Modeling of immunoglobulin uptake by N,N,N',N'-ethylenediaminetetramethylenephosphonic acid-modified zirconia particles under static and dynamic conditions

A matrix developed from N,N,N',N'-ethylenediaminetetramethylenephosphonic acid-modified zirconia beads (further referred to as r_PEZ); 25-38 microm in diameter and with a pore size of 22+/-3 nm, was utilized for the separation of immunoglobulins (Igs). r_PEZ has been shown to bind to various Igs originating from a wide variety of species. To understand the mechanisms controlling the uptake of Igs by r_PEZ, static protein uptake experiments were carried out. The protein uptake profiles were further modeled with a kinetic rate constant model. Individual studies were undertaken for human immunoglobulin A, G and M (HIgA, HIgG and HIgM). The kinetic rate constant model indicated that HIgG binding to r_PEZ was more favorable than its disassociation. The equilibrium rate constants were found to decrease with increasing concentration. The effect of continuous loading in a packed bed system utilizing r_PEZ matrix was evaluated by carrying out frontal studies, using different feed concentrations and linear velocities. The breakthrough profiles obtained for the uptake of HIgG were modeled with the pore diffusion model. The model was found to best describe the breakthrough profiles obtained at a feed concentration of 2.0 mg of HIgG per milliliter. The NTU for the packed bed was found to be equal to 2.     

Identification of the mass transfer mechanisms involved in the transport of human immunoglobulin-G in N,N,N',N'-ethylenediaminetetramethylenephosphonic acid-modified zirconia

Zirconia particles modified with N,N,N',N'-ethylenediaminetetramethylenephosphonic acid (EDTPA), further referred to as r_PEZ, were studied as a support material for use in chromatography. Our previous studies have demonstrated the utility of r_PEZ in the separation of immunoglobulins from biological fluids. In the present study we sought to understand the underlying factors and identify the rate-limiting mechanisms that govern the transport of biomolecules in r_PEZ. Pulse injection techniques were used to elucidate the individual mass transfer parameters. Elution profiles obtained under retained and unretained conditions were approximated by the Gaussian equation and the corresponding HETP contributions were estimated. The dependence of the HETP values on incremental salt concentration in the mobile phase was determined. Resulting data in conjunction with the equations outlined in literature were used to estimate the theoretical number of transfer units for the chromatographic separation process. Our results indicate that surface diffusion probably plays a minor role; however pore diffusion was established to be the rate limiting mechanism for immunoglobulin G adsorption to r_PEZ. The HETP based methodology may be used to estimate the rate limiting mechanisms of mass transfer for any given chromatographic system under appropriate conditions.     

Platinum(II) and palladium(II) complexes with (N,N') and (C,N,N')- ligands derived from pyrazole as anticancer and antimalarial agents: synthesis, characterization and in vitro activities

The study of the reactivity of three 1-(2-dimethylaminoethyl)-1H-pyrazole derivatives of general formula [1-(CH₂)₂NMe₂}-3,5-R₂-pzol] {where pzol represents pyrazole and RH (1a), Me (1b) or Ph (1c)} with [MCl₂(DMSO)₂] (MPt or Pd) under different experimental conditions allowed us to isolate and characterize cis-[M{κ²-N,N′-{[1-(CH₂)₂NMe₂}-3,5-R₂-pzol])}Cl₂] {MMPtPt (2a-2c) or Pd (3a-3c)} and two cyclometallated complexes [M{κ³-C,N,N′-{[1-(CH₂)₂NMe₂}-3-(C₅H₄)-5-Ph-pzol])}Cl] {MPt(II) (4c) or Pd(II) (5c)}. Compounds 4c and 5c arise from the orthometallation of the 3-phenyl ring of ligand 1c. Complex 2a has been further characterized by X-ray crystallography. Ligands and complexes were evaluated for their in vitro antimalarial against Plasmodium falciparum and cytotoxic activities against lung (A549) and breast (MDA MB231 and MCF7) cancer cellular lines. Complexes 2a-2c and 5c exhibited only moderate antimalarial activities against two P. falciparum strains (3D7 and W2). Interestingly, cytotoxicity assays revealed that the platinacycle 4c exhibits a higher toxicity than cisplatin in the three human cell lines and that the complex 2a presents a remarkable cytotoxicity and selectivity in lung (IC₅₀ = 3 μM) versus breast cancer cell lines (IC₅₀ > 20 μM). Thus, complexes 2c and 4c appear to be promising leads, creating a novel family of anticancer agents. Electrophoretic DNA migration studies in presence of the synthesized compounds have been performed, in order to get further insights into their mechanism of action.     

Effect of the zinc chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) on hippocampal mossy fiber calcium signals and on synaptic transmission

An important pool of chelatable zinc is present in the synaptic vesicles of mossy fiber terminals from hippocampal CA3 area, being zinc released following single or repetitive electrical stimulation. Previous studies have suggested different synaptic roles for released mossy fiber zinc, including the inhibition of presynaptic calcium and of postsynaptic N-methyl-D-aspartate (NMDA) and gamma amino-butyric acid (GABAA) receptors. The effect of endogenously released zinc on mossy fiber long-term potentiation (LTP) induction also is not yet established. We have investigated the effect of the permeant zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) on mossy fiber calcium and on synaptic transmission, before and during the application of LTP-inducing stimulation. We have found, using the calcium indicator Fura-2, that single and tetanically-evoked mossy fiber calcium signals are both enhanced in the presence of 20 microM TPEN, while the single field potentials are unaffected. As expected, no effect was observed on the single calcium signals or field potentials obtained at the CA3-CA1 synapses, from the CA1 area, which has a lower concentration of vesicular zinc. These results support the idea that at the hippocampal mossy fiber synapses, released zinc inhibits presynaptic calcium mechanisms. A higher concentration of TPEN (100 microM) significantly reduced mossy fiber synaptic transmission but did not prevent the induction of mossy fiber LTP, suggesting that zinc is not required for the formation of this form of LTP.     

Mechanism of reaction of a suggested superoxide-dismutase mimic, Fe(II)-N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.

The interaction of a recently developed intracellular superoxide dismutase analogue, Fe(II)-N,N,N',N'-tetrakis(2- pyridylmethyl)ethylenediamine (Fe(II)-TPEN), with reactive oxygen species was investigated under in vitro conditions. The complex catalyzed the dismutation of enzyme- or radiolysis-generated superoxide with the production of H2O2; under steady-state conditions the equilibrium was strongly shifted toward Fe(III)-TPEN. Fe(II)-TPEN reacted with H2O2 to generate hydroxyl radicals in a Fenton reaction. The oxidized Fe(III)-TPEN was readily reduced by ascorbate or glutathione. Given the capacity to produce hydroxyl radicals and the reaction with cellular reductants it seems unlikely that Fe-TPEN may find widespread use as an intracellular superoxide dismutase substitute.     

Studies on the kinetic stabilities of the Gd(3+) complexes formed with the N-mono(methylamide), N'-mono(methylamide) and N,N"-bis(methylamide) derivatives of diethylenetriamine-N,N,N',N",N"-pentaacetic acid

High kinetic stability is an important requirement for the Gd(3+) complexes used as contrast enhancement agents in magnetic resonance imaging. The kinetic stabilities of the Gd(3+) complexes formed with DTPA-N-mono(methylamide) (L(3)), DTPA-N'-mono(methylamide) (L(2)) and DTPA-bis(methylamide) (L(1)) are characterized by the rates of the exchange reactions with Eu(3+) and the endogenous Cu(2+) and Zn(2+). The exchange reactions occur via the proton-assisted dissociation of the complexes and direct attack of the exchanging metal ions on the complex. On the basis of the line-shape analysis of the 1H NMR spectra of the LaL(2), obtained in the pH range 2.5-3.5, we assume that for the proton-assisted dissociation of the complexes the formation of an intermediate containing a free iminodiacetate group must be followed with the rupture of the metal-central nitrogen bond. At about pH > or = 5, the reactions between GdL(2) or GdL(3) and Cu(2+) or Zn(2+) proceed predominantly by direct reaction of the reactants, through the formation of dinuclear intermediates. The contribution of the proton-assisted dissociation is highly important for GdL(1), but its reaction with Zn(2+) is significantly slower than the reactions of GdL(2) and GdL(3). The overall rates of dissociation of GdL(1), GdL(2), GdL(3) and Gd(DTPA)(2-) through H(+) (pH 7.4), Cu(2+) (1 x 10(-6) M) and Zn(2+) (1 x 10(-5) M)-assisted reactions are surprisingly very similar. Replacement of one or two carboxylates with amide groups results in significantly decreased stability constants, but has practically no effect on the kinetic stability of the Gd(3+) complexes, indicating the lower reactivity of the amide groups with Cu(2+) and Zn(2+).     

Crystal structure and catecholase-like activity of a mononuclear complex [Cu(EDTB)]2+ (EDTB=N,N,N',N'-tetrakis(2'-benzimidazolyl methyl)-1,2-ethanediamine)

The crystal structure and catecholase-like activity of a mononuclear complex, Cu(EDTB)(NO3)2.C2H5OH (here EDTB stands N,N,N',N'-tetrakis(2'-benzimidazolyl methyl)-1,2-ethanediamine) has been studied in comparison with a binuclear complex Cu2(EDTB)(NO3)4.3H2O. The results show that the reactive rate constants increase with increases of reaction temperature and pH value of intermediate. Electrospray ionization mass spectrum (ESI-MS) shows that tautomerism isomers of catechol with the title complex exist in reaction solution, and catechol is oxidized to quinone, then it is further oxidized resulting in muconic acid and its derivatives via an intradiol mechanism, just like that catalyzed by a mononuclear non-heme iron-containing dioxygenase.     

Inhibition of phospholipase C activity in Drosophila photoreceptors by 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA) and di-bromo BAPTA

In vivo light-induced and basal hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP2) by phospholipase C (PLC) were monitored in Drosophila photoreceptors using genetically targeted PIP2-sensitive ion channels (Kir2.1) as electrophysiological biosensors for PIP2. In cells loaded via patch pipettes with varying concentrations of Ca2+ buffered by 4 mM free BAPTA, light-induced PLC activity, showed an apparent bell-shaped dependence on free Ca2+ (maximum at "100 nM", approximately 10-fold inhibition at <10nM or approximately 1 microM). However, experiments where the total BAPTA concentration was varied whilst free [Ca2+] was maintained constant indicated that inhibition of PLC at higher (>100 nM) nominal Ca2+ concentrations was independent of Ca2+ and due to inhibition by BAPTA itself (IC50 approximately 8 mM). Di-bromo BAPTA (DBB) was yet more potent at inhibiting PLC activity (IC50 approximately 1mM). Both BAPTA and DBB also appeared to induce a modest, but less severe inhibition of basal PLC activity. By contrast, EGTA, failed to inhibit PLC activity when pre-loaded with Ca2+, but like BAPTA, inhibited both basal and light-induced PLC activity when introduced without Ca2+. The results indicate that both BAPTA and DBB inhibit PLC activity independently of their role as Ca2+ chelators, whilst non-physiologically low (<100 nM) levels of Ca2+ suppress both basal and light-induced PLC activity.     

DNA binding, oxidative DNA cleavage, cytotoxicity, and apoptosis-inducing activity of copper(II) complexes with 1,4-tpbd (N,N,N',N'-tetrakis(2-yridylmethyl)benzene-1,4-diamine) ligand

Three new binuclear copper(II) complexes have been synthesized and structurally characterized by X-ray crystallography, [Cu₂(1,4-tpbd)(dafo)₂(MeOH)₂](ClO₄)₄·2.5H₂O (1), [Cu₂(1,4-tpbd) (DMSO)₂(ClO₄)₂](OH)₂·6H₂O (2) and [Cu₂(1,4-tpbd)(OAC)₂(ClO₄)₂]·5H₂O (3) (1,4-tpbd = N,N,N′,N′-tetrakis(2-pyridylmethyl)benzene-1,4-diamine). Complex 1 to 3 shows similar binuclear structure and each Cu atom adopts five-coordinated square-pyramidal geometry. The interactions of the three complexes with CT-DNA (Calf-thymus DNA) have been investigated by UV absorption, fluorescence spectroscopy, circular dichroism spectroscopy and viscosity. Furthermore, the three complexes display oxidative cleavage of supercoiled DNA in the presence of external agents. Complex 3 shows higher DNA affinity and nuclease activity may be attributed to its cis structural configuration and labile acetate and perchlorate anions. The cleavage mechanisms between the complexes and plasmid DNA are likely to involve singlet oxygen or singlet oxygen-like entity as reactive oxygen species. In addition, in vitro cytotoxicity studies on the Hela cell line show that the IC₅₀ values of complexes 1-3 are 14.75, 13.67 and 16.58 μM, respectively. The apoptosis-inducing activity was also assessed by AO/EB (Acridine Orange/Ethidium bromide) staining assay, indicating they have the potential to act as effective metal-based anticancer drugs.     

Effect of N,N'-dicyclohexylcarbodiimide (DCCD) on electron transport particles of Mycobacterium phlei

N,N′-dicyclohexylcarbodiimide (DCCD) was found to uncouple phosphorylation from oxidation with succinate and NAD⁺-linked substrates in the system from Mycobacterium phlei. However, in contrast to the effect of this agent in mammalian mitochondria, DCCD was found to stimulate oxidation with succinate as an electron donor and to inhibit the oxidation of NAD⁺-linked substrates. Furthermore, in the M. phlei system DCCD was found to inhibit the membrane bound latent ATP-ase but had no effect on this activity when the latent ATPase was removed from the membrane vesicles. Reconstitution with the fraction containing latent ATPase activity and the membrane vesicles resulted in inhibition of latent ATPase by DCCD. Studies of the effect of DCCD on the resolved system indicated that DCCD may be associated with membrane vesicles or causes secondary changes in conformation of membrane vesicles. Although DCCD inhibited membrane bound ATPase it did not prevent the addition of the solubilized ATPase to the membrane vesicles. DCCD was found to have no effect on purified succinic dehydrogenase activity but stimulated this activity in the electron transport particles.     

Synthesis of N,N',N"-trisubstituted thiourea derivatives and their antagonist effect on the vanilloid receptor

Twenty-seven N,N',N"-trisubstituted thiourea derivatives were prepared. Among them, 1-[3-(4'-hydroxy-3'-methoxy-phenyl)-propyl]-1,3-diphenethyl-thiourea (8l, IC(50)=0.32 microM), showed 2-fold higher antagonistic activity than that of capsazepine (3, IC(50)=0.65 microM) against the vanilloid receptor in a (45)Ca(2+)-influx assay.     

Synthesis and anticancer activity of [2-hydroxy-1,3-diaminopropane-kappa 2N,N'] platinum(II) complexes

A series of novel platinum(II) complexes involving a carrier with HO- peripheral functional group, 2-hydroxy-1,3-propanediamine (HO-pda), cis-[Pt(HO-dpa)X₂] (X₂ = 2Cl⁻ (1), (2), malonate (3), 1,1-cyclobutane dicarboxylate (CBDCA) (4), 3-hydroxy-1,1-cyclobutanedicarboxylate (HO-CBDCA) (5)), have been synthesized and characterized by elemental analysis and spectroscopic data along with X-ray diffraction for three representative complexes 1, 4 and 5. The Pt(II) is in a square planar environment and is coordinated in cis position by a chelating HO-pda and 2Cl⁻ for 1 and CBDCA for 4 and 5. Pt-N, Pt-Cl and Pt-O distances and coordinate bond angles of N-Pt-N, Cl-Pt-Cl and O-Pt-O are in the normal range. There are two independent molecules in the asymmetric unit of 5, held together by intermolecular hydrogen bonded chain. All the complexes show significant cytotoxicity on the sensitive cell lines SGC-7901, LNcap and A549, and are more active than carboplatin. 4 is also found to be active against the resistant cell A549/ATCC, which suggests that it has less cross-resistance with cisplatin than carboplatin. Moreover 4 shows much greater inhibition of tumor growth than carboplatin in S180-bearing mice, and is therefore worthy of further development as a potential anti-tumor platinum drug.     

Occurrence of aminopropylcadaverine and its aminopropyl derivatives aminopentylnorspermidine and N, N' -bis(3-aminopropyl)cadaverine in Halococcus acetoinfaciens

Abstract Besides putrescine, cadaverine, spermidine, spermine and thermospermine, three novel polyamines were detected in a slightly halophilic eubacterium Halococcus acetoinfaciens (IAM 12094, ATCC 25861). These novel polyamines were found to be N -3-aminopropylcadaverine [NH₂(CH₂)₃NH(CH₂)₅NH₂] and its aminopropyl derivatives: aminopentylnorspermidine [NH₂(CH₂)₃NH(CH₂)₃NH(CH₂)₅NH₂] and N , N ' -bis(3-aminoprophyl)cadaverine [NH₂(CH₂)₃ NH(CH₂)₅NH(CH₂)₃NH₂]. Aminopropylcadaverine was also detected in two other species, Halococcus agglomeratus (IAM 12095, ATCC 25862) and Halococcus nondenitrificans (IAM 12096, ATCC 25863).     

Ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibits the growth of Escherichia coli at alkaline pH

The growth of Escherichia coli was inhibited by ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) in a medium of initial pH 8.8. The growth inhibition was reversed by the addition of CaCl(2). E. coli could grow in the presence of EGTA at pH values below 8. The concentration of free calcium ions increases with a decrease in medium pH because of a decrease in the calcium binding capacity of EGTA. So, although the results suggest that calcium ions are essential for the growth of E. coli, the minimum concentration required is very low.     

N, N' (p-Xylylidene)-bis-aminoguanidine (2HCI) resistant mutants in Saccharomyces cerevisiae and their relation to the amino acid transport system

N,N'-(p-Xylylidene)-bis-aminoguanidine 2HCl proved to be an efficient inducer of non-chromosomal petite mutation in Saccharomyces cerevisiae in either growing or non-growing conditions. A mutant Agr-7 resistant to this compound was obtained. Resistance proved to dominate over its XBAG-sensitive allele in the diploid formed in cross. Mutant Agr-7 is characterized by slow growth rate on YEPG or minimal medium and small colony size. Studies on uptake of amino acids and sugars indicate that the phenomenon of resistance is involved in alteration of the general amino acid permease activity.     

Synthesis, interaction with double-helical DNA and biological activity of the water soluble complex cis-dichloro-1,2-propylenediamine-N,N,N',N'-tetraacetato ruthenium (III) (RAP)

The effects exerted by the new complex cis-dichloro-1,2-propylenediaminetetraacetato ruthenium (III), H[RuCl(2)(PDTA-H(2))] [1, RAP], on DNA and cultured tumor cells (ovarian carcinoma TG cell line) were studied. The comparative study of circular dichroism (CD) spectra obtained from DNA and RAP-DNA system evidences the interaction of the complex with DNA. Compound 1 also interacted with tumor TG cells to slow their proliferation rate. BrdU incorporation was enhanced in cells treated with compound 1, as evidenced by a single-cell electrophoresis method (comet assay), in accordance with RAP-induced DNA damage. DNA migration of compound 1-treated cells was similar to that induced by noxious agents other than cross-linking chemicals. The stability of [RuCl(2)(PDTA-H(2))]-DNA binding is suggested by the high degree of damage that persisted after removal of compound 1 from the culture medium.     

The zinc chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine, increases the level of nonfunctional HIF-1alpha protein in normoxic cells

The hypoxia-inducible factor-1alpha (HIF-1alpha) subunit is activated in response to lack of oxygen. HIF-1alpha-specific prolyl hydroxylase and factor inhibiting HIF-1alpha (FIH-1) catalyze hydroxylation of the proline and asparagine residues of HIF-1alpha, respectively. The hydroxyproline then interacts with ubiquitin E3 ligase, the von Hippel-Lindau protein, leading to degradation of HIF-1alpha by ubiquitin-dependent proteasomes, while the hydroxylation of the asparagine residue prevents recruitment of the coactivator, cAMP-response element-binding protein (CBP), thereby decreasing the transactivation ability of HIF-1alpha. We found that the Zn-specific chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), enhances the activity of HIF-1alpha-proline hydroxylase 2 but the level of HIF-1alpha protein does not fall because TPEN also inhibits ubiquitination. Since the Zn chelator does not prevent FIH-1 from hydroxylating the asparagine residue of HIF-1alpha, its presence leads to the accumulation of HIF-1alpha that is both prolyl and asparaginyl hydroxylated and is therefore nonfunctional. In hypoxic cells, TPEN also prevents HIF-1alpha from interacting with CBP, so reducing expression of HIF-1alpha target genes. As a result, Zn chelation causes the accumulation of nonfunctional HIF-1alpha protein in both normoxia and hypoxia.     

Evidence for the interaction between the calcium indicator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and calcium-binding proteins

Stopped-flow and static difference spectroscopy experiments have shown that the calcium indicator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) interacts with several different calcium-binding proteins (beta-trypsin, parvalbumin, and calmodulin) and with serum albumin under experimental conditions commonly used in biophysical studies. The interaction decreases at high ionic strength. EDTA competes with BAPTA in the interaction with the proteins.     

Conformational properties of N',N'-dimethylamides of N-acetyldehydroalanine and N-acetyl-(Z)-dehydrophenylalanine

Conformational preferences of Ac-deltaAla-NMe2 and Ac-(Z)-deltaPhe-NMe2 were studied and compared with those of their monomethyl counterparts as well as with those of their saturated analogues. X-Ray data and energy calculations revealed a highly conservative conformation of the dehydro dimethylamides, which is located in a high-energy region of the Ramachandran map.