Genetic transformation of Tylophora indica with Agrobacterium rhizogenes A4: growth and tylophorine productivity in different transformed root clones

We have developed an efficient transformation system for Tylophora indica, an important medicinal plant in India, using Agrobacterium rhizogenes strains LBA9402 and A4 to infect excised leaf and stem explants and intact shoots at different sites. The induction of callus and transformed roots was dependent on the bacterial strain, explant type and inoculation site used. Transformed roots were induced only in explants infected with A. rhizogenes strain A4, while an optimal transformation frequency of up to 60% was obtained with intact shoots inoculated at the nodes. The presence of the left-hand transferred DNA (T_L-DNA) in the genome of T. indica roots induced by A. rhizogenes was confirmed by PCR amplification of the rooting locus genes of A. rhizogenes. Root growth and the production of tylophorine, the major alkaloid of the plant, varied substantially among the nine root clones studied. Both parameters increased over time in liquid cultures, with maximum biomass and tylophorine accumulation occurring within 4–6 weeks of growth in fresh medium. Interestingly, in liquid culture, the culture medium also accumulated tylophorine up to concentrations of 9.78±0.21 mg l^–1.     

Effect of arsenic on behaviour of enzymes of sugar metabolism in germinating rice seeds

Arsenic (As) is a potential contaminant of groundwater as well as soil in many parts of the world. The effects of increasing concentration of As (25 μm and 50 μm As₂O₃) in the medium on the content of starch and sugars and activity levels of enzymes involved in starch and sugar metabolism i.e. α-amylase, β-amylase, starch phosphorylase and acid invertase were studied in germinating seeds of two rice cvs. Malviya-36 and Pant-12 during 0–120 h period. As toxicity in situ led to a marked decline in the activities of α-amylase, β-amylase in endosperms as well as embryoaxes of germinating rice seeds. The activity of acid invertase increased in endosperms as well as embryoaxes whereas starch phosphorylase activity declined in endosperms but increased in embryoaxes under As treatment. In endosperms a decline in starch mobilization was observed under As toxicity, however under similar conditions the content of total soluble sugars increased in embryoaxes. The observed inhibition in activities of amylolytic enzymes might contribute to delayed mobilization of endospermic starch which could affect germination of seeds in As polluted environment, while the induced acid invertase activity and increased sugar accumulation in embryoaxes could serve as a possible component for adaptation mechanism of rice seedlings grown under As containing medium.     

Impact of low doses of tritium on the marine mussel, Mytilus edulis: genotoxic effects and tissue-specific bioconcentration

Despite growing scientific, public and regulatory concern over the discharge of radioactive substances, no serious attempts have been made to develop a rationale to evaluate the impact of environmentally relevant radionuclides in the aquatic environment. In this study, we have evaluated the genotoxic effects and tissue-specific concentration of tritium (added as tritiated water, HTO) in the adult life stage of the edible mussel, Mytilus edulis. The genotoxic effects were quantified in terms of the induction of: (a) micronuclei (MN), and (b) DNA single-strand breaks/alkali-labile sites using alkaline single-cell gel electrophoresis (Comet assay) in the haemocytes of exposed animals. The assays were optimised and validated using a range of concentrations (18-56 mgl(-1)) of ethylmethane sulfonate (EMS), a direct-acting reference genotoxic agent, over different exposure periods. Mussels were exposed to a series of concentrations of HTO equivalent to a dose range from 12 to 485 muGyh(-1) for 96 h, and different tissues and organs were then extracted and analysed. The study revealed a dose-dependent increase in the response for both the MN test and the Comet assay and for both EMS and HTO. In addition, HTO delivering dose rates below 500 muGyh(-1) was shown to be capable of inducing genetic damage in the haemocytes of these bivalves. The study also showed that inorganic tritium accumulated differentially in mussel tissues in a dose-dependent manner, with the gut accumulating the highest amount of radioactivity, followed by the gill, mantle, muscle, foot and byssus thread. The faeces and pseudo-faeces accumulated least radioactivity over the exposure period. Differential accumulation of radionuclides has significant implications for biomonitoring programmes, for this and other aquatic organisms. The study also suggests that the generic dose limits recommended by the International Atomic Energy Agency for the protection of aquatic biota might not be applicable to all aquatic organisms.     

Preparation of polyvinyl alcohol-polyacrylamide composite polymer membrane by gamma-irradiation for entrapment of urease

Composite polymer membrane of polyvinyl alcohol (PVA) and acrylamide was prepared on cheesecloth support by gamma-irradiation induced free radical polymerization. The enzyme urease was entrapped in the membrane during polymerization and was cross-linked within the matrix using glutaraldehyde. The membranes could be reused a number of times without significant loss of urease activity.     

Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene

Respiratory syncytial virus (RSV) infection is one of the major causes of respiratory tract infection for which no vaccine or antiviral treatment is available. The RSV NS1 protein seems to antagonize the host interferon (IFN) response; however, its mechanism is unknown. Here, we used a plasmid-borne small interfering RNA targeting the NS1 gene (siNS1) to examine the role of NS1 in modulating RSV infection. RSV replication was reduced in A549 cells, but not IFN-deficient Vero cells, transfected with siNS1. siNS1 induced upregulated expression of IFN-beta and IFN-inducible genes in A549 cells. siNS1-transfected human dendritic cells, upon RSV infection, produced elevated type-1 IFN and induced differentiation of naive CD4+ T cells to T helper type 1 (TH1) cells. Mice treated intranasally with siNS1 nanoparticles before or after infection with RSV showed substantially decreased virus titers in the lung and decreased inflammation and airway reactivity compared to controls. Thus, siNS1 nanoparticles may provide an effective inhibition of RSV infection in humans.     

Production and seeding of protoplasts of <Emphasis Type="Italic">Porphyra okhaensis</Emphasis> (Bangiales,Rhodophyta) in laboratory culture

High yields of viable protoplasts were produced from Porphyra okhaensis H. Joshi, Oza & Tewari following two-step enzymatic digestion (protease pretreatment and cell wall polysaccharides-degrading enzyme treatment) of the thallus. Pretreatment of the tissues with 1% Protease P6 at 20± 1 ^°C for 30 min prior to digestion with cell wall polysaccharide-degrading enzymes increased the protoplast yield two fold compared to tissues that were digested with polysaccharide-degrading enzyme mixture. The polysaccharide-degrading enzymes employed for protoplast isolation from P. okhaensis were Cellulase Onozuka R-10, Macerozyme R-10, abalone acetone powder and agarase. Suitable pH, temperature and duration of enzyme treatment for optimal production of viable protoplasts were pH 6, 20± 1 ^°C and 3 h, respectively. Mannitol (0.8 M) was found to be an excellent osmotic stabilizer. When the tissue of P. okhaensis pretreated with 1% protease solution was digested with commercial enzyme mixture consisting of 2% Cellulase Onozuka R-10, 2% Macerozyme R-10, 1% abalone acetone powder, 50 units of agarase and 0.8 M mannitol in 1% NaCl (adjusted to pH 6.0 with 25 mM MES buffer) with gentle agitation for 3 h at 20± 1 ^°C, 23.2± 0.24× 10⁶ protoplasts g⁻¹ fresh wt. were obtained. The regeneration rate of protoplasts isolated in the present study was found to be 79%. Protoplasts that regenerated cell walls underwent regular cell divisions and developed into leafy gametophytic thallus in the laboratory cultures. Further, the seeding of nylon threads with partially developed protoplasts of P. okhaensis was successful in the laboratory conditions and germlings as long as 3–4 cm were obtained from such seeded threads in one month period in aerated cultures.     

Translation and assembly of CABYR coding region B in fibrous sheath and restriction of calcium binding to coding region A

CABYR is a highly polymorphic, sperm flagellar calcium-binding protein that is tyrosine as well as serine/threonine phosphorylated during capacitation. Six alternative splice variants of human CABYR (I-VI) have previously been identified, involving two coding regions, CR-A and CR-B, separated by an intervening stop codon. It is presently unknown if proteins encoded by the predicted coding region B of CABYR are translated during spermiogenesis, where they localize, or which CABYR isoforms bind calcium. Immunofluorescent and electron microscopic studies using polyclonal antibodies generated to the recombinant c-terminal 198 aa CABYR-B localized the isoforms containing CABYR-B to the ribs and longitudinal columns of the fibrous sheath in the principal piece of the flagellum. Antisera to recombinant CABYR-A and CABYR-B proteins recognized distinct populations of CABYR isoforms encoded by either CR-A alone and/or CR-B as well as a common population of CABYR isoforms. Only the recombinant CABYR-A and not the CABYR-B bound calcium in vitro, which is consistent with the hypothesis that CABYR-A is the only form that binds calcium in sperm. These observations confirmed that, despite the presence of the stop codon in CR-A, splice variants containing CR-B are expressed during spermiogenesis and assemble into the fibrous sheath of the principal piece; however, calcium binding occurs only to those CABYR isoforms containing CABYR-A.     

Characterization of an exopolysaccharide produced by a marine Enterobacter cloacae

An exopolysaccharide producing marine bacterium, Enterobacter cloacae, was isolated from marine sediment collected from Gujarat coast, India. Chemical investigation of exopolysaccharide (EPS 71 a) revealed that this exopolysaccharide was an acidic polysaccliaride containing high amount of uronic acid, fucose and sulfate which is rare for bacterial exopolysaccharides. EPS 71a was found to have fucose, galactose, glucose and glucuronic acid in a molar ratio of 2: 1: 1: 1.     

A novel AP-2 adaptor interaction motif initially identified in the long-splice isoform of synaptojanin 1, SJ170

Phosphoinositides play a fundamental role in clathrin-coat assembly at the cell surface. Several endocytic components and accessory factors contain independently folded phosphoinositide-binding modules that facilitate, in part, membrane placement at the bud site. As the clathrin-coat assembly process progresses toward deeply invaginated buds, focally synthesized phosphoinositides are dephosphorylated, principally through the action of the phosphoinositide polyphosphatase synaptojanin 1. Failure to catabolize polyphosphoinositides retards the fission process and endocytic activity. The long-splice isoform of synaptojanin 1, termed SJ170, contains a carboxyl-terminal extension that harbors interaction motifs for engaging several components of the endocytic machinery. Here, we demonstrate that in addition to DPF and FXDXF sequences, the SJ170 carboxyl terminus contains a novel AP-2 binding sequence, the WXXF motif. The WXXF sequence engages the independently folded alpha-subunit appendage that projects off the heterotetrameric AP-2 adaptor core. The endocytic protein kinases AAK1 and GAK also contain functional WXX(FW) motifs in addition to two DPF repeats, whereas stonin 2 harbors three tandem WXXF repeats. Each of the discrete SJ170 adaptor-interaction motifs bind to appendages relatively weakly but, as tandemly arrayed within the SJ170 extension, can cooperate to bind bivalent AP-2 with good apparent affinity. These interactions likely contribute to the appropriate targeting of certain endocytic components to clathrin bud sites assembling at the cell surface.     

Evidences showing ultraviolet-B radiation-induced damage of DNA in cyanobacteria and its detection by PCR assay

Impact of ultraviolet-B radiation in causing the damages to the DNA of the cyanobacterium, Anabaena strain BT2 has been investigated. Exposure of genomic DNA (in vitro) to UV-B radiation for 1 h did not cause any shift in the absorption peak (lambda(max)) but more than 30% increase in absorbance was noticed in comparison to untreated control DNA (no exposure to UV-B). This increase in absorbance in a way may be comparable to typical hypochromic effect but there was no decrease in absorbance following transfer of UV-B-treated DNA to fluorescent light or in the dark. That the damaging effect of UV-B radiation on native structure of DNA is indeed real was also evident from the PCR-based assay such as RAPD, rDNA amplification, and ARDRA. Template activity of UV-B-treated genomic DNA was drastically inhibited, there was no amplification in RAPD assay after prior exposure of DNA to UV-B for 60 min. Only one band of approximately 400 bp was observed even after 60 min of exposure which suggests that certain segment of DNA strand is resistant to UV-B effects. Similar to the effects on RAPD profile, amplification of rDNA was significantly inhibited following exposure of genomic DNA to UV-B. Our findings clearly demonstrate that UV-B does affect the DNA of cyanobacteria and the killings of these microbes might be due to the irreversible damages caused to DNA by this high energy radiation. It is felt that PCR assay may be conveniently used for screening the damages caused to DNA by UV-B radiation in cyanobacteria and other microorganisms.