Localisation of Plasmodium falciparum uroporphyrinogen III decarboxylase of the heme-biosynthetic pathway in the apicoplast and characterisation of its catalytic properties

Uroporphyrinogen decarboxylase (UROD) is a key enzyme in the heme-biosynthetic pathway and in Plasmodium falciparum it occupies a strategic position in the proposed hybrid pathway for heme biosynthesis involving shuttling of intermediates between different subcellular compartments in the parasite. In the present study, we demonstrate that an N-terminally truncated recombinant P. falciparum UROD (r(Δ)PfUROD) over-expressed and purified from Escherichia coli cells, as well as the native enzyme from the parasite were catalytically less efficient compared with the host enzyme, although they were similar in other enzyme parameters. Molecular modeling of PfUROD based on the known crystal structure of the human enzyme indicated that the protein manifests a distorted triose phosphate isomerase (TIM) barrel fold which is conserved in all the known structures of UROD. The parasite enzyme shares all the conserved or invariant amino acid residues at the active and substrate binding sites, but is rich in lysine residues compared with the host enzyme. Mutation of specific lysine residues corresponding to residues at the dimer interface in human UROD enhanced the catalytic efficiency of the enzyme and dimer stability indicating that the lysine rich nature and weak dimer interface of the wild-type PfUROD could be responsible for its low catalytic efficiency. PfUROD was localised to the apicoplast, indicating the requirement of additional mechanisms for transport of the product coproporphyrinogen to other subcellular sites for its further conversion and ultimate heme formation.

Photo-induced double-strand DNA and site-specific protein cleavage activity of L-histidine (mu-oxo)diiron(III) complexes of heterocyclic bases

Three oxo-bridged diiron(III) complexes of L-histidine and heterocyclic bases [Fe-2(mu-O)(L-his)(2)(B)(2)](ClO4)(2) (1-3), where B is 2,2'-bipyridine (bpy),1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), were prepared and characterized. The bpy complex 1 was structurally characterized by X-ray crystallography. The molecular structure showed a {Fe-2(mu-O)} core in which iron(III) in a FeN4O2 coordination is bound to tridentate monoanionic L-histidine and bidentate bpy ligands. The Fe center dot center dot center dot Fe distance is similar to 3.5 angstrom. The Fe-O-Fe unit is essentially linear, giving a bond angle of similar to 172 degrees. The complexes showed irreversible cyclic voltammetric cathodic response near -0.1 V vs. SCE in H2O-0.1 M KCl. The binuclear units displayed antiferromagnetic interaction between two high-spin (S = 5/2) iron(III) centers giving a -J value of -110 cm(-1). The complexes showed good DNA binding propensity giving a binding constant value of similar to 10(5) M-1. Isothermal titration calorimetric data indicated single binding mode to the DNA. The binding was found to be driven by negative free energy change and enthalpy. The dpq complex 3 showed oxidative double-strand DNA cleavage on exposure to UV-A and visible light. The phen complex 2 displayed single-strand photocleavage of DNA. The DNA double-strand breaks were rationalized from theoretical molecular docking calculations. Mechanistic investigations showed formation of hydroxyl radicals as the reactive species through photodecarboxylation of the L-histidine ligand. The complexes exhibited good binding propensity to bovine serum albumin (BSA) protein in Tris-HCl/NaCl buffer medium. The dpq complex 3 showed UV-A light-induced site-specific oxidative BSA cleavage forming fragments of similar to 45 kDa and similar to 20 kDa molecular weights via SOH pathway.

Theoretical gain optimization studies in CO2-N2 lasers. III. Inclusion of line shape effects

The influence of Lorentz and Doppler line-broadening mechanisms on the small-signal optical gain of lasers and, in particular, gasdynamic lasers, is discussed. A relationship between the critical parameter reflecting the line-broadening mechanisms and some of the important parameters arising out of the gain optimization studies in CO2-N2 gasdynamic lasers is established. Using this relationship, methods by which the deleterious effect of the Doppler mechanisms on small-signal gain can be suppressed are suggested. Journal of Applied Physics is copyrighted by The American Institute of Physics.

Iron(II) and iron(III) complexes of 2,4-dithiobiuret

Ferrous and ferric complexes of 2,4-dithiobiuret (Dtb) of the type Fe(Dtb)m Xn where m, n = 1-3, and X = CI–, Br–, I– and SO 4 2– , and a neutral Fe(Dtb-H)2 complex have been synthesized and characterised by elemental analyses, magnetic susceptibility, i.r., electronic and Mössbauer spectroscopic studies. From its i.r. spectrum Dtb was found to act as a S,S-coordinating bidentate chelate. The magnetic moment, electronic and Massbauer spectra are consistent with a low spin distorted octahedral structure for the ferric complexes and a high spin form for ferrous complexes.

Co(II) and Cr(III) complexes of formate-formamide mixed ligands: synthesis, structures, single crystal-to-single crystal transformation and magnetic behaviour

Solvothermal treatment of an equimolar mixture of Co(NO3)(2)center dot 6H(2)O, HCONH2 and NaN3 in MeOH at 100 degrees C yielded a three-dimensional NaCl type network Co(HCOO)(2)(HCONH2)(2) center dot HCONH2 (1a) containing formamides in the pores of the structure. Solvated pink 1a undergoes single crystal-to-single crystal (SCSC) transformation at 215 degrees C to form the desolvated dark brown product Co(HCOO)(2)-( HCONH2)(2) (1b) with the retention of the original framework. Reversible single crystal-to-single crystal transformation of 1b (brown) to 1a (pink) in the presence of excess formamide was also established at room temperature. The coordination environment around Co(II) in both 1a and 1b is octahedral with a CoN2O4 coordination composition. A similar reaction replacing Co(II) by Cr(III) produced a heterometallic 3D extended network Na[Cr(HCOO)(4)(HCONH2)(2)]center dot 2H(2)O (2a) at 100 degrees C. An increase in reaction temperature to 150 degrees C produced a simple mononuclear complex Cr(HCOO)(3)(HCONH2)(3) center dot 3H(2)O (2b). Variable temperature magnetic studies revealed the presence of a canting phenomena in both 1a and 1b, and hysteresis loop in the field dependent magnetisation plot at 2 K whereas complex 2a is simply paramagnetic in nature.

Anaerobic DNA cleavage activity in red light and photocytotoxicity of (pyridine-2-thiol)cobalt(III) complexes of phenanthroline bases

Cobalt(III) complexes [Co(pnt)(B)(2)](NO3)(2) (1-3) of pyridine-2-thiol (pnt) and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyrido[3,2-d: 2',3'-f]quinoxaline (dpq in 2) and dipyrido[3,2-a:2',3'-c] phenazine (dppz in 3), have been prepared, characterized and their photo-induced anaerobic DNA cleavage activity studied. The crystal structure of 1a as mixed ClO4- and PF6- salt of 1 shows a (CoN5S)-N-III coordination geometry in which the pnt and phen showed N,S- and N,N-donor binding modes, respectively. The complexes exhibit Co(III)/Co(II) redox couple near -0.3 V (vs. SCE) in 20% DMF-Tris-HCl buffer having 0.1 M TBAP. The complexes show binding propensity to calf thymus DNA giving K-b values within 2.2 x 10(4)-7.3 x 10(5) M-1. Thermal melting and viscosity data suggest DNA surface and/or groove binding of the complexes. The complexes show significant anaerobic DNA cleavage activity in red light under argon atmosphere possibly involving sulfide anion radical or thiyl radical species. The DNA cleavage reaction under aerobic medium in red light is found to involve both singlet oxygen and hydroxyl radical pathways. The dppz complex 3 shows non-specific BSA and lysozyme protein cleavage activity in UV-A light of 365 nm via both hydroxyl and singlet oxygen pathways. The dppz complex 3 exhibits photocytotoxicity in HeLa cervical cancer cells giving IC50 values of 767 nM and 19.38 mu M in UV-A light of 365 nm and in the dark, respectively. A significant reduction of the dark toxicity of the dppz base (IC50 = 8.34 mu M in dark) is observed on binding to the cobalt(III) center.

Nucleotidases in plants *1, , *2: III. Effect of metabolites on the enzyme hydrolyzing flavine adenine dinucleotide (FAD) from Phaseolus radiatus

The highly purified enzyme from mung bean seedlings hydrolyzing FAD at pH 9.4 and temperature 49 °, functioned with an initial fast rate followed by a second slower rate. The activity was linear with enzyme concentration over a small range of concentration and was dependent on the time of incubation. Inhibition of enzyme activity with increasing concentrations of AMP was sigmoid;concentrations less than 1 × 10−6 M were without effect, concentrations between 1 × 10−6 and 8 × 10−5 M inhibited by 20% and concentrations beyond 8 × 10−5 Image caused progressive inhibition. Concentrations beyond 1 × 10−3 Image inhibited the activity completely. Preincubation of the enzyme with PCMB or NEM, or aging, or reversible denaturation with urea abolished the inhibitory effect of AMP at concentrations lower than 8 × 10−6 Image . The aged enzyme could be reactivated by ADP.

Photocytotoxic Lanthanum(III) and Gadolinium(III) Complexes of Phenanthroline Bases Showing Light-Induced DNA Cleavage Activity

Lanthanide complexes of formulation [La(B)(2)(NO3)(3)] (1-3) and [Gd(B)(2)(NO3)(3)] (4-6), where B is a N,N-donor phenanthroline base, namely, 1,10-phenanthroline (phen in 1, 4),dipyrido[3,2-d2',3'-f]quinoxaline (dpq in 2,5) and dipyrido[3,2-a2',3'-c]phenazine (dppz in 3, 6), have been prepared, characterized from physicochemical data, and their photoinduced DNA and protein cleavage activity studied The photocytotoxicity of the dppz complexes 3 and 6 has been studied using HeLa cancer cells. The complexes exhibitligand centered bands in the UV region The dppz complexes show thelowest energy band at 380 nm in N,N-dimethylformamide (DMF) The La(III)complexes are diamagnetic. The Gd(III) complexes (4-6) have magneticmoments that correspond to seven unpaired electrons The complexes are1(.)1 electrolytic in aqueous DMF The dpq and dppz complexes in DMFshow ligand-based reductions. The complexes display moderate binding propensity to calf thymus DNA giving binding constant values in the range of 5.7 x 10(4)-5.8 x 10(5) M-1 with a relative order. 3, 6 (dppz)> 2, 5 (dpq) > 1, 4 (phen) The binding data suggest DNA surface and/or groove binding nature of the complexes. The complexes do not show any hydrolytic cleavage of plasmid supercoiled pUC19 DNA. The dpq and dppz complexes efficiently cleave SC DNA to its nicked circular form onexposure to UV-A light of 365 nm at nanomolar complex concentration. Mechanistic studies reveal the involvement of singlet oxygen (O-1(2)) and hydroxyl radical (HO center dot) as the cleavage active species.The complexes show binding propensity to bovine serum albumin (BSA)protein giving K-BSA values of similar to 10(5) M-1. The dppz complexes 3 and 6 show BSA protein cleavage activity in UV-A light of 365 nm The dppz complexes 3 and 6 exhibit significant photocytotoxicity in HeLa cells giving respective IC50 values of 341 nM and 573 nM in UV-A light of 365 nm for an exposure time of 15 min (IC50 > 100 mu M in dark for both the complexes) Control experiments show significant dark and phototoxicity of the dppz base alone (IC50 = 413 nM in light with 4 h incubation in dark and 116 mu M in dark with 24 h incubation). A significant decrease in the dark toxicity of the dppz base is observedon binding to the lanthanide ions while retaining similar phototoxicity.

Reactions of tetrahalogeno-1,2-benzoquinones. Part III. Reaction of tetrachloro-1,2-benzoquinone with tetralones and naphthols: Pathway to the condensates

With a view to understanding the mechanism of the formation of 6-methoxy-2,2-(tetrachloro--phenylenedioxy)-naphthalen-1 (2H)-one (IIIa) in the reaction of 6-methoxy-1-tetralone (Ia) with tetrachloro-1,2-benzoquinone (II), the reaction of (II) with various tetralones and naphthols has been studied. Reaction with either α-tetralone or α-naphthol gives 2,2-(tetrachloro-o-phenylenedioxy)naphthalen-1 (2H)-one (IIIb), whereas reaction with either β-tetralone or β-naphthol gives a mixture of (IIIb) and ,1-(tetrachloro-o-phenylenedioxy)-naphthalen-2 (1H)-one (IX), with the former predominating. Further, reactions of (II) with 7-methoxy-3,4-dihydrophenanthren- 1 (2H)-one and m-methoxyphenol gave respectively 7-methoxy- ,2-(tetrachloro-o- phenylenedioxy)phenanthren-1 (2H)-one (VII) and 3-methoxy-6,6-(tetrachloro-o- phenylenedioxy)cyclohexa-2,4-dien-1-one (VIII). Structures of all these compounds have been proved on the basis of i.r. and n.m.r. data. The pathway to the formation of the condensates (III) is discussed.

Mode of action of antitumour antibiotics-III: Modulation of permeability of nuclear membrane in the presence of the antibiotics

The binding characteristics of the antibiotics to nuclei and their effect on the permeability of nuclear membrane with respect to histones and ribonucleic acids have been investigated. The binding constant for chromomycin A3 was found to be 1.4 × 104M?1 and number of binding sites was equal to 3.48 ± 1.08 × 1012 molecules/nuclei. The antibiotic chromomycin A3 enhanced the uptake of lysine-rich histone, actinomycin D decreased the uptake and ethidium bromide had no effect. Chromomycin A3 also enhanced the release of acid insoluble fraction containing RNA from the nuclei, actinomycin D and ethidium bromide inhibited the release of acid insoluble fraction containing RNA. The relevance of this finding to the role of nuclear envelope in understanding the mechanism of action of the antibiotic has been discussed.

One pot synthesis of polycyclic oxygen aromatics. Part III mechanism of formation

Reaction of 6-Image -butyl-1-bromomethyl-2-(2-tetrahydropyranyloxy)-naphthalene2c with tetrachlorocatechol (TCC) in acetone in presence of K2CO3 gave diastereomers 6c and 7c. A mechanism (Scheme-1) invoking the base induced cleavage of the pyranyl ether 2 to 1,2-naphthoquinone-1-methide 8 as the first step has been postulated. The cleavage of the pyranyl ether linkage in 2 to give dimers 4 and 5 of 1,2-naphthoquinone-1-methide has been demonstrated with different bases. 1,2-Naphthoquinone-1-methide 8, thus generated, undergoes Michael addition with TCC followed by elimination of chloride ions to give a diketone, which further undergoes aldolisation with acetone to give diastereomers 6 and 7. Michael reaction of 8, generated Image from pyranyl ethers 2a-c, with tetrabromocatechol (TBC) under similar-reaction conditions gave the expected monobromo compounds 6h, 6i, 6k, 7n, 7n and 7q. The last step in the proposed mechanism, Image ., aldolisation has also been demonstrated using different ketonic solvents. Thus, reaction of 2a-c with TCC/TBC in diethyl ketone/methyl ethyl ketone under similar reaction conditions gave the expected compounds 6 and 7.

Mixed ligand cobalt(III) complexes of tetradentate N,N′-ethylenebis(acetylacetonimine) and flexidentate isonitroso-β-diketones and isonitrosomonoketones

A series of mixed ligand cobalt(III) complexes having the general formula Co(EA)X [where EA = dianion of N,N′-ethylenebis(acetylacetonimine) and X = anion of isonitroso-acetylacetone, IAA; isonitrosobenzoylacetone, IBA; isonitrosodibenzoylmethane, IDBM; isonitrosoethylacetoacetate, IEA; isonitrosoacetoacetanillide, IAN; isonitrosoethylmethylketone, IEMK; isonitrosobenzylmethylketone, IBMK and isonitrosopropiophenone, IPP] have been synthesised and characterised. A facial-cis-β structure (cis with respect to the coordinated two oxygen atoms of EA) with N,N,N,O,O,O ligational environment has been assigned for the complexes. The characterisation of the complexes has been based upon chemical analysis, electrical conductivity, magnetic moment, IR, PMR and electronic spectra.

Structure-Activity Relationship of Photocytotoxic Iron(III) Complexes of Modified Dipyridophenazine Ligands

Iron(III) complexes FeL(B)] (1-5) of a tetradentate trianionic phenolate-based ligand (L) and modified dipyridophenazine bases (B), namely, dipyrido-6,7,8,9-tetrahydrophenazine (dpqC in 1), dipyrido3,2-a:2',3'-c]phenazine-2-carboxylic acid (dppzc in 2), dipyrido3,2-a:2',3'-c]phenazine-11-sulfonic acid (dppzs in 3), 7-aminodipyrido3,2-a:2',3'-c]phenazine (dppza in 4) and benzoi]dipyridro3,2-a:2',3'-c]phenazine (dppn in 5), have been synthesized, and their photocytotoxic properties studied along with their dipyridophenazine analogue (6). The complexes have a five. electron paramagnetic iron(III) center, and the Fe(III)/Fe(II) redox couple appears at about 0.69 V versus SCE in DMF-0.1 M TBAP. The physicochemical data also suggest that the complexes possess similar structural features as that of its parent complex FeL(dppz)] with FeO3N3 coordination in a distorted octahedral geometry. The DNA-complex and protein-complex interaction studies have revealed that the complexes interact favorably with the biomolecules, the degree of which depends on the nature of the substituents present on the dipyridophenazine ring. Photocleavage Of pUC19 DNA by the complexes has been studied using visible light of 476, 530, and 647 nm wavelengths. Mechanistic investigations with inhibitors show formation of HO center dot radicals via a photoredox pathway. Photocytotoxicity study of the complexes in HeLa cells has shown that the dppn complex (5) is highly active in causing cell death in visible light with sub micromolar IC50 value. The effect of substitutions and the planarity of the phenazine moiety on the cellular uptake are quantified by determining the total Cellular iron content using the inductively coupled plasma-optical emission spectrometry (ICP-OES) technique. The cellular uptake increases marginally with an increase in the hydrophobicity of the dipyridophenazine ligands whereas complex 3 with dppzs shows very high uptake. Insights into the cell death mechanism by the dppn complex 5, obtained through DAFT nuclear staining in HeLa cells, reveal a rapid programmed cell death mechanism following photoactivation of complex 5 with visible light. The effect of substituent on the DNA photocleavage activity of the complexes has been rationalized from the theoretical studies.

Rearrangements in the cerium(IV) and manganese(III) oxidations of substituted naphthalenes and the nih shift mechanism

Ceric ammonium sulphate oxidation of 1- and 1,4- disubstituted naphthalenes gives 2- and/or 2,3- disubstituted 1,4- naphthoquinones through migration of substituents (D, Br, Ph). Similar rearrangements are also observed in the manganese(III) oxidation and also in the anodic oxidation of these substrates. The results are consistent with the proposal that these oxidations go through the formation of radical cation followed by reaction with H2O and further oxidation of the radical to the carbocationic intermediate on the way to the corresponding 1,4-naphthoquinone. Oxidation of 1,4-diphenylnaphthalene gives 2,3-diphenyl-1,4-naphthoquinone or 4-hydroxy- 2,4- diphenyl - 1(4)R - naphthalenone. The results are in accordance with the conclusion that such rearrangements do not require prior formation of arene oxide intermediates, originally proposed for the NM shift mechanism.