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.     

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.     

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.     

Synthesis of 3-O-β-d-xylopyranosyl-l-serine (xylosylserine) andO-β-d-galactopyranosyl-(1-4)-O-β-d-xylopyranosyl-l-serine (galactosylxylosylserine) and use of the synthetic products for detection of galactosyltransferase I activity in rat liver

3-O-β-d-Xylopyranosyl-l-serine (xylosylserine) was synthesized by the following three-step procedure: 1) 2,3,4-tri-O-benzoyl-α-d-xylopyranosyl bromide (benzobromoxylose) was condensed withN-carbobenzoxy-l-serine benzyl ester using the silver triflate-collidine complex as promoter; 2) theN-carbobenzoxy and benzyl ester groups in the resultant glycoside were cleaved by transfer hydrogenation with palladium black as catalyst and ammonium formate as hydrogen donor; and 3) the benzoyl groups were removed with methanolic ammonia. Xylosylserine was obtained in an overall yield of 70%. O-β-d-Galactopyranosyl-(1-4)-O-β-d-xylopyranosyl-(1-3)-l-serine (galactosylxylosylserine) was also synthesized by this methodology and was characterized by 2-dimensional (2D) NMR spectroscopy techniques. The two serine glycosides (xylosylserine and galactosylxylosylserine) were used in detection and partial purification of galactosyltransferase I (UDP-d-galactose:d-xylose galactosyltransferase) from adult rat liver.     

Alteration of protein phosphorylation patterns in cell lines morphologically transformed by human cytomegalovirus

Human fibroblastic cell lines morphologically transformed by either live virus or DNA fragments of human cytomegalovirus had altered plasma membrane protein composition; quantitative changes, and gains and losses in protein composition in comparison to normal parent cell lines were detected. These transformed cell lines showed altered total cell protein phosphorylation patterns when compared to parent cell lines. A two to four fold increase in in vivo protein phosphorylation at serine and threonine residues was observed; no increase in phosphorylation at total cell tyrosine residues was detected. Analysis of the in vivo phosphorylated protein by two dimensional gel electrophoresis revealed some similarities as well as differences in the types of polypeptides phosphorylated between transformed and control cell lines. Increased (two-to sixfold over parent cell extracts) casein kinase and polyamine dependent casein kinase activities were detected in HCMV transformed cell extracts.     

Posttreatment with sodium arsenite is coclastogenic in log phase but not in stationary phase

Summary Posttreatment with sodium arsenite in log phase synergistically increases the chromosomal aberrations induced by ethyl methanesulfonate in Chinese hamster ovary cells, human fibroblasts, and human lymphocytes. However, posttreatment with sodium arsenite in stationary phase has no apparent effect on the clastogenicity of ethyl methanesulfonate. These results indicate that the cycling state of the cell plays a crucial role in the action of arsenite coclastogenicity. One prediction from this finding is that in combined treatment, posttreatment with sodium arsenite should preferentially kill cancer cells.     

国产姜科植物的染色体计数(四)

<正> 本文继续报道对国产姜科植物8属15种所作的染色体计数观察的结果,其中9种是染色体计数的新记录(表1、图1),并初步确定茴香砂仁属Achasma和喙花姜属Rhynchanthus的染色体基数分别为12和11。