Use of rat primary lung cells for studying genotoxicity with the sister-chromatid exchange and micronucleus assays

Primary culture of lung cells from CD rats was established for pulmonary genotoxicity studies using two genetic endpoints, sister-chromatid exchange (SCE) and micronucleus formation (MN). In the cell isolation study, a combined enzyme separation of rat lungs with trypsin (1.3 mg/ml) plus collagenase (50 U/ml) gave the highest yield of viable and colony-forming cells. For the MN assay, the cytokinesis block induced by cytochalasin B (CYB) was employed to enumerate MN in binucleated (BN) cells. Treatment of primary lung cells with 2 micrograms CYB/ml for two days appeared to be optimal for scoring micronuclei in CYB-induced BN cells. By this procedure, mitomycin C (MMC), triethylenemelamine, and benzo[a]pyrene caused a dose-related increase in micronucleated BN cells in vitro without metabolic activation. In the SCE assay, maximum second-division metaphases were obtained after cells were incubated with bromodeoxyuridine for 48-54 h. After this incubation time, high frequencies of SCE induced by MMC and 3-methylcholanthrene after in vitro exposure (without S9 activation) or in vivo exposure were observed. The results indicate that rat primary lung cells can metabolize polycyclic aromatic hydrocarbons and that this lung cell system is potentially useful for the detection of pulmonary genotoxicants.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Distribution and regulation of natriuretic factor-R1C receptor subtypes in mammalian cell lines

The differential distribution of natriuretic peptide receptor subtypes and their distinct properties were assessed in mammalian cellular models which were screened for their ability to produce cGMP upon stimulation by different natriuretic peptides. The ANF-R_1A receptor subtype was distinguished by its selective activation by atrial natriuretic factor (ANF) while the ANF-R_1C was characterized by preferential stimulation by C-type natriuretic peptide (CNP). AT-t20 pituitary cells, bovine adrenal chromaffin cells, and NIH-3T3 fibroblasts mainly express the ANF-R_1C receptor subtype. Other cell lines such as PC12, RASM and GH3 express significant but varying amounts of both ANF-R_1A and ANF-R_1C subtypes. A10 and NIH cells which express high density of ANF-R₂ receptor subtype, also demonstrate a higher sensitivity to CNP over ANF suggesting that they express significant amounts of ANF-R_1C. Studies of the regulation by ATP of guanylyl cyclase activity indicate that both ANF-R_1A and ANF-R_1C subtypes are modulated in the same manner. In the presence of Mn²⁺, ATP inhibits the CNP-stimulated guanylyl cyclase activity while in the presence of Mg²⁺ adenine nucleotides potentiate the stimulation by CNP. In addition, we show that like the ANF-R_1A, the ANF-R_1C guanylyl cyclase activity can be regulated by phosphorylation since preincubation with TPA or FKL attenuates the subsequent stimulation by CNP in cultured cells. The results presented demonstrate that specific cell types express distinct natriuretic peptide receptor subtypes and also that the newly characterized ANF-R_1C subtype is regulated by ATP and serine/threonine kinases in the same way as the ANF-R_1A subtype.Abbreviation ANF atrial natriuretic factor - BNP brain natriuretic peptide - CNP C-type natriuretic peptide - ATP adenosine-5-triphosphate - IBMX 3-isobutyl-1-methylxanthine - TPA 12-O-tetradecanoyl-phorbol-13-acetate - FKL forskolin - PKC calcium-phospholipid-dependent protein kinase - PKA cAMP-dependent protein kinase - PKG cGMP-dependent protein kinase - C-ANF [Cys¹¹⁶]-ANF-(102-116)-NH₂ - CC chromaffin cells     

A METHOD OF OBTAINING AXENIC CULTURES OF TRENTEPOHLIA SPP. (CHLOROPHYTA)1

Axenic cultures of Trentepohlia species are necessary for the study of growth and hysiological characters of the algae. We describe the use of a Sherman micromanipulator to isolate filaments from samples of T. aurea and T. odorata collected from their natural habitats. These filaments were then used as inocula for the establishment of axenic cultures. In the case of T. aurea, further treatment with lactic acid was necessary.     

Natriuretic Peptides Inhibit Nicotine-Induced Whole-Cell Currents and Catecholamine Secretion in Bovine Chromaffin Cells: Evidence for the Involvement of the Atrial Natriuretic Factor R2 Receptors

Abstract: There is increasing evidence that members of the natriuretic peptide family display sympathoinhibitory activity, but it remains uncertain which receptor pathway is implicated. We performed cyclic GMP production studies with chromaffin cells treated with either atrial natriuretic factor (ANF) or C-type natriuretic peptide (CNP) and found that these cells specifically express the ANF-R_1C but not the ANF-R_1A receptor subtype. Evidence for the existence of ANF-R₂ receptors was obtained from patch-clamp experiments where C-ANF, an ANF-R₂-specific agonist, inhibited nicotinic currents in single isolated chromaffin cells. Involvement of ANF-R₂ receptors in the modulation of nicotinic currents was further supported by the significant loss of this inhibitory activity after the cleavage of the disulfide-bridged structure of C-ANF. This linearized form of C-ANF also displayed a lower binding affinity for ANF-R₂ receptors. Like the patch-clamp studies, secretion experiments demonstrated that both CNP and C-ANF are equally effective in reducing nicotine-evoked catecholamine secretion by cultured chromaffin cells, raising the possibility that this effect of CNP is predominantly mediated by the ANF-R₂ and not the ANF-R_1C receptors. Finally, this response appears to be specific to nicotinic agonists because neither histamine- nor KCI-induced secretions were affected by natriuretic peptides. In the present study, we report (1) the presence of ANF-R_1C and ANF-R₂ receptor subtypes in bovine chromaffin cells, (2) the inhibition by natriuretic peptides of nicotinic whole-cell currents as well as nicotine-induced catecholamine secretion, (3) the possible mediation of these effects by the ANF-R₂ class of receptors, and (4) the specificity of this inhibition to nicotinic agonists. Because bovine chromaffin cells release ANF, BNP, and CNP together with catecholamines, all three peptides might exert negative feedback regulation of catecholamine secretion in an autocrine manner by interacting with the nondiscriminating ANF-R₂ receptor subtype.     

Protracted release of the LHRH agonist avorelin (MF 6001) from two depot formulations in dogs and men

Avorelin is a new superagonist of naturalluteinizing-hormone-releasing-hormone. Avorelin hasbeen formulated in high molecular weight polylactic glycolic acid to afford protracted andcontinuous release of the peptide from subcutaneousimplants. Two different formulations (10 and 15 mg)were tested first in dogs and then in men during aclinical phase II trial. Chemical castration wasmaintained for at least 6 months in dogs withboth formulations. A similar duration of activity(approximately 6 months) was observed in men.     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

Effects of actinomycin D,cycloheximide and kinetin on ribonuclease and beta-fructofuranosidase in water stressed tomato cotyledons

Three days old excised tomato cotyledons were subjected to mannitol induced water stress in the presence of actinomycin D and cycloheximide. Within a few hours, in the presence of actinomycin D but not cycloheximide, water stress induced increase in ribonuclease activities and decrease in beta-fructofuranosidase activities. The water stress action in the presence of actinomycin D was reversible by addition of kinetin. It was postulated that water stress had some immediate fundamental action on the protein synthesis sites at the ribosomes.