The effect of CaCl2 and verapamil on the binding of cisplatin to cultures of normal and transformed human fibroblasts

Normal and transformed human fibroblasts were treated for either 1 sec or 1 h with the antitumor drug cis-dichlorodiamine platinum (cisplatin). The dose response of drug binding and cell survival was determined for cells treated with the drug in the presence or absence of 3.0 mM CaCl2. The levels of drug initially bound to both cell types was similar and was not affected by the presence of Ca2+. The dividing non-transformed cells were most sensitive to killing by short treatment with cisplatin compared to the transformed cells or the confluent non-transformed cultures. After 1 h of cisplatin treatment, the levels of drug bound to the cells were significantly less than that recovered after the shorter treatment. This time-dependent loss of cisplatin was inhibited both by CaCl2 and by the calcium channel blocking agent, verapamil. The higher levels of cisplatin bound after 1 h in the presence of these agents, however, did not in all cases result in decreased survival; the effects were dependent on cell type and on whether the cells were dividing or confluent. Analysis of cisplatin binding to cell cultures indicated that initially the cisplatin was weakly attached to the pericellular and substratum attached material but that with time, the drug bound to this material decreased. This time-dependent removal from the extracellular matrix was much less in the transformed cell cultures and was inhibited by calcium. We propose that the major site of interaction of cisplatin with these cells is in the extracellular matrix and with time the cultures alter their extracellular matrix to decrease this binding. This removal process appears to involve calcium or calcium transport since CaCl2 and verapamil both block these changes.     

L-641,953 (R-8-fluoro-dibenzo[b, f]thiepin-3-carboxylic acid-5-oxide): a novel thromboxane-prostaglandin endoperoxide antagonist

The effects of L-641,953 (R-8-fluoro-dibenzo[b, f]thiepin-3-carboxylic acid-5-oxide) have been studied on pulmonary and other smooth muscle preparations in vitro and in vivo. When studied in vitro on guinea-pig tracheal chains, L-641,933 produced significant shifts in the dose-response curves to the prostaglandin endoperoxide analogues, U-44069 (pA2 7.06) and U-46619 (pA2 7.14), and prostaglandin (PG) F2 alpha (pA2 6.33) had minimal activity against contractions induced by histamine (pA2 4.38), 5-hydroxytryptamine (pA2 4.63), and acetylcholine (pA2 4.56) and slightly enhanced relaxation induced by PGE2. When tested on the guinea-pig gall bladder strip in vitro, L-641,953 antagonized contractions induced by U-44069 (pA2 7.03) but was less active against those induced by PGF2 alpha (pA2 6.03), PGE1 (pA2 5.62), and histamine (pA2 4.84). When tested in vitro on the guinea-pig pulmonary artery, L-651-953 significantly antagonized contractions induced by U-44069 (pA2 7.04), U-46619 (pA2 7.14), and PGF2 alpha (pA2 7.16) but was less effective against contractions induced by histamine (pA2 4.19). Schild analysis indicated that L-641,953 was fully competitive against contractions of either the guinea-pig tracheal chain induced by U-46619 or the guinea-pig pulmonary artery induced by U-44069 and U-46619. When tested on human platelets in vitro L-641,953 inhibited aggregation induced by U-44069 (IC50 1.3 X 10(-6) M) but not ADP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Viral and bacterial contamination of mussels (Mytilus edulis) exposed in an unpolluted marine environment

Bacteria indicating faecal contamination, cell-culturable enteroviruses and hepatitis A virus (HAV) were investigated in sea-water and in mussels exposed in an unpolluted marine environment, over a 7-month period with two samplings per month. Of the 16 mussel samples examined, none contained cell-culturable enteroviruses, four showed a low-level contamination by HAV and two did not conform to the current bacteriological norms. No connection was observed between the viral and bacterial contamination. No viral contamination was detected in the sea-water samples, but two gave bacterial counts above current norms.     

T-DNA length variability in mannopine hairy root: more than 50 kilobasepairs of pRi T-DNA can integrate in plant cells

Independent carrot (Daucus carota) hairy root lines were established by inoculation of discs taken from the same carrot with Agrobacterium rhizogenes 8196 and A. tumefaciens C58C1(pRi8196) carrying pRi8196. Several lines were compared with respect to T-DNA length. One of them was found to have integrated sequences covering more than 50 kbp of the Ri plasmid     

Biochemical characterization of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to pTOM5

Transgenic tomato plants expressing antisense RNA to a ripening-related cDNA clone (pTOM5) had yellow ripening fruit and pale coloured flowers. Carotenoid levels in fruit of these plants were reduced by up to 97%. In order to determine the step of carotenoid biosynthesis which was blocked, a cell-free system active in the synthesis of carotenoid intermediates was prepared. Incubations with radiolabelled carotenoid precursors led to the identification of the block between GGDP and phytoene. Analysis of carotenoids in different tissues of transgenic and control plants indicated that although ripe fruit and flower carotenoid levels were reduced in the modified plants, leaf carotenoid levels were not decreased. This implies that the pTOM5 gene product is not involved in carotenoid synthesis in the leaf.     

Agrobacterium rhizogenes mediated transformation of grapevine (Vitis vinifera L.)

Genetically transformed grapevine (Vitis vinifera L.) roots were obtained after inocultation of in vitro grown whole plants (cv. Grenache) with Agrobacterium rhizogenes. The strain used contains two plasmids: the wild-type Ri plasmid pRi 15834 and a Ti-derived plasmid which carries a chimaeric neomycin phosphotrans-ferase gene (NPT II) and the nopaline synthase gene. Expression of the NPT II gene can confer kanamycin resistance to transformed plant cells. Slowly growing axenic root cultures derived from single root tips were obtained. Opine analysis indicated the presence of agropine and/or nopaline in established root cultures. For one culture, the presence of T-DNA was confirmed by dot-blot hybridization with pRi 15834 TL-DNA. Callogenesis was induced by subculturing root fragments on medium supplemented with benzylaminopurine and indoleacetic acid.Transformation of in vitro cultured grapevine cells has recently been reported (baribault T.J. et al., Plant Cell Rep (1989) 8: 137–140). In contrast with the results presented here, expession of the NPT II gene Conferred kanamycin resistance to Vitis vinifera calli that was sufficient for selection of trasformed cells.Abbreviations BAP benzylaminopurine - IAA indoleacetic acid - NAA naphtaleneacetic acid - NPT II neomycin phosphostransferase II - EDTA ethylenediaminetetraacetic acid     

Competence pheromone, oligopeptide permease, and induction of competence in Streptococcus pneumoniae

An unmodified heptadecapeptide pheromone capable of eliciting competence for genetic transformation in Streptococcus pneumoniae has recently been identified and characterized. In considering possible signaltransduction mechanisms for the peptide, the previously characterized Ami oligopeptide permease and the three highly homologous oligopeptide-binding lipoproteins, AmiA. AliA, and AliB, appeared to be good candidates for receptors. We therefore compared the spontaneous transformability of Ami, AliA and AliB mutants to that of an isogenic wild-type strain and we investigated the response of the various mutants to treatment with synthetic competence-stimulating peptide (CSP). Our results clearly demonstrate that neither Ami nor any of the three highly homologous oligopeptide-binding lipoproteins identified so far in S. pneumoniae are required for competence induction following treatment with synthetic CSP. Although the existence of a fourth unidentified oligopeptide-binding lipoprotein and/or a second oligopeptide permease operon could not be completely ruled out, we favour the hypothesis that CSP signal transmission rather involves a two-component regulatory system. Although none of the single or double Ami and Ali mutants tested appeared severely affected for competence, an exceptional aliB plasmid-insertion mutation abolished competence completely. In addition, the triple AmiA-AliA-AliB mutant differed from wild type in showing no sharp peak of competence but exhibiting transformability throughout the exponential phase of growth. These and previous observations are discussed and a general hypothesis is proposed to account for the modulation of competence by peptide permease mutants in S. pneumoniae.     

Zolpidem Displays Heterogeneity in Its Binding to the Nonhuman Primate Benzodiazepine Receptor In Vivo

Abstract: The distinctive pharmacological activity of zolpidem in rats compared with classical benzodiazepines has been related to its differential affinity for benzodiazepine receptor (BZR) subtypes. By contrast, in nonhuman primates the pharmacological activity of zolpidem was found to be quite similar to that of classical BZR agonists. In an attempt to explain this discrepancy, we examined the ability of zolpidem to differentiate BZR subtypes in vivo in primate brain using positron emission tomography. The BZRs were specifically labeled with [¹¹C]flumazenil. Radiotracer displacement by zolpidem was monophasic in cerebellum and neocortex, with in vivo Hill coefficients close to 1. Conversely, displacement of [¹¹C]flumazenil was biphasic in hippocampus, amygdala, septum, insula, striatum, and pons, with Hill coefficients significantly smaller than 1, suggesting two different binding sites for zolpidem. In these cerebral regions, the half-maximal inhibitory doses for the high-affinity binding site were similar to those found in cerebellum and neocortex and ～100-fold higher for the low-affinity binding site. The low-affinity binding site accounted for <32% of the specific [¹¹C]-flumazenil binding. Such zolpidem binding characteristics contrast with those reported for rodents, where three different binding sites were found. Species differences in binding characteristics may explain why zolpidem has a distinctive pharmacological activity in rodents, whereas its pharmacological activity in primates is quite similar to that of classical BZR agonists, except for the absence of severe effects on memory functions, which may be due to the lack of substantial zolpidem affinity for a distinct BZR subtype in cerebral structures belonging to the limbic system.