Ca2+ -dependent inactivation of the mitochondrial Ca2+ uniporter involves proton flux through the ATP synthase

Stimulation of receptors on the surface of animal cells often evokes cellular responses by raising intracellular Ca(2+) concentration. The rise in cytoplasmic Ca(2+) drives a plethora of processes, including neurotransmitter release, muscle contraction, and cell growth and proliferation. Mitochondria help shape intracellular Ca(2+) signals through their ability to rapidly take up significant amounts of Ca(2+) from the cytosol via the uniporter, a Ca(2+)-selective ion channel in the inner mitochondrial membrane. The uniporter is subject to inactivation, whereby a sustained cytoplasmic Ca(2+) rise prevents further Ca(2+) uptake. In spite of its importance in intracellular Ca(2+) signaling, little is known about the mechanism underlying uniporter inactivation. Here, we report that maneuvers that promote matrix alkalinisation significantly reduce inactivation whereas acidification exacerbates it. We further show that the F(1)F(0)-ATP synthase complex is an important source of protons for inactivation of the uniporter. These findings identify a novel molecular mechanism that regulates the activity of this ubiquitous intracellular Ca(2+) channel, with implications for intracellular Ca(2+) signaling and aerobic ATP production.     

Assessment of genetic diversity among okra genotypes using SSR markers

Okra (Abelmoschus esculentus) is an important nutritious vegetable. Despite its high economic and industrial value, very little attention has been paid to assess genetic diversity of okra at molecular level. For effective conservation and proper deployment of germplasm, a study on diversity analysis of okra germplasm was conducted with DNA markers. Microsatellite/Simple sequence repeat (SSR) markers were utilized to evaluate the genetic diversity among 96 accessions of Abelmoschus, of which 92 accessions were of A. esculentus and one accession each of A. tuberculatus, A. moschatus, A. moschatus subspecies tuberosus and A. manihot. A set of 40 SSR primers were tested, of which 30 primers gave reproducible amplification which were used further for diversity analysis. With a mean of 7.1 bands per SSR, DNA amplification with 30 SSRs generated a total 213 bands, of which 60.66 % were recorded polymorphic. Polymorphic information content ranged between 0.11 and 0.80 with an average of 0.52, indicating that the majority of primers were informative. The Jaccard’s coefficient ranged from 0.107 to 0.969. The UPGMA analysis grouped Abelmoschus genotypes into three main clusters at a cut-off of 0.20. Results of present study revealed that sufficient variation exists among the studied accessions and GAO-5 which was found highly diverse can be exploited for okra improvement. The outcome of present research would assist to make use of Ablemoschus germplasm for okra breeding.     

Butanol production by hypersolvent-producing mutant Clostridium beijerinckii BA101 in corn steep water medium containing maltodextrin

Clostridium beijerinckii NCIMB 8052 parent strain and BA101, a hypersolvent-producing mutant, fermented 6% (w/v) glucose, maltodextrin, maltose or xylose in a medium containing corn steep water (CSW) to produce butanol. Batch fermentation in an unoptimized 6% (w/v) maltodextrin plus 1.6% solids CSW medium demonstrated that C. beijerinckii NCIMB 8052 and BA101 produced 10.7 g butanol/L and 14.5 g butanol/L, respectively.     

Computer programs in nucleic acid synthesis: synthetic strategy development using solid-phase chemical techniques with data storage, retrieval and analysis capabilities.

A computer program has been designed to aid development of synthetic strategies for oligonucleotides produced by solid-phase chemical techniques. The program reduces the time required to develop a strategy and a data file from hours to minutes. The program contains inventories, provides cost analyses, and generates and stores other associated data. The program searches an inventory of sequences for that sequence to avoid duplicate synthesis. If the sequence is not in the inventory the program devises a synthetic strategy, calculates the amounts of reagents and labor costs necessary to complete the synthetic oligonucleotide. The program also deducts the reagents from inventory files. Physical data is also calculated. A file is generated in a sequence inventory for storage of the data as well as other data that will be generated during the purification processes. All variable parameters can be easily edited. The programs were designed to provide a cross-referencing feature for data analysis and can use several parameters as a constant.     

Fed-batch fermentation of glucose to acetate by an improved strain ofClostridium thermoaceticum

The fed-batch approach to the production of acetate from glucose by an improved strain ofClostridium thermoaceticum resulted in better performance than the batch fermentation, especially in media containing an excess (3X) of nutrients and trace salts. At pH 6.6, 46 g/l acetic acid was produced in 192 hours with 93% substrate utilization. In contrast, batch fermentation under similar conditions resulted in a maximum of 35 g/l acetic acid with less than 82% substrate utilization.     

Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Various conditions were analyzed and optimized for the preparative elution of proteins from nitrocellulose membranes after transfer from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of elution was best using pyridine or acetonitrile elution solvents, intermediate for buffer containing a mixture of sodium dodecyl sulfate, Triton X-100, and sodium deoxycholate, and negligible for buffers containing any single detergent or chaotropic salt, such as urea or guanidine hydrochloride. The efficiency of elution with any solvent also depended on the molecular weight of the proteins, smaller proteins being more easily removed from membranes. As a general procedure, proteins may be eluted from nitrocellulose membranes by incubation with either 40% acetonitrile or 50% pyridine in 0.1 M ammonium acetate, pH 8.9, for 1-3 h at 5-37 degrees C. The recommended procedures for protein elution appear to offer a rapid, simple, and efficient means of recovering proteins from complex mixtures after separation by SDS-PAGE and transfer to nitrocellulose membranes.     

High concentrations of acetate with a mutant strain ofC. thermoaceticum

Summary Fed-batch fermentation of glucose by a mutant strain ofC.thermoaceticum resulted in acetate concentrations of 83–100 gL^–1. Excess nutrients were required to maintain cell viability, especially when high cell concentrations were used. The strain was tolerant to high levels of Na, Ca and Mg. Product yield was 0.74–0.80 g acetate/g glucose, and the productivity was 0.60–0.85 gL^–1h^–1.     

Salt mediated changes in some enzymes of carbohydrate metabolism in halotolerantCladosporium sphaerospermum

Cladosporium sphaerospermum, isolated from salt pans was halotolerant. When grown in the presence of salt, the activities of invertase, isocitrate lyase, fructose-1,6 diphosphate aldolase and malate dehydrogenase were found to be increased and that of amylase decreased. Both, enzyme activation as well as an increase inde novo synthesis of enzymes were found to be some of the mechanisms of salt mediated changes. This may be one of the adaptive mechanisms, in halotolerantCladosporium sphaerospermum.     

N-Alkane oxidation enzymes of a pseudomonad.

A nicotinamide adenine dinucleotide (NAD)-dependent n-alkane dehydrogenase and an NAD phosphate (reduced form)-dependent alkane hydroxylase have been purified from cell-free extracts of Pseudomonas sp. strain 196Aa grown anaerobically on n-alkane. The n-alkane dehydrogenase (fraction R-3), obtained as a single peak from Bio-Gel P-60, showed an overall 135-fold purification and was demonstrated by infrared spectroscopy and gas chromatography to convert n-decane to 1-decene. The alkene hydroxylase activity in the S-3 fraction, purified 167 times from diethylaminoethyl-cellulose, was shown by the same methodology to convert decene to decanol. Commercial ferredoxin has been shown to increase the alkane dehydrogenase activity. An NAD-, flavine adenine dinucleotide-, and iron-dependent alcohol dehydrogenase was demonstrated in the R-3 fraction. A mechanism for the anaerobic conversion of n-alkane to fatty acid has been proposed.     

Isolation and Molecular Characterization of Arsenite-Tolerant Alishewanella sp. GIDC-5 Originated from Industrial Effluents

An As-hypertolerant Alishewanella sp. GIDC-5 (Accession no. HQ659190) was isolated from an effluent treatment plant of the industrial area near Sachin, Gujarat (India). In vitro studies revealed that GIDC-5 can tolerate 18 mM of arsenite [As(III)] and 220 mM of arsenate [As(V)]. PCR analysis confirmed the presence of arsenite transporters [arsB and ACR3(1)] and arsenite oxidase gene [aioB]. Specific activities of arsenite oxidase and arsenate reductase, located on membrane and cytoplasmic fractions respectively, increased in dose dependent manner with arsenite concentration. Also, specific activities of antioxidant enzymes viz., catalase, ascorbate peroxidase, superoxide dismutase and glutathione S-transferase increased in presence of arsenite. Increased exposure to arsenite changes enzyme activities of the glycolysis, Krebs and glyoxylate branches dramatically. These results reveal that along with ars operon, metabolic adaptation and antioxidant activities participate in As(III) tolerance in Alishewanella sp. GIDC-5.