Inhibition of ornithine decarboxylase in human fibroblast cells by type I and type II interferons

Dilution of human fibroblast GM2767 cell cultures into fresh serum-containing growth medium induces ornithine decarboxylase activity 45-fold over a six-hour interval. When the fibroblast cultures are supplemented with human fibroblast α-, β-, or γ-interferon at the time of dilution into fresh growth medium, the induction of ornithine decarboxylase is inhibited 61%, 90%, and 65%, respectively. β-Interferon is the most effective type of interferon to inhibit induction of ornithine decarboxylase.     

Selective loss of mitochondrial genome can be caused by certain unsaturated fatty acids

Various unsaturated fatty acids had different effectiveness for maintaining the continued replication of functional mitochondria in an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae (KD115). Certain isomers of octadecenoic acid (i.e., cis-9) and eicosatrienoic acid (i.e.,cis-8,11,14) permitted continued replication of mitochondria and provided cultures that contained only 4 to 5% cells that formed petite colonies. On the other hand, cultures grown with cis-12- or cis-13-octadecenoic acid or cis-11,14,17-eicosatrienoic acid, produced a 12- to 16-fold greater frequency of petite mutants (50-60%) after 8 to 10 generations of growth. The production of the petite mutants occurred despite adequate incorporation of these unsaturated fatty acids into cellular phospholipids and an apparently normal ability to undergo the initial steps in the induction of cellular respiration. The evidence suggests that some cellular processes necessary for continued mitochondrial replication depend on the structural features of the fatty acyl chains as well as the overall content of unsaturated fatty acids in membrane phospholipids. Impairment of that process by certain inadequate fatty acids or by an inadequate supply of a suitable fatty acid leads to a permanent loss of the mitochondrial genome from the cells of subsequent generations.     

Catalase synthesis in Escherichia coli is not controlled by catabolite repression

Catabolite repression is not involved in the regulation of catalase gene expression. The presence of glucose in minimal salts media and LB medium did not affect the basal levels of catalase but did enhance catalase synthesis following induction with either hydrogen peroxide or ascorbate. The cofactor for catabolite gene activator protein, cAMP, did not affect either the basal levels or the rate or extent of catalase synthesis. Catalase synthesis occurred normally in an adenylate cyclase mutant where β-galactosidase, a catabolite-sensitive enzyme was not synthesized.     

Activation of a silent gene is accompanied by its demethylation

The phenomenon of gene activation by cell fusion makes it possible to study a gene when it passes from a silent to an active state. The relationship between methylation and activation of the mouse albumin gene has been investigated in two types of hybrid clones: mouse lymphoblastoma--rat hepatoma hybrids where activation is very frequent, and mouse L-cell--rat hepatoma hybrids where activation is a rare event. Analysis of the methylation pattern of seven MspI/HpaII sites that occur along the first 8000 bases of the mouse albumin gene has been performed. The entire 5' region is unmethylated only in albumin-producing cells (adult liver and hepatoma); in non-hepatic cells this region is heavily methylated. In hybrids between rat hepatoma cells and mouse cells of mesenchymal origin, the only regular change is the demethylation of the most 5' site (M1), which is systematically observed in clones where expression of the mouse albumin gene has been activated. Demethylation of this site, like activation of the mouse albumin gene, is gene dosage-dependent; it is systematic in the lymphoblastoma--hepatoma hybrids and rare in L-cell--hepatoma hybrids. We conclude that demethylation of this site is tightly coupled with activation of the gene and may well be a necessary prerequisite for activation.     

An acidic protein which assembles nucleosomes in vitro is the most abundant protein in Xenopus oocyte nuclei

Eggs and oocytes of the frog Xenopus laevis contain an acidic thermostable protein which promotes assembly of nucleosomes from histones and DNA in vitro (Laskey et al., 1978). Analysis of the abundance and intracellular distribution of this protein reveal that it is exclusively localized within the oocyte nucleus where it is the most abundant protein. Its intranuclear concentration is 5 to 7 mg/ml representing 7 to 10% of the total nuclear protein. Micro-injection into oocyte cytoplasm demonstrates that the property of intranuclear migration resides in the mature protein. The injected protein migrates into the nucleus efficiently and becomes distributed throughout the nucleoplasm but it does not associate preferentially with structures containing DNA.     

Hydrogen peroxide release by rat peritoneal macrophages in the presence and absence of tumor cells

The ability of mineral oil-elicited rat peritoneal macrophages to release hydrogen peroxide (H₂O₂) to the extracellular medium was measured in the presence and absence of rat lymphoma cells grown in tissue culture, and in the presence of phorbol myristate acetate (PMA). Horseradish peroxidase (HRP)-catalyzed oxidation of scopoletin or phenol red was used to measure H₂O₂ release during incubation of cells in monolayer culture for periods up to 24 h. Macrophages appeared to release H₂O₂ with or without PMA, although PMA greatly increased the amount of H₂O₂ released in short (1 to 4 h) incubations. Tumor cells did not replace PMA as a triggering agent for H₂O₂ release. Instead, tumor cells inhibited H₂O₂ release. The probable basis for inhibition was competition between macrophages and tumor cells for the supply of oxygen (O₂). Tumor cells did not inhibit H₂O₂ release when the O₂ concentration was held constant. The rates at which macrophages took up O₂ and released H₂O₂ were proportional to the O₂ concentration, as measured with the O₂ electrode. Rates of H₂O₂ release could be calculated from the difference in the rate constants for O₂ uptake measured in the presence of two different extracellular H₂O₂-consuming systems (HRP-scopoletin vs catalase). PMA-stimulated uptake of O₂ and release of H₂O₂ were highest in a small subpopulation of macrophages, obtained at the lowest-density position on gradients of bovine serum albumin. These cells also released H₂O₂ in the absence of PMA. Tumor cells had no effect on the rate constants for O₂ uptake and H₂O₂ release by the unfractionated macrophages or the macrophage subpopulations.     

The kinetics and mechanism of oxidation of maltose and lactose by Tl(III) in acidic media

The kinetics of the cleavage of d-arabino-hexos-2-ulose (1) and of glyoxal (2) with hydrogen peroxide in alkaline water and in 44% (w/w) ethanol-water solutions (pOH 0.5-5) were studied over a temperature range of ?25 to +25°. The relative rate of the competing reactions of 1 with the cleavage in 0.03-1M sodium hydroxide was determined from the rate of formation of hydrogen peroxide in the oxidation of d-glucose to 1 with 2-anthraquinonesulfonic acid in the presence of oxygen at 25 and 40°. The cleavages of both 1 and 2 were first-order with respect to hydrogen peroxide, and also to hydroxyl ion at low alkalinities. The rate of cleavage of 1 reached a maximum at pOH ～2.5, whereas the competing reactions of 1 and the cleavage of 2 were constantly accelerated with increasing hydroxyl-ion concentration. Unlike 2, compound 1 was cleaved more rapidly in ethanol-water than in water. The activation energies of the cleavage of 1 and 2, and the competing reactions of 1, were 49, 57, and 65 kJ.mol^?1, respectively.     

Synthesis of specific membrane proteins is a function of DNA replication and phospholipid synthesis in Caulobacter crescentus

Analysis of the effects on membrane function and protein composition of altering phospholipid synthesis in Caulobacter crescentus showed that, like other bacteria, C. crescentus continues to induce a lactose transport system and to synthesize most membrane proteins. However, we show that the incorporation of a set of outer membrane proteins primarily synthesized in stalked cells is dependent on DNA replication which, in turn, is dependent on membrane phospholipid synthesis. Furthermore, the incorporation of another set of membrane proteins, two of which are synthesized primarily in the swarmer cell, appears to be independent of the replication of the chromosome but to be directly dependent on phospholipid synthesis. We have also found that when phospholipid synthesis is blocked, the synthesis of the flagellar proteins is inhibited and that this effect may be mediated by the primary inhibition of DNA replication. Newton has presented evidence that the synthesis of flagellar proteins is dependent on specific execution points in DNA replication and that this connection serves as a temporal regulator of differential protein synthesis (Osley et al., 1977; Sheffery & Newton, 1981). We suggest here that a direct link between the replicating chromosome and the growing membrane might serve, in turn, to dictate the site of membrane assembly of newly synthesized gene products.     

The effects of cations and trypsin on extraction of chlorophyll-protein complexes by octyl glucoside

The extraction of chlorophyll-protein (CP) complexes from thylakoids by the detergent octyl glucoside is strongly affected by pretreatment of the thylakoids with trypsin or cations. In these experiments, washed thylakoids were incubated in the presence of 0.5 μm to 5 mm Mg²⁺, pelleted, and extracted with octyl glucoside (30 mm). Increasing amounts of Mg²⁺ depressed extractability of all CP complexes, but especially the chlorophyll a + b-containing light-harvesting complex (LHC). This cation effect is observed with other cations which promote thylakoid stacking (5 mm Mn²⁺ or Ca²⁺, 50 mm Na⁺). However, the effect is not merely due to stacking, since low concentrations of Mg²⁺ (0.5 μmto 0.5 mm) have a marked effect on extractability but have no effect on light scattering (OD 550 nm), an indicator of stacking. Furthermore, trypsin treatment of thylakoids stacked with 5 mm Mg²⁺ caused a significant reversal of stacking, but had little effect on extractability. Trypsin treatment of unstacked membranes resulted in increased extractability of all CP complexes, but especially of the LHC. Cation-treated membranes are also significantly different from those “stacked” at pH 4.5. While the latter do show decreased extractability, there is no change in the chlorophyll $\text{a}\text{b}$ ratio of the extract, and the membranes cannot be “unstacked” with trypsin. We conclude that octyl glucoside extractability reflects the lateral interaction of CP complexes with each other and with other components in the same plane of the membrane. It is clear that divalent cations have several effects on thylakoid membranes, not all of which are due to their ability to promote stacking.     

Analysis of dansyl-1-amides in mixtures by mass spectrometry, using metastable defocusing.

Analyses of mixtures of several dansyl-derivatives of biogenic amines were obtained by focusing the metastable ions related to the fragmentation process: molecular ion → dimethylaminonaphthalene ion. The method appears to be useful for the analysis of amines present in biological materials.     

Solvent influence on rate and mechanism of oxidation of ethylenediaminetetraacetatocobaltate(II) by periodate

The kinetics of the oxidation of Co^II(EDTA)²⁻ by IO₄⁻ were studied in various ethanol + water mixtures covering the range 7.9 to 58.0 wt% ethanol, at five different temperatures in the range 15–35 °C. The effect of solvent on the rate and mechanism of the reaction was investigated. An inner-sphere mechanism for the reaction was proposed and supported by the calculated activation parameters.     

Enhancement by nitrite of peroxide-induced degradation of uric acid and 3-N-ribosyluric acid

The oxidation of uric acid and 3-N-ribosyluric acid by hydrogen peroxide and methemoglobin was stimulated by the addition of sodium nitrite, which alone has no effect on the urates. The urates were not oxidized by either hydrogen peroxide alone or hydrogen peroxide and sodium nitrite unless methemoglobin was present. t-Butyl hydroperoxide also oxidized the urates in the presence of methemoglobin, but the reaction was not stimulated by sodium nitrite. The addition of either sodium azide or potassium cyanide reduced the rate of the reaction with either hydrogen peroxide or t-butyl hydroperoxide both in the presence and absence of sodium nitrite. Possible explanations for the stimulation by nitrite of peroxide-induced degradation of urates are presented.     

Experimental access to the molecular and electronic structures of organo-f-element complexes by NMR spectroscopy

The main objective of this survey is to demonstrate that by extensive assessment of variable temperature ¹H NMR data obtained on paramagnetic f-element complexes in solution, not only valuable information on details of the molecular structure, but also on the electronic structure may be deduced. One of the most informative quantities to arrive at is the paramagnetic anisotropy term, χ∥ - χ⊥, of axially symmetric molecules from which, if the bulk susceptibility $\text{χ}$ is also known, the crystal-field sensitive parameters χ∥ and χ⊥ can be derived.The majority of the examples considered belong to the widely studied type [Cp₃^fML_n]^q (Cp = η⁵C₅H₄R); ^fM = Pr(III), Nd(III), Yb(III) and U(IV); n = 0, 1 and 2; q = 0 or ?1) and to the uranocene family. The survey also includes the two sub-classes of novel anionic complexes [Cp₃LnL]^? and [(Cp₃Ln)₂(μ-L)]^?, respectively, and different isomers of the general composition [Cp₃UXY]^q (L = lanthanoid).     

Shape,conformation and stability of fibronectin fragments determined by electron microscopy,circular dichroism and ultracentrifugation

Human plasma fibronectin digested with cathepsin D yields a number of fragments: an N-terminal fibrin-binding 30K³ fragment, a gelatin-binding 40K fragment, a 70K fragment containing the 30K and 40K domains, a central 95/105K fragment and a heparin-binding 140K fragment. The 140K fragment is linked by disulphide bonds and forms the C-terminal ends of the fibronectin. Electron microscopy of rotary-shadowed specimens revealed the 40K, 70K and 95/105K fragments as thin rods (diameter about 2.2 nm) with lengths of 13, 25 and 25 nm. respectively. These dimensions agree with the distances between flexible, proteasesusceptible regions in individual fibronectin strands determined previously. The 140K fragment appeared as a V-shaped structure with two arms 21.5 nm long emerging at a preferred angle of about 70 °. The distribution function of this angle was identical to that observed for the angle between the two arms of intact fibronectin. The free energy required for a distortion of this angle by 60 ° was estimated to be 8 kJ/mol. Reduced and alkylated fibronectin was visualized as single strands (about 55 nm long) with kinks of variable angles corresponding to sites of increased flexibility. The hydrodynamic shapes of the fragments in solution were similar to the shapes observed by electron microscopy, indicating that the individual domains of fibronectin maintain their native structure after proteolytic separation. This was also demonstrated by circular dichroism (c.d.) studies. The c.d. spectrum and the thermal melting curve of native fibronectin were well represented by a linear combination of the contributions of the fragments, indicating conformational independence of the domains. The data support earlier findings that fibronectin consists of two thin strands (length about 60 nm), which are composed of several domains separated by flexible and protease-susceptible intervening regions. This very extended structure agrees with the small sedimentation constant found for fibronectin under conditions where electrostatic interactions between domains are repulsive or depressed. Probably because of such interactions the sedimentation constant is larger at neutral pH and low ionic strength, but even under these conditions a very asymmetric shape is still maintained.     

Copper(II) complexation by D-glucosamine. Spectroscopic and potentiometric studies

The Cu(II) complex formation equilibria of D- glucosamine were studied in aqueous solution by potentiometric and spectroscopic (ESR, CD, absorption spectra) techniques. All data agree that two major species are formed in the pH region 6–9 involving two D-glucosamine ligand molecules bound to the cupric ion via NH₂(CuL₂) or NH₂ and O^? (CuH_?2L₂). In the latter case deprotonated hydroxyls were found to be very effective coordination sites for Cu(II) giving rise to chelate complexes. On the contrary, no complex formation was observed for the Cu(II) N-acetyl-D-glucosamine system.     

Electrostatic facilitation of the reaction catalyzed by the manganese-containing and the iron-containing superoxide dismutases

Both the iron-containing and the manganese-containing superoxide dismutases from Escherichia coli show diminished activity with increasing ionic strength, indicative of electrostatic facilitation of the catalyzed reaction. Since both enzymes bear a net negative charge at the assay pH, as does the substrate, this suggests a cationic locale in the active site region. Acetylation of the enzymes inverted their response to increasing ionic strength. It thus appears that lysine residues provide the observed electrostatic facilitation. A specific inhibition by large monovalent anions was observed with the iron-containing superoxide dismutase and was taken to indicate the presence of a cationic group, within a hydrophobic crevice, at the active site.     

Restricted fragmentation of poliovirus type 1, 2 and 3 RNAs by ribonuclease III.

Cleavage of the genome RNAs of poliovirus type 1, 2 and 3 with the ribonuclease III of Escherichia coli has been investigated with the following results: (1) at or above physiological salt concentration, the RNAs are completely resistant to the action of the enzyme, an observation suggesting that the RNAs lack “primary cleavage sites”; (2) lowering the salt concentration to 0.1 m or below allows RNase III to cleave the RNAs at “secondary sites”. Both large and small fragments can be obtained in a reproducible manner depending on salt conditions chosen for cleavage. Fingerprints of three large fragments of poliovirus type 2 RNA show that they originate from unique segments and represent most if not all sequences of the genome. Based upon binding to poly(U) filters of poly(A)- linked fragments, a physical map of the large fragments of poliovirus type 2 RNA was constructed. The data suggest that RNase III cleavage of single-stranded RNA provides a useful method to fragment the RNA for further studies.