This document describes the one-pot synthesis of three copper(II) complexes with 2,2'-bipyridine and derivatives of a barbiturate ligand. Complex 1 contains 5-hydroxy-hydurilic acid, complex 2 contains alloxanic acid, and complex 3 retains the original barbiturate ligand framework. The complexes were characterized using X-ray crystallography, spectroscopy, and thermal analysis. Their interactions with DNA were studied through absorption and emission titrations as well as circular dichroism. Complexes 1 and 3 were found to cleave plasmid DNA. Their cytotoxicity against T-cell lymphoma cells was also evaluated.
SYNTHESIS AND CHARACTERIZATION OF THERMAL ANALYSIS OF La (II) MACROCYCLIC COM...inventionjournals
The macrocyclic complex compounds of La(II) containing a ligand having tetraoxotetrahydrazin moity are synthesized by template condensation of malonodihydrazide (C3H8N4O2) with different aldehydes. The complexes are characterized on the basis of elemental analysis, UV-visible & IR spectroscopy, magnetic moment and conductance measurement and other physical properties.Antibacterial activity of the derived complex compounds, as well as already used standard compound kanamycin, was tested on fourteen pathogenic bacteria. Given results were then compared to the efficacy of the Antibacterial activity of standard compound kanamycin used for control of these pathogenic bacteria.
The International Journal of Engineering and Science (The IJES)theijes
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
SYNTHESIS AND CHARACTERIZATION OF THERMAL ANALYSIS OF La (II) MACROCYCLIC COM...inventionjournals
The macrocyclic complex compounds of La(II) containing a ligand having tetraoxotetrahydrazin moity are synthesized by template condensation of malonodihydrazide (C3H8N4O2) with different aldehydes. The complexes are characterized on the basis of elemental analysis, UV-visible & IR spectroscopy, magnetic moment and conductance measurement and other physical properties.Antibacterial activity of the derived complex compounds, as well as already used standard compound kanamycin, was tested on fourteen pathogenic bacteria. Given results were then compared to the efficacy of the Antibacterial activity of standard compound kanamycin used for control of these pathogenic bacteria.
The International Journal of Engineering and Science (The IJES)theijes
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
Synthesis, Characterization, Antibacterial and DNA Binding Studies of Mn (II)...IOSRJAC
Mn (II) complex of the Schiff base (L), 3-(2-(2-hydroxy-3-methoxybenzylidene) hydrazinyl) quinoxalin-2(1H)-one (VHQO) was prepared and characterized by IR, UV-VIS, Mass, Elemental analysis, TGDTA, magnetic susceptibility and conductivity measurements. VHQO behaved as monobasic, tridentate ligand and formed 1:2 complex (ML2) with the metal ion (M) coordinating through ring nitrogen, azomethine nitrogen and phenolic oxygen. Thermal analysis of the complex indicated the absence of lattice and coordinated water. Based on the spectral and analytical data, octahedral geometry was assigned to the complex. Antibacterial activity of the complex against gram positive bacteria, Bacillus subtillis, Staphylococcus aureus, and gram negative bacteria, Escherichia coli, Proteus vulgaris, Pseudomonas aeroginosa , and Klebsiella pneumonia was studied. The interaction of the complex with Calf Thymus DNA (CT-DNA) was studied by absorption spectroscopy and the intrinsic binding constant was calculated.
Spectral studies of 5-({4-amino-2-[(Z)-(2-hydroxybenzylidene) amino] pyrimidi...IOSR Journals
Some transition metal ions Complexes with 5-({4-amino-2-[(Z)-(2-hydroxybenzylidene) amino]
pyrimidin-5-yl} methyl)-2,3,4-trimethoxybenzene were prepared and characterized by elemental analyses,
Infrared , magnetic moment, electronic spectra , mass spectra, X-ray powder diffraction, molar conductance
and thermal analysis (TGA). The complexes have general formulae [ML2.2H2O] {where M = Mn (II), Co (II), Ni
(II), Cu (II), Zn (II), Pd (II) and Pt (II). The coordination behavior of the metal ions towards to the investigated
Schiff base takes place through –C=N,-NH2 and –OH groups. The obtained C, H and N elemental analysis data
showed the Metal: Ligand ratio is 1:2 [M: L] ratio. The molar conductance data reveal that all the metal
complexes are non-electrolytic in nature. From the magnetic moments the complexes are paramagnetic except
Zn metal ion complexes have octahedral geometry with coordination number eight. The thermal behavior of
these complexes shows that, the hydrated complexes have loses two water molecules and immediately followed
by decomposition of the anions and ligand molecules in the second and third stage. The Schiff bases and metal
complexes show good activity against some bacteria. The antimicrobial results indicate that, the metal
complexes have better antimicrobial activity as compared to the prepared Schiff base.
New Schiff base ligand (E)-6-(2-(4-
(dimethylamino)benzylideneamino)-2-phenylacetamido)-3,3-
dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic
acid = (HL) Figure(1) was prepared via condensation of
Ampicillin and 4(dimethylamino)benzaldehyde in methanol
.Polydentate mixed ligand complexes were obtained from 1:1:2
molar ratio reactions with metal ions and HL, 2NA on reaction
with MCl2 .nH2O salt yields complexes corresponding to the
formulas [M(L)(NA)2Cl] ,where M =
Fe(II),Co(II),Ni(II),Cu(II),and Zn(II) and NA=nicotinamide.
The 1H-NMR, FT-IR, UV-Vis and elemental analysis
were used for the characterization of the ligand. The complexes
were structurally studied through AAS, FT-IR, UV-Vis,
chloride contents, conductance, and magnetic susceptibility
measurements. All complexes are non-electrolytes in DMSO
solution. Octahedral geometries have been suggested for each
of the complexes. The Schiff base ligands function as
tridentates and the deprotonated enolic form is preferred for
coordination. In order to evaluate the effect of the bactericidal
activity, these synthesized complexes, in comparison to the un
complexed Schiff base has been screened against bacterial
species, Staphy
Effect of Water And Ethyl Alcohol Mixed Solvent System on the Stability of Be...IJERDJOURNAL
ABSTRACT: The stabilities of ternary complexes of metal ions (copper, nickel, zinc and cobalt) with betahydroxy ketone(BHK) derivatives and benzotriazole(BTAZ) derivatives in various mixed solvent systems (Water+Ethyl Alcohol) medium in 0.1 M KNO3 ionic strength at 250C using pH metric titration method have been established. The data reveal that the copper forms more stable complexes, which is followed by zinc and Ni complexes with these ligands. Cobalt form less stable complexes with these ligands. The stabilities of these complexes are further quantified with Δ log K values, intra-molecular equilibrium constants and percentage of stacking interaction in the ternary systems. The observed positive Δ log K values suggest that the flexible side chain alkyl moiety (ethyl group, butyl group) or aromatic moiety (phenyl group) in BHK ligand overlaps with the fixed aromatic moiety of BTAZ ligand in the ternary complex, which results in the enhanced stabilities for the (BHK-Alk)-Metal(II)-BTAZ and (BHK-Ph)-Metal(II)-BTAZ systems. Interestingly, the positive Δ log K values for both BHK-Alk and BHK-Ph ligands in their corresponding ternary complexes are about the same. This suggests the flexible Alkyl or phenyl side chain of BHK is overlapping with the triazole ring, but not the phenoxy ring of the BTAZ ligand.
Targetting cancer with Ru(III/II)-phosphodiesterase inhibitor adducts: A nove...rkkoiri
Lack of specificity and normal tissue toxicity are the two major limitations faced with most of the anticancer
agents in current use. Due to effective biodistribution and multimodal cellular actions, during
recent past, ruthenium complexes have drawn much attention as next generation anticancer agents. This
is because metal center of ruthenium (Ru) effectively binds with the serum transferrin and due to higher
concentration of transferrin receptors on the tumor cells, much of the circulating Ru-transferrin complexes
are delivered preferentially to the tumor site. This enables Ru-complexes to become tumor cell
specific and to execute their anticancer activities in a somewhat targeted manner. Also, there are evidences
to suggest that inhibition of phosphodiesterases leads to increased cyclic guanosine monophosphate
(cGMP) level, which in turn can evoke cell cycle arrest and can induce apoptosis in the tumor
cells. In addition, phosphodiesterase inhibition led increased cGMP level may act as a potent vasodilator
and thus, it is likely to enhance blood flow to the growing tumors in vivo, and thereby it can further facilitate
delivery of the drugs/compounds to the tumor site.
Therefore, it is hypothesized that tagging PDE inhibitors (PDEis) with Ru-complexes could be a relevant
strategy to deliver Ru-complexes-PDEi adduct preferentially to the tumor site. The Ru-complex tagged
entry of PDEi is speculated to initially enable the tumor cells to become a preferential recipient of such
adducts followed by induction of antitumor activities shown by both, the Ru-complex & the PDEi, resulting
into enhanced antitumor activities with a possibility of minimum normal tissue toxicity due to
administration of such complexes.
Synthesis, Characterization, Antibacterial and DNA Binding Studies of Mn (II)...IOSRJAC
Mn (II) complex of the Schiff base (L), 3-(2-(2-hydroxy-3-methoxybenzylidene) hydrazinyl) quinoxalin-2(1H)-one (VHQO) was prepared and characterized by IR, UV-VIS, Mass, Elemental analysis, TGDTA, magnetic susceptibility and conductivity measurements. VHQO behaved as monobasic, tridentate ligand and formed 1:2 complex (ML2) with the metal ion (M) coordinating through ring nitrogen, azomethine nitrogen and phenolic oxygen. Thermal analysis of the complex indicated the absence of lattice and coordinated water. Based on the spectral and analytical data, octahedral geometry was assigned to the complex. Antibacterial activity of the complex against gram positive bacteria, Bacillus subtillis, Staphylococcus aureus, and gram negative bacteria, Escherichia coli, Proteus vulgaris, Pseudomonas aeroginosa , and Klebsiella pneumonia was studied. The interaction of the complex with Calf Thymus DNA (CT-DNA) was studied by absorption spectroscopy and the intrinsic binding constant was calculated.
Spectral studies of 5-({4-amino-2-[(Z)-(2-hydroxybenzylidene) amino] pyrimidi...IOSR Journals
Some transition metal ions Complexes with 5-({4-amino-2-[(Z)-(2-hydroxybenzylidene) amino]
pyrimidin-5-yl} methyl)-2,3,4-trimethoxybenzene were prepared and characterized by elemental analyses,
Infrared , magnetic moment, electronic spectra , mass spectra, X-ray powder diffraction, molar conductance
and thermal analysis (TGA). The complexes have general formulae [ML2.2H2O] {where M = Mn (II), Co (II), Ni
(II), Cu (II), Zn (II), Pd (II) and Pt (II). The coordination behavior of the metal ions towards to the investigated
Schiff base takes place through –C=N,-NH2 and –OH groups. The obtained C, H and N elemental analysis data
showed the Metal: Ligand ratio is 1:2 [M: L] ratio. The molar conductance data reveal that all the metal
complexes are non-electrolytic in nature. From the magnetic moments the complexes are paramagnetic except
Zn metal ion complexes have octahedral geometry with coordination number eight. The thermal behavior of
these complexes shows that, the hydrated complexes have loses two water molecules and immediately followed
by decomposition of the anions and ligand molecules in the second and third stage. The Schiff bases and metal
complexes show good activity against some bacteria. The antimicrobial results indicate that, the metal
complexes have better antimicrobial activity as compared to the prepared Schiff base.
New Schiff base ligand (E)-6-(2-(4-
(dimethylamino)benzylideneamino)-2-phenylacetamido)-3,3-
dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic
acid = (HL) Figure(1) was prepared via condensation of
Ampicillin and 4(dimethylamino)benzaldehyde in methanol
.Polydentate mixed ligand complexes were obtained from 1:1:2
molar ratio reactions with metal ions and HL, 2NA on reaction
with MCl2 .nH2O salt yields complexes corresponding to the
formulas [M(L)(NA)2Cl] ,where M =
Fe(II),Co(II),Ni(II),Cu(II),and Zn(II) and NA=nicotinamide.
The 1H-NMR, FT-IR, UV-Vis and elemental analysis
were used for the characterization of the ligand. The complexes
were structurally studied through AAS, FT-IR, UV-Vis,
chloride contents, conductance, and magnetic susceptibility
measurements. All complexes are non-electrolytes in DMSO
solution. Octahedral geometries have been suggested for each
of the complexes. The Schiff base ligands function as
tridentates and the deprotonated enolic form is preferred for
coordination. In order to evaluate the effect of the bactericidal
activity, these synthesized complexes, in comparison to the un
complexed Schiff base has been screened against bacterial
species, Staphy
Effect of Water And Ethyl Alcohol Mixed Solvent System on the Stability of Be...IJERDJOURNAL
ABSTRACT: The stabilities of ternary complexes of metal ions (copper, nickel, zinc and cobalt) with betahydroxy ketone(BHK) derivatives and benzotriazole(BTAZ) derivatives in various mixed solvent systems (Water+Ethyl Alcohol) medium in 0.1 M KNO3 ionic strength at 250C using pH metric titration method have been established. The data reveal that the copper forms more stable complexes, which is followed by zinc and Ni complexes with these ligands. Cobalt form less stable complexes with these ligands. The stabilities of these complexes are further quantified with Δ log K values, intra-molecular equilibrium constants and percentage of stacking interaction in the ternary systems. The observed positive Δ log K values suggest that the flexible side chain alkyl moiety (ethyl group, butyl group) or aromatic moiety (phenyl group) in BHK ligand overlaps with the fixed aromatic moiety of BTAZ ligand in the ternary complex, which results in the enhanced stabilities for the (BHK-Alk)-Metal(II)-BTAZ and (BHK-Ph)-Metal(II)-BTAZ systems. Interestingly, the positive Δ log K values for both BHK-Alk and BHK-Ph ligands in their corresponding ternary complexes are about the same. This suggests the flexible Alkyl or phenyl side chain of BHK is overlapping with the triazole ring, but not the phenoxy ring of the BTAZ ligand.
Targetting cancer with Ru(III/II)-phosphodiesterase inhibitor adducts: A nove...rkkoiri
Lack of specificity and normal tissue toxicity are the two major limitations faced with most of the anticancer
agents in current use. Due to effective biodistribution and multimodal cellular actions, during
recent past, ruthenium complexes have drawn much attention as next generation anticancer agents. This
is because metal center of ruthenium (Ru) effectively binds with the serum transferrin and due to higher
concentration of transferrin receptors on the tumor cells, much of the circulating Ru-transferrin complexes
are delivered preferentially to the tumor site. This enables Ru-complexes to become tumor cell
specific and to execute their anticancer activities in a somewhat targeted manner. Also, there are evidences
to suggest that inhibition of phosphodiesterases leads to increased cyclic guanosine monophosphate
(cGMP) level, which in turn can evoke cell cycle arrest and can induce apoptosis in the tumor
cells. In addition, phosphodiesterase inhibition led increased cGMP level may act as a potent vasodilator
and thus, it is likely to enhance blood flow to the growing tumors in vivo, and thereby it can further facilitate
delivery of the drugs/compounds to the tumor site.
Therefore, it is hypothesized that tagging PDE inhibitors (PDEis) with Ru-complexes could be a relevant
strategy to deliver Ru-complexes-PDEi adduct preferentially to the tumor site. The Ru-complex tagged
entry of PDEi is speculated to initially enable the tumor cells to become a preferential recipient of such
adducts followed by induction of antitumor activities shown by both, the Ru-complex & the PDEi, resulting
into enhanced antitumor activities with a possibility of minimum normal tissue toxicity due to
administration of such complexes.
Today’s emerging metro Ethernet networks represent a significant shift in the way in which data services are offered to enterprise and residential customers. With Ethernet emerging as the access interface of choice, services such as packet-leased line and virtual private LAN service are being delivered over a wide mix of transport technologies – SONET/SDH, next-gen SONET/SDH, Ethernet/WDM, and...
In recent years, different types of inorganic nanoparticles (iNPs) with unique physicochemical properties have emerged.1−4 Among these, quantum dots (QDs) have proved to be very versatile,finding applications in electroluminescent displays, quantumcomputing, photovoltaics , solar cells,transistors,and biological imaging.For biological imaging applications, QDs are now excellent alternatives to organic chromophores.given that they can have similar sizes, shapes,and surface functional groups. A potentially prolific new direction in inorganic chemistry and nanochemistry could be to combine NPs with small metal complexes to seek synergistic and/or cooperative effects. In this context,combining QDs with coordination complexes is being explored as a new strategy to obtain cooperative systems with improved properties for applications in sensing, biological imaging, and molecular therapy. A prominent area of research in coordination chemistry is the development of metal complexes that can act as artificial nucleases. Overall, these synthetic DNA-cleaving reagents
Synthesis and Characterization of Thermal Analysis of La (Ii) Macrocyclic Com...inventionjournals
The macrocyclic complex compounds of La(II) containing a ligand having tetraoxotetrahydrazin moity are synthesized by template condensation of malonodihydrazide (C3H8N4O2) with different aldehydes. The complexes are characterized on the basis of elemental analysis, UV-visible & IR spectroscopy, magnetic moment and conductance measurement and other physical properties.Antibacterial activity of the derived complex compounds, as well as already used standard compound kanamycin, was tested on fourteen pathogenic bacteria. Given results were then compared to the efficacy of the Antibacterial activity of standard compound kanamycin used for control of these pathogenic bacteria.
This PDF presentation describes briefly my research experiences in synthetic organic. The time period of these research projects range from 1999 to 10/2005. Projects of later positions were also included but not all. Time period, place of work and position were mentioned at the beginning of each project. To noted that all the experimental synthesis, separation/purification, characterization and spectral interpretation were performed independently by me.
SYNTHESIS AND CHARACTERIZATION OF SCHIFF BASE METAL COMPLEXES DERIVED FROM NA...chemsurya
New Schiff base metal complexes having general structural formulae ML2Cl2 where M= Co(II), Ni(II) and Cu(II), and MLCl2 where M=Zn(II), Cd(II) and Hg(II) were synthesized by the condensation of indoline-2,3-dione (Isatin) and naphtho[2,1-b]furan-2-carbohydrazide. Tentative structures for the synthesized compounds have been elucidated based on analytical, IR, 1HNMR, and Mass spectral studies. Further selected compounds were tested for their antimicrobial activity.
Synthesis, spectroscopic, electrochemical, magnetic properties and super oxid...IOSR Journals
Five new mixed-ligand nickel (II) complexes; viz; [Ni (BHM)(PMDT)]1; [Ni(BHM)(dien)] 2; [Ni(BHM)(L1)]3; [Ni(BHM)(L2)] 4, [Ni(BHM)(L3)] 5; where H2BHM= N’-(1E)-(5-bromo-2-hydroxyphenyl) methylidene] benzoylhydrazide, PMDT= N,N,N’,N,”N”-Pentamethyldiethylenetriamine; dien= diethylenetriamine; L1 = N,N- dimethyl-N’ (Pyrid-2-yl-methyl) ethylenediamine; L2= N-methyl-N’-(pyrid-2-ylmethyl)ethylenediamine; L3 = N,N-dimethyl-N’-(6-methyl)pyrid-2-ylmethyl) ethylenediamine, have been synthesized and characterized by using elemental analyses, FAB (fast atomic bombardment), magnetic measurements, electronic absorption , conductivity measurements, cyclic voltammetry (CV) and IR- spectroscopy . All the complexes yielded an irreversible couple that can be assigned to a NiII→ NiI redox process. Infrared spectra, ligand field spectra and magnetic susceptibility measurements agree with the observed octahedral environment. H2BHM is a diprotic tridentate Schiff base ligand (ONO donor atom) whereas L1-L3 possessing N3 coordination sites. The SOD activities have been measured using alkaline DMSO as a source of superoxide radical (O2–) and nitro blue tetrazolium (NBT) as O2– scavenger.
A new Schiff base 4-chlorophenyl)methanimine
(6R,7R)-3-methyl-8-oxo-7-(2-phenylpropanamido)-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylate= (HL)= C23H20
ClN3O4S) has been synthesized from β-lactam antibiotic
(cephalexin mono hydrate(CephH)=(C16H19N3O5S.H2O) and 4-
chlorobenzaldehyde . Figure(1) Metal mixed ligand complexes
of the Schiff base were prepared from chloride salt of
Fe(II),Co(II),Ni(II),Cu(II),Zn(II) and Cd (II), in 50% (v/v)
ethanol –water medium (SacH ) .in aqueous ethanol(1:1)
containing and Saccharin(C7H5NO3S) = sodium hydroxide.
Several physical tools in particular; IR, CHN, 1H NMR, 13C
NMR for ligand and melting point molar conductance, magnetic
moment. and determination the percentage of the metal in the
complexes by flame(AAS). The ligands and there metal
complexes were screened for their antimicrobial activity against
four bacteria (gram + ve) and (gram -ve) {Escherichia coli,
Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus}.
The proposed structure of the complexes using program, Chem
office 3D(2006). The general formula have been given for the
prepared mixed ligand complexes Na2[M(Sac)3(L)], M(II) = Fe
(II), Co(II) , Ni(II), Cu (II), Zn(II) , and Cd(II).
HL= C29H24 ClN3O4S, L= C29H23 ClN3O4S -.
New One-Pot Synthetic Route and Spectroscopic Characterization of Hydroxo-Bri...AJASTJournal
A new convenient and efficient route for the synthesis of two very important hydroxo-bridged stepped-cubane copper complexes viz: [Cu4(bpy)4Cl2(OH)4]Cl2.6H2O (1) and [Cu4(phen)4Cl2(OH)4]Cl2.6H2O (2) have been obtained. This synthetic route from the mononuclear CubpyCl2 complex is easier, more reproducible and afforded the complex in a much higher yield than the other two previously reported procedures which were equally serendipitously discovered. The purity and formation of the complexes were confirmed with elemental (C,H,N) analysis and the details of the UV-Vis, Fourier transform infrared, electrospray ionization mass spectra of both complexes and the single crystal X-ray crystallography of 1 are presented and discussed. X-ray crystallography confirms the absolute structure of the complexes. The complexes were formed via the connection of four copper atoms to four hydroxide bridging ligands and four bipyridyl ligands with two chloride ligands. There are two coordinate environments around two pairs of copper atoms (CuN2ClO2 and CuN2O3) and each copper atom is pentacoordinate with square pyramidal geometry.
A new convenient and efficient route for the synthesis of two very important hydroxo-bridged stepped-cubane copper complexes viz: [Cu4(bpy)4Cl2(OH)4]Cl2.6H2O (1) and [Cu4(phen)4Cl2(OH)4]Cl2.6H2O (2) have been obtained. This synthetic route from the mononuclear CubpyCl2 complex is easier, more reproducible and afforded the complex in a much higher yield than the other two previously reported procedures which were equally serendipitously discovered. The purity and formation of the complexes were confirmed with elemental (C,H,N) analysis and the details of the UV-Vis, Fourier transform infrared, electrospray ionization mass spectra of both complexes and the single crystal X-ray crystallography of 1 are presented and discussed. X-ray crystallography confirms the absolute structure of the complexes. The complexes were formed via the connection of four copper atoms to four hydroxide bridging ligands and four bipyridyl ligands with two chloride ligands. There are two coordinate environments around two pairs of copper atoms (CuN2ClO2 and CuN2O3) and each copper atom is pentacoordinate with square pyramidal geometry.
Synthesis and Application of C-Phenylcalix[4]resorcinarene in Adsorption of C...Jacsonline.Org
Synthesis and Application of C-Phenylcalix[4]resorcinarene in Adsorption of Cr(III) and Pb(II), for more information visit our website http://jacsonline.org/
An overview of the toxic effect of potential human carcinogen Microcystin-LR ...rkkoiri
The worldwide occurrence of cyanobacterial blooms due to water eutrophication evokes
extreme concerns. These blooms produce cyanotoxins which are hazardous to living organisms.
So far among these toxins, Microcystin-LR (MC-LR) is the most toxic and the most
frequently encountered toxin produced by the cyanobacteria in the contaminated aquatic
environment. Microcystin-LR is a potential carcinogen for animals and humans, and the
International Agency for Research on Cancer has classified Microcystin-LR as a possible
human carcinogen. After liver, testis has been considered as one of the most important target
organs of Microcystin-LR toxicity. Microcystin-LR crosses the blood–testis barrier and
interferes with DNA damage repair pathway and also increases expression of the protooncogenes,
genes involved in the response to DNA damage, cell cycle arrest, and apoptosis
in testis. Toxicity of MC-LR disrupts the motility and morphology of sperm and also affects
the hormone levels of male reproductive system. MC-LR treated mice exhibit oxidative
stress in testis through the alteration of antioxidant enzyme activity and also affect the
histopathology of male reproductive system. In the present review, an attempt has been
made to comprehensively address the impact of MC-LR toxicity on testis.
Activation of p53 mediated glycolytic inhibition-oxidative stressapoptosis pa...rkkoiri
There is a general agreement that most of the cancer cells switch over to aerobic glycolysis (Warburg
effect) and upregulate antioxidant enzymes to prevent oxidative stress induced apoptosis. Thus, there is
an evolving view to target these metabolic alterations by novel anticancer agents to restrict tumor
progression in vivo. Previously we have reported that when a non toxic dose (10 mg/kg bw i.p.) of a novel
anticancer ruthenium(II)-complex containing 4-carboxy N-ethylbenzamide; Ru(II)-CNEB, was administered
to the Dalton's lymphoma (DL) bearing mice, it regressed DL growth by inducing apoptosis in the
DL cells. It also inactivated M4-LDH (M4-lactate dehydrogenase), an enzyme that drives anaerobic
glycolysis in the tumor cells. In the present study we have investigated whether this compound is able to
modulate regulation of glycolytic inhibition-apoptosis pathway in the DL cells in vivo. We observed that
Ru(II)-CNEB could decline expression of the inducible form of 6-phosphofructo-2-kinase (iPFK2:
PFKFB3), the master regulator of glycolysis in the DL cells. The complex also activated superoxide dismutase
(the H2O2 producing enzyme) but declined the levels of catalase and glutathione peroxidase (the
two H2O2 degrading enzymes) to impose oxidative stress in the DL cells. This was consistent with the
enhanced p53 level, decline in Bcl2/Bax ratio and activation of caspase 9 in those DL cells. The findings
suggest that Ru(II)-CNEB is able to activate oxidative stress-apoptosis pathway via p53 (a tumor
supressor protein) mediated repression of iPFK2, a key glycolytic regulator, in the DL cells in vivo.
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
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5cl-adba precursor (semi finished )
5cl-adba raw materials
ADBB precursor (semi finished )
ADBB raw materials
APVP powder
5fadb/4f-adb
Jwh018 / Jwh210
Eutylone crystal
Protonitazene (hydrochloride) CAS: 119276-01-6
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Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
Associate Division Director for Ambulatory Operations
The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
2. N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267 257
one-pot-reaction. Complex 1 possesses 5-hydroxy-[5,5′]-bipyrimidinyl- 2.4. Synthesis of complex 1
2,4,6,2′,4′,6′-hexaone (5-hydroxy-hydurilic acid) whereas the other
complex, 2 contains 4-hydroxy-2,5-dioxo-imidazolidine-4-carboxylic A methanolic solution (10 mL) of Cu(bpy)(NO3)2·2H2O(0.361 g,
acid (alloxanic acid) as co-ligand. Since none of the complexes bear the 1 mmol) was added drop wise to a solution of LH4 (0.374 g, 1 mmol) in
framework of LH4, the original ligand must have been transformed 5 mLdimethylformamide(DMF).Thereactionmixturewasthenrefluxed
during the reaction. In order to explore the effect of 2,2′-bipyridyl groups for 2 h. The resulting solution was kept at room temperature for slow
on the transformation of LH4, a reaction between LH4 and copper nitrate evaporation. After 5–6 days, the dark green coloured crystals were
salt was also carried out. This reaction provided complex 3 of type [Cu obtained. These crystals were washed with methanol and dried in air.
(LH3)2(H2O)2], which retained the original ligand framework. Yield: 54%, M.P. 220 °C, elemental analysis calculated for C18H11CuN6O10
In view of the aforementioned reports and owing to the biological (%): C, 40.44; H, 2.05; and N, 15.73. Found (%): C, 41.24; H, 2.17; and
significance of Cu(II) ions [32], the nuclease property of the novel N, 16.21. UV–vis absorptions: λmax (DMSO, 10−4 M), nm (ε/104 M−1
complexes was studied. In addition, the present article embodies the cm−1) 266 (4.0), 312 (2.922) and 620 (0.023). IR (KBr): νmax, cm−1
spectroscopic and single crystal characterization of the newly synthesized 3436 (OH, H2O), 3201 (NH), 3086 (CH, Ph), 1695 (CO), and 1603 (2,2′-bpy).
complexes. The DNA binding and cleavage properties of copper(II)
complexes 1 and 3 have been studied (complex 2 is insoluble in common 2.5. Synthesis of complex 2
organic solvents and was not investigated further). The cytotoxic effects of
complexes 1 and 3 against Dalton's lymphoma cell lines are also reported. After isolation of complex 1, the filtrate thus obtained provided a
bluish brown solid product after two weeks. It was then redissolved in
2. Experimental section MeOH and left for slow evaporation at room temperature. After 24 h,
block shaped blue colour crystals were formed which were found
2.1. Materials insoluble in all common organic solvents. The crystals were then
washed with diethyl ether and dried in air. Yield: 25%, M.P. N250 °C,
Barbituric acid, 2,2′-bipyridine and salicylaldehyde were purchased elemental analysis calculated for C14H10CuN4O6 (%): C, 42.74; H, 2.54;
from Sigma Aldrich Chem. Co and copper(II) nitrate dihydrate was and N, 14.24. Found (%): C, 42.85; H, 3.08; and N, 14.84. IR (KBr): νmax,
purchased from S.D. Fine Chemicals, India and used as received. Solvents cm−1 3303 (NH), 1731 and 1656 (CO), 3037 (CH, 2,2′-bpy), 2929
were purchased from E. Merk and were freshly distilled prior to their (CH), 1266, 1024 (C–O–C), and 3378 (OH, water).
use. The barbiturate ligand (LH4) was synthesized using slight
modification of the reported procedure [33]. Calf thymus (CT) DNA 2.6. Synthesis of complex 3
and supercoiled (SC) plasmid DNA pBR322 (as a solution in Tris buffer
and cesium chloride purified), with a length of 4361 base pairs were A solution of Cu(NO3)2·2H2O (0.241 g, 1 mmol) in MeOH (10 mL)
purchased from Bangalore Genei, India. pUC19 plasmid DNA with a was added drop wise to a solution of LH4 (0.374 g, 1 mmol) in DMF
length of 2686 base pairs was purchased from Fermentas. Restriction (5 mL). The reaction mixture after stirring for 5–6 h at room
enzymes were purchased from New England Biolabs and DNA temperature was left for slow evaporation. Fluorescent block shaped
oligonucleotide primers were purchased from Sigma Aldrich Chem. Co. green colour crystals were grown in solution after 4–5 days. The crystals
were washed with MeOH followed by diethyl ether and then dried
2.2. Physical measurements in air. Yield: 72%, M.P. N250 °C, elemental analysis calculated for
C30H38CuN8O22 (%): C, 38.87; H, 4.10; and N, 12.09. Found (%): C, 39.20;
IR (KBr disc, 400–4000 cm−1) spectra were recorded on a Varian FTIR H, 4.76; and N, 12.98. UV–vis absorptions: λmax (DMSO, 10−4 M), nm
3100 spectrometer; elemental analysis was done on Carbo-Erba 1108 (ε/104 M−1 cm−1) 329 (4.059), 379 (0.088) and 408 (0.056). IR (KBr):
elemental analyzer, UV-visible (UV-vis) spectra were recorded on a νmax, cm−1 3225 (NH), 1705 and 1658 (CO), 3020 (CH, Ph), 2937 (CH),
Shimadzu UV-1601 spectrometer while TGA plots were taken on a DU- 1266, 1039 (C–O–C), and 3409 (OH, water).
PONT9900thermalanalyzingsystem(heatingrate10 °C/min)upto400 °C.
Cyclic voltammetric measurements were performed on a CHI 620c 2.7. X-ray structural studies
Electrochemical Analyzer using glassy carbon as working electrode, a
platinum wire auxiliary electrode, and Ag/Ag+ reference electrode in a Single crystal X-ray diffraction data for the complexes were collected
standard three-electrode configuration. Tetrabutylammonium perchlo- in the temperature range of 100(2) K to 293(2) K on an Enraf Nonius
rate(TBAP)wasusedasthesupportingelectrolyte,andtheconcentrationof MACH 3 diffractometer using graphite monochromatized Mo Kα
solutionsofthecomplexesinDMSOwasmaintainedas10−3 M.ESRspectra radiation (λ = 0.71073 ) from block shaped crystals in the ω–2θ scan
were recorded at 273 K and 77 K on a Varian E-line Century Series ESR mode for complexes 1, 2 and 3. Intensities of these reflections were
spectrometer equipped with a dual cavity and operating at X-band of measured periodically to monitor crystal decay. The crystal structures
100 kHz modulation frequency. Tetracyanoethylenewas used asthe field were solved by direct methods and refined by full matrix least squares
marker (g = 2.00277). The CD measurements of DNA with and without (SHELX-97) [34]. Due to high degree of hydration, thermal motion and
complexeswerecarriedoutwithaJascoJ500spectropolarimetercalibrated disorder, hydrogen atoms of water of crystallization could not be
withammonium(+)-10-camphorsulfonate. located. Drawings were carried out using MERCURY [35] and special
computations were carried out with PLATON [36]. The crystal
2.3. Equipments used for DNA cleavage studies refinement data are collected in Table 1 while selected bond distances
and bond angles are reported in Table 2.
PCR amplification was performed on an Eppendorf Mastercycler ep
gradient S. Polyacrylamide gel electrophoresis was carried out with 2.8. Interaction of complexes 1 and 3 with DNA
20× 30 cm self-cast denaturing polyacrylamide gels (5–20% acrylamide,
7 M urea, 1× TBE (89 mM Tris, 89 mM boric acid, and 2 mM EDTA, pH 2.8.1. Absorption titration
8.3) on CBS Scientific DNA sequencing systems using PowerPac HV The binding of complexes 1 and 3 with DNA was measured in a Na-
power supply from Biorad. Gels were dried on a Whatman 3MM filter phosphate buffer solution (pH 7.2). The absorption ratio at 260 nm
paper using a gel dryer model 583 from Biorad at 80 °C for 30 min. and 280 nm of calf thymus DNA (CT DNA) solutions was found as
Phosphorimaging was performed with a Storm 820 Phosphorimager 1.9:1, demonstrating that DNA is sufficiently free of protein. The
from GE Healthcare. concentration of DNA was then determined by UV-visible absorbance
3. 258 N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267
Table 1
Crystal data for 1, 2 and 3.
Compound 1 2 3
Chemical formula C18H11N6O10Cu C14H10N4O6Cu C30H38N8O22Cu
Formula weight 534.87 393.80 926.22
Temperature 150(2) K 273(2) K 150(2) K
Wavelength 0.71073 A 0.71073 A 0.71073 A
Crystal system Triclinic Monoclinic Triclinic
Space group P-1 P 1 21/n 1 (n = 14) P-1
a(Å) 9.201(2) 13.5470(11) 7.7024(2)
b(Å) 9.201(2) 7.2157(6) 8.9975(4)
c(Å) 14.331(4) 14.8634(12) 13.9532(6)
α(°) 72.31(2) 90.00 99.533(4)
β(°) 72.31(2) 90.187(3) 97.280(3)
γ(°) 73.159(2) 90.00 104.157(3)
Volume(Å3) 1074.9(4) 1452.9(2) 910.42(6)
Z 2 18 2
Absorption coefficient 1.086 mm−1 6.497 mm−1 0.705 mm−1
F(000) 540 918 479
Theta range for data collection 2.98 to 25.00° 2.03 to 23.74° 2.91 to 25.00°
Reflections collected/unique 10974/3727 [R(int) = 0.0859] 11283/1578 [R(int) = 0.0576] 8621/3192 [R(int) = 0.0207]
Completeness to theta 98.4% 71.2% 99.7%
Goodness-of-fit on F2 1.021 0.92 1.021
Final R indices [I N 2σ(I)] R1 = 0.0638, wR2 = 0.1613 R1 = 0.0472, wR2 = 0.1213 R1 = 0.0262, wR2 = 0.0653
R indices (all data) R1 = 0.1058, wR2 = 0.1818 R1 = 0.0795, wR2 = 0.1409 R1 = 0.0329, wR2 = 0.0686
Largest diff. peak and hole 2.003 and −0.959 e. Å3 0.347 and −0.470 e. Å3 0.296 and −0.356 e. Å3
using the molar absorptivity (6600 M−1 cm−1) at 260 nm [37]. The [Complex], the extinction coefficient for the free copper(II) complex
absorption titration of 1 and 3 (100 μM) in Na-phosphate buffer (pH initially, after sequential addition of DNA and extinction coefficient for
7.2) with 10% DMSO against CT DNA were performed by monitoring the copper(II) complex in the fully bound form, respectively [38]. Kb is
the changes in absorption spectra. The titration experiments were the ratio of slope to the intercept.
performed by maintaining the concentration of metal complexes
constant at 100 μM while the concentration of CT DNA was varied 2.8.2. Competitive binding studies
within 25–225 μM. An equal quantity of CT DNA was also added to the Relative binding of the copper complexes to CT DNA was studied by
reference solution to eliminate the absorption by DNA. From the fluorescence spectroscopy using ethidium bromide (EB) bound to CT
absorption data, the intrinsic binding constant Kb was calculated from DNA in a Na phosphate buffer solution (pH 7.2). In a typical experiment,
a plot of [DNA] / (εa − εf) vs. [DNA] using the equation: 20 μL of CT-DNA solution (A260 = 2.0) was added to 2.0 mL of EB buffer
solution (pH 7.2) and the fluorescence intensity was measured upon
½DNAŠ = ðεa −εf Þ = ½DNAŠ = ðεb −εf Þ + ½Kb ðεb −εf ÞŠ
−1 excitation at 510 nm; maximum emission was observed at 600 nm. The
complex concentration was increased by addition of aliquots from a
0.1 mM stock solution until the fluorescence intensity did not decrease
where [DNA] represents the concentration of DNA in base pairs. The
any further. Stern–Volmer quenching constants were calculated using
apparent absorption coefficients εa, εf and εb correspond to Aobsd/
the following equation [39],
Iο = I = 1 + Ksv r;
Table 2
Selected bond lengths (Å) and angles (°).
where Iο and I are the fluorescence intensities in absence and
Complex 1
presence of complexes, respectively, Ksv is a linear Stern–Volmer
Cu1–O2 1.873(9) O2–Cu1–O1 97.27(18) quenching constant and r is the ratio of the total concentration of
Cu1–O1 1.877(19) O2–Cu1–N1 89.43(19) complex to that of DNA. The value of Ksv is given by the ratio of slope
Cu1–N1 1.985(24) O1–Cu1–N1 169.87(20)
Cu1–N2 1.987(7) O2–Cu1–N2 168.93(22)
to intercept in a plot of Iο/I vs. [Complex]/[DNA].
O1–C14 1.418(15) O1–Cu1–N2 92.3(2)
O2–C18 1.288(17) N1–Cu1–N2 81.86(21) 2.8.3. DNA cleavage study
The nuclease activity of the copper(II) complexes was studied
Complex 2
using supercoiled pBR322 and pUC19 plasmid DNA. Electrophoresis in
Cu1–O1 1.881(5) N2–Cu1–O4 95.66(23) native agarose gel was used to quantify the unwinding of plasmid
Cu1–N2 1.971(6) O1–Cu1–N1 94.58(21)
DNA induced by copper(II) complexes. The cleavage reactions on
Cu1–O4 1.973(6) N2–Cu1–N1 81.58(23)
Cu1–N1 1.998(6) O4–Cu1–N1 165.84(23)
pBR322 were carried out for 24 h at 37 °C in a total volume of 25 μL
Cu1–O6i 2.303(5) O1–Cu1–O6i 95.08(19) containing 0.5 μg pBR322 DNA and different concentrations of
O6–Cu1ii 2.303(5) N2–Cu1–O6i 93.07(21) complexes (ranging from 10 to 500 μM) in 5 mM Tris–HCl buffer
O1–Cu1–N2 171.70(21) O4–Cu1–O6i 87.72(19) (pH 7.2), 50 mM NaCl and 10% DMSO. The samples were analyzed by
O1–Cu1–O4 86.27(21) N1–Cu1–O6i 106.25(22)
electrophoresis for 3 h at 50 V on 1% agarose gel in 1× TAE buffer
Complex 3 (40 mM Tris acetate and 1 mM EDTA) pH 8.3. The gel was stained with
a 0.5 μg/ml ethidium bromide and visualized by UV light and then
Cu1–O1 1.963(3) O1–Cu1–O1i 179.99(5)
Cu1–O1i 1.963(3) O1–Cu1–O8i 90.55(6) photographed for analysis. The extent of cleavage was determined
Cu1–O8i 1.976(2) O1i–Cu1–O8i 89.45(6) from the intensities of the bands using the AlphaImager 2200
Cu1–O8 1.976(2) O8i–Cu1–O8 179.99(6) software [40]. However, cleavage study on pUC19 was carried in a
Cu1–O7i 2.415(7) O1–Cu1–O7i 95.43(5) total reaction volume of 10 μL, containing 100 ng (1 μL) of pUC19
Cu1–O7 2.415(7) O8i–Cu1–O7i 89.11(6)
DNA, and different concentrations of complexes 1 and 3 in 5 mM Tris–
4. N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267 259
HCl buffer (pH 7.2) containing 25% DMSO for 24 h at 37 °C. The triphosphate) mix (2 mM), 1 μL Taq buffer (10 mM Tris–HCl, 50 mM
samples were analyzed by electrophoresis for 1.3 h at 75 V on 1% KCl, and 1.5 mM MgCl2) pH 8.3 and 0.5 μL Taq DNA polymerase (5 U/μL)
agarose gel in 1× TAE buffer. The gel was stained with 1:20000 stain G, in a final volume of 10 μL. The primer extension reactions were run
visualized by UV light, and photographed for analysis. under PCR conditions with temperature cycling (30 cycles of denatur-
ation at 94 °C (30 s), annealing at 50 °C (30 s), and extension at 72 °C
2.8.4. Determination of site of DNA cleavage (30 s), followed by a final extension at 72 °C for 5 min). After the
For this study pUC19 plasmid DNA was used. The linearization of completion of PCR reaction, 3 μL of stop solution was added to both the
pUC19 with complexes was studied first and then primer extension tubes and heated at 90 °C for 2 min. The tubes were then cooled in an ice
reactions were carried out to locate the probable site of cleavage. The bath for 2 min and the samples were loaded on a 10% denaturing
details of expected extended product and primers and restriction polyacrylamide gel. The gel was run for 1.5 h at 35 W. The gel was
enzyme combinations used are given in Table 3. soaked on a filter paper and then dried in a gel dryer (80 °C for 30 min)
and exposed overnight to a phosphor screen. The screen was scanned to
2.8.4.1. Restriction digestion of pUC19 visualize the DNA bands.
2.8.4.1.1. Eco-RI restriction digestion. In a reaction tube, 100 ng
(1 μL) of pUC19 DNA, 1 μL of Eco buffer (50 mM Tris–HCl pH 7.5, 2.8.5. In vitro cytotoxicity assay
10 mM MgCl2, 100 mM NaCl, 0.02% Triton X-100 and 0.1 mg/mL BSA), The DL (Dalton's lymphoma: a transplantable T cell lymphoma) cells
0.5 μL of EcoRI restriction enzyme (10 U/μL) and 7.5 μL of deionized were collected from the mouse ascite. The viable DL cells, determined by
water were mixed together. Then the tube was incubated at 37 °C for trypan blue exclusion test, were seeded onto 96 well plates in 100 μL of
1 h. This reaction mixture was used for a control lane for visualizing the RPMI-1640 culture medium supplemented with 10% fetal bovine
linear pUC19 DNA. serum, penicillin G(100 U/mL), and streptomycin(100 μg/mL). The cells
2.8.4.1.2. PvuII restriction digestion. 50 ng of pUC19 (1 μL), 1 μL of were then allowed to grow in a CO2 incubator with 5% CO2 at 37 °C. After
buffer G (10 mM Tris–HCl pH 7.5, 10 mM MgCl2, 50 mM NaCl, 0.1 mg/ 24 h incubation, different concentrations (10−15 to 10−8 M) of the
ml bovine serum albumine), 0.5 μL of PvuII restriction enzyme (10 copper (II) complexes, made by serial dilutions in the culture medium,
units/μL) and 7.5 μL of deionized water were incubated for 1 h at were added and the plates were incubated for another 24 h. Cell viability
37 °C. This PvuII digested DNA was used as template in control primer was determined by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
extension reactions. diphenyltetrazolium bromide) assay, which is based on the ability of
the viable cells to reduce a soluble yellow tetrazolium salt to blue
2.8.4.2. pUC19 linearization with complex 1. 50 ng of pUC19 was formazan crystals [41]. Briefly, after 24 h of the treatment, the MTT dye
incubated with 100 μM of complex 1 at 37 °C for 24 h in 5 mM Tris– (10 μL/100 μL of medium), prepared in phosphate buffered saline (PBS),
HCl buffer (pH 7.2), 25% DMSO. This linearized DNA was used as was added to all the wells. The plates were then incubated for 4 h at
template in primer extension reactions. 37 °C, the medium was discarded and 100 μL of DMSO was added to
each well. Optical density was measured at 570 nm. As described in our
2.8.4.3. 5′-32P-Labeling of primers with T4 polynucleotide kinase (PNK). previous report [42] the percentage of viable cells was determined by
In a reaction tube, 25 pmol (0.25 μL) of the primer, 1 μL of PNK buffer taking the cell counts in the untreated sets as 100%. The semi logarithmic
(70 mM Tris–HCl, 10 mM MgCl2 and 5 mM dithiothreitol) pH 7.6, dose–response plots, constructed using the Graphpad Prism5 software
0.5 μL of [γ-32P] ATP (10 mCi/mL) of specific activity 3000 Ci/mmol, [43], were used to determine the IC50 values as the complex
0.5 μL of PNK enzyme (10 U/μL) and 7.75 μL of deionized water were concentrations that inhibited DL cell growth by 50%.
added together. After incubation for 1 h at 37 °C, stop solution (10 μL)
containing 95% formamide, 1 mg/ml bromophenol blue and 1 mg/ml 3. Results and discussion
xylene cyanol was added. The enzyme was deactivated by incubation
at 90 °C for 2 min and the reaction mixture was loaded on 12% 3.1. Synthesis and characterization
denaturing polyacrylamide gel. The gel was run at 35 W for 1 h. The
labeled DNA was extracted by crush-and-soak using TEN buffer In our earlier study it has been reported that LH4 reacts with Zn(bpy)
(10 mM Tris–HCl pH 8.0, 1 mM EDTA, and 300 mM NaCl) and then (NO3)2 2H2O, and provides a supramolecular structure consisting of two
precipitated using three volumes of cold absolute ethanol. LH− anion and one [Zn(bpy)2·2H2O]2+ cation together with seven co-
3
crystallized water molecules [31]. Enthused by this study, a reaction of
2.8.4.4. Primer extension studies. In a typical primer extension LH4 was carried with another metal precursor 2,2′-bipyridyl-dinitrato-
experiment, two reactions were performed in parallel using Taq DNA copper(II)-dihydrate in anticipation that the Cu(II) ion, due to its
polymerase and linearized pUC19 DNAs as templates. For the control distorted configuration and its redox active nature, may interact with
reaction, pUC19 was linearized with PvuII restriction enzyme, whereas the ligand LH4 in a different way compared to Zn(II) ion. The reaction
the reaction product from incubation of pUC19 with complex 1 was used between Cu(bpy)(NO3)2·2H2O and LH4 in DMF containing MeOH
for locating the cleavage site. Primer extensions were run with both resulted initially in a dark green solution, from which two products
templates using 1 μL 32P-labeled primer, 2 μL dNTP (deoxynucleotide crystallized, a dark green complex 1 and a blue coloured complex 2.
Table 3
Primers of pUC19 and restriction enzymes used for primer extension studies.
Primer 5′–3′ Sequence Comment Restriction enzyme used Length of extended
to prepare template DNA product (bp)
P1 GTAAAACGACGGCCAGT M13/pUC fwd 379–395 PvuII 249
P2 AACAGCTATGACCATC M13/pUC rev 476–461 PvuII 170
P3 GGAGACGGTCACAGC pUC19 fwd 50–64 PvuII 256
P4 TCGGAACAGGAGAGC pUC19 rev 1000–986 PvuII 372
P5 GGTACCTGTCCGCC pUC19 fwd 1016–1029 BSaI 750
P6 AAGCATCTTACGGATG pUC19 rev 2162–2147 BSaI 396
P7 CAATAACCCTGATAAATGC pUC19 rev 2531–2513 ScaI 354
P8 CACATTTCCCCGAAAAGT pUC19 fwd 2592–2610 PvuII 400
5. 260 N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267
These complexes were characterized initially by their IR spectra.
Complex 1 showed sharp peaks at 1695 and 1602 cm−1, whereas
complex 2 showed a distinct peak at 1731 cm−1 in addition to other
major peaks at 1665 and 1611 cm−1. Thus, the IR spectra suggested
that both complexes 1 and 2 contain ligands of different constitution.
This was confirmed by solving their X-ray crystal structure, which
demonstrated that the original framework of LH4 was no longer
present. The ligand LH4 was transformed into two different com-
pounds which in situ coordinated with Cu(II)-2,2′-bipyridine to give
two new complexes (Scheme 1).
In addition, LH4 was allowed to react with copper nitrate in the
absence of the bipyridyl ligand. This resulted in formation of green
coloured complex 3, which was characterized by the presence of two
molecules of the original barbiturate derivative LH4 acting as Cu(II)
ligands.
3.2. Structural description of complexes
Complex 1 consists of a tetra coordinated Cu(II) ion having a
N2O2 coordination core (Fig. 1a), involving 2 nitrogen atoms from 2,2′-
bipyridine and 2 oxygen atoms from 5-hydroxy-hydurilic acid. It
crystallizes into a triclinic P-1 space group and Cu–N and Cu–O distances
are lying in the reported range (Table 2). It has a Kitaigorodskii Packing
Index (KPI) of 70.2% which shows compact packing with few solvent
Fig. 1. (a) Molecular structure of 1 (30% probability ellipsoid), hydrogen atoms are
accessible voids [44]. Several H-donor and acceptor functional groups
omitted for clarity and (b) a perspective of water clusters in crystal lattice of 1.
present on the skeleton of the ligand form ten hydrogen bonds (S-1).
The formation of six non-conventional hydrogen bonds involves C–H as
H-donor and oxygen as H-acceptor whereas four conventional H-bonds
are formed using N–H as donors and O as acceptors. The co-crystallized Like complex 1, complex 3 also crystallizes in a triclinic P-1 space
water molecules are arranged in a C3 chain water cluster in packing group. Its Cu(II) ion is surrounded by 4 oxygen atoms, two originate
diagram forming a water hexamer (Fig. 1b). from two monodentate ligands as LH−, whereas two other oxygen
3
Complex 2 is monoclinic with P-1 space group. It consists of a penta- atoms are from two coordinated water molecules (Fig. 3a). Complex 3
coordinated Cu(II) ion with N2O3 coordination core from 2,2′-bipyridyl exhibits square planar geometry and also contains four co-crystallized
(2N) and alloxanic acid (3O) (Fig. 2a). The Cu–O and Cu–N bond water molecules (Fig. 3b) which stabilize the structure by formation
distances (Table 2) are found in range as reported for other penta- of hydrogen bonds (S-2).
coordinated Cu(II) complexes [45]. The assembly of monomeric unit
leads to a helical 1D polymeric framework (Fig. 2b). The study of weak 3.3. UV–vis spectroscopy and electrochemical studies
interactions using PLATON indicates the presence of seven hydrogen
bonds in crystal packing of complex 2. Five non-conventional hydrogen The complexes were characterized by UV–vis spectroscopy and
bonds involve C–H donor groups, and two conventional hydrogen bonds their electrochemical properties were determined. The paramagnetic
involve O–H as donors (Fig. 2c). copper(II) complexes 1 and 3 in solution (10−4 M in DMSO) exhibited
O
O
HN NH O
HN NH
H
O O N N O
Cu(bpy)(NO3)2.2H2O O
O O O Cu O
N
DMF, MeOH O O N Cu
N O
HN N
HN N O
O N O
H
O
1 2
Cu(NO3)2.2H2O
O O
NH
DMF, MeOH HN
NH H2O HN O
O
O Cu O
O O
H2O HN
NH O
O NH
N
H O O
3
Scheme 1. Synthetic strategy for complexes 1-3.
6. N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267 261
Fig. 2. (a) Molecular structure of a single unit of 2 (30% probability ellipsoid), hydrogen atoms are omitted for clarity, (b) zigzag polymeric structure of 2 and (c) conventional
hydrogen bonds in crystal lattice of 2.
a broad d–d band in the range of 590–690 nm with a molar extinction complex 2 in powder displayed well resolved four lines at liquid N2
coefficient of 145–235 M−1 cm−1. However, intense absorption bands temperature. The axial g and A tensor values with g∥ N g⊥ suggest that
are observed at 260–270 nm, which are attributed to π–π* transitions. dx2–y2 is a ground state while g0 values are calculated using the
Absorption bands observed in the region of 300–400 nm are assigned relationship g0 = (g∥ + 2g⊥) [46]. The values of calculated ESR
to n–π* transition overlapping with ligand to metal charge transfer parameters are shown in Table S-6. Although the ratio g∥/|A∥| is
(LMCT) transition between the heterocyclic base and the metal ion. normally taken as an indication of the stereochemistry of the copper
Since, the DNA binding and cleavage studies were carried out in (II) complexes, yet it is suggested that this ratio may be an empirical
aqueous medium, UV–vis spectra of complexes 1 and 3 were also indication of the tetrahedral distortion of a square planar geometry
recorded in DMSO/water (v/v, 1:10) mixture. It showed (S-3) that [47]. The values of hyperfine splitting lower than 135 cm−1 are
complexes retain their structures in DMSO as well as in DMSO/water observed for square planar structures and those higher than 150 cm−1
mixture. for tetrahedrally distorted complexes. The data shown in Table S-6 are
The complexes display a quasi-reversible cyclic voltammetric found in consistence with earlier reports as well as structure observed
response in the range of 0.2 to 0.5 V (vs. silver reference electrode) in from their X-ray diffraction studies.
DMSO (10−4 M). The redox peak is assigned to Cu(II)/Cu(III) couple in
view of reported redox potential data (S-4). 3.5. Thermo-gravimetric studies
3.4. Electron spin resonance Thermo gravimetric analysis (TGA) (S-7) of the complexes showed
that the loss of crystallized water molecules starts at ~90 °C in each
The ESR spectra of complexes 1 and 3 in DMSO at 66 K displayed complex. The weight loss continues up to 165 °C in complex 1 and the
the typical four-line pattern as expected from 63Cu nucleus (S-5). magnitude of the weight loss (%) corresponds to three water
Three parallel hyperfine lines were well resolved in both complexes molecules (observed 11.1, calculated 10.4). In complex 2 weight
while the fourth line overlapped with g⊥ signal. The spectrum of loss of 4.2% corresponds to removal of one water molecule (calcd.
7. 262 N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267
Fig. 3. (a) Molecular structure of 3 (30% probability ellipsoid), hydrogen atoms are omitted for clarity and (b) a perspective of water clusters in crystal lattice of 3.
3.8%) and it continues up to 150 °C. However, in complex 3 six water
molecules (two coordinated and four co-crystallized) are lost
between 90 and 130 °C (observed weight loss 12.3%, calculated
weight loss 11.6%). The TGA data thus showed that the water
molecules are bound weakly in the lattice of complex 3 as compared
to complexes 1 and 2 [48].
3.6. DNA binding studies
In general, intercalation of a complex into DNA results in a
hypochromic red shift of its absorption band. This may occur due to
strong stacking interactions between the aromatic chromophore of
the complex and the base pairs of the DNA. On increasing the
concentration of CT DNA, the hypochromicity increased in the ligand-
centred (LC) band of complex 1. In contrast, a hyperchromic effect
was observed in LC band of complex 3 (Fig. 4). The copper(II)
complexes can bind to the double-stranded DNA in different modes
on the basis of their structure, charge and type of ligands. As DNA
double helix possesses many hydrogen bonding ligands accessible
both in the minor and major grooves, it is likely that the N–H group of
barbiturate ligand might be forming hydrogen bonds with DNA.
Hence, it may contribute to the hyperchromic shift in its absorption
spectrum. In order to compare the binding strength of the complexes
with CT DNA, the intrinsic binding constants Kb were obtained from
the ratio of slope to the intercept from the plots of [DNA] / (εa − εf) vs.
[DNA]. The calculated Kb values of 1.9 × 106 M−1 and 1.7 × 105 M−1 for
complexes 1 and 3 respectively show that DNA binds complex 1
stronger than complex 3.
3.7. Competitive binding with ethidium bromide
The ability of a complex to affect the fluorescence intensity of EB-
DNA adduct is a reliable tool for the measurement of its affinity
towards DNA. Intense fluorescent light is emitted from EB in presence
of DNA owing to its strong intercalation between adjacent DNA base
pairs. A complex binds with DNA by the displacement of EB bound to
DNA. Consequently, the intensity of emission is reduced as emission
from free EB is readily quenched by surrounding water molecule [49].
Fig. 4. UV–vis absorption spectra of (a) [complex 1] = 25 μM in the absence and in
The emission quenching from DNA bound ethidium bromide is due to presence of increasing amounts of DNA = 0–225 μM and (b) [complex 3] = 25 μM in the
displacement of ethidium bromide from the DNA helix. The emission absence and in presence of increasing amounts of DNA = 0–225 μM. Arrow shows the
spectra of EB-DNA system in the presence and absence of copper absorbance changes upon increasing DNA concentrations.
8. N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267 263
Fig. 5. Emission spectra of EB bound to DNA in the absence (—) and in the presence of, [complex 1] 0–4 μM, [EB] 10 μM, [DNA] 10 μM. Arrow shows changes in the emission intensity
upon addition of increasing concentration of the complex.
complexes 1 and 3 are shown in Figs. 5 and 6. The addition of shows two conservative CD bands in the UV region, a positive band at
complexes to DNA pretreated with EB shows appreciable reductions 278 nm due to base stacking and a negative band at 246 nm due to
in emission intensity. On the addition of 4 μM of complex 1 to 10 μM of poly nucleotide helicity [50]. The changes in CD pattern of DNA
CT DNA pretreated with EB, ~ 80% displacement of ethidium bromide observed after interaction with these complexes is considered to
was observed. This suggests that complex 1 is a good intercalator. assign the corresponding changes in structure of DNA [51]. Simple
However, complex 3 brings about only ~ 40% displacement of groove binding and electrostatic interaction of small molecules show
ethidium bromide at the same concentrations of both CT DNA and less or no perturbation on the base-stacking and helicity bands, while
the complex. The quenching plots of Iο/I vs. [Complex]/[DNA] (insets intercalation enhances the intensities of both bands and stabilizes the
in Figs. 5 and 6) are in good agreement with the linear Stern–Volmer right-handed B conformation of CT DNA, as observed for classical
equation. Stern–Volmer quenching constants (Ksv) were calculated to intercalator methylene blue [52].
be 3.8 and 1.2 for complex 1 and complex 3 respectively. CD spectral variations of calf thymus DNA (50.0 μM, in 0.1 mM Na-
phosphate buffer (pH = 7.4), were recorded in the presence of
3.8. CD spectral studies increasing amounts of complexes 1 and 3 until [complex]/[DNA]
molar ratios approached approximately 0.4. By addition of complex 1,
Circular dichroism measurements were conducted in order to a blue shift of the positive CD band of DNA was observed (Fig. 7a).
determine the extent of changes which occur in DNA conformation These findings indicate that a subtle change of the DNA double helix
upon binding of complexes 1 or 3. The B form conformation of DNA occurs owing to the interaction of the metal complex with DNA [53].
Fig. 6. Emission spectra of EB bound to DNA in the absence (—) and in the presence of, [complex 3] 0–4 μM, [EB] 10 μM, [DNA] 10 μM. Arrow shows changes in the emission intensity
upon addition of increasing concentration of the complex.
9. 264 N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267
Fig. 8. (a) Gel electrophoresis diagram showing the cleavage of SC pBR322 DNA (0.5 μg) by
complex 1 on 12 h of incubation in 50 mM Tris–HCL buffer (pH 7.2): lane 1, DNA control;
lane 2, DNA + 10 μM; lane 3, DNA + 25 μM; lane 4, DNA + 50 μM; lane 5, DNA + 100 μM;
and lane 6, DNA + 500 μM, (b) cleavage of supercoiled pBR322 DNA showing the decrease
in SC DNA and the formation of NC DNA with increasing concentration of complex 1.
activity in the physiological pH range. Though, future experiments
will be needed to characterize the cleavage mechanism in detail.
However, a preliminary experiment showed that neither Cu(bpy)
(NO3)2·2H2O nor LH4 separately caused DNA cleavage (S-8).
3.10. Determination of site of DNA cleavage
The gel electrophoretic separation of plasmid pUC19 DNA induced
Fig. 7. Circular dichroism spectra of CT-DNA (50 μM) in the absence (—) and presence of by complexes 1 and 3 and EcoRI is shown in Figs. 10 and 11.
complex 1 (a) and complex 3 (b) in 0.1 mM Na-phosphate buffer. Complexes 1 and 3 linearize pUC19 at concentrations of 25 μM and
10 μM respectively on incubation for 24 h in a medium of Tris–HCl/
NaCl pH 7.2 containing 25% DMSO. The intensity of linear form
Therefore, it can be inferred that complex 1 tightly binds to DNA. increases with the increase in the concentration of complexes.
However, binding of DNA with complex 3 induces a decrease in the Primer extension reactions were performed to assay the site of
intensity of both positive and negative bands with a red shift in the DNA cleavage by complex 1. In these experiments, 5′-32P-labeled
position of the band [54]. primers annealed to template DNA are extended from their 3′-end
with Taq DNA polymerase until the 5′-end of the template DNA is
3.9. DNA cleavage study reached. This “end” in the template DNA can either be generated by
a restriction enzyme (linearization of the plasmid DNA) or by Cu-
The DNA-cleaving ability of the copper(II) complexes has been complex catalyzed cleavage of the DNA. A set of 8 primers in
studied by the relaxation of supercoiled pBR322 DNA to the nicked combination with three restriction enzymes was used to probe the full
circular DNA. When circular plasmid DNA is subjected to electropho- length of the 2686 base-pair long pUC19 plasmid (Fig. 12). Primer
resis, relatively fast migration is observed for intact supercoiled form extension of 32P-labeled primer 8 from the DNA template that had
(S form). However, if scission of DNA occurs at one strand (nicking), been treated with complex 1 yielded an extension product that was
the supercoiled DNA will relax to generate a slower-moving open/ much shorter than the product generated from a control reaction with
nicked circular (NC form). If both strands are cleaved, a linear form (L) PvuII-digested DNA (S-9). No stop was observed on DNA treated with
will be generated [55]. The gel electrophoretic mobility assay (Figs. 8 complex 1 in other primer extension reactions using the primers of
and 9) showed that both copper(II) complexes convert supercoiled Table 3. A likely explanation for this observation is that complex 1
(SC) plasmid pBR322 DNA into nicked circular (NC) DNA after cleaves pUC19 specifically within the ca 400 bp region between the
incubation at 37 °C for 24 h in a medium of Tris–HCl/NaCl pH 7.2. primer 8 binding site and the first PvuII cleavage site (position 306,
Complex 1 converts more than 90% of SC form into NC form at a see Fig. 12).
concentration of 100 μM, whereas, for a similar level of conversion,
only 50 μM of complex 3 is required. Thus, both complexes show 3.11. Evaluation of cytotoxicity in vitro
nuclease activity without addition of any external oxidizing or
reducing agent unlike most of the other Cu(II)-based nucleases. In presence of copper(II) complexes, IC50 values were determined
These results suggest that the copper(II) complexes show nuclease against Dalton's lymphoma (DL) cell lines. The MTT assay measures
10. N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267 265
Fig. 11. Gel electrophoresis diagram showing the cleavage of pUC19 DNA (0.1 μg) by
complex 3 on 24 h of incubation in 5 mM Tris–HCL buffer (pH 7.2): lane 1, marker; lane
2, DNA control; lane 3, EcoRI treated DNA; lane 4, DMSO control; lane 5, DNA + 10 μM;
lane 6, DNA + 25 μM; lane 7, DNA + 50 μM.
Fig. 9. Gel electrophoresis diagram showing the cleavage of SC pBR322 DNA (0.5 μg) by and the IC50 values are determined using the GraphPad Prism5
complex 3 on 12 h of incubation in 50 mM Tris–HCL buffer (pH 7.2): lane 1, DNA software.
control; lane 2, DNA + 10 μM; lane 3, DNA + 25 μM; lane 4, DNA + 50 μM; and lane 5,
The results thus obtained suggested that after 24 h of incubation,
DNA + 100 μM, (b) cleavage of supercoiled pBR322 DNA showing the decrease in SC
DNA and the formation of NC DNA with increasing concentration of complex 3. the copper(II) complexes are cytotoxic against DL cells with an IC50
values ~9.0 nm and 0.6 nm for complexes 1 and 3 respectively. Copper
(II) complexes decreased viability of DL cells in a concentration-
mitochondrial dehydrogenase activity as an indication of cell viability. dependent manner (with increasing concentration from 10−15 M to
It has been carried out with the copper complexes using murine 10−8 M). A ~40% decrease in cell viability is observed in the presence
Dalton's lymphoma cells which are T cell lymphoma of spontaneous of Cu(II) complexes as compared to control. The values of IC50
origin in the thymus. Dalton's lymphoma cells have often been indicate that complex 3 is a stronger cytotoxic agent than complex 1
successfully used to identify the anticancer potential of newly when tested against DL cell (S-10). These values are found to be
synthesized compounds both in vitro and in vivo [56]. Hence, the significantly higher than the IC50 value of cisplatin against DL cell
effect of Cu(II) complexes on the viability of DL cell lines has been lines [57].
measured after 24 h of treatment as a function of concentration. The
experiments have been performed in triplicates for all the complexes
4. Conclusion
Three new copper(II) complexes of different geometry were
prepared and characterized. Complexes 1 and 2 bearing 2,2′-bipyridyl
as terminal ligand were isolated in a one pot synthesis as a result
of transformation of the original barbiturate ligand LH4 in the
presence of Cu(bpy)(NO3)2·H2O. The complexes contain a significant
number of co-crystallized water molecules in their crystal lattice
which stabilize the corresponding supramolecular structures through
H-bonds.
Complexes 1 and 3 bind with the calf thymus DNA strongly though
the binding constant for complex 1 is little higher than that of
complex 3. These complexes also transform supercoiled DNA to
nicked and linear forms under physiological conditions and possess
considerable chemical nuclease activity. In contrast to DNA binding
results, DNA cleavage studies indicated that complex 3 is a better
nuclease in comparison to complex 1. The better binding affinity of
complex 1 with DNA could be due to the presence of 2,2′-bipyridine
ligand which reportedly intercalates well with DNA. However, the
better nuclease property of complex 3 could be attributed to the
presence of ligand LH4 bearing various H donor and acceptor
functionalities in its structure. The findings also suggest that the
DNA cleavage property of the described complexes is region-specific.
Such molecules may offer new prospects for controlled manipulation
Fig. 10. Gel electrophoresis diagram showing the cleavage of pUC19 DNA (0.1 μg) by
complex 1 on 24 h of incubation in 5 mM Tris–HCL buffer (pH 7.2): lane 1, marker; lane
of the genome and therefore, can be of great interest in biotechnology
2, DNA control; lane 3, EcoRI treated DNA; lane 4, DMSO control; lane 5, DNA + 10 μM; and therapeutics. Both complexes 1 and 3 are also active against
lane 6, DNA + 25 μM; lane 7, DNA + 50 μM. Dalton's lymphoma cell lines at nano-molar concentrations.
11. 266 N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267
Fig. 12. Schematic representation of pUC19 plasmid target with location of cleavage sites of restriction enzymes and lengths of primer extension products using appropriate primers.
Arrows represent the direction of primer extension.
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