Application of flow karyotyping in prenatal detection of chromosome aberrations

This paper describes the application of bivariate flow karyotyping to (1) classification of chromosomes isolated from cultures of cells taken by amniocentesis and (2) detection of numerical and structural aberrations. Chromosomes were isolated from primary cultures 2-5 wk after amniocentesis, stained with Hoechst 33258 and chromomycin A3, and analyzed using dual beam flow cytometry. Information about chromosome DNA content and DNA base composition was derived from the locations of the peaks in the flow karyotypes, each peak being produced by one or more chromosome types with similar DNA content and DNA base composition. Information about the relative frequency of each chromosome type was determined on the basis of the relative volume of the peak for that chromosome type. Cytogenetic information determined on the basis of flow karyotypes was compared with that obtained by visual analysis following G-banding. Variability among the peak means and volumes in flow karyotypes was determined from analyses of 50 normal amniocyte cultures. Numerical aberrations involving chromosomes 21, 18, and Y were detected correctly in all of 28 analyses, including eight in a blind study. Structural aberrations involving chromosomes 1, 2, 3, 6, 9-12, 13, 14, 15, 21, and 22 were detected in all of seven cultures in a blind study. Flow karyotypes proved to be insensitive to small, normally occurring chromosome polymorphisms detected by banding analysis. In addition, a few samples were erroneously scored as having numerical aberrations.     

Movements of some indigenous riverine fish in Sri Lanka

The migratory movements of reproductive potamodromus fish established in man-made tropical lakes in the dry zone of Sri Lanka were determined. It was shown that the nine indigenous riverine species do not spawn in these lakes but move into the upstream channels when sexually mature. The pattern of upstream movement was found to be species-specific although most species showed a diurnal periodicity. It is suggested that the development of totally lacustrine forms of riverine species is very unlikely to occur in the man-made lakes of Sri Lanka because of their relatively short duration time.     

Comparison of crude and affinity purified cytosolic epoxide hydrolases from hepatic tissue of control and clofibrate-fed mice

An affinity purification procedure was developed for the cytosolic epoxide hydrolase based upon the selective binding of the enzyme to immobilized methoxycitronellyl thiol. Several elution systems were examined, but the most successful system employed selective elution with a chalcone oxide. This affinity system allowed the purification of the cytosolic epoxide hydrolase activity from livers of both control and clofibrate-fed mice. A variety of biochemical techniques including pH dependence, substrate preference, kinetics, inhibition, amino acid analysis, peptide mapping, Western blotting, analytical isoelectric focusing, and gel permeation chromatography failed to distinguish between the enzymes purified from control and clofibrate-fed animals. The quantitative removal of the cytosolic epoxide hydrolase acting on trans-stilbene oxide from 100,000g supernatants, allowed analysis of remaining activities acting differentially on cis-stilbene oxide and benzo[a]pyrene 4,5-oxide. Such analysis indicated the existence of a novel epoxide hydrolase activity in the cytosol of mouse liver preparations.     

Inhibition of oxidative phosphorylation by Ca or Sr: A competition with Mg for the formation of adenine nucleotide complexes

Intramitochondrial Sr²⁺, similar to Ca²⁺, inhibits oxidative phosphorylation in intact rat-liver mitochondria. Both Ca²⁺ and Sr²⁺ also inhibit the hydrolytic activity of the ATPase in submitochondrial particles. Half-maximal inhibition of ATPase activity was attained at a concentration of 2.5 mM Ca²⁺ or 5.0 mM Sr²⁺ when the concentration of Mg²⁺ in the medium was 1.0 mM. The inhibition of ATPase activity by both cations was strongly decreased by increasing the Mg²⁺ concentration in the reaction medium. In addition, kinetical data and the determination of the concentration of MgATP, the substrate of the ATPase, in the presence of different concentrations of Ca²⁺ or Sr²⁺ strongly indicate that these cations inhibit ATP hydrolysis by competing with Mg²⁺ for the formation of MgATP. On the basis of a good agreement between these results with submitochondrial particles and the results of titrations of oxidative phosphorylation with carboxyatractyloside or oligomycin in mitochondria loaded with Sr²⁺ it can be concluded that intramitochondrial Ca²⁺ or Sr²⁺ inhibits oxidative phosphorylation in intact mitochondria by decreasing the availability of adenine nucleotides to both the ADP/ATP carrier and the ATP synthase.     

Pigment patterns in mutants affecting the biosynthesis of pteridines and xanthommatin in Drosophila melanogaster

Eye-color mutants of Drosophila melanogaster have been analyzed for their pigment content and related metabolites. Xanthommatin and dihydroxanthommatin (pigments causing brown eye color) were measured after selective extraction in acidified butanol. Pteridines (pigments causing red eye color) were quantitated after separation of 28 spots by thin-layer chromatography, most of which are pteridines and a few of which are fluorescent metabolites from the xanthommatin pathway. Pigment patterns have been studied in 45 loci. The pteridine pathway ramifies into two double branches giving rise to isoxanthopterin, drosopterins, and biopterin as final products. The regulatory relationship among the branches and the metabolic blockage of the mutants are discussed. The Hn locus is proposed to regulate pteridine synthesis in a step between pyruvoyltetrahydropterin and dihydropterin. The results also indicate that the synthesis and accumulation of xanthommatin in the eyes might be related to the synthesis of pteridines.Support for this work was provided to J.F. in part by a grant from the Ministerio de Universidades e Investigación (Spain) and to F.J.S. by a grant from the Ministerio de Educación y Ciencia (Spain).     

Primary productivity and related parameters in three different types of inland waters in Sri Lanka

Gross and net primary production together with chlorophyll-a biomass were investigated with respect to depth and diurnal changes in three categories of inland waters (reservoirs, temporary ponds, brackish water lagoons) in Sri Lanka. Ten field sites, in both the dry and wet zones of the island, were investigated. Bimodal productivity profiles were recorded in two of the three reservoirs studied. The diel pattern of net photosynthetic rate varied between sites although peak photosynthetic efficiency occurred at solar noon. Surface photoinhibition was characteristic of the reservoirs and brackish water lagoons but not of the temporary ponds. Mean gross primary production was 3.02 g C m^–2 d^–1 but was higher in the temporary ponds than in the reservoirs. The gross primary production in the brackish water Koggala Lagoon at 0.08 g C m^–2 d^–1 is a record low for tropical lagoons and was 2.5 times less than the two other lagoons investigated. Variability in net primary production between sites was similar to the variation in gross production with a relatively low mean value for tropical inland waters of 0.495 C m^–2 d^–1. Mean maximum photosynthetic rate was 0.30 mg C m^–3 h^–1 but was lower in the reservoirs than in the temporary ponds and lagoons.     

Evidence for the presence of muscarinic acetylcholine receptors in bovine brain coated vesicles

Evidence obtained from quinuclidinylbenzilate binding determinations suggested that muscarinic acetylcholine receptor molecules are present in purified bovine brain coated vesicles. Immunoprecipitates formed from coated vesicles with polyclonal antibodies to clathrin bound on the surface of fixed Staphylococcus aureus cells also showed quinuclidinylbenzilate binding activity. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Muscarinic receptor sites for quinuclidinylbenzilate binding to coated vesicles displayed a Kd of 25 pM and a Bmax of about 191 fmol/mg of protein. Binding competition experiments using atropine, N-methylscopolamine, oxotremorine, and carbamylcholine confirmed the typical muscarinic nature of the binding site. Ranking order of potency for the receptors was: atropine greater than N-methylscopolamine greater than oxotremorine greater than carbachol Analysis of data using a two-site model revealed 13% high-affinity sites for oxotremorine, 66% high-affinity sites for carbachol, and 62% for the antagonist N-methylscopolamine. Heterogeneity of binding affinities for muscarinic drugs detected in the coated vesicles may be related to the presence of coated vesicle subpopulations in brain tissue, (Kohtz, D. S., Kohtz, J. D., Schook, W. J., and Puszkin, S. (1985) J. Cell Biol. 101, 48a; Pfeffer, S. R., and Kelly, R. B. (1985) Cell 40, 949-957).     

Active and passive respiratory mechanics in anesthetized dogs

In six spontaneously breathing anesthetized dogs (pentobarbital sodium, 30 mg/kg) airflow, volume, and tracheal and esophageal pressures were measured. The active and passive mechanical properties of the total respiratory system, lung, and chest wall were calculated. The average passive values of respiratory system, lung, and chest wall elastances amounted to, respectively, 50.1, 32.3, and 17.7 cmH2O X l-1. Resistive pressure-vs.-flow relationships for the relaxed respiratory system, lung, and chest wall were also determined; a linear relationship was found for the former (the total passive intrinsic resistance averaged 4.1 cmH2O X l-1 X s), whereas power functions best described the others: the pulmonary pressure-flow relationship exhibited an upward concavity, which for the chest wall presented an upward convexity. The average active elastance and resistance of the respiratory system were, respectively, 64.0 cmH2O X l-1 and 5.4 cmH2O X l-1 X s. The greater active impedance reflects pressure losses due to force-length and force-velocity properties of the inspiratory muscles and those due to distortion of the respiratory system from its relaxed configuration.     

The biological activity of cyclopropyl analogs of all-trans- and 13-cis-retinoic acid in the rat vaginal smear assay

The biological activity of a series of cyclopropyl analogs of all-trans- and 13-cis-retinoic acid has been evaluated in the vaginal smear assay carried out in vitamin A-deficient rats. These analogs were designed to probe the role of the 13-cis isomer in the actions of the parent all-trans-retinoic acid by blocking the interconversion of these two compounds. Although relatively less active, the potency of some of the cyclopropyl analogs suggests that 13-cis-retinoic acid is a fully active metabolite of all-trans-retinoic acid. Since 13-cis-retinoic acid represents a small percentage of the retinoic acid metabolites, the physiological significance of this activity is still unclear. Possible reasons for the reduced activity of the cyclopropyl analogs, as well as an aromatic analog of retinoic acid, are discussed.     

Effects of hyperthermia on DNA interstrand crosslinking after treatment with BCNU in 9L rat brain tumor cells

The effects of hyperthermia (42 degrees C) on 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-mediated DNA interstrand crosslink formation were investigated in 9L rat brain tumor cells using the technique of alkaline elution. When cells were treated with 60 microM BCNU for 1 hr at 37 degrees C and incubated for 6 hr in drug-free medium at 42 degrees C, there was a 50% increase in crosslinking; and when cells were treated at 42 degrees C and incubated at 37 degrees C, there was a 45% increase in crosslinking compared with the results for cells treated and incubated at 37 degrees C. When cells were treated and incubated at 42 degrees C, there was a 129% increase in DNA crosslinking. The same relative order of results was found for cell survival. These results suggest that hyperthermia can increase DNA interstrand crosslink formation and the consequent cell death through two independent mechanisms: an increase in the amount of initial alkylation because of the increased rate of hydrolysis of BCNU at higher temperatures, and the effect of heat on DNA structure that leads to an increase in the number of crosslinks formed.