Measurement of internal pH in the coccolithophoreEmiliania huxleyi using 2′,7′-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein acetoxymethylester and digital imaging microscopy

Internal pH (pH_i) was determined inEmiliania huxleyi (Lohmann) using the probe 2,7-bis-(2-carboxyethyl)-5(and-6)carboxyfluoresceinacetoxymethylester (BCEF-AM) and digital imaging microscopy. The probe BCECF-AM was taken up and hydrolysed to the free acid by the cells. A linear relationship was established between pH_i and the 490/450 fluorescence ratio of BCECF-AM over the pH range 6.0 to 8.0 using the ionophore nigericin. Two distinct pH domains were identified within the cell, the cytoplasmic domain (approx. pH 7.0) and the chloroplast domain (approx. pH 8.0). The average pH_i was 7.29 (±0.11) for cells in the presence of 2 mM HCO ₃ ^– . In the absence of HCO ₃ ^– the pH_i was decreased by 0.8 pH unit. The importance of these changes in pH_i is considered in relation to inorganic-carbon uptake.Abbreviations AM acetoxymethylester - BCECF 2,7-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - pH_i intracellular pH     

A new use of β-Ala-Lys (AMCA) as a transport reporter for PEPT1 and PEPT2 in renal brush border membrane vesicles from the outer cortex and outer medulla

Integral membrane proteins PEPT1 and PEPT2 are essential for reabsorbing almost all hydrolysed or filtered di- and tripeptides alongside a wide range of peptidomimetic drugs in the kidney. The aim of this study was to investigate the potential use of the fluorophore-conjugated dipeptide β-Ala-Lys (AMCA) as a biosensor for measuring peptide transport activity in brush border membrane vesicles isolated from the outer cortex (BBMV-OC) and outer medulla (BBMV-OM) (representing PEPT1 and PEPT2 respectively). The vesicles were isolated using a dual magnesium precipitation and centrifugation technique. Intravesicular fluorescence accumulation was measured after incubating extra-vesicular media at pH 6.6 and different concentrations of β-Ala-Lys (AMCA) with vesicles pre-equilibrated at pH 7.4. Both BBMV-OC and BMMV-OM showed accumulation of an intravesicular fluorescence signal after 20 min incubation. Changing the extra-vesicular pH to 7.4 caused a significant reduction in the β-Ala-Lys (AMCA) uptake into BBMV-OC at concentrations > 100 μM. When different concentrations of dipeptide, Gly-Gln was added, there was a significant inhibition of 100 μM β-Ala-Lys (AMCA) uptake into BBMV-OC and BMMV-OM, reaching 69% and 80%, respectively. Kinetic analysis of β-Ala-Lys (AMCA) at 20 min showed that the K_m and V_max were 783.7 ± 115.7 μM and 2191.2 ± 133.9 ΔF/min/mg for BBMV-OC, while BMMV-OM showed significantly higher affinity, but lower capacity at K_m = 93.6 ± 21.9 μM and V_max = 935.8 ± 50.2 ΔF/min/mg. These findings demonstrate the applicability of β-Ala-Lys (AMCA) as a biosensor to measure the transport activity of the renal-type PEPT1 and PEPT2 in BBMV-OC and BMMV-OM respectively.     

Polymorphism of Δ3,5-Δ2,4-dienoyl-coenzyme A isomerase (the ECH1 gene product protein) in human striated muscle tissue

Two polymorphic variants of the ECH1 gene product protein (delta3,5-delta2,4-dienoyl-coenzyme A isomerase) have been revealed by proteomics methods in samples of human striated muscle tissue. These variants are identical in molecular weight (29.7 kD) but different in pI values (6.57 and 6.75) and in amino acid substitution (41 E-->A) confirmed by mass spectrometry. The same type of polymorphism has been detected in samples of different tissues of the same person, so these variants are considered (also based on other data) to be allelic. The rates of these alleles in two representative cohorts of Moscow and Minsk residents are similar.     

The enzymic conversion of linoleic acid into 9-(nona-1′,3′-dienoxy)non-8-enoic acid, a novel unsaturated ether derivative isolated from homogenates of Solanum tuberosum tubers

1. A major component of the lipids in aqueous (pH7.5) homogenates of tuber tissue from Solanum tuberosum was isolated and characterized as 9-(nona-1',3'-dienoxy)non-8-enoic acid. 2. This novel unsaturated ether fatty acid derivative, which contains a butadienylvinyl ether function, has the structure: [Formula: see text] and is formed from linoleic acid by a sequence of enzymic reactions. 3. A precursor of the unsaturated ether derivative is 9-d-hydroperoxyoctadeca-10,12-dienoic acid, formed by the action of S. tuberosum lipoxygenase on linoleic acid. 4. An enzyme that converts the fatty acid hydroperoxide into the unsaturated ether derivative was isolated from S. tuberosum. The pH optimum of this enzyme is approx. 9, although the overall conversion of linoleic acid into the ether derivative is maximal at pH7.5. 5. An unusual feature of this pathway is the insertion of an oxygen atom into the alkyl chain of a fatty acid. 6. This novel mechanism may play a role in the breakdown of polyunsaturated fatty acids to volatile products in plants.     

Conversion of allyl 2-acetamido-2-deoxy-β-d-glucopyranoside into 2-methyl-(3,4,6-tri-O-benzoyl-1,2-dideoxy-α-d- galactopyrano)-[2′,1′:4,5]-2-oxazoline

2-Methyl-(3,4,6-tri-O-benzoyl-1,2-dideoxy-α-d-galactopyrano)-[2′,1′:4,5]-2-oxazoline (7) was prepared from 1-propenyl 2-acetamido-3,4,6-tri-O-benzoyl-2- deoxy-β-d-galactopyranoside (6). The latter was prepared from allyl 2-acetamido-2-deoxy-β-d-glucopyranoside (1) through selective benzoylation at O-3 and O-6, conversion into the 4-p-bromobenzenesulfonate 4, inversion of configuration at C-4 to afford allyl 2-acetamido-3,4,6-tri-O-benzoyl-β-d-galactopyranoside (5), and subsequent isomerization with palladium-charcoal to give 6.     

Dinuclear platinum complexes with<Emphasis Type="Italic"> N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>′-bis(aminoalkyl)-1,4-diaminoanthraquinones as linking ligands. Part II. Cellular processing in A2780 cisplatin-resistant human ovarian carcinoma cells: new insights into the mechanism of resistance

The cellular processing of three fluorescent N,N-bis(aminoalkyl)-1,4-diaminoanthraquinones (aminoalkyl=2-aminoethyl, 3-aminoprop-1-yl or 4-aminobut-1-yl) and their dinuclear platinum complexes in A2780 human ovarian carcinoma cells with acquired resistance to cisplatin has been monitored over time by time-lapse fluorescence microscopy. The results were compared with the previously reported observations in the parent A2780 cell line. The cellular distribution pattern for the free ligands is similar in sensitive and resistant cells, whereas significant differences in cellular distribution were observed in the case of the platinum complexes. In the cisplatin-resistant cell line the platinum complexes were found to be sequestrated in acidic vesicles in the cytosol from the very beginning of the incubation. This sequestration was not observed in the case of sensitive cells. Platinum accumulation in vesicles possibly presents a mechanism of resistance to platinum complexes. This mechanism appears to be unrelated to the mechanism of deactivation of platinum compounds by glutathione. Encapsulation of the dinuclear platinum complexes in lysosomal vesicles provides a plausible explanation for the decreased activity of these compounds in the resistant cell line, as compared to the sensitive cell line.Abbreviations AQ2 N,N-bis(2-aminoethyl)-1,4-diaminoanthracene-9,10-dione - AQ3 N,N-bis(3-aminoprop-1-yl)-1,4-diaminoanthracene-9,10-dione - AQ4 N,N-bis(4-aminobut-1-yl)-1,4-diaminoanthracene-9,10-dione - l-BSO l-buthionine-S,R-sulfoximine - dien diethylenetriamine - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide - PAQ2 [{trans-PtCl(NH₃)₂}₂(-AQ2)](NO₃)₂ - PAQ3 [{trans-PtCl(NH₃)₂}₂(-AQ3)](NO₃)₂ - PAQ4 [{trans-PtCl(NH₃)₂}₂(-AQ4)](NO₃)₂ - PAM [{Pt(dien)}₂(-AQ2)](NO₃)₄ - PBS phosphate buffered saline