Quantitative study of natural antioxidant systems for cellular nitrofurantoin toxicity

The toxicity of nitrofurantoin was studied on human WI-38 fibroblasts: this chemical was lethal when added at concentrations higher than 5·10⁻⁵ M in the culture medium. The protection afforded by anitoxidants was then tested: α-tocopherol gave at 10⁻⁴ M a light protection in contrast to ascorbic acid which even became toxic at high concentrations. We also tested catalase, superoxide dismutase and glutathione peroxidase introduced intracellularly by the microinjection technique. On a molecular basis, glutathione peroxidase was 23-times more efficient than catalase and 3000-times more than superoxide dismutase. The results also showed that a similar range of enzyme concentrations was found for the protection against high oxygen pressure. This suggests that, in the case of both oxygen and nitrofurantoin toxicity, the peroxide derivatives are the most toxic intermediates of the free radical attacks.     

Cellular and molecular markers of anteroposterior and dorsoventral organisation in the vitellogenic follicles of adult Sarcophaga bullata (Diptera) and dorsoventral orientation of follicles in the ovary

Summary Polar organisation in the follicles of adult Sarcophaga bullata is reflected in the nurse cell-oocyte axis and in the orientation of the two polar cell pairs in the follicular epithelium. The internal organisation of the nurse cell chamber contributes to polarity but not to dorsoventral asymmetry. Dorsoventral asymmetry is correlated with the eccentric position of the germinal vesicle and the orientation of the polar cell pairs; no other follicle cell specialisations are seen. In an ovary, follicles are preferentially orientated with the dorsal side to the centre of the ovary. Cytoskeletal and some haemolymph proteins are molecular markers of polarity. Thus, in pre-vitellogenic stages, tubulin immunoreactivity is higher in the oocyte than in the nurse cells, actin immunoreactivity is the same over the cystocytes and larval serum proteins are restricted to the poles. During vitellogenesis, both actin and tubulin become more concentrated in the nurse cells and larval serum protein 1 accumulated in the polar cells during border cell migration when yolk polypeptides also accumulate in the oocyte. At the end of vitellogenesis a lipophorin is taken up by the oocyte. No molecular marker of dorsoventral asymmetry was identified.     

Effect of uncoupling treatments on FSH-induced hyperpolarization of immature rat Sertoli cells from Sertoli cell-enriched cultures

Sertoli cell-enriched cultures isolated from immature rat testes by enzymic treatments were investigated by intracellular microelectrode recordings. The hyperpolarization of cells induced by FSH was independent of the age of the rats (7-37 days) and was unchanged by exposure to a hormone-free medium or to a glycine buffer of pH 3. It was reduced by treatments which decreased the electrical coupling between cells either by an increase of intracellular calcium [i.e. calcium ionophore (A 23187, 5 x 10(-6) M), general anaesthetic (heptanol, 3.5 mM) and uncoupler of oxidative phosphorylations (carbonylcyanide m-chlorophenylhydrazone-CCmP, 10(-6) M)] or by a decrease of extracellular calcium [i.e. 0Ca + EGTA (1 mM) medium]. These effects were partly or totally reversed by a recovery period in a drug-free medium. Similar results were obtained by an exposure to trypsin (0.05%) followed by a second mechanical dispersion, but new cell hyperpolarization was induced by a new exposure to FSH. This electrophysiological study suggests an initial effect of FSH on the junctional complex between Sertoli cells, then the control by calcium of this complex.     

Influence of the Molecular Structure of Steroids on Their Ability to Interrupt Gap Junctional Communication

17β-estradiol propionate was found to reduce the gap junctional communication in a concentration range similar to that of testosterone propionate, in primary cultures of rat Sertoli cells and cardiac myocytes. Uncoupling was reversible on washing out and occurred without concomitant rise in the intracellular calcium concentration. Esterification was a prerequisite for the activity of extracellularly applied steroid compounds (for example, testosterone was ineffective even at external concentrations up to 100 μm, whereas its intracellular application at 1 μm totally interrupted intercellular communication), but their uncoupling efficiency did not depend on the nature of the ester chain nor on its position on the steroid nucleus. The derivatives of two other androgen hormones (derivatives of the androstane nucleus) were also efficient as junctional uncouplers. Among five steroid molecules belonging to the pregnane family, only one (pregnanediol diacetate) interrupted the junctional communication. Neither cholic acid nor cholesteryl acetate or ouabain showed this effect. Altogether, no correlation with the presence or position of double bonds nor with the trans- or cis-fusion of the A and B rings could be recognized. These results suggest that this reversible, nondeleterious uncoupling effect of steroids is independent of the shape of the molecules and is more probably related to their size and liposolubility, that condition their insertion into the lipid bilayer. Their incorporation into the membrane could disturb the activity of the membrane proteins by a physical mechanism. Received: 10 April 1995/Revised: 27 October 1995     

Germline genomic structure of the B10.A mouseTcra-V2 gene subfamily

 The number of mouse Tcra-V gene segments varies from one individual to another and is estimated to be about 100. Southern blot analysis revealed that most of the Tcra-V are organized in clusters composed of copies of Tcra-V belonging to different subfamilies. We analyzed in detail a Tcra-V subfamily and looked for new Tcra-V in order to improve the knowledge of the mouse Tcra locus organization. A series of genomic clones derived from the B10.A mouse strain enclosing these clusters was used to determined the structure of all the Tcra-V2. We were able to identify ten Tcra-V2. This study showed that the Tcra-V2 can be organized into three structural subgroups. The distribution of the genes along the Tcra locus, plus their structural organization, indicates that successive duplications occurred during the processes of expansion and contraction of the Tcra-V gene subfamilies. Several Tcra-V2 are also identical, indicating recent duplications. The most divergent Tcra-V2 differ by 7.4% nucleotides, leading to 5.2% differences in amino acid contents. Received: 8 August 1995 / Revised: 24 April 1996     

Cytoskeleton modifications induced by phenobarbital, 2-acetylaminofluorene and 4-acetylaminofluorene in normal and initiated/selected hepatocytes: Relation with the “resistant” phenotype

Initiatedlselected (ISH) and normal (NH) rat hepatocytes were used to study cytoskeleton modifications induced by three liver acting chemicals: 2-AAF, a liver complete carcinogen; PB, a liver tumor promoter; and 4-AAF, a noncarcinogen analogue of 2-AAF. Cytoskeleton alterations were visualized by disappearance of F-actin fibers and tubulin depolymerization. The three drugs induced actin fragmentation in normal hepatocytes; a net loss of actin protein was observed with PB. They also induced varied tubulin depolymerization. The principal difference between chemicals is that 2-AAF led to non-reversible effects, in comparison with PB and 4-AAF which induced reversible damages on cytoskeleton. By contrast to normal hepatocytes, the cytoskeleton of ISH obtained from rats subjected to the resistant hepatocyte protocol was much less susceptible to the effect of the three chemicals. Moreover, we observed a lack of LDH release in the culture medium and a very rapid inducibility of GST activity after exposure of ISH to drugs. The moderate effect of the three chemicals on actin and tubdin in ISH could thus be explained by the resistant metabolic profile of these cells.Abbreviations TPA 12-O-tetradecanoyl-phorbol-13-acetate - PB phenobarbital - 2-AAF 2-acetylaminofluorene - 4-AAF 4-acetylaminofluorene - GSH reduced glutathione - GST glutathione-S-transferase - LDH lactatedehydrogenase - NH normal hepatocytes - ISH initiated/selected hepatocytes - BSA bovine serum albumin     

Protease-nexin: A cellular component that links thrombin and plasminogen activator and mediates their binding to cells

This report identifies a component of normal human fibroblasts that forms a covalent linkage with thrombin and urokinase (urinary plasminogen activator) and mediates most of the specific cellular binding of these proteases. This component, here named protease-nexin (PN), is both associated with the cell surface and released into the culture medium. In several ways PN resembles antithrombin III (AT3), a prominent inhibitor of thrombin in serum: PN links thrombin, probably via an ester bond; PN does not link thrombin blocked at its catalytic site serine; PN has a high-affinity heparin-binding site; and heparin greatly accelerates the rate of linkage between soluble PN and thrombin. Despite these similarities, PN and AT3 are distinct; they differ in size and are not immunologically cross-reactive. Whereas AT3 regulates the proteolytic activity of thrombin in serum, PN may regulate the activity of serine proteases at and near the cell surface.     

Electrogenic pump activity in red beet: Its relation to ATP levels and to cation influx

Summary In storage tissue ofBeta vulgaris L., carbonyl cyanidem-chlorophenylhydrazone or cyanide+salicylhydroxamic acid reduce cell electropotentials from about –200 to below –100 mV. The relationship between potential and cellular ATP level is examined during treatment with different concentrations of inhibitiors. At low ATP levels the potential rises sharply with increases in ATP, but above an ATP level of approximately 50% of the uninhibited level the potential changes very little with ATP concentration. A plot of membrane potentialvs.⁸⁶Pb⁺ influx or of potentialvs. net K⁺ uptake indicates that as the level of inhibition is decreased, the potential tends to reach a limit while cation influx and net uptake continue to increase. Resistance measurements, although subject to difficulties of interpretation, indicate no change in conductance with potential, ion flux, or ATP level. Thus the membrane potential should directly reflect electrogenic pump activity, attributed to active uncoupled H⁺ efflux. K⁺ uptake can occur against its electrochemical gradient and is attributed to a coupled K⁺ influx/H⁺ efflux pump. The results show that the electrogenic pump activity is independent of the K⁺/H⁺ exchange rate. Thus electrogenic H⁺ efflux and K⁺/H⁺ exchange may represent different transport systems, or different modes of operation of a single pump with variable stoichiometry.     

Improved Isolation of SlaA and SlaB S-layer proteins in Sulfolobus acidocaldarius

The Sulfolobus acidocaldarius S-layer is composed of two main proteins: SlaA, which forms the ordered structure of the S-layer matrix, and SlaB, which supports and anchors the S-layer into the tetraether lipid membrane. While SlaA has previously been purified by exploiting its thermotolerance and high resistance to detergents, SlaB has resisted isolation, particularly from the cell membrane. Removal of proteins other than those of the S-layer is especially difficult if large batch-scale culture volumes are unavailable. Here, we describe a benchtop-scale protocol for the purification of SlaA from S. acidocaldarius, enabling isolation of SlaB using size exclusion chromatography (gel filtration). Using this protocol, we were able to identify for the first time tetraether lipids strongly attached to SlaB via heat- and detergent-resistant interactions.