Reactivity of the heme-dioxygen complex of the inducible nitric oxide synthase in the presence of alternative substrates

Single turnover reactions of the inducible nitric oxide synthase oxygenase domain (iNOSoxy) in the presence of several non alpha-amino acid N-hydroxyguanidines and guanidines were studied by stopped-flow visible spectroscopy, and compared with reactions using the native substrates L-arginine (L-arg) or N(omega)-hydroxy-L-arginine (NOHA). In experiments containing dihydrobiopterin, a catalytically incompetent pterin, and each of the studied substrates, L-arg, butylguanidine (BuGua), para-fluorophenylguanidine (FPhGua), NOHA, N-butyl- and N-(para-fluorophenyl)-N'-hydroxyguanidines (BuNOHG and FPhNOHG), the formation of a iron(II) heme-dioxygen intermediate (Fe(II)O2) was always observed. The Fe(II)O2 species then decayed to iron(III) iNOSoxy at rates that were dependent on the nature of the substrate. Identical reactions containing the catalytically competent cofactor tetrahydrobiopterin (BH4), iNOSoxy and the three N-hydroxyguanidines, all exhibited an initial formation of an Fe(II)O2 species that was successively converted to an Fe(III)NO complex and eventually to high-spin iron(III) iNOSoxy. The formation and decay kinetics of the Fe(III)NO complex did not vary greatly as a function of the N-hydroxyguanidine structure, but the formation of Fe(III)NO was substoichiometric in the cases of BuNOHG and FPhNOHG. Reactions between BH4-containing iNOSoxy and BuGua exhibited kinetics similar to those of the corresponding reaction with L-arginine, with formation of an Fe(II)O2 intermediate that was directly converted to high-spin iron(III) iNOSoxy. In contrast, no Fe(II)O2 intermediate was observed in the reaction of BH4-containing iNOSoxy and FPhGua. Multi-turnover reaction of iNOS with FPhGua did not lead to formation of NO or to hydroxylation of the substrate, contrary to reactions with BuGua or L-arg. Our results reveal how different structural and chemical properties of NOS substrate analogues can impact on the kinetics and reactivity of the Fe(II)O2 intermediate, and support an important role for substrate pKa during NOS oxygen activation.     

Differential effects of cyclopolyamines on the stability and conformation of triplex DNA

Linear polyamines are excellent promoters of triplex DNA formation. The effects of structural rigidization of polyamines on triplex DNA stability are not known at present. We wished to develop a series of polyamine analogs as secondary ligands for triplex DNA stabilization for antigene applications. To accomplish this goal, we synthesized cyclopolyamines by interconnecting the two amino or imino groups of linear polyamines with a --(CH2)n-bridge (n=3,4,5). Melting temperature (Tm) data showed that [4,3]-spermine and [4,4]-spermine stabilized poly(dA) x 2poly(dT) triplex at >25 microM concentrations (Tm = 71 degrees C at 100 microM). The dTm/dlog [polyamine] values for these compounds were 26 and 40, respectively. [4,3]-Spermine and [4,4]-spermine also stabilized triplex DNA formed by a purine-motif triplex-forming oligonucleotide, TG3TG4TG4TG3T with its target duplex, as determined by Tm, circular dichroism (CD) spectroscopy, and electrophoretic mobility shift assay (EMSA). In contrast, [4,4]-putrescine and [4,5]-putrescine as well as [4,5]-spermine had no triplex DNA stabilizing effect. CD spectra also showed triplex DNA aggregation and psi-DNA formation at >100 microM [4,3]-spermine. These data demonstrate that structural rigidization of linear polyamines has a profound effect on their ability to stabilize triplex DNA and provoke conformational transitions.     

Differential effects of antimitotic agents on the stability and behavior of cytoplasmic and ciliary microtubules

Summary When sea urchin gastrulae are treated with colchicine or hydrostatic pressure the cytoplasmic microtubules disappear, but the ciliary microtubules which make up the ciliary axoneme (9+2) remain. With calcium-free sea water the cytoplasmic microtubules are reduced in number yet the 9+2 complex in the cilia is unaffected. Furthermore during the administration of any of these agents the cilia continue to beat so that functionally as well as morphologically the ciliary microtubules are normal even though the cytoplasmic microtubules are broken down and their presumed function in development is interrupted.Available evidence indicates that these two types of microtubules appear to be made up of similar subunits. Since there are morphological connections between the microtubules of the ciliary axoneme, and since the ciliary microtubules appear to stain more intensely than the cytoplasmic microtubules, we conclude that the ciliary microtubules are stabilized either by the addition of material or through interactions between adjacent tubules or both.Supported by Grant #5T 1-GM-707 from the National Institutes of Health to ProfessorKeith R.Porter.     

mRNA stability and plasmid copy number effects on gene expression from an inducible promoter system

The effects of mRNA stability and plasmid copy number on gene expression in Escherichia coli were evaluated by constructing multicopy (pMB1-based) and low-copy (F-based) plasmids containing an arabinose-inducible promoter system, the lacZ reporter gene, and mRNA-stabilizing 5' hairpin structures. Product formation and cell growth were evaluated under a number of inducer concentrations. The introduction of a 5' hairpin into the untranslated region of the mRNA resulted in significantly higher gene expression from the multicopy plasmids at low inducer concentrations and increased gene expression from the low-copy plasmids across all inducer concentrations investigated. With high inducer concentrations, expression from high-copy plasmids significantly slowed cell growth, whereas expression from the low-copy plasmids had little effect on growth rate. At inducer concentrations between 1 x 10(-4) and 4 x 10(-4)%, the productivity of low-copy plasmids containing the 5'-hairpin was equal to or greater than that from multicopy plasmids. Together, these two gene expression strategies may find important use in metabolic engineering and heterologous gene expression.     

Differential effects of Parkinson's disease-associated mutations on stability and folding of DJ-1

Mutations in the PARK7/DJ-1 gene cause autosomal-recessive Parkinson's disease. In some patients the gene is deleted. The molecular basis of disease in patients with point mutations is less obvious. We have investigated the molecular properties of [L166P]DJ-1 and the novel variant [E64D]DJ-1. When transfected into non-neuronal and neuronal cell lines, steady-state expression levels of [L166P]DJ-1 were dramatically lower than wild-type [WT]DJ-1 and [E64D]DJ-1. Cycloheximide and pulse-chase experiments revealed that the decreased expression levels of [L166P]DJ-1 were because of accelerated protein turnover. Proteasomal degradation was not the major pathway of DJ-1 breakdown because treatment with the proteasome inhibitor MG-132 caused only minimal accumulation of DJ-1, even of the very unstable [L166P]DJ-1 mutant. Because of the structural resemblance of DJ-1 with bacterial cysteine proteases, we considered an autoproteolytic mechanism. However, neither pharmacological inhibition nor site-directed mutagenesis of the putative active site residue Cys-106 stabilized DJ-1. To gain further insight into the structural defects of DJ-1 mutants, human [WT]DJ-1 and both mutants were expressed in Escherichia coli. As in eukaryotic cells, expression levels of [L166P]DJ-1 were dramatically reduced compared with [WT]DJ-1 and [E64D]DJ-1. Circular dichroism spectrometry revealed that the solution structures of [WT]DJ-1 and [E64D]DJ-1 are rich in beta-strand and alpha-helix conformation. Alpha-helices were more susceptible to thermal denaturation than the beta-sheet, and [WT]DJ-1 was more flexible in this regard than [E64D]DJ-1. Thus, structural defects of [E64D]DJ-1 only become apparent upon denaturing conditions, whereas the L166P mutation causes a drastic defect that leads to excessive degradation.     

Assessing the impact of inductive electronic effects on the metrical parameters and reactivity of a series of ferrous complexes

Reported are four iron(II) complexes with N-benzyl-N,N′-bis(2-pyridylmethyl)-1,2-ethanediamine (L^H) and three electronically modified derivatives: N-(4-methoxy)benzyl-N,N′-bis(2-pyridylmethyl)-1,2-ethanediamine (L^OMe), N-(4-chloro)benzyl-N,N′-bis(2-pyridylmethyl)-1,2-ethanediamine (L^Cl), and N-(4-nitro)benzyl-N,N′-bis(2-pyridylmethyl)-1,2-ethanediamine (L^NO₂). The four ligands react with FeCl₂ to form a series of mononuclear species with the general formula [Fe(L^R)Cl₂]. The cis-α conformation of the ligand places the amine N-donors trans to the Fe-Cl bonds. The identity of the 4-benzyl substituent has profound influences on the lengths of the iron-ligand bonds, the optical spectra, and the redox activities of the [Fe(L^R)Cl₂] compounds.     

Differential effects of different statins on endothelin-1 gene expression and endothelial NOS phosphorylation in porcine aortic endothelial cells

It has been reported that 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitors (statins) produce a variety of cardiovascular protective effects independent of their ability to lower total and low-density lipoprotein cholesterol. Recent studies have also reported that statins produce pleiotropic effects through improved endothelial function, enhanced fibrinolysis, and antithrombotic actions. In the present study, we examined the effects of pitavastatin, pravastatin, atorvastatin, and cerivastatin on endothelin (ET)-1 production in cultured porcine aortic endothelial cells (PAECs). Treatment with cerivastatin but not pitavastatin, pravastatin, or atorvastatin decreased basal and TNF-alpha-stimulated ET-1 release from PAECs in a dose-dependent manner (1-10 microM). Northern blot analysis showed that cerivastatin markedly suppressed prepro ET-1 mRNA expression in both conditions. In addition, these inhibitory effects of cerivastatin on ET-1 release and prepro ET-1 mRNA expression were completely abolished by simultaneous treatment with 200 microM mevalonate. Furthermore, cerivastatin did not have any effects on endothelial nitric oxide synthase (eNOS) protein levels, but induced eNOS phosphorylation at Ser1177. From these findings, it is most likely that cerivastatin suppresses ET-1 production, possibly through an increase in eNOS activity and the subsequent nitric oxide production in PAECs. These findings also suggest that cerivastatin may have beneficial effects on ET-1-related diseases.     

Differential Vicia villosa agglutinin reactivity identifies three distinct dystroglycan complexes in skeletal muscle.

We present evidence for the expression of three alpha-dystroglycan glycoforms in skeletal muscle cells, including two minor glycoforms marked by either patent or latent reactivity with the N-acetylgalactosamine-specific lectin Vicia villosa agglutinin. Both minor glycoforms co-isolated with beta-dystroglycan, but not with other dystrophin/utrophin-glycoprotein complex components, suggesting that they may perform distinct or modified cellular functions. We also confirmed that both patent and latent V. villosa agglutinin-reactive alpha-dystroglycan glycoforms are expressed in C2C12 myotubes. However, we found that the combined effect of saturating concentrations of V. villosa agglutinin and laminin-1 were strictly additive with respect to acetylcholine receptor cluster formation in C2C12 myotubes, which suggests that laminin-1 and V. villosa agglutinin do not compete for the same binding site on the cell surface. Finally, although beta-N-acetylhexosaminidase digestion dramatically inhibited agrin-, V. villosa agglutinin-, and laminin-1-induced acetylcholine receptor clustering in C2C12 myotubes, treatment with this enzyme had no effect on the amount of alpha-dystroglycan that was bound to V. villosa agglutinin-agarose. We conclude that alpha-dystroglycan is not the V. villosa agglutinin receptor implicated in acetylcholine receptor cluster formation. However, our data provide new support for the hypothesis that different glycoforms of alpha-dystroglycan may perform distinct functions even within the same cell.     

The effect of axial ligands on the reactivity and stability of the oxoferryl moiety in model complexes of Compound I of heme-dependent enzymes

 The contribution of simple inorganic model complexes to the understanding of related processes in biomolecules is demonstrated by a series of Compound I analogs of heme-dependent enzymes. The oxoiron(IV) porphyrin cation radical state in these synthetic complexes is stable enough to be studied by spectroscopic methods as a function of only one variable, the axial ligand trans to the oxoiron bond. Complementary information from kinetic investigations of the reactivity in epoxidation of olefins enables the separation of the thermodynamic and kinetic effects of the axial ligands. The results clearly indicate that epoxidation by these complexes proceeds by two distinguishable steps, which are affected differently by the axial ligands. The first step is electron transfer from the olefin to the ferryl moiety, probably followed by intramolecular charge rearrangement and product release. It is proposed that part of the enhanced oxygenation activities of cytochrome P-450 monooxygenases and chloroperoxidases is due to a lowering of the energy barrier for the second step via participation of their redox-active cysteinate ligand in charge rearrangement. Received and accepted: 7 May 1996     

Differential effects of zinc and magnesium ions on mineralization activity of phosphatidylserine calcium phosphate complexes

Mg²⁺ and Zn²⁺ are present in the mineral of matrix vesicles (MVs) and biological apatites, and are known to influence the onset and progression of mineral formation by amorphous calcium phosphate (ACP) and hydroxyapatite (HAP). However, neither has been studied systematically for its effect on mineral formation by phosphatidylserine-Ca²⁺-Pi complexes (PS-CPLX), an important constituent of the MV nucleation core. Presented here are studies on the effects of increasing levels of Mg²⁺ and Zn²⁺ on the process of mineral formation, either when present in synthetic cartilage lymph (SCL), or when incorporated during the formation of PS-CPLX. Pure HAP and PS-CPLX proved to be powerful nucleators, but ACP took much longer to induce mineral formation. In SCL, Mg²⁺ and Zn²⁺ had significantly different inhibitory effects on the onset and amount of mineral formation; HAP and PS-CPLX were less affected than ACP. Mg²⁺ and Zn²⁺ caused similar reductions in the rate and length of rapid mineral formation, but Zn²⁺ was a more potent inhibitor on a molar basis. When incorporated into PS-CPLX, Mg²⁺ and Zn²⁺ caused significantly different effects than when present in SCL. Even low, subphysiological levels of Mg²⁺ altered the inherent structure of PS-CPLX and markedly reduced its ability to induce and propagate mineral formation. Incorporated Zn²⁺ caused significantly less effect, low (<20 μM) levels causing almost no inhibition. Levels of Zn²⁺ present in MVs do not appear to inhibit their nucleational activity.     

Synthesis and reactivity of tin amide complexes

A terminal tin amide and a terminal tin anilide complex, (BDI)SnN(iPr₂) and (BDI)SnN(H)Ar (Ar = 2,6-ⁱPr₂C₆H₃), respectively, have been synthesized utilizing the bulky β-diketiminate ligand, [{N(2,6-ⁱPr₂C₆H₃)C(Me)}₂CH]⁻, or BDI, to stabilize the low coordinate divalent tin metal center. Only (BDI)SnN(iPr₂) reacts with phenylacetylene to yield (BDI)SnCCPh, but both species react with methyl triflate to give (BDI)SnOTf and carbon dioxide, resulting in the formation of (BDI)SnOC(O)N(iPr₂) and (BDI)SnOC(O)N(H)Ar.     

Differential effects of chelating agents on cytosolic glucocorticoid receptor stability and nuclear binding in vitro

The effect of several metal chelators (EDTA, EGTA, and 1,10 phenanthroline) on rat liver glucocorticoid receptor properties in vitro was investigated. At 4 degrees C 10 mM EDTA (unlike 10 mM EGTA and 10 mM 1,10 phenanthroline) had a significant stabilizing effect on unbound hepatic glucocorticoid receptors. At higher temperature (25 degrees C) 10 mM EGTA appeared to act as a chemical stabilizer of unbound receptors. 1,10 Phenanthroline had no stabilizing effect at either temperature. Scatchard analysis indicated that the alteration in receptor binding after incubation at 4 and 25 degrees C in the presence and absence of chelating agents was due to a change in the number of steroid binding sites rather than perturbation of receptor affinity. Unlike results obtained with unbound receptors, all three chelating agents appeared to enhance prebound glucocorticoid-receptor complex inactivation. Interestingly these chelating reagents also significantly altered glucocorticoid-receptor complex binding to isolated nuclei.     

Experimental reactivity and antigenic activity of the bacterial-fungal antigen complexes

The immunogenic and reactogenic properties of monovaccines prepared from Staphylococcus aureus, S. epidermidis and Candida albicans, as well as those of a associated polyvaccine prepared from these infective agents, were experimentally studied on rabbits. The monovaccines and the associated bacterial-fungal vaccines were found to be safe and faintly reactogenic; in the blood serum of vaccinated rabbits a growth in the titer of agglutinins and its preservation at a high level for 4 months were noted.     

Differential effects of phenobarbital on the constitutive and inducible expression of P450 2B and 3A subfamilies in sheep tissues.

The activity and expression of cytochromes P450 were determined in liver, kidneys, lungs, duodenum, jejunum, ileum, and caecum of adult Lacaune sheep. High expression of total P450, benzphetamine and erythromycin demethylase activities, and P450 2B isoforms, as two distinct proteins that were detected and called P4502 Bm and P4502 Bx, was found in the lungs (in addition to liver). By contrast, the P450 3A subfamily was only expressed in liver and duodenal mucosa of untreated sheep. Phenobarbital (PB) treatment led to significant increases in all measured hepatic parameters and in total P450 of each investigated organ with the exception of ileum and caecum. Benzphetamine demethylase activity increased in liver and kidneys, correlating with the expression of the two P450 2B proteins, which were also induced in duodenum and ileum. By contrast, benzphetamine demethylase activity and expression of the P450 2B isoforms in lungs were unchanged by PB treatment. Erythromycin demethylation activity and P450 3A subfamily expression was increased only in liver of PB-treated sheep.