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.     

Purification of RNA-free plasmid DNA using alkaline extraction followed by Ultrogel A2 column chromatography

A procedure for extracting RNA-free plasmid DNA from bacterial cells is described. The method is simple and rapid enough to obtain pure plasmid DNA in 8 to 10 h after plasmid amplification. The protocol uses the alkaline extraction procedure described by Birnboim and Doly (1979, Nucl. Acid Res. 7, 1513-1523). Plasmid DNA is then separated from high-molecular-weight RNA by ammonium acetate precipitation and from low-molecular-weight RNA contaminants by Ultrogel A2 column chromatography. The plasmid DNA obtained by this inexpensive technique is sufficiently pure to be used for restriction endonuclease analysis, 5'-end labeling, S1 mapping, DNA sequencing, and colony hydridization.     

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     

Molecular characterization of mouse-virulent poliovirus type 1 Mahoney mutants: involvement of residues of polypeptides VP1 and VP2 located on the inner surface of the capsid protein shell.

Most poliovirus (PV) strains, including PV PV-1/Mahoney, are unable to cause paralysis in mice. Determinants for restriction of PV-1/Mahoney in mice have been identified by manipulating PV-1 cDNA and located on the viral capsid protein VP1. These determinants consist of a highly exposed amino acid sequence on the capsid surface corresponding to the B-C loop (M. Murray, J. Bradley, X. Yang, E. Wimmer, E. Moss, and V. Racaniello, Science 241:213-215, 1988; A. Martin, C. Wychowski, T. Couderc, R. Crainic, J. Hogle, and M. Girard, EMBO J. 7:2839-2847, 1988) and of residues belonging to the N-terminal sequence located on the inner surface of the protein shell (E. Moss and V. Racaniello, EMBO J. 10:1067-1074, 1991). Using an in vivo approach, we isolated two mouse-neurovirulent PV-1 mutants in the mouse central nervous system after a single passage of PV-1/Mahoney inoculated by the intracerebral route. Both mutants were subjected to two additional passages in mice, plaque purified, and subsequently characterized. The two cloned mutants, Mah-NK13 and Mah-NL32, retained phenotypic characteristics of the parental PV-1/Mahoney, including epitope map, heat lability, and temperature sensitivity. Mah-NK13 exhibited slightly smaller plaques than did the parental virus. The nucleotide sequences of the mutant genomes were determined, and mutations were identified. Mutations were independently introduced into the parental PV-1/Mahoney genome by single-site mutagenesis. Mutated PV-1/Mahoney viruses were then tested for their neurovirulence in mice. A single amino acid substitution in the capsid proteins VP1 (Thr-22-->Ile) and VP2 (Ser-31-->Thr) identified in the Mah-NK13 and Mah-NL32 genomes, respectively, conferred the mouse-virulent phenotype to the mouse-avirulent PV-1/Mahoney. Ile-22 in VP1 was responsible for the small-plaque phenotype of Mah-NK13. Both mutations arose during the first passage in the mouse central nervous system. We thus identified a new mouse adaptation determinant on capsid protein VP1, and we showed that at least one other capsid protein, VP2, could also express a mouse adaptation determinant. Both determinants are located in the inside of the three-dimensional structure of the viral capsid. They may be involved in the early steps of mouse nerve cell infection subsequent to receptor attachment.     

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     

Recovery of the in vitro reactivity of splenic cells of CBA/N mice toward type 2 thymus-independent antigens

CBA/N mice bearing a chromosome X linked immunological deficiency (Xid) cannot respond to type 2 thymus independent antigens (TI-2). However, when their spleen cells are in vitro simultaneously stimulated by both a TI-2 (Fluorescein conjugated polyacrylamide, Flu-PAA) antigen and a type 1 thymus independent (Trinitrophenyl conjugated Brucella abortus, TNP-BA) antigen, their capacity to respond to the TI-2 antigen is recovered. On the contrary, thymus dependent (TD) Sheep red blood cells (SRBC) antigen did not produce any significant increase of the anti-TI-2 response.     

Potential oncogenic effects of basic fibroblast growth factor requires cooperation between CUG and AUG-initiated forms.

Normal adult bovine aortic endothelial cells were infected with various recombinant retroviruses expressing one, two, or three human basic fibroblast growth factor (bFGF) proteins normally synthesized by an alternative use of translation initiation codons. We show here that the constitutive expression of the AUG-initiated from (18 kDa) leads the transfected cells to form colonies in soft agar. The expression of the high molar weight (HMW) forms (22.5 and 21 kDa) initiated at one of the two CUG initiation codons allows cell immortalization, whereas the tumorigenic potential is reached when the three forms are constitutively expressed. Furthermore, we provide evidence that constitutive expression of (HMW) bFGF forms has a down-regulation effect on bFGF synthesis from the gene naturally active in parental endothelial cells.