Intragenomic variation in ribosomal RNA gene of the sea urchin <Emphasis Type="Italic">Lytechinus variegatus</Emphasis>

The first series of studies on the rDNA satellite of the sea urchin, Lytechinus variegatus, based on saturation hybridization of rRNA-rDNA and renaturation kinetics, showed that repeat length of rRNA gene was of about 8 kb in which there was no provision for NTS. The EM denaturation mapping, however, revealed (1) that the gene was 75% larger (longer) than 8 kb, within which there was a NTS whose length varied in repeating units, (3) and there was a region of high GC almost in the middle of the transcribed part. The suggestion of length and sequential heterogeneity in the gene copies coming from the first denaturation mapping prompted further studies with techniques so that the conclusions of the previous results could be stated with finality. The results that emanated from further studies established that the rDNA repeat length of L. variegatuswas of about 12 kb and that the NTS ranged from 3.8 to 6.4 kb. Earlier demonstration of a moderately high-GC segment within the transcribed part was also confirmed by sequence analysis. However, the stipulations on the NTS regarding sequential and length heterogeneity, still awaits elucidation by sequence analysis.     

Different population histories of the Mundari- and Mon-Khmer-speaking Austro-Asiatic tribes inferred from the mtDNA 9-bp deletion/insertion polymorphism in Indian populations

Length variation in the human mtDNA intergenic region between the cytochrome oxidase II (COII) and tRNA lysine (tRNA^lys) genes has been widely studied in world populations. Specifically, Austronesian populations of the Pacific and Austro-Asiatic populations of southeast Asia most frequently carry the 9-bp deletion in that region implying their shared common ancestry in haplogroup B. Furthermore, multiple independent origins of the 9-bp deletion at the background of other mtDNA haplogroups has been shown in populations of Africa, Europe, Australia, and India. We have analyzed 3293 Indian individuals belonging to 58 populations, representing different caste, tribal, and religious groups, for the length variation in the 9-bp motif. The 9-bp deletion (one copy) and insertion (three copies) alleles were observed in 2.51% (2.15% deletion and 0.36% insertion) of the individuals. The maximum frequency of the deletion (45.8%) was observed in the Nicobarese in association with the haplogroup B5a D-loop motif that is common throughout southeast Asia. The low polymorphism in the D-loop sequence of the Nicobarese B5a samples suggests their recent origin and a founder effect, probably involving migration from southeast Asia. Interestingly, none of the 302 (except one Munda sample, which has 9-bp insertion) from Mundari-speaking Austro-Asiatic populations from the Indian mainland showed the length polymorphism of the 9-bp motif, pointing either to their independent origin from the Mon-Khmeric-speaking Nicobarese or to an extensive admixture with neighboring Indo-European-speaking populations. Consistent with previous reports, the Indo-European and Dravidic populations of India showed low frequency of the 9-bp deletion/insertion. More than 18 independent origins of the deletion or insertion mutation could be inferred in the phylogenetic analysis of the D-loop sequences.     

Engineering tomato for resistance to tomato leaf curl disease using viral <Emphasis Type="Italic">rep</Emphasis> gene sequences

Transgenic tomato resistant to tomato leaf curl disease (ToLCD) using replicase (rep) gene sequences of Tomato leaf curl virus in antisense orientation were developed via Agrobacterium-mediated transformation. A binary vector carrying the antisense rep gene (untranslatable full length sequence, 1086 bp) along with the npt II gene was used for transformation. High level of resistance and inheritability of the transgene was observed up to T₂ stage following challenge inoculation with the virus. The mechanism of resistance appears RNA-mediated, since the plants carried the untranslatable antisense rep gene. Progeny analysis of these plants showed classical Mendelian pattern of inheritance in two of the six transgenic lines having single transgene insertion.     

Single cell kinetics of intracellular, nonviral, nucleic acid delivery vehicle acidification and trafficking

Mechanistic understanding of the intracellular trafficking of nonviral nucleic acid delivery vehicles remains elusive. A live, single cell-based assay is described here that is used to investigate and quantitate the spatiotemporal, intracellular pH microenvironment of polymeric-based nucleic acid delivery vehicles. Polycations such as polyethylenimine (PEI), poly-l-lysine (PLL), beta-cyclodextrin-containing polymers lacking or possessing imidazole termini (CDP or CDP-imid), and cyclodextrin-grafted PEI (CD-PEI) are used to deliver an oligonucleotide containing a single fluorophore with two emission lines that can be employed to measure the pH. Delivery vehicles were also sterically stabilized by addition of poly(ethylene glycol) (PEG) and investigated. The intracellular trafficking data obtained via this new methodology show that vectors such as PEI and CDP-imid can buffer the endocytic vesicles while PLL and CDP do not. Additionally, the PEGylated vectors reveal the same buffering capacity as their unstabilized variants. Here, the live cell, spatiotemporal mapping of these behaviors is demonstrated and, when combined with cell uptake and luciferase expression data, shows that there is not a correlation between buffering capacity and gene expression.     

A comparative evaluation of oxygen mass transfer and broth viscosity using Cephalosporin-C production as a case strategy

The production of Cephalosporin-C (CPC) a secondary metabolite, using a mold Acremonium chrysogenum was studied in a lab scale Internal loop air lift reactor. Cephalosporin-C production process is a highly aerobic fermentation process. Volumetric gas–liquid mass transfer coefficient (k_La) and viscosity (η) were evaluated, during the growth and production phases of the microbial physiology. An attempt has been made to correlate the broth viscosity, η and volumetric oxygen transfer coefficient, k_La during the Cephalosporin-C production in an air lift reactor. The impact of biomass concentration and mycelial morphology on broth viscosity has been also evaluated. The broth exhibits a typical non-Newtonian fermentation broth. Rheology parameters like consistency index and fluidity index are also studied.     

Suitability of aquaculture effluent solids mixed with cardboard as a feedstock for vermicomposting

Recirculating aquaculture systems are highly intensive culture systems that actively filter and reuse water, thus minimizing water requirements and creating relatively small volumes of concentrated waste (compared to flow-through aquaculture systems). Vermicomposting, which uses earthworms to stabilize and transform organic wastes into valuable end-products, has been proposed as an alternative treatment technology for high-moisture-content organic wastes from agricultural, industrial and municipal sources. This study was conducted to determine if the effluent solids from a large recirculating aquaculture facility (Blue Ridge Aquaculture, Martinsville, Virginia) were suitable for vermicomposting using the earthworm Eisenia fetida. In two separate experiments, worms were fed mixtures of solids removed from aquaculture effluent (sludge) and shredded. Mixtures containing 0%, 5%, 10%, 15%, 20%, 25%, and 50% aquaculture sludge (dry weight basis) were fed to the worms over a 12-week period and their growth (biomass) was measured. Worm mortality, which occurred only in the first experiment, was not influenced by feedstock sludge concentration. In both experiments worm growth rates tended to increase with increasing sludge concentration, with the highest growth rate occurring with feedstocks containing 50% aquaculture sludge. Effluent solids from recirculating aquaculture systems mixed with shredded cardboard appear to be suitable feedstocks for vermicomposting.     

Glycolipids and other constituents from Desmodium gangeticum with antileishmanial and immunomodulatory activities

Nineteen compounds of various classes, such as flavonoid glycosides, pterocarpanoids, lipids, glycolipids, and alkaloids, were isolated and identified from the Desmodium gangeticum whole plant. Aminoglucosyl glycerolipid (8) is reported here for the first time. Its structure has been elucidated by spectroscopic and degradation studies. This novel compound exhibited in vitro antileishmanial and immunomodulatory activities, as it enhanced nitric oxide (NO) production and provided resistance against infection established in peritoneal macrophages by the protozoan parasite Leishmania donovani. Another known compound, glycosphingolipid (cerebroside) (7) was found to possess significant in vitro antileishmanial and immunomodulatory activities against the same parasite. Other compounds were found to be inactive.     

Differential apoptotic and redox regulatory activities of curcumin and its derivatives

We have synthesized different bioconjugates of curcumin, which were tested for their pro- and antioxidant properties. In the present study five representative derivatives of curcumin, i.e., 4,4'-di-(O-acetyl) curcumin, 4,4'-di-(O-glycinoyl) curcumin, 4,4'-di-(O-glycinoyl-di-N-piperoyl) curcumin, 4,4'-di-(O-piperoyl) curcumin, and 4,4'-(O,O-cystinoyl)-3,3'-dimethoxydiphenyl-1,6-heptadiene-3,5-dione, were used for testing their apoptotic potential on tumor cells. Dipiperoyl and diglycinoyl derivatives showed higher apoptotic activity at lower concentrations, whereas diacetyl curcumin had slightly lower apoptotic activity on tumor cells. On the other hand, diglycinoyl-dipiperoyl and cystinoyl heptadiene derivatives had lost their apoptotic potential significantly. The apoptotic activity of these derivatives correlated very well with the generation of ROS by the tumor cells, whereas GSH levels remained unaltered. Our studies also indicate downregulation of Bcl-2 and participation of caspase-3 in the apoptotic death of tumor cells.