Recognition of G-U mismatches by tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III).

The coordination complex tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III) [Rh(DIP)3(3+)], which promotes RNA cleavage upon photoactivation, has been shown to target specifically guanine-uracil (G-U) mismatches in double-helical regions of folded RNAs. Photoactivated cleavage by Rh(DIP)3(3+) has been examined on a series of RNAs that contain G-U mismatches, yeast tRNA(Phe) and yeast tRNA(Asp), as well as on 5S rRNAs from Xenopus oocytes and Escherichia coli. In addition, a "microhelix" was synthesized, which consists of seven base pairs of the acceptor stem of yeast tRNA(Phe) connected by a six-nucleotide loop and contains a mismatch involving residues G4 and U69. A U4.G69 variant of this sequence was also constructed, and cleavage by Rh(DIP)3(3+) was examined. In each of these cases, specific cleavage is observed at the residue which lies to the 3'-side of the wobble-paired U; some cleavage by the rhodium complex is also evident in several structured RNA loops. The remarkable site selectivity for G-U mismatches within double-helical regions is attributed to shape-selective binding by the rhodium complex. This binding furthermore depends upon the orientation of the G-U mismatch, which produces different stacking interactions between the G-U base pair with the Watson-Crick base pair following it on the 5'-side of U compared to the Watson-Crick pair preceding it on the 3'-side of U. Rh(DIP)3(3+) therefore serves as a unique probe of G-U mismatches and may be useful both as a model and in probing RNA-protein interactions as well as in identifying G-U mismatches within double-helical regions of folded RNAs.     

Delineation of structural domains in eukaryotic 5S rRNA with a rhodium probe.

The three-dimensional folding of Xenopus oocyte 5S rRNA has been examined using the coordination complex Rh(phen)2phi3+ (phen = phenanthroline; phi = phenanthrenequinone diimine) as a structural probe. Rh(phen)2phi3+ binds neither double-helical RNA nor unstructured single-stranded regions of RNA. Instead, the complex targets through photoactivated cleavage sites of tertiary interaction which are open in the major groove and accessible to stacking. The sites targeted by the rhodium complex have been mapped on the wild-type Xenopus oocyte RNA, on a truncated RNA representing the arm of the molecule comprised of helix IV-loop E-helix V, and on several single-nucleotide mutants of the 5S rRNA. On the wild-type 5S rRNA, strong cleavage is found at residues U73, A74, A101, and U102 in the E loop and U80 and G81 in helix IV; additional sites are evident at A22 and A56 in the B loop, C29 and A32 in helix III, and C34, C39, A42, and C44 in the C loop. Given the similarity observed in cleavage between the full 5S RNA and the truncated fragment as well as the absence of any long-range effects on cleavage in mutant RNAs, the results do not support models which involve long-range tertiary interactions. Cleavage results with Rh(phen)2phi3+ do, however, indicate that the apposition of several noncanonical bases as well as stem--loop junctions may result in intimately stacked structures with opened major grooves. In particular, on the basis of cleavage results on mutant RNAs, both loops C and E represent structures where the strands constituting each loop are not independent of one another but are intrinsically structured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ entry in T cells is activated by emptying the inositol 1,4,5-triphosphate sensitive Ca2+ pool

Using alpha-linolenic acid (ALA), one of several polyunsaturated fatty acids (PUFAs) that have previously been shown to both mobilize intracellular Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pool independently of IP3 production and inhibit Ca2+ influx, the relationship between Ca2+ mobilization from intracellular stores and Ca2+ influx in T cells (JURKAT) was studied. JURKAT cells were treated with 30 microM ALA to deplete the IP3-sensitive Ca2+ pool. When the intracellular free Ca2+ concentration [( Ca2+]i) returned to basal level, fatty acid free bovine serum albumin (BSA) was added to remove extracellular and membrane bound ALA. This resulted in a sustained increase in [Ca2+]i in the absence of inositol phosphates' formation. This sustained increase in [Ca2+]i was insensitive to protein kinase C activation but was inhibited by Ni2+ ions. The extent of Ca2+ influx was found to be correlated to the amount of Ca2+ initially discharged from the IP3-sensitive Ca2+ pool by sub-optimal concentrations of ALA. Ligation of the CD3 complex of the T cell antigen receptor with an anti-CD3 antibody (OKT3) during the sustained [Ca2+]i increased (induced by a sub-optimal concentration of ALA), produced a greater response. No increase in the sustained response was observed when the CD3 complex was activated in cells pretreated with an optimal concentration of ALA. In summary, Ca2+ entry in T cells is activated by emptying of the IP3-sensitive Ca2+ pool which can be dissociated from inositol phosphate production. The rate of Ca2+ influx appears to be closely correlated to the initial discharge of Ca2+ from the IP3-sensitive Ca2+ pool, suggesting that Ca2+ may first enter the depleted pool and then is released into the cytosol.     

Influence of BA and sucrose on the competence and determination of pepper (Capsicum annuum L. var. Sweet Banana) hypocotyl cultures during shoot formation

Summary The simultaneous presence of 6-benzyladenine (BA) and sucrose in a Murashige and Skoog medium (SIM) during the initial stages of shoot initiation have been found to be obligatory for high-frequency shoot formation in the Capsicum annuum L. var. Sweet Banana upper hypocotyl explants. The explants are determined for shoot formation following a minimum of 8 days of culture on SIM. Deprivation of exogenous sucrose from day 6 to day 20 of culture had no effect on the shoot forming response of the explants. BA and sucrose appear to act independently on different aspects of the competence of explants to respond to SIM during shoot initiation.Abbreviations BA N⁶-benzyladenine - MS Murashige and Skoog medium - SIM shoot induction medium - HFM hormone free medium - SUC sucrose minus medium     

The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton.

The GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, motility, and cytokinesis. We recently reported on a p150 serine/threonine kinase (termed ROK alpha) binding RhoA only in its active GTP-bound state and on its cDNA; introduction of RhoA into HeLa cells resulted in translocation of the cytoplasmic kinase to plasma membranes, consistent with ROK alpha being a target for RhoA (T. Leung, E. Manser, L. Tan, and L. Lim, J. Biol. Chem. 256:29051-29054, 1995). Reanalysis of the cDNA revealed that ROK alpha contains an additional N-terminal region. We also isolated another cDNA which encoded a protein (ROK beta) with 90% identity to ROK alpha in the kinase domain. Both ROK alpha and ROK beta, which had a molecular mass of 160 kDa, contained a highly conserved cysteine/histidine-rich domain located within a putative pleckstrin homology domain. The kinases bound RhoA, RhoB, and RhoC but not Rac1 and Cdc42. The Rho-binding domain comprises about 30 amino acids. Mutations within this domain caused partial or complete loss of Rho binding. The morphological effects of ROK alpha were investigated by microinjecting HeLa cells with DNA constructs encoding various forms of ROK alpha. Full-length ROK alpha promoted formation of stress fibers and focal adhesion complexes, consistent with its being an effector of RhoA. ROK alpha truncated at the C terminus promoted this formation and also extensive condensation of actin microfilaments and nuclear disruption. The proteins exhibited protein kinase activity which was required for stress fiber formation; the kinase-dead ROK alpha K112A and N-terminally truncated mutants showed no such promotion. The latter mutant instead induced disassembly of stress fibers and focal adhesion complexes, accompanied by cell spreading. These effects were mediated by the C-terminal region containing Rho-binding, cysteine/histidine-rich, and pleckstrin homology domains. Thus, the multidomained ROK alpha appears to be involved in reorganization of the cytoskeleton, with the N and C termini acting as positive and negative regulators, respectively, of the kinase domain whose activity is crucial for formation of stress fibers and focal adhesion complexes.     

Photoinactivation of photosystem II by cumulative exposure to short light pulses during the induction period of photosynthesis

Photoinactivation of Photosystem (PS) II in vivo was investigated by cumulative exposure of pea, rice and spinach leaves to light pulses of variable duration from 2 to 100 s, separated by dark intervals of 30 min. During each light pulse, photosynthetic induction occurred to an extent depending on the time of illumination, but steady-state photosynthesis had not been achieved. During photosynthetic induction, it is clearly demonstrated that reciprocity of irradiance and duration of illumination did not hold: hence the same cumulative photon exposure (mol m^–2) does not necessarily give the same extent of photoinactivation of PS II. This contrasts with the situation of steady-state photosynthesis where the photoinactivation of PS II exhibited reciprocity of irradiance and duration of illumination (Park et al. (1995) Planta 196: 401–411). We suggest that, for reciprocity to hold between irradiance and duration of illumination, there must be a balance between photochemical (qP) and non-photochemical (NPQ) quenching at all irradiances. The index of susceptibility to light stress, which represents an intrinsic ability of PS II to balance photochemical and non-photochemical quenching, is defined by the quotient (1-qP)/NPQ. Although constant in steady-state photosynthesis under a wide range of irradiance (Park et al. (1995). Plant Cell Physiol 36: 1163–1169), this index of susceptibility for spinach leaves declined extremely rapidly during photosynthetic induction at a given irradiance, and, at a given cumulative photon exposure, was dependent on irradiance. During photosynthetic induction, only limited photoprotective strategies are developed: while the transthylakoid pH gradient conferred some degree of photoprotection, neither D1 protein turnover nor the xanthophyll cycle was operative. Thus, PS II is more easily photoinactivated during photosynthetic induction, a phenomenon that may have relevance for understorey leaves experiencing infrequent, short sunflecks.Abbreviations D1 protein psbA gene product - DTT dithiothreitol - F_v, F_m, F_o variable, maximum, and initial (corresponding to open traps) chlorophyll fluorescence yield, respectively - NPQ non-photochemical quenching - PS Photosystem - Q_A primary quinone acceptor of PS II - qP photochemical quenching coefficient     

Poliovirus neutralization by antibodies to internal epitopes of VP4 and VP1 results from reversible exposure of these sequences at physiological temperature.

Antisera were raised against peptide sequences that are normally internal in the poliovirus virion. These antisera contain neutralizing activity, but this neutralizing activity is dependent on coincubation of the virus and antisera at 37 degrees C. Immunoprecipitation analyses demonstrate that the neutralization is due to exposure of these normally internal sequences at 37 degrees C and subsequent antibody binding. Exposure of these sequences is reversible. These data demonstrate that the poliovirus particle is a dynamic entity that is capable of undergoing conformational alterations at physiological temperatures. This conformational flexibility provides an explanation for earlier observations of virus neutralization by antibodies to internal epitopes which can be accommodated within the framework of existing models for antibody-mediated neutralization of viral infectivity. Analogies between the sequences which are reversibly exposed at 37 degrees C with those which are irreversibly exposed upon receptor binding suggest that the observed conformational dynamics also may play a role in cell entry.     

The effect of propargylic substitution on the activation and aromatization of enediynes

The thiol activation and aromatization of bicyclo[7.1.0]enediynes was found to be dependent of the nature of the propargyl substituent. These effects are correlated to antitumor activity.