M. tuberculosis sliding β-clamp does not interact directly with the NAD+-dependent DNA ligase

The sliding β-clamp, an important component of the DNA replication and repair machinery, is drawing increasing attention as a therapeutic target. We report the crystal structure of the M. tuberculosis β-clamp (Mtbβ-clamp) to 3.0 Å resolution. The protein crystallized in the space group C222₁ with cell-dimensions a = 72.7, b = 234.9 & c = 125.1 Å respectively. Mtbβ-clamp is a dimer, and exhibits head-to-tail association similar to other bacterial clamps. Each monomer folds into three domains with similar structures respectively and associates with its dimeric partner through 6 salt-bridges and about 21 polar interactions. Affinity experiments involving a blunt DNA duplex, primed-DNA and nicked DNA respectively show that Mtbβ-clamp binds specifically to primed DNA about 1.8 times stronger compared to the other two substrates and with an apparent K_d of 300 nM. In bacteria like E. coli, the β-clamp is known to interact with subunits of the clamp loader, NAD⁺ -dependent DNA ligase (LigA) and other partners. We tested the interactions of the Mtbβ-clamp with MtbLigA and the γ-clamp loader subunit through radioactive gel shift assays, size exclusion chromatography, yeast-two hybrid experiments and also functionally. Intriguingly while Mtbβ-clamp interacts in vitro with the γ-clamp loader, it does not interact with MtbLigA unlike in bacteria like E. coli where it does. Modeling studies involving earlier peptide complexes reveal that the peptide-binding site is largely conserved despite lower sequence identity between bacterial clamps. Overall the results suggest that other as-yet-unidentified factors may mediate interactions between the clamp, LigA and DNA in mycobacteria.     

Long-Term Engraftment of Human Natural T Regulatory Cells in NOD/SCID IL2rγcnull Mice by Expression of Human IL-2

Regulatory T cells are essential to maintain immune homeostasis and prevent autoimmunity. Therapy with in vitro expanded human nT_Regs is being tested to prevent graft versus host disease, which is a major cause for morbidity and mortality associated with hematopoietic stem cell transplantation. Their usefulness in therapy will depend on their capacity to survive, migrate appropriately and retain suppressive activity when introduced into a transplant recipient. The lack of a suitable animal model for studying the in vivo reconstitutive capability of human nT_Regs is a major impediment for investigating the behavior of adoptively transferred nT_Regs in vivo. We show that injection of a plasmid encoding human IL-2 is necessary and sufficient for long term engraftment of in vitro expanded nT_Regs in NOD-SCID IL2rγc^null mice. We also demonstrate that these in vivo reconstituted T_Regs traffic to different organs of the body and retain suppressive function. Finally, in an IL-2 accelerated GVHD model, we show that these in vivo reconstituted T_Regs are capable of preventing severe xenogenic response of human PBMCs. Thus, this novel ‘hu-T_Reg mouse’ model offers a pre-clinical platform to study the in vivo function and stability of human nT_Regs and their ability to modulate autoimmune diseases and GVHD.     

Direct somatic embryogenesis from <Emphasis Type="Italic">Coffea arabica</Emphasis> L. and <Emphasis Type="Italic">Coffea canephora</Emphasis> P ex Fr. under the influence of ethylene action inhibitor-silver nitrate

For the first time direct somatic embryogenesis from hypocotyl explants of in vitro regenerated plantlets of C. arabica and C. canephora was achieved on modified MS medium containing 10 – 70 μM silver nitrate supplemented with 1.1 μM N⁶ benzyladenine and 2.85 μM indole-3-acetic acid. A maximum of 144.1±7.3 and 68.7±3.3 embryos per explant were produced at 40 μM silver nitrate in C. canephora and C. arabica respectively. Only yellow friable embryogenic callus obtained from the cut edges of most of leaf explants of both C. arabica and C. canephora at all concentrations of silver nitrate were tried in this experiment. Formation of secondary embryos from stage I primary embryos (small yellow, round, globular embryos) was more (28.23±1.3) at 60 μM silver nitrate in C. canephora, while 40 μM silver nitrate supported more of secondary embryo formation in C. arabica (40.5±1.2). When stage II (green globular round matured embryos) and stage III primary embryos (tubular stage embryos) were used, secondary embryo formation was very small and many of these embryos developed into plantlets and some of them even rooted. By using these protocols within 45 – 60 days it is possible to get secondary embryos from primary embryos and direct somatic embryos from hypocotyls of in vitro plantlets in both these Coffea species.     

Involvement and interaction of various signaling compounds on the plant metabolic events during defense response,resistance to stress factors,formation of secondary metabolites and their molecular aspects

Plants are a nearly unlimited source of phytochemicals. The plants produce various secondary metabolites, which are useful in its interaction with the environment, various stress factors and development of resistance against pathogen attack. A wide array of external stimuli are capable of triggering changes in the plant cell which leads to a cascade of reactions, ultimately resulting in the formation and accumulation of secondary metabolites which helps the plant to overcome the stress factors. The biotic and abiotic elicitors can result in an enhancement of the secondary metabolite production. The stimuli are perceived by receptors, which then result in the activation of the secondary messengers. These then transmit the signals into the cell through the signal transduction pathways leading to gene expression and biochemical changes. There is interplay of the signaling molecules also which regulates the entire pathway. This review is oriented towards the factors, which influence signal transduction pathway(s) with special reference to polyamines, calcium, jasmonates, salicylates, nitric oxide and ethylene. The interplay of these components to elicit a defense response is discussed. Molecular aspects of disease resistance and regulation of plant secondary metabolism has also been presented.     

Biotransformation of Digitoxin in Cell Cultures of Capsicum frutescens in the Presence of β-cyclodextrin

Cell suspension cultures of Capsicum frutescens accumulated digoxin, purpureaglycoside A and other unknown derivatives when digitoxin, a cardiac glycoside, was used as a precursor. The feeding of digitoxin complexed with β-cyclodextrin increased the accumulation of digoxin, purpureaglycoside A and other unknown derivatives. Control cultures (without digitoxin) did not produce any of these metabolites. The growth of cells was affected by both digitoxin as well as digitoxin- β-cyclodextrin. The accumulation of purpureaglycoside A and digoxin reached a maximum of 1241 and 374 μg 100 ml -1 culture on the 6th and 2nd day, respectively, which was 3.9 and 4.5 fold higher than cultures treated with digitoxin alone (sampled on the 13th day). The other unknown derivatives formed in digitoxin- β-cyclodextrin fed cultures were 15 times higher than digitoxin alone fed C. frutescens cultures. The addition of glucose to digitoxin- β-cyclodextrin treated cultures increased the accumulation of purpureaglycoside A which reached a maximum of 3589 μg 100 ml -1 culture after 12 h incubation, which was a 2.9 fold increase over cultures treated with digitoxin- β-cyclodextrin alone.     

Performance of hairy root cultures of Cichorium intybus L. In bioreactors of different configurations

Summary A transformed root culture of Cichorium intybus L. cv. Lucknow Local grown in different configurations of bioreactors was examined. The roots grown in an acoustic mist bioreactor showed the best performance in terms of increased specific growth rate (0.072d⁻¹) and esculin content (18.5gl⁻¹), the latter of which was comparable to that of shake flask data. C. intybus hairy root cultures grown in an acoustic mist bioreactor produced nearly twice as much esculin as compared to roots grown in bubble column and nutrient sprinkle bioreactors. Studies relating to on-line estimation of conductivity and osmolarity to predict the growth of hairy root cultures are also discussed. The results demonstrate the efficacy and the advantages of an acoustic mist bioreactor for the cultivation of hairy root cultures, especially with reference to C. intybus hairy roots.     

in vitro propagation of Ochreinauclea missionis (Wall. EX G. Don), an ethnomedicinal endemic and threatened tree

Summary Biotechnology has offered a nonconventional method of plant propagation and has been intensively applied as a conservation strategy for sustaining biodiversity for rare plants. In vitro conservation through micropropagation of Ochreinauclea missionis, a rare, endemic and medicinal tree species of Western Ghats in Karnataka region of India is reported. Multiple shoots were initiated from nodal explants on Murashige and Skoog (MS) medium supplemented with 8.8 μM 6-benzylaminopurine (BA) and 0.3% (w/v) activated charcoal. Shoots were elongated in MS medium with a combination of 2.2 μM BA and 5.3 μM α-naphthaleneacetic acid (NAA) or growth regulator-free medium. Individual shoots with a minimum of one node were excised and rooted in vitro on MS medium with 0.3% activated charcoal or ex vitro rooted by treatment with 49 μM indole-3-butyric acid (IBA) for 30 min. Regenerants acclimated in Soil-rite exhibited 65% survival in the greenhouse.     

Abnormality in Translational Regulation of Catalase Expression in Disorders of Peroxisomal Biogenesis

Abstract: Peroxisomal disorders are a newly described group of inherited neurological diseases. In disorders of peroxisomal biogenesis, e.g., Zellweger syndrome, owing to the lack of peroxisomes, catalase, a peroxisomal enzyme, is found to be present in the cytoplasm instead. We observed higher catalase activity (7.59 ± 0.41 mU/mg of protein) in cultured skin fibroblasts from Zellweger patients than in control fibroblasts (4.45 ± 0.29 mU/mg of protein). Moreover, we also found that the majority of the catalase in Zellweger cells was present in the inactive form. The specific activities following reactivation in Zellweger and control cells were 12.1 and 4.9 mU/mg of protein, respectively. To understand the molecular basis of higher levels of catalase in Zellweger than control cells, we examined the rate of synthesis and turnover of catalase and levels of catalase mRNA and protein levels in Zellweger cells as compared with control cells. The initial rates of synthesis of catalase in Zellweger (1.68 ± 0.15 mU/mg of protein) and control (1.51 ± 0.14 mU/mg of protein) cells were similar. The rates of turnover of catalase in Zellweger (t_1/2 = 47 ± 8 h) and control (t_1/2 = 49 ± 7 h) were also similar. Consistent with the enzyme activity, the levels of catalase protein were higher in Zellweger cells as compared with control cells. On the other hand, there was no difference in the level of catalase mRNA between control and Zellweger cells. Although the rate of synthesis in Zellweger and control cells were initially similar, it was down-regulated to a lower level at ～72 h of culture in control fibroblasts as compared with Zellweger cells, which continued to synthesize catalase at the same rate up to 5 days in culture. The presence of similar levels of mRNA in control and Zellweger cells and continued synthesis of catalase in Zellweger cells at a higher level as compared with control cells suggest a loss of regulation at the translational level.     

Decavanadate possesses α-adrenergic agonist activity and a structural motif common with trans-β form of noradrenaline

Decavanadate, an inorganic polymer of vanadate, produced contraction of rat aortic rings at a relatively high concentration compared to phenylephrine, an agonist of -adrenergic receptor. This effect was blocked by two known a-adrenergic receptor antagonists, prazosin and phenoxybenzamine. Decavanadate, formed by possible dimerization of V5 under acid conditions, possessed a structural feature of two pairs of unshared oxygen atoms at a distance of 3.12 Å, not found in its constituents of V4 or V5. A structural motif of O..O..O using such oxygen atoms is recognized in decavanadate. This matches with a similar motif of N..O..O that uses the essential amino and hydroxyl groups of the side-chain and the m-hydroxyl group in trans-b form of noradrenaline. The interaction of such a structural motif with the membrane receptor is likely to be the basis of the unusual noradrenaline-mimic action of decavanadate.