Enhancement of catalytic activity of enzymes by heating in anhydrous organic solvents: 3D structure of a modified serine proteinase at high resolution.

For the first time, it is demonstrated that exposure of an enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes, namely, proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin were exposed to acetonitrile at 70 degrees C for three hr. The activities of these enzymes were found to be considerably enhanced.

Prediction of metal precipitates in tannery sludge leachate based on thermodynamic calculations.

Thermodynamic calculations were performed in order to predict the formation of metal precipitates during the tannery sludge leaching. Gibbs free energy deltaG=RTln(Qc/Kc) of precipitation reaction was used to examine the formation of precipitates during the leaching process. The values of activity equilibrium constant (Ka) of various precipitation reactions were adopted from the literature.

Acid and alkaline treatments for enhancing the growth of rhizobia in sludge.

Wastewater sludges have been proposed as an effective media for the production of rhizobia. The effect of total suspended solid (TSS) concentrations and pretreatments of sludge on the growth of Sinorhizobium meliloti were investigated. Acid (pH 2.

Assessment of toxicity reduction after metal removal in bioleached sewage sludge.

Sewage sludge can be applied to land to supply and recycle organic matter and nutrients. Trace elements in sludge, however, may accumulate in the soil with repeated sludge applications. Reducing metal content may therefore reduce the adverse effects of sludge application.

The gene for the axonal cell adhesion molecule TAX-1 is amplified and aberrantly expressed in malignant gliomas.

The human TAX-1 gene encodes a Mr 135,000 glycoprotein that is transiently expressed on the surface of a subset of neurons during development and is involved in neurite outgrowth. The TAX-1 gene has been mapped to a region on chromosome 1 that has been implicated in microcephaly and the Van der Woude syndrome. Using restriction landmark genome scanning to search for amplified genes in gliomas, we found TAX-1 to be amplified in 2 high-grade gliomas among a group of 26 gliomas investigated.

Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells.

An expression construct containing the cDNA encoding a modified aequorea green fluorescent protein (GFP) ligated to the 5'-end of the rat androgen receptor (AR) cDNA (GFP-AR) was used to study the intracellular dynamics of the receptor movement in living cells. In three different cell lines, ie. PC3, HeLa, and COS1, unliganded GFP-AR was seen mostly in the cytoplasm and rapidly (within 15-60 min) moved to the nuclear compartment after androgen treatment.

Enhancement of catalytic efficiency of enzymes through exposure to anhydrous organic solvent at 70 degrees C. Three-dimensional structure of a treated serine proteinase at 2.2 A resolution.

The enzyme behavior in anhydrous media has important applications in biotechnology. So far chemical modifications and protein engineering have been used to alter the catalytic power of the enzymes. For the first time, it is demonstrated that an exposure of enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region.

Ribozyme-mediated cleavage of the estrogen receptor messenger RNA and inhibition of receptor function in target cells.

Estrogen receptor (ER) functions as a ligand-activated transcription factor for estrogen-regulated genes. Because of the critical role of the ER in the proliferation of certain estrogen-dependent cancer cell types such as the mammary tumor, inhibitors of estrogen action at the level of receptor function are of major clinical interest. Here we describe developments of two ribozymes that can selectively degrade the human ER mRNA and inhibit trans-activation of an artificial promoter containing the estrogen response element.

Stimulation of fructose 1,6-bisphosphate production in melanoma cells by alpha-melanocyte-stimulating hormone-- 31P/13C-NMR and 32P-labeling studies.

In a 31P-NMR spectroscopic study of cultured M2R mouse melanoma cells, we previously demonstrated the acute stimulation of three peaks in the phosphomonoester region of the spectrum by [Ahx4, DPhe7]alpha-melanotropin (concomitant with an increase in cellular adenosine 3',5'-phosphate (cAMP) and a decrease in ATP [Degani, H., DeJordy, J. O.

Mechanisms of progesterone receptor export from nuclei: role of nuclear localization signal, nuclear export signal, and ran guanosine triphosphate.

Steroid hormone receptors are, in most cases, mainly nuclear proteins that undergo a continuous nucleocytoplasmic shuttling. The mechanism of the nuclear export of these proteins remains largely unknown. To approach this problem experimentally in vivo, we have prepared cell lines permanently coexpressing the wild-type nuclear progesterone receptor (PR) and a cytoplasmic receptor mutant deleted of its nuclear localization signal (NLS) [(deltaNLS)PR].

Carbodi-imide coupling of enzymes to the reversibly soluble insoluble polymer Eudragit S-100.

The coupling of proteins and enzymes to soluble-insoluble polymers by carbodi-imide can be performed by using numerous variations of the protocol. This protocol has been investigated for the coupling of five different enzymes, namely wheatgerm acid phosphatase, beta-glucosidase, beta-galactosidase, trypsin and xylanase, to an enteric methacrylate polymer Eudragit S-100. The following results were found.

Synthesis and anti-inflammatory activity of some potential cyclic phenothiazines.

Some new schiff's bases (IVa-IVe), thiazolidinones (Va-Ve), delta 2-triazolines (VIa-VIe) and formazans (VIIa-VIIe) of 2-chlorophenothiazine have been synthesized and screened against Carrageenin induced oedema in albino rats. Some compounds of the series have shown promising activity. The most active compound is 2-chloro-10[5-(2-fluorophenyl-2-oxo-4 thiazolidin-1-yl)-amino acetyl] phenothiazine was found to be most potent.

GAC1, a new member of the leucine-rich repeat superfamily on chromosome band 1q32.1, is amplified and overexpressed in malignant gliomas.

We have used two-dimensional electrophoresis of enzyme-digested genomic DNA to identify a novel gene GAC1, which maps at 1q32.1 and which is overexpressed in malignant gliomas in which it is amplified. GAC1 encodes a protein which belongs to the leucine-rich repeat superfamily.

Chemical modification and chemical cross-linking for protein/enzyme stabilization.

The protein function as well as its stability is governed by the amino acid sequence which in turn defines the collective noncovalent interactions leading to its specific conformation. Hence, it is not surprising that chemical modification with monofunctional and bifunctional reagents (the latter is called chemical cross-linking) causes structural changes (sometimes even subtle) which can result in significant changes in the stability. This review, while recapitulating the early lessons, analyses recent work (including work from authors' laboratory) involving these twin approaches for protein stabilization.

Isolation, phenotypic characterization, and phylogenetic position of a novel, facultatively autotrophic, moderately thermophilic bacterium, Thiobacillus thermosulfatus sp. nov.

Thiobacillus thermosulfatus ATCC 51520T (T = type strain) was isolated from sewage sludge samples enriched with elemental sulfur. The cells of this organism were gram negative, rod shaped, motile, facultatively autotrophic, and strictly aerobic and contained polyphosphate inclusions and polyhedral bodies. During growth on thiosulfate, the following intermediates were produced: tetrathionate, trithionate, and sulfate, and the pH was lowered from neutrality to around 2.

Simultaneous extraction of cellular lipids and water-soluble metabolites: evaluation by NMR spectroscopy.

A method for simultaneous extraction of lipids and water-soluble metabolites from a single cell sample was developed and optimized for NMR spectroscopy. Intermediary metabolites in cultured M2R mouse melanoma cells and changes therein in response to challenge with melanotropin were studied by 31P and 13C NMR. Cells were extracted with methanol, chloroform, and water (1:1:1, v/v/v).

Microbial ecology of simultaneous thermophilic microbial leaching and digestion of sewage sludge.

The microbial population encountered during a simultaneous thermophilic microbial leaching and digestion process at 50 degrees C, based on microbial sulfur oxidation, was investigated. The cell count of the sulfuric acid producer Thiobacillus thermosulfatus increased, followed by a decrease. In the absence of sulfur (control: conventional thermophilic digestion), Thiobacillus thermosulfatus population decreased under the detection limit.

Immobilization of Aspergillus niger xylanase on magnetic latex beads.

Xylanase from Pectinex 3XL was purified 70-fold by precipitation with an enteric polymer, Eudragit S-100. The purified xylanase was immobilized on magnetic latex beads via carbodi-imide coupling. The immobilized preparation showed 80% of the total activity bound to the beads.

Purification and characterization of an acid phosphatase from Arachis hypogaea.

An acid phosphatase from Arachis hypogaea (peanuts) has been purified. The electrophoretically homogeneous enzyme preparation is free of any phophodiesterase activity. The enzyme has a molecular weight of 120,000.

A rapid, high-yield purification of L-alanine:4,5-dioxovalerate transaminase from rat kidney mitochondria using an improved enzyme assay method.

The present report documents an improved enzyme assay method for the mammalian L-alanine:4,5-dioxovalerate transaminase which is of significant utility in work with crude tissue homogenates, cell cultures, or purified enzyme preparations. We also describe a new and rapid purification procedure for this enzyme from rat kidney mitochondria. The three-step procedure involves the use of digitonin and lubrol for mitochondrial matrix preparation and L-alanine-Sepharose 4B column chromatography followed by gel filtration on Sepharose 6B.

Comparison of Microbial Sulfuric Acid Production in Sewage Sludge from Added Sulfur and Thiosulfate.

Microbial leaching is one of the most attractive methods of removing toxic metals from sewage sludge. Sulfuric acid produced by indigenous microflora by the oxidation of elemental sulfur and sulfur compounds solubilizes toxic metals. The oxidation of sulfur compounds can be achieved by the direct oxidation to sulfates or by indirect oxidation, through the production and accumulation of soluble intermediate (S O , S O , S O ) compounds.

An insoluble aggregate of commercially available bovine serum albumin shows antiproteolytic activity.

Chemical aggregation of bovine serum albumin by extensive chemical crosslinking with glutaraldehyde yielded an insoluble protein preparation with significant antiproteolytic activity. This was presumably due to the presence of alpha 2-macroglobulin in bovine serum albumin preparations. Chemical crosslinking of bovine serum albumin under optimum conditions was found to increase its antitryptic activity by about four times.

Screening of pollen grains vis-à-vis whole plants of oilseed brassicas for tolerance to salt.

Pollen grains and whole plants of 11 cultivars of oilseed brassicas (B. juncea,B. campestris,B.

Simultaneous determination of native and subunit molecular weights of proteins by pore limit electrophoresis and restricted use of sodium dodecyl sulfate.

In the present communication, we describe a method for the determination of both the native and subunit molecular weight of a protein by a single pore limit electrophoretic run. When native proteins and sodium dodecyl sulfate (SDS)-heat denatured proteins were electrophoresed in a 4-28% gradient-polyacrylamide gel, in the presence of 0.02% SDS in running buffer, their respective molecular weights (native and subunit) could be determined by comparing the migration distances of unknown and standard proteins.