Oxidative DNA cleavage induced by an iron(III) flavonoid complex: synthesis, crystal structure and characterization of chlorobis(flavonolato)(methanol) iron(III) complex

A flavonol iron(III) complex, [Fe(flavonolato)(2)Cl(MeOH)], has been prepared. The compound has been characterized by X-ray crystallography, spectroscopy, magnetism and electronic paramagnetic resonance (EPR) at X- and Q-band. The geometrical environment around the metal is best described as rhombic distorted octahedral. This distortion has also been inferred from the magnetic measurements and from the EPR spectra at different temperatures, E/D(rhombicity parameter) approximately 0.06. The DNA cleavage activity of the iron(III) complex with and without ascorbate/hydrogen peroxide is reported. Mechanisms of the oxidative cleavage have been proposed when DNA strand scission is performed both with and without ascorbate/hydrogen peroxide activation.     

Studies of fibronectin synthesized by cultured chick hepatocytes

We have adapted a chick embryo liver cell system for studying the synthesis of proteins secreted by hepatocytes. In primary liver cell cultures maintained for several days in arginine-deficient medium containing ornithine (0.7 mM) and carbamyl phosphate (1 mM), only hepatocytes demonstrated normal morphological and biosynthetic characteristics, indicating that they possessed a functional ornithine cycle as a source of arginine production. Non-parenchymal liver cells, such as fibroblasts, which lack the ornithine cycle were excluded. Hepatocytes in arginine-deficient or arginine-containing medium synthesized fibronectin (Fn) over several days at a constant rate of 3 micrograms +/- 1 microgram/mg cell protein per day, with fibronectin representing approximately 3% of the total secreted hepatocyte proteins during any culture period after the first 24 h. Pulse-chase experiments indicated that Fn synthesis and secretion was relatively rapid (t1/2 = 45 min) and represented approximately 95% of the intracellularly labelled Fn. This Fn is secreted predominantly as a 450 kD dimer with a subunit size that is indistinguishable from the plasma form as assessed by one-dimensional electrophoretic analysis. Continuous exposure of hepatocytes to insulin caused a moderate decrease (26%) in Fn synthesis, whereas there was no effect of short-term exposure. In contrast, dexamethasone stimulated Fn production 2-3-fold, consistent with its known ability to stimulate hepatocyte production of acute phase proteins. Under these conditions, electrophoretic analyses showed that an increased quantity of intact hepatocyte Fn was produced having the same molecular size of plasma Fn.     

Airway smooth muscle: contraction and beyond

Airway smooth muscle (ASM), an important tissue involved in the regulation of bronchomotor tone, exists in the trachea and in the bronchial tree up to the terminal bronchioles. The physiological relevance of ASM in healthy airways remains unclear. Evidence, however, suggests that ASM undergoes marked phenotypic modulation in lung development and in disease states such as asthma, chronic bronchitis and emphysema. The shortening of ASM regulates airway luminal diameter and modulates airway resistance, which can be augmented by cytokines as well as extracellular matrix alterations. ASM may also serve immunomodulatory functions, which are mediated by the secretion of pro-inflammatory mediators such as cytokines and chemokines. In addition, ASM mass increases in chronic airway diseases and may represent either a pathologic or an injury-repair response due to chronic inflammation. This review will present evidence that ASM, a "passive" contractile tissue, may become an "active participant" in modulating inflammation in chronic lung diseases. Cell facts 1. Found in the trachea and along the bronchial tree. 2. Critically important in regulating bronchomotor tone of the airways. 3. Differentiation state is associated with the expression of various "contractile proteins." 4. Displays phenotypic modulation of mechanical, synthetic and proliferative responses. 5. Secretes cytokines, chemokines and extracellular matrix proteins. 6. May serve as a potential new target for the treatment of chronic lung diseases.     

TNF-[alpha] modulates murine tracheal rings responsiveness to G-protein-coupled receptor agonists and KCl.

Although the mechanisms that underlie airway hyperresponsiveness in asthma are complex and involve a variety of factors, evidence now suggests that intrinsic abnormalities in airway smooth muscle (ASM) may play an important role. We previously reported that TNF-alpha, a cytokine involved in asthma, augments G-protein-coupled receptor (GPCR) agonist-evoked calcium responses in cultured ASM cells. Here we have extended our previous studies by investigating whether TNF-alpha also modulates the contractile and relaxant responses to GPCR activation using cultured murine tracheal rings. We found that in tracheal rings treated with 50 ng/ml TNF-alpha, carbachol-induced isometric force was significantly increased by 30% compared with those treated with diluent alone (P < 0.05). TNF-alpha also augmented KCl-induced force generation by 70% compared with rings treated with diluent alone (P < 0.01). The enhancing effect of TNF-alpha on carbachol-induced isometric force generation was completely abrogated in the tracheal rings obtained from TNF-alpha receptor (TNFR)1-deficient mice and in control rings treated with a TNF-alpha mutant that solely activates TNFR2. TNF-alpha also attenuated relaxation responsiveness to isoproterenol but not to PGE2 or forskolin. TNF-alpha modulatory effects on GPCR-induced ASM responsiveness were completely abrogated by pertussis toxin, an inhibitor of Gialpha proteins. Taken together, these data suggest that TNF-alpha may participate in the development of airway hyperresponsiveness in asthma via the modulation of ASM responsiveness to both contractile and beta-adrenoceptor GPCR agonists.     

Translational competence of ribosomes released from a premature termination codon is modulated by NMD factors

In addition to their well-documented roles in the promotion of nonsense-mediated mRNA decay (NMD), yeast Upf proteins (Upf1, Upf2/Nmd2, and Upf3) also manifest translational regulatory functions, at least in vitro, including roles in premature translation termination and subsequent reinitiation. Here, we find that all upfΔ strains also fail to reinitiate translation after encountering a premature termination codon (PTC) in vivo, a result that led us to seek a unifying mechanism for all of these translation phenomena. Comparisons of the in vitro translational activities of wild-type (WT) and upf1Δ extracts were utilized to test for a Upf1 role in post-termination ribosome reutilization. Relative to WT extracts, non-nucleased extracts lacking Upf1 had approximately twofold decreased activity for the translation of synthetic CAN1/LUC mRNA, a defect paralleled by fewer ribosomes per mRNA and reduced efficiency of the 60S joining step at initiation. These deficiencies could be complemented by purified FLAG-Upf1, or 60S subunits, and appeared to reflect diminished cycling of ribosomes from endogenous PTC-containing mRNAs to exogenously added synthetic mRNA in the same extracts. This hypothesis was tested, and supported, by experiments in which nucleased WT or upf1Δ extracts were first challenged with high concentrations of synthetic mRNAs that were templates for either normal or premature translation termination and then assayed for their capacity to translate a normal mRNA. Our results indicate that Upf1 plays a key role in a mechanism coupling termination and ribosome release at a PTC to subsequent ribosome reutilization for another round of translation initiation.     

Mutational analysis of the helicase-like domain of Thermotoga maritima reverse gyrase

Reverse gyrase is a unique type IA topoisomerase that is able to introduce positive supercoils into DNA in an ATP-dependent process. ATP is bound to the helicase-like domain of the enzyme that contains most of the conserved motifs found in helicases of the SF1 and SF2 superfamilies. In this paper, we have investigated the role of the conserved helicase motifs I, II, V, VI, and Q by generating mutants of the Thermotoga maritima reverse gyrase. We show that mutations in motifs I, II, V, and VI completely eliminate the supercoiling activity of reverse gyrase and that a mutation in the Q motif significantly reduces this activity. Further analysis revealed that for most mutants, the DNA binding and cleavage properties are not significantly changed compared with the wild type enzyme, whereas their ATPase activity is impaired. These results clearly show that the helicase motifs are tightly involved in the coupling of ATP hydrolysis to the topoisomerase activity. The zinc finger motif located at the N-terminal end of reverse gyrases was also mutated. Our results indicate that this motif plays an important role in DNA binding.     

Separation and analysis of the major forms of plasma fibronectin

Human plasma fibronectin exists in circulation in multiple molecular forms that are distinguishable by SDS-polyacrylamide gel electrophoresis (zone I, approx. 450 kDa dimers; zone II, 190-235 kDa; Zone III, 146-175 kDa). (Chen, A.B., Amrani, D.L. and Mosesson, M.W. (1977) Biochim. Biophys. Acta 493, 310-322). We report here on investigations of plasma fibronectin that had been purified from the 'heparin-precipitable fraction' of plasma by DEAE-cellulose chromatography using buffers containing a chaotropic salt (KSCN). Zone I fibronectin and zone II fibronectin were subsequently separated by Sepharose CL-6B chromatography in the presence of 0.3 M KSCN. Electrophoresis of reduced zone I fibronectin dimers showed the presence of three types of subunits (i.e., 220 kDa, 215 kDa, 207 kDa), evidently all having the same NH2-terminal sequence. Subunits of this size were also found in reduced zone II fibronectin, as well as another polypeptide of 190 kDa, the latter amounting to under 5% of the total. Unreduced zone I fibronectin was resolved by gel electrophoresis into a doublet. The upper component amounted to approx. 90% of the total and was comprised of 220 kDa and/or 215 kDa subunits; the lower component contained 207 kDa plus a 220 kDa or 215 kDa subunit. Scanning transmission electron microscopy indicated that under physiologic conditions zone II fibronectin molecules, like those in zone I, exist as pleiomorphic, loosely folded structures (approx. 16 X 8-12 nm) that are somewhat smaller than dimeric zone I molecules (approx. 24 X 16 nm). Circular dichroic spectral analyses suggests that both types have similarly folded local domains. Affinity chromatography experiments revealed a relative decrease in the binding of zone II fibronectin to gelatin but no difference from zone I fibronectin with respect to heparin or fibrin binding.     

Self-processing 2A-polyproteins--a system for co-ordinate expression of multiple proteins in transgenic plants

Achieving co-ordinate, high-level and stable expression of multiple transgenes in plants is currently difficult. Expression levels are notoriously variable and influenced by factors that act independently on transgenes at different genetic loci. Instability of expression due to loss, re-arrangement or silencing of transgenes may occur, and is exacerbated by increasing numbers of transgenic loci and repeated use of homologous sequences. Even linking two or more genes within a T-DNA does not necessarily result in co-ordinate expression. Linking proteins in a single open reading frame--a polyprotein--is a strategy for co-ordinate expression used by many viruses. After translation, polyproteins are processed into constituent polypeptides, usually by proteinases encoded within the polyprotein itself. However, in foot-and-mouth disease virus (FMDV), a sequence (2A) of just 16-20 amino acids appears to have the unique capability to mediate cleavage at its own C-terminus by an apparently enzyme-independent, novel type of reaction. This sequence can also mediate cleavage in a heterologous protein context in a range of eukaryotic expression systems. We have constructed a plasmid in which the 2A sequence is inserted between the reporter genes chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS), maintaining a single open reading frame. Here we report that expression of this construct in wheatgerm lysate and transgenic plants results in efficient cleavage of the polyprotein and co-ordinate expression of active CAT and GUS. Self-processing polyproteins using the FMDV 2A sequence could therefore provide a system for ensuring co-ordinated, stable expression of multiple introduced proteins in plant cells.