Isoelectric focusing--polynucleotide/polyacrylamide-gel electrophoresis. A technique to separate and characterize nuclease activities.

Individual native nuclease activities from human leucocytes are separated by using two-dimensional gel electrophoresis in an apparatus that allows the simultaneous running of 28 gels. Proteins are separated by isoelectric focusing in a disc gel, followed by electrophoresis into a slab gel containing DNA. Protein denaturants are avoided in the second dimension by the use of a running pH well above the optimal pH for DNAase (deoxyribonuclease) activity. Electrophoresed gels are incubated in appropriate buffers to activate nuclease activity. After staining for intact DNA, the positions of active enzymes, unobscured by the presence of other proteins, are revealed as colourless spots in a reddish-purple field. The technique is easy to use and is sensitive to 50pg of DNAase I. Versatility is provided by the use of either acidic or basic electrophoresis running buffers and by the use of specific gel incubation conditions to reveal different sets of enzyme activities. Two DNAases active at pH 7.4 in the presence of Mg2+ and Ca2+, and sixteen DNAases active at acidic pH and not requiring metals, are detected. Treatment of the human enzymes with specific glycosidases reveals that many of the human DNAases are glycoproteins containing negatively charged moieties and may be derived from modification of parent activities.     

甘蔗和烟草幼叶原生质体的核酸含量与核酸酶活性及其影响因素

与幼叶组织相比,酶法新鲜分离的甘蔗和烟草幼叶原生质体内的ＲＮＡ、ＤＮＡ及总核酸含量均降低。其原因可能是刚游离的原生质体内酸性和碱性ＲＮＡ酶与ＤＮＡ酶等活性提高所致。甘蔗叶原生质体内的核酸降低量和ＲＮＡ酶与ＤＮＡ酶活性的增加程度均高于烟草。随用作渗透压稳定剂的甘露醇浓度增加,甘蔗和烟草叶原生质体的ＲＮＡ酶和ＤＮＡ酶活性均相应提高。其中以甘蔗叶原生质体的核酸酶活性增加水平较明显。在细胞壁降解产物的作用下,除了甘蔗原生质体内的ＲＮＡ酶活性略被促进外,其ＤＮＡ酶和烟草叶原生质体内的核酸酶均不受影响     

Peripheral hyaline blebs (podosomes) of macrophages

The plasmalemma and hyaline ectoplasm together constitute the sensory and motor organ of macrophages. The purpose of this study was to isolate this cell fraction in order to analyze it biochemically and functionally. Brief sonification of warmed rabbit lung macrophages caused release of heterodisperse hyaline blebs and filopodia, which were easily collected by differential centrifugation. Viewed in the electron microscope, these structures consisted of membrane-bounded sacs principally containing actin filaments. Some contained secondary lysosomes. They were enriched threefold over whole cell homogenates in specific adenylate cyclase activity and in trichloroacetic-acid-precipitable (125)I when derived from cells labeled with 125(I) by means of a lactoperoxidase-catalyzed reaction. These markers were found to have identical isopycnic densitites when macrophage homogenates were subjected to sedimentation in a focusing sucrose density gradient system, and these markers had densities distinct from those of other cytoplasmic organelles. These markers were therefore assumed to be associated with macrophage plasma membranes. The specific β- glucuronidase activity of the bleb fraction was similar to that of homogenates, but the blebs had considerably lower specific succinic dehydrogenase activity and RNA content, and DNA was undetectable. Electrophoresis of blebs solubilized in sodium dodecyl sulfate on polyacrylamide gels revealed polypeptides co-migrating with macrophage actin-binding protein, myosin, and actin; blebs also had EDTA-activated adenosine triphosphatase activity characteristic of myosin. The concentrations of actin-binding protein and myosin were higher in blebs than in cells or cytoplasmic extracts, whereas actin concentrations were similar (relative to extracts) or only slightly greater (than in cells). Blebs and intact cells had high lactate dehydrogenase activities in the presence but not the absence of Triton X-100. Blebs and cells oxidased 1-[(14)C]glucose, and the rate of glucose oxidation was increased substantially in the presence of latex beads. We conclude that intact sacs of plasmalemma encasing contractile proteins and cytoplasmic enzymes can be isolated from macrophages. They are enriched in myosin and actin-binding protein, indicating that the contractile apparatus is regulated in the cell periphery. These structures have the capacity to respond to environmental signals. We suggest the name "podosomes" for them because of their resemblance to macrophage pseudopodia. We propose that podosome formation results from rapid dissolution of the cortical gel when the membrane is in an actively extended configuration.     

Purification and characterization of membrane-bound phospholipase C specific for phosphoinositides from human platelets

Two peaks (mPLC-I and mPLC-II) of phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity were resolved when 1% sodium cholate extract from particulate fractions of human platelet was chromatographed on a heparin-Sepharose column. The major peak of enzyme activity (mPLC-II) was purified to homogeneity by a combination of Fast Q-Sepharose, heparin-Sepharose, Ultrogel AcA-44, Mono Q, Superose 6-12 combination column, and Superose 12 column chromatographies. The specific activity increased 2,700-fold as compared with that of the starting particulate fraction. The purified mPLC-II had an estimated molecular weight of 61,000 on sodium dodecyl sulfate-polyacrylamide gels. The minor peak of enzyme activity (mPLC-I) was partially purified to 430-fold. Both enzymes hydrolyzed PIP2 at low Ca2+ concentration (0.1-10 microM) and exhibited higher Vmax for PIP2 than for phosphatidylinositol. PIP2-hydrolyzing activities of both enzymes were enhanced by various detergents and lipids, such as deoxycholate, cholate, phosphatidylethanolamine, and dimyristoylphosphatidylcholine. The mPLC-I and mPLC-II activities were increased by Ca2+, but not by Mg2+, while Hg2+, Fe2+, Cu2+, and La3+ were inhibitory. GTP-binding proteins (Gi, Go, and Ki-ras protein) had no significant effects on the mPLC-II activity.     

Generation of alloreactive cytotoxic T lymphocytes: production of T cell and macrophage helper factors in addition to IL 1 and IL 2 by peritoneal cells from mice immunized to Listeria monocytogenes

We investigate the production and biological activity of soluble helper factors produced by peritoneal T cells and macrophage derived from mice primed in vivo with Listeria monocytogenes. Supernatant fluids from co-cultures of these immune T cells and activated macrophages contained Interleukin 1 (IL 1) and Interleukin 2 (IL 2), and had the ability to assist the generation of cytotoxic T lymphocytes (CTL) from a population of nylon wool nonadherent spleen cells sensitized to allogeneic heat-treated thymocytes. The ability to assist CTL development involved T cell and macrophage factors in addition to IL 1 and IL 2. Immune T cells cultured alone produced a factor, devoid of significant IL 2 activity, that assisted CTL development only if adherent cells were present in the responding population. Activated macrophage produced a 38,000 dalton component, distinct from IL 1 on the basis of m.w., that assisted the development of CTL from nylon wool nonadherent splenic cells. Supernatants fluids from co-cultures of immune T cells and allogeneic, nonactivated macrophage contained a CTL helper factor but did not contain IL 1 or IL 2 activities. In contrast, supernatant fluids from co-cultures of immune T cells and syngeneic, nonactivated macrophage contained all 3 activities. This suggests a genetic restriction for the production of IL 1 and IL 2 that does not restrict the production of a CTL helper factor. These results demonstrate that T cell- and macrophage-derived helper factors distinct from IL 1 and IL 2 participate in the development of CTL.     

Granules of human CD3+ large granular lymphocytes contain a macrophage regulating factor(s) that induces macrophage H2O2 production and tumoricidal activity but decreases cell surface Ia antigen expression.

CTL (cytotoxic T lymphocytes) and LGL (large granular lymphocytes) exocytose cytoplasmic granules on activation after recognition of their target, releasing granule-associated molecules. We have previously suggested that this process could release immunoregulatory molecules. In this study we investigated whether normal human LGL granules contained a factor regulating different macrophage activity. Human CD3+ LGL cells were generated by activating peripheral blood lymphocytes (PBL) for 10-12 days with recombinant human IL-2 (rhIL-2), and granules were isolated from disrupted cell homogenate by Percoll gradient fractionation. Solubilized granules were tested for macrophage-activating factor (MAF) activity in three different macrophage assays. When M-CSF-differentiated murine bone marrow-derived macrophages were incubated 9 hr with human LGL granules, they were fully activated to lyse the TNF-resistant P815 tumor cells. The granule-MAF showed a synergistic effect with rhIL-1 beta, rmTNF-alpha, and rmIFN-tau in the cytolytic assay. In addition, proteose-peptone-elicited murine peritoneal macrophages profoundly increased H2O2 production after activation with human LGL granules. However, unlike IFN-tau, no increase in peritoneal macrophage Ia antigen expression was detected after incubation with granules. Moreover, granule-MAF suppressed Ia induction by IFN-tau. These results confirm that human CD3+ LGL granules contain a molecule(s) capable of regulating macrophage function.     

细虫草胞外多糖对小鼠腹腔巨噬细胞免疫功能研究

本实验在体外条件下,以人工发酵培养的细虫草胞外多糖OgE、OgE-F1和OgE-F2作用于小鼠腹腔巨噬细胞RAW264.7,通过测定其对巨噬细胞的增殖率、代谢MTT活力、NO分泌和吞噬能力的影响,评价细虫草胞外多糖的免疫调节活性。结果表明,细虫草多糖对巨噬细胞无细胞毒性,且能促进巨噬细胞代谢MTT活力;在0.2mg/mL^1.0mg/mL浓度范围内,多糖呈剂量依赖性的促进巨噬细胞分泌NO水平和吞噬能力。本研究表明,细虫草多糖能有效地增强小鼠巨噬细胞的活性,潜在地可改善小鼠的先天性免疫调节。     

Tetrameric manganese superoxide dismutases from anaerobic Actinomyces

Superoxide dismutase was isolated from each of the anaerobically grown organisms Actinomyces naeslundii, Actinomyces strain E1S.25D, and Actinomyces odontolyticus. The enzymes were 100,000-110,000 mol wt acidic proteins (pI 4.3-4.6) and contained Mn and Zn, but no detectable Fe. The Mn and Zn content varied with the enzyme source. A. naeslundii superoxide dismutase, specific activity 2200 U/mg, contained 2.3 g atoms Mn and 1.4 g atoms Zn per mole tetramer whereas A. odontolyticus SOD, specific activity 700 U/mg, contained 1.4 g atoms Mn and 1.8 g atoms Zn per mole tetramer. Actinomyces strain E1S.25D, specific activity 1300 U/mg, contained 1.8 g atoms Mn and 1.2 g atoms Zn per mole tetramer. The amino acid compositions of the enzymes were comparable except for arginine, lysine, and tryptophan content. The enzymatic activity of each enzyme was stable in 5 mM H2O2 at 23 degrees C for 2 h. The enzymes were only modestly inhibited by 20 mM NaN3. The enzymatic activity was increased at low ionic strength but was markedly decreased at increased ionic strength with each salt tested except sodium perchlorate, which caused marked inhibition even at low ionic strength. Polyclonal antibodies to A. naeslundii and Actinomyces strain E1S.25D precipitated and inactivated their respective antigens whereas the precipitated A. odontolyticus superoxide dismutase-antibody complex retained virtually full catalytic activity. Immunological studies revealed that the native A. naeslundii and Actinomyces strain E1S.25D MnSODs share common epitopes and cross-reacted with precipitin lines of complete identity in Ouchterlony double diffusion gels. Antibody to the A. odontolyticus enzyme displayed only partial cross-reactivity with superoxide dismutase from the two other Actinomyces. Western blotting of the denatured antigens revealed reactivities of the antibodies that differed only slightly from the results of the Ouchterlony gels.     

Distribution of lysosomal enzymes in different types of rat liver cells.

Eight lysosomal enzymes were measured in different types of rat liver cells. Hepatocytes were purified by low speed centrifugation of a cell suspension obtained by treating the perfused liver with collagenase. Nonparenchymal cells (NPC) were purified by centrifugation after treating the initial cell suspension with pronase, which selectively destroys the parenchymal cells (PC). Kupffer cells were found to attach selectively to tissue culture dishes after overnight culture of an NPC suspension. The specific activity of lysosomal enzymes was generally higher in NPC than in hepatocytes, but the different enzymes were concentrated to different degrees in the NPC. Specific activity of acid phosphatase was 1.7 times higher in NPC than in hepatocytes. Specific activity of acid DNAase, on the other hand, was 8 times higher in NPC than in hepatocytes. Other enzymes showed intermediate values. Assuming that 30% of the liver cells are nonparenchymal it may be calculated that from 7% (acid phosphatase) to 25% (acid DNAase) of the hepatic lysosomal enzymes are present in the NPC. The pattern of lysosomal enzymes in cultured Kupffer cells was similar to that of the NPC from which the Kupffer cells were derived. Cathepsin D and β-glucuronidase were, however, elevated in Kupffer cells as compared with NPC. The enzyme pattern in Kupffer cells was almost identical with that of rat peritoneal macrophages.     

Cathepsin B and D activity in stimulated peritoneal macrophages

Summary Highly sensitive and specific synthetic substrates were used to quantitate cathepsin B and D activity in peritoneal macrophages in response to stimulation in vivo with mineral oil and thioglycollate. After intraperitoneal instillation of mineral oil the activity of cathepsin B increased significantly (to 15 300 units/mg protein versus 7 340 in saline controls), reaching values approaching those found in alveolar macrophages (18 400 units/mg protein). Significantly greater stimulation of enzyme activity was obtained after intraperitoneal instillation of thioglycollate (23 600 units/mg protein). Cathepsin D activity also increased significantly after both mineral oil and thioglycollate. However, the increase was moderate (from 806 to about 1 200 units/mg protein), remaining still more than six times lower-than in alveolar macrophages. The data are the first to demonstrate that cathepsin B activity can be stimulated in vivo in peritoneal macrophages by instillation of agents that induce acute inflammation. They also point to a differential control of expression of cathepsin B and D activity in both peritoneal and alveolar macrophages in spite of the common lysosomal origin of the two enzymes.Abbreviations Cbz -N-benxyloxycarbonyl - 2NA 2-naphthylamine - EDTA ethylenediamine tetraacetate - DMSO dimethylsulfoxide - PBS phosphate-buffered saline - PM peritoneal macrophage - AM alveolar macrophage     

Methanol dissimilation in Xanthobacter H4-14: activities, induction and comparison to Pseudomonas AM1 and Paracoccus denitrificans

Methanol dissimilatory enzymes detected in the methanol autotroph Xanthobacter H4-14 were a typical phenazine methosulphate-linked methanol dehydrogenase, a NAD+-linked formate dehydrogenase, and a dye-linked formaldehyde dehydrogenase that could be assayed only by activity stains of polyacrylamide gels. This same methanol dehydrogenase activity was found in ethanol-grown cells and was apparently utilized for ethanol oxidation. Formaldehyde dehydrogenase activities were investigated in Paracoccus denitrificans, Xanthobacter H4-14, and Pseudomonas AM1. P. denitrificans contained a previously reported NAD+-linked, GSH-dependent activity, but both Xanthobacter H4-14 and Pseudomonas AM1 contained numerous activities detected by activity stains of polyacrylamide gels. Induction studies showed that in Xanthobacter H4-14, a 10 kDal polypeptide, probably a dehydrogenase-associated cytochrome c, was co-induced with methanol dehydrogenase, but the formaldehyde and formate dehydrogenases were not co-regulated. Analogous induction experiments revealed similar patterns in P. denitrificans, but no evidence for co-regulation of dissimilatory activities in Pseudomonas AM1.     

Studies on deoxyribonucleases from Haemophilus influenzae on DNA agarose affinity chromatography. Two-step purification of ATP-dependent deoxyribonuclease.

In a first part of this report, purification and characterization of several nucleased from lysates of Haemophilus influenzae are described. The enzymes bind to DNA with agarose columns and are removed by elution with phosphate buffer. Among the considered enzymes, the exonucleases 1 and 3, and endonuclease, a DNA polymerase and a restriction enzyme were recovered mixed by raising the phosphate concentration from 0.1 to 0.3 M, while the ATP-dependent DNAase recovered well purified, by raising the phosphate concentration to 0.45 M. After a rechromatography, on a second DNA with agarose column, of the peak of the ATP-dependent DNAase, the specific activity tested with 3H-labeled DNA was 125 units/mg of protein, representing a 300-fold purification of the original crude extract. In a second part, we have investigated the inactivation, at various pH, of transforming DNA of Haemophilus influenzae wild strain Rd with the different eluted fractions of the column, in order to determine the importance of contamination with other enzymatic activities, and also in order to confirm the nature of theisolated enzymes with a biological method. Finally, with enzymatic extracts of mutant strain Rd com minus 56, a strain which integrates shorter than normal pieces of DNA and which is suspected to possess and "activated specific endonuclease" able to recognize even small conformational modifications in paired structures, we tried to detect this activity on artificially constructed heteroduplex regions in DNA.     

Multiple DNA repair activities for 3''-deoxyribose fragments in Escherichia coli.

Escherichia coli contains multiple enzymes that hydrolyze deoxyribose fragments (phosphoglycolaldehyde, PGA) from the 3' termini of a synthetic DNA substrate. The major such activities are the main bacterial apurinic endonucleases, exonuclease III and endonuclease IV. In a double mutant deficient in both of these oxidation repair enzymes, Mg++-dependent 3'-PGA diesterase was detected at 3% the level found in wild-type bacteria. Gel filtration fractionated this residual diesterase activity into two peaks of Mr 40,000-52,000 (Pool A) and Mr 22,000-30,000 (Pool B) with differing abilities to remove 3'-phosphates from DNA. These multiple repair activities were resolved in 3'-PGA diesterase activity gels. The exonuclease III and endonuclease IV bands were identified using the purified proteins and by their specific absence from strains defective for the respective structural genes. Gel filtration Pool B yielded two activity bands of apparent Mr 25,000 and 28,000, but Pool A did not form a new band in the activity gels. Incubation of activity gels in different transition metals or boiling of the samples before electrophoresis also served to distinguish the various activities. The possible identities of the novel E. coli 3'-PGA diesterases and the importance of multiple repair enzymes for 3' damages are discussed.     

Subcellular redistribution of newly synthesized macrophage lysosomal enzymes. Correlation between delivery to the lysosomes and maturation

Cultured mouse peritoneal macrophages were fractionated by two methods at various times after pulse labeling with [35S]methionine. The lysosomal enzymes beta-glucuronidase and beta-galactosidase were isolated from each fraction by immunoprecipitation and electrophoresis on sodium dodecyl sulfate-acrylamide gels. Two distinct peaks of label were obtained on Percoll density gradients. An early appearing peak of low density, containing the precursor forms of both enzymes, co-sedimented with markers for the endoplasmic reticulum, the Golgi apparatus, and the plasma membrane. With time, immunoprecipitable label cosedimented with the bulk of the lysosomal enzyme activity at high density and corresponded to the mature forms of the lysosomal enzymes. By differential centrifugation, newly synthesized enzymes were found predominantly in small particle fractions, unlike the bulk of the lysosomal enzymic activity which was found in larger particle fractions. With increasing time, newly synthesized enzymes were transferred to assume a distribution similar to that of lysosomal enzymic activity. The results suggest that transport of newly synthesized enzymes to lysosomes and conversion to mature forms are closely linked events. Conversion of lysosomal precursors to mature forms occurs either in a prelysosomal vesicle or shortly after reaching the lysosome. The two enzymes follow similar subcellular pathways at similar rates. Also, the macrophage system appears suitable for direct analysis of newly synthesized lysosomal enzymes during subcellular transport.     

Characterization of DNA metabolizing enzymes in situ following polyacrylamide gel electrophoresis

We have detected the in situ activities of DNA glycosylase, endonuclease, exonuclease, DNA polymerase, and DNA ligase using a novel polyacrylamide activity gel electrophoresis procedure. DNA metabolizing enzymes were resolved through either native or SDS-polyacrylamide gels containing defined 32P-labeled oligonucleotides annealed to M13 DNA. After electrophoresis, these enzymes catalyzed in situ reactions and their [32P]DNA products were resolved from the gel by a second dimension of electrophoresis through a denaturing DNA sequencing gel. Detection of modified (degraded or elongated) oligonucleotide chains was used to locate various enzyme activities. The catalytic and physical properties of Novikoff hepatoma DNA polymerase beta were found to be similar under both in vitro and in situ conditions. With 3'-terminally matched and mismatched [32P]DNA substrates in the same activity gel, DNA polymerase and/or 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I (large fragment), DNA polymerase III (holoenzyme), and exonuclease III were detected and characterized. In addition, use of matched and mismatched DNA primers permitted the uncoupling of mismatch excision and chain extension steps. Activities first detected in nondenaturing activity gels as either multifunctional or multimeric enzymes were also identified in denaturing activity gels, and assignment of activities to specific polypeptides suggested subunit composition. Furthermore, DNA substrates cast within polyacrylamide gels were successfully modified by the exogenous enzymes polynucleotide kinase and alkaline phosphatase before and after in situ detection of E. coli DNA ligase activity, respectively. Several restriction endonucleases and the tripeptide (Lys-Trp-Lys), which acts as an apurinic/apyrimidinic endonuclease, were able to diffuse into gels and modify DNA. This ability to create intermediate substrates within activity gels could prove extremely useful in delineating the steps of DNA replication and repair pathways.     

Experimental effect of lysozyme on macrophage metabolism and resistance to infection

The peritoneal macrophages of mice treated with lysozyme were studied by cytochemical assay. In single and repeated doses of 0.5-5 mg/kg lysozyme induced an increase in macrophage metabolism. This was evident from an increased activity of succinate dehydrogenase, NADP X N-DH and the enzymes catalyzing glycolysis typical of these cells (lactate dehydrogenase and alpha-glycerophosphate). The changes in the activity of the enzymatic systems were most pronounced in minute and less mature macrophages after repeated administrations of the drugs. In a dose of 50 mg/kg lysozyme somewhat decreased the activity of a number of the enzymes. In the doses optimal for the macrophage activity lysozyme had a low effect on the infection resistance and slightly increased the cephotaxim efficiency in experimental staphylococcal infection. This may be mainly due to the immunomodulating effect of lysozyme and its low effect on the large macrophages having the bactericidal effect.     

Augmentation of an antitumor CTL response In vivo by inhibition of suppressor macrophage nitric oxide

Evidence is provided that inhibition of macrophage NO production can augment in vivo CTL responses. Specifically, administration of NG-monomethyl-l -arginine (NGMMA) via osmotic pumps increases the tumor-specific CTL response against the P815 mastocytoma in the peritoneal cavity of preimmunized mice. Both the magnitude and duration of the CTL response were increased. That the augmented CTL response resulted from inhibition of the NO synthase pathway is supported by the finding that macrophage NO production from NGMMA-treated mice was reduced. Also, in vitro inhibition of NO production by peritoneal exudate cells from P815 tumor-challenged mice augmented the secondary CTL response observed. Cell proliferation was augmented by NGMMA in these cultures, suggesting that macrophage NO may suppress CTL by inhibiting clonal expansion. NO-mediated inhibition was observed in vivo in this experimental system, even though the CTL response is not suppressed, in that tumor rejection occurs. Therefore, the present results are consistent with the conclusion that macrophage NO-mediated inhibition of the CTL response is a side effect of activating macrophages rather than resulting from the action of a distinct subset of what have long been termed suppressor macrophages. Most important, the results indicate that NO-mediated suppressor macrophage activity can be an important CTL immunoregulatory element in vivo.     

Characterization of deglycosylated human urinary colony-stimulating factor (CSF-1)

1. Isolation of CSF-1 from human urine was performed through five purification steps. These include concentration by dialysis, silica gel absorption, hydrophobic chromatography and phenyl-Sepharose CL-6B, Fast Protein Liquid Chromatography (FPLC) and finally preparative electrophoresis on polyacrylamide gels. These methods have been reported in a previous paper (Tao et al., 1987). 2. The isolated CSF-1 which exhibits a one band pattern on SDS-PAGE under non-reducing conditions after Coomassie Blue and silver stainings. CSF-1 was purified 100,000-fold and has a specific activity of 2.16 x 10(7) units/mg protein. Its apparent Mr is 57,000 with an isoelectric point pI = 5.8-6.0 CSF-1 is a glycoprotein with 40% of carbohydrate (w/w). 3. An almost complete removal of the carbohydrate moiety from CSF-1 was obtained after treatment with trifluoromethanesulfonic (TFMS) acid followed by gel filtration on Sephadex G-25 (Fine). The deglycosylated (DG) CSF-1 possesses an apparent Mr of 38,000 and an isoelectric point, pI: 6.2 as compared to native-CSF-1 (N-CSF-1), Mr = 57,000 and pI = 5.8 respectively. 4. The TFMS treatment did not alter the activities of CSF-1 as shown by biological assay and receptor binding assay. The thermostability experiment revealed that DG-CSF-1 was less stable than N-CSF-1. The circular dichroism spectra (CD) of N-CSF-1 and DG-CSF-1 were different. 5. The features of interaction of iodinated-N-CSF-1 and iodinated-DG-CSF-1 with single cell suspensions from human peritoneal macrophage were studied. The binding activity of peritoneal macrophage was the highest among all cells examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hb switching in chickens

We have taken advantage of the preferential digestion of active genes by DNAase I to investigate the chromosomal structure of embryonic and adult β-globin genes during erythropoiesis in chick embryos, and in particular to examine the question of hemoglobin switching during development. DNA in isolated red cell nuclei was mildly digested with DNAase I to about 10–15 kb, purified and restricted with a variety of restriction enzymes. The DNA was then separated on agarose gels, transferred to nitrocellulose filters and hybridized with an adult-specific β-globin cDNA clone or a genomic clone containing the genes coding for both an embryonic and an adult β-globin chain. Preferential sensitivity of the respective globin genes was monitored by the disappearance of specific restriction bands after DNAase I digestion of nuclei. In embryonic red cells, both adult and embryonic β-globin genes are very sensitive to DNAase I; however, in adult erythroid lines, the embryonic β-globin gene becomes relatively more resistant but the adult gene remains highly sensitive. Controls showed that all globin genes were resistant to DNAase I in brain nuclei and nuclei from lymphoid cells. Thus the switch from embryonic to adult globin expression is associated with an apparent change in the chromosome structure of the embryonic globin gene as reflected in the gene becoming less accessible to DNAase I in adult red cell nuclei. Our results also show that the chromosomal structure of both adult and embryonic genes is altered in embryonic red cell nuclei; thus the nonexpressed globin gene (that is, the adult gene in embryonic red cells) has already been “recognized” to some degree and marked by the erythroid compartment. The sensitivity of the adult globin gene in embryonic cells may represent a “pre-activation” state of the chromosome.     

双歧杆菌对腹腔巨噬细胞ERK1/2蛋白激酶通道的影响

目的探讨双歧杆菌对巨噬细胞。ERK1／2蛋白激酶通道的调控。方法收集SD大鼠腹腔巨噬细胞,提取细胞浆蛋白及核蛋白,采用Western印迹分析双歧杆菌对巨噬细胞ERK蛋白激酶水平的影响。结果双歧杆菌呈浓度依赖性诱导巨噬细胞ERK1／2的活化。10^3／ml的双歧杆菌即可增加ERK1／2磷酸化。用10^3／ml的双歧杆菌刺激巨噬细胞,ERK1／2磷酸化30min达到高峰。特异性ERK蛋白激酶抑制剂PD98059能显著降低双歧杆菌诱导的巨噬细胞ERK1／2的活化。结论双歧杆菌可以通过ERK蛋白激酶通道调控巨噬细胞。