Activation requirements for antigen- and mitogen-induced interferon-gamma release from cytotoxic T lymphocytes

We have studied the activation signals that regulate interferon-gamma (IFN-gamma) secretion from murine cytotoxic T lymphocytes (CTL) upon binding mitogen or antigen. CTL clones were found to require at least 1 hr of stimulation with concanavalin A (Con A) in order to produce detectable levels of IFN-gamma. Full activation of IFN-gamma synthesis in CTL clones occurred after stimulation for 2 hr or more, and in those cultures CTL continued to produce high levels of IFN-gamma even after the effects of Con A had been neutralized. Splenic T cells and uncloned long-term CTL lines required a longer period of stimulation than cloned CTL for Con A-induced IFN-gamma secretion. The relationship between IFN-gamma secretion and cytotoxic activity was studied in an antigen-specific system. These studies reveal marked differences in the types of effector responses generated by CTL upon contact with antigen, demonstrating that some antigen-bearing cells promote high levels of IFN-gamma secretion and are poorly lysed by CTL, whereas other cell lines are lysed with high efficiency by CTL but induce low levels of IFN-gamma secretion.     

Kinetics of the interaction of hemin liposomes with heme binding proteins

As a model for the transport of hemin across biological membranes, sonicated phosphatidylcholine liposomes with incorporated hemin were characterized. The interaction of the hemin liposomes with the heme binding proteins albumin, apomyoglobin, and hemopexin was examined as a function of liposome charge and cholesterol content. In all cases, there was an almost complete transfer of hemin from liposome to protein; a rapid phase and a slow phase were observed for the transfer. For negatively charged liposomes (with 11% dicetyl phosphate), the rapid and slow phases showed observed rates of transfer of ca. 2 and 0.01 s-1, respectively, for all three proteins. The presence of cholesterol in the liposomes decreased the observed rates by a factor of 2, and positively charged liposomes (with 11% stearylamine) showed about one-fifth the observed rates of negatively charged liposomes. The observed rates were independent of protein concentration, indicating that the rate-determining step is hemin efflux from the lipid bilayer. The hemin interaction with the phospholipid bilayer is suggested to be primarily hydrophobic with some electrostatic character. The two phases are suggested to arise from two different populations of hemin within the liposomes and are interpreted as arising from two different orientations of hemin within the bilayer.     

The structural basis of ankyrin function. II. Identification of two functional domains

Human erythrocyte ankyrin was cleaved by restricted proteolysis at 0 degrees C into two distinct chemical domains. The site on ankyrin that binds spectrin was found to be within a 55,000-dalton domain by spectrin affinity chromatography and co-sedimentation with spectrin in a sucrose gradient. A 32,000-dalton fragment of this domain was prepared (tryptic digest, 0 degrees C, 24 h), separated by gel filtration, and shown to inhibit spectrin binding to the membrane. By comparison with previous two-dimensional peptide maps, the spectrin-binding site was located within this 32,000-dalton fragment near the end of the molecule. The band 3-binding site was identified within an 82,000-dalton domain by binding to a band 3 affinity column. Gel electrophoresis in the absence of detergents confirmed these results and demonstrated that a peptide from the cytoplasmic portion of band 3 retained the capacity to bind the 82,000-dalton domain. The binding properties of the structural domains of ankyrin were correlated with a determination of the affinity constant of the intact molecule. Ankyrin bound with a high affinity to the cytoplasmic portion of band 3 (KD = 8 X 10(-8) M) and to spectrin tetramer (KD = 1 X 10(-7) M) but less so to spectrin dimer (KD = 1 X 10(-6) M). These findings are summarized in a preliminary structural and functional model of ankyrin's role in linking spectrin to the membrane.     

Interactions of porphyrins with nucleic acids

The interactions of nucleic acids with water-soluble porphyrins and metalloporphyrins have been investigated by stopped-flow and temperature-jump techniques. Both natural DNA (calf thymus) and synthetic homopolymers [poly(dG-dC) and poly(dA-dT)] have been employed. The porphyrins studied belong to the tetrakis(4-N-methylpyridyl)porphine (H2TMpyP-4) series and can be divided into two groups: (i) those which have no axial ligands when bound to nucleic acids [e.g., Ni(II), Cu(II), and the nonmetallic derivatives] and (ii) those which maintain axial ligands upon binding [e.g., Mn(III), Fe(III), Co(III), and Zn(II) derivatives]. The reaction of both axially and nonaxially liganded porphyrins at AT sites is too rapid to be measured by the kinetic methods utilized, whereas at GC sites the interaction of the nonaxially liganded porphyrins is in the millisecond time range and can be monitored by both stopped-flow and temperature-jump techniques. These results corroborate previous static studies, utilizing visible spectroscopy and circular dichroism, which indicate that the formation of an intercalated complex occurs only at GC base pair sites with porphyrins which do not possess axial ligands. With all the porphyrins investigated, the complexes formed at AT sites are envisioned as being of an "external" type involving some degree of overlap between the porphyrin and the bases of the duplex. In relaxation experiments of poly-(dG-dC) with H2TMpyP-4, a large, reproducible effect is observed which can be analyzed as a single exponential. Rate constants for association and dissociation of the H2TMpyP-4/poly(dG-dC) complex are 3.7 X 10(5) M-1 s-1 and 1.8 s-1, respectively. Relaxation studies of mixtures of poly(dA-dT) and poly(dG-dC) with H2TMpyP-4 indicate that the transfer of the porphyrin from one homopolymer to another occurs via a mechanism involving dissociation rather than direct transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences between ATP-mediated cytotoxicity and cell-mediated cytotoxicity.

There has been considerable recent interest in the possible role of extracellular ATP in cell-mediated cytotoxicity. To investigate the similarities and differences between ATP-mediated lysis and CTL-mediated lysis, we studied in detail the ATP-mediated lysis of EL-4 cells, which are highly susceptible to lysis by extracellular ATP. ATP-mediated lysis was time and concentration dependent; most lysis occurred within 4 to 6 h of incubation. The kinetics of ATP- and cell-mediated lysis were similar. However, in contrast to CTL-mediated lysis, ATP-mediated lysis of EL-4 cells did not appear to be accompanied by characteristic chromosomal degradation (apoptosis). In order to compare these cytotoxic processes in greater detail, ATP-resistant clones were derived by growing EL-4 cells continuously in the presence of high concentrations of ATP. These cloned EL-4 lines showed marked resistance to ATP-mediated cytotoxicity across a wide range of concentrations but were as susceptible as the parent EL-4 cells to cell-mediated cytotoxicity by an alloreactive effector population from a MLC. Thus, there appear to be a number of differences between ATP-mediated and cell-mediated cytotoxicity in this system; most notable is the identification of cell lines that are resistant to ATP but susceptible to CTL-mediated lysis.     

Fixation and embedding variables in the immuno-electron microscopic study of rat heymann nephritis

Summary Fixation and embedding variables were compared in immuno-electron microscopic localization of rat IgG in an autologous immune complextype nephritis. Specimens from kidney cortex were fixed for 3, 6 or 9 h in the following fixatives made in 0.1 M phosphate buffer at pH 7.44% paraformaldehyde, 2.5% glutaraldehyde, periodate-lysine-paraformaldehyde or modified Karnovsky's fixative. Localization of IgG was performed on tissue sections cut with a tissue chopper, cryostat or sliding microtome, using agarose, Ames O.C.T. Compound or polyethylene glycol respectively as cutting matrixes. The sections were incubated in peroxidase-labelled antirat IgG antiserum (diluted 120 with phosphate-buffered saline) for 60 h. Peroxidase activity was then revealed and the sections embedded in Epon. Exact localization of IgG throughout the sections and good ultrastructure were achieved when paraformaldehyde and agarose were used. Periodate-lysine-paraformaldehyde proved almost as useful as paraformaldehyde in connection with agarose in respect of peroxidase reaction and ultrastructure. Fixatives containing glutaraldehyde gave a mostly weak and unevenly distributed peroxidase reaction product. In the cryostat sections breaking of the tissue structure could not be avoided. When polyethylene glycol was used as cutting matrix no peroxidase reaction was achieved.     

Catalysis of the disproportionation of superoxide by metalloporphyrins.

The efficiencies of several metalloporphyrin complexes at catalyzing the disproportionation of superoxide have been determined at pH 10 in both carbonate and borate buffer systems. Catalytic rate constants were obtained for the iron(III) and cobalt(III) derivatives of tetrakis(4-N-methylpyridyl) porphine, for tetraphenylporphinesulfonatoferrate(III) and for hemin. In addition, the effects of added bovine serum albumin and imidazole were studied. The order of catalytic efficiency is FeTMpyP greater than FeTMpyP(Im)2 greater than FeTPPS(Im)2 approximately FeTPPS approximately FeTPPS.BSA approximately Fe(EDTA) greater than or approximately CoTMpyP greater than hemin(Im)2 greater than or approximately hemin.     

含锰固氮酶组分Ⅰ蛋白的纯化和特性

棕色固氮菌（Azotobacter vinelandii Lipmann)突变种UW3能在无Mo的无氨培养基中固氮生长,低浓度的Mn对UW3突变种生长有促进作用,从在Mn中生长的UW3菌体中分离得到的部分纯固氮酶组分Ⅰ蛋白含量有Mn和Fe原子（Fe/Mo/Mn为10．41：0．19：1．00）并有OP MoFe蛋白一半的还原乙炔和质子的活性。这种蛋白的吸收光谱和圆二色谱与MoFe蛋白存在明显的差异,含Mn蛋白的亚基分子量都与MoFe蛋白的α亚基相近。初步结果表明,这种含Mn蛋白可胡是一种固氮酶组分Ⅰ蛋白。     

固氮酶铬铁蛋白的晶体生长

在合适的结晶条件下 ,从含Cr无氨培养基中生长的固氮菌 (AzotobactervinelandiiLipmann)突变种UW3 中纯化出的CrFe蛋白可从溶液中析出深棕色斜四棱柱晶体 ,晶体最大的两条对角线长度分别可达 0 .2 5mm和 0 .12mm。PEG 80 0 0、MgCl2 、NaCl、Tris和Hepes缓冲液的浓度及结晶方法等对该蛋白的出晶率、晶核数目、晶体大小和质量都有明显影响。CrFe蛋白结晶所需的上述化合物的最适浓度与在Mn中生长的固氮菌突变种UW3 的MnFe蛋白和缺失nifZ固氮菌突变种的ΔnifZMoFe蛋白结晶所需的最适浓度有所不同。结果表明 ,该蛋白晶体可能为CrFe蛋白的晶体