Effect of chloramphenicol on ribonucleic acid synthesis in liver cells in suspension

1. Chloramphenicol has a stimulatory effect on the incorporation of radioactive phosphate into the RNA of perfused rat-liver slices, whole liver homogenates or the liver-cell suspensions, and no effect on the incorporation of [(14)C]adenine and [(14)C]uracil into the RNA of the tissue slices. 2. Chloramphenicol completely inhibits the incorporation of labelled adenine and uracil into the RNA of the cell suspensions, or into the RNA of homogenates derived from the whole liver tissues. 3. Chloramphenicol has at most a slight inhibitory effect on the transport of labelled adenine or uracil in the hepatic cells in suspension; in the slices, the transport of these bases is not inhibited at all. 4. The above observations indicate that: (a) unlike the tissue slices, hepatic cells in suspension are permeable to chloramphenicol; (b) in the presence of chloramphenicol, for reasons that are not clear, the conversion of the base into the appropriate nucleotide does not proceed.     

A novel immunohistochemical technique for demonstration of specific binding of human monoclonal antibodies to human cryostat tissue sections

We describe a novel immunohistochemical technique which permits the detection of specific binding of human monoclonal antibodies (MAb) to cryostat sections of human tissues. The technique overcomes the problem of background staining caused by the presence of endogenous immunoglobulins in tissue sections. This is achieved by the formation of a molecular complex of the primary antibody (a human MAb), horseradish peroxidase-conjugated goat anti-human immunoglobulin, and normal human serum. This complex is then incubated with cryostat sections of human tissue, and binding of the complex is demonstrated using diaminobenzidine/hydrogen peroxide. The method is suitable for immunohistochemical screening of small samples of tissue culture supernatant for the presence of human MAb of potential interest, and for determining the pattern of binding of such MAb to a wide range of normal and pathological human tissues.     

Effect of the essential oil fromAchillea fragrantissima onEscherichia coli cells

Escherichia coli cells treated with the essential oil from the plantAchillea fragrantissima released five polypeptides as well as K⁺ ions into the incubation medium. The oil also inhibited the respiration ofE. coli cells and reduced their ATP content. Electron micrographs showed that oil-treated cells were permeable to uranyl acetate. The effect of the essential oil on the cell membrane is discussed.     

Microbial mats and physicochemistry in a saltern in the Bretagne (France) and in a laboratory scale saltern model

Abstract A saltern near La Baule (Bretagne, France) was remodeled in a programmable temperature and humidity controlled walk-in environmental chamber resembling the characteristics of the original saltern. The saltern showed different types of microbial mats predominantly composed of algae, oxy- and anoxyphotobacteria, and associated chemoorganotrophic bacteria, fungi and animals. Well-developed microbial mats were found up to a salinity of 10% during the three or four months in summer when salinity gradients and NaCl precipitation were established. The main phototrophic organisms were diatoms, the cyanobacteria Aphanothece, Microcoleus, Spirulina , and Oscillatoria , and Chromatiaceae. At higher salinity, Halobacterium sp., diatoms, and Dunaliella were dominant. Typical microbial mats and saltern-typical invertebrate, algal and bacterial species also developed in the saltern model, building up a stable community. The ionic composition of the brines and physicochemical parameters were similar to those determined for the original saltern. Different photosynthetic organisms, e.g. a filamentous purple bacterium and a hypersaline Chloroflexus -like organism, could be enriched within the microbial mats by changing the light regime.     

Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils

Stimulation of human neutrophils with f-met-leu-phe, platelet-activating factor, or leukotriene B4 resulted in an increase in [Ca2+]i. The [Ca2+]i rise was greater in the presence than absence of external Ca2+; the component that was dependent on external Ca2+ was blocked by Ni2+, or could be reconstituted by addition of external Ca2+ following discharge of the internal Ca2+ store. These measurements of [Ca2+]i responses provide only indirect evidence for agonist-stimulated Ca2+ entry, and here we have used an alternative approach to demonstrate directly agonist-stimulated divalent cation entry. In the presence of extracellular Mn2+, f-met-leu-phe, leukotriene B4, and platelet-activating factor stimulate a quench in fluorescence of fura-2-loaded human neutrophils. This quench was due to stimulated Mn2+ influx and was blocked by Ni2+. When Mn2+ was added in the continued presence of agonist, after discharge of the internal store of Ca2+, a stimulated quench was seen; this result shows that an elevated [Ca2+]i is not needed for the stimulation of Mn2+ entry. Depolarization by high [K+] or addition of the L-type Ca2+ channel agonist, BAY-R-5417, had little or no effect on either [Ca2+]i or Mn2+ entry. These results show that agonists stimulate divalent cation entry (Ca2+ or Mn2+) by a mechanism independent of changes in [Ca2+]i and unrelated to voltage-dependent Ca2+ channels.     

The influence of self-MHC and non-MHC antigens on the selection of an antigen-specific T cell receptor repertoire

We have examined the influence of self-Ag on TCR expression and specificity in the immune response to the Ag pigeon cytochrome c. Previous work has shown that most Ek-restricted cytochrome c-specific T cells from B10 background mice express TCR alpha beta-heterodimers encoded by V beta 3 and V alpha 11 genes, but that T cells expressing V beta 3 proteins are eliminated due to self-tolerance in Mls-2a mouse strains. Thus, EK-restricted cytochrome c-specific T cells from Mls-2a mice fail to express any V beta 3. In the current study the influence of self-MHC and non-MHC Ag on TCR usage in the immune response to cytochrome c was further examined. First, it was demonstrated that the absence of V beta 3 expression in Mls-2a mice does not alter Ir gene function. Specifically, Mls-2a/Eb haplotype V beta 3- [C3H.SW x B10.A(5R)]F1 mice were high responders to cytochrome c despite the fact that previous structure function analyses have shown a very close correlation between Eb-restricted cytochrome c recognition and V beta 3 expression. This demonstration of the plasticity of TCR expression suggests that relatively few Ir gene defects result from tolerance induced by self-Ag. We also examined differences in V alpha 11 expression among cytochrome c-specific T cells from various H-2k haplotype mouse strains. In particular, the low level of expression of V alpha 11 in cytochrome c-specific T cells from C57BR (H-2k) mice was shown not to be due to self-tolerance. Rather, evidence for limited strain polymorphism of V alpha 11 genes, plus the fact that cytochrome c-specific T cells from F1 hybrids between H-2k, Mls-2b identical C57BR and B10.BR mice express high levels of V alpha 11, suggested the possibility that the variable V alpha 11 usage in the cytochrome c-specific responses of these two strains reflected differences in positive selection during ontogeny by non-MHC non-Mls self-Ag.     

Solubilization of covalently bound extensin from capsicum cell walls

Acidified sodium chlorite cleaves isodityrosine and solubilizes covalently bound hydroxyproline-rich material from cell walls. This has been taken as evidence that isodityrosine acts as a cross-link holding the hydroxyproline-rich glycoprotein extensin in the cell wall. However, acidified chlorite was found to cleave peptide bonds in salt-soluble extensin and in bovine serum albumin (BSA). This invalidates the use of conventional acidified chlorite treatment to provide evidence for isodityrosine cross-links. The ratio of BSA:chlorite was important in determining peptidyl cleavage. At a ratio of 0.75:1.00 (mole amino acid residues/mole chlorite), or higher, peptidyl cleavage was not detected. Furthermore, in samples where a low concentration of radioactive extensin was present, BSA substantially protected the peptide bonds of the extensin against peptidyl cleavage during treatment with acidified chlorite, while not preventing the cleavage of isodityrosine. Therefore, acidified sodium chlorite plus BSA was a more specific reagent for the cleavage of isodityrosine than was acidified chlorite alone. This modified treatment solubilized in intact form the `covalently bound' extensin from cell walls of Capsicum frutescens (chili pepper) suspension cultures, providing new evidence compatible with the view that extensin molecules are held in the cell wall by isodityrosine cross-links.     

Pattern of expression of ecotropic murine leukemia virus in gonads of inoculated SWR/J mice.

An ecotropic murine leukemia virus (MuLV) isolate has recently been shown to be able to infect the germ line or the early embryo or both when inoculated at birth to SWR/J females (J. J. Panthier, H. Condamine, and F. Jacob, Proc. Natl. Acad. Sci. USA 85:1156-1160, 1988). We have used this isolate to further study this phenomenon. By using the techniques of RNA-RNA in situ hybridization, immunocytochemistry, and transmission electron microscopy, the identities of two important cell types that are infected by ecotropic MuLV in the gonads of inoculated mice were determined. These cells are the thecal cells surrounding the follicles in the ovary and the Leydig cells in the testis. Both types actively synthesize viral RNA and express a viral antigen. Furthermore, we documented the production of viral particles by the thecal cells. The expression of ecotropic MuLV by nonlymphoid cells in vivo may play a key role in the vertical transmission of these viruses by females as well as in their horizontal transmission.     

SK&F 96365, a novel inhibitor of receptor-mediated calcium entry.

A novel inhibitor of receptor-mediated calcium entry (RMCE) is described. SK&F 96365 (1-(beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H- imidazole hydrochloride) is structurally distinct from the known 'calcium antagonists' and shows selectivity in blocking RMCE compared with receptor-mediated internal Ca2+ release. Human platelets, neutrophils and endothelial cells were loaded with the fluorescent Ca2(+)-indicator dyes quin2 or fura-2, in order to measure Ca2+ or Mn2+ entry through RMCE as well as Ca2+ release from internal stores. The IC50 (concn. producing 50% inhibition) for inhibition of RMCE by SK&F 96365 in platelets stimulated with ADP or thrombin was 8.5 microM or 11.7 microM respectively; these concentrations of SK&F 96365 did not affect internal Ca2+ release. Similar effects of SK&F 96365 were observed in suspensions of neutrophils and in single endothelial cells. SK&F 96365 also inhibited agonist-stimulated Mn2+ entry in platelets and neutrophils. The effects of SK&F 96365 were independent of cell type and of agonist, as would be expected for a compound that modulates post-receptor events. Voltage-gated Ca2+ entry in fura-2-loaded GH3 (pituitary) cells and rabbit ear-artery smooth-muscle cells held under voltage-clamp was also inhibited by SK&F 96365; however, the ATP-gated Ca2(+)-permeable channel of rabbit ear-artery smooth-muscle cells was unaffected by SK&F 96365. Thus SK&F 96365 (unlike the 'organic Ca2+ antagonists') shows no selectivity between voltage-gated Ca2+ entry and RMCE, although the lack of effect on ATP-gated channels indicates that it discriminates between different types of RMCE. The effects of SK&F 96365 on functional responses of cells thought to be dependent on Ca2+ entry via RMCE were also studied. Under conditions where platelet aggregation is dependent on stimulated Ca2+ entry via RMCE, the response was blocked by SK&F 96365 with an IC50 of 15.9 microM, which is similar to the IC50 of 8-12 microM observed for inhibition of RMCE. Adhesion and chemotaxis of neutrophils were also inhibited by SK&F 96365. SK&F 96365 is a useful tool to distinguish RMCE from internal Ca2+ release, and to probe the role of RMCE in mediating functional responses of cells. However, SK&F 96365 is not as potent (IC50 around 10 microM) or selective (also inhibits voltage-gated Ca2+ entry) as would be desirable, so caution must be exercised when using this compound.