Radioisotopic assays of rates of carbon monoxide conversion by anaerobic thermophilic prokaryotes

The rate of CO conversion by a pure culture of a thermophilic CO-oxidizing, H2-producing bacterium Carboxydocella sp. strain 1503 was determined by the radioisotopic method. The overall daily uptake of 14CO by the bacterium was estimated at 38-56 micromol CO per 1 ml of the culture. A radioisotopic method was developed to separate and quantitatively determine the products of anaerobic CO conversion by microbial communities in hot springs. The new method was first tested on the microbial community from a sample obtained from a hot spring in Kamchatka. The potential rate of CO conversion by the anaerobic microbial community was found to be 40.75 nmol CO/cm3 sediment per day. 85% of the utilized 14CO was oxidized to carbon dioxide; 14.5% was incorporated into dissolved organic matter, including 0.2% that went into volatile fatty acids; 0.5% was used for cell bio mass production; and only just over 0.001% was converted to methane.     

Radioisotopic measurement of femtomolar amounts of NAD(P)H in the assay of enzymatic activity at a single cell level

A radioisotopic method for the assay of reduced or oxidized pyridine nucleotides, based on the interconversion of 2-[U-14C]ketoglutarate or 2-keto[3,4-3H]glutarate and labelled L-glutamate in the reaction catalyzed by glutamate dehydrogenase, was applied to the measurement of lactate dehydrogenase activity in rat pancreatic islet homogenates. Using the tritiated tracer, the limit of sensitivity of the procedure for NAD(P)H assay was close to 1.0 fmol/sample, and lactate dehydrogenase activity could be measured in as little as 0.0005 islet/sample i.e., at a single cell level. This radioisotopic procedure, which can be used for the assay of various metabolites and enzymic activities, thus provides a tool for investigating the heterogeneity in metabolic behaviour of individual cells.     

Cytolytic activity of human tonsillar lymphocytes]

The radioisotopic method was applied to the study of cytolytic activity of human tonsil lymphocytes against chick erythrocytes, intact and loaded with streptococcus and staphylococcus antigens. Lymphocytes proved to be much more active in the lysis of target cells treated with microbial antigens than of the intact ones. The degree of erythrocytolysis dispension of the tonsillar cells and their treatment with antiglobulin serum.     

A sensitive urea-silver stain method for detecting trace quantities of separated proteins in polyacrylamide gels

An ultrasensitive method using a urea-silver staining procedure to detect trace quantities of proteins in polyacrylamide gels (PAGE) is described. This technique is sensitive enough to detect picogram quantities of proteins resolved on sodium dodecyl sulfate-polyacrylamide gels. The major advantages of our method are that it provides a clear background and it is more sensitive than other techniques allowing it to substitute for radioisotopic techniques in some cases.     

Hydroxyindole-O-methyltransferase activity assay using high-performance liquid chromatography with fluorometric detection: determination of melatonin enzymatically formed from N-acetylserotonin and S-adenosyl-l-methionine

A reliable, sensitive and rapid assay has been developed for determining the activity of hydroxyindole-O-methyltransferase (HIOMT; S-adenosyl-l-methionine:N-acetylserotonin-O-methyltransferase; EC 2.1.1.4), which catalyzes the final step in the melatonin (N-acetyl-5-methoxytryptamine) biosynthetic pathway. This method is based on the separation and detection of melatonin formed enzymatically from N-acetylserotonin and S-adenosyl-l-methionine, by high-performance liquid chromatography with fluorometric detection. The detection limit for melatonin formed per sample was as low as 150 fmol, indicating that the sensitivity of this assay was comparable to that of a radioisotopic assay. The assay was applied to the determination of HIOMT activity in rat pineal gland. The HIOMT activity obtained in this study was comparable with, or slightly lower than those reported previously using radioisotopic assays.     

The attraction of paleomagnetism

The majority of fossil primates and human archeological sites are recovered from sedimentary rocks, but obtaining radioisotopic dates directly from rocks of this type is generally difficult. Paleomagnetism offers an alternative dating approach. Specific applications of paleomagnetism in the field of anthropology range from dating late Eocene fossil anthropoids to determining human archeological site use and occupation. This single dating technique is not restricted to a specific time range, but can be used across the entire span of primate evolution. When linked with other dating techniques, such as radioisotopic or astrochronologic methods, paleomagnetism offers a high level of temporal resolution in unraveling questions of the past.     

Radioisotopic tracing of carbon monoxide conversion by anaerobic thermophilic prokaryotes

The rate of CO conversion by a pure culture of a thermophilic CO-oxidizing, H₂-producing bacterium Carboxydocella sp. strain 1503 was determined by the radioisotopic method. The overall daily uptake of ¹⁴CO by the bacterium was estimated at 38–56 μmol CO per 1 ml of the culture. A radioisotopic method was developed to separate and quantitatively determine the products of anaerobic CO conversion by microbial communities in hot springs. The new method was first tested on the microbial community from a sample obtained from a hot spring in Kamchatka. The potential rate of CO conversion by the anaerobic microbial community was found to be 40.75 nmol CO/cm³ sediment per day. 85% of the utilized ¹⁴CO was oxidized to carbon dioxide; 14.5% was incorporated into dissolved organic matter, including 0.2% that went into volatile fatty acids; 0.5% was used for cell biomass production; and only just over 0.001% was converted to methane.     

Microbiological and isotopic-geochemical investigations of meromictic lakes in Khakasia in winter

Microbiological and isotopic-geochemical investigations of the brackish meromictic lakes Shira and Shunet were performed in the steppe region of Khakasia in winter. Measurements made with a submersed sensor demonstrated that one-meter ice transmits light in a quantity sufficient for oxygenic and anoxygenic photosynthesis. As in the summer season, in the community of phototrophic bacteria found in Lake Shira, the purple sulfur bacteria Amoebobacter purpureus dominated, whereas, in Lake Shunet, the green sulfur bacteria Pelodictyon luteolum were predominant. Photosynthetic production, measured using the radioisotopic method, was several times lower than that in summer. The rates of sulfate reduction and production and oxidation of methane in the water column and bottom sediments were also lower than those recorded in summer. The process of anaerobic methane oxidation in the sediments was an exception, being more intense in winter than in summer. The data from radioisotopic measurements of the rates of microbial processes correlate well with the results of determination of the isotopic composition of organic and mineral carbon (delta13C) and hydrogen sulfide and sulfate (delta34S) and suggest considerable seasonal variations in the activity of the microbial community in the water bodies investigated.     

Gas chromatographic determination of monosaccharide transport rates across human erythrocyte membranes

The conditions for the determination of rates of monosaccharide transport across human erythrocyte membranes by gas chromatography are presented and compared with measurements undertaken by other techniques. The use of gas chromatography in the measurement of transport rates represents an advantage in some types of transport experiments over light-scattering measurements but is comparable to radioisotope procedures for determining transport. When radioisotopic sugars are not readily available, gas chromatographic measurements may constitute the method of choice.     

THE METABOLISM OF TRITIATED DOPAMINE IN REGIONS OF THE RAT BRAIN IN VIVO—I

Abstract— A simple combination of acid- and base-exchange resin columns enabled a more complete separation of amine, acid and neutral metabolites of catecholamines from individual small brain samples to be made. Fractions containing individual catecholamines or metabolites were obtained in aqueous eluates suitable for fluorimetric or radioisotopic analysis. With consistent intraventricular injection and brain dissection techniques, this separation method enables a study of the metabolism of catecholamines in regions of the rat brain and the effects of drugs on this metabolism.     

Assay of human platelet guanylate cyclase activity by high-performance liquid chromatography with fluorescence derivatization

A selective and sensitive high-performance liquid chromatography (HPLC) method with fluorescence derivatization for the assay of guanylate cyclase (GC) activity is described. GTP and cGMP, which are the substrate and the product of GC, respectively, and other guanine-containing compounds are selectively converted by the reaction with (3,4-dimethoxyphenyl)glyoxal to the fluorescent derivatives. The derivatives were separated by reversed-phase HPLC. The limit of detection at a signal-to-noise ratio of 3 for cGMP was 10 fmol on the column. The sensitivity of this method was less than that of the conventional radioisotopic method, but this method is simple and convenient. Human platelet GC activity was measured, and the effects of some compounds were investigated.     

Sequencing reactions in microtiter plates

We describe a simple and inexpensive method of performing sequencing reactions for 24 single-strand M13 DNA clones in microtiter plates. To simplify elevated temperature incubations during sequencing reactions, two heating blocks were designed to accommodate microtiter plates and fit within common laboratory heating modules. With only slight modification of standard fluorescent and radioisotopic sequencing methods, the sequencing reactions for 24 clones can be done in as little as 40 minutes.     

Silver staining DNA in polyacrylamide gels

This protocol describes a simple silver staining method used to visualize DNA fragments and other organic molecules with unsurpassed detail following traditional polyacrylamide gel electrophoresis (PAGE). Sensitivity rivals radioisotopic methods and DNA in the picogram range can be reliably detected. The described protocol is fast (approximately 1 h) and is implemented using readily available chemicals and materials. To achieve the sensitivity and visual clarity expected, quality reagents and clean handling are important. The updated protocol described here is based on the widely used method of Bassam et al. (1991), but provides improved image contrast and less risk of staining artefacts.     

Diagnosis of deep vein thrombosis using autologous indium-III-labelled platelets.

Forty-eight patients who had undergone surgical reduction of a fractured neck of femur or in whom deep vein thrombosis was suspected clinically were studied by ascending phlebography and imaging after injection of autologous indium-111-labelled platelets to assess the accuracy and value of the radioisotopic technique in diagnosing deep vein thrombosis. Imaging was performed with a wide-field gammacamera linked with data display facilities. Phlebography showed thrombi in 26 out of 54 limbs examined and a thrombus in the inferior vena cava of one patient; imaging the labelled platelets showed the thrombi in 24 of the 26 limbs and the thrombus in the inferior vena cava. The accumulation of indium-111 at sites corresponding to those at which venous thrombi have been shown phlebographically indicates that this radioisotopic technique is a useful addition to methods already available for the detection of deep vein thrombosis.     

Rapid scanning of 32 P-labeled acrylamide gels

We describe a radioisotopic assay for ornithine-δ-transaminase using precursor ornithine-U-¹⁴C. We quantitate the product Δ¹-pyrroline-5-carboxylate-¹⁴C by cation-exchange chromatography. The sensitivity of the method allows for measurement of enzyme activity in extracts prepared from 10⁵ mammalian cells.     

Effect of Some Treatments of Milk on the Exchangeability of Colloidal Calcium in Milk with Soluble Calcium

Effect of heating, freeze-drying, frozen storage and coagulation by chymosin on the exchangeability of colloidal calcium with soluble calcium in milk was investigated by the radioisotopic method previously reported. Heating at 100°C for 30 min caused a slight decrease of the exchangeability. Frozen storage at ?20°C for 120 days resulted in a marked decrease of the exchangeability accompanying a decrease of soluble calcium. Coagulation by chymosin gave no significant effect on the amount of soluble calcium or on the exchangeability of colloidal calcium.     

Concentration of prostaglandins and cyclic adenosine-3',5'-monophosphate in the tissues of rats]

The content of prostaglandines (PG) and cyclic 3',5'-adenosine monphosphate (cAMP) was investigated in rat tissues by the radioisotopic method of competitive binding. Maximum quantities of both PG and cAMP were revealed in the same most actively functioning organs: the brain, incretory glands, small intestine. Fatty tissue showed minimum quantities of these substances. Results indicate a close functional relationship between the PG synthesis and adenylatecyclase activity in the body tissues.     

The role of Ca2+-ATpase and its hydrophobic component in the release of Ca2+ from skeletal muscle sarcoplasmic reticulum

The Ca2+ permeability of proteoliposomes containing Ca2+-ATPase of sarcoplasmic reticulum and its hydrophobic fragment was investigated, using the method of synthetic penetrant ions and the radioisotopic method. The former method was used to determine the diffusional membrane potential formed by Ca2+ concentration gradient. It was demonstrated that Ca2+-ATPase, whose active center is oriented outside, has and asymmetric conductivity, i. e., it facilitates the rapid efflux of Ca2+ from proteoliposomes. This efflux is stimulated by the membrane potential positive inside. The hydrophobic fragment of Ca2+-ATPase forms a Ca2+-channel with a high conductivity for Ca2+. This channel is responsible for the Ca2+ efflux from sarcoplasmic reticulum.     

Analysis of cyclic nucleotide phosphodiesterase by isoelectric focusing coupled to a specific activity stain

The procedure described allowed a convenient analysis of cyclic nucleotide phosphodiesterase. The different phosphodiesterase forms present in a crude cytosolic preparation from rat heart were separated by isoelectric focusing on a polyacrylamide gel plate. Phosphodiesterase activity bands were rendered evident by a specific staining method. They were then characterized by means of their substrate specificity and their sensitivity to selective phosphodiesterase inhibitors. The correspondence between the stain bands and the previously described activity peaks, obtained by a preparative technique and detected by radioisotopic enzyme assay, was also investigated.     

A radioisotopic assay for polyamine oxidase

A new radioisotopic assay for polyamine oxidase with N1-acetylspermine as substrate is presented. A modified method for the chemical synthesis of radioactive N1-acetylspermine, which gave a good yield, is also described. The reaction mixture, containing N1-[14C]acetylspermine and tissue homogenate, was incubated for the enzyme reaction and applied to a minicolumn of Amberlite CG-50. The reaction product 3-[14C]-acetamidopropanal did not adsorb to the column, but passed through it; thus the eluate could be directly subjected to liquid scintillation counting. The blank levels were low and relatively constant even with crude tissue homogenates. The detection limit obtained was 0.05 nmol per tube. This method is simple, highly sensitive, and highly specific.