Studies on human blood lymphocytes with iC3b (type 3) complement receptors. I. Granular, Fc-IgG receptor positive and negative subsets in healthy subjects and patients with systemic lupus erythematosus

By using the OKM1 monoclonal antibody and the fluorescence-activated cell sorter to identify lymphocytes bearing iC3b (type 3) complement receptors, two principal populations of OKM1+ lymphocytes have been identified in human peripheral blood. One subset exhibited azurophilic granules and Fc receptors for IgG stained by Leu-11. The other population did not display FcR, but was enriched in cells reacting with OKT3 and OKT8 (low intensity). In healthy subjects, approximately 60% of CR3+ lymphocytes were granular FcR-bearing cells and only 18% co-expressed OKT3 determinants. In patients with systemic lupus erythematosus (SLE), CR3+ lymphocytes were predominantly FcR negative cells and 71% lacked granules. Only 33% reacted with Leu-11, but 50% co-expressed OKT3, 44% reacted with OKT8+, and 15% were OKT4+. We tested the hypothesis that agranular OKT3+ Leu-11- lymphocytes, such as those found in SLE patients, contained the precursors of natural killer (NK) cells. Leu-11+ cells were removed from normal lymphocytes by complement lysis, and the remaining cells were treated with recombinant IFN-alpha, IFN-gamma, or IL 2. These procedures were ineffective in generating typical NK effector cells. Our studies do not support the hypothesis that CR3+ Leu-11- lymphocytes are the precursors of granular Leu-11+ NK cells.     

Vicilin genes ofPisum

Summary We describe four genomic clones of pea 7S storage protein gene, one of which corresponds to convicilin, and the others to vicilin. Hybridization studies exploiting these clones, and previously identified cDNA clones, have enabled us to define six different loci. Three of these loci have been mapped to positions on chromosome 7.     

Relationship between CO and transit times for dye and thermal indicators in central circulation

We investigated two factors that may influence the estimation of lung water by the thermal-dye double-indicator-dilution method: 1) changes in cardiac output (CO), and 2) thermal equilibration with cardiac tissue. In theory, the difference between mean transit times of thermal and dye indicators (delta MTT) is proportional to the extravascular volume of distribution of the thermal indicator (VODev) and inversely related to CO. The delta MTT also includes a time element DT due to the difference in response times of the measuring instruments such that delta MTT = VODev/CO + DT. In nine anesthetized dogs we recorded 286 aortic thermal and dye curves following left atrial (LA) and right atrial (RA) injections as CO was increased from 2.35 to 6.65 ml X s-1 X kg-1 by isoproterenol infusion, and a regression of delta MTT on CO-1 was performed. DT was measured in vitro for comparison with the y-intercept. In six of nine dogs the slope of the regression for LA injections was not different from zero, indicating that there is no measurable volume of distribution for thermal indicator in cardiac tissue. For RA injections the relationship between delta MTT and CO-1 was linear in all experiments, with an average correlation coefficient of 0.97 +/- 0.01 (SE), indicating that the VODev was constant over a threefold increase in CO. Although the in vitro measurement of DT agreed closely with the average of the y-intercepts of the regressions, small between-subject differences in DT can lead to apparent flow-related changes in extravascular thermal volume computed in the conventional fashion using the in vitro estimate of DT.     

Functional studies on the precursors of human lymphokine-activated killer cells

Human peripheral blood lymphocytes cultured for 4 days in the interleukin 2 (IL-2)-containing cell-free supernatant of the MLA144 cell line (MLA144CM) are cytolytic to NK-susceptible and NK-resistant tumor target cells. This lymphokine-activated killer (LAK) activity is dependent on IL-2 as development of LAK activity is inhibited in the presence of a monoclonal antibody (MoAb) reacting with the IL-2 receptor (anti-Tac). Addition of cyclosporin A (CyA) to mixed lymphocyte cultures inhibits the development of allospecific cytotoxic activity and inhibits the development of IL-2 responsiveness. However, development of LAK activity is unaffected by the inclusion of CyA in the cultures, showing that the LAK precursor can be functionally distinguished from the allospecific cytotoxic precursor cell. Development of LAK activity does not require mature NK cells as shown by the generation of LAK activity from NK inactive human thymocytes and lymph node cells. In addition, depletion of NK activity from human PBL does not impair the development of LAK activity.     

Photochemical demonstration of stacked C.C+ base pairs in a novel DNA secondary structure

The secondary structure of the alternating polydeoxynucleotide sequence poly[d(C-T)] was studied as a function of pH by ultraviolet absorbance and circular dichroism spectroscopy and by the analysis of UV-induced photoproducts. As the pH was lowered, poly[d(C-T)] underwent a conformational transition that was characterized by changes in the long-wavelength region (280-320 nm) of the CD spectrum. These changes have previously been interpreted as evidence for the formation of a core of stacked, protonated C X C+ base pairs in a double-helical complex of poly[d(C-T)], with the thymidyl residues being looped out into the solvent [Gray, D. M., Vaughan, M., Ratliff, R. L., & Hayes, F. N. (1980) Nucleic Acids Res. 8, 3695-3707]. In the present work, poly[d(C-T)] was labeled with [U-14C]cytosine and [methyl-3H]thymine and irradiated at pH values both above and below the conformational transition point (monitored by CD spectroscopy). The distribution of radioactivity in uracil means value of uracil dimers, uracil means value of thymine dimers (the deamination products of cytosine means value of cytosine and cytosine means value of thymine dimers, respectively), and thymine-means value of thymine dimers was then determined. As the pH was decreased, we found an increase in the yield of uracil means value of uracil dimers and a decrease in the yield of uracil means value of thymine dimers, which occurred concomitantly with the change in the CD spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Low-Temperature Acclimation and Oxygen Stress on Tocopheron Production in Euglena gracilis Z

The effects of low-temperature acclimation and oxygen stress on tocopheron production were examined in the unicellular phytoflagellate Euglena gracilis Z. Cells were cultured photoheterotrophically at 27.5 ± 1°C with 5% carbon dioxide-95% air and 740 microeinsteins m⁻² s⁻¹ (photosynthetically active radiation) and served as controls. Low-temperature acclimation (12.5 ± 1°C) and high-oxygen stress (5% carbon dioxide-95% oxygen) were individually examined in the mass culturing of the algae. Chromatographic analyses demonstrated a six-to sevenfold enhancement of α-tocopherol production in temperature-stressed cells, along with a concomitant decline in the levels of α-tocotrienol and the absence of other tocopherol homologs. Oxygen-stressed cultures demonstrated the presence of high levels of α-tocopherylquinone; α-tocopheron and its homologs and precursors were absent or declined markedly. These findings are discussed in terms of the feasibility of microbial production of natural tocopherols. In addition, these results lend themselves to speculation regarding the biological role(s) of tocopherols as antioxidants and free radical scavengers in reducing photo-induced oxidative damage or lipid peroxidation toxicities or both in photosynthetically active E. gracilis Z.     

Interferon-gamma modulates protein kinase C activity in murine peritoneal macrophages

The content of Ca2+-, phospholipid-dependent protein kinase activity (protein kinase C) in murine peritoneal macrophages treated with recombinant interferon-gamma (IFN-gamma) has been investigated. Protein kinase C activity was solubilized by nonionic detergent extraction of sonicated cells and separated by high performance liquid chromatography on a TSK 4000 SW gel filtration column. The enzyme eluted from the column in a molecular weight range of 60-80 X 10(3) and was identified by virtue of Ca2+ and phospholipid requirements. Macrophages treated with recombinant IFN-gamma exhibited a substantial increase in total protein kinase activity which could be accounted for entirely by increased protein kinase C activity. This activity was enhanced as much as 5-fold over that seen in untreated macrophages and was specific for IFN-gamma in that other agents known to signal changes in macrophage function had no effect. The time required for the elevation of kinase activity was identical to that required for induction of other functions by IFN-gamma in macrophages. These observations suggest that protein kinase C may be a focus of regulatory action in IFN-gamma-mediated macrophage activation.     

Complex subcellular distributions of enzymatic markers in intestinal epithelial cells

Summary Current procedures for isolating intestinal epithelial cell surface and intracellular membranes are based on the assumption that each organelle is marked by some unique constitutent. This assumption seemed inconsistent with the dynamic picture of subcellular organization emerging from studies of membrane turnover and recycling. Therefore, we have designed an alternative fractionation which is independent ofa priori marker assignments. We subjected mucosal homogenates to a sequence of separations based on sedimentation coefficient, equilibrium density, and partitioning in aqueous polymer twophase systems. The resulting distributions of protein and enzymatic markers define a total of 17 physically and biochemically distinct membrane populations. Among these are: basal-lateral membranes, with Na,K-ATPase enriched 21-fold; brush-border membranes, with alkaline phosphatase enriched as much as 38-fold; two populations apparently derived from the endoplasmic reticulum; a series of five populations believed to have been derived from the Golgi complex; and a series of five acid phosphatase-rich populations which we cannot identify unequivocally. Each of the five enzymatic markers we have followed is associated with a multiplicity of membrane populations. Basallateral, endoplasmic reticulum, and Golgi membranes contain alkaline phosphatase at the same specific activity as the initial homogenate. Similarly, Na,K-ATPase appears to be associated branes at specific activities two-to seven-fold that of the initial homogenate.     

Comparison of goose-type,chicken-type,and phage-type lysozymes illustrates the changes that occur in both amino acid sequence and three-dimensional structure during evolution

Summary The three-dimensional structure of goose-type lysozyme (GEWL), determined by x-ray crystallography and refined at high resolution, has similarities to the structures of hen (chicken) eggwhite lysozyme (HEWL) and bacteriophage T4 lysozyme (T4L). The nature of the structural correspondence suggests that all three classes of lysozyme diverged from a common evolutionary precursor, even though their amino acid sequences appear to be unrelated (Grütter et al. 1983).In this paper we make detailed comparisons of goose-type, chicken-type, and phage-type lysozymes. The lysozymes have undergone conformational changes at both the blobal and the local level. As in the globins, there are corresponding -helices that have rigid-body displacements relative to each other, but in some cases corresponding helices have increased or decreased in length, and in other cases there are helices in one structure that have no counterpart in another.Independent of the overall structural correspondence among the three lysozyme backbones is another, distinct correspondence between a set of three consecutive -helices in GEWL and three consecutive -helices in T4L. This structural correspondence could be due, in part, to a common energetically favorable contact between the first and the third helices.There are similarities in the active sites of the three lysozymes, but also one striking difference. Glu 73 (GEWL) spatially corresponds to Glu 35 (HEWL) and to Glu 11 (T4L). On the other hand, there are two aspartates in the GEWL active site, Asp 86 and Asp 97, neither of which corresponds exactly to Asp 52 (HEWL) or Asp 20 (T4L). (The discrepancy in the location of the carboxyl groups is about 10 Å for Asp 86 and 4 Å for Asp 97.) This lack of structural correspondence may reflect some differences in the mechanisms of action of three lysozymes. When the amino acid sequences of the three lysozyme types are aligned according to their structural correspondence, there is still no apparent relationship between the sequences except for possible weak matching in the vicinity of the active sites.     

Regulation of haemolysin synthesis in E. coli determined by HLY genes of human origin

Summary We have previously reported the secretion of a 107K polypeptide into the medium from a haemolytic E. coli K12 strain (Mackman and Holland 1984a). In addition, we demonstrated that haemolysin production was correlated with the presence of this polypeptide in the growth medium in a large number of E. coli isolates of human and animal origin (Mackman and Holland 1984b).In this paper we confirm that the 107K polypeptide is indeed haemolysin: both haemolytic activity and the 107K polypeptide show a similar pattern of accumulation during the growth cycle; identical levels are produced in three different growth media; they have the same half-life in minimal medium. The results also show that the expression of haemolysin is not influenced by the growth medium or subject to catabolite repression. However, expression is apparently switched off as cells enter the late exponential phase of growth. Finally, we present data indicating that the previously reported variation in haemolysin production in different media is entirely due to the instability of the haemoolysin itself. Degradation of the 107K polypeptide in the medium was accompanied by the accumulation of a major breakdown product of 60K.