Cell membrane stability and biochemical response of cultured cells of groundnut under polyethylene glycol-induced water stress

Cell membrane stability (CMS) in suspension cultures of two groundnut cultivars was studied under polyethylene glycol(PEG)-induced water stress. There was a negative relationship between PEG concentration in the medium and membrane stability measured as electrolyte leakage. The CMS values in the cell cultures correlated well with the whole plant tissue and permitted the differentiation of cultivars based on their known response to drought stress. The cell membrane stability was lower (more electrolyte leakage) when cells were grown in culture as compared to the intact plant tissue. Kadiri-3, the drought tolerant cultivar maintained higher CMS than JL-24, the drought susceptible one. With increasing PEG levels the concentration of Potassium in cultured cells declined in both cultivars. However, Kadiri-3 maintained higher K values than JL-24 accompanied with greater cell membrane stability. Total soluble sugars also increased with increasing stress in both cultivars; the increase being higher in Kadiri-3. There was no significant change in the total free amino acids but proline accumulated markedly in both varieties. However, no relationship was found between proline levels and CMS. The results demonstrated that CMS test can also be used under in vitro conditions to differentiate the drought tolerant and susceptible cultivars and the cellular K level has a positive relationship with membrane stability.     

Efficient plant regeneration from shoot apices of sorghum

An efficient and rapid regeneration protocol was developed using shoot apices from germinating seedlings of two cultivars of sorghum, SPV-462 and M35-1, as explants. A vertical slit given from the base of each dissected apex enhanced the efficiency of callusing response by two fold. MS medium containing 0.5 mg dm⁻³ each of 2,4-D and kinetin was most effective in producing friable and embryogenic calli. Scanning electron microscopy of these calli detected somatic embryogenesis. Calli thus induced gave rise to approximately 42 green shoots per callus in both the genotypes when transferred to regeneration medium containing 1.5 mg dm⁻³ kinetin.     

Superfluous glutamine synthetase activity in Chinese Hamster Ovary cells selected under glutamine limitation is growth limiting in glutamine-replete conditions and can be inhibited by serine

Passaging and expansion of animal cells in lean maintenance medium could result in periods of limitation of some nutrients. Over time, such stresses could possibly result in selection of cells with metabolic changes and contribute to heterogeneity. Here, we investigate whether selection of Chinese Hamster Ovary (CHO) cells under glutamine limitation results in changes in growth under glutamine-replete conditions. In glutamine-limiting medium, compared to control cells passaged in glutamine-rich medium, the selected cells showed higher glutamine synthetase (GS) activity and attained a higher peak viable cell density (PVCD). Surprisingly, in glutamine-replete conditions, selected cells still showed a higher GS activity but a lower PVCD. We show that in glutamine-replete medium, PVCD of selected cells was restored on (a) inhibition of GS activity with methionine sulfoximine, (b) supplementation of aspartate—without affecting GS activity, and (c) supplementation of serine, which is reported to inhibit GS in vitro. Consistent with the reported effect of serine, inhibition of GS activity was observed upon serine supplementation along with reduced growth of cells under glutamine-limiting conditions. The latter observation is important for the design of glutamine-free culture medium and feed used for GS-CHO and GS-NS0. In summary, we show that CHO cells selected under glutamine limitation have superfluous GS activity in glutamine-replete medium, which negatively affects their PVCD. This may be due to its effect on availability of aspartate which was the limiting nutrient for the growth of selected cells in glutamine-replete conditions.     

The RECQL4 protein,deficient in Rothmund–Thomson syndrome is active on telomeric D-loops containing DNA metabolism blocking lesions

Telomeres are critical for cell survival and functional integrity. Oxidative DNA damage induces telomeric instability and cellular senescence that are associated with normal aging and segmental premature aging disorders such as Werner Syndrome and Rothmund–Thomson Syndrome, caused by mutations in WRN and RECQL4 helicases respectively. Characterizing the metabolic roles of RECQL4 and WRN in telomere maintenance is crucial in understanding the pathogenesis of their associated disorders. We have previously shown that WRN and RECQL4 display a preference in vitro to unwind telomeric DNA substrates containing the oxidative lesion 8-oxoguanine. Here, we show that RECQL4 helicase has a preferential activity in vitro on telomeric substrates containing thymine glycol, a critical lesion that blocks DNA metabolism, and can be modestly stimulated further on a D-loop structure by TRF2, a telomeric shelterin protein. Unlike that reported for telomeric D-loops containing 8-oxoguanine, RECQL4 does not cooperate with WRN to unwind telomeric D-loops with thymine glycol, suggesting RECQL4 helicase is selective for the type of oxidative lesion. RECQL4's function at the telomere is not yet understood, and our findings suggest a novel role for RECQL4 in the repair of thymine glycol lesions to promote efficient telomeric maintenance.     

Membrane-Active Macromolecules Resensitize NDM-1 Gram-Negative Clinical Isolates to Tetracycline Antibiotics

Gram-negative ‘superbugs’ such as New Delhi metallo-beta-lactamase-1 (bla _NDM-1) producing pathogens have become world’s major public health threats. Development of molecular strategies that can rehabilitate the ‘old antibiotics’ and halt the antibiotic resistance is a promising approach to target them. We report membrane-active macromolecules (MAMs) that restore the antibacterial efficacy (enhancement by >80-1250 fold) of tetracycline antibiotics towards bla _NDM-1 Klebsiella pneumonia and bla _NDM-1 Escherichia coli clinical isolates. Organismic studies showed that bacteria had an increased and faster uptake of tetracycline in the presence of MAMs which is attributed to the mechanism of re-sensitization. Moreover, bacteria did not develop resistance to MAMs and MAMs stalled the development of bacterial resistance to tetracycline. MAMs displayed membrane-active properties such as dissipation of membrane potential and membrane-permeabilization that enabled higher uptake of tetracycline in bacteria. In-vivo toxicity studies displayed good safety profiles and preliminary in-vivo antibacterial efficacy studies showed that mice treated with MAMs in combination with antibiotics had significantly decreased bacterial burden compared to the untreated mice. This report of re-instating the efficacy of the antibiotics towards bla _NDM-1 pathogens using membrane-active molecules advocates their potential for synergistic co-delivery of antibiotics to combat Gram-negative superbugs.     

Diversity and variability in seed characters and growth of Pongamia pinnata (L.) Pierre accessions

A thorough and extensive wild germplasm exploration survey was undertaken and 50 high yielding candidate plus trees (CPTs) of Pongamia pinnata (L.) Pierre from different locations from a latitudinal and longitudinal spread between 12°41′ and 22°E longitude and 77° and 84°40′N latitude covering 11 locations in an area spread of 150,000 km² were collected for evaluating genetic association and variability in seed and growth characters. There were significant differences observed in seed morphology and oil content as was in plant height, and number of branches in the progeny trial. Plant height and number of branches exhibited much higher values of both phenotypic and genotypic variance than observed in the seed characters. Among seed characters oil content exhibited highest broad sense heritability of more than 93% followed by seed length (90.0%). In contrast seed width showed the second highest genetic advance of 5.64% following the highest genetic advance of 10.15% exhibited by oil content. Hierarchical clustering by Ward’s Minimum Variance Cluster Analysis method showed phylogeographic patterns of genetic diversity. K means clustering revealed that trees from different geographic regions were grouped together in a cluster and as were trees from the same geographical area placed in different clusters suggesting that geographical diversity did not go hand in hand with genetic diversity. In addition clustering identified promising accessions with favourable traits for future establishment of orchards.     

Studies on fermentative production of rifamycin using Amycolatopsis mediterranei

The fermentative production of rifamycin by Amycolatopsis mediterranei (MTCC17) has been studied. Both qualitative and quantitative aspects of the fermentation were determined by optimizing cultural conditions and medium design to improve the production of rifamycin. A pH value of 7.0, a temperature of 26°C, an aeration rate of 250rev/min for a 50ml volume, a level of inoculum of 10% grown aeration for 48h and a fermentation period of 11days were found to be optimum. Among the nitrogen sources, and culture conditions, peanut meal and aeration were found to be critical for rifamycin production respectively. The above mentioned exercise increased the yields of rifamycin from 350mg/l to 2000mg/l.