Enzymatic cleavage of carotenoids

1. 1.|Carotene 15,15′-dioxygenase (EC 1.13.11.21) has been isolated from the intestine of guinea pig and rabbit and purified 38- and 30-fold, respectively, but subjecting the intestinal homogenate to protamine sulfate treatment, (NH4)2SO4 fractionation and acetone precipitation. 2. 2.|The guinea pig enzyme showed a pH optimum at 8.5, an optimum substrate concentration of 200 nmoles of β,β-carotene per 25 ml of reaction mixture, an apparent Km with β,β-carotene as substrate of 9.5 · 10−6 M and a V 3.3 nmoles of retinal formation/mg protein per h. The reaction was linear upto 3 h and the reaction rate increased linearly with increase in enzyme protein concentration. The enzyme was activated by GSH and Fe2+ and inhibited by sodium dodecylsulfate, sulfhydryl binding and iron chelating agents. 3. 3.|The reaction catalysed by guinea pig enzyme was strictly stoichiometric. 4. 4.|Rabbit enzyme showed a close similarity with guinea pig enzyme with respect to time course, optimum substrate concentration, activation by Fe2+ and GSH, inhibition by sodium dodecylsulfate, iron chelating and sulfhydryl binding agents. However, it showed a slightly lower pH optimum (pH 7.8). 5. 5.|The enzyme from guinea pig and rabbit showed remarkable similarity with respect to cleavage of carotenoids. The enzyme from both the species was more specific for β,β-carotene but could also cleave a number of other carotenoids at the 15,15′-double bond. 6. 6.|10′-Apo-β-carotenal and 10′-apo-β-carotenol were readily cleaved compared with other apo-β-carotenals and apo-β-carotenols tested. 7. 7.|It has been conclusively shown for the first time that mono-ring substituted carotenoids are also cleaved at the 15,15′-double bond.

Pregnancy suppression by active immunization against gestation-specific riboflavin carrier protein

A riboflavin carrier protein isolated from chickens cross-reacts with a gestation-specific rodent carrier for riboflavin. Active immunization of female rats of proved fertility with the purified chicken carrier protein completely yet reversibly suppressed early pregnancy without impairing implantation per se. Concurrently there were no discernible adverse effects on maternal health in terms of weight gain, vitamin status, and fertility.

Characterization of Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease, reveals a unique mode of DNA-binding, helical distortion and cleavage, compared to a canonical LAGLIDADG homing endonuclease

Mycobacterium leprae, which has undergone reductive evolution leaving behind a minimal set of essential genes, has retained intervening sequences in four of its genes implicating a vital role for them in the survival of the leprosy bacillus. A single in-frame intervening sequence has been found embedded within its recA gene. Comparison of M. leprae recA intervening sequence with the known intervening sequences indicated that it has the consensus amino acid sequence necessary for being a LAGLIDADG-type homing endonuclease. In light of massive gene decay and function loss in the leprosy bacillus, we sought to investigate whether its recA intervening sequence encodes a catalytically active homing endonuclease. Here we show that the purified M. leprae RecA intein (PI-MleI) binds to cognate DNA and displays endonuclease activity in the presence of alternative divalent cations, Mg2+ or Mn2+. A combination of approaches including four complementary footprinting assays such as DNase I, Cu/phenanthroline, methylation protection and KMnO4, enhancement of 2-aminopurine fluorescence and mapping of the cleavage site revealed that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage site and generates two staggered double-strand breaks. Taken together, these results implicate that PI-MleI possess a modular structure with separate domains for DNA target recognition and cleavage, each with distinct sequence preferences. From a biological standpoint, it is tempting to speculate that our findings have implications for understanding the evolution of LAGLIDADG family of homing endonucleases

A new mode of ring cleavage of 2,3-dihydroxybenzoic acid in Tecoma stans (L.). Partial purification and properties of 2,3-dihydroxybenzoate 2,3-oxygenase.

2,3-Dihydroxybenzoic acid has been shown to be oxidized via the 3-oxoadipate pathway in the leaves of Tecoma stans. The formation of 2-carboxy-cis,cis-muconic acid, a muconolactone, 3-oxoadipic acid and carbon dioxide during its metabolism has been demonstrated using an extract of Tecoma leaves. The first reaction of the pathway, viz., the conversion of 2,3-dihydroxybenzoate to 2-carboxy-cis,cis-muconic acid has been shown to be catalysed by an enzyme designated as 2,3-dihydroxybenzoate 2,3-oxygenase. The enzyme has been partially purified and a few of its properties studied. The enzyme is very labile with a half-life of 3--4 h. It is maximally active with 2,3-dihydroxybenzoate as the substrate and does not exhibit any activity with catechol, 4-methyl catechol, 3,4-dihydroxybenzoic acid, etc. However, 2,3-dihydroxy-p-toluate and 2,3-dihydroxy-p-cumate are also oxidized by the enzyme by about 38% and 28% respectively, compared to 2,3-dihydroxybenzoate. Sulfhydryl reagents inhibit the enzyme reaction and the inhibition can be prevented by preincubation of the enzyme with the substrate. Substrate also affords protection to the enzyme against thermal inactivation. Sulfhydryl compounds strongly inhibit the reaction and the inhibition cannot be prevented by preincubation of the enzyme with its substrates. Data on the effect of metal ions as well as metal chelating agents suggest that copper is the metal cofactor of the enzyme. Evidence is presented which suggests that iron may not be participating in the overall catalytic mechanism.

Photocytotoxic 3d-Metal Scorpionates with a 1,8-Naphthalimide Chromophore Showing Photoinduced DNA and Protein Cleavage Activity

Ternary 3d-metal complexes of formulation [M(Tp(Ph))(py-nap)](ClO4)(1-3), where M is Co(II) (1), Cu(II) (2), and Zn(II) (3); Tp(Ph) is anionic tris (3-phenylpyrazolyl)borate; and py-nap is a pyridyl ligand with a conjugated 1,8-naphthalimide moiety, have been prepared from the reaction of metal perchlorate with KTp(Ph) and py-nap in CH2Cl2. The complexes have been characterized from analytical and physicochemical data. The complexes are stable in solution as evidenced from the electrospray ionization mass spectrometry data. The complexes show good binding propensity with calf thymus (CT) DNA, giving binding constant (K-b) values of similar to 10(5) M-1 and a molecular ``light-switch'' effect that results in an enhancement of the emission intensity of the naphthalimide chromophore on binding to CT DNA. The complexes do not show any hydrolytic cleavage of DNA. They show poor chemical nuclease activity in the presence of 3-mercaptopropionic acid or hydrogen peroxide (H2O2). The Co(II) and Cu(II) complexes exhibit oxidative pUC19 DNA cleavage activity in UV-A light of 365 rim. The Zn(II) complex shows moderate DNA photocleavage activity at 365 nm. The Cu(II)complex 2 displays photoinduced DNA cleavage activity in red light of 647.1 nm and 676 rim and near-IR light of >750 rim. A mechanistic studyin UV-A and visible light suggests the involvement of the hydroxyl radical as the reactive species in the DNA photocleavage reactions. The complexes also show good bovine serum albumin (BSA) protein binding propensity, giving K-BSA values of similar to 10(5) M-1. Complexes 1 and 2 display significant photoinduced BSA cleavage activity in UV-A light. The Co(II) complex 1 shows a significant photocytotoxic effect in HeLa cervical cancer cells on exposure to UV-A light of 365 nm, giving an IC50 value of 32 mu M. The IC50 value for the py-nap ligand alone is 41.42 mu m in UV-A light. The IC50 value is >200 mu M in the dark, indicating poor dark toxicity of 1. The Cu(II) complex 2 exhibits moderate photocytotoxicity and significant dark toxicity, giving IC50 values of 18.6 mu m and 29.7 mu m in UV-A light and in the dark, respectively.

New ternary copper(II) complexes of L-alanine and heterocyclic bases: DNA binding and oxidative DNA cleavage activity

Four new ternary copper(II) complexes of alpha-amino acid having polypyridyl bases of general formulation [Cu(L-ala)(B)(H2O)](X)(1-4), where L-ala is L-alanine, B is an N,N-donor heterocyclic base, viz. 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2) and 5,6-phenanthroline dione (dione, 3), dipyrido[3,2:2',3'-f] quinoxaline (dpq, 4), and X = ClO4-/NO3- are synthesized, characterized by various spectroscopic and X-ray crystallographic methods. The complexes show a distorted square-pyramidal (4 + 1) CuN3O2 coordination geometry. The one-electron paramagnetic complexes (1-4) display a low energy d-d band near 600 nm in aqueous medium and show a quasi-reversible cyclic voltammetric response due to one-electron Cu(II)/Cu(I) reduction near - 100 mV (versus SCE) in DMF-0.1 M TBAP. Binding interactions of the complexes with calf thymus DNA (CT-DNA) were investigated by UV-Vis absorption titration, ethidium bromide displacement assay, viscometric titration experiment and DNA melting studies. All the complexes barring the complexes 1 and 3 are avid binder to the CT-DNA in the DNA minor groove giving an order: 4 > 2 >>>1, 3. The complexes 2 and 4 show appreciable chemical nuclease activity in the presence of 3-mercaptopropionic acid (MPA) as a reducing agent. Hydroxyl radical was investigated to be the DNA cleavage active species. Control experiments in the presence of distamycin-A show primarily minor groove-binding propensity for the complexes 2 and 4 to the DNA.

Suppression of the zero-order term in off-axis digital holography through nonlinear filtering

We present experimental validation of a new reconstruction method for off-axis digital holographic microscopy (DHM). This method effectively suppresses the object autocorrelation,namely, the zero-order term,from holographic data,thereby improving the reconstruction bandwidth of complex wavefronts. The algorithm is based on nonlinear filtering and can be applied to standard DHM setups with realistic recording conditions.We study the robustness of the technique under different experimental configurations,and quantitatively demonstrate its enhancement capabilities on phase signals.

Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not

Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their ctive center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

Photocytotoxic Oxovanadium(IV) Complexes Showing Light-Induced DNA and Protein Cleavage Activity

Oxovanadium(IV) complexes [VO(L)(B)]Cl-2 (1-3), where L is bis(2-benzimidazolylmethyl)amine and B is 1,10-phenanthroline(phen),dipyrido[3,2-d:2',3'-f]quinoxaline(dpq) or dipyrido[3,2-a:2',3'-c]phenazine (dppz), have been prepared, characterized, and their photo-induced DNA and protein cleavage activity studied. The photocytotoxicity of complex 3 has been studied using adenocarcinoma A549 cells, The phen complex 1, structurally characterized by single-crystal X-ray crystallography, shows the presence of a vanadyl group in six-coordinate VON5 coordination geometry. The ligands L and phen display tridentate and bidentate N-donor chelating binding modes, respectively. The complexes exhibit a d-d band near 740 nm in 15% DMF-Tris-HCl buffer (pH 7.2). The phen and dpq complexes display an irreversible cathodic cyclic voltammetric response near -0.8 V in 20% DMF-Tris-HCl buffer having 0.1 M KCl as supporting electrolyte. The dppz complex 3 exhibits a quasi-reversible voltammogram near -0.6 V (vs SCE) that is assignable to the V(IV)-V(III)couple. The complexes bind to calf thymus DNA giving binding constant values in the range of 6.6 x 10(4)-2.9 x 10(5) M-1. The binding site size, thermal melting and viscosity binding data suggest DNA surface and/or groove binding nature of the complexes. The complexes show poor ``chemical nuclease'' activity in dark in the presence of 3-mercaptopropionic acid or hydrogen peroxide. The dpq and dppz complexes are efficient photocleavers of plasmid DNA in UV-A light of 365 nm via a mechanistic pathway that involves formation of both singlet oxygen and hydroxyl radicals. The complexes show significant photocleavage of DNA in near-IR light (>750 nm) via hydroxyl radical pathway. Among the three complexes, the dppz complex 3 shows significant BSA and lysozyme protein cleavage activity in UV-A light of 365 nm via hydroxyl radical pathway. The dppz complex 3 also exhibits photocytotoxicity in non-small cell lung carcinoma/human lung adenocarcinoma A549 cells giving IC50 value of 17 mu M in visible light(IC50 = 175 mu M in dark).

The RecA intein of Mycobacterium tuberculosis promotes cleavage of ectopic DNA sites. Implications for the dispersal of inteins in natural populations

The RecA intein of Mycobacterium tuberculosis, a novel double-stranded DNA endonuclease, requires both Mn(2+) and ATP for efficient cleavage of the inteinless recA allele. In this study, we show that Mg(2+) alone was sufficient to stimulate PI-MtuI to cleave double-stranded DNA at ectopic sites. In the absence of Mg(2+), PI-MtuI formed complexes with topologically different forms of DNA containing ectopic recognition sequences with equal affinity but failed to cleave DNA. We observed that PI-MtuI was able to inflict double-strand breaks robustly within the ectopic recognition sequence to generate either a blunt end or 1-2-nucleotide 3'-hydroxyl overhangs. Mutational analyses of the presumptive metal ion-binding ligands (Asp(122), Asp(222), and Glu(220)) together with immunoprecipitation assays provided compelling evidence to link both the Mg(2+)- and Mn(2+) and ATP-dependent endonuclease activities to PI-MtuI. The kinetic mechanism of PI-MtuI promoted cleavage of ectopic DNA sites proceeded through a sequential mechanism with transient accumulation of nicked circular duplex DNA as an intermediate. Together, these data suggest that PI-MtuI, like group II introns, might mediate ectopic DNA transposition and hence its lateral transfer in natural populations.

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.

DNA cleavage in red light promoted by copper(II) complexes of -amino acids and photoactive phenanthroline bases

Ternary copper(II) complexes [Cu(L-trp)(B)(H2O)](NO3) ( 1–3) and [Cu(L-phe)(B)(H2O)](NO3) ( 4–6) of L-tryptophan (L-trp) and L-phenylalanine (L-phe) having phenanthroline bases (B), viz. 1,10-phenanthroline (phen, 1 and 4), dipyrido[3,2-d:2,3-f]quinoxaline (dpq, 2 and 5) and dipyrido[3,2-a:2,3-c]phenazine (dppz, 3 and 6), were prepared and characterized by physico-chemical techniques. Complexes 3 and 6 were structurally characterized by X-ray crystallography and show the presence of a square pyramidal (4 + 1) CuN3O2 coordination geometry in which the N,O-donor amino acid (L-trp or L-phe) and N,N-donor phenanthroline base bind at the equatorial plane with an aqua ligand coordinated at the elongated axial site. Complex 3 shows significant distortion from the square pyramidal geometry and a strong intramolecular – stacking interaction between the pendant indole ring of L-trp and the planar dppz aromatic moiety. All the complexes display good binding propensity to the calf thymus DNA giving an order: 3, 6 (dppz) > 2, 5 (dpq) > 1, 4 (phen). The binding constant (Kb) values are in the range of 2.1 × 104–1.1 × 106 mol-1 with the binding site size (s) values of 0.17–0.63. The phen and dpq complexes are minor groove binders while the dppz analogues bind at the DNA major groove. Theoretical DNA docking studies on 2 and 3 show the close proximity of two photosensitizers, viz. the indole moiety of L-trp and the quinoxaline/phenazine of the dpq/dppz bases, to the complementary DNA strands. Complexes 2 and 3 show oxidative DNA double strand breaks (dsb) of supercoiled (SC) DNA forming a significant quantity of linear DNA along with the nicked circular (NC) form on photoexposure to UV-A light of 365 nm and red light of 647.1 nm (Ar–Kr laser). Complexes 1, 5 and 6 show only single strand breaks (ssb) forming NC DNA. The red light induced DNA cleavage involves metal-assisted photosensitization of L-trp and dpq/dppz base resulting in the formation of a reactive singlet oxygen (1O2) species.

DNA looping and translocation provide an optimal cleavage mechanism for the type III restriction enzymes

EcoP15I is a type III restriction enzyme that requires two recognition sites in a defined orientation separated by up to 3.5 kbp to efficiently cleave DNA. The mechanism through which site- bound EcoP15I enzymes communicate between the two sites is unclear. Here, we use atomic force microscopy to study EcoP15I-DNA pre-cleavage complexes. From the number and size distribution of loops formed, we conclude that the loops observed do not result from translocation, but are instead formed by a contact between site- bound EcoP15I and a nonspecific region of DNA. This conclusion is confirmed by a theoretical polymer model. It is further shown that translocation must play some role, because when translocation is blocked by a Lac repressor protein, DNA cleavage is similarly blocked. On the basis of these results, we present a model for restriction by type III restriction enzymes and highlight the similarities between this and other classes of restriction enzymes.

Experimental Demonstration of H{infty} Control based Active Vibration Suppression in Composite Fin-tip of Aircraft using Optimally Placed Piezoelectric Patch Actuators

The goal of this study is the multi-mode structural vibration control in the composite fin-tip of an aircraft. Structural model of the composite fin-tip with surface bonded piezoelectric actuators is developed using the finite element method. The finite element model is updated experimentally to reflect the natural frequencies and mode shapes accurately. A model order reduction technique is employed for reducing the finite element structural matrices before developing the controller. Particle swarm based evolutionary optimization technique is used for optimal placement of piezoelectric patch actuators and accelerometer sensors to suppress vibration. H{infty} based active vibration controllers are designed directly in the discrete domain and implemented using dSpace® (DS-1005) electronic signal processing boards. Significant vibration suppression in the multiple bending modes of interest is experimentally demonstrated for sinusoidal and band limited white noise forcing functions.