Phosphorylation of peripherin, an intermediate filament protein, in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons

Peripherin, an intermediate filament protein, described recently, is expressed in well defined neuronal populations. We studied the phosphorylation, in vivo, of this protein in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons labelled with [32P]-orthophosphate. The autoradiograms of proteins separated on two-dimensional polyacrylamide gels were compared with the Coomassie-blue stainings. The results show that peripherin occurs as a mixture of phosphorylated and non-phosphorylated isoforms, and that these forms coexist in both differentiated and non-differentiated cells. We demonstrate by cleavage at the unique tryptophan residue, a characteristic shared by most other intermediate filament proteins (IFP), that the phosphorylation sites are located on the amino-terminal half of peripherin as it is for vimentin and desmin. These results are discussed in relation to the organization of the filamentous network constituted by peripherin.     

Histamine stimulates calcium-mediated protein phosphorylation in a colonic epithelial cell line

Protein phosphorylation responses in intact enterocytes were examined by stimulating 32Pi-labeled T84 cell monolayers with histamine and resolving proteins by two-dimensional gel electrophoresis. Histamine increases 32P-incorporation into two acidic proteins of Mr 83,000 and of Mr 29,000, designated p83 and p29. Labeling of p83 and p29 is also increased in cells exposed to ionomycin, but not in cells exposed to vasoactive intestinal peptide under conditions resulting in cAMP-mediated secretion and cAMP-stimulated protein phosphorylation. When T84 cell fractions are incubated with [gamma-32P]ATP, labeling of p83 is stimulated by Ca++, but not by cAMP. Thus, histamine stimulates Ca++-mediated protein phosphorylation during the regulation of Cl- secretion.     

Can ribozymes be used to regulate procaryote gene expression?

The in vivo activity of ribozymes designed against mRNA coding for E. coli beta-galactosidase was tested both in intramolecular and in intermolecular conditions. When recombinant M13 phage DNA carrying on the same molecule the information for both the ribozyme and the target was transfected into bacterial cells, ribozyme activity was observed. Conversely, a ribozyme coded by a recombinant M13 vector, but targeted against an mRNA transcribed from the F episome including the remaining part of the beta-galactosidase gene, was inefficient.     

Beta-lactamase-catalyzed aminolysis of depsipeptides: proof of the nonexistence of a specific D-phenylalanine/enzyme complex by double-label isotope trapping

The steady-state kinetics of the Enterobacter cloacae P99 beta-lactamase-catalyzed aminolysis of the depsipeptide m-[[(phenylacetyl)glycyl]oxy]benzoic acid by D-phenylalanine were consistent with an ordered sequential mechanism with D-phenylalanine binding first [Pazhanisamy, S., Govardhan, C. P., & Pratt, R. F. (1989) Biochemistry (first of three papers in this issue)]. In terms of this mechanism, the kinetics data required that in 20 mM MOPS buffer, pH 7.5, the dissociation constant of the initially formed enzyme/D-phenylalanine complex be around 1.3 mM; at pH 9.0 in 0.1 M carbonate buffer, the complex should be somewhat more stable. Attempts to detect this complex in a binary mixture by spectroscopic methods (fluorescence, circular dichroic, and nuclear magnetic resonance spectra) failed. Kinetic methods were also unsuccessful--the presence of 20 mM D-phenylalanine did not appear to affect beta-lactamase activity nor inhibition of the enzyme by phenylmethanesulfonyl fluoride, phenylboronic acid, or (3-dansylamidophenyl)boronic acid. Equilibrium dialysis experiments appeared to indicate that the dissociation constant of any binary enzyme/D-phenylalanine complex must be somewhat higher than the kinetics allowed (greater than 2 mM). Since the kinetics also required that, at high depsipeptide concentrations, and again with the assumption of the ordered sequential mechanism, the reaction of the enzyme/D-phenylalanine complex to aminolysis products be faster than its reversion to enzyme and D-phenylalanine, a double-label isotope-trapping experiment was performed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Perturbation in liver mitochondrial Ca2+ homeostasis in experimental iron overload: a possible factor in cell injury

The functional state of isolated mitochondria and specifically the integrity of the inner membrane, were investigated in the liver of rats made siderotic by dietary supplementation with carbonyl iron. The concentration of iron in the hepatic tissue increased progressively up to nearly 40 days and reached a steady-state level. When the iron content reached a threshold value (higher than 90 nmol/mg protein) the occurrence of in vivo lipid peroxidation in the mitochondrial membrane was detected. This process did not result in gross alterations in the mitochondrial membrane, as indicated by electron microscopy, phosphorylative capability and membrane potential measurements. On the contrary, the induction of lipoperoxidative reaction appeared to be associated with the activation of Ca2+ release from mitochondria. This was shown to occur as a consequence of rather subtle modifications in the inner membrane structure via a specific efflux route, which appeared to be linked to the oxidation level of mitochondrial pyridine nucleotides. The induction of this Ca2+ release from iron-treated mitochondria resulted in enhancement of Ca2+ cycling, a process which dissipates energy to reaccumulate into mitochondria the released Ca2+. The perturbation in mitochondrial Ca2+ homeostasis reported here may be a factor in the onset of cell damage in this experimental model of hepatic iron overload.     

Nonelectrolyte permeability of liposomes of hydroxyfatty acid-containing phosphatidylcholines

Two phosphatidylcholines containing hydroxylated fatty acids, 1-palmitoyl-2-[5-hydroxy-6,8,11,14-eicosatetraenoyl]-sn-glycero-3- phosphocholine (1-palm-2-5HETE PC) and 1-palmitoyl-2-[15(S)-hydroxy-5,8,11,13- eicosatetraenoyl]-sn-glycero-3-phosphocholine (1-palm-2-15HETE PC), and one phosphatidylcholine containing nonhydroxylated fatty acids, 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (1-palm-2-arach PC) were synthesized. Permeation of small nonelectrolytes (glycerol, 1,2-propanediol, urea, methylurea, propionamide and dimethylformamide) was assessed in multilamellar liposomes containing these synthetic PCs plus egg yolk phosphatidycholine (EPC) in the presence and absence of cholesterol. In liposomes containing 23% cholesterol, 69.3% EPC and 7.7% of either 1-palm-2-5HETE PC or 1-palm-2-15HETE PC the permeability to small nonelectrolytes was 60 to 400% greater than in liposomes containing 23% cholesterol and 77% EPC. The HETE-containing PCs also increased permeability in liposomes without cholesterol but the effects were less striking. Addition of the synthetic PCs did not affect the energy of activation of permeation.     

Molecular aspects of the removal of ferritin-bound iron by DL-dihydrolipoate

The removal of ferritin-bound iron by the physiologic dithiol DL-dihydrolipoate was studied over the pH range 5.5-9.0. A novel method was devised for the determination of iron removal, making it possible to study the actual release of iron from ferritin, regardless of the oxidation state or complexation form. The overall iron-removal process appears to depend upon a balance between the deprotonation of the dithiol and the protolytic dissolution of the iron core inside the ferritin molecule. The amount of iron removed at equilibrium increases with the pH, at any of the dihydrolipoate/ferritin iron ratios tested. The formation of the binuclear iron-dithiol complex [Fe2(dihydrolipoate)3]-3 is not strictly required for iron mobilization, but it seems to affect the efficiency of the dithiol in iron mobilization by providing a stable complexation form for the released iron outside the ferritin protein shell. Comparison of the release of ferritin-bound iron by free and immobilized dihydrolipoate indicates that mobility of the dithiol is mandatory for the removal process to take place.     

A major single-stranded DNA binding protein from ovaries of the frog, Xenopus laevis, is lactate dehydrogenase

The most abundant single-stranded DNA binding protein (SSB) found in ovaries of the frog, Xenopus laevis, was purified to electrophoretic homogeneity. Under physiological conditions, the purified SSB lowered the Tm of poly[d(A-T)] and stimulated DNA synthesis by the homologous DNA polymerase DNA primase alpha complex on single-stranded DNA templates. These properties are characteristic of a bona fide single-stranded DNA binding protein. The Stokes radius of native SSB was calculated to be 45 A, corresponding to a molecular mass of about 140 kDa. On SDS polyacrylamide gels, the SSB migrated as a single band with a molecular mass of 36 kDa. We assumed, therefore, that the SSB was a tetramer of 36 kDa subunits. We subsequently discovered that the SSB was LDH, D-lactate dehydrogenase, EC 1.1.1.28. Purified SSB has high LDH specific activity. Following electrophoresis on SDS polyacrylamide gels, the 36 kDa subunits were renatured and exhibited LDH activity. The amino-acid composition of X. laevis SSB/LDH was similar to that of LDH from other species and to other reported single-stranded DNA binding proteins. Mammalian SSB/LDH also preferentially bound single-stranded DNA. Mammalian SSB/LDH bound to RNA as demonstrated by affinity chromatography on poly(A)-agarose and by its effect on translation of mRNA in vitro.     

Influence of lactate on isoproterenol-induced lipolysis and beta-adrenoceptors distribution in human fat cells

The influence of lactate on human adipocytes lipolysis and the possible relationship between lactate-induced metabolic effects and beta-adrenoceptor binding sites were investigated. beta-sites were identified in membranes with (125I)-cyanopindolol and in intact cells with (125I)-cyanopindolol and (3H)-CGP 12177. Lactate reduced isoproterenol-induced lipolysis in a dose-response fashion and such inhibition became significant only at 16 mmol/l lactate. Exposure of human fat cells to 16 mmol/l lactate significantly reduced beta-adrenoceptors density on crude membranes. When the binding assay was performed on intact cells using (125I)-cyanopindolol at 37 degrees C, the radioligand identified the same number of receptors, regardless of the presence of lactate in the preincubation medium. When (3H)-CGP 12177 was used, it bound to about 35% less receptors in lactate pre-treated cells than in control. Seemingly, at 37 degrees C, because of its lipophilicity, (125I)-cyanopindolol can cross the plasma membrane and bind to intracellular sites whereas, (3H)-CGP 1277, due to its hydrophilicity, identifies surface receptors only. Thus, the present in vitro study provides evidence that high levels of lactate, similar to the concentrations usually achieved in overt lactic acidosis, are able per se to inhibit human lipolysis and to redistribute beta-adrenoceptors from cell surface to a domain not accessible to hydrophilic ligands.     

Comparison of chemical analyses of boat and helicopter-collected water samples

Helicopters can be used to collect water samples from many lakes over a wide geographic area within a relatively short time period. Here we report the results from an experiment in which sequential water samples from a lake were collected first from a nonmotorized boat and then immediately afterward from a helicopter. No significant differences were found between the means of the measurement of 20 chemical parameters for the two methods of collection. When compared to obtaining samples from a boat, collection of samples from a helicopter platform had no effect on the content of the water samples.