Escherichia coli, Bacillus megaterium, and three species of yersiniae grew rapidly without significant production of soluble siderophores in a defined iron-sufficient medium (20 microM Fe3+). In iron-deficient medium (0.1 to 0.3 microM Fe3+) all organisms showed reduced growth, and there was extensive production of siderophores by E. coli and B. megaterium. Release of soluble siderophores by Yersinia pestis, Y. pseudotuberculosis, or Y. enterocolitica in this medium was not detected. Citrate (1 mM) inhibited growth of yersiniae in iron-deficient medium, indicating that the organisms lack an inducible Fe3+-citrate transport mechanism. Uptake of 59Fe3+ by all yersiniae was an energy-dependent saturable process, showing increased accumulation after adaptation to iron-deficient medium. Growth of Y. pseudotuberculosis and Y. enterocolitica but not Y. pestis on iron-limited solid medium was enhanced to varying degrees by exogenous siderophores (desferal, schizokinen, aerobactin, and enterochelin). Only hemin (0.1 pmol) or a combination of inorganic iron plus protoporphyrin IX promoted growth of Y. pestis on agar rendered highly iron deficient with egg white conalbumin (10 microM). Growth of Y. pseudotuberculosis and Y. enterocolitica was stimulated on this medium by Fe3+ or hemin. These results indicate that hemin can serve as a sole source of iron for yersiniae and that the organisms possess an efficient cell-bound transport system for Fe3+. (+info)
Ferriporphyrins and endothelium: a 2-edged sword-promotion of oxidation and induction of cytoprotectants.
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Heme arginate infusions blunt the symptoms of patients with acute intermittent porphyria without evidence of the vascular or thrombotic side effects reported for hematin. To provide a rationale for heme arginate's safety, the present study examined the effects of various ferriporphyrins to sensitize human endothelial cells to free radical injury and to induce heme oxygenase and ferritin expression. Heme arginate, unlike hematin, did not amplify oxidant-induced cytotoxicity mediated by hydrogen peroxide (5.3 +/- 2.4 versus 62.3 +/- 5.3% (51)Cr release, P <.0001) or by activated neutrophils (14.4 +/- 2.9 versus 41.1 +/- 6.0%, P <.0001). Nevertheless, heme arginate efficiently entered endothelial cells similarly to hematin, since both markedly induced heme oxygenase mRNA (more than 20-fold increase) and enzyme activity. Even with efficient permeation, endothelial cell ferritin content was only minimally increased by heme arginate compared with a 10-fold induction by hematin; presumably less free iron was derived from heme arginate despite up-regulation of heme oxygenase. Hematin is potentially vasculopathic by its marked catalysis of oxidation of low-density lipoprotein (LDL) to endothelial-toxic moieties. Heme arginate was significantly less catalytic. Heme arginate-conditioned LDL was less than half as cytotoxic to endothelial cells as hematin-conditioned LDL (P <.004). It is concluded that heme arginate may be less vasculotoxic than hematin since it is an effective heme oxygenase gene regulator but a less efficient free-radical catalyst. (+info)
Characterization of chloroquine-hematin mu-oxo dimer binding by isothermal titration calorimetry.
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Numerous studies indicate that a key feature of chloroquine's (CQ) antimalarial activity is its interaction with hematin. We now characterize this CQ-hematin interaction in detail using isothermal titration calorimetry (ITC). Between pH 5.6 and 9.0, association constants (K(a) values) for enthalpy-driven CQ-hematin mu-oxo dimer binding fell in the narrow range of 2.3-4.4 x 10(5) M(-1). It is therefore probable that CQ-hematin mu-oxo dimer binding affinity does not diminish at the pH range (4.8-5.4) of the parasite food vacuole. The binding affinity was unaffected by high salt concentrations, suggesting that ionic interactions do not contribute significantly to this complexation. With increasing ionic strength, the entropic penalty of CQ-hematin mu-oxo dimer binding decreased accompanied by increased hematin mu-oxo dimer aggregation. A stoichiometry (n) of 1:4 in the pH range 6.5-9.0 indicates that CQ binds to two hematin mu-oxo dimers. At pH 5.6, a stoichiometry of 1:8 suggests that CQ binds to an aggregate of four hematin mu-oxo dimers. This work adds further evidence supporting the hypothesis that CQ impedes hematin monomer incorporation into hemozoin by producing a forward shift in the hematin monomer-hematin mu-oxo dimer equilibrium, contributing to a destructive accumulation of soluble forms of hematin in the parasite and leading to its death by hematin poisoning. (+info)
Ferritin from the obligate anaerobe Porphyromonas gingivalis: purification, gene cloning and mutant studies.
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Porphyromonas gingivalis is an obligate anaerobe that utilizes haem, transferrin and haemoglobin efficiently as sources of iron for growth, and has the ability to store haem on its cell surface, resulting in black pigmentation of colonies on blood agar plates. However, little is known about intracellular iron storage in this organism. Ferritin is one of the intracellular iron-storage proteins and may also contribute to the protection of organisms against oxidative stresses generated by intracellular free iron. A ferritin-like protein was purified from P. gingivalis and the encoding gene (ftn) was cloned from chromosomal DNA using information on its amino-terminal amino acid sequence. Comparison of the amino acid sequence deduced from the nucleotide sequence of ftn with those of known ferritins and bacterioferritins identified the protein as a ferritin and positioned it between proteins from the Proteobacteria and Thermotogales. The P. gingivalis ferritin was found to contain non-haem iron, thus confirming its identity. Construction and characterization of a P. gingivalis ferritin-deficient mutant revealed that the ferritin was particularly important for the bacterium to survive under iron-depleted conditions (both haemin and transferrin starvation), indicating that intracellular iron is stored in ferritin regardless of the iron source and that the iron stored in ferritin is utilized under iron-restricted conditions. However, the ferritin appeared not to contribute to protection against oxidative stresses caused by peroxides and atmospheric oxygen. (+info)
Ferryl-oxo heme intermediate in the antimalarial mode of action of artemisinin.
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Fourier transform infrared (FTIR) and resonance Raman (RR) spectroscopies have been employed to investigate the reductive cleavage of the O-O bond of the endoperoxide moiety of the antimalarial drug artemisinin and its analog trioxane alcohol by hemin dimer. We have recorded FTIR spectra in the nu(O-O) and nu(as)(Fe-O-Fe) regions of artemisinin and of the hemin dimer that show the cleavage of the endoperoxide and that of the hemin dimer, respectively. We observed similar results in the trioxane alcohol/hemin dimer reaction. The RR spectrum of the artemisinin/hemin dimer reaction displays a vibrational mode at 850 cm(-1) that shifts to 818 cm(-1) when the experiment is repeated with (18)O-O(18) endoperoxide enriched trioxane alcohol. The frequency of this vibration and the magnitude of the (18)O-O(18) isotopic shift led us to assign the 850 cm(-1) mode to the Fe(IV) = O stretching vibration of a ferryl-xoxo heme intermediate that occurs in the artemisinin/hemin dimer and trioxane alcohol/hemin reactions. These results provide the first direct characterization of the antimalarial mode of action of artemisinin and its trioxane analog, and suggest that artemisinin appears to react with heme molecules that have been incorporated into hemozoin and subsequently the heme performs cytochrome P450-type chemistry. (+info)
Modulatory effects of carbon monoxide on baroreflex activation in nucleus tractus solitarii of rats.
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Recent studies suggest that carbon monoxide (CO), which is produced in significant quantities in many brain regions, may function as a neurotransmitter. Heme oxygenase catalyzes the metabolism of heme to CO and biliverdin; however, the physiological role of CO in central cardiovascular regulation was not well understood. In the present study, we evaluated the baroreflex response of CO in the nucleus tractus solitarii (NTS) of rats. Male Sprague-Dawley rats were anesthetized with urethane, and blood pressure and heart rate were monitored intra-arterially. Unilateral microinjection (60 nL) of hematin, a heme molecule cleaved by heme oxygenase to yield CO, into the NTS produced prominent dose-related depressor and bradycardic effects. Baroreflex responses were elicited by increasing doses of phenylephrine (10 to 30 microg/kg IV) before and after intra-NTS administration of zinc deuteroporphyrin 2,4-bis-glycol (ZnDPBG) (1 nmol), an inhibitor of heme oxygenase activity, or vehicle alone. The reflex bradycardia elicited by phenylephrine was significantly inhibited by pretreatment with ZnDPBG. Furthermore, the inhibitory effect of ZnDPBG on baroreflex activation was dose dependent. These results suggest CO formed by brain heme oxygenase plays a significant role in central cardiovascular regulation and that inhibition of heme oxygenase attenuated baroreflex activation. (+info)
p53-mediated differentiation of the erythroleukemia cell line K562.
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The tumor suppressor gene p53 can mediate both apoptosis and cell cycle arrest. In addition, p53 also influences differentiation. To further characterize the differentiation inducing properties of p53, we overexpressed a temperature-inducible p53 mutant (ptsp53Val135) in the erythroleukemia cell line K562. The results show that wild-type p53 and hemin synergistically induce erythroid differentiation of K562 cells, indicating that p53 plays a role in the molecular regulation of differentiation. However, wild-type p53 did not affect phorbol 12-myristate 13-acetate-dependent appearance of the megakaryocyte-related cell surface antigens CD9 and CD61, suggesting that p53 does not generally affect phenotypic modulation. The cyclin-dependent kinase inhibitor p21, a transcriptional target of p53, halts the cell cycle in G1 and has also been implicated in the regulation of differentiation and apoptosis. However, transiently overexpressed p21 did neither induce differentiation nor affect the cell cycle distribution or viability of K562 cells, suggesting that targets downstream of p53 other than p21 are critical for the p53-mediated differentiation response. (+info)
Hematin polymerization assay as a high-throughput screen for identification of new antimalarial pharmacophores.
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Hematin polymerization is a parasite-specific process that enables the detoxification of heme following its release in the lysosomal digestive vacuole during hemoglobin degradation, and represents both an essential and a unique pharmacological drug target. We have developed a high-throughput in vitro microassay of hematin polymerization based on the detection of (14)C-labeled hematin incorporated into polymeric hemozoin (malaria pigment). The assay uses 96-well filtration microplates and requires 12 h and a Wallac 1450 MicroBeta liquid scintillation counter. The robustness of the assay allowed the rapid screening and evaluation of more than 100, 000 compounds. Random screening was complemented by the development of a pharmacophore hypothesis using the "Catalyst" program and a large amount of data available on the inhibitory activity of a large library of 4-aminoquinolines. Using these methods, we identified "hit" compounds belonging to several chemical structural classes that had potential antimalarial activity. Follow-up evaluation of the antimalarial activity of these compounds in culture and in the Plasmodium berghei murine model further identified compounds with actual antimalarial activity. Of particular interest was a triarylcarbinol (Ro 06-9075) and a related benzophenone (Ro 22-8014) that showed oral activity in the murine model. These compounds are chemically accessible and could form the basis of a new antimalarial medicinal chemistry program. (+info)