Phosphorylation of nuclear matrix proteins in isolated regenerating rat liver nuclei

The phosphorylation of nuclear matrix proteins from normal and regenerating rat liver nuclei was examined using an $\text{in vitro}$ system of isolated nuclei and γ-³²P-ATP. Phosphorylation of the nuclear matrix proteins was 2–3 fold higher than that of the total nuclear proteins in normal nuclei. The level of phosphorylation of the matrix proteins was enhanced an additional three fold at a period in liver regeneration (12 hours) just preceding the onset of DNA synthesis.     

Effect of ozone and nitrogen dioxide on the agglutination of rat alveolar macrophages by concanavalin A

$\text{In vitro}$ exposure of rat alveolar macrophages to 0.5 ppm ozone for 60 minutes results in a 76% decrease in agglutination by concanavalin A. A decrease in the agglutinability of rat alveolar macrophages by concanavalin A was also observed following inhalation of 0.5 or 1.0 ppm ozone for two hours. In contradistinction, $\text{in vitro}$ exposure of rat alveolar macrophages to 2.4 ppm nitrogen dioxide for 60 minutes produced a 64% increase in agglutination by concanavalin A; and increased agglutinability was also noted following inhalation of 12.1 ppm nitrogen dioxide for two hours. Agglutination was almost completely inhibited by alpha-methyl-mannose. Neither pollutant significantly altered the binding of ³H-concanavalin A to rat alveolar macrophages. These two air pollutants, both of which are known to potentiate respiratory tract infections, appear to affect the response of the alveolar macrophage membrane to concanavalin A in a dissimilar fashion.     

Mass spectrometric identification of various molecular forms of dynorphin in bovine adrenal medulla

Deliberate miscompartmentalization of liver outer mitochondrial membrane (OMM) proteins and liver mitochondrial proteins has been achieved by polyethylene-glycol mediated OMM vesicle-hepatocyte or mitochondrial-hepatocyte fusion. Reductively methylated OMM and mitochondrial proteins (³H) are destroyed at rates remarkably similar to those for OMM ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 60–70 h) or mitochondrial proteins ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 84–104 h) in liver $\text{in vivo}$ when studied over 4–5 days in hepatocyte monolayers cultured in conditions giving stabilized endogenous protein catabolic rates mimicking endogenous $\text{in vivo}$ rates. Destruction of transplanted OMM proteins is partially sensitive to chloroquine, supporting some lysosomally mediated autophagic destruction of long-lived transplanted OMM proteins in hepatocyte monolayers.     

U.V. induced covalent crosslinking of E.coli ribosomal RNA to specific proteins

$\text{E.}\text{coli}$ 70S ribosomes uniformly labeled $\text{in}\text{vivo}$ with ³²PO₄ were subjected to varying doses of u.v. radiation and then to the combined action of the RNases A and T₁. Following these treatments the ribosomal proteins were separated by trichloroacetic acid precipitation from the noncovalently attached RNA degradation fragments. Subsequent two-dimensional gel electrophoresis and autoradiography of these proteins revealed that significant ³²PO₄ was associated with unique ribosomal proteins, L2 was among these.     

Bacterial lipopolysaccharides and mycoplasmal lipoglycans: A comparison between their abilities to induce macrophage-mediated tumor cell killing and Limulus amebocyte lysate clotting

The ability of various bacterial lipopolysaccharides and mycoplasmal lipopolysaccharides (lipoglycans) to induce macrophage-mediated tumor cell killing and $\text{Limulus}$ amebocyte lysate clotting was determined. Lipoglycans from the mycoplasma $\text{Acholeplasma}\text{axantum}$ or $\text{Acholeplasma}\text{granularum}$ had no activity or 10⁴ to 10⁵ less activity than lipopolysaccharides from $\text{Escherichia}\text{coli}$ 0128:B12, $\text{Escherichia}\text{coli}$ K235, or $\text{Salmonella}\text{minnesota}$ R595 in causing $\text{Limulus}$ lysate clotting and tumor cell killing by peritoneal macrophages from normal or bacillus Calmette-Guérin-infected mice. Previous studies have shown that the lipid A portion of bacterial lipopolysaccharide is responsible for the effects on macrophage-mediated tumor cell killing and $\text{Limulus}$ lysate clotting. The known differences in the lipid structures of bacterial lipopolysaccharides and mycoplasmal lipopolysaccharides (lipoglycans) may account for the noted differences in the biologic potencies observed here.     

ATP-dependent Ca2+ accumulation in intracellular membranes of guinea pig macrophages after saponin treatment

Ca²⁺ transport system in the intracellular membranes was studied by using saponin-treated macrophages of the guinea pig, in which the plasma membranes could be selectively destroyed. Saponin-treated macrophages could accumulate 3.1 nmoles $\text{Ca}{}^{\mathrm{2+}}\text{4 × 10}{}^6$ macrophages in the presence of Mg-ATP and sodium azide with an apparent affinity constant of 6 × 10⁶ M^?1. In the absence of sodium azide, the value of Ca²⁺ uptake of saponin-treated macrophages was 95 nmoles/4 × 10⁶ macrophages, and its affinity constant for Ca²⁺ was 3 × 10⁵ M^?1. Saponin-treated macrophages may be suitable for the studies of Ca²⁺ transport systems in the intracellular membranes.     

Identification of ubiquinone binding proteins in ubiquinol-cytochrome c reductase by arylazido ubiquinone derivative

A functionally active $\text{arylazido-1-[}{}^{14}\text{C]-β-alanine}$ ubiquinone derivative has been synthesized for the identification of the ubiquinone binding protein in ubiquinol-cytochrome $\text{c}$ reductase. After photolysis, the ¹⁴C activity was found to be specifically associated to proteins with mobilities relative to cytochrome $\text{c}$ of 0.841 and 0.475 in the sodium dodecylsulfate polyacrylamide gel electrophoresis of the Weber and Osborn system. These two proteins have previously been identified as $\text{b}$ cytochromes. The ¹⁴C activity distribution pattern was observed to be identical in the presence or absence of phospholipids during the photolysis. Antimycin A also produces no change in the ¹⁴C activity distribution among the proteins of this enzyme complex.     

Tunicamycin inhibits glycosylation of precursor polyprotein encoded by env gene of Rauscher murine leukemia virus.

The molecular weight of the precursor polyprotein to the envelope proteins of Rauscher murine leukemia virus is reduced from 85,000 to 68,000 daltons when synthesized in the presence of tunicamycin, a specific inhibitor of the synthesis of oligosaccharides that attach to glycoproteins via asparagine residues. The unglycosylated precursor protein (Pr68^$\text{env}$) is synthesized at a rate comparable to that of the normal carbohydrate-containing envelope precursor (gPr85^$\text{env}$). Pr68^$\text{env}$ is not proteolytically processed and remains undegraded in the cell. Thus, most if not all of the carbohydrate content of gPr85^$\text{env}$ is N-linked, and glycosylation appears to be necessary for normal processing of precursor proteins into viral particles.     

Comparison of monocyte and alveolar macrophage antibody-dependent cellular cytotoxicity and Fc-receptor activity

The cytotoxic potential of rabbit peripheral blood monocytes and alveolar macrophages in antibody-dependent cellular cytotoxicity (ADCC) toward both erythrocyte (RBC_ox) and tumor cell (CEM T-lymphoblast) targets was examined. ADCC was measured in a 4-hr ⁵¹Cr-release assay. Alveolar macrophages were more efficient at killing the tumor cell targets (optimally sensitized with rabbit antisera) than monocytes at similar effector cell/target cell ($\text{E}\text{T}$) ratios. Tumor cell targets sensitized with seven different antisera (anti-CEM) were lysed by alveolar macrophages but not by the monocytes. In marked contrast, the monocytes were more effective at lysing the sensitized erythrocyte target cells. The degree of cytolysis of RBCox and CEM was dependent on the $\text{E}\text{T}$ ratio and the degree of sensitization of these target cells. It was demonstrated that the effector cell selectivity in ADCC was directly related to their ability or inability to bind the sensitized target cells as determined by Fc-receptor rosette formation. The transition from monocyte to macrophage may, therefore, have resulted in an alteration in the criteria necessary for Fc-receptor binding to antibody-sensitized target cells and subsequent ADCC.     

Reversible redox titrations of cytochrome c and cytochrome c oxidase using detergent solubilized electrochemically generated mediator-titrants

The repetitive, $\text{reversible}$ equilibrium redox cycling of cytochrome $\text{c}$, cytochrome $\text{c}$ oxidase, or mixtures thereof has been made possible by the use of the oxidant, ferricinium ion. This ion is electrochemically generated by the use of non-ionic detergent solubilized ferrocene which is apparently incorporated as micelles and readily electron transfers with an electrode. The $\text{ferricinium-ferrocene}$ couple equilibrates rapidly with these heme proteins. Electrochemically generated benzylviologen radical cations are used as the reductant. The E^O′ values for cytochrome $\text{c}$ oxidase at pH 7.0 are 209 ± 15 mv (2e^?) and 340 ± 15 mv (2e^?).     

Collagenase from rabbit pulmonary alveolar macrophages.

Rabbit pulmonary alveolar macrophages produce a collagenase which lyses labeled collagen gels, specifically cleaves collagen types I, II and III, is inhibited by ethylenediaminetetraacetate, cysteine, dithiothreitol and serum but is not inhibited by a serine protease inhibitor. Alveolar macrophage collagenase activity can be enhanced by $\text{in vivo}$ BCG activation, $\text{in vitro}$ latex, silica or mycobacterium activation and by $\text{in vitro}$ uncovering of latent enzymatic activity with trypsin treatment. The production of collagenase by unactivated alveolar macrophages and the presence of “latent” collagenase in culture media of alveolar macrophages are examples of significant differences between alveolar and peritoneal macrophages.     

Distribution of free sulfhydryl and disulfide groups among platelet membrane proteins

Reactive sulfhydryl and disulfide groups were identified in platelet membrane proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet membranes treated with $\text{N-}\text{ethyl}\text{[1?}{}^{14}\text{C}\text{]}\text{maleimide}$, phenyl[²⁰³Hg]mercuric acetate and $\text{p-}\text{chloro}\text{[}{}^{203}\text{Hg}\text{]}\text{mercuribenzoate}$ showed similar patterns of distribution of sulfhydryl groups among the sodium dodecyl sulfate-solubilized membrane proteins. Four major and two minor polypeptides ranging in molecular weight from > 200 000 to 20 000 were found to have reactive SH groups. Reduction of membrane proteins by sulfite coupled with subsequent mercaptide formation of the resultant monothiols led to the identification of four polypeptides with disulfide bonds. Reaction of platelet membranes with ¹⁴C-labeled 5,5′-dithio-bis(2-nitrobenzoic acid) resulted in changes in the distribution profile of the solubilized membrane proteins suggestive of a polymerization process dependent upon 5,5′-dithio-bis(2-nitrobenzoic acid)-induced intermolecular disulfide interchange.     

Release of the chloride-dependent arginine aminopeptidase from PMN leukocytes and macrophaces during phagocytosis

The zymosan particles induced a time-dependent release of the chloride-dependent arginine aminopeptidase from rat peritoneal macrophages during $\text{in}$$\text{vitro}$ incubations. Intraperitoneal injections of zymosan, a streptococcal cell preparation and a $\text{Micrococcu}$-suspension caused the release of the chloride-activated arginine aminopeptidase into the peritoneal fluid. The arginine aminopeptidases obtained both from the cell cultivation media and the peritoneal washes were partly purified. The enzymes were similar with regard to the following properties: chloride activation with an optimum at physiological concentrations; strong inhibition by 10^?6M $\text{p}$-chloromercuribenzoate; similar elution properties and preferential hydrolysis of mainly the $\text{N}$-L-aminoacyl-2-naphthylamines of arginine and lysine. The chloride-activated arginine aminopeptidase released into the media in $\text{in}$$\text{vitro}$ conditions was inactivated in contrast to the enzyme released into the peritoneal fluid as a result of the intraperitoneal injections. The timing of the release of the chloride-activated arginine aminopeptidase both $\text{in}$ and $\text{in}$$\text{vitro}$ suggests that the enzyme plays a role in the initial phases of inflammation.     

Effects of prostaglandin PGE2 on the synthesis of placental proteins and human placental lactogen (HPL)

The biosynthesis of placental proteins and placental lactogen (HPL) was studied $\text{in vitro}$ in 10–12 week, 16–18 week and term human placenta in the presence and absence of PGE_2α. The highest ¹⁴C-leucine incorporation was detected in 10 to 12 weeks old placentas. Addition of PGE_2α to the induction medium depressed the rate of incorporation of ¹⁴C-leucine into placental proteins on a dose dependent manner. Placentas most sensitive to this action of PGE_2α were those obtained at 18 weeks gestation followed by placentas at term. $\text{In vivo}$ application of PGE_2α for tharapeutic induction of abortions resulted in the marked inhibition of placental protein synthesis $\text{in vitro}$.     

Selective killing of Leishmania infected mouse macrophages by 6-methylpurine 2′-deoxyriboside

6-methylpurine 2′-deoxyriboside killed mouse macrophages infected with amastigotes of $\text{Leishmania donovani}$ and $\text{Leishmania mexicana}$, but did not affect the growth of non-parasitized cells. $\text{Leishmania}$ extracts cleaved the non-toxic 6-methylpurine 2′-deoxyriboside to 6-methylpurine, a potent adenine antimetabolite for mammalian cells. By eliminating macrophages latently infected with $\text{Leishmania donovani}$ amastigotes, 6-methylpurine 2′-deoxyriboside could augment the effects of leishmanicidal agents $\text{in vivo}$.     

Hormonal regulation of macrophage collagenase activity.

Whereas peritoneal macrophages from nonpregnant guinea pigs were stimulated $\text{in vitro}$ by endotoxin to produce collagenase on the second day of culture, those from pregnant guinea pigs were incapable of this response. However, if the cells from pregnant animals were preincubated for one day prior to endotoxin stimulation, collagenase activity could be detected. Injection of either estrogen or progesterone into guinea pigs at doses comparable to those found during pregnancy prior to removal of the peritoneal cells also inhibited the $\text{in vitro}$ stimulation of collagenase production. The addition of these hormones $\text{in vitro}$ revealed that at 5 × 10^?6 M estrogen and progesterone inhibited 53% and 100% respectively of the collagenase activity. Addition of both hormones at a final concentration of 5 × 10^?7 M of each inhibited 87% of the activity indicating a synergistic effect since this concentration of either hormone alone was ineffective.     

Comparative kinetic-ionic strength study of two differently charged cytochrome C: effects are limited to overall charge.

The reduction kinetics of two differently charged cytochromes $\text{c}$, horse cytochrome $\text{c}$ and $\text{Rhodosprillum rubrum}$ cytochrome $\text{c}{}_2$, by ferrous EDTA^2? were studied as a function of ionic strength. Since both proteins have nearly the same heme edge region, but have very different overall surface charge, this comparative study served as a direct test of the utility of small nonbinding non-physiological redox agents in the study of the charge of electron transfer sites of redox proteins. Calculations based on the ionic strength-kinetic data yielded protein charges of +10 and +2.3 for cytochrome $\text{c}$ and cytochrome $\text{c}{}_2$ respectively and compared well with values of +9 and +3 for the overall charge of the proteins based on acidic and basic amino acid residues. It is concluded that ionic strength effects upon the redox kinetics with such nonbinding nonphysiological redox agents reflect the influence of the overall protein charge and not the localized charge of the presumed site of electron transfer.     

Partial purification and properties of a non-ligandin (3H) 3-methylcholanthrene binding protein from liver cytosol

(³H) 3-Methylcholanthrene binds $\text{in vivo}$ to a macromolecule in addition to the previously reported binding to ligandin in liver cytosol. The properties of this second molecule are identical to those of the glucocorticosteroid receptor (Binder II) through 400 fold purification over the cytosol proteins (elution position from DEAE-Sephadex A-50 columns, molecular weight by gel filtration and pI value by isoelectrofocusing). The carcinogen, probably a metabolite, binds very strongly or covalently to the macromolecule $\text{in vivo}$, but non-covalently $\text{in vitro}$ in the absence of microsomes. Large amounts of unlabeled carcinogen administered $\text{in vivo}$ do not compete significantly with subsequent (³H) dexamethasone binding to the hormone receptor fraction $\text{in vitro}$. Methylcholanthrene and dexamethasone do not compete for binding sites $\text{in vitro}$ on isolated unlabeled Binder II leading to the conclusion that the glucocorticosteroid receptor and the methylcholanthrene binding protein are distinct entities.     

Phosphorylation and acetylation of nonhistone chromosomal proteins of the brain of rats of various ages and their modulation by calcium and estradiol.

$\text{In}\text{vitro}$ phosphorylation and acetylation of nonhistone chromosomal (NHC) proteins and their modulation by Ca⁺⁺ and estradiol were studied by incubating slices of cerebral cortex of 2-, 15- and 84-week female rats with ³²Pi and ¹⁴C-Na-acetate. Phosphorylation pattern of NHC proteins is unique for each age. Ca⁺⁺ and estradiol stimulate phosphorylation of different NHC proteins which is also age-specific. Acetylation of NHC proteins decreases precipitously with age. No unique NHC protein is acetylated preferentially at any age, nor does Ca⁺⁺ stimulate acetylation. Estradiol, however, stimulates acetylation of a few NHC proteins. It is suggested that phosphorylation of NHC proteins and its modulation by effectors may be more important for gene expression than their acetylation.     

Replacement of the B protein requirement of the E. coli quinolinate synthetase system by chemically-generated iminoaspartate

Two proteins (A and B) from $\text{Escherichia}\text{,}\text{coli}$ are required for $\text{in}\text{,}\text{vitro}$ synthesis of the NAD⁺ precursor, quinolinate, from L-aspartate and dihydroxyacetone phosphate. The requirement for B protein and L-aspartate in this system can be replaced by millimolar concentrations of oxaloacetate and ammonia if they are added together. This finding supports the concept that the B protein (L-aspartate oxidase) functions to form iminoaspartate which is condensed with dihydroxyacetone phosphate by the A protein to form quinolinate.