Homology between antigenic determinants of bovine clathrin and clathrin from the brown alga Laminaria digitata

Coated vesicles, essential organelles of intracellular membrane traffic, have been extensively studied in animal and higher plant cells. In the algae, cytological studies only have been performed which demonstrate the presence of such coated vesicles with their surrounding clathrin lattice. The present work has been carried out on coated vesicles isolated for the first time from the brown algae Laminaria digitata. For comparison of the antigenic characteristics of clathrin prepared from the Bovine brain or adrenocortical cells and the clathrin prepared from algae, polyclonal antibodies have been raised to a purified Bovine brain clathrin in Goat and to Bovine adrenocortical clathrin in Rabbit. The positive immunological responses of the coated vesicles and the clathrin from Algae to these antibodies, evidence an homology between antigenic determinants of clathrin from animal and vegetal cells.     

Insulin action in normal and protein kinase C-deficient rat hepatoma cells. Effects on protein phosphorylation, protein kinase activities, and ornithine decarboxylase activities and messenger ribonucleic acid levels

Insulin and tumor-promoting phorbol esters such as phorbol 12-myristate 13-acetate (PMA) share some biological activities in normal hepatocytes and in some lines of cultured hepatoma cells. To investigate the possibility that some of these common effects might involve a common pathway, we examined the effects of insulin and PMA on several biological processes in normal and protein kinase C-deficient H4IIE rat hepatoma cells. Protein kinase C deficiency was achieved by preincubating the cells in high concentrations of PMA, and was documented by direct enzyme measurement in soluble and particulate cellular fractions, and by analysis of immunoreactive protein kinase C concentrations in whole cellular homogenates. In the protein kinase C-deficient cells, the following actions of insulin remained at near normal levels: stimulated phosphorylation of the ribosomal protein S6; activation of a ribosomal S6 protein kinase; and increases in ornithine decarboxylase activity and mRNA accumulation. PMA stimulated all of these responses in the normal cells, but none of them in the PMA-pretreated cells. We conclude that insulin can exert some of its actions in a normal manner in protein kinase C-deficient H4IIE hepatoma cells (ATCC CRL 1548) and that some of the actions insulin holds in common with PMA may be due to common activation of one or more distal pathways. A candidate for such a distal step is activation of the ribosomal protein S6 protein kinase.     

An immunocytochemical method for the localization of fibronectin in Araldite-embedded specimens

Summary The detection of fibronectin (FN) in osmium-fixed and Araldite-embedded frog skin fragments was studied using a modification of Baskin's procedure (Baskin et al. 1979). Following the removal of Araldite from the semi-thin sections (0.5–1.0 m) with ethanol-NaOH solution, the sections were bleached with hydrogen peroxide. FN was detected by indirect immunoperoxidase method. For precise localization of FN, careful attention was paid to the temperature, antibody concentrations and the quality of the ethanol-NaOH solution. Our results were in agreement with those that we had obtained previously for polyethylene glycol (PEG) sections, suggesting that the present procedure is useful for the detection of FN in Araldite-embedded biological specimens.     

Isolation of a Cellodextrinase from Bacteroides succinogenes

An enzyme which released the cellobiose group from p-nitrophenyl cellobioside was isolated from the periplasmic space of Bacteroides succinogenes grown on Avicel crystalline cellulose in a continuous cultivation system and separated from endoglucanases by column chromatography. The molecular weight of the enzyme was approximately 40,000, as estimated by gel filtration. The enzyme has an isoelectric point of 4.9. The enzyme exhibited low hydrolytic activity on acid-swollen cellulose and practically no activity on carboxymethyl cellulose, Avicel cellulose, and cellobiose, but it hydrolyzed p-nitrophenyl lactoside and released cellobiose from cellotriose and from higher cello-oligosaccharides. These data demonstrate that the enzyme is a cellodextrinase with an exotype of function.     

Plasmid DNA adsorbed to pH-sensitive liposomes efficiently transforms the target cells

We have previously reported that plasmid DNA entrapped in the pH-sensitive immunoliposomes effectively transforms the target cells (Proc. Natl. Acad. Sci. USA, in press). In the present study, we demonstrate that DNA adsorbed on the same liposome also transforms the target cells. The transformation activity is antibody dependent, as liposomes containing no targeting antibody had reduced activity. The activity could be significantly inhibited by excess non-specific DNA (salmon sperm). Since some DNA are likely adsorbed to the liposomes during the entrapment process, the activity of the entrapped DNA is partially accounted for by the adsorbed DNA. The possibility of developing a simple DNA-mediated transfection protocol using liposome adsorbed DNA is discussed.     

An antibody to the receptor for insulin-like growth factor I inhibits the growth of MCF-7 cells in tissue culture

Alpha IR-3, a monoclonal antibody to the insulin-like growth factor I receptor which blocks insulin-like growth factor I binding and inhibits its activity, inhibits the binding of 125I-insulin-like growth factor I to MCF-7 cells (an estrogen dependent human breast carcinoma cell line) with an IC-50 of approximately 100 ng/ml. It also inhibits the growth of MCF-7 cells cultured in 5% calf serum with approximately the same IC-50. Inhibition of growth occurs both when cells are cultured in the presence and absence of estrogen and is more pronounced when cells are grown at a low density. These findings demonstrate a requirement for insulin-like growth factor I for optimal growth of MCF-7 cells and suggest that it is an autocrine growth factor in these cells.     

Scanning calorimetry reveals a new phase transition in L-alpha-dipalmitoylphosphatidylcholine.

We report a new phase transition in fully hydrated dispersions of dipalmitoylphosphatidylcholine (DPPC). This new transition, called the sub-subtransition, exhibits a transition enthalpy of 0.25 kcal/mol with a Tm at 6.8 degrees C. Unlike the subtransition, no extended low temperature incubation is required to observe the sub-subtransition. This new sub-subgel (SGII) phase may be a precursor to the subgel (SGI) phase, and this discovery is discussed in relation to the current knowledge regarding the polymorphic gel phases of both ester- and ether-linked lipids with identical acyl chains.     

Removal of sodium inactivation and block of sodium channels by chloramine-T in crayfish and squid giant axons.

Modification of sodium channels by chloramine-T was examined in voltage clamped internally perfused crayfish and squid giant axons using the double sucrose gap and axial wire technique, respectively. Freshly prepared chloramine-T solution exerted two major actions on sodium channels: (a) an irreversible removal of the fast Na inactivation, and (b) a reversible block of the Na current. Both effects were observed when chloramine-T was applied internally or externally (5-10 mM) to axons. The first effect was studied in crayfish axons. We found that the removal of the fast Na inactivation did not depend on the states of the channel since the channel could be modified by chloramine-T at holding potential (from -80 to -100 mV) or at depolarized potential of -30 mV. After removal of fast Na inactivation, the slow inactivation mechanism was still present, and more channels could undergo slow inactivation. This result indicates that in crayfish axons the transition through the fast inactivated state is not a prerequisite for the slow inactivation to occur. During chloramine-T treatment, a distinct blocking phase occurred, which recovered upon washing out the drug. This second effect of chloramine-T was studied in detail in squid axons. After 24 h, chloramine-T solution lost its ability to remove fast inactivation but retained its blocking action. After removal of the fast Na inactivation, both fresh and aged chloramine-T solutions blocked the Na currents with a similar potency and in a voltage-dependent manner, being more pronounced at lower depolarizing potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uniformly oriented gramicidin channels embedded in thick monodomain lecithin multilayers.

Phosphatidylcholine multilayers, containing 20% water by total sample weight and gramicidin/lipid molar ratios up to 1:40 were aligned by low temperature annealing (less than 60 degrees C) and mechanical stressing. We were able to obtain large (greater than 80 micron thick X 40 mm2 area) monodomain defect-free multilayers containing approximately 10(17) uniformly oriented gramicidin channels. The alignment of lipid multilayers was monitored by conoscopy and polarized microscopy. The smectic defects, which appeared during the alignment process, were identified and dissolved. The incorporation of gramicidin into the multilayers in the form of transmembrane channels was indicated by its circular dichroic (CD) spectrum. A well-defined CD spectrum of uniformly oriented gramicidin channels was obtained. The oriented samples will allow spectroscopic studies of the ion channel in its conducting state and diffraction studies of the channel-channel organization in the membrane.