Culture and regeneration of mesophyll-derived protoplasts of sorghum [Sorghum bicolor (L.) Moench]

 A protocol for plant regeneration from mesophyll/protoplasts of sorghum [Sorghum bicolor (L.) Moench] was developed. The yield of intact protoplasts, their subsequent divisions and regeneration were genotype-dependent. The genotype 296B was always more responsive than IS 32266. For 296B, the sixth leaf from 18-day-old plants kept in dark for 2 days before harvesting was found to be the most suitable source of viable protoplasts. The first division was observed 10–12 days after plating, and the second division after 12–14 days. The maximum plating efficiency was 4.8% in 296 B, followed by 2.48% in IS 32266. Microcolonies were visible after 25–30 days, and microcalli after 60–75 days. Whole plants were obtained after 6–8 weeks of culture of microcalli on MS medium containing 0.2 mg l^–1 kinetin and 2 mg l^–1 BAP. The frequency of regeneration in 296B and IS 32266 was 12.80% and 10.58%, respectively. Ten plants transferred to pots in the glasshouse established well. The seeds collected from glasshouse-grown plants were sown in the field where plants were grown to maturity. Received: 7 October 1998 / Revision received: 13 January 1999 / Accepted: 20 January 1999     

Role of endogenous purine metabolism in thidiazuron-induced somatic embryogenesis of peanut (Arachis hypogaea L.)

Somatic embryogenesis was induced at the hypocotyledonary notch region of intact peanut (Arachis hypogaea L.) seedings cultured on a medium containing 10 mol·L^–1 thidiazuron (TDZ). Inclusion of the purine analogs 2,6-diaminopurine (DAP), azaadenine or azaguanine to the thidiazuron amended medium inhibited the embryogenic response of the seedlings. DAP-mediated inhibition was not overcome by the addition of adenine sulphate. Inhibition of the embryogenic response by DAP provides evidence that the TDZ-induced accumulation of purine cytokinins is an essential component of TDZ-induced somatic embryogenesis process. Analyses of the endogenous level of purine metabolites indicated that supplementation of the media with TDZ resulted in an overall increase in the endogenous cytokinins while DAP inhibited the purine recycling resulting in decreased levels of endogenous adenine and zeatin.     

Protection of Energy Transfer Process in Isolated Phycobilisome by "White Light" from Ultraviolet-B Induced Damage in the Cyanobacterium Spirulina Platensis

Effect of UV-B (1.9 W m^-2) alone or in combination with supplemental "white light". WL (20 W m^-2) exposure was studied on the energy transfer process of intact phycobilisomes isolated from Spirulina platensis. Exposure of UV-B or supplemental irradiation induced a decrease in room temperature fluorescence intensity and caused a shift towards shorter wavelengths. The low temperature fluorescence measurements showed that UV-B impairs energy transfer from phycocyanin to allophycocyanin B and the extent of damage may be reduced by the exposure to supplemental WL. This revised version was published online in June 2006 with corrections to the Cover Date.     

Seizure-induced plasticity of h channels in entorhinal cortical layer III pyramidal neurons

The entorhinal cortex (EC) provides the predominant excitatory drive to the hippocampal CA1 and subicular neurons in chronic epilepsy. Discerning the mechanisms underlying signal integration within EC neurons is essential for understanding network excitability alterations involving the hippocampus during epilepsy. Twenty-four hours following a single seizure episode when there were no behavioral or electrographic seizures, we found enhanced spontaneous activity still present in the rat EC in vivo and in vitro. The increased excitability was accompanied by a profound reduction in I(h) in EC layer III neurons and a significant decline in HCN1 and HCN2 subunits that encode for h channels. Consequently, dendritic excitability was enhanced, resulting in increased neuronal firing despite hyperpolarized membrane potentials. The loss of I(h) and the increased neuronal excitability persisted for 1 week following seizures. Our results suggest that dendritic I(h) plays an important role in determining the excitability of EC layer III neurons and their associated neural networks.     

Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2

We examined expression of sphingosine 1-phosphate (S1P) receptors and sphingosine kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S1P-induced 20-kDa myosin light chain (MLC₂₀) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P₁ and S1P₂ receptors. S1P activated G_q, G₁₃, and all G_i isoforms and stimulated PLC-1, PLC-3, and Rho kinase activities. PLC- activity was partially inhibited by pertussis toxin (PTX), G or G_q antibody, PLC-1 or PLC-3 antibody, and by expression of G_q or G_i minigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of G₁₃ or G_q minigene and abolished by expression of both. S1P stimulated Ca²⁺ release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC₅₀ 1 nM). Initial contraction and MLC₂₀ phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and G_q or G antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC₂₀ phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73122 or ML-9. These results indicate that S1P induces 1) initial contraction mediated by S1P₂ and S1P₁ involving concurrent activation of PLC-1 and PLC-3 via G_q and G_i, respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca²⁺ release and MLCK-mediated MLC₂₀ phosphorylation, and 2) sustained contraction exclusively mediated by S1P₂ involving activation of RhoA via G_q and G₁₃, resulting in Rho kinase- and PKC-dependent MLC₂₀ phosphorylation. muscle contraction; signal transduction     

Synthesis of fluorinated analogues of SJG-136 and their DNA-binding potential

A series of C2-exo-fluorounsaturated pyrrolobenzodiazepines (PBDs) have been synthesized. These C2-exo-fluorounsaturated PBD dimers have exhibited remarkable DNA-binding affinity.     

Artificial neural network as an alternative to multiple regression analysis in optimizing formulation parameters of cytarabine liposomes

The objective of the study was to optimize the formulation parameters of cytarabine liposomes by using artificial neural networks (ANN) and multiple regression analysis using 3(3) factorial design (FD). As model formulations, 27 formulations were prepared. The formulation variables, drug (cytarabine)/lipid (phosphatidyl choline [PC] and cholesterol [Chol]) molar ratio (X1), PC/Chol in percentage ratio of total lipids (X2), and the volume of hydration medium (X3) were selected as the independent variables; and the percentage drug entrapment (PDE) was selected as the dependent variable. A set of causal factors was used as tutorial data for ANN and fed into a computer. The optimization was performed by minimizing the generalized distance between the predicted values of each response and the optimized one that was obtained individually. In case of 3(3) factorial design, a second-order full-model polynomial equation and a reduced model were established by subjecting the transformed values of independent variables to multiple regression analysis, and contour plots were drawn using the equation. The optimization methods developed by both ANN and FD were validated by preparing another 5 liposomal formulations. The predetermined PDE and the experimental data were compared with predicted data by paired t test, no statistically significant difference was observed. ANN showed less error compared with multiple regression analysis. These findings demonstrate that ANN provides more accurate prediction and is quite useful in the optimization of pharmaceutical formulations when compared with the multiple regression analysis method.     

Identification of the G protein-activating sequence of the single-transmembrane natriuretic peptide receptor C (NPR-C)

Rat natriuretic peptideclearance receptor (NPR-C) contains four sequences capable ofinhibiting adenylyl cyclase. We have undertaken mutational and deletionstudies on the intracellular domain of rat NPR-C to determine which ofthese sequences is functionally relevant. Nine mutant receptors wereconstructed by deletion of 11 or 28 COOH-terminal residues or bysite-directed mutagenesis of basic residues in a 17-amino acidsequence, R⁴⁶⁹RNHQEESNIGKHRELR⁴⁸⁵,corresponding to the main active peptide. Substitution of arginine residues (R⁴⁶⁹R⁴⁷⁰) flanking theNH₂ terminus abolished G_i1 and G_i2and PLC- activities and inhibition of adenylyl cyclase. Substitutionof one or two basic residues (H⁴⁸¹ and/or R⁴⁸²or R⁴⁸⁵) in the COOH-terminal motif(H⁴⁸¹RELR⁴⁸⁵) greatly decreased or abolished Gprotein and PLC- activities and inhibition of adenylyl cyclase. Thisimplies that sequences NH₂-terminal to the motif orCOOH-terminal to R⁴⁷⁰ could not sustain receptor activityin situ, although they exhibited activity when used as syntheticpeptides. Deletion of the 11 COOH-terminal residues (E⁴⁸⁶to A⁴⁹⁶) suggested an autoinhibitory function for thissequence. We conclude that the 17-amino acid sequence (R⁴⁶⁹to R⁴⁸⁵) in the middle region of the intracellular domainof NPR-C is both necessary and sufficient for activation of G proteinsand effector enzymes.