Somatic embryogenesis and plant regeneration from cell suspension culture of niger (Guizotia abyssinica Cass.)

Somatic embryogenesis was achieved from cell suspension cultures of niger (Guizotia abyssinica Cass.). Initially, friable embryogenic calluses were induced from cotyledonary leaves of niger on Murashige and Skoog (MS) agar medium containing 5 μM 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 μM kinetin (KIN). Cell suspension cultures were established by using embryogenic calluses in MS liquid medium containing 5 μM 2,4-D and 0.5 μM KIN. Initiation of somatic embryogenesis and development up to globular stage from embryogenic cell clumps occurred in the liquid medium itself. Thereafter embryogenic cell aggregates were transferred to MS agar medium supplemented with 3 μM KIN for embryo differentiation, whereas maturation of somatic embryos occurred in MS agar medium containing 10 μM abscisic acid.     

Replitase: complete machinery for DNA synthesis

Replication of nuclear DNA in eukaryotes presents a tremendous challenge, not only due to the size and complexity of the genome, but also because of the time constraint imposed by a limited duration of S phase during which the entire genome has to be duplicated accurately and only once per cell division cycle. A challenge of this magnitude can only be met by the close coupling of DNA precursor synthesis to replication. Prokaryotic systems provide evidence for multienzyme and multiprotein complexes involved in DNA precursor synthesis and DNA replication. In addition, fractionation of nuclear proteins from proliferating mammalian cells shows co-sedimentation of enzymes involved in DNA replication with those required for synthesis of deoxynucleoside triphosphates (dNTPs). Such complexes can be isolated only from cells that are in S phase, but not from cells in G(0)/G(1) phases of cell cycle. The kinetics of deoxynucleotide metabolism supporting DNA replication in intact and permeabilized cells reveals close coupling and allosteric interaction between the enzymes of dNTP synthesis and DNA replication. These interactions contribute to channeling and compartmentation of deoxynucleotides in the microvicinity of DNA replication. A multienzyme and multiprotein megacomplex with these unique properties is called "replitase." In this article, we summarize some of the relevant evidence to date that supports the concept of replitase in mammalian cells, which originated from the observations in Dr. Pardee's laboratory. In addition, we show that androgen receptor (AR), which plays a critical role in proliferation and viability of prostate cancer cells, is associated with replitase, and that identification of constituents of replitase in androgen-dependent versus androgen-independent prostate cancer cells may provide insights into androgen-regulated events that control proliferation of prostate cancer cells and potentially offer an effective strategy for the treatment of prostate cancer.     

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.     

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.     

Diabetic endothelial dysfunction: the role of poly(ADP-ribose) polymerase activation

Diabetic patients frequently suffer from retinopathy, nephropathy, neuropathy and accelerated atherosclerosis. The loss of endothelial function precedes these vascular alterations. Here we report that activation of poly(ADP-ribose) polymerase (PARP) is an important factor in the pathogenesis of endothelial dysfunction in diabetes. Destruction of islet cells with streptozotocin in mice induced hyperglycemia, intravascular oxidant production, DNA strand breakage, PARP activation and a selective loss of endothelium-dependent vasodilation. Treatment with a novel potent PARP inhibitor, starting after the time of islet destruction, maintained normal vascular responsiveness, despite the persistence of severe hyperglycemia. Endothelial cells incubated in high glucose exhibited production of reactive nitrogen and oxygen species, consequent single-strand DNA breakage, PARP activation and associated metabolic and functional impairment. Basal and high-glucose-induced nuclear factor-kappaB activation were suppressed in the PARP-deficient cells. Our results indicate that PARP may be a novel drug target for the therapy of diabetic endothelial dysfunction.     

A flavone and an unusual 23-carbon terpenoid from Andrographis paniculata

Phytochemical investigation of the roots and aerial parts of Andrographis paniculata Nees yielded a new flavone, 5-hydroxy-7,2',6'-trimethoxyflavone and an unusual 23-carbon terpenoid, 14-deoxy-15-isopropylidene-11,12-didehydroandrographolide together with five known flavonoids and four known diterpenoids. The structures of these compounds were determined on the basis of spectral and chemical studies.     

Heterologous desensitization of response mediated by selective PKC-dependent phosphorylation of G(i-1) and G(i-2)

This study examined the ability of protein kinase C (PKC) toinduce heterologous desensitization by targeting specific G proteinsand limiting their ability to transduce signals in smooth muscle.Activation of PKC by pretreatment of intestinal smooth muscle cellswith phorbol 12-myristate 13-acetate, cholecystokinin octapeptide, orthe phosphatase 1 and phosphatase 2A inhibitor, calyculin A,selectively phosphorylated G_i-1 and G_i-2,but not G_i-3 or G_o, and blockedinhibition of adenylyl cyclase mediated by somatostatin receptorscoupled to G_i-1 and opioid receptors coupled toG_i-2, but not by muscarinic M₂ and adenosineA₁ receptors coupled to G_i-3. Phosphorylationof G_i-1 and G_i-2 and blockade of cyclaseinhibition were reversed by calphostin C and bisindolylmaleimide, andadditively by selective inhibitors of PKC and PKC. Blockade ofinhibition was prevented by downregulation of PKC. Phosphorylation ofG-subunits by PKC also affected responses mediated by-subunits. Pretreatment of muscle cells withcANP-(4-23), a selective agonist of the natriureticpeptide clearance receptor, NPR-C, which activates phospholipase C(PLC)-3 via the -subunits of G_i-1 andG_i-2, inhibited the PLC- response to somatostatin and[D-Pen^2,5]enkephalin. The inhibition waspartly reversed by calphostin C. Short-term activation of PKC had noeffect on receptor binding or effector enzyme (adenylyl cyclase orPLC-) activity. We conclude that selective phosphorylation ofG_i-1 and G_i-2 by PKC partly accounts forheterologous desensitization of responses mediated by the - and-subunits of both G proteins. The desensitization reflects adecrease in reassociation and thus availability of heterotrimeric G proteins.