Preparation of selenium yeasts I. Preparation of selenium-enriched Saccharomyces cerevisiae.

Selenium (Se) is an essential micronutrient for human and animal organisms. Organic selenium complexes and selenium-containing amino acids are considered the most bioavailable. Under appropriate conditions yeasts are capable of accumulating large amounts of trace elements, such as selenium, and incorporating them into organic compounds. It has been found that introduction of water-soluble selenium salt as a component of the culture medium for yeasts produced by conventional batch processing results in a substantial amount of selenium being absorbed by the yeast. Using a culture medium supplemented with 30 microg/mL sodium-selenite added during the exponential growth phase results in selenium-accumulation in the range of 1200-1400 microg/g dried baker's yeast (Saccharomyces cerevisiae) measured by ICP-AES method. In our previous studies it was shown that higher amounts of sodium-selenite in the culture medium have a strong inhibitory effect on the growth of this yeast. As a consequence of variations in cultivation conditions we obtained selenium yeast with different inorganic selenium content. The most important parameters influencing incorporated forms of selenium are pH value and dissolved oxygen level in the culture medium, and depending on these the selenium consumption rate of the yeast. A 0.40-0.50 mg/g h-1 specific selenium consumption rate was found to be appropriate to obtain selenium-enriched bakers' yeast of a high quality. Under suitable conditions the undesirable inorganic selenium content of the yeast could be suppressed to as low as 5-6% at the expense, however, of approximately a 20% decrease in the final biomass.     

Preparation of thianucleoside derivatives.

1'-Deoxy-4'-thioribonucleoside 1 was synthesized from acyclic allylic alcohol 2 by 8 steps. TMSOTf and SnCl4 were found to be a good mediator for the key coupling of thiafuranose part with pyrimidine base to give beta-isomer preferentially.     

Preparation of allosteric ribonuclease.

A method for the preparation of allosteric ribonuclease from bovine pancreas is described. The effects of freeze-drying ribonuclease from acid and alkaline solutions on plots of velocity versus substrate concentration for the hydrolysis of 2':3'-cyclic CMP are examined. Comparison of these plots with the plots obtained with severeal commercial enzyme preparations indicates that the conformation of the enzyme is dependent on the method of preparation. Aging experiments demonstrate that further conformational changes occur at different rates, depending on the methods of storage. Results suggest that the allosteric behaviour of ribonuclease has not always been observed with commercial preparations, owing to variations in methods of preparation and storage of the enzyme.     

Preparation of oligonucleotide-peptide conjugates.

A procedure for preparing oligonucleotide-peptide conjugates is presented. It is based on appending a maleimide group to the oligonucleotide for selective coupling to the thiol side chain of a cysteine residue in the peptide. A convenient chromatographic purification procedure, based on Fmoc-on/Fmoc-off, is described.     

Iodoglucagon. Preparation and characterization.

Iodinated derivatives of glucagon containing an average of 1 to 5 g-atoms of 127I per mol have been prepared by reacting the hormone with increasing amounts of iodine monochloride. Their iodoamino acid composition has been determined by ion-exchange chromatography and electrophoresis, following hydrolysis by pronase. Iodination of the two tyrosyl residues occurs first and is nearly complete after addition of a 4-fold molar excess of ICl. Iodination of the single histidyl residue is a later event and does not exceed an average of one atom per residue. Hydrolysis of iodoglucagon by trypsin and subsequent separation of the iodotyrosyl peptides shows that iodine is equally distributed between tyrosyl residues 10 and 13. Crude iodoglucagon containing an average of 1 g-atom of iodine per mol has been resolved into several components of differing iodine content and iodoamino acid composition by chromatography on DEAE-cellulose. Monoiodoglucagon isolated by this procedure shows a single band when analyzed by polyacrylamide gel electrophoresis. Iodoglucagons containing an average of 1 to 4 g-atoms of iodine per mol are more potent than native glucagon in their ability to stimulate adenylate cyclase activity and to bind to glucagon receptors of liver cell membranes of the rat. The maximal increase in biological potency occurring upon iodination is about 5-fold with respect to adenylate cyclase activity, and 2-fold with respect to binding to receptors; tetra and triiodinated derivatives show, respectively, the highest potency. Similar effects occur whether inactivation by liver membranes is inhibited or not, indicating an enhancement in the intrinsic affinity of iodoglucagon for the receptors. Iodination beyong 4 g-atoms per mol slightly decreases the affinity of the hormone for adenylate cyclase and for the receptors. Iodination causes a 2-20 fold decrease in the ability of liver plasma membranes and of blood plasma to inactivate glucagon in vitro; these effects correlate with the degree of iodination. With liver microsomal membranes, a decrease in glucagon inactivation occurs only at iodine contents exceeding 4 g-atoms per mol, and lower degrees of iodination result in opposite effects. Monoiodination causes a 4-6-fold increase in the plasma concentration of glucagon within the first 18 min following a single intrvenous injection of the hormone to rats. More extensive iodination results, in addition, in a marked decrease in the rate of dissappearance of glucagon from the blood. The immunological reactivity of glucagon is little affected by monoidination, but strongly depressed by higher degrees of iodination...     

Preparation of coenocytes for freeze-etching.

Successful freeze-etching of a coenocyte has been accomplished with glutaraldehyde stabilization followed by infiltration with cryoprotectant. Hyphae of the coenocytic water mold Achlya were stabilized with 5% glutaraldehyde in phosphate buffer. Gradual infiltration by dropwise addition of the cryoprotectant (25% glycerol, 10% ethylene glycol, distilled water, v/v) is accomplished over a period of 8-10 hr on a shaker. Subsequent freeze-etching is carried out by standard procedures.     

Preparation of multiply deuterium-labeled cortisol.

Cortisol labeled with four deuterium atoms at chemically stable sites ([9,11,12,12-(2)H4]cortisol, cortisol-d4) was prepared by hydrogen-deuterium exchange and reductive deuteration reactions. After protecting the C-17 dihydroxyacetone side chain of cortisone (cortisone-BMD), hydrogen-deuterium exchange was carried out with 6.5% NaOD in MeOD, which was followed by protection of the C-3 carbonyl as the semicarbazone. Subsequent reductive deuteration at C-11 with NaBD4 followed by removal of exchangeable deuterium under the same exchange-reaction conditions in a medium of 6.5% NaOH in MeOH and deprotection afforded the desired cortisol-d4 with high isotopic content (d3, 21.2%; d4, 78.1%; d5, 0.74%). The method was applied to the synthesis of cortisol labeled with nine deuterium atoms [( 1,1,9,11,12,12,19,19,19-(2)H9]cortisol, cortisol-d9) starting from [1,1,19,19,19-(2)H5]cortisone (cortisone-d5).     

Preparation and isolation of dityrosine.

A new procedure for the isolation of dityrosine has been developed. Tyrosine was oxidized by means of incubation both with hydrogen peroxide and horse-radish peroxidase. The reaction mixture was separated by permeation chromatography on Sephadex G-10 being monitored at 280 and 310 nm spectrophotometrically. The dityrosine fraction was freeze-dried and purified on a cation-exchange column (in acidic citrate buffer). The purified fraction was desalted and freeze-dried. The yield was 96 mg of homogenous dityrosine per 1 g of D,L-tyrosine. Some physico-chemical constants of the preparation were measured (optical characteristics with U.V. and I.R. spectra, fluorescence spectra, chromatography on an amino acid analyzer).     

Preparation of micelle-forming polymer-drug conjugates.

Adriamycin, a hydrophobic anticancer drug, was conjugated with poly(ethylene oxide)-poly(aspartic acid) block copolymers composed of various lengths of each block copolymer segment ranging from 1000 to 12,000 in molecular weight and from 10 to 80 units, respectively. Conjugation was achieved without precipitation by adjusting the ratio of adriamycin to aspartic acid residues of the block copolymer and the quantity of DMF used for the reaction. Thus obtained conjugates showed high water solubility irrespective of a large amount of the conjugated adriamycin. Furthermore, these conjugates were found to form micellar structures with a hydrophobic inner core and a hydrophilic outer shell. This micellar architecture may be utilized for effective drug targeting.     

Preparation of rat enterocyte mitochondria.

Rat enterocyte mitochondria were prepared with respiratory control ratios of 4 or 5 and occasionally 6. When EGTA was excluded from the mitochondrial incubation medium the calculated P/O ratios were high, especially those based on the first addition of ADP. These ratios were lowered by increasing the EGTA concentration from 1 mM to 2 mM in the mitochondrial preparation medium and including 1 mM-EGTA in the incubation medium. The use of EDTA in the enterocyte isolation medium led to the mitochondria requiring added cytochrome c. Substituting EGTA for EDTA abolished this requirement. The mitochondrial fraction consisted of two components, an upper cream-coloured layer rich in DNA and a lower brown-coloured layer poor in DNA. Both components were capable of oxidative phosphorylation with succinate or the glutamate/malate couple as substrates. The mitochondrial yield was assessed by assaying succinate dehydrogenase activity, and the contamination of the mitochondrial fraction by other cell organelles was assessed by assays for appropriate marker enzymes.     

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Preparation of selenium yeasts I. Preparation of selenium-enriched Saccharomyces cerevisiae

Selenium (Se) is an essential micronutrient for human and animal organisms. Organic selenium complexes and selenium-containing amino acids are considered the most bioavailable.Under appropriate conditions yeasts are capable of accumulating large amounts of trace elements, such as selenium, and incorporating them into organic compounds. It has been found that introduction of water-soluble selenium salt as a component of the culture medium for yeasts produced by conventional batch processing results in a substantial amount of selenium being absorbed by the yeast.Using a culture medium supplemented with 30 μg/mL sodium-selenite added during the exponential growth phase results in selenium-accumulation in the range of 1200–1400 μg/g dried baker's yeast (Saccharomyces cerevisiae) measured by ICP-AES method. In our previous studies it was shown that higher amounts of sodium-selenite in the culture medium have a strong inhibitory effect on the growth of this yeast. As a consequence of variations in cultivation conditions we obtained selenium yeast with different inorganic selenium content. The most important parameters influencing incorporated forms of selenium are pH value and dissolved oxygen level in the culture medium, and depending on these the selenium consumption rate of the yeast. A 0.40–0.50 mg/g h-1 specific selenium consumption rate was found to be appropriate to obtain selenium-enriched bakers' yeast of a high quality. Under suitable conditions the undesirable inorganic selenium content of the yeast could be suppressed to as low as 5–6% at the expense, however, of approximately a 20% decrease in the final biomass.     

Human epidermal transglutaminase. Preparation and properties.

A transglutaminase from human hair follicle-free epidermis was purified to homogeneity using gel filtration and ion exchange chromatography. The enzyme had an apparent Mr = 51,000 +/- 2,000 by sodium dodecyl sulfate electrophoresis, 100,000 +/- 5,000 by discontinuous gel electrophoresis, and 50,000 +/- 2,000 by gel filtration in Bio-Gel A-0.5m agarose. The enzyme cross-linked Factor XIII-free fibrinogen forming gamma dimers and alpha polymers. Either calcium or strontium was necessary for enzyme activity. In the presence of calcium, enzyme activity was increased by heating at 56 degrees or by treating with dimethylsulfoxide. Activation required calcium and occurred in the presence of serine protease inhibitors. The activated and native enzyme had apparently identical mobilities in acrylamide disc electrophoresis and sodium dodecyl sulfate electrophoresis. The Km values for two substrates in the reaction, casein and putrescine, were very similar for the native and the activated enzyme. The activated enzyme had a larger elution volume on Bio-Gel A-0.5m in the presence of calcium than did the native enzyme. The detailed mechanism of activation remains to be determined.     

Preparation and properties of fluorescein-labelled hyaluronate.

Hyaluronate has been labelled with fluorescein groups by two procedures. Products with degrees of substitution ((d.s.) between 0.05 and 0.001 were obtained. Physico-chemical analysis (viscometry, gel chromatography, and measurements of sedimentation and diffusion coefficients) of the parent compound and the products showed that the labelling procedures caused only a limited degradation of the polysaccharide.