Perturbation of growth and differentiation of Friend murine erythroleukemia cells by 5-bromodeoxyuridine incorporation in early S phase.

Cultured Friend murine erythroleukemia cells (Friend cells) are induced to undergo erythroid differentiation when grown in the presence of dimethylsulfoxide (DMSO) and other compounds. The effects of unifilar substitution of bromouracil (BU) for thymidine in the DNA (BU-DNA) of Friend cells were examined. Cells were grown in the presence of 5-bromodeoxy-uridine (BrdU) for one generation, then centrifuged and resuspended in medium containing DMSO without BrdU. These cells exhibited a delay in the appearance of heme-producing, benzidine-reative (B+) cells and a decreased rate of cell proliferation in comparison to the control not containing BU-DNA. A transient inhibition of entry into S phase was observed when control cells or cells containing BU-DNA were grown in the presence of DMSO) for 10 to 20 hours. This transient inhibition was increased in the BrdU culture. Thus BU-substitution in Friend cells alters other cellular functions in addition to erythroid differentiation. The rate of increase in the percent of cells committed to differentiate (those forming B+ colonies in plasma clots) was similar in the BrdU and control cultures until 40 to 50 hours. After this time, a delay in the appearance of committed cells was observed in the BrdU culture. The effect of BrdU on the appearance of B+ cells was more pronounced and occurred earlier than its effect on the rate of commitment. Therefore, the delay in the appearance of B+ cells in the BrdU culture was due primarily to perturbation of post-commitment events such as the accumulation of hemoglobin. We also examined the effect on growth and differentiation after BrdU was incorporated during different intervals of S phase in cells synchronized by centrifugal elutriation or by double thymidine block and hydroxyurea treatment. The delay in the appearance of B+ cells and inhibition of cell proliferation were only observed when BrdU was incorporated in the first half of S phase. BrdU (10 muM) had no effect on growth or differentiation when present during late S or G1 and G2. These results, using two very different methods to achieve cell synchrony, indicate that the effects of BrdU on growth and differentiation described above are due to its incorporation into DNA sequences replicating during early S.     

Structural requirements for diacylglycerols to mimic tumor-promoting phobol diester action on the epidermal growth factor receptor

The structural requirements for diacylglycerols to mimic the action of tumor-promoting phorbol diesters on the epidermal growth factor (EGF) receptor of A431 human epidermoid carcinoma cells were investigated. Five biological effects were considered: inhibition of high affinity 125I-EGF binding, change in the phosphorylation state of the EGF receptor, inhibition of the EGF-dependent tyrosine phosphorylation of the EGF receptor, inhibition of [3H]phorbol 12 beta, 13 alpha-dibutyrate binding, and stimulation of calcium- and phospholipid-dependent protein kinase (C-kinase) in vitro. A marked effect of the acyl chain length, 3-10 carbons, of symmetric sn-1,2-diacylglycerols was observed on their ability to mimic the effect of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). sn-1,2-Dipropanoylglycerol did not mimic the effects of PMA, but sn-1,2-didecanoylglycerol potently mimicked PMA action. A correlation was found between the ability of these diacylglycerols to stimulate the activity of C-kinase in vitro and to mimic the effects of PMA on the EGF receptor in intact cells. Analogues of sn-1,2-dioctanoylglycerol in which the 3' hydroxyl group was substituted with hydrogen, thio or chloro moieties were inactive when assayed for their ability to stimulate C-kinase in vitro and mimic PMA action in intact cells. We conclude that the hydroxyl group of a diacylglycerol is vital for the interaction with the phorbol diester receptor. The stringent correlation between the potency of the 11 diacylglycerol analogues tested to modulate C-kinase in vitro and to mimic PMA action in vivo provides strong evidence for the hypothesis that C-kinase plays a central role in the regulation of A431 cell EGF receptors by tumor-promoting phorbol diesters.     

Polyphyly of Asian Tree Toads,Genus Pedostibes Günther, 1876 (Anura: Bufonidae), and the Description of a New Genus from Southeast Asia

The Asian Tree Toad genus Pedostibes, as currently understood, exhibits a conspicuously disjunct distribution, posing several immediate questions relating to the biogeography and taxonomy of this poorly known group. The type species, P. tuberculosus and P. kempi, are known only from India, whereas P. hosii, P. rugosus, and P. everetti are restricted to Southeast Asia. Several studies have shown that these allopatric groups are polyphyletic, with the Indian Pedostibes embedded within a primarily South Asian clade of toads, containing the genera Adenomus, Xanthophryne, and Duttaphrynus. Southeast Asian Pedostibes on the other hand, are nested within a Southeast Asian clade, which is the sister lineage to the Southeast Asian river toad genus Phrynoidis. We demonstrate that Indian and Southeast Asian Pedostibes are not only allopatric and polyphyletic, but also exhibit significant differences in morphology and reproductive mode, indicating that the Southeast Asian species’ are not congeneric with the true Pedostibes of India. As a taxonomic solution, we describe a new genus, Rentapia gen. nov. to accommodate the Southeast Asian species.     

Further characterisation of the [35S]-TBPS binding site of the GABA receptor complex in locust (Schistocerca gregaria) ganglia membranes

1. Using homogenates of supraoesophageal ganglia from locust we observed specific binding of [35S]-TBPS which was linear with protein concentration up to 7 mg/ml, showed a pH optimum at pH 9.0 and was linear with NaCl concentration up to 350 mM. 2. Kinetic analysis of the binding showed positive cooperativity as a result of changes in on and off-rates with occupation of the binding site by the ligand. The apparent KD = 417 nM and Bmax = 1083 fmol/mg of membrane protein were calculated using a computer program for dose-response curve fitting. 3. The binding was enhanced by GABA, pentobarbital and benzodiazepines. Picrotoxinin had no effect on the binding at 0.1 mM. Only the cage convulsants TBPS and IBP were able to displace the binding. 4. Whilst the characteristics of the binding are similar to those reported for house fly thorax and abdomen preparations they are significantly different from those reported for house fly head, cockroach nerve cord and rat brain.     

Gastrointestinal microflora,food components and colon cancer prevention

Evidence that the intestinal microbiota is intrinsically linked with overall health, including cancer risk, is emerging. Moreover, its composition is not fixed but can be influenced by several dietary components. Dietary modifiers, including the consumption of live bacteria (probiotics) and indigestible or limited digestible food constituents such as oligosaccharides (prebiotics) and polyphenols or both (synbiotics), are recognized modifiers of the numbers and types of microbes and have been reported to reduce colon cancer risk experimentally. Microorganisms also have the ability to generate bioactive compounds from food components. Examples include equol from isoflavones, enterodiol and enterolactone from lignans and urolithins from ellagic acid, which have also been demonstrated to retard experimentally induced cancers. The gastrointestinal microbiota can also influence both sides of the energy balance equation, namely, as a factor influencing energy utilization from the diet and as a factor that influences host genes that regulate energy expenditure and storage. Because of the link between obesity and cancer incidence and mortality, this complex complexion deserves greater attention. Overall, a dynamic interrelationship exists between the intestinal microbiota and colon cancer risk, which can be modified by dietary components and eating behaviors.     

Membrane targeting of RecA during genetic transformation

Recombination in prokaryotes and eukaryotes is mediated by the RecA family of proteins. Although the interactions between RecA and DNA are well studied, the cellular location of these interactions is not known. Using genetic transformation of Streptococcus pneumoniae as a model system, there was increased expression of a protein, colligrin, and RecA, products of the rec locus during genetic transfer. These proteins formed a complex and were found associated with the membranes of genetically competent cells. With immunoelectron microscopy and subcellular fractionation, we showed that the induction of competence led to the translocation of RecA and colligrin to the membrane and to the formation of clusters of RecA in a colligrin-dependent step. Based on the behaviour of colligrin and RecA during genetic exchange and the numerous proteins in prokaryotes and eukaryotes with domains similar to colligrin, we suggest that there may exist a family of proteins, which gathers macromolecules at specific sites in biological membranes.