Levels and 75Se-labeling of specific proteins as a consequence of dietary selenium concentration in mice and rats

Selenium-labeled proteins (SLP) distinct from glutathione peroxidase (GSH-PX) recently have been purified and partially characterized. Antisera to two SLP, a 56-kDa and a 14-kDa protein, were generated in rabbits and used to examine expression of these proteins as a consequence of dietary selenium concentration (0.02, 0.2, 2.0 ppm) in mice and rats. Additionally, the kinetics of 75Se labeling in plasma, liver, kidney, and mammary gland were examined over a 40-hr time period as a function of dietary selenium concentration. A plasma 57-kDa protein was labeled by 30 min after 75Se injection and reached maximum labeling by 4 hr. The cellular 56-kDa and 14-kDa proteins, as well as GSH-Px, labeled progressively over 40 hr starting between 1 and 4 hr after injection. In general, the 56-kDa and GSH-Px followed similar labeling patterns, whereas the 14-kDa protein was labeled less and was not labeled in discernible quantities until 40 hr. The extent of labeling of all proteins was inversely proportional to the dietary selenium concentration and was probably a reflection of different endogenous selenium body pools. The most important observation was generated by the immunoblot data. The amount of 56-kDa and 14-kDa proteins as detected and measured on immunoblots was not a function of dietary selenium concentration. This result suggests that the synthesis and maintenance of the 56-kDa and 14-kDa proteins are not selenium dependent, a characteristic which distinguishes the two proteins from GSH-Px. The single exception to the above results was the 40% decrease of liver 14-kDa protein concentration in carcinogen-treated rats fed 2.0 ppm of selenium. An organic selenium compound, selenobetaine, did not lead to a decrease under similar conditions. In 15 rat mammary tumors induced by 7,12-dimethylbenzanthracene and analyzed on immunoblots, the SLP-56 was undetected in 5 cases and appeared as two bands (56,000 Da, 50,000 Da) in 10 cases. This latter result raises the possibility that the expression of SLP-56 may be altered in mammary tumors as compared with normal mammary gland.     

Proteomic detection of proteins involved in perchlorate and chlorate metabolism

Mass spectrometry and a time-course cell lysis method were used to study proteins involved in perchlorate and chlorate metabolism in pure bacterial cultures and environmental samples. The bacterial cultures used included Dechlorosoma sp. KJ, Dechloromonas hortensis, Pseudomonas chloritidismutans ASK-1, and Pseudomonas stutzeri. The environmental samples included an anaerobic sludge enrichment culture from a sewage treatment plant, a sample of a biomass-covered activated carbon matrix from a bioreactor used for treating perchlorate-contaminated drinking water, and a waste water effluent sample from a paper mill. The approach focused on detection of perchlorate (and chlorate) reductase and chlorite dismutase proteins, which are the two central enzymes in the perchlorate (or chlorate) reduction pathways. In addition, acetate-metabolizing enzymes in pure bacterial samples and housekeeping proteins from perchlorate (or chlorate)-reducing microorganisms in environmental samples were also identified.     

An Omega(n2/ log n) speed-up of TBR heuristics for the gene-duplication problem

The gene-duplication problem is to infer a species supertree from gene trees that are confounded by complex histories of gene duplications. This problem is NP-hard and thus requires efficient and effective heuristics. Existing heuristics perform a stepwise search of the tree space, where each step is guided by an exact solution to an instance of a local search problem. We improve on the time complexity of the local search problem by a factor of n2= log n, where n is the size of the resulting species supertree. Typically, several thousand instances of the local search problem are solved throughout a stepwise heuristic search. Hence, our improvement makes the gene-duplication problem much more tractable for large-scale phylogenetic analyses.     

The dynamic nature of contact networks in infectious disease epidemiology

Although contact network models have yielded important insights into infectious disease transmission and control throughout the last decade, researchers have just begun to explore the dynamic nature of contact patterns and their epidemiological significance. Most network models have assumed that contacts are static through time. Developing more realistic models of the social interactions that underlie the spread of infectious diseases thus remains an important challenge for both data gatherers and modelers. In this article, we review some recent data-driven and process-driven approaches that capture the dynamics of human contact, and discuss future challenges for the field.     

Seasonal variations in harvest index and bacoside A contents amongst accessions of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.) Wettst. collected from wild populations

Bacoside A, a major active principle of Bacopa monnieri known for its cognitive effects is a mixture of saponins like bacoside A3, bacopaside II, isomer of bacopasaponin C and bacopasaponin C. Seasonal changes in biomass and bacoside A levels in fourteen accessions of B. monnieri were evaluated after maintaining these at a common site at Thapar University campus, Patiala (30°19′36.12″N and 76°24′1.08″E) for 1 year. Harvestable biomass and total bacoside A contents varied significantly between the accessions and also in a particular accession during different seasons of the year. The maximum dry weight of plant (biomass 1.64 g) and bacoside A levels (6.82 mg/plant) were recorded in accession BM1. Harvestable biomass was highest during summer in accessions BM1 and BM7 (FW 4.2 g/plant), whereas bacoside A levels were also highest during summer and in accession BM1 (6.82 mg/plant). The lowest bacoside A level (0.06 mg/plant) was recorded in accession BM14 during winter. Principal component analysis showed that samples of summer were positively correlated with both the components suggesting an appropriate time for the harvest.     

The interaction of tetanus toxin with intact bovine adrenal chromaffin cells: binding of toxin and subsequent inhibition of catecholamine release.

Tetanus toxin (about 1 nM) inhibits 70% of the nicotine-evoked release of catecholamines from intact adrenal medullary chromaffin cells after 20 h of incubation and 30% of the K(+)-evoked release. Inhibition of Ca(2+)-evoked release from detergent-permeabilized cells requires higher concentrations of toxin (about 1 microM) toxin, but is maximal after 12 min. Preincubation of the intact cells with ganglioside GT1 in the absence of toxin also inhibits evoked secretion. 125I-labelled toxin bound specifically to these cells; the binding capacity was greater at pH 6 (about 1 pmol toxin/mg cell protein) than at pH 7.4 (about 0.25 pmol). In both cases there were at least two binding components: one of high affinity (Kd about 1 nM) accounting for about 20% of total binding and one of lower affinity (Kd 10-20 nM). Preincubation of the cells with ganglioside increased the binding capacity, but did not affect the Kd of the lower affinity component. Similar observations could be made when binding was measured immunocytochemically. Extraction of gangliosides from chromaffin cells and overlay experiments with radiolabelled toxin showed that, as well as GM3, the major ganglioside component of chromaffin cell membranes, a ganglioside having the chromatographic mobility of GT1 was a major ligand for toxin.     

Recovery of urokinase from integrated mammalian cell culture cryogel bioreactor and purification of the enzyme using p-aminobenzamidine affinity chromatography

An integrated product recovery system was developed to separate urokinase from the cell culture broth of human kidney cells HT1080. Supermacroporous monolithic cryogels provided ideal matrices with respect to surface and flow properties for use as cell culture scaffold as well as for affinity chromatographic capture step of the enzyme in the integrated system. The urokinase was produced continuously in the reactor running for 4 weeks with continuous circulation of 500 ml of culture medium. The enzyme activity in the culture medium reached to 280 Plough units (PU)/mg protein. Cu(II)-iminodiacetic acid (IDA)-polyacrylamide (pAAm) cryogel column was used to capture urokinase by integrating with the gelatin-coupled pAAm-cryogel bioreactor for HT1080 cell culture. After removing the urokinase capture column from the integrated system the bound protein was eluted. The metal affinity capture step gave 4.5-fold purification of the enzyme thus achieving a specific activity of 1300 PU/mg protein. The enzyme eluate from Cu(II)-IDA-pAAm cryogel capture column was further purified on benzamidine-Sepharose affinity column. This step finally led to a homogeneous preparation of different forms of urokinase in two different elution peaks with a best urokinase activity of 13 550 PU/mg of protein. As compared to initial activity in the cell culture broth, about 26.2- and 48.4-fold increase in specific activity was achieved with enzyme yields corresponding to 32% and 35% in two different peak fractions, respectively. Native electrophoresis and SDS-PAGE showed multiple protein bands corresponding to different forms of the urokinase, which were confirmed by Western blotting and zymography.     

Mutagenicity associated with O6-methylguanine-DNA damage and mechanism of nucleotide flipping by AGT during repair

Methylated guanine damage at O6 position (i.e. O6MG) is dangerous due to its mutagenic and carcinogenic character that often gives rise to G:C-A:T mutation. However, the reason for this mutagenicity is not known precisely and has been a matter of controversy. Further, although it is known that O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6MG paired with cytosine in DNA, the complete mechanism of target recognition and repair is not known completely. All these aspects of DNA damage and repair have been addressed here by employing high level density functional theory in gas phase and aqueous medium. It is found that the actual cause of O6MG mediated mutation may arise due to the fact that DNA polymerases incorporate thymine opposite to O6MG, misreading the resulting O6MG:T complex as an A:T base pair due to their analogous binding energies and structural alignments. It is further revealed that AGT mediated nucleotide flipping occurs in two successive steps. The intercalation of the finger residue Arg128 into the DNA double helix and its interaction with the O6MG:C base pair followed by rotation of the O6MG nucleotide are found to be crucial for the damage recognition and nucleotide flipping.