Mutagenicity,sister chromatid exchange inducibility andin vitro cell transforming ability of particulates from Athens air

Airborne particulates were collected over a period of twelve months by the use of Hi-Vol samplers in the basin of Athens, Greece. N-Hexane extracts were tested in a battery ofin vitro tests for their ability to induce mutation in bacteria as well as mutation, sister chromatid exchange and morphological transformation in cultured mammalian cells. Positive results were found for mutagenicity withSalmonella strain TA98 in the Ames assay, for sister chromatid exchange induction in CHO cells and for transformation in BALB/c 3T3 cells in culture. They also showed weak non-doserelated induction of ouabain resistance in BALB/c 3T3 cells. The contribution of oxidizing and nitrating agents found in the Athens atmosphere, together with sunlight UV irradiation in the formation of direct acting mutagens and potential carcinogens from ambient polycyclic aromatic hydrocarbons, is suggested.Abbreviations FCS fetal calf serum - FPG fluorescent-plus-Giemsa technique - oua^R ouabain resistant - PAH polycyclic aromatic hydrocarbon - SCE sister chromatid exchange - TSP total suspended particulate     

An improved purification procedure and properties of casein kinase II from brain

A simple and short purification procedure applicable to casein kinase II has been developed, for fully characterizing the enzyme from calf cerebral cortex cytosol. The procedure consists of four chromatographic steps: DEAE-cellulose, phosphocellulose, phosvitin-Sepharose and ATP-agarose which yields 87% pure casein kinase II. The purified enzyme shows three major bands with apparent molecular masses of 42, 38, and 27 kDa by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and is self-autophosphorylated on its 27 kDa polypeptide. The enzyme shows all the characteristics described for casein kinase II from other sources: it is independent of cyclic nucleotides, calcium/phospholipids, and double-stranded poly(I).poly(C); it can utilize both ATP and GTP as phosphoryl donors and can phosphorylate both casein and phosvitin but not histone. The kinetic studies establish that theK _m for ATP is 12.5 M and 25.1 M when using phosvitin and casein respectively as phosphoryl acceptors. TheK _m for phosvitin is 0.91 mg/ml and for casein 1.43 mg/ml, while theV _max is 315 nmol/min/per mg protein and 479 nmol/min/per mg protein for phosvitin and casein respectively. The activity of the kinase is highly stimulated by KCl or NaCl, and almost completely inhibited by heparin concentrations of 1 g/ml (92%). This inhibition is reduced to only 33% in the presence of optimal KCl concentrations (150 mM). Spermine stimulates enzyme activity, whilst hemin produces a slight inhibition.     

Polystyrene substratum for bulk culture of anchorage dependent cells

Twisted ribbons made of polystyrene were used as a packing material for the cultivation of anchorage dependent cells. Normal human fibroblast cells grown on this support in a laboratory scale reactor reached densities of about 5–7×10⁵ cells/ml. The cells adhered strongly to the carrier and no cell detachment was observed upon transfer to serum free medium. The properties of this packing material and its potential use are discussed.     

The phylogenic expression of plasmolipin in the vertebrate nervous system

Plasmolipin is a plasma membrane proteolipid is a major myelin membrane component (Cochary et al., 1990). In this study we report the phylogenic expression of plasmolipin in the vertebrate nervous system. Using Western blot analysis with polyclonal antibodies, we have analyzed membrane fractions, including myelin, from elasmobranchs, teleosts, amphibians, reptiles, birds and mammals. On the basis of immune detection, plasmolipin appears to be restricted to the mammalian nervous system. Comparison of the central and peripheral nervous systems of mammals showed only minor differences in the level of plasmolipin in these two regions. Within mammals, little quantitative differences were observed when rat, human and bovine membrane fractions were compared. The late evolutionary expression of plasmolipin which results in its restriction to mammals makes it unique among the (major) myelin proteins. The potential physiologic significance of these data are discussed.Abbreviations EDTA Ethylene diamine N.,NN tetracetic acid - EGTA Ethylene glycol bis-(B-Aminoethyl Ether) N,,NN tetracetic acid - MES ([N-Morpholino] ethane sulfonic acid) DCCD, N, Dicyclohexyl carbodiimide     

Quantitative gap junction alterations in mammalian heart cells quickly frozen or chemically fixed after electrical uncoupling

Summary The gap junction morphology was quantified in freeze-fracture replicas prepared from rat auricles that had been either quickly frozen at 6 K or chemically fixed by glutaraldehyde, in a state of normal cell-to-cell conduction or in a state of electrical uncoupling. The general appearance of the gap junctions was similar after both preparative procedures. A quantitative analysis of three gap junctional dimensions provided the following measurements in the quickly frozen conducting auricles (mean±sd): (a) P-face particles' diameter 8.27±0.74 nm (n =5709), (b) P-face particles' center-to-center distance 10.78±2.12 nm (n=4800), and (c) E-face pits' distance 9.99±2.19 nm (n=1600). Corresponding values obtained from chemically fixed tissues were decreased by about 3% for the particle's diameter and about 5% for the particles' and pits' distances. Electrical uncoupling by the action of either 1 mM 2–4-dinitrophenol (DNP), or 3.5 mMn-Heptan-1-ol (heptanol), induced a decrease of the particle's diameter, which amounted to –0.69±0.01 nm (mean ±se) in the quickly frozen preparations and –0.71±0.01 nm in the chemically fixed ones. The particles' distance was decreased by –0.96±0.04 nm in the quickly frozen samples and by –0.90 ±0.03 nm in the chemically fixed ones and the E-face pits' distance was similarly reduced. All differences were statistically significant (P<0.001 for all dimensions). Electrical recoupling after the heptanol effect promoted a return of these gap junctional dimensions towards normal values, which was about 50% complete within 20 min. It is concluded that very similar morphological alterations of the gap junctional structure are induced in the mammalian heart by different treatments promoting electrical uncoupling and that these conformational changes appear independently of the preparative procedure. The suggestion that the observed decrease of the particles' diameter is genuinely related to the closing mechanism of the unit cell-to-cell channel set in thei centers is thus confirmed.     

The kinetics of the first wave of spermatogenesis in the newborn male mouse

A combination of autoradiography and air-dried techniques was used to calculate the duration of the major meiotic stages in the first wave of spermatogenesis in the newborn mouse. The data indicated that the entry into meiosis occurred asynchronously over 2 days, and the time required for each stage and the total cycle was constant. These time intervals were nearly identical with those estimated for adult animals in the present study and by other authors.     

Effects of the calcium channel blocker diltiazem on the excitability of mouse oocytes

Effects of the organic Ca antagonist diltiazem on Ca channels were studied in ovulated and unfertilized oocytes of the mouse by using intracellular recording techniques. The resting potential was not affected by diltiazem. The threshold level of the Ca action potential shifted slightly toward positive voltages with diltiazem concentration, but the shift was not statistically significant. The overshoot and maximum rate of rise of the Ca action potential were inhibited by the drug in a dose-dependent manner, but higher amounts of diltiazem were necessary to cause similar blocking effects on Ca channels in mouse oocytes than in other differentiated cells. Increases of external concentration of Ca²⁺ antagonized the degree of diltiazem inhibition. However, the sequence of block of Ca²⁺, Sr²⁺, and Ba²⁺ currents was different for diltiazem vs Cd²⁺. It is suggested that diltiazem inhibition can not be explained by simple competitive scheme, ie, antagonism between diltiazem and permeant cations does not occur at the same binding site associated with the Ca channel in mouse oocytes.     

Effects of incubation in an atmosphere of 20% CO2 in air on the syrian hamster embryo clonal transformation assay

Summary An atmosphere containing 10% CO₂ has been generally accepted as optimal for the growth of Syrian hamster embryo cells in a clonal transformation assay. Data presented in this paper show that 10% CO₂ may not be the optimum environment for this assay. Using 10 or 20% (analytically measured) CO₂ in air (1 atm pressure), hamster embryo cell pools were examined for clonal growth characteristics and transformability using five known carcinogens and a single noncarcinogenic compound. At 10% CO₂, only 2 of 11 pools weee transformed by the five carcinogens but not by the noncarcinogen. At 20% CO₂, six of seven pools were transformed by the five carcinogens and not by the noncarcinogen. Further, the transformation frequencies were found to be greater in cultures incubated in an atmosphere consisting of 20% CO₂ in air. The data also show that 20% CO₂ increased the cloning efficiency of these cells. A comparison of the 10 and 20% CO₂ data to results reported from other conflicting interlaboratory results with this assay system may be due, in part, to variations of CO₂ concentrations. In some instances, the CO₂ levels indicated by incubator flow meters vary considerably from analytically determined CO₂ values. To prevent these CO₂ discrepancies and their resultant effects on transformation and cloning efficiency, methods for monitoring the CO₂ environment other than flow meters are recommended. The observation of increased cloning efficiencies and transformation rates strongly suggests that culture incubation at 20% CO₂ is a preferred environment for the conduct of this assay.