Protein synthesis by attached pulmonary macrophages. Effect of phagocytosis

We studied the effect of phagocytosis of polystyrene latex beads on protein synthesis by pulmonary macrophages. To do this we determine the specific radioactivity of extracellular and intracellular free phenylalanine and of phenylalanine released from tRNA and used this information in calculating the rates of protein synthesis. Phagocytosis resulted in an increased rate of protein synthesis irrespective of which precursor specific radioactivity was used in the calculation. The rate of protein synthesis was increased per microgram polyribosomal RNA; but there was no increase in the amount of polyribosomal RNA in phagocytizing macrophages. The increase in the rate of protein synthesis (1.4-fold) was almost identical to the increase (1.3-fold) in the rate of ribosome transit in phagocytizing compared to nonphagocytizing macrophages. The decreased ribosome transit time during phagocytosis occurred without a fall in the average molecular weight of macrophage proteins. We conclude that phagocytosis increases the rate of protein synthesis in attached pulmonary macrophages and that this increased rate of synthesis can be accounted for almost completely by an increased rate of polypeptide chain elongation and/or termination.     

The effect of aging on cell-free protein synthesis in the free-living nematode Turbatrix aceti

The age-related reduction in cell-free synthesis in the free-living nematode Turbatrix aceti is due to a defect in the ribosomes. Addition of young ribosomal wash or use of young medium does not improve the activity of old, run-off ribosomes in the presence of phenylalanine and poly(U). It appears that some of the old ribosomes are incapable of binding the EF-1-GTP-aminoacyl-tRNA complex. These ineffective ribosomes are present in the 80 S (monosomal) fraction. Old ribosomes obtained from polysomes appear to bind normally.     

Adhesion,phagocytosis and cell surface energy. The binding of fixed human erythrocytes to rat macrophages and polymethylpentene

Fixed human erythrocytes were used as model particles for the study of adhesion and phagocytosis by rat peritoneal macrophages. Erythrocytes were fixed with various concentrations of glutaraldehyde or tannic acid, or were treated with neuraminidase. Adhesion and phagocytosis of these cells were measured. In addition, the surface energy of these erythrocytes and macrophages was estimated by the contact angle technique. Free energies of adhesion, based on the cell surface energies, were correlated with both adhesion and phagocytosis.     

Translocation of nucleolar phosphoprotein B23 ( ) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

The hormonal control of protein N-glycosylation in the developing rabbit mammary gland and its effect upon transferrin synthesis and secretion

Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [³H]leucine or [³H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [³H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+ prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [³H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [³H]mannose and [¹⁴C]leucine confirmed this. The decreased incorporation of [³H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does.     

The stability of rabbit reticulocyte polysomes at high hydrostatic pressure

The application of high hydrostatic pressure to an in vitro rabbit reticulocyte, polypeptide-synthesizing system has been shown to inhibit synthesis either partially or totally depending upon the magnitude of pressure utilized (Scheck, A.C. and Landau, J.V. (1982) Biochim. Biophys. Acta 718, 21–25). This paper shows that the total inhibition of synthesis seen at 670 atm is similar to the inhibition of elongation produced by cycloheximide in that the polysome profiles remain intact. Partial inhibition at 300 atm shows a reduced rate of ribosome run-off with elongation being affected to the same, or greater extent than initiation. In no instance was disassociation of polysomes seen as a causative factor in the inhibition of synthesis by high pressure.     

Protein synthesis in cell-free extracts of Dictyostelium discoideum

We have developed an in vitro translation system for the lower eukaryote Dictyostelium discoideum. Active extracts using endogenous mRNA support protein synthesis with optimal Mg²⁺ and K⁺ concentrations of 5 mM and 120 mM, respectively. [³⁵S]Methionine incorporation is linear for more than 2 h. Polypeptides synthesized from endogenous mRNA have sizes ranging from less than 20 to over 100 kDa. Heat-shock proteins are synthesized in vitro in extracts prepared from heat-shocked cells. Possible uses of this system for study of translational control during growth and differentiation are discussed.     

Role of flagellum and chemotactic motility of Vibrio anguillarum for phagocytosis by and intracellular survival in fish macrophages

The role of the flagellum and chemotactic motility of Vibrio anguillarum for phagocytosis by and intracellular survival in fish macrophages was determined using a wild-type strain, a mutant without the flagellum, a mutant with a truncated flagellum and a non-chemotactic mutant. For all strains, the numbers of intracellular bacteria were relatively low and fell steadily during the observation period. The presence of a flagellum did not influence the uptake by the macrophages, but the smooth swimming phenotype of a non-chemotactic mutant increased its intracellular presence. We suggest that this is due to an increased collision between the mutant and the macrophage, due to a higher average speed of the non-chemotactic mutant.     

The effect of high hydrostatic pressure on eukaryotic protein synthesis

The pressure response of two eukaryotic protein synthesizing systems has been characterized. The rabbit reticulocyte system has been tested, both in vivo and in vitro, using endogenous polysomes and polyuridylic acid (poly U). In addition, the poly U-directed polyphenylalanine synthesizing system obtained from wheat germ was utilized. The effect of pressure on eukaryotic protein synthesis has been found to be basically similar to that observed in prokaryotic systems, although the response of the eukaryotic protein synthesizing system is somewhat more complex signifying a greater influence of overlapping reactions. Magnesium was found to affect eukaryotic systems in much the same way as has been reported for prokaryotic systems, i.e., increasing the Mg²⁺ concentration in a protein synthesizing system increases the barotolerance exhibited by that system. Under conditions of high Mg²⁺ concentration, however, extreme (up to 160%) stimulation of protein synthesis at lower pressure levels was observed in the eukaryotic systems. Such high stimulation is not apparent in prokaryotic systems. The poly U-directed wheat germ system exhibited the most barotolerant polypeptide synthesis ever seen in our laboratory. This extreme barotolerance was only slightly decreased when the system was tested at reduced concentrations of magnesium.     

Enhancement of phagocytosis by calcitonin gene-related peptide (CGRP) in cultured mouse peritoneal macrophages

Calcitonin gene-related peptide (CGRP) is widely distributed in sensory neurons and nerve fibers. To clarify the function of CGRP on the immune system, the effect of CGRP on phagocytosis by peritoneal mactophages was examined by means of flow cytofluorometry. CGRP enhanced phagocytosis of latex beads in a dose-dependent manner. Because the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) enhanced the CGRP-induced enhancement of phagocytosis, the enhancement might be mediated by cAMP. In the presence of mannan, the phagocytosis was suppressed and the CGRP-induced enhancement was also blocked, suggesting that mannose receptors on macrophages were involved in mediating the phagocytosis of latex beads, and CGRP enhanced the mannose receptor-mediated phagocytosis. The present results indicate that CGRP can modulate the function of macrophages in nerve terminals of sensory neurons during the development and maintenance of inflammation.     

Transferrin synthesis by macrophages: up-regulation by γ-interferon and effect on lymphocyte proliferation

Abstract We have investigated transferrin synthesis by human and mouse lymphoid and myeloid cells. It was found that transferrin synthesis is a property of mouse but not human macrophages, whereas in man T lymphocytes synthesised transferrin. Synthesis by mouse macrophages showed a dose-dependent increase in response to γ-interferon (γ-IFN), but iron added as ferric nitrilotriacetate had no effect. Macrophage-derived transferrin was found to contain iron already bound to it and was able to support Con A-stimulated mouse lymphocyte proliferation.     

Fate of ecto-NAD+ glycohydrolase during phagocytosis of normal and mannosylated latex beads by murine macrophages

In order to gain a better understanding of the role of ecto-NAD+ glycohydrolase, an enzyme predominantly associated with phagocytic cells, we have studied its fate in murine macrophages (splenic, resident peritoneal and Kupffer cells) during phagocytosis of opsonized on mannosylated latex beads. In parallel, we have also monitored nucleotide pyrophosphatase, another ecto-enzyme of macrophages. Phagosomes were isolated by flotation in a discontinuous sucrose gradient and the enzyme activities were determined with fluorometric methods. Low levels of NAD+ glycohydrolase and nucleotide pyrophosphatase could be measured associated with the phagosomal fractions, eg, respectively less than 4.5% and 10% in spleen macrophages. The phagosomal activities originate from the plasma membrane, ie they were latent and inactivation of ecto-NAD+ glycohydrolase with the diazonium salt of sulfanilic acid resulted in a marked decrease of this enzyme activity in the phagosomal fractions. Pre-labelling of the cell surface by [3H]-galactosylation indicated that NAD+ glycohydrolase is internalized to a lesser extent than an average surface-membrane unit. These results indicate that if ecto-NAD+ glycohydrolase of macrophages can be internalized to a limited extent during phagocytosis of opsonized or mannosylated latex beads, this enzyme appears to be predominantly excluded from the surface area involved in the uptake of such particles.     

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Inhibition of hepatocytic protein degradation by methylaminopurines and inhibitors of protein synthesis

Nutritional control of protein degradation in isolated rat hepatocytes can take place in the absence of protein synthesis. Suppression of degradation by amino acids (step-up) is unaffected and the enhanced degradation seen upon amino acid deprivation (step-down) is only partially inhibited by cycloheximide at a concentration (10^?3 M) which inhibits protein synthesis virtually completely. Protein degradation per se is, however, inhibited by cycloheximide as well as by puromycin, apparently at least in part by mechanisms additional or unrelated to their effect on protein synthesis. Several puromycin analogues (methylaminopurines) are stronger inhibitors of protein degradation than of protein synthesis, most notably puromycin aminonucleoside and 6-dimethylaminopurine riboside (N⁶, N⁶-dimethyladenosine). The latter compounds appear to specifically inhibit cellular autophagy, since neither the degradation of endocytosed protein (asialofetuin) nor the extralysosoma (amino acid-, propylamine- and leupeptin-resistant) degradation are affected.     

Retention of actin synthesis in liver under conditions that inhibit synthesis of almost all other proteins

As briefly reported [(1986) Fed. Proc. 45, 1771, Abstr. 1690], rats fed a protein-free diet for a few days often show a marked inhibition of protein synthesis in liver cytosol. However the synthesis of a protein of molecular mass approximately 42 kDa is fully retained. We show here on the basis of its molecular mass, number of bands on isoelectric focusing, isoelectric point and immunological reactivity that this protein is actin and also that actin mRNA is not degraded by micrococcal nuclease under conditions which degrade the bulk of other mRNAs.     

CD47 promotes both phosphatidylserine-independent and phosphatidylserine-dependent phagocytosis of apoptotic murine thymocytes by non-activated macrophages

The ubiquitously expressed cell surface glycoprotein CD47 on host cells can inhibit phagocytosis of unopsonized or opsonized viable host target cells. Here we studied the role of target cell CD47 in macrophage uptake of viable or apoptotic murine thymocytes. As expected, IgG-opsonized viable CD47^−/− thymocytes were taken up more efficiently than equally opsonized Wt thymocytes. However IgG-opsonized apoptotic thymocytes from Wt and CD47^−/− mice were taken up equally. Although uptake of apoptotic thymocytes by non-activated bone marrow-derived macrophages was phosphatidylserine (PS)-independent, while uptake by non-activated resident peritoneal macrophages was PS-dependent, both macrophage populations showed a reduced uptake of non-opsonized apoptotic CD47^−/− thymocytes, as compared with the uptake of apoptotic Wt thymocytes. This difference was only seen with non-activated macrophages, and not with β-1,3-glucan-activated macrophages. CD47 promoted binding of thymocytes to macrophages, which did not require F-actin polymerization. CD47 became clustered on apoptotic thymocytes, both co-localized with or separated from, clustered PS and cholesterol-rich GM-1 domains. Thus, CD47 does not inhibit, but rather support, both PS-independent and PS-dependent uptake of apoptotic cells in the murine system. This mechanism only comes into play in non-activated macrophages.     

Protein synthesis by intracellular symbionts in two closely interrelated aphid species

An aphid endosymbiont in vivo synthesizes symbionin almost exclusively which is not produced in vitro by the same symbiont. While symbionin produced by the endosymbiont of the pea aphid is an acidic protein with a molecular weight of 63,000, that by the symbiont of the kondo aphid, the closest relative to the former, is a distinct, less acidic, molecule. While the two endosymbionts in vivo in old insects synthesize about 11 protein species in common, they produce many different proteins when incubated extracellularly.     

Modulation of protein kinase C activity by NaF in bone marrow derived macrophages

Stimulation of murine bone marrow derived macrophages with NaF, prelabeled with [1-¹⁴C]oleate and [³H]inositol, increased the production of inositol phosphates and the release of 1,2-[¹⁴C]diacylglycerol (DAG). Moreover, NaF also induced activation of protein kinase C. These results indicate that bone marrow derived macrophages exhibit a phosphatidyl-4,5-bisphosphate phospholipase C activity, sensitive to NaF, which might be modulated by G-proteins. Activation of protein kinase C could have been mediated by NaF-induced release of DAG.     

The C-terminal end of P proteins mediates ribosome inactivation by trichosanthin but does not affect the pokeweed antiviral protein activity

Ribosome inactivating proteins (RIPs) inhibit protein synthesis depurinating a conserved residue in the sarcin/ricin loop of ribosomes. Some RIPs are only active against eukaryotic ribosomes, but other RIPs inactivate with similar efficiency prokaryotic and eukaryotic ribosomes, suggesting that different RIPs would interact with different proteins. The SRL in Trypanosoma cruzi ribosomes is located on a 178b RNA molecule named 28Sδ. In addition, T. cruzi ribosomes are remarkably resistant to TCS. In spite of these peculiarities, we show that TCS specifically depurinate the predicted A⁵¹ residue on 28Sδ. We also demonstrated that the C-terminal end of ribosomal P proteins is needed for full activity of the toxin. In contrast to TCS, PAP inactivated efficiently T.cruzi ribosomes, and most importantly, does not require from the C-terminal end of P proteins. These results could explain, at least partially, the different selectivity of these toxins against prokaryotic and eukaryotic ribosomes.     

Translation of poly(U) on ribosomes from Streptomyces aureofaciens

Slowly cooled cells of Streptomyces aureofaciens contained mainly tight-couple ribosomes. Maximum rate of polyphenylalanine synthesis on ribosomes of S. aureofaciens was observed at 40°C, while cultures grew optimally at 28°C. Ribosomes of S. aureofaciens differed from those of E. coli in the amount of poly(U) required for maximum synthetic activity. The polyphenylalanine-synthesizing activity of E. coli ribosomes was about 3-times higher than that of S. aureofaciens ribosomes. The addition of protein S1 of E. coli or the homologous protein from S. aureofaciens had no stimulatory effect on the translation of poly(U). In order to localize alteration(s) of S. aureofaciens ribosomes in the elongation step of polypeptide synthesis we developed an in vitro system derived from purified elongation factors and ribosomal subunits. The enzymatic binding of Phe-tRNA to ribosomes of S. aureofaciens was significantly lower than the binding to ribosomes of E. coli. This alteration was mainly connected with the function of S. aureofaciens 50 S subunits. These subunits were not deficient in their ability to associate with 30 S subunits or with protein SL5 which is homologous to L7/L12 of E. coli.