Plant regeneration from peduncle segments of oil seed Brassica species: Influence of silver nitrate and silver thiosulfate

Cultured peduncle segments of B. juncea, B. campestris, B. napus, B. nigra and B. carinata produced shoot buds on Murashige and Skoog medium supplemented with benzyladenine and 1-naphthalene acetic acid. Supplementation of the media with 30 μm silver nitrate or silver thiosulfate enhanced the frequency of shoot regeneration. The regenerated shoots could be rooted at a frequency of 95% and transferred to soil where 75% survived and set seed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Gametophytes,embryogeny and pericarp ofMicrostylis wallichii lindl. (Orchidaceae)

The anther wall is 4-layered thick. Its development is of the Monocotyledonous type. Simultaneous cytokinesis results in decussate, isobilateral, linear and tetrahedral tetrads. At anthesis, the microspores are 2-celled. The mature ovules are anatropous, bitegmic and tenuinucellate. Both the integuments are dermal in origin and 2-layered. The inner integument alone forms the micropyle. Development of the female gametophyte is of the Monosporic type. Double fertilization occurs but the primary endosperm nucleus degenerates without any division. Development of embryo corresponds to the variation of the Onagrad type. The mature embryo lacks differentiation. The seeds are minute and non-endospermic. The seed coat is formed entirely by the outer layer of outer integument. There are three sterile and three fertile valves in the ovary. In the prefertilization stages valves consist of parenchymatous cells. After fertilization, the sterile valves become sclerenchymatous whereas the fertile valves remain parenchymatous.     

Fuel smoke condensate induced DNA damage in human lymphocytes and protection by turmeric (Curcuma longa)

Summary Twigs-dry leaves smoke condensate (TDS) was investigated for its DNA damaging activity in human peripheral lymphocytes, by using a sensitive method, fluorescence analysis of DNA unwinding (FADU). An aqueous turmeric component (Aq.T) was studied as a protective agent. TDS at one to 100 folds dilution induced 55% DNA damage at 20 min, while 12-0-tetradecanoylphorbol-13-acetate (TPA) at 10 ng/ml induced only 25% damage. Aq.T at 300 ng/1 afforded 90% protection to DNA against TPA and 65% against TPA. The mechanism of Aq.T protection was investigated by using (i) inhibitors of arachidonate cascade, viz., indomethacin (28 M), NDGA (10 M), DBAP (36 M), (ii) antioxidant enzymes viz., CAT (0.2 U/l), SOD (0.6 U/1), (iii) antioxidants - BHA, curcumin (40 M), mixed gangliosides (20 nM) and protease inhibitor TLCK (100 M). These compounds offered the following extents of protection to DNA against TDS: indomethacin-40%, NDGA-83%, DBAP-70%, SOD-38%, CAT-40%, BHA-38%, curcumin-60%, mixed gangliosides-88%, TLCK-85%. Against TPA as clastogenic agent, the extents of protection were: indomethacin-73%, NDGA-32%, DBAP-72%, SOD-60%, CAT, BHA-negligible, curcumin-23%, mixed gangliosides - 60%, TLCK - 59%. These results indicate that (i) TDS and TPA induce DNA damage possibly by different mechanisms, (ii) Aq.T is a more effective protectant against TDS whereas it is on par with other inhibitors against TPA.Abbreviations FADU Fluoroscence Analysis of DNA Unwinding - Aq.T Aqueous component of turmeric - TDS Twigs-Dry leaves Smoke condensate - PBS Phosphate Buffered Saline, 20 mM, 150 mM NaCl, pH 7.4 - TPA 12-O-Tetradecanoyl Phorbol-13-Acetate - NDGA Nordihydroguaiaretic Acid - DBAP 2,4-Dibromo Acetophenone - CAT Catalase - SOD Superoxide Dismutase - BHA Butylated Hydroxyanisole - TLCK Tosyl Lysyl Chloromethyl Ketone - ROS Reactive Oxygen Species - PAH Polycyclic Aromatic Hydrocarbons - DMSO Dimethyl Sulfoxide - Buffer B 250 mM m-inositol, 10 mM sodium phosphate, 1 mM magnesium chloride, pH 7.3 - BSC Beedi Smoke Condensate - CSC Cigarette Smoke Condensate     

Target cell induced activation of NK cells in vitro: cytokine production and enhancement of cytotoxic function

This study examines the effect of fixed AK-5 tumour cells on rat NK cells. Co-culture of NK cells with fixed tumour cells augmented the cytotoxicity of NK cells against NK-sensitive targets, YAC-1 and AK-5, and induced the secretion of IFN-gamma by NK cells. Antibody against IFN-gamma suppressed the anti-tumour activity of NK cells, whereas the addition of T cells during co-culture enhanced this activity. However, macrophages and B cells had no significant effect when present during co-culture with NK cells. All the inducible cytotoxicity was contained within the NK (CD161+) and NKT (CD3+, CD161+) subsets of lymphocytes. However, in the presence of T cells, the cytolytic potential of NKT cells was higher than that of NK cells alone. The augmentation of cytotoxic activity of NK cells by AK-5 cells in presence of T cells was dependent on IL-2 and IFN-gamma secretion. NK cell activation was blocked by specific antibodies to IL-2 and IFN-gamma in the presence of T cells. Interaction between fixed AK-5 cells with NK and T cell populations induced the expression of Fas-L and perforin in NK cells. These data demonstrate that fixed AK-5 cells initiated cytokine synthesis by NK cells, and the enhanced cytotoxic activity in the presence of T cells was induced as a consequence of the products secreted by activated T lymphocytes. The present observations reflect the possible interactions taking place in vivo after the transplantation of AK-5 tumour in animals. They also suggest direct activation of NK cells after their interaction with the tumour cells.     

PID15, a novel 6?kDa secreted peptide,mediates Naja naja venom phospholipase A2 induced apoptosis in isolated human peripheral lymphocytes

Background

Snake venoms are a complex mixture of active principles mainly peptides and proteins also including amino acids, nucleotides, free lipids, carbohydrates and metallic elements bound to proteins that interfere in several biological systems. In this study, we aimed to understand the mode of action of the apoptosis inducing ability of Naja naja venom phospholipase A₂ (NV-PLA₂) using isolated human peripheral lymphocytes.

Results

Human peripheral lymphocytes when incubated with Naja naja venom phospholipase A₂ (NV-PLA₂) induced up to 68% DNA fragmentation. The dialysed conditioned media obtained by incubating lymphocytes with NV-PLA₂ at 15^th min induced 44% DNA fragmentation, referred to as cmlp-active. Cmlp-active showed 20.5% increased protein concentration than the corresponding control condition media cmlp-c-15. Test for creatine kinase activity in cmlp-active proved negative and negligible amount of lactate dehydrogenase did not show significant DNA fragmentation. Fractionation of cmlp-active on Sephadex G-25 showed two peaks, major peak induced 38% DNA fragmentation, which was further rechromatographed on Sephadex G-25. The single peak obtained was named PID15 (Phospholipase A ₂ Induced DNA fragmentation factor secreted at 15 ^th min). Q-Tof MS/MS analysis of PID-15 showed it is a 6 kDa peptide. PID15 sequence analysis gave 40 amino acids in the following order, msilpcknvs iwvikdtaas dkevvlgsdr aikflylatg. The homology search for the sequence revealed it to be an Apoptosis Inducing Factor (AIF).

Conclusion

Results indicate that the secretion of PID15 is dependent on concentration of NV-PLA₂ treatment, incubation time and also on temperature and the probable membrane origin of PID15 and not of cytosolic origin with apoptosis inducing ability.     

Binding of synthetic B knobs to fibrinogen changes the character of fibrin and inhibits its ability to activate tissue plasminogen activator and its destruction by plasmin

Synthetic peptides corresponding to the amino-terminal sequence of the beta chain of fibrin increase the turbidity of fibrin clots, whether they are generated by the direct interaction of thrombin and fibrinogen or by the reassociation of fibrin monomers. The turbidity of batroxobin-induced clots, which are characteristically "fine," is increased even more dramatically. Pentapeptides are more effective than tetrapeptides. Surprisingly, the same peptides also delay fibrinolysis, whether activated by exogenously added plasmin or from the fibrin-enhanced stimulation of tissue plasminogen activator (tPA) activation of plasminogen. The peptides have only a very slight effect on the plasmic hydrolysis of a chromogenic peptide, either by the direct addition of plasmin or by plasmin generated from plasminogen by tPA. The synthetic peptides mimicking the B knobs appear to exert their action in two ways. First, they render fibrin less vulnerable to attack by plasmin. Second, they delay the fibrin activation of tPA. The latter is attributed to their ability to prevent the binding of the authentic B knob, which itself is located at the end of a flexible 50-residue tether and which needs time to find its elusive "hole". We propose that, when after a while the tethered knob does become inserted, it locks the betaC domain in a conformation that allows access to tPA-plasminogen-binding sites, whereas the untethered synthetic knobs restrict the fibrin to a conformation in which those sites remain inaccessible. Thus, although the interaction involving the A knob and gammaC hole is the basis for the polymerization of fibrin, the comparable but delayed interaction involving the B knob and the betaC hole is ultimately directed at preparing the clot for its eventual destruction.     

In silico evolution of functional modules in biochemical networks

Understanding the large reaction networks found in biological systems is a daunting task. One approach is to divide a network into more manageable smaller modules, thus simplifying the problem. This is a common strategy used in engineering. However, the process of identifying biological modules is still in its infancy and very little is understood about the range and capabilities of motif structures found in biological modules. In order to delineate these modules, a library of functional motifs has been generated via in silico evolution techniques. On the basis of their functional forms, networks were evolved from four broad areas: oscillators, bistable switches, homeostatic systems and frequency filters. Some of these motifs were constructed from simple mass action kinetics, others were based on Michaelis-Menten kinetics as found in protein/protein networks and the remainder were based on Hill equations as found in gene/protein interaction networks. The purpose of the study is to explore the capabilities of different network architectures and the rich variety of functional forms that can be generated. Ultimately, the library may be used to delineate functional motifs in real biological networks.     

Structure of Ceramide-1-Phosphate at the Air-Water Solution Interface in the Absence and Presence of Ca

Ceramide-1-phosphate, the phosphorylated form of ceramide, gained attention recently due to its diverse intracellular roles, in particular in inflammation mediated by cPLA₂α. However, surprisingly little is known about the physical chemical properties of this lipid and its potential impact on physiological function. For example, the presence of Ca²⁺ is indispensable for the interaction of Cer-1-P with the C2 domain of cPLA₂α. We report on the structure and morphology of Cer-1-P in monomolecular layers at the air/water solution interface in the absence and presence of Ca²⁺ using diverse biophysical techniques, including synchrotron x-ray reflectivity and grazing angle diffraction, to gain insight into the role and function of Cer-1-P in biomembranes. We show that relatively small changes in pH and the presence of monovalent cations dramatically affect the behavior of Cer-1-P. On pure water Cer-1-P forms a solid monolayer despite the negative charge of the phosphomonoester headgroup. In contrast, pH 7.2 buffer yields a considerably less solid-like monolayer, indicating that charge-charge repulsion becomes important at higher pH. Calcium was found to bind strongly to the headgroup of Cer-1-P even in the presence of a 100-fold larger Na⁺ concentration. Analysis of the x-ray reflectivity data allowed us to estimate how much Ca²⁺ is bound to the headgroup, ∼0.5 Ca²⁺ and ∼1.0 Ca²⁺ ions per Cer-1-P molecule for the water and buffer subphase respectively. These results can be qualitatively understood based on the molecular structure of Cer-1-P and the electrostatic/hydrogen-bond interactions of its phosphomonoester headgroup. Biological implications of our results are also discussed.     

Unusual binding interactions in PDZ domain crystal structures help explain binding mechanisms

PDZ domains most commonly bind the C‐terminus of their protein targets. Typically the C‐terminal four residues of the protein target are considered as the binding motif, particularly the C‐terminal residue (P0) and third‐last residue (P‐2) that form the major contacts with the PDZ domain's “binding groove”. We solved crystal structures of seven human PDZ domains, including five of the seven PDLIM family members. The structures of GRASP, PDLIM2, PDLIM5, and PDLIM7 show a binding mode with only the C‐terminal P0 residue bound in the binding groove. Importantly, in some cases, the P‐2 residue formed interactions outside of the binding groove, providing insight into the influence of residues remote from the binding groove on selectivity. In the GRASP structure, we observed both canonical and noncanonical binding in the two molecules present in the asymmetric unit making a direct comparison of these binding modes possible. In addition, structures of the PDZ domains from PDLIM1 and PDLIM4 also presented here allow comparison with canonical binding for the PDLIM PDZ domain family. Although influenced by crystal packing arrangements, the structures nevertheless show that changes in the positions of PDZ domain side‐chains and the αB helix allow noncanonical binding interactions. These interactions may be indicative of intermediate states between unbound and fully bound PDZ domain and target protein. The noncanonical “perpendicular” binding observed potentially represents the general form of a kinetic intermediate. Comparison with canonical binding suggests that the rearrangement during binding involves both the PDZ domain and its ligand.