during the early stages of endothelial cell infection, probably mediating multiple roles in entry, signal transduction, and viral-gene expression.     

The Effect of Polyhydramnios on Cervical Length in Twins: A Controlled Intervention Study in Complicated Monochorionic Pregnancies

Objective

To test the hypothesis that cervical shortening in polyhydramnios reflects the degree of excess amniotic fluid, and increases with normalisation of amniotic fluid volume.

Study Design

Prospective cohort study of 40 women with monochorionic twins undergoing interventional procedures between 16–26 weeks. Cervical length was assessed via transvaginal sonography pre-procedure, 1 and 24 hours post-procedure, and results compared between amnioreduction and control procedures. Amniotic fluid index (AFI) was measured pre- and post-procedure.

Results

Pre-procedural cervical length correlated with AFI (linear fit = 5.07 -0.04x, R² = 0.17, P = 0.03) in patients with polyhydramnios (n = 28). Drainage of 2000ml fluid (range 700–3500ml), reduced AFI from 42cm to 21cm (P<0.001). Their pre-procedural cervical length did not change at one (mean Δ:−0.1cm, 95%CI, −0.4 to 0.2) or 24 hours (0.2cm, −0.1 to 0.6) after amnioreduction. There was no change in cervical length at control procedures.

Conclusion

Cervical shortening in twins with polyhydramnios does not appear to be an acute process; cervical length can be measured before or after therapeutic procedures.     

In vitro withanolide production by Withania somnifera L. cultures

In vitro multiple shoots, root, callus and cell suspension cultures of Withania somnifera exhibited the potentiality to produce pharmacologically active withanolides. Multiple shoots cultures exhibited an increase in withanolide A accumulation compared to shoots of the mother plant. In vitro generated root cultures as well as callus and suspension cultures also produced withanolides albeit at lower levels.     

TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita

Patients with dyskeratosis congenita (DC), a heterogeneous inherited bone marrow failure syndrome, have abnormalities in telomere biology, including very short telomeres and germline mutations in DKC1, TERC, TERT, or NOP10, but approximately 60% of DC patients lack an identifiable mutation. With the very short telomere phenotype and a highly penetrant, rare disease model, a linkage scan was performed on a family with autosomal-dominant DC and no mutations in DKCI, TERC, or TERT. Evidence favoring linkage was found at 2p24 and 14q11.2, and this led to the identification of TINF2 (14q11.2) mutations, K280E, in the proband and her five affected relatives and TINF2 R282H in three additional unrelated DC probands, including one with Revesz syndrome; a fifth DC proband had a R282S mutation. TINF2 mutations were not present in unaffected relatives, DC probands with mutations in DKC1, TERC, or TERT or 298 control subjects. We demonstrate that a fifth gene, TINF2, is mutated in classical DC and, for the first time, in Revesz syndrome. This represents the first shelterin complex mutation linked to human disease and confirms the role of very short telomeres as a diagnostic test for DC.     

Sequential liquid-liquid extraction of d-amino acid oxidase from Trigonopsis variabilis

A method for isolation of d-amino acid oxidase (DAAO) from disrupted Trigonopsis variabilis cells has been developed. In an aqueous two-phase system consisting of PEG6000 (220 g l^–1), potassium phosphate (110 g l^–1, K₂HPO₄ + KH₂PO₄ = 10.1:1, mol mol^–1) and dl-methionine (11 g l^–1), the major portion of cellular proteins (87%) was partitioned into the salt phase. By sequential extraction, 48% of DAAO was recovered in PEG phase, giving a yield of 211 U mg protein^–1.     

D-Aspartate oxidase and D-amino acid oxidase are localised in the peroxisomes of terrestrial gastropods

D-Aspartate oxidase and D-amino acid oxidase were found in high activity in the tissues of representative species of terrestrial gastropods. Analytical subcellular fractionation demonstrated that both of these oxidases co-localised with the peroxisome markers, acyl-CoA oxidase and catalase, in the digestive gland homogenate. Electron microscopy of peak peroxisome fractions showed particles of uniform size with generally well preserved variably electron-dense matrices bounded by an apparently single limiting membrane. Many of the particles exhibited a core region of enhanced electron density. Catalase cytochemistry of peak fractions confirmed the peroxisome identity of the organelles. Peroxisome-enriched subcellular fractions were used to investigate the properties of gastropod D-aspartate oxidase and D-amino acid oxidase activities. The substrate and inhibitor specificities of the two activities demonstrated that two distinct enzymes were present analogous to, but not identical to, the equivalent mammalian peroxisomal enzymes.