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PubMed1985

Phosphorylation of glycophorin A in membranes of intact human erythrocytes.

The qualitative and quantitative contribution of glycophorin A phosphorylation to the general and specific pattern of membrane protein phosphorylation in intact erythrocytes pre-incubated with 32Pi was examined. Intense 32P-labeled bands at 88,000 and 38,000 Mr were identified as phosphorylated glycophorin A dimer and monomer respectively on the basis of several criteria. Quantitatively, phosphorylated glycophorin A dimer accounted for about 70% of 32P in the band 3 region. This value is at least three times that previously reported. The results of ancillary experiments involving selective extraction of ghosts in acidified chloroform/methanol solvents and electrophoresis in the presence of detergents make it unlikely that the 32P associated with glycophorin A was due to bound polyphosphoinositides.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

Isometric force redevelopment of skinned muscle fibers from rabbit activated with and without Ca2+.

Fiber isometric tension redevelopment rate (kTR) was measured during submaximal and maximal activations in glycerinated fibers from rabbit psoas muscle. In fibers either containing endogenous skeletal troponin C (sTnC) or reconstituted with either purified cardiac troponin C (cTnC) or sTnC, graded activation was achieved by varying [Ca2+]. Some fibers were first partially, then fully, reconstituted with a modified form of cTnC (aTnC) that enables active force generation and shortening in the absence of Ca2+. kTR was derived from the half-time of tension redevelopment. In control fibers with endogenous sTnC, kTR increased nonlinearly with [Ca2+], and maximal kTR was 15.3 +/- 3.6 s-1 (mean +/- SD; n = 26 determinations on 25 fibers) at pCa 4.0. During submaximal activations by Ca2+, kTR in cTnC reconstituted fibers was approximately threefold faster than control, despite the lower (60%) maximum Ca(2+)-activated force after reconstitution. To obtain submaximal force with aTnC, eight fibers were treated to fully extract endogenous sTnC, then reconstituted with a mixture of a TnC and cTnC (aTnC:cTnC molar ratio 1:8.5). A second extraction selectively removed cTnC. In such fibers containing aTnC only, neither force nor kTR was affected by changes in [Ca2+]. Force was 22 +/- 7% of maximum control (mean +/- SD; n = 15) at pCa 9.2 vs. 24 +/- 8% (mean +/- SD; n = 8) at pCa 4.0, whereas kTR was 98 +/- 14% of maximum control (mean +/- SD; n = 15) at pCa 9.2 vs. 96 +/- 15% (mean +/- SD; n = 8) at pCa 4.0. Maximal reconstitution of fibers with aTnC alone increased force at pCa 9.2 to 69 +/- 5% of maximum control (mean + SD; n = 22 determinations on 13 fibers) and caused a small but significant reduction of kTR to 78 +/- 8% of maximum control (mean +/- SD; n = 22 determinations on 13 fibers); neither force nor krR was significantly affected by Ca>2(pCa 4.0). Taken together, we interpret our results to indicate that kTR reflects the dynamics of activation of individual thin filament regulatory units and that modulation of kTR by Ca> is effected primarily by Ca>+ binding to TnC.

Animals↗
PubMed2004

Subunit A of the E. coli ATP synthase: reconstitution and high resolution NMR with protein purified in a mixed polarity solvent.

Subunit a of the Escherichia coli ATP synthase, a 30 kDa integral membrane protein, was purified to homogeneity by a novel procedure incorporating selective extraction into a monophasic mixture of chloroform, methanol and water, followed by Ni-NTA chromatography in the mixed solvent. Pure subunit a was reconstituted with subunits b and c and phospholipids to form a functional proton-translocating unit. Nuclear magnetic resonance (NMR) spectra of the pure subunit a in the mixed solvent show good chemical shift dispersion and demonstrate the potential of the solvent mixture for NMR studies of the large membrane proteins that are currently intractable in aqueous detergent solutions.

Amino Acid Sequence↗
PubMed1998

A novel centromere monospecific serum to a human autoepitope on the histone H3-like protein CENP-A.

Centromere autoantibodies are commonly found in the serum of patients with some systemic autoimmune diseases. Previous studies have shown that a major human centromere autoantigen is the histone H3-like protein CENP-A. Although the human cDNA has been cloned, native CENP-A has been neither isolated nor expressed in Escherichia coli, and specific antibodies to this chromatin-associated centromere protein are not available yet. In this report, a highly charged peptide on CENP-A (residues 3-17) was used to generate a monospecific antibody that reacts by immunoblots with the 17 kDa centromeric protein. Immunofluorescence analysis showed reactivity of this anti-CENP-A serum in several but not all mammalian culture cells analyzed, suggesting that the sequence of this histone-like centromere protein could be more variable throughout evolution than originally thought. Selective extractions of human placenta nuclear proteins and immunoblot analysis indicated that CENP-A behaves in a similar way to the core histone polypeptides after nuclease digestion of chromatin. Also, immunoblot analysis demonstrated that the CENP-A peptide used as immunogen is a target region on the CENP-A molecule in several but not all CREST patients analyzed with high titers of autoantibodies to the centromere. Lastly, we found that in Jurkat cells induced to apoptosis, CENP-A remains associated with the centromere, in contrast to other human autoantigens studied during apoptosis.

Amino Acid Sequence↗
PubMed2000

Simultaneous determination of acidic and non-acidic pesticides in natural waters by liquid chromatography-mass spectrometry.

There is increasing interest and demand for real multi-residue methods able to simultaneously determine pesticides with a broad spectrum of chemical characteristics in environmental and biological matrices. A method based on solid-phase extraction with a Carbograph 4 cartridge and liquid chromatography with electrospray mass spectrometry (LC-ES-MS) enabling simultaneous determination of non-acidic and acidic pesticides in real water samples is described. On repeatedly (n=5) extracting 4 l of drinking water (spike level 50 ng/l), 2 l of ground water (spike level 100 ng/l) and 1 l of river water (spike level 200 ng/l), recovery of 26 base/neutral pesticides and 13 acidic pesticides were equal to or better than 80%, except for carbendazim (67%), butocarboxim (73%), aldicarb (75%) and molinate (77%). Relative standard deviations ranged between 4 and 15%. Final extracts containing acidic and non-acidic pesticides were analyzed in a single chromatographic run while the ES-MS system was operated in both positive and negative ion modes. With the aim of finding the best operating conditions, in terms of sensitivity, the pH of the LC eluent was varied in the 2.9-8.4 range. Altogether, the best results were obtained by using an LC eluent containing 1 mmol/l formic acid. Over the entire pH range considered, well shaped peaks for both basic and acidic analytes were achieved by the use of a new generation LC column. By extracting selected ion current profiles from the total ion current mass chromatogram relative to analysis of 4 l of drinking water spiked with 50 ng/l of each of the 39 analytes, estimated limits of detection ranged between 0.05 and 1.5 ng/l, except for propyzamide (8 ng/l) and 2,4-DB (3 ng/l).

Acids↗
PubMed2000

Fractionation of apple procyanidins according to their degree of polymerization by normal-phase high-performance liquid chromatography.

A new method was developed for the fractionation of procyanidin oligomers according to their degree of polymerization. Monomeric flavan-3-ols and low molecular mass procyanidins were selectively extracted from the lyophilized powder of apple condensed tannins (ACTs) by methyl acetate extraction. Sequentially, the separation of each oligomer from dimer to pentamer in this extract was carried out by normal-phase high-performance liquid chromatography using a silica-beads packed column. The best separation was achieved with a mobile phase system containing hexane; (1) hexane-methanol-ethyl acetate, (2) hexane-acetone. These sequential treatments can be easily adapted to large-scale fractionation.

Biflavonoids↗
PubMed1982

Determination of vitamin D3 in cod-liver oil by high-performance liquid chromatography.

The present British Pharmacopoeia monograph for cod-liver oil requires a bioassay for the vitamin D3 content which is both time-consuming and complex. Alternative assays employing chromatographic procedures have been described but all these involve prior saponification of the oil. A selective extraction for vitamin D3 without the need for saponification is reported in this paper. The extraction utilizes only chromatographic assay using argentation on reversed-phase silica, with vitamin D2 as the internal standard. Reproducibility of injection gave a coefficient of variation of 0.6%, and repeatability of extraction for six samples gave a coefficient of variation of 6.8%.

Animals↗
PubMed1976

Specific ion-exchange chromatography and fluorimetric assay for urinary 3-O-methyldopamine.

A technique for the selective extraction of 3-O-methyldopamine, normetanephrine and metanephrine from a single urine sample has been investigated. After hydrolysis of the conjugates, the diluted mixture is passed through a Dowex 50W-X2 column and the methoxylated amines are eluted by means of concentrated ammonia. The eluate, containing metanephrine, normetanephrine and 3-O-methyldopamine is evaporated, and a solution of the residue in borate buffer is fractionated under strictly controlled conditions on an Amberlite CG-50 column. The three amines so separated are estimated by specific fluorimetric methods. The extraction recovery is 80 +/- 3% for pure solutions and 78 +/- 4% for 3-O-methyldopamine added to urine. The fluorimetric procedure, carried out under well-defined conditions, allows the estimation of 10 ng of 3-O-methethyldopamine. The spectral characteristics of the fluorescent derivative are similar to those obtained with dopamine, so that it can be assumed that iodine oxidation of 3-O-methyldopamine demethylates this compound and oxidises the resulting dopamine to the dopamine fluorophore (5,6-dihydroxy-indole). Of the compounds that might interfere in the fluorimetric procedure, dopamine, DOPA and alpha-methyl-DOPA are destroyed by the ammoniacal elution from the Dowex column and 3-O-methyl-DOPA is eliminated in the effluent from the Amberlite column. The elimination of interfering compounds and the improved separation on Amberlite ensure high specificity for this procedure. We have applied the method to normal urine and to pathological urines from patients with adrenergic tumours or untreated and treated parkinsonian subjects; vital information has been obtained on the prognosis of adrenergic tumours. The presence of large amounts of dopamine, normetanephrine and/or metanephrine does not affect the assay for 3-O-methyldopamine. The method is also applicable to rat and dog urine, and can be applied to tissue extracts with little modification.

Adrenal Gland Neoplasms↗
PubMed1983

Quantitative analysis of minaprine and some of its metabolites with application to kinetic studies in rats.

A simple and rapid high-performance liquid chromatographic method is described for the quantitative analysis of the psychotropic drug minaprine and three of its metabolites (M1, M3 and M11), including one as yet undetected metabolite (M11) known as a monoamine oxidase type A inhibitor in vitro. After selective extraction all four compounds were separated on a reversed-phase muBondapak C18 column using sodium acetate (0.03 M)-acetonitrile-methanol (88:7:5) (pH 3.3) as the mobile phase. The eluted compounds were detected with a UV detector at 254 nm. The sensitivity of the method is 0.02 microgram per millilitre of body fluid or per gram of tissue for M1 and M11 and 0.05 microgram per minaprine and M3. The method has been applied successfully to the determination of minaprine and the metabolites in plasma and brain and is compared here with an gas-liquid chromatographic method with an electron-capture detector previously developed for the detection of minaprine and M11. M11 was identified in rat urine by gas chromatography-mass spectrometry.

Animals↗
PubMed1991

High-performance liquid chromatographic evaluation of Med 15 and its metabolites Med 5 and tolmetin in rat plasma.

A simple and reliable high-performance liquid chromatographic method is described for the quantitative analysis of the new non-steroidal anti-inflammatory agent Med 15 and its metabolites Med 5 and tolmetin in rat plasma. After selective extraction the three analytes and an internal standard (p-phenyl-phenol) were separated on a reversed-phase Ultrasphere 5 micron column using potassium dihydrogenphosphate (0.05 M)-acetonitrile (52:48) (pH 4.7) as the mobile phase. The analytes were detected at 313 nm; the sensitivity of the method proved to be 0.05 microgram/ml for all three compounds. The method has been applied to investigate Med 15 pharmacokinetics in rats.

Animals↗
PubMed2003

Equilibrium partition of polycyclic aromatic hydrocarbons in a cloud-point extraction process.

A cloud-point extraction (CPE) process using the nonionic surfactant Tergitol 15-S-7, a secondary ethoxylated alcohol, to extract selected polycyclic aromatic hydrocarbons (PAHs) from aqueous solutions is investigated. The CPE process is facilitated at the ambient temperature, ca. 22 degrees C, by the reduction of the cloud-point temperature of the surfactant solution by addition of sodium sulfate. It is observed that the preconcentration factor could be enhanced either by increasing the salt concentration or by decreasing the initial surfactant concentration in the micellar solution. A high preconcentration factor of about 40 was achieved at 1 wt% surfactant concentration with the addition of 0.6 M Na(2)SO(4). It is also noted that the equilibrium partition coefficients of the model PAHs are nearly independent of surfactant concentrations, up to 3 wt%, in this study. Correlations between the equilibrium partition coefficients K(p) of the PAHs and their octanol-water partition coefficients K(ow), as well as K(p) and the molar volume V(x) of these PAHs, indicate that the partition processes of the PAHs in the CPE processes are mainly governed by their hydrophobic affinities to the surfactant aggregates. Furthermore, the effect of added Na(2)SO(4) on the equilibrium partition coefficients is also studied. It is shown that addition of more Na(2)SO(4) to the surfactant solution gives more partition of the PAHs into the surfactant-rich phase.

Journal Article↗
PubMed2000

The regulation of prostaglandin and arachidonoyl-CoA formation from arachidonic acid in rabbit kidney medulla microsomes by palmitoyl-CoA.

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). In the present study, we investigated the effects of palmitic acid (PA) and palmitoyl-CoA (PA-CoA) on the PG and AA-CoA formation from high and low concentrations of AA (60 and 5 microM) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 60 or 5 microM [14C]-AA in 0.1 M-Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl2 and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. PA (10-100 microM) had no effect on the PG and AA-CoA formation from either 60 or 5 microM AA. PA-CoA (10-100 microM) was without effect on the PG and AA-CoA formation from 60 microM AA, whereas it markedly decreased the PG formation (6-40%) and increased the AA-CoA formation (1.1-2.3-fold) from 5 microM AA, showing that the effects of PA-CoA on the PG and AA-CoA formation change depending on the AA concentration. These results suggest that PA-CoA, but not PA, may regulate the PG and AA-CoA formation at low substrate concentrations (close to the physiological concentration of AA), and that this in-vitro method using 5 microM AA may be useful for clarifying the homeostatic control of the metabolic fate of AA into these two enzymatic pathways.

Acyl Coenzyme A↗
PubMed2001

Partitioning of trace metals before and after biological removal of metals from sediments.

Metal removal by biological solubilization in three strongly contaminated sediments was carried out in a two-liter stirred bioreactor. Biological treatment yielded metal removal efficiencies in the range of 11-30%, 43-57%, 60-79%, 61-90%, 18-21%, 0-10% for Pb, Cu, Zn, Cd, Ni and Cr, respectively. The treated sediments were then rinsed with a NaCl solution (0.5 M), resulting in an increase by nearly 47% in Pb removal for the three sediments, while for other metals (Cu, Zn, Cd, Ni, Cr), the NaCl rinse did not seem to allow any significant increase in metal solubilization. A standard procedure of sequential selective extraction (SSE) was applied to the sediments before and after each treatment. With regard to Pb, Zn and Cd, the carbonate bound fractions (2/3 sediments) represented 18-42% of metals prior to treatment, while the iron and manganese oxides bound fraction constituted 39-60% of metals for the three sediments. Between 90 and 100% of Pb, Zn and Cd removed by the process came from the fractions bound to carbonates and from those bound to Fe and Mn oxides. The organic matter and sulfide bound fractions contained 65-72% of total Cu present before treatment and the process removed, on average, 63% Cu present in this fraction. In contrast, Ni and Cr were found mainly in the residual fractions (50-80%). Finally, this biological treatment did not solubilize Cr appreciably, while removal of Ni mostly originated from the carbonate and Fe/Mn oxides fractions (70-80%).

Carbonates↗
PubMed2003

Heavy metals extraction by microemulsions.

The objective of this study is the heavy metal extraction by microemulsion, using regional vegetable oils as surfactants. Firstly, the main parameters, which have influence in the microemulsion region, such as: nature of cosurfactant, influence of cosurfactant (C)/surfactant (S) ratio and salinity were studied, with the objective of choosing the best extraction system. The extraction/reextraction process by microemulsion consists of two stages. In the first one, the heavy metal ion present in the aqueous phase is extracted by the microemulsion. In a second step, the reextraction process occurs: the microemulsion phase, rich in metal, is acidified and the metal is recovered in a new aqueous phase, with higher concentration. The used system had the following parameters: surfactant-saponified coconut oil; cosurfactant-n-butanol; oil phase-kerosene; C/S ratio=4; salinity-2% (NaCl); temperature of 27+/-1 degrees C; water phase-aqueous solution that varied according to the heavy metal in study (Cr, Cu, Fe, Mn, Ni and Pb). A methodology of experimental planning was used (Scheffé Net) to study the behavior of the extraction in a chosen domain. The extraction was accomplished in one step and yielded extraction percentage higher than 98% for all metals. In the reextraction HCl-8M was used as reextraction agent and the influence of the pH and time were verified. This work showed the great efficiency of the microemulsion, indicating that it is possible to extract selectively the heavy metals from the aqueous phase.

Emulsions↗
PubMed2003

Development of a bioluminescent ATP assay to quantify mammalian and bacterial cell number from a mixed population.

Modification of biomaterial surfaces is one approach aimed at improving cellular interactions and the subsequent integration of medical devices into the body. However, by optimising surfaces to enhance mammalian cell adhesion there is the potential risk that adhesion of bacteria will also be increased. Bacterial colonisation of biomaterials can be problematic as infection often results in morbidity and the consequent removal of the failing device from the body. Currently, quantifying cellular adhesion from a mixed population of bacterial and mammalian cells can only be determined by slow and laborious methods such as microscopy. ATP is a key molecule in the metabolism of both mammalian and bacterial cells and can be used to quantify cell numbers. In this study, we have modified a bioluminescence-based ATP assay to enable the differential measurement of both mammalian and bacterial cell ATP levels within the same culture. Mixed populations of Staphylococcus epidermidis and 3T3 fibroblasts were assessed both in suspension and adhered to a surface. ATP levels from cultures in suspension were selectively extracted and measured from both cell types, revealing a linear trend that would enable the differentiation of cell numbers from a mixed population. The application of the assay to adhered mixed cultures also allowed differences in ATP levels from both cell types to be distinguished. The data presented reveals that this assay would be useful for the rapid screening of cellular adhesion to modified surfaces although, its use in detecting subtle differences in ATP levels may be limited due to natural interactions between the two cell types.

3T3 Cells↗
PubMed2001

Factors controlling metal distributions in the surface sediments of the Erdek Bay, Sea of Marmara, Turkey.

A geochemical study of the Erdek Bay sediments in the Sea of Marmara, Turkey, was carried out to investigate the heavy metal distributions and sources. The bay has an average depth of 34 m and a two-layer water column stratification, with a pycnocline at 20 m. The distribution of "total" metal (Fe, Mn, Pb, Cu, Ni, Cr, Zn and Hg) concentrations, together with sequential selective extraction analyses, shows that the relatively high metal concentrations are mainly due to land-based natural inputs from the erosion products of mineralized zones and mafic-ultramafic rocks delivered to the bay by two rivers from the south. However, some Pb, Zn and Cu occur in the Fe-Mn-oxyhydroxide and organic fractions, suggesting, in part, some anthropogenic inputs in addition to the predominantly natural inputs. The vertical distribution of metal/A1 ratios in a gravity core in the bay shows no significant change with depth, except for an early diagenetic enrichment at 0.7-1.6 m below sea floor (mbsf) above a 4750-3500-year-old sapropelic layer. This distribution is a further indication of the insignificant antropogenic metal pollution in the bay.

Environmental Monitoring↗
PubMed2003

Structure of the skin barrier and its modulation by vesicular formulations.

The natural function of the skin is to protect the body from unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. Since the lipids regions in the stratum corneum form the only continuous structure, substances applied onto the skin always have to pass these regions. For this reason the organization in the lipid domains is considered to be very important for the skin barrier function. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid phase behavior is different from that of other biological membranes. In stratum corneum crystalline phases are predominantly present, but most probably a subpopulation of lipids forms a liquid phase. Both the crystalline nature and the presence of a 13 nm lamellar phase are considered to be crucial for the skin barrier function. Since it is impossible to selectively extract individual lipid classes from the stratum corneum, the lipid organization has been studied in vitro using isolated lipid mixtures. These studies revealed that mixtures prepared with isolated stratum corneum lipids mimic to a high extent stratum corneum lipid phase behavior. This indicates that proteins do not play an important role in the stratum corneum lipid phase behavior. Furthermore, it was noticed that mixtures prepared only with ceramides and cholesterol already form the 13 nm lamellar phase. In the presence of free fatty acids the lattice density of the structure increases. In stratum corneum the ceramide fraction consists of various ceramide subclasses and the formation of the 13 nm lamellar phase is also affected by the ceramide composition. Particularly the presence of ceramide 1 is crucial. Based on these findings a molecular model has recently been proposed for the organization of the 13 nm lamellar phase, referred to as "the sandwich model", in which crystalline and liquid domains coexist. The major problem for topical drug delivery is the low diffusion rate of drugs across the stratum corneum. Therefore, several methods have been assessed to increase the permeation rate of drugs temporarily and locally. One of the approaches is the application of drugs in formulations containing vesicles. In order to unravel the mechanisms involved in increasing the drug transport across the skin, information on the effect of vesicles on drug permeation rate, the permeation pathway and perturbations of the skin ultrastructure is of importance. In the second part of this paper the possible interactions between vesicles and skin are described, focusing on differences between the effects of gel-state vesicles, liquid-state vesicles and elastic vesicles.

Administration, Cutaneous↗
PubMed1997

An alternative purification protocol for producing hepatitis B virus X antigen on a preparative scale in Escherichia coli.

A truncated variant of the hepatitis B virus X gene (HBx) was cloned into the fusion expression vector of pGEX-3X (Pharmacia), resulting in a GST-HBx fusion gene construction (pGEX-3XXBF). This plasmid was transformed into and expressed by the Escherichia coli strain DH5. More than 80% of the expressed fusion protein was found in the insoluble fraction (inclusion body) of the cell lysate. The fusion protein was selectively extracted from the inclusion bodies with 8 M urea at pH 6.5, and it was refolded by diluting 3-fold with deionized distilled water at 4 degrees C. The in vitro cleavage of the refolded fusion protein by factor Xa at about 2-3 mg ml-1 in the presence of 2.66 M urea at pH 6.5 was complete. The final steps of purification involved precipitation of the cleaved proteins with ammonium sulphate, solubilization in guanidine hydrochloride and separation on a Superdex 75 FPLC column. With this approach, following an inclusion body strategy and a beneficial in vitro refolding, a predominantly hydrophobic and highly disulphide-bonded protein was produced in preparative scale for subsequent diagnostic use.

Amino Acid Sequence↗