Influence of ashing techniques on the analysis of trace elements in biological samples

Dry ashing and wet ashing are two commonly used methods for the preparation of biological materials for trace element analysis by atomic absorption spectrophotometry. In this paper, National Bureau of Standards (NBS) bovine liver was dry ashed at 450°C for 24 h in silica glass (Vycor) or procelain crucibles; the resulting ash was dissolved in either concentrated nitric or hydrochloric acid. Dry ashing efficiency was evaluated by comparing iron, copper, zinc, and manganese concentrations of the samples with the values certified by NBS. Highest recoveries were obtained by dry ashing in silica glass (Vycor) crucibles. Dissolving the resultant ash in either hydrochloric or nitric acids did not significantly alter the results. A comparison between dry and wet ashing shows the latter method to be superior for the preparation of biological tissues for analysis of iron, copper, zinc, and manganese.     

Isolation,characterization and localization of bovine adrenal medullary myosin

Summary Myosin was isolated in high purity from the bovine adrenal medulla by gel filtration and ion exchange chromatography. The purified myosin was analyzed by electrophoresis in gels containing SDS and found to contain a 200,000 molecular weight heavy chain and major light chains of molecular weights 20,000 and 17,000 in a 111 molar ratio. At high ionic strength the myosin had high Ca-ATPase and K-EDTA-ATPase activities and low Mg-ATPase activity. At low ionic strength, the Mg-ATPase was activated to a low level by rabbit muscle actin. The myosin was found to decorate F-actin in the absence, but not the presence of ATP. In low ionic strength solutions, the myosin assembled into characteristic bipolar filaments.The distribution of this myosin in the adrenal medulla and of cross-reacting myosin in several other bovine tissues was determined with the use of antimedullary myosin immunoglobulin G as a specific stain that was detected by direct and indirect immunofluorescence. In the medulla strong staining was seen between the chords of chromaffin cells indicating the presence of a highly muscular vasculature that may perform functions analogous to those of the myoepithelium of exocrine glands. The chromaffin cells showed weak positive staining around the nuclei and in a pattern radiating toward adjacent blood vessels. Cells of the inner zone of the adrenal cortex showed strong staining in the peripheral cytoplasm while cells in the intermediate and outer zones did not stain. In a blood smear, platelets and the cytoplasm of leukocytes stained strongly while erythrocytes did not stain. In striated muscle and the gray and white matter of the cerebrum only the capillaries and larger vessels stained. In the liver the phagocytic cells bordering vascular sinuses stained strongly while the hepatocytes were separated from one another by a 2 micron trilaminar band possibly representing the microfilament web surrounding the bile canaliculi and associated with junctional complexes.The results suggest that myosin is present in several highly differentiated, non-motile tissue cells where it may play a role in secretion or other specialized functions.The author gratefully acknowledges the support and encouragement received from Francis D. Carlson (Johns Hopkins University) and Harvey B. Pollard (National Institutes of Health) in whose laboratories the majority of this work was performed, as well as additional advice and assistance from John Cebra, Richard Cone, William F. Harrington, Shin Lin, Robert Wyllie and the members of their laboratories     

Forest leaf litter decomposition in the vicinity of a zinc smelter

Summary Concentrations of about 26,000 ppm Zn, 10,000 ppm Fe, 2,300 ppm Pb, 900 ppm Cd, 340 ppm Cu, and 0.40% S were measured in the O₂ litter horizon about 1 km from a zinc smelter at Palmerton, Pennsylvania. Samples taken about 6 km east of the smelter had concentrations of about 15,000 ppm Zn, 6,500 ppm Fe, 970 ppm Pb, 250 ppm Cd, 170 ppm Cu, and 0.26% S. Samples from a control area about 40 km east of the smelter had concentrations of 2,800 ppm Fe, 650 ppm Zn, 260 ppm Pb, 50 ppm Cu, 9 ppm Cd, and 0.13% S.Litter bags were used to estimate first-year weight loss in sassafras leaves and a mixture of chestnut oak/red oak leaves in the three sites. At the end of one year, average weight loss for sassafras was 39.3% in the control site, 21.8% at 6 km, and 17.5% at the 1 km site. For the chestnut oak/red oak mixture, average weight loss was 36.8% (40 km), 25.7% (6 km), and 19.1% (1 km). Numbers and diversity of soil microarthropods inhabiting the litter bags showed a corresponding decline at sites near the smelter. Concentrations of Ca, Cd, Cr, Cu, Fe, Mg, Mn, N, Na, Ni, P, Pb, S and Zn in the decomposing litter were also measured.The average amount of organic matter on the forest floor was estimated to be 3.8 kg/m² in the control site, about 3.8 kg/m² at 6 km, and about 8.1 kg/m² 1 km from the smelter. Average thickness of the litter horizons in these three sites was 6.0 cm (40 km), 7.0 cm (6 km), and 12.4 cm (1 km), suggesting a long-term depression of decomposition and mineral cycling near the smelter.     

Microbial Activities in Undecompressed and Decompressed Deep-Seawater Samples

Microbial transformations of ¹⁴C-labeled substrates (sodium glutamate, Casamino Acids, glucose, and sodium acetate) were measured in undecompressed seawater samples collected from depths of 1,800 to 6,000 m, during 14- to 21-day incubation periods at in situ temperature (3°C). Each substrate was tested at two concentrations (ca. 0.5 and 5.0 μg/ml) and two in situ pressures. The data were compared to 1-atmosphere (ca. 1.013 × 10² kPa) controls. The rates of ¹⁴C incorporation and ¹⁴CO₂ production as well as the amounts of total substrate utilization were generally lower at pressure than in the decompressed controls but were significantly different for each of the four substrates used. The utilization of acetate was the least affected by pressure; rates were similar to those measured at 1 atmosphere in two out of four experiments. In contrast, transformation rates of the amino acids at pressure averaged to only 38% of those in the controls. A single but reproducible “barophilic” response was observed with glucose as a substrate in samples collected from a depth of 4,500 m at a specific area in the northwestern Atlantic Ocean. Except for this latter set of experiments, the transformation of all substrates showed an increased lag period at pressure as compared to the 1-atmosphere controls.     

Mating strategy shifts in male green treefrogs (Hyla cinerea): An experimental study

Four field experiments were designed to study calling and satellite mating strategies in the green treefrog, Hyla cinerea. (1) The calling male was removed from 19 satellite associations and 11 of the 19 satellite males began calling. (2) After the calling male was removed from 10 satellite associations, a speaker broadcast synthetic mating calls. All of the satellite males oriented to the speaker. (3) Synthetic mating calls were played back to 14 calling males. Eleven males stopped calling and oriented to the speaker during at least one trial. (4) Sequences of synthetic mating calls and synthetic encounter calls were broadcast to 12 calling males. Nine males became satellites when the speaker emitted mating calls; none did so when encounter calls were presented.     

Human and rat chromosomal localization of two genes for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by analysis of somatic cell hybrids and in situ hybridization

Two genes encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were localized in human and rat chromosomes. PFKFB1 (previously PFRX), which encodes the liver and muscle isozymes, was assigned to Xq22-q31 in the rat and to Xq27–q28 in the human by in situ hybridization using probes generated by the polymerase chain reaction. PFKFB2, which encodes the heart isozyme of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was assigned to chromosome 13 in the rat and to chromosome 1 in the human by hybridization of DNA from somatic cell hybrids. By in situ hybridization, this gene was localized to the regions 13q24–25 in the rat and 1q31 in the human.     

Characterization of murine cell lines from Diethylstilbestrol-Induced uterine endometrial Adenocarcinomas

Neonatal treatment with estrogens is associated with development of uterine adenocarcinomas in CD-1 mice. Treatment with the synthetic estrogen diethylstilbestrol (DES) on Days 1 to 5 after birth results in 90% incidence of these hormonedependent lesions in 18-mo.-old mice. Three cell lines were established from these DES-associated tumors. Each of these cell lines exhibited morphologic and ultrastructural characteristics of transformed epithelial cells, including an increased nuclear:ytoplasmic ratio, enlarged and irregular nuclei with multiple nucleoli and areas of chromatin condensation, positive staining for cytokeratin, desmosomes, and microvilli. After subcutaneous injection into nude mice, all three cell lines formed solid tumors within 4 wk. Although the primary uterine tumors and tumor transplants in nude mice had been shown to be estrogen-dependent and estrogen-receptor positive, neither the monolayer growth nor the tumorigenicity of any of the three cell lines in this study was enhanced by or dependent on estrogen. Estrogen receptor levels were low in early and intermediate passage cells. Allele-specific oligonucleotide hybridization analysis of PCR-amplified cell line DNA revealed no point mutations in the 12th, 13th, or 61st codons of the K-ras or H-ras protooncogenes. Southern analysis revealed no changes in genomic organization of the putative tumor suppressor gene DCC, but demonstrated a three-to four-fold amplification of the c-myc gene in one cell line. Expression of c-myc RNA was concomitantly increased in the same cell line. These three transformed cell lines represent the end point in the process of hormone-associated tumorigenesis and as such should prove useful in investigating the molecular changes and the mechanisms involved in hormonal carcinogenesis.     

Phylogenetic relationships among various helical bacteria

Helical bacteria from the generaSpirillum, Oceanospirillum, Aquaspirillum, andAzospirillum—as well asSerpens flexibilis—were characterized by oligonucleotide cataloging of 16S rRNA in order to establish their phylogenetic relationships to one another and to Gramnegative bacteria in general. The various genera of helical bacteria are not specifically related to one another (to the exclusion of nonhelical bacteria) and, where tested, the individual genera as presently constituted are not phylogenetically coherent (with the possible exception ofOceanospirillum, which may form a deep grouping).