A rapid method for monitoring intracellular lipid production using1H NMR spectroscopy

Summary A method has been developed for rapid mondtoring of lipid production in the intact yeast cells ofRhodotorula gracilis using¹H NMR spectroscopy.     

Glycerol production by immobilised cells of Pichia farinosa

Summary Cells ofPichia farinosa were immobilised in calcium alginate and K-Carrageenan and their ability to produce glycerol from glucose under aerobic conditions with acidic as well as alkaline pH was investigated. An average glycerol production rate of 0.07 g/l.h was obtained with immobilised cells (IMC) in shake flasks. Continuous glycerol production in a fluidised bed reactor (FBR) under steady state operation gave a glycerol concentration of 13.5 g/l in the product stream.     

Production of fungal rennet byMucor miehei using solid state fermentation

Summary Investigations have been carried out on the production of fungal rennet using a thermophilic strain ofMucor miehei under solid state fermentation conditions. A high milk clotting enzyme activity (58000 Soxhlet units/g) was achieved when optimum conditions were used. Further, a high ratio of 6.6:1 between milk clotting and proteolytic activities for this enzyme was obtained. Cheese prepared using this enzyme was also found to be acceptable in organoleptic quality. Large scale production of the enzyme in trays using the optimum conditions gave milk-clotting enzyme activities comparable to those in flask experiments.     

Production of extracellular lipase by the hexachlorocyclohexane utilizing Pseudomonas strain Ptm+

Extracelluar lipase activity was detected in a culture of Pseudomonas strain Ptm⁺ growing on hexachlorocyclohexane. Lipase activity was associated with growth of the bacterium and reached a peak at early idiophase (22 h). Following the lipase activity, bioemulsifier was produced, with maximum activity at mid-idiophase (32 h). The extracellular lipase is probably involved in the extracellular synthesis of the bioemulsifier in the culture broth.     

Comparative studies on the metabolism of aniline and chloroanilines by Pseudomonas multivorans strain An 1

Summary Pseudomonas multivorans strain An 1 used aniline but not chloroanilines as the sole source of carbon and energy for growth. The aniline-adapted cells, however, were able to oxygenate chloroanilines. Relative oxygenation rates for aniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline, and 3,4-dichloroaniline were 100, 46, 66, 20, and 3%, respectively.The first intermediates in the metabolism of chloroanilines were chlorocatechols. 3-Chlorocatechol accumulated during growth of the organism in the presence of 2-chloroaniline, whereas 4-chlorocatechol was an intermediate metabolite of 3-chloroaniline and 4-chloroaniline.Chloroanilines were able to induce synthesis of the aniline oxygenating enzyme system of Pseudomonas multivorans strain An 1. In continuous culture experiments, induction of this enzyme system appeared to depend on cell density, concentration, toxicity, and pK-values of aniline or chloroanilines.Studies with ¹⁴C-labelled 3-chloroaniline and 4-chloroaniline showed that the turnover of chloroanilines did not cease with the formation of chlorocatechols, because radioactivity was detected in the CO₂ released and in bacterial cell components. The results suggest that the turnover of chloroanilines is due to metabolism rather than to cometabolism.     

Regulation of the utilization of 4-hydroxybenzoate and 4-hydroxycinnamate in batch and continuous cultures of Pseudomonas testosteroni

Pseudomonas testosteroni metabolized 4-hydroxycinnamate by an initial cleavage of the side chain to yield acetate and the aromatic moiety, 4-hydroxybenzaldehyde. The latter was further oxidized via 4-hydroxybenzoate to protocatechuate, which underwent meta cleavage. During growth of the organism on 4-hydroxycinnamate, the for acetate showed an undulating pattern, which was attributed to alternating induction and repression of enzymes involved in the oxidation of acetate. Repression was caused either by 4-hydroxybenzoate or by its later metabolites, formate and pyruvate.In batch culture, P. testosteroni oxidized mixtures of 4-hydroxybenzoate and 4-hydroxycinnamate in a diauxic pattern. The capacity to oxidize 4-hydroxycinnamate appeared in the cells before 4-hydroxybenzoate was exhausted, indicating that the enzymes catalysing the conversion of 4-hydroxycinnamate into 4-hydroxybenzoate. were induced despite the presence of 4-hydroxybenzoate. The induction of these early enzymes of 4-hydroxycinnamate catabolism started when the molar concentration ratio of 4-hydroxybenzoate to 4-hydroxycinnamate fell below a value of 0.3.In continuous culture of P. testosteroni on a mixture of 4-hydroxybenzoate and 4-hydroxycinnamate, both substrates were almost completely utilized up to a dilution rate of about 0.5/h. At higher dilution rates, 4-hydroxycinnamate was decreasingly utilized so that eventually at a dilution rate of 0.74/h, its effluent concentration equalled its influent concentration. At D _M, a utilization ratio of 1.23 in favour of 4-hydroxybenzoate was found to become established in the culture. The of the cells for acetate was maximal at a dilution rate of 0.38/h and decreased before 4-hydroxycinnamate utilization was at its peak at 0.59/h. This suggested that it was mainly the aromatic moiety of 4-hydroxycinnamate which was metabolized at high dilution rates. The failure to utilize acetate at high dilution rates was apparently due to the repression of its catabolic enzymes by later metabolites of 4-hydroxybenzoate and to the relatively low concentration of acetate in the fermenter. This low concentration, due to the continuous washout of acetate, prevented it from relieving the repression.Abbreviations 4HB 4-hydroxybenzoate - 4HC 4-hydroxycinnamate - D _M dilution rate allowing maximal cell output rate - OD optical density     

Origin and diversification of Indian Ceropegieae (Apocynaceae) and its possible relation to the Indian monsoon

The Indian subcontinent has experienced a major shift in climatic regime from a wet tropical regime to increased seasonal rainfall, since the late Miocene. This shift has been attributed to the intensification of monsoons, which led to opening up of dry habitats in humid forests and formation of deciduous forests. We explored the role of this climatic shift in the origin and diversification of dry‐adapted plant genera Ceropegia and Brachystelma (Ceropegiae, Asclepiadoideae, Apocynaceae). We sampled Ceropegia and Brachystelma from across India and used five markers (two nuclear and three plastid regions) to reconstruct a global phylogeny of this group. Indian members of the tribe Ceropegiae were derived from Africa through at least four independent dispersal events. All dispersal events occurred in late Miocene after establishment of a monsoon climate. One of these early dispersing lineages underwent rapid radiation in peninsular India, giving rise to around 50 species. Thus, both dispersal and diversification events coincided with the intensification of monsoons and concomitant aridification. The role of environment in the evolution of floral characteristics and root type in the Indian radiation is also discussed. This is one of the first reports on a dry‐adapted endemic radiation of plants in India.     

Modulation of G protein-coupled adenosine receptors by strategically functionalized agonists and antagonists immobilized on gold nanoparticles

Gold nanoparticles (AuNPs) allow the tuning of pharmacokinetic and pharmacodynamic properties by active or passive targeting of drugs for cancer and other diseases. We have functionalized gold nanoparticles by tethering specific ligands, agonists and antagonists, of adenosine receptors (ARs) to the gold surface as models for cell surface interactions with G protein-coupled receptors (GPCRs). The AuNP conjugates with chain-extended AR ligands alone (PEGylated nucleosides and nonnucleosides, anchored to the Au via thioctic acid) were found to be insoluble in water due to hydrophobic entities in the ligand. Therefore, we added a second, biologically inactive pendant moiety to increase the water solubility, consisting of a PEGylated chain terminating in a carboxylic or phosphate group. The purity and stability of the immobilized biologically active ligand were examined by ultrafiltration and HPLC. Pharmacological receptor binding studies on these GPCR ligand-derivatized AuNPs (2–5 nm in diameter), performed using membranes of mammalian cells stably expressing human A₁, A_2A, and A₃ARs, showed that the desired selectivity was retained with K _i values (nanomolar) of A₃AR agonist 21b and A_2AAR antagonists 24 and 26a of 14 (A₃), 34 (A_2A), and 69 (A_2A), respectively. The corresponding monomers displayed K _i values of 37, 61, and 1,420 nM, respectively. In conclusion, we have synthesized stable, water-soluble AuNP derivatives of tethered A₃ and A_2AAR ligands that retain the biological properties of their monomeric ligands and are intended for therapeutic and imaging applications. This is the first prototypical application to gold carriers of small molecule (nonpeptide) GPCR ligands, which are under investigation for treatment of cancer and inflammatory diseases.     

Biosensors for the Detection of Organophosphorous Pesticides

Cytokinins are master regulators of plant growth and development. They are involved in the regulation of many important physiological and metabolic processes. Recent progress in cytokinin research at the molecular level, including identification of related genes and cytokinin receptors, plus elucidation of signal transduction, has greatly increased our understanding of cytokinin actions. Although still in its infant stage, molecular breeding of crops with altered cytokinin metabolism, when combined with the transgenic approach, has shown very promising potential for application to agriculture. In this review we briefly introduce recent progress in cytokinin molecular biology, discuss applications of cytokinin genetic engineering to agriculture, and present implications and future research directions.