Reactions of the UVRABC excision nuclease with DNA damaged by diamminedichloroplatinum(II).

Mutants of Escherichia coli, which are blocked in excision repair (uvrA6, uvrB5, or uvrC34) are exceptionally sensitive to the antitumor drug cis-Pt(II)(NH3)2Cl2 (cis-DDP) but not the trans isomer. Plasmid DNA, damaged by either the cis or trans compound and treated with the UVRABC excision nuclease was cut as shown by conversion of supercoiled DNA to relaxed forms. All three protein products of the uvrA, uvrB, and uvrC genes were required for incision. End-labeled fragments damaged with cis-DDP and reacted with the UVRABC nuclease were cut at the 8th phosphodiester bond 5' and at the 4th phosphodiester bond 3' to adjacent GG's. DNA treated with trans-DDP was not cut appreciably at adjacent GG's by the repair enzyme as subsequent analysis of reaction products after enzyme digestion gave a pattern similar to those obtained with control untreated fragments. The results indicate that the UVRABC nuclease may promote cell survival by the removal of adjacent GG's which are crosslinked by cis-Pt(II)(NH3)2Cl2.     

Anatomical and physiological characteristics of individual neurones in the central antennal pathway of insects

Signals of tens up to hundreds of thousands of (mostly olfactory) receptor cells on an insect antenna are switched to a comparatively low number of neurones in the antennal lobe of the deutocerebrum in circumscribed units of neuropile, the glomeruli. Each glomerulus is connected via its output neurone to two separate neuropiles (calyces of mushroom body, and lateral lobe) of the protocerebrum. Local interneurones interconnect between the glomeruli. Certain modes of convergence between receptors and central neurones provide for a very high sensitivity of the latter to certain odours and their sensitivity for complex odour stimuli, and in many cases for a marked multimodality. Anatomical and physiological data are given especially for pheromone sensitive neurones and their projections.     

Radiosensitization, pharmacokinetics, and toxicity of a 2-nitroimidazole nucleoside (RA-263)

A 2-nitroimidazole nucleoside, 1-(2',3'-dideoxy-alpha-D-erythro-hex-2'-enopyranosyl)-2-nitroimida zole (RA-263), has been investigated for its radiosensitization, pharmacokinetics, and toxicity properties. The in vitro radiosensitization tests against hypoxic Chinese hamster (V-79) cells demonstrated that RA-263 was a more potent radiosensitizer than misonidazole and at 2 mM concentration approached the oxic curve. Significant in vitro radiosensitization activity was also observed in EMT6 mammary tumor cells. The in vitro cytotoxicity data suggested that RA-263 is considerably more toxic to hypoxic cells than misonidazole. The increased cytotoxicity may be related to its higher depletion of nonprotein thiols (NPSH) than misonidazole. The combined effects of radiosensitization and hypoxic cell toxicity were measured by preincubation of the V-79 cells for 4 h under hypoxic conditions before irradiation. The results demonstrated a synergistic response by causing a significant decrease in the extrapolation number with loss of shoulder of the radiation survival curves. The in vivo radiosensitization experiments conducted by the in vivo-in vitro cloning assay with the EMT6 mammary tumor indicate that RA-263 is an effective sensitizer. Pharmacokinetic data suggested that RA-263 was eliminated from plasma by a rapid alpha phase and a slower beta phase with T 1/2 of 36 and 72 min, respectively. The concentration in the brain was approximately one-sixth of tumor concentration, suggesting that RA-263 is excluded from the CNS. Moreover, RA-263 was two times less toxic than misonidazole on equimolar basis by acute LD50 tests. This agent was also significantly less mutagenic than misonidazole in a strain of Escherichia coli.     

In vitro studies on the subcellular location of glucosidase I and glucosidase II in dog pancreas

When programmed with yeast prepro--factor mRNA, the heterologous reticulocyte/dog pancreas translation system synthesizes two pheromone related polypeptides, a cytosolically located primary translation product (pp--F_cyt, 21 kDa) and a membrane-specific and multiply glycosylated e-factor precursor (pp--F₃, 27.5 kDa). Glycosylation of the membrane specific pp--F₃ species is competitively inhibited by synthetic peptides containing the consensus sequence Asn-Xaa-Thr as indicated by a shift of its molecular mass from 27.5 kDa to about 19.5 kDa (pp--F₀) , whereas the primary translation product pp--Fcyt is not affected. Likewise, only the glycosylated pp--F₃ structure is digested by Endo H yielding a polypeptide with a molecular mass between PP--F₀ and pp--Fcyt. These observations strongly suggest that the primary translation product is proteolytically processed during/on its translocation into the lumen of the microsomal vesicles. We believe that this proteolytic processing is due to the cleavage of a signal sequence from the pp--Fcyt species, although this interpretation contradicts previous data from other groups. The distinct effect exerted by various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-dNM, 1-deoxymannojirimycin) on the electrophoretic mobility of the pp--F₃ polypeptide indicates that its oligosaccharide chains are processed to presumbly Man₉-GlcNAc₂ structures under thein vitro conditions of translation. This oligosaccharide processing is most likely to involve the action of glucosidase I and glucosidase II as follows from the specificity of the glycosidase inhibitors applied and the differences of the molecular mass observed in their presence. In addition, several arguments suggest that both trimming enzymes are located in the lumen of the microsomal vesicles derived from endoplasmic reticulum membranes.Abbreviations dNM 1-deoxynojirimycin - N-Me-dNM N-methyl-dNM - dMM 1-deoxymannojirimycin - CCCP carbonylcyanide m-chlorophenyl hydrazone     

Crystal-associated matrix components in rat incisor enamel

Summary Sections of glutaraldehyde-OsO₄-fixed, plastic-embedded rat incisor enamel were left untreated, stained, decalcifed (1% formic acid in 10% sodium citrate), or decalcified-stained. The presence of apatite crystals was monitored with electron diffraction. After brief decalcification and staining, apatite crystals and matrix components were visualized in the same field. The ghost was continuous with crystal fragments, and the coat appeared as a dense line next to crystals and ghosts. Position of ghosts and crystals at the ameloblast-enamel junction (AEJ) of the secretion zone suggested that there may be a lag of no more than 1/5 min between the elaboration of ghost and crystal. A major change in enamel morphology occurs between the AEJ and the deep enamel of the secretion zone. The ghost becomes thinner, the coat more pronounced, and the crystal enlarges. There is only little change from the deep secretion to the maturation zone enamel.     

CO2 sensitive receptors on labial palps ofRhodogastria moths (Lepidoptera: Arctiidae): physiology,fine structure and central projection

Summary The tips of the labial palps ofRhodogastria possess a pit housing uniform sensilla (Fig. 1), histologically characterized by wall-pores and receptor cells with lamellated outer dendrites (Fig. 2). The receptor cell axons project to glomeruli in the deutocerebrum (cf. Fig. 3) which are not innervated by antennal receptors. From their histology as well as from their central projection these sense organs are identical with palpal pit organs of other Lepidoptera (Lee et al. 1985; Kent et al. 1986; Lee and Altner 1986).Physiologically, the palp-pit receptors respond uniformly; they are most excitable by stimulation with carbon dioxide (Fig. 6) while they exhibit relatively moderate responses to various odorants (Fig. 4). The responses to CO₂ (Fig. 7) show a steep dose-response characteristic. In ambient atmosphere (i.e., ca. 0.03% CO₂) the cells are in an excited condition already; the seeming spontaneous activity exhibited in air is decreased if the preparation is kept under N₂ or O₂ or CO₂-free air (Figs. 7, 10). There is hardly any adaptation of the responses to continuous or repeated stimulation (Fig. 8). Perhaps CO₂ sensitivity is correlated with sensilla characterized by both wall-pores and lamellated dendrites. Pilot tests indicate that CO₂ perception might be widespread in the Lepidoptera (cf. Fig. 12), but the biological significance remains obscure.     

UDP-glucose:digitoxin 16′-O-glucosyltransferase from suspension-cultured Digitalis lanata cells

Suspension cultures from several cell lines of Digitalis lanata, as well as cultures from 6 other plant species were checked for their ability to form purpurea-glycoside A from digitoxin. An in-vitro assay for the UDP-glucose:digitoxin 16-O-glucosyltransferase (DGT, EC 2.4.1.-) has been established based on an HPLC method. The enzyme is located in the soluble fraction. Its pH optimum is at 7.4. No enzyme activity was found in either purified vacuole preparations or lysed vacuoles. Ascorbate (10 mM) increased the transferase activity about 4-fold. Of the sugar nucleotides tested, only UDP-glucose served as a glucosyl donor. Digitoxin, digoxin, -acetyldigitoxin, and -acetyldigoxin are substrates for the glucosyltransferase. The role of the DGT during the biotransformation of cardenolides in Digitalis lanata cell suspension cultures is discussed.Abbreviation DGT UDP-glucose:digitoxin 16-C-glucosyltransferase     

Synthesis and characterization of lipid-linked mannosyl oligosaccharides in Volvox carteri f. nagariensis

Particulate membrane fractions from Volvox carteri catalyze the transfer of mannose from GDP-mannose to dolichyl diphosphate-[¹⁴C]chitobiose to form lipid-linked oligosaccharides up to a dolichyl diphospnate-chitobiose-(mannose)₅ structure. Mannosylation of the chitobiosyl lipid requires divalent cations and detergents as solubilizing agents. Depending on the nature of the detergent, the oligosaccharide pattern differs markedly: With deoxycholate or the zwitterionic detergent 314 a lipid-linked trisaccharide accumulates. The nonionic Triton X-100, however, gives rise to a spectrum of compounds up to a heptasaccharide. Enzyme digestion of the tri- and pentasaccharide structure, obtained after mild acid hydrolysis of the corresponding [¹⁴C]glycolipids, revealed that the first mannose is bound via a β-glycosidic linkage to the chitobiosyl core, whereas the outer mannose residues are linked as α-mannosides. Our studies indicate that, in agreement with recent findings in other organisms, the innermost α-mannosidic residues are donated directly from GDP-mannose. The structure of oligosaccharides synthesized by Volvox membranes is thus consistent with results from other eucaryotic species, suggesting a common pathway of N-glycosylation of glycoproteins.     

The early effect of sublethal X-irradiation of phagocytic cells in mouse blood and the influence of cystamine as measured by chemiluminescence

The metabolic burst accompanying phagocytosis of granulocytes (PMN) leads to the generation of activated oxygen species such as O-2, H2O2, 1O2 and OH; which give rise to chemiluminescence (CL) in the presence of luminol. Reliable CL-measurements of stimulated PMN can be carried out in freshly drawn mouse blood, when photon counts are related to the number of PMN. Effects of low dose total body X-irradiation were studied using C57B1/6 mice. It was found that 24 and 48 hours after irradiation (0.24-0.95 Gy) CL of whole blood was slightly decreased. If however CL-counts were related to the number of PMN, an enhanced CL per single granulocyte was recorded. The administration of cystamine leads to an immune stimulating effect of unirradiated animals. In animals, who received 0.95 Gy a distinct radioprotective effect of cystamine can be observed.