Base-sequence dependence of noncovalent complex formation and reactivity of benzo[a]pyrene diol epoxide with polynucleotides

The base-sequence selectivity of the noncovalent binding of (+/-)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyr ene (BPDE) to a series of synthetic polynucleotides in aqueous solutions (5 mM sodium cacodylate buffer, 20 mM NaCl, pH 7.0, 22 degrees C) was investigated. The magnitude of a red-shifted absorbance at 353 nm, attributed to intercalative complex formation, was utilized to determine values of the association constant Kic. Intercalation in the alternating pyridine-purine polymers poly(dA-dT).(dA-dT) (Kic = 20,000 M-1), poly(dG-dC).(dG-dC) (4200 M-1), and poly(dA-dC).(dG-dT) (9600 M-1) is distinctly favored over intercalation in their nonalternating counterparts poly(dA).(dT) (780 M-1), poly(dG).(dC) (1800 M-1), and poly(dA-dG).(dT-dC) (5400 M-1). Methylation at the 5-position of cytosine gives rise to a significant enhancement of intercalative binding, and Kic is 22,000 M-1 in poly(dG-m5dG).(dG-m5dC). In a number of these polynucleotides, values of Kic for pyrene qualitatively follow those exhibited by BPDE, suggesting that the pyrenyl residue in BPDE is a primary factor in determining the extent of intercalation. Both BPDE and pyrene exhibit a distinct preference for intercalating within dA-dT and dG-m5dC sequences. The catalysis of the chemical reactions of BPDE (hydrolysis to tetrols and covalent adduct formation) is enhanced significantly in the presence of each of the polynucleotides studied, particularly in the dG-containing polymers. A model in which catalysis is mediated by physical complex formation accounts well for the experimentally observed enhancement in reaction rates of BPDE in the alternating polynucleotides; however, in the nonalternating polymers a different or more complex catalysis mechanism may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of hyperoxia-induced pulmonary vascular paralysis: effect of antioxidant pretreatment

We designed experiments using isolated rabbit lungs to determine the effect of hyperoxia on the pulmonary vasoconstriction caused by the infusion of the lipid peroxide tert-butyl hydroperoxide (t-bu-OOH), which produces vasoconstriction by stimulating the pulmonary synthesis of thromboxane. Exposure to 48-60 h of 100% O2 at 1 ATA markedly reduced the increase in pulmonary artery pressure caused by t-bu-OOH infusion. We also investigated whether the mechanism for the attenuated vasoconstriction was due to altered production of arachidonate mediators or oxidant-induced damage to the contractile mechanism. In addition to infusing t-bu-OOH, which selectively stimulates thromboxane production, we also infused Intralipid, an esterified fatty acid emulsion that stimulates production of both thromboxane and prostacyclin. These experiments were done to study the effect of hyperoxia on prostacyclin synthesis. To determine if antioxidant therapy would prevent the changes in mediator production and vascular reactivity caused by hyperoxia, we pretreated animals with the antioxidants butylated hydroxyanisole (BHA) or vitamin E. The lack of vascular reactivity to t-bu-OOH was not due to a decrease in thromboxane synthesis or an increase in prostacyclin synthesis. Hyperoxia did not affect thromboxane synthesis during basal conditions or after stimulation of synthesis by t-bu-OOH. 100% O2 also did not effect the basal synthesis of prostacyclin by the lung. Hyperoxia did, however, markedly reduce prostacyclin synthesis when it was stimulated by Intralipid infusion. Antioxidant pretreatment did not reverse the inhibition of prostacyclin synthesis but did prevent the loss of vascular reactivity caused by hyperoxia. Thus hyperoxia causes vascular paralysis through oxidant-induced injury to the pulmonary vasculature.     

Fluorescence and photoelectron studies of the intercalative binding of benz(a)anthracene metabolite models to DNA

An analog of the peptidyl transferase inhibitor sparsomycin was a competitive inhibitor (Ki = 1.8 microM) of peptidyl-puromycin synthesis on E. coli polysomes. Preincubation of polysomes with the compound enhanced the degree of inhibition of peptide bond formation. A model for the involvement of a histidine residue in peptidyl transferase activity is presented as a result of our observations which include direct association of [3H] labelled analog with 70S ribosomes. The correct oxidation state of sulfur in the compound was necessary for the "preincubation effect" and entry of the compound into bacterial cells.     

Mechanisms by which endothelin 1 induces pulmonary vasoconstriction in the rabbit.

To investigate the mechanisms by which endothelin 1 (ET-1) causes pulmonary vasoconstriction, we studied the effect of synthetic ET-1 on pulmonary vascular tone in the buffer-perfused isolated rabbit lung. In nanomolar concentrations (1.2-8 nM), ET-1 causes a dose-dependent increase in pulmonary arterial pressure that persists for greater than or equal to 1 h (increase in pressure 19 +/- 2 mmHg with ET-1 vs. 2 +/- 1 with vehicle, P less than 0.0001). Reduction of calcium availability with verapamil, cadmium, or a calcium-free buffer significantly blunts the increase in pressure caused by ET-1. Pretreatment with a calcium-free buffer plus the chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA) completely eliminates the vasoconstriction. Three different inhibitors of protein kinase C, phloretin, staurosporine, and dihydrosphingosine, significantly diminish the response to ET-1. Indomethacin and a thromboxane synthase inhibitor partially decrease the response to the highest concentration of ET-1. Isoproterenol and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) are significantly more effective in preventing the vasoconstriction caused by ET-1 than are nitroprusside and guanosine 5'-cyclic monophosphate (cGMP) analogues. ET-1 in doses of 1.2-8 nM is a potent pulmonary vasoconstrictor in the isolated rabbit lung. ET-1 appears to cause pulmonary vasoconstriction by increasing calcium entry and by activating protein kinase C. Vasodilators that increase cAMP are substantially more effective in preventing the increase in pressure than are drugs that increase cGMP.     

Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA-binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.     

Spectroscopic and kinetic characterization of nonenzymic and aldose reductase mediated covalent NADP-glycolaldehyde adduct formation

Reaction of glycolaldehyde with the binary E-NADP complex of bovine kidney aldose reductase (ALR2) produces an enzyme-bound chromophore whose absorbance (lambd max 341 nm) and fluorescence (lambda ex max 341 nm; lambda emit max 421 nm) properties are distinct from those of NADPH or E.NADPH yet are consistent with the proposed covalent adduct structure [1,4-dihydro-4-(1-hydroxy-2-oxoethyl)nicotinamide adenine dinucleotide phosphate]. The kinetics of adduct formation, both in solution and at the enzyme active site, support a mechanism involving rate-determining enolization of glycolaldehyde at high [NADP+] or [E.NADP]. At low [NADP+] or [E.NADP] the reaction is second-order overall, but the ALR2-mediated reaction displays saturation by glycolaldehyde due to competition of the aldehyde (plus hydrate) and enol for E.NADP. Measurement of the pre-steady-state burst of E-adduct formation confirms that glycolaldehyde enol is the reactive species and gives a value of 1.3 x 10(-6) for Kenol = [enol]/[( aldehyde] + [hydrate]), similar to that determined by trapping the enol with I3-. At the ALR2 active site, the rate of adduct formation is enhanced 79,000-fold and the adduct is stabilized greater than or equal to 13,000-fold relative to the reaction with NADP+ in solution. A portion of this enhancement is ascribed to specific interaction of NADP+ with the enzyme since the 3-acetylpyridine analogue, (AP)ADP+, gives values that are 15-200-fold lower. Additional evidence for strong interaction of ALR2 with both NADP+ and NADPH is reported. Yet, because dissociation of adduct is slow, catalysis of the overall adduct formation reaction by ALR2 is less than or equal to 67-fold.     

The fauna of benthic sediments from the organically enriched Oslofjord,Norway

The macrobenthic fauna of the organically enriched Oslofjord was sampled in Sept.–Nov. 1977. Seventy-six stations were sampled using a stratified random plan. A total of 146 species were identified, dominated by polychaetes, molluscs and echinoderms. The faunal data were analysed by a variety of methods including diversity indices, log-normal distribution of individuals among species, factor analysis and numerical classification. The primary aim was the detection of pollution-induced disturbance and to delimit the extent of pollution on the benthic fauna of Oslofjord. All of the methods used showed a gradient from heavy pollution at the innermost part of the fjord where few species occurred, together with high dominance and low diversity, to normal unpolluted conditions at the Drøbak sill with high species diversity. From the classification analysis a combination of both site and species data revealed a trend in species groupings which were broadly similar to data from other Scandinavian fjords and from Scotland. Hydrographical and biological data taken at the turn of the century and in the 1960's compared with the present data show declining conditions in Oslofjord, due primarily to organic enrichment combined with a naturally poor water exchange which leads to stagnation of the water mass.     

Ultraviolet photoelectron studies of 5-halouracils

Ultravoilet photoelectron spectroscopy has been employed to exmamin the valence electronic structure of 5-fluorouracil, 5-chlorouracil, 5-bromouracil, and 5-iodouracil. Photoelectron bands associted with the three highest π orbitals and the two oxygen atom lone-pair orbitals were assigned by a comparison to similar bands observed in the photoelectron spectrum of uraciul. Bands arising from the halogen atom lone-pair orbitals were assigned by comparing the present results with photoelectron spectra measured for halobenzenes, and by considering the linear dependence of halogen atom lone-pair ionization potentials upon halogen atom electronegativities. The present spectroscopic results have been compared with results from studies of association constants of 5-halouracil–adenine complexes. This examination in dicates that the complex association constants incresase as the ionization potentials of the highest occupied π orbital and the halogen atom lone-pair orbitals of th halouracils decrease.     

Delphinidin Reduces Cell Proliferation and Induces Apoptosis of Non-Small-Cell Lung Cancer Cells by Targeting EGFR/VEGFR2 Signaling Pathways

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) have emerged as two effective clinical targets for non-small-cell lung cancer (NSCLC). In the present study, we found that delphinidin, an anthocyanidin, present in pigmented fruits and vegetables, is a potent inhibitor of both EGFR and VEGFR2 in NSCLC cells that overexpress EGFR/VEGFR2. Using these cells, we next determined the effects of delphinidin on cell growth and apoptosis in vitro and on tumor growth and angiogenesis in vivo. Delphinidin (5-60 µM) treatment of NSCLC cells inhibited the activation of PI3K, and phosphorylation of AKT and MAPKs. Additionally, treatment of NSCLC cells with delphinidin resulted in inhibition of cell growth without having significant toxic effects on normal human bronchial epithelial cells. Specifically, treatment of NCI-H441 and SK-MES-1 cells with delphindin (5-60 µM) resulted in (i) cleavage of PARP protein, (ii) activation of caspase-3 and -9, (iii) downregulation of anti-apoptotic proteins (Bcl2, Bcl-xL and Mcl-1), (iv) upregulation of pro-apoptotic proteins (Bax and Bak), and (v) decreased expression of PCNA and cyclin D1. Furthermore, in athymic nude mice subcutaneously implanted with human NSCLC cells, delphinidin treatment caused a (i) significant inhibition of tumor growth, (ii) decrease in the expression of markers for cell proliferation (Ki67 and PCNA) and angiogenesis (CD31 and VEGF), and (iii) induction of apoptosis, when compared with control mice. Based on these observations, we suggest that delphinidin, alone or as an adjuvant to current therapies, could be used for the management of NSCLC, especially those that overexpress EGFR and VEGFR2.