Effects of N-acetylglucosamine and platelet inhibitors on the synergistic interaction of platelets and aggregating agents in the presence of wheat germ agglutinin

Low concentrations of wheat germ agglutinin (4 micrograms/ml) have been shown to act synergistically to induce platelet aggregation with epinephrine, collagen, arachidonate and ionophore A23187. Aggregation ceased on the addition of the haptenic sugar N-acetylglucosamine at any time following the onset of aggregation with these agonists and a small degree of disaggregation was observed during the reversible first wave with the biphasic aggregating agents epinephrine and ADP. Cyclooxygenase inhibitors such as indomethacin and aspirin blocked the second wave of aggregation with the biphasic aggregating agents epinephrine and ADP but a synergistic response continued to be shown with the first wave in the presence of these inhibitors. Release of [14C]serotonin and the mobilization of [3H]arachidonate by epinephrine and collagen were markedly stimulated in the presence of wheat germ agglutinin but there was no increase of either radiolabel in the case of ADP. Platelet shape change, but not aggregation, occurred with low levels of wheat germ agglutinin and the synergistic response with ADP, collagen or ionophore A23187 occurred without further shape change. Wheat germ agglutinin did not affect the basal or stimulated levels of cyclic AMP. The membrane fluidity of platelets was not affected by the lectin or by thrombin as shown by the lack of change in fluorescence polarization with diphenylhexatriene. It is suggested that the binding of wheat germ agglutinin to the platelet surface induces platelet activation by mechanisms similar to those of other agonists and that it may affect the distribution of membrane-bound Ca2+ by a reversible perturbation of the platelet membrane.     

Contributions to the cytology of endosperm in some angiosperms-VIII.Chrysanthemum carinatum L.

Cytological studies ofChrysanthemum carinatum have shown that the endosperm was free nuclear in the beginning, but, as the wall formation commenced very early, it was cellular for most of its life. The endosperm, in general, was triploid with 3n=27 chromosomes, but polyploidy (6n) and aneuploidy also occurred in a small number of cells.Although most of the nuclei in the main part of the endosperm were uniform in size and shape, they showed some variation in the haustorial region. The possible cause of this variation has been suggested to be endoduplication.Mitotic aberrations such as bridges, laggards and multipolar divisional stages appear to be the probable causes for the failure of endosperm in some of the immature seeds, resulting in a low seed set.     

Cryopreservation of Cymbidium eburneum Lindl. and C. hookerianum Rchb. f., two threatened and vulnerable orchids via encapsulation–dehydration

A successful cryopreservation protocol for the long-term conservation of protocorms of two threatened and vulnerable orchids, Cymbidium eburneum Lindl. and Cymbidium hookerianum Rchb. f., was developed using encapsulation–dehydration. Protocorms were osmoprotected in liquid Murashige and Skoog medium (MS) containing 0.7 M sucrose for 20 h at 25?±?2°C on a rotary shaker, and incorporated into an encapsulation matrix [consisting of 3% (w/v) sodium alginate and 100 mM CaCl₂]. The encapsulated protocorms, which were desiccated in a laminar airflow cabinet for 6 h, were able to withstand cryostorage in liquid nitrogen. Maximum regeneration into complete plantlets (72% for C. eburneum and 70% for C. hookerianum) of the cryostored, encapsulated protocorms was obtained using MS medium containing 3% sucrose and 0.8% agar. Using this protocol of cryopreservation, long-term preservation for ex situ conservation of these two threatened orchids can be accomplished.     

Molecular structure,vibrational spectroscopic,NBO and HOMO–LUMO studies of 2-amino 6-bromo 3-formylchromone

A systematic quantum mechanical study of the possible conformations and vibrational spectra of 2-amino 6-bromo 3-formylchromone has been reported. The equilibrium geometry, harmonic vibrational frequencies, infrared intensities and activities of Raman scattering were calculated by Hartree–Fock and density functional theory employing Becke's three-parameter (local, non-local and HF) hybrid exchange functionals with Lee–Yang–Parr co-relational (B3LYP) functionals using 6-311++G(d,p) basis set with complete relaxation in the potential energy surface. The calculated wavenumbers after proper scaling show a very good agreement with the observed values. The electrostatic potential mapped onto isodensity surface has been obtained. The natural bond orbital analysis has been carried out in order to study the intra-molecular bonding, interactions among bonds and delocalisation of unpaired electrons. The highest occupied molecular orbital–lowest unoccupied molecular orbital studies have been conducted in order to determine the way the molecule interacts with other species.     

Evidence of salicylic acid pathway with EDS1 and PAD4 proteins by molecular dynamics simulation for grape improvement

Biotic stress is a major cause of heavy loss in grape productivity. In order to develop biotic stress-resistant grape varieties, the key defense genes along with its pathway have to be deciphered. In angiosperm plants, lipase-like protein phytoalexin deficient 4 (PAD4) is well known to be essential for systemic resistance against biotic stress. PAD4 functions together with its interacting partner protein enhanced disease susceptibility 1 (EDS1) to promote salicylic acid (SA)-dependent and SA-independent defense pathway. Existence and structure of key protein of systemic resistance EDS1 and PAD4 are not known in grapes. Before SA pathway studies are taken in grape, molecular evidence of EDS1: PAD4 complex is to be established. To establish this, EDS1 protein sequence was retrieved from NCBI and homologous PAD4 protein was generated using Arabidopsis thaliana as template and conserved domains were confirmed. In this study, computational methods were used to model EDS1 and PAD4 and simulated the interactions of EDS1 and PAD4. Since no structural details of the proteins were available, homology modeling was employed to construct three-dimensional structures. Further, molecular dynamic simulations were performed to study the dynamic behavior of the EDS1 and PAD4. The modeled proteins were validated and subjected to molecular docking analysis. Molecular evidence of stable complex of EDS1:PAD4 in grape supporting SA defense pathway in response to biotic stress is reported in this study. If SA defense pathway genes are explored, then markers of genes involved can play pivotal role in grape variety development especially against biotic stress leading to higher productivity.     

Comparative study of key phosphorus and nitrogen metabolizing enzymes in mycorrhizal and non-mycorrhizal plants of Dendrobium chrysanthum Wall. ex Lindl.

The role of mycorrhizal fungi in overcoming nutrient limitation to plant growth by enhancing nutrient acquisition, especially phosphorus (P) and nitrogen (N), is well documented. However, in orchids, the importance of mycorrhizal fungi in nutrient acquisition is not as extensively studied as in other plants. Therefore, an in vitro culture system to study the effects of mycorrhizal association on P and N metabolizing enzymes, viz., acid phosphatase, alkaline phosphatase, nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) in Dendrobium chrysanthum was developed. After 90 days of mycorrhizal treatment, activities of acid phosphatase, alkaline phosphatase, NR, NiR and GS were higher in mycorrhizal plantlets than in control plantlets. The hardened plantlets that were initially treated with mycorrhiza under in vitro conditions also showed higher activities of the enzymes investigated. These mycorrhizal plantlets showed higher survival (96.33 %), shoot length (3.7 cm) and shoot fresh weight (0.359 g) as compared to control after 120 days of hardening. The results presented in this study suggest that mycorrhizal association might have increased the assimilation of P and N in D. chrysanthum plantlets, indicating the importance of mycorrhiza in orchids.     

α-Synuclein Membrane Association Is Regulated by the Rab3a Recycling Machinery and Presynaptic Activity

α-Synuclein is an abundant presynaptic protein and a primary component of Lewy bodies in Parkinson disease. Although its pathogenic role remains unclear, in healthy nerve terminals α-synuclein undergoes a cycle of membrane binding and dissociation. An α-synuclein binding assay was used to screen for vesicle proteins involved in α-synuclein membrane interactions and showed that antibodies directed to the Ras-related GTPase Rab3a and its chaperone RabGDI abrogated α-synuclein membrane binding. Biochemical analyses, including density gradient sedimentation and co-immunoprecipitation, suggested that α-synuclein interacts with membrane-associated GTP-bound Rab3a but not to cytosolic GDP-Rab3a. Accumulation of membrane-bound α-synuclein was induced by the expression of a GTPase-deficient Rab3a mutant, by a dominant-negative GDP dissociation inhibitor mutant unable to recycle Rab3a off membranes, and by Hsp90 inhibitors, radicicol and geldanamycin, which are known to inhibit Rab3a dissociation from membranes. Thus, all treatments that inhibited Rab3a recycling also increased α-synuclein sequestration on intracellular membranes. Our results suggest that membrane-bound GTP-Rab3a stabilizes α-synuclein on synaptic vesicles and that the GDP dissociation inhibitor·Hsp90 complex that controls Rab3a membrane dissociation also regulates α-synuclein dissociation during synaptic activity.     

In-silico analysis of caspase-3 and -7 proteases from blood-parasitic Schistosoma species (Trematoda) and their human host

Proteolytic enzymes of the caspase family, which reside as latent precursors in most nucleated metazoan cells, are core effectors of apoptosis. Of them, the executioner caspases- 3 and -7 exist within the cytosol as inactive dimers and are activated by a process called dimerization. Caspase inhibition is looked upon as a promising approach for treating multiple diseases. Though caspases have been extensively studied in the human system, their role in eukaryotic pathogens and parasites of human hosts has not drawn enough attention. In protein sequence analysis, caspases of blood flukes (Schistosoma spp) were revealed to have a low sequence identity with their counterparts in human and other mammalian hosts, which encouraged us to analyse interacting domains that participate in dimerization of caspases in the parasite and to reveal differences, if any, between the host-parasite systems. Significant differences in the molecular surface arrangement of the dimer interfaces reveal that in schistosomal caspases only eight out of forty dimer conformations are similar to human caspase structures. Thus, the parasite-specific dimer conformations (that are different from caspases of the host) may emerge as potential drug targets of therapeutic value against schistosomal infections. Three important factors namely, the size of amino acids, secondary structures and geometrical arrangement of interacting domains influence the pattern of caspase dimer formation, which, in turn, is manifested in varied structural conformations of caspases in the parasite and its human hosts.