Isolation and partial characterization of sheep plasma α1-mucoprotein

1. 1. Sheep plasma α1-mucoprotein was isolated in an electrophoretically homogeneous state by a combination of ammonium sulphate saturation, isoelectric precipitation and preparative agar electrophoresis in a yield of approx. 150 mg/l of plasma. 2. 2. The mucoprotein was water-soluble, non-coagulable on heating at 100°, not precipitable by 1.8 M perchloric acid, 10% trichloroacetic acid but precipitable by saturated ammonium sulphate solution, 0.6 M sulfosalicylic acid and 5% phosphotungstic acid in 2 N HCl. It had E1 cm1 % value of 9.57 at 278 mμ in water, refractive-index index increment 1.9·10-4 (g/l) in water, isoelectric point at pH 4.45 (sodium acetate-acetic acid buffer) and was homogeneous in pH range 4.0-11.5 but at pH values 2.6 and 3.5 showed some dissociation. 3. 3. The mucoprotein had the following chemical composition: Nitrogen, 12.4%; polypeptide, 77.4%; total hexose (only mannose and galactose), 7.1%; fucose, 1.0%; glucosamine, 4.9% and sialic acid, 4.8%. It had no N-terminal amino acid.

Ultrasonic propagation through aqueous solutions of acetates

Using the pulse method in the range of 2 to 26Mc's the ultrasonic absorption, velocity and the adiabatic compressibility have been studied in eleven aqueous acetate solutions up to a concentration of 1 mole/litre. The substances studied are the acetates of lithium, sodium, potassium, ammonium, magnesium, calcium, strontium, barium, zinc, cadmium and lead. Absorption in mercuric acetate has been studied only at 2 and 6 Mc/s. Two regions of relaxation are noticed, one below 10 Mc/s and the other between 10 and 26 Mc/s. The first relaxation is ascribed to the dissociation reaction of the salt and the second one to the monomerdimer reaction of the acetic acid formed by the hydrolysis of the salt in water.

Crystal And Molecular-Structure Of 8-Acetoxy-6-(2,4-Dimethoxy-5-Bromophenyl)-3-Methyl-Tricyclo[5,2,1,03,8 ]Decan-2-One

The crystal and molecular structure has been determined by the heavy-atom method and refined by the least-squares procedure to R= 8"3 % for 2033 photographically observed reflexions. The compound crystallizes in the space group P]" with two molecules in a unit cell of dimensions a = 11"68 + 0-02, b = 12"91 +0"02, c= 10"43+0"02/~, e= 114"7+ 1, fl=90-2+ 1 and 7,= 118.3+ 1 °. The unit cell also contains one molecule of the solvent, benzene. The 'cage' part of the molecule exhibits a large number of elongated bonds and strained internal valency angles. The bridgehead angle in the bicyclic heptane ring system is 89 °. The acetate group at C(16) and the methyl group at C(15) are cis to each other.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of the putative SAICAR synthetase (PH0239) from Pyrococcus horikoshii OT3

The study of proteins involved in de novo biosynthesis of purine nucleotides is central in the development of antibiotics and anticancer drugs. In view of this, a protein from the hyperthermophile Pyrococcus horikoshii OT3 was isolated, purified and crystallized using the microbatch method. Its primary structure was found to be similar to that of SAICAR synthetase, which catalyses the seventh step of de novo purine biosynthesis. A diffraction-quality crystal was obtained using Hampton Research Crystal Screen II condition No. 34, consisting of 0.05 M cadmium sulfate hydrate, 0.1 M HEPES buffer pH 7.5 and 1.0 M sodium acetate trihydrate, with 40%(v/v) 1,4-butanediol as an additive. The crystal belonged to space group P3(1), with unit-cell parameters a = b = 95.62, c = 149.13 angstrom. Assuming the presence of a hexamer in the asymmetric unit resulted in a Matthews coefficient (V-M) of 2.3 angstrom(3) Da(-1), corresponding to a solvent content of about 46%. A detailed study of this protein will yield insights into structural stability at high temperatures and should be highly relevant to the development of antibiotics and anticancer drugs targeting the biosynthesis of purine nucleotides.

Template-Assisted Sol-Gel Synthesis of Nanocrystalline BaTiO3

Nanocrystalline perovskite barium titanate with an average particle size less than similar to 10 nm is produced using sol-gel route involving ligand-assisted templating. BaTiO3 is obtained by the controlled hydrolysis and condensation reaction of barium acetate (Ba(CH3COO)(2)) with titanium tetra chloride (TiCl4) in the reverse micelles of dodecylamine (DDA) which is used as the template. Our attempts to produce mesoporous BaTiO3 have resulted in the formation of nanocrystalline BaTiO3. The synthesis of nanostructured BaTiO3 is carried out using the ligand-assisted templating approach which proceeds through the sol-gel route. Dodecylamine is used as the template. The sol-gel process in general presents inherent advantages because the nanostructure of the desired materials can be controlled together with their porous structure. Ligand-assisted templating approach involves the formation of covalent bond between the inorganic analogue and the template. Ba(CH3COO)(2) and TiCl4 are used as barium-source and titanium-source respectively. The reaction between Ba(CH3COO)(2) and TiCl4 is found to take place deliberately on the pre-assembled species which acts as the template or occurring with in them which in turn will lead to the generation of the desired nanoscale structure (nanopores or nanoparticles).

Nucleotide-sequence of 5S RNA from mycobacterium-smegmatis

32P labelled 5S RNA isolated fromMycobacterium smegmatis was digested withT 1 and pancreatic ribonucleases separately and fingerprinted by two dimensional high voltage electrophoresis on thin-layer DEAE-cellulose plates. The radioactive spots were sequenced and their molar yields were determined. The chain length of the 5S RNA was found to be 120. It showed resemblances to both prokaryotic and eukaryotic 5S RNAs.

Sorghum proteinase inhibitors: purification and some biochemical properties

An investigation has been carried out on the proteinase inhibitors of grain sorghum (Sorghum bicolor (L.) Moench). One of the inhibitors has been isolated in a pure form and characterized. The proteinase inhibitor was extracted from the acetone-defatted sorghum meal and purified by selective thermal denaturation, ammonium sulfate fractionation, Sephadex gel filtration and DEAE-cellulose chromatography (DEAE-preparation II). This preparation was demonstrated to be a mixture of three inhibitor components by polyacrylamide disc gel electrophoresis. Further resolution of this mixture into Inhibitors I to III was achieved by QAE-Sephadex chromatography. Sorghum Inhibitor III was homogeneous by the criteria of disc gel electrophoresis and has been more fully characterized. A molecular weight of 25,000 was obtained for Inhibitor III by gel filtration and was in agreement with the value calculated from the amino acid composition of the inhibitor. The N-terminal amino acid residue of Inhibitor III, a single chain protein, was isoleucine. Sorghum proteinase inhibitors inhibit specifically the serine proteinases and are inactive towards the other classes of proteinases. Inhibitor III is primarily a chymotrypsin inhibitor, whereas Inhibitors I and II inhibit both trypsin and chymotrypsin.

Environmental and climatic dependences of stable isotope ratios in tree rings on different temporal scales

This work examines stable isotope ratios of carbon, oxygen and hydrogen in annual growth rings of trees. Isotopic composition in wood cellulose is used as a tool to study past climate. The method benefits from the accurate and precise dating provided by dendrochronology. In this study the origin, nature and the strength of climatic correlations are studied on different temporal scales and at different sites in Finland. The origin of carbon isotopic signal is in photosynthetic fractionation. The basic physical and chemical fractionations involved are reasonably well understood. This was confirmed by measuring instantaneous photosynthetic discrimination on Scots pine (Pinus sylvestris L.). The internal conductance of CO2 was recognized to have a significant impact on the observed fractionation, and further investigations are suggested to quantify its role in controlling the isotopic signal of photosynthates. Isotopic composition of the produced biomass can potentially be affected by variety of external factors that induce physiological changes in trees. Response of carbon isotopic signal in tree ring cellulose to changes in resource availability was assessed in a manipulation experiment. It showed that the signal was relatively stable despite of changes in water and nitrogen availability to the tree. Palaeoclimatic reconstructions are typically based on functions describing empirical relationship between isotopic and climatic parameters. These empirical relationships may change depending on the site conditions, species and timeframe studied. Annual variation in Scots pine tree ring carbon and oxygen isotopic composition was studied in northern and in central eastern Finland and annual variation in tree ring latewood carbon, oxygen and hydrogen isotopic ratio in Oak (Quercus robur L.) was studied in southern Finland. In all of the studied sites at least one of the studied isotope ratios was shown to record climate strongly enough to be used in climatic reconstructions. Using the observed relationships, four-century-long climate reconstructions from living Scots pine were created for northern and central eastern Finland. Also temporal stability of the relationships between three proxy indicators, tree ring growth and carbon and oxygen isotopic composition was studied during the four-hundred-year period. Isotope ratios measured from tree rings in Finland were shown to be sensitive indicators of climate. Increasing understanding of environmental controls and physiological mechanisms affecting tree ring isotopic composition will make possible more accurate interpretation of isotope data. This study also demonstrated that by measuring multiple isotopes and physical proxies from the same tree rings, additional information on tree physiology can be obtained. Thus isotopic ratios measured from tree ring cellulose provide means to improve the reliability of climate reconstructions.

Antibodies raised against denatured DNA bind to double-stranded DNA

Antibodies raised against denatured DNA complexed with methylated bovine serum albumin have been reported to react with ssDNA but not with dsDNA. Using a highly sensitive avidin-biotin microELISA, we report that such antibodies also bind to dsDNA. Antibodies which reacted with ssDNA and dsDNA were found to be IgG type. The antibodies did not react with tRNA and rRNA. The binding of antibodies to dsDNA was partially inhibited dy individual deoxyribonucleotides. ssDNA as well as dsDNA inhibited the binding of antibodies to dsDNA. The binding of these antibodies to supercoiled and relaxed forms of pBR322 DNA was demonstrated by gel retardation assay. The cross-reaction with ssDNA was observed even after affinity purification on native DNA-cellulose. The antibodies were also shown to bind to poly(dA-dT)·poly(dA-dT)

Effect of phenoxy acids and their derivatives on lipid metabolism in groundnut (Arachis hypogaea) leaves

The effect of four phenoxy compounds [2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid, 4-chlorophenoxyacetic acid 2-(dimethylamino)ethyl ester (centrophenoxine), and 4-chlorophenoxy ethyl 2-(dimethylamino) ethyl ether (neophenoxine)] on lipid metabolism in groundnut (Arachis hypogaea) leaves was investigated under nonphotosynthetic conditions. In experiments with leaf disks, the uptake of [1-14C]acetate, [32P]orthophosphate, [35S]sulfate and [methyl-14C]choline was substantially inhibited by all the phenoxy compounds except neophenoxine. When the incorporation of these precursors into lipids was measured and expressed as percentage of total uptake, there was significant inhibition of incorporation of [1-14C]acetate and [32P]orthophosphate into lipids by all the compounds except neophenoxine. The incorporation of [methyl-14C]choline was unaffected by all except centrophenoxine which showed stastically significant stimulation. [35S]Sulfate incorporation into lipids was markedly inhibited only by centrophenoxine. The fatty acid synthetase of isolated chloroplasts assayed in the absence of light was inhibited 20–50% by the phenoxy compounds at 0.5 mM concentration. This inhibition showed a dependence on time of preincubation with the herbicide suggesting an interaction with the enzyme. It was, however, reversible and excess substrate did not prevent the inhibition, suggesting that the herbicide interaction may not be at the active site. sn-Glycerol-3-phosphate acyltransferase in the chloroplast and microsomal fractions was inhibited by 2,4-D while the phosphatidic acid phosphatase was insensitive to all the phenoxy compounds. It is concluded that phenoxy compounds affect precursor uptake, their incorporation into lipids, and the chloroplast fatty acid synthetase. The free acids were the most potent compounds while the ester (centrophenoxine) was less effective and the ether (neophenoxine) was completely ineffective in their influence on lipid metabolism.

A simple synthesis and characterization of CuS nanocrystals

Water-soluble CuS nanocrystals and nanorods were prepared by reacting copper acetate with thioacetamide in the presence of different surfactants and capping agents. The size of the nanocrystals varied from 3-20 nm depending on the reaction parameters such as concentration, temperature, solvent and the capping agents. The formation of nanocrystals was studied by using UV-visible absorption spectroscopy.

Simultaneous effect of lead and cadmium on granulosa cells: A cellular model for ovarian toxicity

Lead (Pb) and cadmium (Cd) are known reproductive toxicants, which accumulate in granulosa cells of the ovary. Female Charles foster rats were treated with sodium acetate (control), lead acetate and cadmium acetate either alone or in combination at a dose 0.05 mg/kg body weight intra-peritoneally for 15 days daily. Animals were killed at proestrous stage and granulosa cells were isolated from the ovaries. Binding of I-125-luteinizing hormone (I-125-LH), I-125-follicle stimulating hormone (I-125-FSH) and 17 beta-hydroxysteroid dehydrogenase activity were measured. As these receptors are localized on the surface of the cell membrane, we also estimated the membrane parameters of these cells. Our results demonstrated that both lead and cadmium caused a significant reduction in gonadotropin binding, which altered steroidogenic enzyme activity of granulosa cells. These changes exhibited a positive correlation with membrane changes of the granulosa cells.

Using Water as a Design Element in Crystal Engineering. Host-Guest Compounds of Hydrated 3,5-Dihydroxybenzoic Acid

Thirteen host guest compounds of 3,5-dihydroxybenzoic acid (DHBA) have been structurally characterized. Water molecules occupy the peripheries of a hexagonal void, created with DHBA molecules, and act as ``hooks'' to connect the guest molecules with the host-framework via hydrogen bonding. The ``water hook'' is an OH group acting as a donor. Consequently, the guest molecules were chosen so that they contain good hydrogen bond acceptor functionalities. A number of multicomponent hydrates were isolated with stoichiometries (DHBA)(x)(H2O). (guest),. Of these, compounds with the following as guests were obtained as crystals that were good enough for single crystal work: ethyl acetate (EtOAc), diethyl oxalate, dimethyl oxalate, di(n-propyl) oxalate, diethyl malonate, diethyl succinate, chloroacetonitrile, N,N-dimethyl formamide (DMF), acetone, dimethyl sulfoxide (DMSO), 1-propanol, and 2-butanol. From 2-butanol, a hemihydrate, (DHBA)(2)(H2O), was also obtained concomitantly. Further to guest stabilization, water acts as a good mediator of effective crystal packing and also determines the topology of the host framework. En the present series of compounds, the role of water is wide ranging, and it is not easy to classify it specifically as a host or as a guest.

Purification and properties of trehalase from the thermophilic fungus Humicola lanuginosa

Trehalase (?,?-Trehalosee gludohydrolase, EC 3.2.1.28) was partially solubilized from the thermophilic fungus Humicola lanuginosa RM-B, and purified 184-fold. The purified enzyme was optimally active at 50°C in acetate buffer at pH 5.5. It was highly specific for ?,?-trehalose and had an apparent Km = 0.4 mM at 50°C. None of the other disaccharides tested either inhibited or activated the enzyme. The molecular weight of the enzyme was around 170000. Trehalase from mycelium grown at 40 and 50°C had similar properties. The purified enzyme, in contrast to that in the crude-cell free extract, was less stable. At low concentration, purified trehalase was afforded protection against heat-inactivation by �protective factor(s)� present in mycelial extracts. The �protective factor(s)� was sensitive to proteolytic digestion. It was not diffusable and was stable to boiling for at least 30 min. Bovine serum albumin and casein also protected the enzyme from heat-inactivation.

Purification and characterization of a beta-glucosidase and endocellulase from Humicola insolens

A .beta.-glucosidase and an endocellulase were purified from the culture filtrates of a thermophilic cellulolytic fungus Humicola insolens. Both the preparations were homogeneous by PAGE, ultracentrifugation and gel filtration (Mr 45,000). Ouchterlony immunodiffusion showed complete cross reactivity between the antibodies and the two enzyme antigens, indicating the presence of a common epitope on the two enzyme proteins. The two enzymes, however, differ in their amino acid composition and their substrate specificity. .beta.-Glucosidase acts on p-nitrophenyl .beta.-D-glucopyranoside and hydrolyses cellulose to release mainly glucose and small amounts of cellobiose from the non-reducing end. On the other hand, endocellulase hydrolyses cellulose to release cellopentaose, cellotetraose, cellotriose along with cellobiose and glucose and also hydrolyses larch wood xylan.