The effect of D-penicillamine on myeloperoxidase: formation of compound III and inhibition of the chlorinating activity

The inhibitory effect of the anti-arthritic drug D-penicillamine on the formation of hypochlorite (HOCl) by myeloperoxidase from H2O2 and Cl- was investigated. When D-penicillamine was added to myeloperoxidase under turnover conditions, Compound III was formed, the superoxide derivative of the enzyme. Compound III was not formed when D-penicillamine was added in the presence of EDTA or in the absence of oxygen. However, when H2O2 was added to myeloperoxidase, D-penicillamine and EDTA, Compound III was formed. Therefore it is concluded that formation of Compound III is initiated by metal-catalysed oxidation of the thiol group of this anti-arthritic drug, resulting in formation of superoxide anions. Once Compound III is formed, a chain reaction is started via which the thiol groups of other D-penicillamine molecules are oxidized to disulphides. Concomitantly, Compound I of myeloperoxidase would be reduced to Compound II and superoxide anions would be generated from oxygen. This conclusion is supported by experiments which showed that formation of Compound III of myeloperoxidase by D-penicillamine depended on the chloride concentration. Thus, an enzyme intermediate which is active in chlorination (i.e. Compound I) participated in the generation of superoxide anions from the anti-arthritic drug. From the results described in this paper it is proposed that D-penicillamine may exert its therapeutic effect in the treatment of rheumatoid arthritis by scavenging HOCl and by converting myeloperoxidase to Compound III, which is inactive in the formation of HOCl.     

Maintenance of transposable element copy number in natural populations of Drosophila melanogaster and D. simulans

To investigate the main forces controlling the containment of transposable elements (TE) in natural populations, we analyzed the copia, mdg1, and 412 elements in various populations of Drosophila melanogaster and D. simulans. A lower proportion of insertion sites on the X chromosome in comparison with the autosomes suggests that selection against the detrimental effects of TE insertions is the major force containing TE copies in populations of Drosophila. This selection effect hypothesis is strengthened by the absence of the negative correlation between recombination rate and TE copy number along the chromosomes, which was expected under the alternative ectopic exchange model (selection against the deleterious rearrangements promoted by recombination between TE insertions). A cline in 412 copy number in relation to latitude was observed among the natural populations of D. simulans, with very high numbers existing in some local populations (around 60 copies in a sample from Canberra, Australia). An apparent absence of selection effects in this Canberra sample and a value of transposition rate equal to 1–2 × 10^-3 whatever the population and its copy number agree with the idea of recent but temporarily drastic TE movements in local populations. The high values of transposition rate in D. simulans clearly disfavor the hypothesis that the low amount of transposable elements in this species could result from a low transposition rate. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Metal-ligand vibrations of cyanoferric myeloperoxidase and cyanoferric horseradish peroxidase: evidence for a constrained heme pocket in myeloperoxidase

The low-frequency FeCN vibrations of cyanoferric myeloperoxidase (MPO) and horseradish peroxidase (HRP) have been measured by resonance Raman spectroscopy. The ordering of the frequencies of the predominantly FeC stretching and FeCN bending normal vibrational modes in the two peroxidases differs. These normal mode vibrations are identified by their wavenumber shifts upon isotopic substitution of the cyanide ligand. For MPO, the stretching mode nu 1 (361 cm-1) occurs at a lower frequency than the bending mode delta 2 (454 cm-1). For HRP, the order is reversed as nu 1 (456 cm-1) is at a higher frequency than delta 2 (404 cm-1). Normal coordinate analyses and model complexes have been used to address the origin of this behavior. The nu 1 stretching frequencies in cyanide complexes of iron porphyrin and iron chlorin model compounds are similar to one another and to that of HRP. Thus, the inverted order and altered frequencies of the nu 1 and delta 2 vibrations in MPO, relative to those in HRP and the model compounds, are not inherent to the proposed iron chlorin prosthetic group in MPO but, rather, are attributed to distinct distal environmental effects in the MPO active site. The normal coordinate analyses for MPO and HRP showed that the nu 1 and delta 2 vibrational frequencies are not pure; the potential energy distributions for these modes respond not only to the geometry but also to the force constants of the nu(FeC) and delta(FeCN) internal coordinates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.