Addition Of Hemin Research Articles

OnpA, an Unusual Flavin-Dependent Monooxygenase Containing a Cytochrome b 5 Domain

ortho-Nitrophenol 2-monooxygenase (EC 1.14.13.31) from Alcaligenes sp. strain NyZ215 catalyzes monooxygenation of ortho-nitrophenol to form catechol via ortho-benzoquinone. Sequence analysis of this onpA-encoded enzyme revealed that it contained a flavin-binding monooxygenase domain and a heme-binding cytochrome b(5) domain. OnpA was purified to homogeneity as a His-tagged protein and was considered a monomer, as determined by gel filtration. FAD and heme were identified by high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry (HPLC-MS) as cofactors in this enzyme, and quantitative analysis indicated that 1 mol of the purified recombinant OnpA contained 0.66 mol of FAD and 0.20 mol of heme. However, the enzyme activity of OnpA was increased by 60% and 450% after addition of FAD and hemin, respectively, suggesting that the optimal stoichiometry was 1:1:1. In addition, site-directed mutagenesis experiments confirmed that two highly conserved histidines located in the cytochrome b(5) domain were associated with binding of the heme, and the cytochrome b(5) domain was involved in the OnpA activity. These results indicate that OnpA is an unusual FAD-dependent monooxygenase containing a fused cytochrome b(5) domain that is essential for its activity. Therefore, we here demonstrate a link between cytochrome b(5) and flavin-dependent monooxygenases.

Label-free and amplified electrochemical detection of cytokine based on hairpin aptamer and catalytic DNAzyme

In this work, by incorporating a specific DNAzyme sequence into a hairpin aptamer probe, we describe a label-free and sensitive method for electrochemical detection of cytokines using recombinant human IFN-γ as the model analyte. The hairpin aptamer probes are immobilized on a gold electrode through self-assembly. The presence of IFN-γ opens the hairpin structure and forms the hemin/G-quadruplex peroxidase-mimicking DNAzyme with subsequent addition of hemin. The peroxidase-mimicking DNAzyme catalyzes the electro-reduction of H(2)O(2) and amplifies the current response for IFN-γ detection, which enables the monitoring of IFN-γ at the sub-nanomolar level. The proposed sensor also shows high selectivity towards the target analyte. Our strategy thus opens new opportunities for label-free and amplified detection of different types of cytokines.

EYA3 May Be Required for Globin Gene Expression in Erythroid Differentiation

Abstract 4797 Background:Extensive studies have led to a considerable understanding of the cellular and molecular control of haemoglobin production during red blood cell differentiation, however, identification of the genes expressed as part of the erythroid differentiation programme remains an important goal because of the insights that these data will bring to erythrocyte biology and disease. Previous results using SAGE identified 93 differentially-expressed genes during erythroid development. One of these genes, EYA3, a homologue gene of Eyes Absent 3 in Drosophila, is a transcription cofactor with intrinsic phosphatase activity and its expression was observed to be high at the end of CD34+ cell differentiation and in human bone marrow. Aim:To evaluate globin gene, fetal hemoglobin (HbF) expressions and apoptosis levels in the erythroleukemic K562 cell line after EYA3 gene silencing and induction with hemin. Methods:Four different cultures from human K562 cells (1×105cells/mL in DMEM, 10% FBS, penicillin/streptomycin, 5% CO2, 37°C) were transfected with control or EYA3 knockdown lentiviruses (MOI=2.5). After proliferation and selection of successfully transfected cells with puromicin (2.0 ug/mL), cells were treated with 30μM hemin and collected after 0, 24, 48, 72 and 96h for gene expression and flow cytometry analyses. EYA3, LXN, α, and g-gene expression was measured by qRT-PCR and normalized using the Genorm program. HbF expression and apoptosis were evaluated by flow cytometry. Results:Analysis of globin gene expression showed that α-globin gene was downregulated in EYA3 silenced K562 culture cells compared with the control culture in 72h after hemin addition (1.791±0.1735; 0.7404±0.1709, respectively, **P<0.001, n=4). g-globin gene expression was found to be downregulated in K562 EYA3 silenced culture after 24h (1.350±0.1296; 0.5285±0.1736, respectively, *P<0.05, n=4) and 72h (1.554±0.1042; 0.6889±0.1535, respectively, **P<0.001, n=4). HbF expression was found to be downregulated in the same culture compared to the control culture at 72h after hemin addition (3568±41.00; 1947±206.50, respectively, *P<0.05, n=4). Apoptosis levels were found increased in EYA3 silenced K562 culture cells compared with the control culture in 72h after hemin addition (4.7±0.10; 8.55±0.55, respectively, *P<0.001, n=4). Conclusions:Our results show that silencing EYA causes modifications in the expression pattern of α- and g-globin gene expression as well as in HbF expression pattern and apoptosis levels in a model of erythroid differentiation. Further studies should be performed in primary erythroid cell cultures using siRNA-based gene silencing and overexpression of these genes to determine how these genes are involved in the mechanisms of globin gene regulation.Support by FAPESP, CNPq and INCTS Disclosures:No relevant conflicts of interest to declare.

Plasma Hemoglobin and Heme Trigger Weibel Palade Body Exocytosis and Vaso-Occlusion in Transgenic Sickle Mice

Abstract 896Sickle cell disease (SCD) is a devastating hemolytic disease characterized by recurring episodes of painful vaso-occlusive crises and endothelial dysfunction. We hypothesize that hemoglobin (Hb) or hemin (ferri-protoporphyrin IX), released from HbS derived from hemolyzed sickle red blood cells, is fundamental to vaso-occlusion and vasculopathy in SCD. Utilizing intravital microscopy and dorsal skin fold chambers implanted on mice we previously demonstrated transient stasis in subcutaneous venules in response to hypoxia in transgenic sickle, but not normal mice. In the current studies we used NY1DD sickle mice; Townes-AA, -AS, and -SS mice; and C57 normal mice to test whether Hb or heme induce vaso-occlusion in the absence of a hypoxic stimulus. Four groups of 3–6 mice were given a bolus infusion (0.012 ml/g i.v.) of the following: 1) saline; 2) stroma-free HbA (32 umols heme/kg); 3) hemin (32 umols/kg); or 4) water (to induce intravascular hemolysis). In NY1DD mice, Hb, hemin or water induced a range of 36–46% stasis at 1 and 4 hours post-infusion compared to only 4–7% stasis at 1 and 4 hours in saline controls (Fig 1A, p<0.001 for all groups vs saline). In contrast, Hb, hemin or saline induced only 0–6% stasis in C57 mice (Fig 1B, p<0.001 for Hb or hemin in C57 vs NY1DD). Similarly, Townes-AA, -AS and –SS mice had a range of 5–15%, 27–37% and 40–50% stasis, respectively, in response to HbA (p<0.05 for all Townes pairs). [Display omitted] [Display omitted] The response to Hb or hemin was dose dependent with as little as 0.32 umols heme/kg, equivalent to ∼5 uM heme, a level found in SCD patients. These data support that heme, derived from Hb, promotes vascular stasis in sickle mice. The plasma of SCD patients is deficient in the two primary Hb/heme scavenging proteins haptoglobin (Hp) and hemopexin (Hpx). Therefore we infused equimolar Hp-Hb or Hpx-hemin into NY1DD mice and inhibited Hb- and heme-induced stasis at 1 hour by 79 and 88%, respectively (p<0.001).To examine whether nitrogen derivatives contribute to the mechanism of Hb- or heme-induced stasis, we measured plasma nitrite and nitrate (NOx) in NY1DD sickle mice 4 hours after infusion of Hb or hemin. NOx levels decreased in mice infused with Hb, but not in mice infused with hemin suggesting that NO consumption does not play a role in heme-induced stasis.Previous studies have demonstrated a role for P-selectin in vaso-occlusion in sickle mice. We tested the ability of heme to trigger Weibel Palade body (WPB) exocytosis in cultured primary human umbilical vein endothelial cells (HUVEC). Cells were treated with 10 uM hemin for 2, 5, 10, 15, 30 or 60 minutes and then fixed and stained (without permeabilization) for surface expression of P-selectin and von Willebrand factor (vWF). Cells treated with 100 uM histamine and vehicle served as positive and negative controls, respectively. There was rapid expression of P-selectin as well as vWF strings on the surface of HUVEC within 5 minutes of hemin addition. This is the first report that heme is an agonist for WPB exocytosis. Recently, heme has been shown to be an extracellular inflammatory signaling molecule with strict binding specificity for toll-like receptor-4 (TLR4). A specific small molecule inhibitor of TLR4 (TAK-242) completely prevented heme-induced P-selectin expression in vitro. In vivo the pulmonary veins and arteries of sickle mice injected with hemin expressed surface P-selectin within 15 minutes. Supporting this novel mechanism, blocking antibodies to P-selectin or the drug TAK-242 inhibited heme-induced stasis and thus provide a potential therapy for vaso-occlusion.These data strongly support that heme, released from hemolyzed sickle red blood cells, is fundamental to vaso-occlusion and vasculopathy in SCD.We speculate that removal of Hb and heme with Hp and Hpx, or as we've previously shown, detoxifying heme with heme oxygenase-1, would decrease the oxidative stress, inflammation and vaso-occlusion in SCD that cause endothelial cell dysfunction. Novel therapies focusing on the consequences of endothelial cell/heme interactions such as TLR4 or P-selectin antagonists, in addition to Hp and Hpx modulators should be considered in SCD. Disclosures:Belcher:Sangart, Inc: Research Funding. Nguyen:Sangart Inc: Research Funding. Chen:Sangart, Inc: Research Funding. Vercellotti:Sangart, Inc: Research Funding.

Identification and analysis of the Shewanella oneidensis major oxygen-independent coproporphyrinogen III oxidase gene

Shewanella oneidenesis MR-1 is a facultative anaerobe that can use a large number of electron acceptors including metal oxides. During anaerobic respiration, S. oneidensis MR-1 synthesizes a large number of c cytochromes that give the organism its characteristic orange color. Using a modified mariner transposon, a number of S. oneidensis mutants deficient in anaerobic respiration were generated. One mutant, BG163, exhibited reduced pigmentation and was deficient in c cytochromes normally synthesized under anaerobic condition. The deficiencies in BG163 were due to insertional inactivation of hemN1, which exhibits a high degree of similarity to genes encoding anaerobic coproporphyrinogen III oxidases that are involved in heme biosynthesis. The ability of BG163 to synthesize c cytochromes under anaerobic conditions, and to grow anaerobically with different electron acceptors was restored by the introduction of hemN1 on a plasmid. Complementation of the mutant was also achieved by the addition of hemin to the growth medium. The genome sequence of S. oneidensis contains three putative anaerobic coproporphyrinogen III oxidase genes. The protein encoded by hemN1 appears to be the major enzyme that is involved in anaerobic heme synthesis of S. oneidensis. The other two putative anaerobic coproporphyrinogen III oxidase genes may play a minor role in this process.

Highly Effective Colorimetric and Visual Detection of ATP by a DNAzyme–Aptamer Sensor

A new and simple method was developed to detect adenosine triphosphate (ATP) by using a DNAzyme-aptamer sensor. The DNAzyme used was a single-stranded DNA that could combine with hemin. The aptamer, a single, short nucleic acid sequence that can specifically bind with many targets, was an anti-ATP aptamer. Two DNA sequences were designed: i) a functional chain (Chain A) consisting of two parts, i.e., the anti-ATP aptamer (recognition part) and the DNAzyme (signal transduction part) and ii) a blocker chain (Chain B), which could partially hybridize with Chain A. The hybridized chains A and B were unfolded by the addition of ATP and hemin, and the blocker chain and the complex of the functional chain with ATP and hemin were in solution. The DNAzyme in the functional chain formed a G-quadruplex with hemin and then catalyzed the oxidation by H(2) O(2) of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS(2-) ) to the colored ABTS(.-) radical. The color change caused by this reaction could be clearly observed by naked eye, and the absorbance was recorded at 414 nm. The detection limit was 1×10(-6) M.

The mitochondrial protein frataxin is essential for heme biosynthesis in plants

Frataxin, a conserved mitochondrial protein implicated in cellular iron homeostasis, has been involved as the iron chaperone that delivers iron for the Fe-S cluster and heme biosynthesis. However, its role in iron metabolism remains unclear, especially in photosynthetic organisms. In previous work, we found that frataxin deficiency in Arabidopsis results in decreased activity of the mitochondrial Fe-S proteins aconitase and succinate dehydrogenase, despite the increased expression of the respective genes, indicating an important role for Arabidopsis thaliana frataxin homolog (AtFH). In this work, we explore the hypothesis that AtFH can participate in heme formation in plants. For this purpose, we used two Arabidopsis lines, atfh-1 and as-AtFH, with deficiency in the expression of AtFH. Both lines present alteration in several transcripts from the heme biosynthetic route with a decrease in total heme content and a deficiency in catalase activity that was rescued with the addition of exogenous hemin. Our data substantiate the hypothesis that AtFH, apart from its role in protecting bioavailable iron within mitochondria and the biogenesis of Fe-S groups, also plays a role in the biosynthesis of heme groups in plants.

Heme induces DNA damage and hyperproliferation of colonic epithelial cells via hydrogen peroxide produced by heme oxygenase: A possible mechanism of heme‐induced colon cancer

Epidemiological and animal model studies have suggested that high intake of heme, present in red meat, is associated with an increased risk of colon cancer. However, the mechanisms underlying this association are not clear. This study aimed to investigate whether heme induces DNA damage and cell proliferation of colonic epithelial cells via hydrogen peroxide produced by heme oxygenase (HO). We examined the effects of zinc protoporphyrin (ZnPP; a HO inhibitor) and catalase on DNA damage, cell proliferation, and IL-8 production induced by the addition of hemin (1-10 microM) to human colonic epithelial Caco-2 cells. DNA damage was determined with a comet assay, and cell proliferation was evaluated with 5-bromo-2'-deoxyuridine incorporation assay. Both ZnPP and exogenous catalase inhibited the hemin-induced DNA damage and cell hyperproliferation dose-dependently. IL-8 messenger RNA expression and IL-8 production in the epithelial cells increased following the hemin treatment, but the production was inhibited by ZnPP and catalase. These results indicate that hemin has genotoxic and hyperproliferative effects on Caco-2 cells by HO and hydrogen peroxide. The mechanism might explain why a high intake of heme is associated with increased risk of colon cancer.

Structure–function study of peroxidase-like G-quadruplex-hemin complexes

The structure-function relationship of G-quadruplex-hemin complexes with peroxidase activity was studied by comparing peroxidase activity and circular dichroism (CD) spectra of 22 oligonucleotides with the sequence of d(G(2)T(n))(3)G(2), d(G(3)T(n))(3)G(3) (n = 1-4) and dG(3)T(i)G(3)T(j)G(3)T(k)G(3). According to the experimental results, some conclusions can be drawn, such as the addition of hemin may promote the conversion of some G-quadruplexes from antiparallel structures to parallel structures; the formation of G-quadruplexes is a crucial factor in determining the peroxidase activity of G-quadruplex-hemin complexes; and the complexes formed by hemin and parallel G-quadruplexes have much higher peroxidase activity than those formed by hemin and antiparallel G-quadruplexes.

合成廃水を用いたメタン発酵プロセスにおけるプロピオン酸酸化の安定化要因の探索

Factors affecting propionate oxidation in a methanogenic microbial community derived from an anaerobic-digester sludge of cattle waste were investigated. The sludge was inoculated into a propionate-containing artificial wastewater medium, and cultures were incubated at mesophilic (30°C) and thermophilic (55°C) temperatures by a subculture technique. During subculture, however, the rate of propionate oxidation rapidly declined. To stabilize propionate oxidation, the use of some additives was examined. The addition of sulfate, pyruvate, fumarate, hemin, or yeast extract did not prevent the decrease in the rate of propionate oxidation. The addition sludge extract (supernatant fluid fraction of sludge autoclaved at 121°C) efficiently stabilized propionate oxidation. Although, the addition of dry powder of the sludge extract prepared at 110°C stabilized methane fermentation, that of sludge extract ash did not stabilize the process. Moreover, the addition of a low-molecular-weight fraction (below ca. 14,000) obtained by the dialysis of the sludge extract also stabilized the process. The present results indicate that effective components for stabilizing propionate oxidation in the sludge extract are low-molecular-weight organic compounds.

Colorimetric Split G-Quadruplex Probes for Nucleic Acid Sensing: Improving Reconstituted DNAzyme’s Catalytic Efficiency via Probe Remodeling

Split G-rich DNA probes can assemble into active peroxidase-mimicking DNAzymes in the presence of bioanalytes such as DNA, thereby providing a simple and cheap means to detect analytes in biological samples. A comprehensive study designed to reveal the salient probe architectural features and reaction conditions that facilitate facile reconstitution into enzymatically proficient enzymes unveiled these important findings: (a) The loops that connect the G3-tracts in a G-quadruplex structure can be replaced with a stem-loop or loop-stem-loop motif without destabilizing the resulting quadruplex structure; endowing the split G-rich probes with regions of limited complementarity leads to more proficient reconstituted enzymes. (b) The addition of hemin to antiparallel G-quadruplex DNAzymes lead to a blue shift in the CD spectra of the G-quadruplex DNAzymes. (c) The architectures of the DNA motifs that lie adjacent to the G-quadruplex structure influence both the stability and the enzymatic proficiency of the reconstituted enzymes. (d) The nature of the monovalent cation that is present in excess is a key determinant of the turnover number of the G-quadruplex DNAzyme; decomposition of G-quadruplex DNAzymes is slower in buffers that contain ammonium ions than those that contain sodium or potassium ions. These findings are important for the design of bioassays that use peroxidase-mimicking G-quadruplexes as detection labels.

Functional Characterization of the Canine Heme-Regulated eIF2αKinase: Regulation of Protein Synthesis

The heme-regulated inhibitor (HRI) negatively regulates protein synthesis by phosphorylating eukaryotic initiation factor-2α (eIF2α) thereby inhibiting protein translation. The importance of HRI in regulating hemoglobin synthesis in erythroid cells makes it an attractive molecular target in need of further characterization. In this work, we have cloned and expressed the canine form of the HRI kinase. The canine nucleotide sequence has 86%, 82%, and 81% identity to the human, mouse, and rat HRI, respectively. It was noted that an isoleucine residue in the ATP binding site of human, rat, and mouse HRI is replaced by a valine in the canine kinase. The expression of canine HRI protein by in vitro translation using wheat germ lysate or in Sf9 cells using a baculovirus expression system was increased by the addition of hemin. Following purification, the canine protein was found to be 72 kD and showed kinase activity determined by its ability to phosphorylate a synthetic peptide substrate. Quercetin, a kinase inhibitor known to inhibit mouse and human HRI, inhibits canine HRI in a concentration-dependent manner. Additionally, quercetin is able to increase de novo protein synthesis in canine reticulocytes. We conclude that the canine is a suitable model species for studying the role of HRI in erythropoiesis.

Leishmaniarequires Rab7-mediated degradation of endocytosed hemoglobin for their growth

Leishmania is unable to synthesize heme and must acquire it from exogenous source, the mechanism of which is not known. We have shown that Leishmania endocytoses hemoglobin (Hb) and subsequently degrade it probably to generate heme. To understand how internalized Hb is degraded, we have cloned and expressed Rab7 homolog from Leishmania donovani. Interestingly, Rab7 in Leishmania is found to be localized both on early and late endocytic compartment and regulates both uptake and degradation of endocytosed Hb demonstrating that Rab7 in Leishmania play a very unique role connecting both early and late events of Hb endocytosis. Our data also indicate that overexpression of Rab7:WT in Leishmania induces transport of Hb to lysosomes and rapidly degrade internalized Hb. Whereas Hb transport to lysosomes and its degradation is significantly inhibited in cells overexpressing Rab7:T21N, a GDP locked mutant of Rab7. Moreover, cells overexpressing Rab7:T21N grow at a slower rate (<50%) compared with control Leishmania. Addition of exogenous hemin recovers the growth of Rab7:T21N mutant cells almost to the control level, suggesting that intracellular heme generated by Rab7-mediated Hb degradation is required for optimal growth of the parasites. Thus, our results identify a potential target which might be exploited to suppress the growth of Leishmania.

Neudesin, a Neurotrophic Factor, Is a Novel Extracellular Heme‐binding Protein.

Neudesin is a secreted protein with neurotrophic activity in neurons and undifferentiated neural cells. We report here that neudesin is an extracellular heme‐binding protein and that its neurotrophic activity is dependent on the binding of heme to its cytochrome b5‐like heme/steroid‐binding domain. At first, we found that at least a portion of the purified recombinant neudesin appeared to bind hemin because the purified neudesin solution was tinged with green and had a sharp absorbance peak at 402nm. The addition of exogenous hemin extensively increased the amount of hemin‐bound neudesin. In contrast, neudesinΔHBD, a mutant lacking the heme‐binding domain, could not bind hemin. The neurotrophic activity of the recombinant neudesin that bound exogenous hemin (neudesin‐hemin) was significantly greater than that of the recombinant neudesin in either primary cultured neurons or Neuro2a cells, suggesting that the activity of neudesin depends on hemin. The inhibition of endogenous neudesin by RNA interference significantly decreased cell survival in Neuro2a cells. This indicates that endogenous neudesin possibly contains hemin. The experiment with anti‐neudesin antibody suggested that the endogenous neudesin detected in the culture medium of Neuro2a cells was associated with hemin because it was not retained on a heme‐affinity column at all. Neudesin is the first extracellular heme‐binding protein that shows signal transducing activity by itself. The present findings may shed new light on the function of extracellular heme‐binding proteins.

Neurotrophic Activity of Neudesin, a Novel Extracellular Heme-binding Protein, Is Dependent on the Binding of Heme to Its Cytochrome b5-like Heme/Steroid-binding Domain

Neudesin is a secreted protein with neurotrophic activity in neurons and undifferentiated neural cells. We report here that neudesin is an extracellular heme-binding protein and that its neurotrophic activity is dependent on the binding of heme to its cytochrome b(5)-like heme/steroid-binding domain. At first, we found that at least a portion of the purified recombinant neudesin appeared to bind hemin because the purified neudesin solution was tinged with green and had a sharp absorbance peak at 402 nm. The addition of exogenous hemin extensively increased the amount of hemin-bound neudesin. In contrast, neudesinDeltaHBD, a mutant lacking the heme-binding domain, could not bind hemin. The neurotrophic activity of the recombinant neudesin that bound exogenous hemin (neudesin-hemin) was significantly greater than that of the recombinant neudesin in either primary cultured neurons or Neuro2a cells, suggesting that the activity of neudesin depends on hemin. The neurotrophic activity of neudesin was enhanced by the binding of Fe(III)-protoporphyrin IX, but neither Fe(II)-protoporphyrin IX nor protoporphyrin IX alone. The inhibition of endogenous neudesin by RNA interference significantly decreased cell survival in Neuro2a cells. This indicates that endogenous neudesin possibly contains hemin. The experiment with anti-neudesin antibody suggested that the endogenous neudesin detected in the culture medium of Neuro2a cells was associated with hemin because it was not retained on a heme-affinity column at all. Neudesin is the first extracellular heme-binding protein that shows signal transducing activity by itself. The present findings may shed new light on the function of extracellular heme-binding proteins.

Hemin Interactions and Alterations of the Subcellular Localization of Prion Protein

Hemin (iron protoporphyrin IX) is a crucial component of many physiological processes acting either as a prosthetic group or as an intracellular messenger. Some unnatural, synthetic porphyrins have potent anti-scrapie activity and can interact with normal prion protein (PrPC). These observations raised the possibility that hemin, as a natural porphyrin, is a physiological ligand for PrPC. Accordingly, we evaluated PrPC interactions with hemin. When hemin (3-10 microM) was added to the medium of cultured cells, clusters of PrPC formed on the cell surface, and the detergent solubility of PrPC decreased. The addition of hemin also induced PrPC internalization and turnover. The ability of hemin to bind directly to PrPC was demonstrated by hemin-agarose affinity chromatography and UV-visible spectroscopy. Multiple hemin molecules bound primarily to the N-terminal third of PrPC, with reduced binding to PrPC lacking residues 34-94. These hemin-PrPC interactions suggest that PrPC may participate in hemin homeostasis, sensing, and/or uptake and that hemin might affect PrPC functions.

Anti-Glycophorin C Induces a Loss of Mitochondrial Membrane Potential but Fails To Activate Apoptotic Caspases.

The human erythrocyte transmembrane sialoglycoprotein, glycophorin C (GYPC), plays a functional role in regulating red cell shape and mechanical stability. Antibodies to GYPC cause hemolytic disease of the fetus and newborn (HDFN) that is associated with classical Fcγ receptor-mediated phagocytosis. However, in vitro clonogenic studies with cord blood progenitor cells suggest that anti-GYPC also suppresses erythropoiesis, which is consistent with the observations of severe and early fetal anemia and late onset neonatal anemia [Transfus Med 2005; 15:125–32]. The mechanism of the suppressive effect on erythropoiesis is unknown. The K562 erythroleukemic cell line treated with anti-GYPC is a potential model system to study the suppressive effect of anti-GYPC. The present in vitro studies were designed to confirm the effect of anti-GYPC on K562 cell growth and viability, and to evaluate changes in mitochondrial membrane potential, phosphatidylserine (PS) expression, propidium iodide (PI) binding, and caspase activation. K562 cells fail to grow in the presence of anti-GYPC confirming earlier CFU-E/BFU-E studies [Brit J Haematol 2006; 133:443–4], and increased the exofacial expression of PS/PI over time. This process was caspase-independent as demonstrated by the failure of Z-VAD, a caspase inhibitor, to reverse growth inhibition and PS/PI expression. A loss of mitochondrial membrane potential was demonstrated using JC-1, a cationic dye that is sensitive to potential-dependent accumulation or loss in mitochondria. There was a 50% increase in K562 cell mitochondrial membrane potential disruption after 2 days of culture with anti-GYPC (see figure). Morphological examination of May Grunwalde Giemsa-stained K562 cells treated with anti-GYPC for 2 days showed a decrease in mitotic activity compared to isotype treated cells. By day 4, the anti-GYPC treated cells were showing evidence of plasma membrane damage and cell death resulting from fragmentation and dissolution of the cytoplasm. The addition of hemin, an oxidative form of iron protoporphyrin IX known to induce erythroid differentiation of K562 cells, to anti-GYPC treated cells reversed growth inhibition by 45% but did not prevent the loss of mitochondrial membrane potential. Overall, although caspases appear to be unimportant in anti-GYPC induced cell death, the mitchondria play an important role as the early events leading to antibody-mediated suppression of erythropoiesis. [Display omitted]

In Vitro and In Vivo Activities of Synthetic Trioxolanes against Major Human Schistosome Species

Schistosomiasis is a parasitic disease that remains of considerable public health significance in tropical and subtropical environments. Since the mainstay of schistosomiasis control is chemotherapy with a single drug, praziquantel, drug resistance is a concern. Here, we present new data on the antischistosomal properties of representative synthetic 1,2,4-trioxolanes (OZs). Exposure of adult Schistosoma mansoni for 24 h to a medium containing 20 mug/ml OZ209 reduced worm motor activity, induced tegumental alterations, and killed worms within 72 h. While exposure of S. mansoni to OZ78 had no apparent effect, addition of hemin reduced worm motor activity and caused tegumental damage. Administration of single 200-mg/kg of body weight oral doses of OZ78, OZ209, and OZ288 to mice harboring a juvenile S. mansoni infection resulted in worm burden reductions of 82.0 to 95.4%. In the adult infection model in mice, single 400-mg/kg doses of these compounds resulted in a maximum total worm burden reduction of 52.2%. High worm burden reductions (71.7 to 86.5%) were observed after administration of single 200-mg/kg doses of OZ78 and OZ288 to hamsters infected with either juvenile or adult S. mansoni. A single 200-mg/kg dose of OZ78 to hamsters infected with adult Schistosoma japonicum resulted in total and female worm burden reductions of 94.2 to 100%. Our results, along with the low toxicity, metabolic stability, and good pharmacokinetic properties of the OZs, indicate the potential for the development of novel broad-spectrum antischistosomal OZ drug candidates.

Reduction of lactate production in Lactococcus lactis, a combined strategy: metabolic engineering by introducing foreign alanine dehydrogenase gene and hemin addition

An approach to decreasing the lactate production in Lactococcus lactis by metabolic engineering is presented. The inhibitory effects of a low pH due to the accumulation of lactate on cell growth and nisin production in L. lactis are well known. To avoid such inhibitory effects, a new strategy by rerouting carbon flow was considered. In an effort to suppress lactate production, a new gene was introduced into L. lactis to create a novel pathway for alanine synthesis to reroute the metabolic flow of lactate. Alanine dehydrogenase (E.C.1.4.1.1) encoded by alaD from Bacillus sphaericus was expressed in L. lactis. The enzyme was expressed to a specific activity of nearly 0.39 U/mg protein in the transformant. Hemin addition was also considered to decrease the lactate production in L. lactis. The effect of hemin on the alanine production in the transformant was investigated. This study showed that using the combined strategy, stronger effects on lactate and alanine productions were observed in the transformant.

Protoporphyrin IX Down-Regulates DMT1 Associated Protein (DAP) in K562 Cells.

The final steps of heme biosynthesis include the transport of coproporphyrin with the transport step probably mediated by the peripheral benzodiazepine receptor (PBR). Within the mitochondria copropoprhyrin is then converted to protoporphyrin IX (PPIX) which in turn is converted to hemin with insertion of iron by ferrochelatase. PBR is ubiquitously expressed and has been implicated in steriodogenesis, apoptosis, erythroid differentiation, and inflammation. Interestingly, PPIX is among several high affinity ligands for PBR. Various cytosolic proteins that interact with PBR have also been defined including PBR associated protein 7 (PAP7). The various PBR ligands including PPIX may affect the binding of these proteins to PBR. We have demonstrated (Blood , Nov 2004; 104: 53) that DAP, a protein highly homologous to PAP7, binds to the C-terminus of DMT1 and may have a role in regulation of intracellular iron transport. We, therefore, examined the effects of PPIX on the functions of DAP and other proteins that affect cellular iron metabolism. DAP is 526 amino acid protein with a nuclear localization signal domain (aa 212–229) and a Golgi localization domain (aa 380–524), and is distributed in the cytoplasm, Golgi apparatus, and nuclei of K562 cells. K562 cells were grown in the presence of 5 μM PPIX for 24 hours and then the expression of DAP, transferrin receptor 1 (TfR1), and ferritin examined by western blot analysis. In addition, cells were grown in medium of either normal iron content (3.5 μM from ferri-transferrin), high iron content (217 μM from the addition of ferric ammonium citrate), or low iron content (by the addition of 50 mM desferroxamine). Under all three iron conditions PPIX induced differentiation but down-regulated ferritin expression and up-regulated TfR1 expression. Additionally, PPIX had a striking effect on DAP expression markedly decreasing DAP levels but only in cells grown either in normal or low iron medium. In addition, PPIX affected the expression of the iron transporter DMT1in parallel with DAP. As PPIX induced erythroid differentiation of K562 cells we examined the effects of hemin which can also induce differentiation of K562 cells. In contrast to PPIX, hemin caused strong down-regulation of TfR and up-regulation of ferritin and DAP. The down-regulation of DAP induced by PPIX was restored by the addition of hemin. These results indicate that PPIX affects DAP expression and other important elements involved in cellular iron metabolism and that these effects are partially modified by the iron status of the cell.