Nucleotide sequence of psbQ gene for 16-kDa protein of oxygen-evolving complex from Arabidopsis thaliana and regulation of its expression.

The psbQ gene encoding a 16-kDa polypeptide of the oxygen-evolving complex of photosystem II has been isolated from Arabidopsis thaliana and characterized. The gene consists of a 28 nucleotide long leader sequence, two introns and three exons encoding a 223-amino-acid precursor polypeptide. The first 75 amino acids act as a transit peptide for the translocation of the polypeptide into the thylakoid lumen. Expression studies show that the gene is light-inducible and expresses only in green tissues with high steady-state mRNA levels in leaves. Using this gene as a probe, restriction fragment length polymorphism between two ecotypes, Columbia and Estland, has also been detected.     

Mechanism of racemization of amino acids by aspartate aminotransferase.

Aspartate aminotransferase (mitochondrial isoenzyme from chicken) has been found to racemize very slowly dicarboxylic amino acid substrates in the presence of their cognate oxo acids [Kochhar, S. & Christen, P. (1988) Eur. J. Biochem. 175, 433-438]. Tyrosine, phenylalanine and alanine are racemized at the same rate although they undergo the transamination reaction 3-5 orders of magnitude more slowly than the dicarboxylic substrates. Similarly, the truncated enzyme aspartate aminotransferase-(27/32-410) catalyzes the racemization at the same rate as the native enzyme, while its rate of transamination is decreased to 3% of that of the native enzyme. Apparently, the rate-limiting step in racemization is not immediately linked to the transamination cycle. Decreasing the water concentration in the reaction medium by adding methanol at 0 degrees C drastically reduces the rate of racemization without affecting the rate of transamination. On the basis of these and additional kinetic data and the model of the three-dimensional structure of the active site, we conclude that a water molecule is responsible for the protonation of C alpha of the coenzyme-substrate intermediate from the wrong side. The diffusion of the water molecule into the interior of the enzyme appears to be the rate-limiting step in aspartate-aminotransferase-catalyzed racemization.     

Lipoprotein receptor ‘CK’-dependent signalling in human platelets

The study addressed to understand whether or not lipoproteins at low concentrations could modulate Receptor-C dependent platelet signalling revealed that LDL, like exogneous cholesterol, had the capacity to initiate PLD-dependent platelet signalling in a dose dependent fashion and this effect was inhibited in presence of HDL; cAMP; DTT; Zn⁺⁺ and butanol whereas cGMP had no effect upon this PLD-dependent signalling. Further Receptor C from platelet in the purified-form displayed LDL-or cholesterol-dependent autophosphorylation at the tyrosine residues and this Receptor-C tyrosine kinase (Receptor-C_k) activity contributed to the observed LDL-or cholesterol-dependent PLD activity in human platelets. Based upon these results coupled with earlier results, an attempt was made to define the lipoprotein-dependent platelet signalling pathway.     

An active-site carboxyl group in liquefying α-amylase: specific chemical modification

Bacillus amyloliquefaciens -amylase activity is pH-dependent and the plot log (Vmax/Km) versus pH implicated a carboxyl group of aspartic acid/glutamic acid at the active site. Chemical modification of -amylase with EDC confirmed this view. Further, analysis of inactivation kinetics showed that modification of a single carboxyl group led to complete loss of the enzymic activity.     

An analysis of the action of light on betalain synthesis in the seedling of Amaranthus caudatus,var. viridis

Accumulation of betalain (amaranthin) in the seedling of Amaranthus caudatus, var. viridis, is inducible by light. Since the apparent lag-phase of amaranthin accumulation after the onset of light is of the order of 3 h, light induction experiments could be performed up to 3 h after the onset of light without interference with actual synthesis. The intricate induction phenomena can be explained as follows: The inductive light operates through phytochrome and through a blue/UV photoreceptor (cryptochrome). A phytochrome-dependent High Irradiance Reaction is of minor importance. However, there is a strong, specific interaction between the light effects mediated through phytochrome and cryptochrome in the sense that the extent of the reversible response — (response obtained with a particular light treatment terminated with a saturating red light pulse) minus (response obtained with the same light treatment when terminated with a saturating 756 nm light pulse) —increases with increasing P_fr level and total fluence rate during the induction period. It is concluded that light induced amaranthin synthesis is, in fact, a convenient biochemical model system of photomorphogenesis in the case when phytochrome and cryptochrome operate simultaneously in mediating photomorphogenesis. Abbreviations: see Table 1 and 2 Materials and Medthods     

Changes in plasma membrane and microfilaments accompanying morphologic differentiation in Chinese hamster ovary (CHO) cells.

Studies on the application of the techniques of counter-current distribution (CCD) in aqueous two-phase systems and multiple sedimentation for the fractionation of metaphase chromosomes are presented. The two-phase systems were composed of aqueous solutions of Dextran 500 and poly(ethylene)glycol 6000 (PEG). It has been found that different groups of chromosomes differ in their distribution between the two phases and that the introduction of PEG with covalently attached positively or negatively charged groups provides a means of steering the distribution of chromosomes. A rough fractionation of chromosomes on the basis of size is possible by the technique of multiple sedimentation and this, in combination with CCD, yields 10 fractions of chromosomes. Partition and CCD in aqueous two-phase system separate chromosomes according to their surface properties and may prove useful for isolation of individual chromosomes in bulk.     

ParA encoded on chromosome II of Deinococcus radiodurans binds to nucleoid and inhibits cell division in Escherichia coli

Bacterial genome segregation and cell division has been studied mostly in bacteria harbouring single circular chromosome and low-copy plasmids. Deinococcus radiodurans, a radiation-resistant bacterium, harbours multipartite genome system. Chromosome I encodes majority of the functions required for normal growth while other replicons encode mostly the proteins involved in secondary functions. Here, we report the characterization of putative P-loop ATPase (ParA2) encoded on chromosome II of D. radiodurans. Recombinant ParA2 was found to be a DNA-binding ATPase. E. coli cells expressing ParA2 showed cell division inhibition and mislocalization of FtsZ-YFP and those expressing ParA2-CFP showed multiple CFP foci formation on the nucleoid. Although, in trans expression of ParA2 failed to complement SlmA loss per se, it could induce unequal cell division in slmAminCDE double mutant. These results suggested that ParA2 is a nucleoid-binding protein, which could inhibits cell division in E. coli by affecting the correct localization of FtsZ and thereby cytokinesis. Helping slmAminCDE mutant to produce minicells, a phenotype associated with mutations in the ‘Min’ proteins, further indicated the possibility of ParA2 regulating cell division by bringing nucleoid compaction at the vicinity of septum growth.     

A plasma global metabolic profiling approach applied to an exercise study monitoring the effects of glucose, galactose and fructose drinks during post-exercise recovery

A global metabolic profiling methodology based on gas chromatography coupled to time-of-flight mass spectrometry (GC-TOFMS) for human plasma was applied to a human exercise study focused on the effects of beverages containing glucose, galactose, or fructose taken after exercise and throughout a recovery period of 6 h and 45 min. One group of 10 well trained male cyclists performed 3 experimental sessions on separate days (randomized, single center). After performing a standardized depletion protocol on a bicycle, subjects consumed one of three different beverages: maltodextrin (MD)+glucose (2:1 ratio), MD+galactose (2:1), and MD+fructose (2:1), consumed at an average of ～1.25 g of carbohydrate (CHO) ingested per minute. Blood was taken straight after exercise and every 45 min within the recovery phase. With the resulting blood plasma, insulin, free fatty acid (FFA) profile, glucose, and GC-TOFMS global metabolic profiling measurements were performed. The resulting profiling data was able to match the results obtained from the other clinical measurements with the addition of being able to follow many different metabolites throughout the recovery period. The data quality was assessed, with all the labelled internal standards yielding values of <15% CV for all samples (n=335), apart from the labelled sucrose which gave a value of 15.19%. Differences between recovery treatments including the appearance of galactonic acid from the galactose based beverage were also highlighted.