In vitro induction of multiple shoots and plant regeneration inVigna

Summary Cotyledonary nodes, excised cotyledons, and hypocotyl segments of six varieties ofVigna mungo andV. radiata have been tested for their morphogenic potential on media containing a range of hormonal combinations including benzyladenine, kinetin, thidiazuron (TDZ), and zeatin. Multiple shoots developed on cotyledonary node explants in all varieties tested on basal medium containing cytokinin. Presence of both the cotyledons, either full or half, resulted in a maximum number of shoots produced. Shoot bud regeneration was achieved via meristem formation on excised cotyledons on Murashige-skoog basal medium with B₅ vitamins supplemented with TDZ. Mature plants had normal phenotypes.V. mungo var. PS1 andV. radiata var. Pusa 105 were found to be the most responsive varieties for shoot regneration. The histology ofin vitro organogenesis was studied.     

Pressure enhances thermal stability of DNA polymerase from three thermophilic organisms

DNA polymerases derived from three thermophilic microorganisms, Pyrococcus strain ES4, Pyrococcus furiosus, and Thermus aquaticus, were stabilized in vitro by hydrostatic pressure at denaturing temperatures of 111°C, 107.5°C, and 100°C (respectively). Inactivation rates, as determined by enzyme activity measurements, were measured at 3, 45, and 89 MPa. Half-lives of P. strain ES4, P. furiosus, and T. aquaticus DNA polymerases increased from 5.0, 6.9, and 5.2 minutes (respectively) at 3 MPa to 12, 36, and 13 minutes (respectively) at 45 MPa. A pressure of 89 MPa further increased the half-lives of P. strain ES4 and T. aquaticus DNA polymerases to 26 and 39 minutes, while the half-life of P. furiosus DNA polymerase did not increase significantly from that at 45 MPa. The decay constant for P. strain ES4 and T. aquaticus polymerases decreased exponentially with increasing pressure, reflecting an observed change in volume for enzyme inactivation of 61 and 73 cm³/mol, respectively. Stabilization by pressure may result from pressure effects on thermal unfolding or pressure retardation of unimolecular inactivation of the unfolded state. Regardless of the mechanism, pressure stabilization of proteins could explain the previously observed extension of the maximum temperature for survival of P. strain ES4 and increase the survival of thermophiles in thermally variable deep-sea environments such as hydrothermal vents. Received: September 12, 1997 / Accepted: February 24, 1998     

Inhibition of Methyltransferases Accelerates Degradation of cFLIP and Sensitizes B-Cell Lymphoma Cells to TRAIL-Induced Apoptosis

Non-Hodgkin lymphomas (NHLs) are characterized by specific abnormalities that alter cell cycle regulation, DNA damage response, and apoptotic signaling. It is believed that cancer cells are particularly sensitive to cell death induced by tumor necrosis factor α–related apoptosis-inducing ligand (TRAIL). However, many cancer cells show blocked TRAIL signaling due to up-regulated expression of anti-apoptotic factors, such as cFLIP. This hurdle to TRAIL’s tumor cytotoxicity might be overcome by combining TRAIL-based therapy with drugs that reverse blockages of its apoptotic signaling. In this study, we investigated the impact of a pan-methyltransferase inhibitor (3-deazaneplanocin A, or DZNep) on TRAIL-induced apoptosis in aggressive B-cell NHLs: mantle cell, Burkitt, and diffuse large B-cell lymphomas. We characterized TRAIL apoptosis regulation and caspase activation in several NHL-derived cell lines pre-treated with DZNep. We found that DZNep increased cancer cell sensitivity to TRAIL signaling by promoting caspase-8 processing through accelerated cFLIP degradation. No change in cFLIP mRNA level indicated independence of promoter methylation alterations in methyltransferase activity induced by DZNep profoundly affected cFLIP mRNA stability and protein stability. This appears to be in part through increased levels of cFLIP-targeting microRNAs (miR-512-3p and miR-346). However, additional microRNAs and cFLIP-regulating mechanisms appear to be involved in DZNep-mediated enhanced response to extrinsic apoptotic stimuli. The capacity of DZNep to target cFLIP expression on multiple levels underscores DZNep’s potential in TRAIL-based therapies for B-cell NHLs.     

Anthocyanin production in a callus line of <Emphasis Type="Italic">Panax sikkimensis</Emphasis> Ban

A root-derived callus line of Panax sikkimensis that stably accumulates anthocyanins was established by small cell aggregate selection method. The selected line showed a growth index of 221.36 and an anthocyanin content of 2.76 mg/g fw (7.076% dw) in 50–60 d of growth on a modified MS medium containing 4.5 μM 2,4-dichlorophenoxy acetic acid and 1.2 μM kinetin under 16-h light and 8-h dark photoperiodic conditions. Incubation under continuous light increased the growth index to 435.57 but led to a marginal dilution of anthocyanin content to 2.192 mg/g fw (6.928% dw). The purple-red pigment had absorption maximum at 528 nm. The selected callus line has shown sustained growth and productivity for more than 6 yr now. Interestingly, pigment accumulation in the selected line did not hinder the ginsenoside production in the callus tissue (0.9–1.2% fw).     

Growth kinetics and ginsenosides production in transformed hairy roots of American ginseng—Panax quinquefolium L

A thin, profusely branched, fast growing hairy root line of Panax quinquefolium (American ginseng) was established by co-culturing epicotyl explants with a wild type strain of Agrobacterium rhizogenes. The transformed roots grew by over 10-fold from the initial inoculum within 8 weeks. The crude ginsenosides content in the roots was about 0.2 g/g dry wt level up to the 10th week of culture. Ginsenosides Rb2, Rd, Re, Rf and Rg1 constituted 47–49% of the crude saponin fraction between 6 and 8 weeks of growth whereas, Rc ginsenoside was accumulated only after 9th weeks when the biomass started receding. PCR amplification analysis of the hairy roots confirmed their transgenic nature by showing the presence of Ri-TL DNA with rolA, rolB and rolC genes in their genome.     

Study on the effects of chloride depletion on photosystem II using different chloride depletion methods

Chloride is an indispensable factor for the functioning of oxygen evolving complex (OEC) and has protective and activating effects on photosystem II. In this study we have investigated mainly by EPR, the properties of chloride-sufficient, chloride-deficient and chloride-depleted thylakoid membranes and photosystem II enriched membranes from spinach. The results on the effects of different chloride depletion methods on the structural and functional aspects of photosystem II showed that chloride-depletion by treating PS II membranes with high pH is a relatively harsh way causing a significant and irreparable damage to the PS II donor side. Damage to the acceptor side of PS II was recovered almost fully in chloride-deficient as well as chloride-depleted PS II membranes.