In vitro shoot multiplication and plantlet regeneration from nodal explants of <Emphasis Type="Italic">Cassia angustifolia </Emphasis>(Vahl.): a medicinal plant

An efficient, rapid and reproducible plant regeneration protocol was successfully developed for Cassia angustifolia using nodal explants excised from 14-day-old aseptic seedlings. Of the two cytokinins, 6-benzyladenine (BA) and thidiazuron (TDZ) evaluated as supplements to Murashige and Skoog (MS) medium, TDZ at an optimal concentration of 5.0 μM was effective in inducing multiple shoots. The highest rate of shoot multiplication was achieved on MS medium supplemented with 5.0 μM TDZ and 1.0 μM indole-3-acetic acid (IAA) at pH 5.8. The regenerated shoots when subcultured on hormone free MS medium considerably increased the rate of shoot multiplication and shoot length by end of fourth subculture passage. Rooting was achieved on the isolated shoots using MS medium with 60 μM indole -3- butyric acid (IBA) and 1% activated charcoal for 1 week and subsequently transferring the shootlets to half strength MS liquid media without IBA and activated charcoal. The in vitro raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in greenhouse.     

Mapping QTLs with digenic epistasis under multiple environments and predicting heterosis based on QTL effects

Mixed linear model approach was proposed for mapping QTLs with the digenic epistasis and QTL by environment (QE) interaction as well as additive and dominant effects. Monte Carlo simulations indicated that the proposed method could provide unbiased estimations for both positions and genetic main effects of QTLs, as well as unbiased predictions for QE interaction effects. A method was suggested for predicting heterosis based on individual QTL effects. The immortalized F₂ (IF₂) population constructed by random mating among RI or DH lines is appropriate for mapping QTLs with epistasis and their QE interaction. Based on the models and methodology proposed, we developed a QTL mapping software, QTLMapper 2.0 on the basis of QTLmapper 1.0, which is suitable for analyzing populations of DH, RIL, F₂ and IF₂. Data of thousand grain weight of IF₂ population with 240 lines derived from elite hybrid rice Shanyou 63 were analyzed as a worked example.     

Fine structure of the gap junction in the tunicate heart

Summary The plasma membranes of the tunicate heart exhibit an abundance of macular gap junctions distributed widely over the membrane surface. A study of these junctions by the freeze-etch technique was undertaken in an effort to elucidate the fine structure of this important membrane modification in a primitive heart. In cross or near-cross fractured junctions the junctional particles in contiguous membranes appear to be paired in register and to meet in the midline. In favorable face views, the junctional particles are seen to be disposed in hexagonal array. The individual particles display a distinct rosette-like substructure consistent with a six-membered ring of globular protein molecules clustered around a central channel. Similar junctional-type particles can be found in nonjunctional areas of membrane suggesting that the transport mechanism which they may represent is not restricted to the gap junction.Career Investigator of the American Heart AssociationWe wish to thank Dr. J.B. Jillett for use of the facilities of the Portobello Marine Biological Station; Mr. W.S. Bertaud, Physics and Engineering Laboratory, D.S.I.R., Lower Hutt, who kindly supervised the preparation of some of the freeze-etch replicas; Dr. R.H. Millar of the Dunstaffnage Marine Research Laboratory, Oban, Argyll, Scotland, who identified the tunicate used in the present (and previous) study; Prof. W.D. Trotter who made facilities in the Department of Anatomy, University of Otago Medical School, Dunedin, available to one of us (V.L.); and Mrs. S.M. O'Kane for excellent technical assistance. Generous support from the American Heart Association (to V.L.) and from the Medical Research Council of New Zealand (to D.G.R.) is gratefully acknowledged     

Ethylene biosynthesis in isolated vacuoles of Vicia faba L. — requirement for membrane integrity

The ability of vacuoles prepared from V. faba leaves to convert 1-aminocyclopropane-1-carboxylic acid to C₂H₄ was destroyed when vacuoles were lysed by passage through a hypodermic needle, freezing and thawing, osmotic shock, treatment with ethanol or with a detergent. Ethylene synthesis in the vacuolar fraction was also inhibited by the uncouplers carbonyl cyanide m-chlorophenyl hydrazone and dinitrophenol and by the ionophores valinomycin, nigericin, and A23187. Ethylene formation increased with increasing pH of the incubation medium over the pH range of 5.0–7.5. These observations support the hypothesis that C₂H₄ biosynthesis in vacuolar preparations is dependent on membrane integrity, possibly because of the requirement for a transmembrane ion gradient.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CCCP carbonyl cyanide m-chlorophenyl hydrazone     

Homology modeling of histidine-containing phosphocarrier protein and eosinophil-derived neurotoxin: Construction of models and comparison with experiment

Homology modeling methods have been used to construct models of two proteins—the histidine-containing phosphocarrier protein (HPr) from Mycoplasma capricolum and human eosinophil-derived neurotoxin (EDN). Comparison of the models with the subsequently determined X-ray crystal structures indicates that the core regions of both proteins are reasonably well reproduced, although the template structures are closer to the X-ray structures in these regions—possible enhancements are discussed. The conformations of most of the side chains in the core of HPr are well reproduced in the modeled structure. As expected, the conformations of surface side chains in this protein differ significantly from the X-ray structure. The loop regions of EDN were incorrectly modeled—reasons for this and possible enhancements are discussed. © 1995 Wiley-Liss, Inc.     

Detection of native-like models for amino acid sequences of unknown three-dimensional structure in a data base of known protein conformations.

We present an approach which can be used to identify native-like folds in a data base of protein conformations in the absence of any sequence homology to proteins in the data base. The method is based on a knowledge-based force field derived from a set of known protein conformations. A given sequence is mounted on all conformations in the data base and the associated energies are calculated. Using several conformations and sequences from the globin family we show that the native conformation is identified correctly. In fact the resolution of the force field is high enough to discriminate between a native fold and several closely related conformations. We then apply the procedure to several globins of known sequence but unknown three dimensional structure. The homology of these sequences to globins of known structures in the data base ranges from 49 to 17%. With one exception we find that for all globin sequences one of the known globin folds is identified as the most favorable conformation. These results are obtained using a force field derived from a data base devoid of globins of known structure. We briefly discuss useful applications in protein structural research and future development of our approach.     

Introducing “best single template” models as reference baseline for the Continuous Automated Model Evaluation (CAMEO)

Critical blind assessment of structure prediction techniques is crucial for the scientific community to establish the state of the art, identify bottlenecks, and guide future developments. In Critical Assessment of Techniques in Structure Prediction (CASP), human experts assess the performance of participating methods in relation to the difficulty of the prediction task in a biennial experiment on approximately 100 targets. Yet, the development of automated computational modeling methods requires more frequent evaluation cycles and larger sets of data. The “Continuous Automated Model EvaluatiOn (CAMEO)” platform complements CASP by conducting fully automated blind prediction evaluations based on the weekly pre-release of sequences of those structures, which are going to be published in the next release of the Protein Data Bank (PDB). Each week, CAMEO publishes benchmarking results for predictions corresponding to a set of about 20 targets collected during a 4-day prediction window. CAMEO benchmarking data are generated consistently for all methods at the same point in time, enabling developers to cross-validate their method's performance, and referring to their results in publications. Many successful participants of CASP have used CAMEO—either by directly benchmarking their methods within the system or by comparing their own performance to CAMEO reference data. CAMEO offers a variety of scores reflecting different aspects of structure modeling, for example, binding site accuracy, homo-oligomer interface quality, or accuracy of local model confidence estimates. By introducing the \"bestSingleTemplate\" method based on structure superpositions as a reference for the accuracy of 3D modeling predictions, CAMEO facilitates objective comparison of techniques and fosters the development of advanced methods.     

Molecular evolution and structural analysis of the Ca(2+) release-activated Ca(2+) channel subunit, Orai

Depletion of intracellular Ca(2+) stores evokes Ca(2+) entry across the plasma membrane by inducing Ca(2+) release-activated Ca(2+) (CRAC) currents in many cell types. Recently, Orai and STIM proteins were identified as the molecular identities of the CRAC channel subunit and the endoplasmic reticulum Ca(2+) sensor, respectively. Here, extensive database searching and phylogenetic analysis revealed several lineage-specific duplication events in the Orai protein family, which may account for the evolutionary origins of distinct functional properties among mammalian Orai proteins. Based on similarity to key structural domains and essential residues for channel functions in Orai proteins, database searching also identifies a putative primordial Orai sequence in hyperthermophilic archaeons. Furthermore, modern Orai appears to acquire new structural domains as early as Urochodata, before divergence into vertebrates. The evolutionary patterns of structural domains might be related to distinct functional properties of Drosophila and mammalian CRAC currents. Interestingly, Orai proteins display two conserved internal repeats located at transmembrane segments 1 and 3, both of which contain key amino acids essential for channel function. These findings demonstrate biochemical and physiological relevance of Orai proteins in light of different evolutionary origins and will provide novel insights into future structural and functional studies of Orai proteins.