Structural determinants of cationic amphiphilic amines which induce clear cytoplasmic vacuoles in cultured cells

Disobutamide (D), an antiarrhythmic cationic amphiphilic amine (CAA), was withdrawn from clinical testing when clear cytoplasmic vacuoles (CCV) were found in the rat and dog during toxicity studies. To delineate the structural determinants of amines that induce CCV, we exposed cultured rat urinary bladder carcinoma and rabbit aorta muscle cells to numerous cationic drugs and chemicals and examined cells by phase light microscopy. The cationic moiety of these CAA was responsible for the induction of CCV. The very potent inducers were compounds that had two strongly basic amine (cationic) centers. The bis tertiary amines were particularly potent inducers. Aliphatic diamines of minimal lipophilicity-induced CCV, thus showing that an "amphiphilic" structural feature, though present in many CAA drugs, is not necessary for CCV induction. The distance between the two cationic centers was irrelevant to the induction of CCV. These results support the concept that CCV are a manifestation of intracellular (e.g., intralysosomal) drug storage. These structural delineations will be useful in future drug design and for further understanding of drug-cell interactions. Based on these findings, we were able to synthesize an antiarrhythmic CAA which did not induce CCV.     

Nonphotosynthetic CO(2) Fixation by Alfalfa (Medicago sativa L.) Roots and Nodules

The dependence of alfalfa (Medicago sativa L.) root and nodule nonphotosynthetic CO₂ fixation on the supply of currently produced photosynthate and nodule nitrogenase activity was examined at various times after phloem-girdling and exposure of nodules to Ar:O₂. Phloemgirdling was effected 20 hours and exposure to Ar:O₂ was effected 2 to 3 hours before initiation of experiments. Nodule and root CO₂ fixation rates of phloem-girdled plants were reduced to 38 and 50%, respectively, of those of control plants. Exposure to Ar:O₂ decreased nodule CO₂ fixation rates to 45%, respiration rates to 55%, and nitrogenase activities to 51% of those of the controls. The products of nodule CO₂ fixation were exported through the xylem to the shoot mainly as amino acids within 30 to 60 minutes after exposure to ¹⁴CO₂. In contrast to nodules, roots exported very little radioactivity, and most of the ¹⁴C was exported as organic acids. The nonphotosynthetic CO₂ fixation rate of roots and nodules averaged 26% of the gross respiration rate, i.e. the sum of net respiration and nonphotosynthetic CO₂ assimilation. Nodules fixed CO₂ at a rate 5.6 times that of roots, but since nodules comprised a small portion of root system mass, roots accounted for 76% of the nodulated root system CO₂ fixation. The results of this study showed that exposure of nodules to Ar:O₂ reduced nodule-specific respiration and nitrogenase activity by similar amounts, and that phloem-girdling significantly reduced nodule CO₂ fixation, nitrogenase activity, nodule-specific respiration, and transport of ¹⁴C photoassimilate to nodules. These results indicate that nodule CO₂ fixation in alfalfa is associated with N assimilation.     

Changing Activity of Glucose-6-Phosphate Dehydrogenase from Pea Chloroplasts during Photosynthetic Induction

Light inactivation of glucose 6-phosphate dehydrogenase is rapid and occurs before photosynthetic O₂ evolution is measureable in intact chloroplasts. Likewise, dark activation is rapid. The major light induced change in the kinetic parameters of glucose 6-phosphate dehydrogenase is in maximal velocity.     

Expression of the β-subunit of β-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein -conglycinin, encoded by a multigene family. -Conglycinin consists of three subunits; , , and . A genomic clone for a -subunit of -conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this -subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The -subunit expressed in seeds of petunia and tobacco was recognized by anti--conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the -subunit were produced. There was approximately a twofold variation in the accumulation of the -subunit protein in the mature seeds of transgenic petunia plants, each containing a single -subunit gene. However, the level of protein accumulation in mature seeds and the amount of -subunit mRNA in developing seeds was not correlated. Accumulation of the -subunit protein in transgenic seeds was less than the -subunit protein that accumulated in transgenic petunia seeds containing a single -subunit gene and less than the amount of the -subunit in mature soybean seeds which contain 8–13 -subunit genes. In transgenic tobacco plants, the accumulation of the -subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.Abbreviations kb kilobase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Growth potential in onlay bone grafts: a comparison of vascularized and free calvarial bone and suture bone grafts

Vascularized bone grafts are characterized by a viable cell population with osteogenic potential. These features suggest that continued growth can be anticipated following vascularized membranous bone transfer in a growing craniofacial skeleton. The present paper compares the potential for appositional bone growth in vascularized and free calvarial onlay bone grafts. In seven 8-week-old beagles, growth was assessed by direct caliper measurements of graft dimensions intraoperatively and 16 weeks postoperatively. Vascularized grafts demonstrated a 50 to 60 percent increase in size in all dimensions compared to 10 to 20 percent growth in free grafts (p less than 0.01). Microradiography revealed preservation of calvarial bony architecture and minimal resorption in vascularized grafts, while triple-fluorochrome labeling confirmed subperiosteal appositional bone formation. Free grafts were characterized by significant resorption and a delay in subperiosteal bone formation.     

The relative toxicity of a spore preparation of Bacillus thuringiensis var. israelensis against fourth instar larvae of Aedes aegypti and Toxorhynchites amboinensis: suspension feeding compared with enemas and forced feeding

The toxic effect of a spore preparation of Bacillus thuringiensis var. israelensis Berliner Serotype H-14 (Bti) on 4th instar larvae of Aedes aegypti L. and Toxorhynchites amboinensis (Doleschall) was observed when given either in a suspension feeding test or when injected orally as a forced feeding or via the anus as an enema. The A. aegypti larvae showed the greater sensitivity to Bti both because they greatly concentrate the toxin by filter feeding and they are more sensitive to Bti than are the larvae of T. amboinensis. The latter appeared approximately two-fold less sensitive to Bti than the former after taking into account their greater body weight.

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Résumé La toxicité sur des larves de 4ème stade de A. aegypti et T. amboinensis, d'une préparation de spores de B. thuringiensis var. israelensis Berliner sérotype H-14, a été examinée après: injection orale par alimentation forcée, injection anale comme lavement, — le témoin étant une alimentation à partir d'une suspension de spores.Les larves de A. aegypti ont présenté la plus grande sensibilité au Bti d'une part parce qu'elles concentrent beaucoup la toxine avec leur alimentation par filtration, et parce qu'elles sont plus sensibles sensu stricto au Bti. Même en tenant compte de leur poids plus élevé, T. amboinensis est apparu comme deux fois moins sensible au Bti.

     

Bombesin induces c-fos and c-myc expression in quiescent Swiss 3T3 cells. Comparative study with other mitogens

We have studied the effect of the potent mitogen bombesin on the expression of c-fos and c-myc genes in quiescent mouse fibroblasts. We have demonstrated that bombesin rapidly induces a transient expression of c-fos mRNA followed by a more protracted elevation in c-myc mRNA levels. The intensity of the induction of expression of both proto-oncogenes depended on the dose of bombesin used. Prolonged treatment of the cells with TPA, which causes a selective decrease in protein kinase C activity, partially inhibited the induction of c-fos and c-myc gene expression by bombesin, similar to what has been observed with PDGF. However, a dramatic inhibition of the mitogenic response to bombesin--but not to PDGF--was found in TPA-treated cells. In contrast, TPA-treated cells showed an increased response to EGF with regard to proto-oncogene expression. The role of protein kinase C and Ca2+-dependent pathways in proto-oncogene induction by bombesin is discussed.     

Susceptibility of protein kinase C to oxidative inactivation: loss of both phosphotransferase activity and phorbol diester binding

Exposure of protein kinase C to low concentrations of either N-chlorosuccinimide or H2O2 resulted in rapid and parallel loss of phosphotransferase activity and phorbol ester binding. This oxidative inactivation of protein kinase C also occurred in intact cells exposed to a low concentration of H2O2. With H2O2 treatment the rate of inactivation of protein kinase C in the cytosol of MCF-7 cells was rather slower than that which occurred in the cytosol of PYS cells. However, in both cell types, the oxidative inactivation of membrane-associated protein kinase C occurred rapidly in comparison to the enzyme in the cytosol. Prior treatment of cells with phorbol ester to induce membrane association (stabilization) of protein kinase C, followed by exposure to H2O2, resulted in increased inactivation of protein kinase C, suggesting that membrane association of protein kinase C increases its susceptibility to oxidative inactivation.