MOPS Buffer pH Research Articles

Uptake of copper from hemopexin (HPX) in the absence and presence of heme by hepatic and mammary epithelial cells in culture

Hemopexin (HPX) captures heme units in the blood in conditions such as hemolysis. The resulting heme‐HPX complex is transported mainly to hepatocytes, taken up by receptor mediated endocytosis, and degraded, the heme degradation being initiated by heme oxygenases that release the iron for recycling. This process also provides a safe way to dispose of heme, which would otherwise be toxic [1]. Uptake of the heme‐HPX complex also brings copper into cells, and that copper appears to mediate a coordinate response resulting in induction of heme oxygenase and metallothionein [1]. We recently isolated HPX from porcine blood plasma, and found copper bound to it very tightly (that could not be removed by extensive dialysis with 100 uM histidine (pH 7). EPR analysis showed that Cu(II) was bound to oxygen and nitrogen ligands in HPX. The UV‐Vis spectrum of the same sample showed a typical heme absorbance peak. Treatment with chelating Sepharose however removed not only the copper but also the heme. These findings led us to wonder whether perhaps HPX might normally have a role in the distribution and transport of copper to cells from the circulation. To examine this, we obtained human HPX purified from blood plasma by Athens Research and Technology (Georgia), dialyzed it at pH 5 to remove any heme, added traces of radioactive 67Cu (as the NTA complex), and applied it to size exclusion chromatography on Sephadex G100 equilibrated with MOPS buffer pH 7.4, containing electrolytes to make it isotonic for tissue culture studies. The 67Cu eluted with the HPX protein peak, and no low molecular weight 67Cu was detected, indicating it had all stuck to the HPX. The peak fractions were pooled and incubated with human hepatic (HepG2) or mammary epithelial cells (PMC42) for various lengths of time. 67Cu radioactivity in the cells (washed with medium containing EDTA) was then determined, and rates of uptake (percent dose/h/mg cell protein) were calculated. For both cell types, the uptake was linear over several hours, but were 2–3 times faster with the hepatic cells. In subsequent experiments, the effects of heme on copper uptake were also measured. When heme was added to the 67Cu‐prelabeled HPX at a molar ratio of 1.1:1.0 (heme:HPX), we expected the rate of copper uptake to increase stimulated, since adding heme to HPX would allow receptor‐mediated endocytosis of the complex. Instead, heme addition markedly reduced copper uptake. The potential effects of three different endocytosis inhibitors were also examined with mixed results. However, some significant inhibition of uptake was observed, including when HPX with 67Cu but no added heme was administered. These findings indicate that copper attached to HPX can be delivered to hepatic and non‐hepatic cells even in the absence of heme and suggest that copper may have more than one role in the function of this plasma protein.

Design, synthesis, and biological evaluation of aromatic ester prodrugs of 1-(3'-hydroxypropyl)-2-methyl-3-hydroxypyridin-4-one (CP41) as orally active iron chelators.

In order to improve chelation efficacy and to minimise toxicity, eleven aromatic ester prodrugs of 1-(3'-hydroxypropyl)-2-methyl-3-hydroxypyridin-4-one (CP41) have been synthesised. The distribution coefficients of these ester prodrugs between 1-octanol and MOPS buffer pH 7.4 were measured together with their rates of hydrolysis at pH 2 and pH 7.4, in rat blood and liver homogenate. The biliary metabolic profiles of selected ester prodrugs were investigated in rats. The in vivo iron mobilisation efficacy of these ester prodrugs has been compared with that of the parent drug using a 59Fe-ferritin loaded rat model. The hydrolytic rates of these esters vary appreciably, esters with heteroaromatic acid moieties being less stable than the corresponding benzoyl analogues. Many prodrugs were found to enhance the ability of the parent hydroxypyridinone to facilitate 59Fe excretion, the optimal effect being observed with the 4-methylbenzoyl ester derivative 8d. However, not all prodrugs provide an increased efficacy, indicating that lipophilicity is not the only factor which influences drug efficacy. Furthermore no clear correlation between lipophilicity, susceptibility towards hydrolysis and efficacy was detected.

The role of Phe329 in binding of cationic triarylmethane dyes to human butyrylcholinesterase

Cationic triarylmethane dyes (TAM +)s which are used as colorants in industry and as frequent tools and reagents in analytical, cell biological and biomedical research have been recently characterized as reversible inhibitors of human butyrylcholinesterase. In this study, the inhibitory effects of two TAM +s, malachite green (MG) and methyl green (MeG) on five human BChE mutants (A277V, P285L, H77L, A328F and F329A) were studied spectrophotometrically at 25 °C in 50 mM MOPS buffer pH 8, using butyrylthiocholine as substrate. The kinetic results obtained with mutant enzymes were compared to those obtained with recombinant wild type BChE. MG and MeG were found to act as competitive/linear mixed inhibitors of recombinant wild type BChE and all BChE mutants except the F329A mutant. Both dyes caused complex nonlinear inhibition of F329A mutant, pointing to multisite binding. K i values for MG and MeG, estimated by nonlinear regression analysis, were 3.8 and 27 μM, respectively, as compared to the 50- to 150-fold lower values observed with recombinant wild type BChE. The observed significant differences in kinetic pattern and K i values between recombinant wild type BChE and F329A mutant suggest that phenylalanine at position 329 in human BChE is a critical residue in MG and MeG binding to enzyme.

Development of a capillary electrophoresis method for the determination of orphenadrine citrate in tablets in the presence of paracetamol

A validated method using capillary electrophoresis was developed, for the determination of orphenadrine citrate in its tablet formulations, in the presence of paracetamol. The method employs a running buffer of 30mM pentane sulfonate sodium, dissolved in 20mM MOPS buffer pH 7.7. Samples were injected using hydrodynamic sample injection mode (25mbar, for 25s), using positive polarity of 25kV, at a constant temperature of 30°C. Samples of orphenadrine citrate alone or in mixture solutions with paracetamol were exposed to various degradation conditions, and were electrophoresed using the recommended condition. The method was found to be specific, linear (r2=0.994), precise, accurate, and robust, with an LOQ of 0.02mg/mL. The proposed method was successfully applied for measurement of the percentage per label of orphenadrine citrate in commercially available tablets.

Intracellular acidification delays hormonal G2/M transition and inhibits G2/M transition triggered by thiophosphorylated MAPK in Xenopus oocytes

Xenopus oocyte maturation is analogous to G2/M transition and characterized by germinal vesicle breakdown (GVBD), spindle formation, activation of MPF and Mos-Xp42(Mpk1) pathways. It is accompanied prior to GVBD by a transient increase in intracellular pH. We determined that a well known acidifying compound, NH(4)Cl, delayed progesterone-induced GVBD in a dose-dependent manner. GVBD(50) was delayed up to 2.3-fold by 10 mM NH(4)Cl. Cyclin B2 phosphorylation, Cdk1 Tyr15 dephosphorylation as well as p39(Mos) accumulation, Xp42(Mpk1) and p90(Rsk) phosphorylation induced by progesterone were also delayed by incubation of oocyte in NH(4)Cl. The delay induced by NH(4)Cl was prevented by injection of MOPS buffer pH 7.7. In contrast to acidifying medium, alkalyzing treatment such as Tris buffer pH 9 injections, accelerated GVBD, MPF and Xp42(Mpk1) activation, indicating that pHi changes control early steps of G2/M dynamics. When injected in an immature recipient oocyte, egg cytoplasm triggers GVBD through MPF auto-amplification, independently of protein synthesis. In these conditions, GVBD and Xp42(Mpk1) activation were delayed by high concentration of NH(4)Cl, which never prevented or delayed MPF activation. Strickingly, NH(4)Cl strongly inhibited thiophosphorylated active MAPK-induced GVBD and MPF activation. Nevertheless, Tris pH 9 did not have any effects on egg cytoplasm- or active MAPK-induced GVBD. Taken together, our results suggest that dynamic of early events driving Xp42(Mpk1) and MPF activation induced by progesterone may be negatively or positively regulated by pH(i) changes. However Xp42(Mpk1) pathway was inhibited by acidification alone. Finally, MPF auto-amplification loop was not sensitive to pH(i) changes.

In vitro synthesis of (13)---glucan (callose) and cellulose by detergent extracts of membranes from cell suspension cultures of hybrid aspen

The aim of this work was to optimize the conditions for in vitro synthesis of (1→3)-β-D-glucan (callose) and cellulose, using detergent extracts of membranes from hybrid aspen (Populus tremula × tremuloides) cells grown as suspension cultures. Callose was the only product synthesized when CHAPS extracts were used as a source of enzyme. The optimal reaction mixture for callose synthesis contained 100 mM Mops buffer pH 7.0, 1 mM UDP-glucose, 8 mM Ca2+, and 20 mM cellobiose. The use of digitonin to extract the membrane-bound proteins was required for cellulose synthesis. Yields as high as 50% of the total in vitro products were obtained when cells were harvested in the stationary phase of the growth curve, callose being the other product. The optimal mixture for cellulose synthesis consisted of 100 mM Mops buffer pH 7.0, 1 mM UDP-glucose, 1 mM Ca2+, 8 mM Mg2+, and 20 mM cellobiose. The in vitroβ-glucans were identified by hydrolysis of radioactive products, using specific enzymes. 13C-Nuclear magnetic resonance spectroscopy and transmission electron microscopy were also used for callose characterization. The (1→3)-β-D-glucan systematically had a microfibrillar morphology, but the size and organization of the microfibrils were affected by the nature of the detergent used for enzyme extraction. The discussion of the results is included in a short review of the field that also compares the data obtained with those available in the literature. The results presented show that the hybrid aspen is a promising model for in vitro studies on callose and cellulose synthesis.

Catalytic and DNA-Hydrolyzing Activities of Purified Immunoglobulins IgG from Patients Sera with Autoimmune Disease by Pseudobiospecific Chromatography Using Histidyl-Aminohexyl-Sepharose

Catalytic autoimmune antibodies from patients with antiphospholipid (aPL) antibodies were purified using histidyl-aminohexyl-sepharose gel. The sera were loaded on the columns equilibrated with 25 mM MOPS buffer pH 7.4 and the absorbed proteins were eluted by adding 0.2 M NaCl in the equilibrating buffer. Antibodies purity was evaluated by SDS-PAGE. The purified immunoglobulins G from patients with (aPL) sera by histidyl-aminohexyl-sepharose show DNA-degrading activity of the plasmid pUC19 DNA and catalytic activity in hydrolyzing the peptide substrate Pro-Phe-Arg-7-amido-4-methylcoumarin.

Experimental modulation of capsule size in Cryptococcus neoformans.

Experimental modulation of capsule size is an important technique for the study of the virulence of the encapsulated pathogen Cryptococcus neoformans. In this paper, we summarize the techniques available for experimental modulation of capsule size in this yeast and describe improved methods to induce capsule size changes. The response of the yeast to the various stimuli is highly dependent on the cryptococcal strain. A high CO2 atmosphere and a low iron concentration have been used classically to increase capsule size. Unfortunately, these stimuli are not reliable for inducing capsular enlargement in all strains. Recently we have identified new and simpler conditions for inducing capsule enlargement that consistently elicited this effect. Specifically, we noted that mammalian serum or diluted Sabouraud broth in MOPS buffer pH 7.3 efficiently induced capsule growth. Media that slowed the growth rate of the yeast correlated with an increase in capsule size. Finally, we summarize the most commonly used media that induce capsule growth in C. neoformans.

Synthesis, physicochemical properties and biological evaluation of ester prodrugs of 3-hydroxypyridin-4-ones: design of orally active chelators with clinical potential

The synthesis of a range of hydrophobic ester prodrugs of 3-hydroxypyridin-4-ones with potential for oral administration is described. The distribution coefficient values of a range of these ester prodrugs and the corresponding alcohols in 1-octanol and MOPS buffer pH 7.4 are presented together with their rates of hydrolysis at pH 2, pH 7.4, in rat blood and liver homogenate. In vivo iron mobilisation efficacy of the pivaloyl and benzoyl prodrugs has been compared with their parent drugs using a 59Fe-ferritin loaded rat model. Both classes of prodrug enhanced the ability of the parent hydroxypyridinone to facilitate the excretion of 59Fe. The influence of the pivaloyl function was more marked than that of the benzoyl function. The optimal effect was observed with 1-[2′-(pivaloyloxy)ethyl]-2-methyl-3-hydroxy-4(1 H)-pyridinone 25. However, not all the prodrugs provide increased efficacy which suggests that lipophilicity is not the only factor which influences the drug efficacy. The metabolism of the compound may have a dominating influence on the overall efficacy.

Synthesis, physicochemical properties and biological evaluation of aromatic ester prodrugs of 1-(2'-hydroxyethyl)-2-ethyl-3-hydroxypyridin-4-one (CP102): orally active iron chelators with clinical potential.

The synthesis of seven aromatic ester derivatives of 1-(2'-hydroxyethyl)-2-ethyl-3-hydroxypyridin-4-one is described. These ester prodrugs have been designed to target iron chelators to the liver, the major iron storage organ. In principle this should improve chelation efficacy and minimize toxicity. The distribution coefficients of these ester prodrugs between 1-octanol and MOPS buffer pH 7.4 were measured together with their rates of hydrolysis at pH 2 and pH 7.4, in rat blood and liver homogenate. Esters with heteroaromatic acid moieties were found to be less stable than benzoyl analogues. The in-vivo iron mobilisation efficacy of these ester prodrugs has been compared with that of the parent drug using a 59Fe-ferritin loaded rat model. Many prodrugs were found to enhance the ability of the parent hydroxypyridinone to facilitate 59Fe excretion. However, not all prodrugs provided increased efficacy, demonstrating that lipophilicity is not the only factor which influences drug efficacy. Furthermore, no clear correlation between efficacy and susceptibility to hydrolysis was detected. The picolinic and nicotinic ester derivatives appear to offer the best potential as prodrugs as they have a relatively low LogP value and yet lead to enhanced efficacy over the parent hydroxypyridinone.