The large bat Helitron DNA transposase forms a compact monomeric assembly that buries and protects its covalently bound 5′-transposon end

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Lymphoid cell subclasses in rejecting renal allograft in the rat

We have quantitated the frequency of lymphoid cell subsets in rejecting renal allografts and in the spleen of the allograft recipient during drug-unmodified rejection in the rat. The number of inflammatory (white) cells in the graft was approximately similar to the number of white cells responding to the allograft in the recipient spleen. The inflammatory population of the graft consisted of lymphoid cells and mononuclear phagocytes, with increasing numbers of macrophages toward the end of rejection. Analysis of allograft cellular dispersates with monoclonal antibodies directed to the lymphoid cell subsets demonstrated that although the majority of allograft-infiltrating lymphocytes were T cells, a sizable B-cell proliferation and immunoglobulin synthesis was associated with the inflammatory response of rejection. Within the T-cell subset, the T suppressor/killer cells predominated in the graft whereas the predominant lymphoid cell subset responding to the allograft in the recipient spleen was the T helper cell.     

A biologically active thrombin cleavage product of human serum spreading factor

Purified human serum spreading factor preparations consisting of two immunologically-related, biologically-active proteins of molecular weights approximately 65,000 and 75,000 were incubated with purified hydrolytic enzymes: papain, neuraminidase and thrombin. Biologically active products of the enzymatic digestions were obtained in each case. Digestion of serum spreading factor preparations with thrombin produced a single active form of molecular weight approximately 57,000. Generation of a single molecular weight form of serum spreading factor by thrombin cleavage of the two higher molecular weight forms should simplify studies of the biochemistry and biology of this protein, and may represent a reaction of physiological significance.     

Light-dependent rubidium transport in intact Halobacterium halobium cells

The uptake of rubidium in intact Halobacterium halobium cells was followed, and found to be light-dependent. The exchange process is slow, the steady-state rate of ⁸⁶Rb⁺/Rb⁺ exchange being given by k = 6.3 · 10^?4 min^?1. Starved cells exhibited a faster rate than unstarved cells. The influx of ⁸⁶Rb⁺ was almost completely blocked in the presence of proton conductors (CCCP, FCCP, and SF 6847), and was sensitive to the presence of the permeant cation TPMP⁺. Valinomycin very slightly increased the rate of uptake, while 1 · 10^?6 M nigericin showed significant inhibition. On the other hand, release of ⁸⁶Rb⁺ was not light-dependent, although still affected by uncouplers, TPMP⁺, and nigericin. These experimental observations may be explained in terms of a passive flux driven by an electrical potential difference, and influenced by positive isotope interaction within the membrane. In carefully matched influx-efflux studies, the extent of the positive isotope interaction was measured. Using the formal treatment of Kedem and Essig, the ratio (exchange resistance)/(resistance to net flow) for ⁸⁶Rb⁺ was found to be 1.7.     

Steady-state kinetic studies on the actin activation of skeletal muscle heavy meromyosin subfragments: Effects of skeletal,smooth and non-muscle tropomyosins

The addition of either smooth muscle or brain tropomyosin to skeletal muscle actoheavy meromyosin (HMM) or acto-myosin subfragment-1 (SF1) produces an activation of the actin-activated ATPase activity up to 100%. This contrasts with the opposite, inhibitory effect produced by skeletal muscle tropomyosin. The degree of activation or inhibition depends on the ionic conditions, which influence the affinities of tropomyosin and HMM or SF1 for actin as well as on the molar ratio of actin to myosin.Enzyme kinetic analysis indicates that the inhibitory effect of skeletal muscle tropomyosin results from an approximately six- to tenfold increase in the apparent affinity (K_app) of the myosin head for the F-actin-tropomyosin complex with a concomitant six- to tenfold reduction in the maximal turnover rate (V_max). Thus, there is no direct competition of skeletal muscle tropomyosin and myosin for the same site on actin. Brain tropomyosin has an opposite effect, decreasing the apparent affinity with concomitant increase in the V_max.The effect of smooth muscle tropomyosin is more complex. At high ratios of myosin to actin this tropomyosin produces the same change in the K_app as skeletal muscle tropomyosin but yields a value of V_max that is about twofold higher. At lower molar ratios (below about 1 to 5 myosin subfragments to actin) the activating effect of this tropomyosin remains unchanged while the apparent affinity decreases to that observed for pure F-actin.On the basis of these data as well as from experiments carried out at fixed actin and varying SF1 concentrations, it is concluded that tropomyosins act in general as allosteric un-competitive inhibitors or activators of actomyosin by increasing or reducing the co-operative activation of myosin by actin at the level of product release.     

Effects of metabolic inhibitors on cell lethality and mutation induction in Chinese hamster cells. I. Inhibitors of de novo purine synthesis and a comparison with the effects of caffeine

The effect of pre- and posttreatment incubation of UV-irradiated and ethyl methanesulphonate (EMS) treated cells with non-toxic concentrations of inhibitors of de novo purine synthesis (dnPS) on expression of potentially lethal and premutational damage at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 cells has been examined. The concentrations of inhibitors used were shown to profoundly perturb de novo DNA synthesis, by measurements of [¹⁴C]formate uptake, and cell cycle progression by flow cytofluorimetry. Postincubation in 6-methyl mercaptopurine ribonucleoside (MMPR) usually but not invariably potentiated the cytotoxic effects of UV and EMS but azaserine (AZS) and methotrexate (MTX) were without effect. No effects on mutant frequencies were observed on posttreatment with any of these agents. Caffeine produced the least effect on dnPS, but invariably potentiated lethal damage. This potentiation of lethal damage is not mediated by dnPS inhibition as has been suggested for Chinese hamster ovary (CHO) cells.     

3,5-Di-tert-butyl-4-hydroxybenzylidenemalononitrile. Effects of pH on its binding to liposomes and evidence for formation of a ternary complex with valinomycin and potassium ion

1. The properties of 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) were studied chemically and spectroscopically. Two molecular species of SF6847 were identified: the undissociated form (SFH; ?₃₆₃, 10 mM^?1) and the dissociated form (SF^?; ?₄₅₄, 35 mM^?1). The pK_a value of the molecule was determined to be 6.9.2. On the basis of these properties the interactions of SF6847 with liposomes and valinomycin · K⁺ were studied. The partition constants of SFH (Kⁿ_p and SF^? (K^?_p) to liposomes were determined separately; Kⁿ_p was 56 mM^?1 and was independent of the pH of the medium, whereas K^?_p dependend greatly on the pH, being 1.2 mM^?1 at pH 7.0 and 2.9 mM^?1 at pH 8.0. Using these values, the partition constant of total SF6847 (K_p) was calculated and found to be essentially the same as that calculated from the kinetics of proton uptake. It was concluded that the amount of SF^? bound to liposomes is rate limiting for proton uptake.3. The effects of membrane potential on partition constants were studied. The K^?_p decreased greatly upon generation of a membrane potential negative inside the liposomes but increased upon generation of a membrane potential positive inside the liposomes.4. The interaction of SF6847 with valinomycin in aqueous solution and in liposomes was demonstrated only in the presence of potassium ion. Potassium ion could not be replaced by sodium ion. Evidence was obtained for the formation of the ternary complex valinomycin · K⁺ · SF^? in liposomes and in hexane. It was concluded that SF^? became more soluble in the liposomal membranes on formation of this ternary complex. All these results support our proposed mechanism for the proton uptake cycle (Yamaguchi, A. and Anraku, Y. (1978) Biochim. Biophys. Acta 501, 136–149).     

Heterogeneity in E. coli RecBCD Helicase-DNA Binding and Base Pair Melting

E. coli RecBCD, a helicase/nuclease involved in double stranded (ds) DNA break repair, binds to a dsDNA end and melts out several DNA base pairs (bp) using only its binding free energy. We examined RecBCD-DNA initiation complexes using thermodynamic and structural approaches. Measurements of enthalpy changes for RecBCD binding to DNA ends possessing pre-melted ssDNA tails of increasing length suggest that RecBCD interacts with ssDNA as long as 17–18 nucleotides and can melt at least 10–11 bp upon binding a blunt DNA end. Cryo-EM structures of RecBCD alone and in complex with a blunt-ended dsDNA show significant conformational heterogeneities associated with the RecB nuclease domain (RecB^Nuc) and the RecD subunit. In the absence of DNA, 56% of RecBCD molecules show no density for the RecB nuclease domain, RecB^Nuc, and all RecBCD molecules show only partial density for RecD. DNA binding reduces these conformational heterogeneities, with 63% of the molecules showing density for both RecD and RecB^Nuc. This suggests that the RecB^Nuc domain is dynamic and influenced by DNA binding. The major RecBCD-DNA structural class in which RecB^Nuc is docked onto RecC shows melting of at least 11 bp from a blunt DNA end, much larger than previously observed. A second structural class in which RecB^Nuc is not docked shows only four bp melted suggesting that RecBCD complexes transition between states with different extents of DNA melting and that the extent of melting regulates initiation of helicase activity.     

Lysoglycerophospholipids in chronic inflammatory disorders: The PLA2/LPC and ATX/LPA axes

Lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA), the most prominent lysoglycerophospholipids, are emerging as a novel class of inflammatory lipids, joining thromboxanes, leukotrienes and prostaglandins with which they share metabolic pathways and regulatory mechanisms. Enzymes that participate in LPC and LPA metabolism, such as the phospholipase A₂ superfamily (PLA₂) and autotaxin (ATX, ENPP2), play central roles in regulating LPC and LPA levels and consequently their actions. LPC/LPA biosynthetic pathways will be briefly presented and LPC/LPA signaling properties and their possible functions in the regulation of the immune system and chronic inflammation will be reviewed. Furthermore, implications of exacerbated LPC and/or LPA signaling in the context of chronic inflammatory diseases, namely rheumatoid arthritis, multiple sclerosis, pulmonary fibrosis and hepatitis, will be discussed. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.