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PubMed1999

Radioactive fingerprinting of microorganisms that oxidize atmospheric methane in different soils.

Microorganisms that oxidize atmospheric methane in soils were characterized by radioactive labelling with (14)CH(4) followed by analysis of radiolabelled phospholipid ester-linked fatty acids ((14)C-PLFAs). The radioactive fingerprinting technique was used to compare active methanotrophs in soil samples from Greenland, Denmark, the United States, and Brazil. The (14)C-PLFA fingerprints indicated that closely related methanotrophic bacteria were responsible for the oxidation of atmospheric methane in the soils. Significant amounts of labelled PLFAs produced by the unknown soil methanotrophs coeluted with a group of fatty acids that included i17:0, a17:0, and 17:1omega8c (up to 9.0% of the total (14)C-PLFAs). These PLFAs are not known to be significant constituents of methanotrophic bacteria. The major PLFAs of the soil methanotrophs (73.5 to 89.0% of the total PLFAs) coeluted with 18:1 and 18:0 fatty acids (e.g., 18:1omega9, 18:1omega7, and 18:0). The (14)C-PLFAs fingerprints of the soil methanotrophs that oxidized atmospheric methane did not change after long-term methane enrichment at 170 ppm CH(4). The (14)C-PLFA fingerprints of the soil methanotrophs were different from the PLFA profiles of type I and type II methanotrophic bacteria described previously. Some similarity at the PLFA level was observed between the unknown soil methanotrophs and the PLFA phenotype of the type II methanotrophs. Methanotrophs in Arctic, temperate, and tropical regions assimilated between 20 and 54% of the atmospheric methane that was metabolized. The lowest relative assimilation (percent) was observed for methanotrophs in agricultural soil, whereas the highest assimilation was observed for methanotrophs in rain forest soil. The results suggest that methanotrophs with relatively high carbon conversion efficiencies and very similar PLFA compositions dominate atmospheric methane metabolism in different soils. The characteristics of the methane metabolism and the (14)C-PLFA fingerprints excluded any significant role of autotrophic ammonia oxidizers in the metabolism of atmospheric methane.

Carbon Radioisotopes↗
PubMed1973

Method for Simultaneous Measurement of Radioactive and Inactive CO(2) Evolved from Soil Samples During Incubation with Labeled Substrates.

An apparatus is described which allows the simultaneous, continuous, and highly sensitive analysis of inactive and radioactive CO(2) evolved from C-supplemented soils or other materials. The apparatus consists of a control unit, a commercially available conductometric CO(2) analyzer, and fraction collector. A number of model experiments were conducted to demonstrate the potentials of the apparatus. These included analysis of the time course of priming action, when C-glucose was added to soil, separation of CO(2) respiration peaks caused by simultaneous degradation of radioactive and inactive soil supplements, and study of the effects of a fungicide, Benomyl, on degradation of C-labeled glucose. In the last experiment, partial degradation of the fungicide could also be followed.

Journal Article↗
PubMed1979

In vitro adherence of radioactively labeled Escherichia coli in normal and cystitis-prone females.

Numerous investigators report data obtained using an in vitro quantitative assay for measuring bacterial adherence to epithelial cells. We found this assay to contain significant sources of error in the large variation in number of bacteria bound per cell and in the dependence on the investigator's visual counting of bacteria bound per cell. In the modified assay described here, we eliminated the need for visual counting of bacteria by incorporating the use of radioactively labeled Escherichia coli. This allowed quantitation of bacterial adherence to as many as 50,000 vaginal cells, whereas the visual counting system limits the determination to perhaps 50 cells. We feel that the use of radioactively labeled bacteria in place of the visual counting system increases the validity and sensitivity of this assay. Using the modified method, we found no statistically significant differences among values for adherence of E. coli type 04 to the vaginal cells of control and cystitis-prone women at either pH 6.4 or 4.0.

Adhesiveness↗
PubMed1990

Relationship between the acceptor/donor radioactivity ratio and cross-linking in bacterial peptidoglycan: application to surface synthesis during the division cycle.

The relationship between the experimental measurement of the cross-linking of bacterial peptidoglycan and the mode of its insertion is analyzed. The cross-linking value, in practice and in theory, is independent of the pattern of strand insertion. Since the measure of the mode or pattern of insertion is the acceptor/donor radioactivity ratio (ADRR), no correction need be made for the ADRR. The measurement of cross-linking using radioactivity is independent of the labeling time, the specific activity of the label, and the mode of strand insertion. It is not concluded, however, that cross-linking does increase during the division cycle.

Bacteria↗
PubMed1985

Detection of adenovirus in nasopharyngeal specimens by radioactive and nonradioactive DNA probes.

The presence of adenovirus DNA in clinical specimens was analyzed by nucleic acid hybridization assays by both radioactive and enzymatic detection systems. The sensitivity of the hybridization tests was in the range of 10 to 100 pg of homologous adenovirus DNA. Minimal background was noticed with unrelated viral and nonviral DNA. Twenty-four nasopharyngeal mucus aspirate specimens, collected from children with acute respiratory infection, were assayed in the hybridization tests and also by an enzyme immunoassay for adenovirus hexon antigen which was used as a reference test. Sixteen specimens positive by the enzyme immunoassay also were positive in the two nucleic acid hybridization tests, and the remaining eight specimens were negative in all of the tests. The results indicate that nucleid acid hybridization tests with both radioactive and nonradioactive probes can be used for diagnosis of microbial infections.

Adenoviruses, Human↗
PubMed1975

Effect of intra-articular inj-ction of radioactive colloids of erbium and yttrium on the growth of rabbit legs.

The effect of the intra-articular injection of radioactive erbium 169 and yttrium 90 on the growth of the leg in rabbits has been studied. I colloid state these isotopes are used clinically for synovial ablation. These beta emitters slow down bone growth in proportion to the amount of radioactivity injected. If the joint has previously been damaged by an inflammatory arthritis the effect of the radiation on the bone growth is reduced.

Animals↗
PubMed1984

Routes of clearance of radioactive water from the rabbit vitreous.

The movement of water to and from the vitreous across the retina and pigment epithelium is important in relation to an understanding of such conditions as retinal detachment and its surgical cure, central serous retinopathy, and retinal oedema. Experiments were carried out to determine the main routes for removal of water injected into the vitreous and to see if the removal could be explained on the basis of diffusion or whether bulk flow was also implicated. 25 microCi of 3H2O in 25 microliter were injected into a central position in the vitreous humour of living rabbits under general anaesthesia. For 9 animals blood was collected from one of the 4 vortex veins draining the choroid and the radioactivity in the samples measured. For another 6 rabbits similarly injected the radioactivity in samples of aqueous humour was determined. The percentage of injectate recovered from the vortex vein blood ranged between 13% and 38%, mean 25 +/- 3%. The percentage of injectate recovered from the aqueous humour ranged between 1.2% and 5.2%, mean 2.8 +/- 0.6%. Analysis of the time course of isotope activity in the samples revealed a mean transit time from the mid vitreous to the choroid of 32 +/- 2 minutes, and from the mid vitreous to the anterior chamber of 84 +/- 3 minutes. By means of a computer model it was calculated that diffusion alone could effect this transfer; if active transport were involved in the transport of 3H2O to the choroid, this was not a limiting factor under the conditions of the experiment.

Animals↗
PubMed1965

QUANTITATIVE SEPARATION OF RADIOACTIVE STEROLS AND BILE ACIDS IN HUMAN FAECES.

A method is described for the quantitative separation of radioactive bile acids and neutral sterols in human faeces on a potassium hydroxide-treated silicic acid column. Results obtained with faecal extracts containing metabolites derived from radioactive cholesterol, cholic acid, and chenodeoxycholic acid show that the efficiency of extraction is at least 96% and that mutual contamination between the bile-acid and sterol fractions is between 0.7 and 3.8%. The method gives better results than one based on separation of the faecal steroids into saponifiable and unsaponifiable material.

Bile Acids and Salts↗
PubMed1968

Method for the determination of unsaturated iron-binding capacity of serum using radioactive iron and magnesium carbonate.

A method for measuring the unsaturated iron-binding capacity of serum using radioactive iron with magnesium carbonate as the adsorbing substance is described. The radioactive iron-magnesium carbonate method, compared with the methods of Caraway (1963), Herbert, Gottlieb, Lau, Fisher, Grevirtz, and Wasserman (1966), and Bothwell, Jacobs, and Kamener (1959), was shorter and simpler and equally reproducible.

Adsorption↗
PubMed1969

Semi-automated micromethod for estimating the unsaturated iron-binding capacity of serum using radioactive iron.

A semi-automated micromethod for the determination of the unsaturated iron-binding capacity (UIBC) of serum using radioactive iron and magnesium carbonate as an adsorbing substance is presented. The method has the advantage of simplicity and speed. The determination of UIBC is carried out on 0.1 ml of serum in duplicate, using autodiluters, at the rate of up to 200 sera per day. The reproducibility of the presented method is comparable to the reproducibility of other methods for the determination of iron-binding capacity. As the method is a simple one it can easily be introduced into laboratories with facilities for radioactive counting.

Autoanalysis↗
PubMed1979

An improved method for the measurement of red cell survival using non-radioactive chromium.

An improved technique for the measurement of red cell survival is described in which non-radioactive 50Cr is used in patients for whom radioactive labels are not advisable. The 50Cr was measured by neutron activation analysis using a high resolution germanium (lithium) diode gamma-ray spectrometer. In adult controls, the values obtained were in the range accepted as normal for the 51Cr method, and double labelling experiments showed close agreement. The application of this technique to the measurement of red cell survival in pregnant women suspected of having haemolytic anemia is described.

Adolescent↗
PubMed1993

K-ras point mutations in routinely processed tissues: non-radioactive screening by single strand conformational polymorphism analysis.

AIMS: To develop a non-radioactive method to screen routinely fixed, paraffin wax embedded specimens for the occurrence of point mutations; to evaluate the single strand conformational polymorphism (SSCP) analysis technique for the detection of K-ras point mutations as a result of electrophoretic mobility shifts. METHODS: DNA was extracted from archival specimens of colon cancer and from established colon cancer cell lines with known point mutations. A K-ras gene fragment containing codons 12 and 13 of exon 1 was amplified with the polymerase chain reaction (PCR). Denatured DNA fragments were run on 10% polyacrylamide gels under non-denaturing conditions. After electrophoresis DNA was blotted and the single stranded DNA was detected using a digoxigenin labelled ras probe. The nature of the detected point mutations was identified and confirmed by sequencing and hybridisation with oligonucleotides using 32P labelling. RESULTS: Wild type and aberrant alleles were detected caused by mobility shifts after electrophoresis of the PCR products. Commonly occurring mutations in the K-ras gene--in the first two positions of codon 12--could easily be detected in DNA from archival paraffin wax embedded colon cancer tissue. In all the colon tumour samples studied wild type gene alleles were also found, presumably derived from normal cells in the specimen. CONCLUSIONS: The SSCP method permits rapid non-radioactive screening of adenomas or carcinomas for the occurrence of point mutations in the K-ras gene. But if a mutation is detected by an electrophoretic mobility shift, its identification requires confirmation by sequencing or oligonucleotide hybridisation.

Colonic Neoplasms↗
PubMed1994

Rapid detection of medium chain acyl-CoA dehydrogenase gene mutations by non-radioactive, single strand conformation polymorphism minigels.

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is a common inherited metabolic disorder affecting fatty acid beta oxidation. Identification of carriers is important since the disease can be fatal and is readily treatable once diagnosed. Twelve molecular defects have been identified in the MCAD gene; however, a single highly prevalent mutation, A985G, accounts for > 90% of mutant alleles in the white population. In order to facilitate the molecular diagnosis of MCAD deficiency, oligonucleotide primers were designed to amplify the exon regions encompassing the 12 mutations enzymatically, and PCR products were then screened with a single strand conformation polymorphism (SSCP) based method. Minigels were used allowing much faster run times, and silver staining was used after gel electrophoresis to eliminate the need for radioisotopic labelling strategies. Our non-radioactive, minigel SSCP approach showed that normals can be readily distinguished from heterozygotes and homozygotes for all three of the 12 known MCAD mutations which were detected in our sampling of 48 persons. In addition, each band pattern is characteristic for a specific mutation, including those mapping in the same PCR product like A985G and T1124C. When necessary, the molecular defect was confirmed using either restriction enzyme digestion of PCR products or by direct DNA sequence analysis or both. This rapid, non-radioactive approach can become routine for molecular diagnosis of MCAD deficiency and other genetic disorders.

Acyl-CoA Dehydrogenase↗
PubMed1997

Characterisation of CAH alleles with non-radioactive DNA single strand conformation polymorphism analysis of the CYP21 gene.

The major cause of congenital adrenal hyperplasia (CAH), a common recessive genetic disease, is the deficiency of steroid 21-hydroxylase (21OH), a microsomal enzyme encoded by the CYP21 gene. Although several CAH causing mutations have been identified in the CYP21 gene of patients with 21OH deficiency, genotyping of the 21OH locus is quite complex because of the high frequency of gene conversion and the presence of multiple mutations on single CAH alleles. In order to perform the complete characterisation of the CYP21 gene coding region more simply, we developed a highly sensitive, non-radioactive method allowing DNA single strand conformation polymorphism (DNA-SSCP) analysis. This method was applied to the characterisation of all the exons and intron-exon junctions of the CYP21 gene in five patients affected by the simple virilising form and one affected by the salt wasting form. In all samples showing SSCP signals, direct sequence analysis showed the presence of more than one single sequence variant. In particular, four mutations which are already known to cause the disease, 16 polymorphisms, and one newly identified C to T transition at position 849 were detected. A random sequence analysis, performed on 31 out of 81 exons showing a normal SSCP pattern, shows the method to be highly sensitive: no sequence variant was detected, thus confirming the validity of this non-radioactive DNA-SSCP analysis in characterising the CYP21 gene in patients with steroid 21OH deficiency. Notwithstanding the complete characterisation of all exons and exon/intron junctions of the CYP21 gene, no complete genotype/phenotype correlation was found in the panel of patients analysed, thus suggesting that characterisation of CAH alleles must be extended to outside the coding region of the CYP21 gene, most probably into the promoter region.

Adrenal Hyperplasia, Congenital↗
PubMed1993

Distribution of radioactivity from 14C-formaldehyde in pregnant mice and their fetuses.

The distribution of 14C after the administration of 14C-formaldehyde was studied in pregnant mice by a whole body low temperature autoradiographic technique. The concentrations of formaldehyde and its metabolites in maternal and fetal blood and tissues were determined in unsectioned tissues by liquid scintillation spectrophotometry. The binding of 14C from 14C-formaldehyde to cells and DNA in maternal and fetal mouse liver was also measured. Radioactivity of 14C deriving from 14C-formaldehyde was found immediately after injection, and showed strong accumulation and retention three hours after injection. The organs that had high concentrations at all studied survival intervals were maternal liver, intestinal mucosa, bone marrow, kidneys, and salivary glands. Considerable amounts of radioactivity were found in the fetuses at six hours after injection, and the concentrations were almost the same as in the maternal tissues. The elimination of 14C-formaldehyde and metabolites from the placenta and fetus occurred more slowly than from maternal tissue.

Animals↗
PubMed1975

Excretion of radioactive diadzein and equol as monosulfates and disulfates in the urine of the laying hen.

The phytoestrogen, diadzein, was injected intramuscularly as [4-14-C]daidzein into two laying hens. The radioactive materials in the urine for the succeeding 23 (hen 1) or 14 (hen 2) days were fractionated on a DEAE-Sephadex column by a gradient of NaCl and the fractions thus separated were further analyzed by solvent partition, susceptability to enzymic cleavage and thin-layer chromatography. The sic following components were identified and quantitated: [14-C]diadzein, [14-C]equol, [14-C]diadzein monosulfate, [14-C]equol monosulfate, [14-C]diazein disulfate, and [14-C]equol disulfate. The urine from hen 2 yielded also the sulfate of an unidentified conversion product of [14-C]daidzein. Repeared tests for glucuronides of [14-C]daidzein or its conversion products gave negative results, excluding the possibility that any appreciable proportion of the radioactivity in the urine was in the form of beta-glucuronide. It is concluded that the diadzein and the equol excretion in the urine of the laying hen are present for the most part as monosulfates and disulfates.

Animals↗
PubMed1977

Translocation of spin-labelled radioactive lipids from isolated guinea pig liver microsomal to mitochondrial membranes.

Translocation of membrane-bound labelled lipids from guinea pig liver microsomal to mitochondrial membranes was studied. When microsomal membranes containing known amounts and composition of spin-labelled radioactive lipids were incubated with unlabelled mitochondrial membranes, reisolated mitochondria contained spin- and radioactive-lipids in an amount which could not be accounted for by the microsomal contamination of reisolated mitochondria, establishing therefore the translocation of labelled lipids from microsomal to mitochondrial membranes. The effect of addition of crude 105000 X g supernatant on the translocation was studied. The translocation of labelled lipids in aged membranes was also described. The rate of loss of paramagnetism in microsomal and reisolated mitochondrial membranes was measured and found to be different, supporting the conclusion that the translocation of labelled lipids between membranes took place. Data from these studies suggested that the translocatndrial membranes probably occurred. Furthermore, our results suggest that the mechanism by which lipid translocation takes place does not necessarily involve soluble cytosol proteins. In view of experimental data, possible mechanisms for the translocation of lipids between biological membranes were discussed.

Animals↗
PubMed1978

Cytosol protein-independent translocation of isomeric spin-labelled radioactive lipids from isolated guinea pig liver microsomal to mitochondrial membranes.

Intermembranous translocation of membrane-bound radioactive lipids covalently labelled with 5-, 12, and 16-doxyl stearic acid was studied. Guinea pig liver microsomal membranes containing known amounts of isomeric spin-labelled radioactive phosphatidic acid, phosphatidylcholine, and diglycerides were incubated with unlabelled mitochondria; reisolated mitochondria contained around 28-31% of microsomal labelled lipids above the microsomal contamination. The effect of adding crude or 'pH 5.1' 105 000 X g cytosol supernatant on the amount and composition of translocated labelled lipids was studied. While the translocation of labelled phosphatidylcholine was slightly stimulated by the addition of these cytosol supernatants, no significant increase of the amount of translocated labelled phosphatidic acic and diglycerides was observed by this addition. In view of these results, a probable mechanism for the cytosol protein-independent translocation of lipids between biological membranes is proposed.

Animals↗