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.     

Mating patterns of different Adh genotypes of Drosophila melanogaster I. Differences in mating ability

The mating ability of the different Adh genotypes of D. melanogaster, from three natural populations, was estimated as the number of females inseminated by a single male in a 24-hour period. The data indicate that populations of D. melanogaster vary in mating properties, but a common tendency was found in all of them: the heterozygous individuals, especially the males, show a relative advantage with respect to homozygotes. On the other hand, our results suggest that the differences in mating patterns observed between populations may be correlated with the different degree of Adh polymorphism found in them.     

Cytogenetics of the European plethodontid salamanders of the genus Hydromantes (Amphibia,Urodela)

A karyological analysis was carried out on different European species of the genus Hydromantes (Plethodontidae). All the species examined share the same chromosome number (2n=28) and, with the exception represented by pair XIV, morphologically similar karyotypes. While the karyotypes display a similar distribution — mainly centromeric and pericentric — of C-heterochromatin, quantitative variations in pericentric heterochromatin are observed among species. In the continental species Hydromantes italicus and ambrosii as well as in the eastern Sardinian species imperialis, flavus and specie nova, pair XIV consists of heteromorphic sex chromosomes of the XX/XY type. It is proposed that the differentiation of the Y might have taken place through the occurrence of a structural rearrangement, such as a pericentric inversion, starting from a hypothetical, homomorphic pair XIV. A sex-related heteromorphism is not found in the western Sardinian species H. genei. A further karyological differentiation among these species concerns the position of the nucleolus organizing region (NOR), which is located on chromosome XII (H. italicus and ambrosii) or on chromosome X, close to the centromere (H. genei, H. imperialis and H. specie nova), or in an intercalary position (H. flavus). The location and the number of the 5 S DNA sites have been conserved during species divergence. On the basis of these karyological data, as well as of results obtained through a preliminary restriction enzyme analysis of the ribosomal and genomic DNAs, the phyletic relationships among the European Hydromantes species are discussed.     

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).     

The benzodiazepine receptor ligand, methyl beta-carboline-3-carboxylate, is both sedative and proconvulsant in chicks

Certain pharmacological properties of methyl beta-carboline-3-carboxylate (beta-CCM), a benzodiazepine receptor ligand, have been investigated in chicks. Although beta-CCM has been established previously as an "inverse agonist" of benzodiazepine receptors in rodents, having effects opposite to those of benzodiazepines in a variety of tests, in chicks this compound had a different pharmacological profile. Firstly, in contrast to the overt convulsant action of beta-CCM in other species, beta-CCM (0.05-40 mg/kg) did not produce convulsions by itself in chicks, but it was only proconvulsant. Secondly and most surprisingly, beta-CCM, like diazepam, produced in chicks a sedation which could be blocked by the benzodiazepine receptor antagonist Ro 15-1788. Thus it appears that beta-CCM can function both as an agonist and as an inverse agonist in this animal.     

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).