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Microprocessor, which consists of a ribonuclease III DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) maturation by cleaving primary miRNA transcripts (pri-miRNAs). We recently demonstrated that the DGCR8 dimer recognizes the apical elements of pri-miRNAs, including the UGU motif, to accurately locate and orient Microprocessor on pri-miRNAs. However, the mechanism underlying the selective RNA binding remains unknown. In this study, we find that hemin, a ferric ion-containing porphyrin, enhances the specific interaction between the apical UGU motif and the DGCR8 dimer, allowing Microprocessor to achieve high efficiency and fidelity of pri-miRNA processing in vitro. Furthermore, by generating a DGCR8 mutant cell line and carrying out rescue experiments, we discover that hemin preferentially stimulates the expression of miRNAs possessing the UGU motif, thereby conferring differential regulation of miRNA maturation. Our findings reveal the molecular action mechanism of hemin in pri-miRNA processing and establish a novel function of hemin in inducing specific RNA-protein interaction.

Hydrogen gas (H2) is an endogenous gaseous molecule in plants. Although its reputation is as a "biologically inert gas", recent results suggested that H2 has therapeutic antioxidant properties in animals and plays fundamental roles in plant responses to environmental stresses. However, whether H2 regulates root morphological patterns is largely unknown. In this report, hydrogen-rich water (HRW) was used to characterize H2 physiological roles and possible signaling transduction pathways in the promotion of adventitious root (AR) formation in cucumber explants. Our results showed that a 50% concentration of HRW was able to mimic the effect of hemin, an inducer of a carbon monoxide (CO) synthetic enzyme, and heme oxygenase-1 (HO-1), in restoring AR formation in comparison with the inhibition effect conferred by auxin-depletion treatment alone. It was further shown that the inducible effect of HRW could be further blocked by the co-treatment with N-1-naphthylphtalamic acid (NPA; an auxin transport inhibitor). The HRW-induced response, at least partially, was HO-1-dependent. This conclusion was supported by the fact that the exposure of cucumber explants to HRW up-regulates cucumber HO-1 gene expression and its protein levels. HRW-mediated induction of representative target genes related to auxin signaling and AR formation, such as CsDNAJ-1, CsCDPK1/5, CsCDC6, CsAUX22B-like, and CsAUX22D-like, and thereafter AR formation (particularly in the AR length) was differentially sensitive to the HO-1 inhibitor zinc protoporphyrin IX (ZnPP). Above blocking actions were clearly reversed by CO, further confirming that the above response was HO-1/CO-specific. However, the addition of a well-known antioxidant, ascorbic acid (AsA), failed to influence AR formation triggered by HRW, thus ruling out the involvement of redox homeostasis in this process. Together, these results indicated that HRW-induced adventitious rooting is, at least partially, correlated with the HO-1/CO-mediated responses. We also suggested that exogenous HRW treatment on plants might be a good option to induce root organogenesis., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Although hepatitis C virus (HCV) affects approximately 130-170 million people worldwide, no vaccines are available. HCV is an important cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma, leading to the need for liver transplantation. In this study, curcumin, a constituent used in traditional Chinese medicine, has been evaluated for its anti-HCV activity and mechanism, using a human hepatoma cell line containing the HCV genotype 1b subgenomic replicon. Below the concentration of 20% cytotoxicity, curcumin dose-dependently inhibited HCV replication by luciferase reporter gene assay, HCV RNA detection and HCV protein analysis. Under the same conditions, curcumin also dose-dependently induced heme oxygenase-1 with the highest induction at 24 h. Hemin, a heme oxygenase-1 inducer, also inhibited HCV protein expression in a dose-dependent manner. The knockdown of heme oxygenase-1 partially reversed the curcumin-inhibited HCV protein expression. In addition to the heme oxygenase-1 induction, signaling molecule activities of AKT, extracellular signal-regulated kinases (ERK) and nuclear factor-κB (NF-κB) were inhibited by curcumin. Using specific inhibitors of PI3K-AKT, MEK-ERK and NF-κB, the results suggested that only PI3K-AKT inhibition is positively involved in curcumin-inhibited HCV replication. Inhibition of ERK and NF-κB was likely to promote HCV protein expression. In summary, curcumin inhibited HCV replication by heme oxygenase-1 induction and AKT pathway inhibition. Although curcumin also inhibits ERK and NF-κB activities, it slightly increased the HCV protein expression. This result may provide information when curcumin is used as an adjuvant in anti-HCV therapy.

Porphyromonas gingivalis is a Gram-negative obligately anaerobic bacterium associated with several forms of periodontal disease, most closely with chronic periodontitis. Previous studies demonstrated that OxyR plays an important role in the aerotolerance of P. gingivalis by upregulating the expression of oxidative-stress genes. Increases in oxygen tension and in H(2)O(2) both induce activation of OxyR. It is also known that P. gingivalis requires hemin as an iron source for its growth. In this study, we found that a hemin-limited growth environment significantly enhanced OxyR activity in P. gingivalis. As a result, expression of sod, dps, and ahpC was also upregulated. Using a chromatin immunoprecipitation quantitative PCR (qPCR) analysis, DNA binding of activated OxyR to the promoter of the sod gene was enhanced in P. gingivalis grown under hemin-limited conditions compared to excess-hemin conditions. Cellular tolerance of H(2)O(2) was also enhanced when hemin was limited in the growth medium of P. gingivalis. Our work supports a model in which hemin serves as a signal for the regulation of OxyR activity and indicates that P. gingivalis coordinately regulates expression of oxidative-stress-related genes by this hemin concentration-dependent pathway.

Intracerebral hemorrhage (ICH) is a subtype of stoke that may cause significant morbidity and mortality. Brain injury due to ICH initially occurs within the first few hours as a result of mass effect due to hematoma formation. However, there is increasing interest in the mechanisms of secondary brain injury as many patients continue to deteriorate clinically despite no signs of rehemorrhage or hematoma expansion. This continued insult after primary hemorrhage is believed to be mediated by the cytotoxic, excitotoxic, oxidative, and inflammatory effects of intraparenchymal blood. The main factors responsible for this injury are thrombin and erythrocyte contents such as hemoglobin. Therapies including thrombin inhibitors, N-methyl-D-aspartate antagonists, chelators to bind free iron, and antiinflammatory drugs are currently under investigation for reducing this secondary brain injury. This review will discuss the molecular mechanisms of brain injury as a result of intraparenchymal blood, potential targets for therapeutic intervention, and treatment strategies currently in development.

This study represents the identification of a single amino acid residue that has the major responsibility for the isomeric orientation of a heme b cofactor in a ferriheme protein. The insertion of hemin b into the asymmetric environment of a protein pocket facilitates two cofactor orientations, A and B, which is often called "heme rotational disorder". The proteins studied herein are nitrophorins, a class of ferriheme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus, in this case NP2 and NP7. NMR spectroscopy (pH* 5.5) of the imidazole complex of NP7 revealed solely the A orientation, whereas NP2 shows primarily the B orientation ( approximately 1:5 A:B). The glutamate 27 residue in NP7 is an obvious difference in the heme pocket compared to those of NP1-4, all of which present a valine residue [valine 24 (NP2 and NP3) or valine 25 (NP1 and NP4)] at the same position. Consequently, the mutant NP2(V24E) was prepared and shown to reverse the heme orientation to exclusively A, whereas NP7(E27V) revealed an approximately 1:3 A:B ratio. The reversal A <--> B following the change glutamine <--> valine was further indicated in circular dichroism (CD) spectroscopy with a positive (A) or negative (B) Deltaepsilon of the heme Soret band. Moreover, CD spectroscopy was applied to the mutant NP7(E27Q) and indicated mainly the A orientation, which allows us to conclude that the steric hindrance provided by the glutamate residue is responsible for the heme orientation rather then the carboxylate charge.

Triggering differentiation has been employed as a strategy to inhibit cell proliferation and accelerate apoptosis in malignant cells. To better understand the mechanisms underlying differentiation-mediated regulation of apoptosis, we have studied the effects of PKC pathway with an activator of the protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), during hemin-induced erythroid differentiation of K562 erythroleukemia cells. K562 cell line has been used as a model of common progenitor of erythroblasts and magakaryocytes and can be differentiated into erythroid and megakaryocytic lineages by hemin and TPA, respectively. TPA induced almost complete loss of proliferation during megakaryocytic differentiation in K562 cells. However, upon hemin-mediated erythroid differentiation, the growth rate was slightly decreased at the subtoxic concentrations. Cotreatment with TPA at the hemin-treated K562 cells produced a concentration-dependent increase in cell injuries with apoptotic changes and significantly diminished the erythroid phenotype. To better understand the events implicated, we have used the PKC inhibitors such as bisindolylmaleimide II, RO318220, and the PKCbeta inhibitor. Our data showed that TPA-potentiated apoptosis in hemin-treated K562 cells was rescued by the application of the PKC inhibitors. Taken together, our results suggested the involvement of PKC in TPA-potentiated apoptosis induction during hemin-mediated erythroid differentiation in K562 cells.

Purpose: Nrf2 (nuclear factor-E2 related factor-2), nuclear factor-kappaB and mitogen-activated protein kinase, including extracellular regulated kinase, c-jun N-terminal kinase and p38 mitogen-activated protein kinase, are crucial for signal transduction. We previously reported that heme oxygenase-1 over expression induced by the heme oxygenase-1 inducer hemin protected testes from torsion-detorsion injury. In this study we elucidated which of these enzymes is involved in hemin induced heme oxygenase-1 over expression in testicular tissues after torsion-detorsion injury., Materials and Methods: Adult male Sprague-Dawley rats (National Science Council, Taipei, Taiwan, Republic of China) were allocated to undergo testicular torsion-detorsion immediately followed by injection of normal saline, hemin or hemin plus tin protoporphyrin, a heme oxygenase-1 inhibitor. Another set of rats that underwent sham operation were used as controls. Testes were harvested 0 and 30 minutes after detorsion and enzyme expression was analyzed., Results: Hemin alone caused no significant effects on the expression of Nrf2, nuclear factor-kappaB, extracellular regulated kinase and c-jun N-terminal kinase. Similarly testicular torsion-detorsion injury caused no significant effects on Nrf2 expression. In the presence of torsion-detorsion injury hemin significantly up-regulated Nrf2 expression. Moreover, this effect was not affected by tin protoporphyrin. Unlike Nrf2, the expression of nuclear factor-kappaB, extracellular regulated kinase and c-jun N-terminal kinase was significantly up-regulated by testicular torsion-detorsion. Hemin significantly attenuated the testicular torsion-detorsion induced up-regulation of nuclear factor-kappaB and extracellular regulated kinase but not c-jun N-terminal kinase. The effects of hemin on nuclear factor-kappaB and extracellular regulated kinase were significantly reversed by tin protoporphyrin. However, the expression of p38 mitogen-activated protein kinase in rodent testes was not affected by these interventions., Conclusions: Hemin induced heme oxygenase-1 over expression in rodent testes after torsion-detorsion injury involves Nrf2, nuclear factor-kappaB and extracellular regulated kinase.

Gelatinase B or matrix metalloproteinase-9 is involved in inflammation and in autoimmune and vascular diseases. In contrast to the constitutive and homeostatic matrix metalloproteinase-2, matrix metalloproteinase-9 is an inducible enzyme. Furthermore, it needs tight regulation, and a major control mechanism of its enzymatic activity is the activation of the latent enzyme by proteolysis of the 87 residue propeptide. Activated matrix metalloproteinase-9 is detected in many vascular or hematological disease states, including in an experimental model for cerebral malaria with Plasmodium berghei ANKA. However, insight into its activation mechanism is incomplete. In view of the association with hemorrhagic and hemolytic diseases, it was studied whether and how hemoglobin and its derivatives might activate pro-matrix metalloproteinase-9. Incubation of matrix metalloproteinase-9 with hemin or beta-hematin, the core constituent of hemozoin or malaria pigment, leads to differential autocatalysis of the propeptide, mediated by allosteric interaction with the hemopexin domain. The cleavage catalyzed by beta-hematin coincides with the first cleavage by stromelysin-1/matrix metalloproteinase-3, and preincubation of matrix metalloproteinase-9 with beta-hematin enhances the activation rate by matrix metalloproteinase-3 at least 6-fold. These findings suggest that reduction of hemorrhage and hemolysis might prevent matrix metalloproteinase-9-mediated inflammatory and vascular damages.

Childhood malaria caused by Plasmodium falciparum is often characterized by severe anemia at low parasite burdens; the mechanism(s) responsible for this pathology remain to be defined. We have reported, based on clinical observations and in vitro models, that complement control proteins on erythrocytes such as CR1, the immune adherence receptor specific for C3b, may be reduced in childhood malaria, suggesting a possible role for complement in erythrocyte destruction. Intravascular lysis of iE by P. falciparum leads to release of erythrocyte breakdown products such as hemoglobin and hematin, which have inflammatory properties. In the present article, we demonstrate that in serum and in anticoagulated whole blood, moderate concentrations of hematin activate the alternative pathway of complement and promote deposition of C3 activation and breakdown products on erythrocytes. The degree of C3 fragment deposition is directly correlated with erythrocyte CR1 levels, and erythrocytes opsonized with large amounts of C3dg form rosettes with Raji cells, which express CR2, the C3dg receptor which is expressed on several types of B cells in the spleen. Thus, the reaction mediated by hematin promotes opsonization and possible clearance of the youngest (highest CR1) erythrocytes. A mAb specific for C3b, previously demonstrated to inhibit the alternative pathway of complement, completely blocks the C3 fragment deposition reaction. Use of this mAb in nonhuman primate models of malaria may provide insight into mechanisms of erythrocyte destruction and thus aid in the development of targeted therapies based on inhibiting the alternative pathway of complement.

Transgenic sickle mice expressing betaS hemoglobin have activated vascular endothelium that exhibits enhanced expression of NF-kappaB and adhesion molecules that promote vascular stasis in sickle, but not in normal, mice in response to hypoxia/reoxygenation. Sickle mice hemolyze rbcs in vivo as demonstrated by increased reticulocyte counts, plasma hemoglobin and bilirubin, and reduced plasma haptoglobin. The heme content is elevated in sickle organs, which promotes vascular inflammation and heme oxygenase-1 expression. Treatment of sickle mice with hemin further increases heme oxygenase-1 expression and inhibits hypoxia/reoxygenation-induced stasis, leukocyte-endothelium interactions, and NF-kappaB, VCAM-1, and ICAM-1 expression. Heme oxygenase inhibition by tin protoporphyrin exacerbates stasis in sickle mice. Furthermore, treatment of sickle mice with the heme oxygenase enzymatic product carbon monoxide or biliverdin inhibits stasis and NF-kappaB, VCAM-1, and ICAM-1 expression. Local administration of heme oxygenase-1 adenovirus to subcutaneous skin increases heme oxygenase-1 and inhibits hypoxia/reoxygenation-induced stasis in the skin of sickle mice. Heme oxygenase-1 plays a vital role in the inhibition of vaso-occlusion in transgenic sickle mice.

Hemin upregulates heme oxygenase-1 (HO-1), a stress-induced enzyme implicated in protection from a variety of injuries while its related isoform HO-2 is constitutively expressed. The role of hemin or HO-1 in the pancreas and their potential modulation of pancreatic injury are unknown. We show that HO-1 is induced in pancreatitis caused by caerulein and more prominently in severe pancreatitis caused by feeding a choline-deficient diet (CDD). Intraperitoneal hemin administration dramatically increases peritoneal and pancreas macrophages that overexpress HO-1 in association with pancreatic induction of the chemoattractants monocyte chemotactic protein-1 and macrophage inflammatory protein-1alpha but not RANTES or macrophage inflammatory protein-2. Hemin administration before CDD feeding protected 8 of 8 mice from lethality while 7 of 16 controls died. Protection is mediated by HO-1-overexpressing macrophages since hemin-primed macrophages home to the pancreas after transfer to naive mice and protect from CDD-induced pancreatitis. Suppression of hemin-primed peritoneal cell HO-1 using HO-1-specific small interfering RNA prior to cell transfer abolishes protection from CDD-induced pancreatitis. Similarly, hemin pretreatment in caerulein-induced pancreatitis reduces serum amylase and lipase, decreases pancreatic trypsin generation, and protects from lung injury. Therefore, hemin-like compounds or hemin-activated macrophages may offer novel therapeutic approaches for preventing acute pancreatitis and its pulmonary complication via upregulation of HO-1.

Brazilian purpuric fever is a severe vascular disease caused by an invasive clone of Haemophilus influenzae biogroup aegyptius, which normally causes self-limiting eye infections. A previous genome subtraction procedure resulted in the isolation of a DNA fragment, which encodes a putative IgA1 protease, specific to the F3031 Brazilian purpuric fever type strain. Cloning and sequencing of the entire F3031 iga1 gene showed that the subtracted DNA fragment encompasses the iga1 region encoding the active site and the cleavage specificity determinant of the protein, which are different from the cognate regions of the proteases produced by other H. influenzae strains. Western and IgA cleavage assays together with clustering analysis showed that the F3031 IgA1 protease is most similar to the type 2 proteases produced by H. influenzae type c and e strains. Analysis of the promoter region of the F3031 iga1 gene revealed the presence of Fur binding sites. However, real-time PCR analysis and transcriptional fusion assays showed that the expression of iga1 is not regulated by iron or hemin under the conditions tested.

The extended postictal state is characterized by neurological problems in patients. Inadequate blood supply to the brain and impaired cerebral autoregulation may contribute to seizure-induced neuronal damage. Recent evidence in newborn pigs indicates that activation of the antioxidative enzyme heme oxygenase (HO) at the onset of seizures is necessary for increased cerebral blood flow during the ictal episode and for normal cerebral vascular functioning during the immediate postictal period. We hypothesized that seizures cause prolonged postictal cerebral vascular dysfunction that can be accentuated by HO inhibition and rescued by HO overexpression. Cerebral vascular responses to endothelium-dependent (hypercapnia, bradykinin) and -independent (isoproterenol, sodium nitroprusside) stimuli were assessed 48 h after bicuculline-induced seizures in: 1) saline-control newborn piglets, 2) HO-inhibited animals (HO was inhibited by tin protoporphyrin, SnPP, 3 mg/kg iv), and 3) HO-overexpressing piglets (HO-1 was upregulated by cobalt protoporphyrin, CoPP, 50 mg/kg ip). Extended alterations of HO expression in cerebral microvessels were confirmed by measuring CO production and inducible HO (HO-1) and constitutive HO (HO-2) proteins. Our data provide evidence that seizures cause a severe, sustained, postictal cerebral vascular dysfunction as reflected by impaired vascular reactivity to physiologically relevant dilators. During the delayed postictal state, vascular reactivity to all dilator stimuli was reduced in saline control and, to a greater extent, in HO-inhibited animals. In CoPP-treated piglets, no reduction in postictal cerebral vascular reactivity was observed. These findings may indicate that CoPP prevents postictal cerebral vascular dysfunction by upregulating HO-1, a finding that might have implications for preventing postictal neurological complications.

This study investigated the role of heme oxygenase (HO)-1 in the cardiac tissue injury of acute ischemia/reperfusion (I/R) in diabetic streptozotocin (STZ)-induced hyperglycemic rats. The effects of 1) hemin, an inducer of HO expression and activity, and 2) zinc protoporphyrin IX (ZnPP-IX), an inhibitor of HO activity, have also been investigated on the tissue injury by I/R and some mediators released in these circumstances. STZ hyperglycemic rats had impaired levels of HO-1 within the cardiac tissue and increased myocardial infarct size (IS) following I/R, as compared with the nondiabetic rats. In these rats, administration of hemin 4 mg/kg 18 h before I/R increases the levels of HO-1 within the tissue. However, the values of HO-1 assayed in these circumstances were significantly lower (P < 0.01) than those assayed in nondiabetic animals subjected to the same procedures; IS was much more extended (P < 0.01) than in the parent nondiabetic group. STZ hyperglycemic rats also predisposed the heart to produce high levels of the cytokines interleukin (IL)-1beta and CXCL8. Subsequent I/R further increased (P < 0.01) the cytokine production, an effect partly prevented by hemin treatment. This recovered the huge number of infiltrated polymorphonuclear (PMN) leukocytes within the cardiac tissue associated with the STZ hyperglycemic state and I/R damage.

Monocyte activation, apoptosis and differentiation are hallmarks of most inflammatory vascular disorders. We studied the effects of heme oxygenase-1 (HO-1) induced by its substrate hemin on apoptosis, caspase-3 expression and the differentiation of freshly isolated human monocytes. Hemin induced HO-1 in a dose- and time-dependent fashion as measured by semi-quantitative RT-PCR and flow cytometry. Apoptosis was markedly suppressed by hemin in cells rendered apoptotic by serum deprivation or dexamethasone as determined by flow cytometric detection of annexin V binding or transmission electron microscopy (TEM). The specific HO-1 inhibitor zinc protoporphyrin (ZnPP) reversed the effects of hemin on monocyte apoptosis and diminished cell lifespan. Surprisingly, the cytoprotective effects of hemin were positively correlated with caspase-3 up-regulation. Hemin-induced apoptosis suppression was enhanced by the caspase-3 inhibitor DEVD-CHO, indicating that caspase-3 was active in a pro-apoptotic fashion. Hemin inhibited CD95 as a putative cytoprotective mechanism. Morphological studies and detection of CD86 showed that monocytes differentiated into macrophages in response to hemin after relatively long incubation times, a phenomenon that might be provoked by caspase-3-regulated pathways. Our results confirm a similar cytoprotective effect of hemin/HO-1 for monocytes as has been shown for other cells, despite caspase-3 up-regulation. The fact that HO-1 may adversely affect monocyte survival and differentiation could be of particular significance in future therapies for occlusive vascular diseases or transplant rejection.

Porphyromonas gingivalis appears to be a major contributor to periodontal disease, especially soft tissue destruction, which is reflected by the ability to cause invasive, spreading lesions, and tissue inflammation in a murine abscess model. This study investigated the role of hemin on the regulation of growth and virulence of P. gingivalis strains. P. gingivalis strains W50, A7A1-28, 3079, 381, W50/BEI, and NG4B19 were grown in broth and on blood agar plates. P. gingivalis cells grown under iron-depleted conditions for multiple passages showed significantly decreased lesion size in mice, in contrast to cells grown under iron-normal (5 microg/ml) and iron-elevated conditions. Statistically significant (P < 0.01) decreases in gingipain enzyme activity were found among the strains grown under iron-depleted conditions. P. gingivalis grown in the presence of blood induced significantly different lesion type, lesion size, lesion onset, and mortality. Elevated hemin resulted in increased cell-associated iron in P. gingivalis, which increased the capacity of the microorganism to survive at times of iron deprivation. These results indicate that hemin or iron availability regulates multiple aspects related to P. gingivalis virulence, including growth, survival, gingipain levels, and iron accumulation.

Oxidative stress and ferrous metabolism are important in the pathogenesis in Parkinson's disease. In dopaminergic neurons, several stress proteins are upregulated under oxidative stress. To clarify this mechanism, we investigated hemin-related signal transduction and the induction of oxidative stress-related proteins in SH-SY5Y cells. We identified phosphatidylinositol 3-kinase (PI3K) and Nrf2 as important molecules in the induction of heme oxygenase-1, thioredoxin, and peroxiredoxin-I. PI3K-related signal controlled Nrf2 activation, and consequently, PI3K inhibitors blocked the nuclear translocation of Nrf2 and induction of stress proteins. These observations suggest that PI3K and Nrf2 are key molecules in maintaining suitable conditions under oxidative stress and ferrous metabolism.

Background: Hemorrhagic shock-related leukocyte adherence to endothelial cells is a key step in microvascular injury-related organ damage. Heme-oxygenase-1 (HO-1) metabolizes heme, a potent cytotoxic agent, to carbon monoxide and biliverdin. We hypothesized that changing HO-1 expression would change leukocyte adherence after hemorrhagic shock., Methods: Rats were administered hemin, zinc protoporphyrin, or vehicle 6 hours before surgery. HO-1 expression was determined by reverse-transcriptase polymerase chain reaction in various tissues. Shock was induced in urethane-anesthetized animals by decreasing mean arterial blood pressure to 40 mm Hg for 60 minutes, followed by standard resuscitation measures. Leukocyte adherence was measured by intravital microscopy in rat mesenteric venules., Results: Hemin, hemorrhagic shock, and the combination resulted in significantly increased HO-1 expression, whereas zinc-protoporphyrin (ZNPP) resulted in significantly decreased leukocyte adherence. After hemorrhagic shock and hemin administration, leukocyte adherence was significantly decreased 60 minutes into resuscitation (7.92 +/- 2.29 vs. 4.84 +/- 0.71 cells/100 microm, p < 0.05) and significantly increased after ZNPP plus shock (14.08 +/- 3.95, p

A correlation between endogenous hemin and pro-oxidant activity was revealed in serum of beta-thalassemia/hemoglobin E disease (beta-thal/Hb E), which is the most common prevalent type of thalassemia in Thailand. The technique of low temperature electron spin resonance spectroscopy was used for characterization and quantification of high spin ferric heme, which had been identified as hemin (iron (III)-protoporphyrin IX). Hemin was present at levels ranging from 50 to 280 microM in serum of beta-thal/Hb E but not detectable in serum of non-thalassemia. Pro-oxidant activity in serum of beta-thal/Hb E was demonstrated by luminol-mediated chemiluminescence, a sensitive method for screening of free radical generation in vitro. In the presence of H2O2, the chemiluminescence intensity (CL) was about 20 fold enhanced in serum of beta-thal/Hb E, indicating its extensive pro-oxidant activity. The CL showed a good correlation with serum heroin, r = 0.778 (p < 0.001), while the correlations with total serum iron and serum ferritin were 0.260 (p = 0.259) and 0.519 (p = 0.004), respectively. Our finding suggested that serum hemin readily catalyzed free radical reactions and it may contribute a major pro-oxidant in blood circulation of beta-thal/Hb E.

Object: The mechanisms involved in brain edema formation following intracerebral hemorrhage (ICH) have not been fully elucidated. The authors have found that red blood cell lysis plays an important role in edema development after ICH. In the present study, they sought to determine whether degradation products of hemoglobin cause brain edema., Methods: Hemoglobin, hemin, bilirubin, or FeCl2 were infused with stereotactic guidance into the right basal ganglia of Sprague-Dawley rats. The animals were killed 24 hours later to determine brain water and ion contents. Western blot analysis and immunohistochemistry were applied for heme oxygenase-1 (HO-1) measurement. The effects of an HO inhibitor, tin-protoporphyrin (SnPP), and the iron chelator deferoxamine, on hemoglobin-induced brain edema were also examined. Intracerebral infusion of hemoglobin, hemin, bilirubin, or FeCl2 caused an increase in brain water content at 24 hours. The HO-1 was upregulated after hemoglobin infusion and HO inhibition by SnPP-attenuated hemoglobin-induced edema. Brain edema induced by hemoglobin was also attenuated by the intraperitoneal injection of 500 mg/kg deferoxamine., Conclusions: Hemoglobin causes brain edema, at least in part, through its degradation products. Limiting hemoglobin degradation coupled with the use of iron chelators may be a novel therapeutic approach to limit brain edema after ICH.

H(+)-ATPase is considered essential for growth of Lactococcus lactis. However, media containing hemin restored the aerobic growth of an H(+)-ATPase-negative mutant, suggesting that hemin complements proton extrusion. We show that inverted membrane vesicles prepared from hemin-grown L. lactis cells are capable of coupling NADH oxidation to proton translocation.

The contraction of three-dimensional type I collagen gels is regarded as a model of contraction during wound healing and tissue remodeling. Because such a process could contribute to vessel narrowing, we hypothesized that endothelial cells may be able to mediate gel contraction. To demonstrate this, type I collagen was extracted from rat tail tendon and used to prepare collagen gels. Bovine arterial endothelial cells (BAECs) or human pulmonary artery endothelial cells (HPAECs) were then plated on the top of the gels in serum-free Ham's F-12 medium or 2% fetal calf serum-endothelium growth medium-2 (FCS-EGM2), respectively. After 48 hours of attachment, gels were released and floated in 0.2% FCS-Ham's F-12 medium (BAECs) or 2% FCS-EGM2 (HPAECs). Gel size was measured with an image analyzer daily for 5 consecutive days. Gels were then digested with collagenase to quantify DNA and hydroxyproline. BAECs contracted the gels in a time-dependent manner over the 5 days. Contraction was dependent on cell density (gel size was 100% of initial size after 5 days with no cells vs. 66.4%+/-0.5% with 0.9x10(4) cells/cm2 and 22.1%+/-0.3% with 7.5x10(4) cells/cm2) and was inversely related to collagen concentration (gel size was 22.3%+/-0.05%, 46.4%+/-0.9%, 72.3%+/-0.4%, and 87.4% +/-0.3% of initial size for gels prepared with 0.5 mg/mL, 0.75 mg/mL, 1 mg/mL, and 2 mg/mL of collagen, respectively). Hemin (a precursor for CO) and cytochalasin D inhibited collagen gel contraction mediated by both bovine and human endothelial cells without changing cell number or hydroxyproline content. In contrast, prostaglandin E2, an inhibitor, and transforming growth factor-beta1, a stimulator of fibroblast-mediated gel contraction, had no effect on endothelial cell-mediated contraction. These findings demonstrate that endothelial cells are able to contract native type I collagen gels and that this process can be modulated by exogenous mediators. Such a capability may cause remodeling of subjacent matrix of endothelial cells and may contribute to vessel narrowing.

Treatment of HeLa cells or human skin fibroblast cells with hemin led to a time- and dose-dependent rapid induction of c-fos mRNA. This induction was absent in the cells treated with actinomycin D, indicating that the c-fos induction by hemin occurs at the level of transcription. Metalloporphyrins, including zinc-, cobalt-, and tin-protoporphyrin, ferric ion, and protoporphyrin also induced c-fos mRNA. Transient reporter assay with the reporter constructs of the human c-fos gene promoter up to -404 bp connected to the luciferase gene showed high activity but no induction by hemin, suggesting that cis-acting elements, including the serum response element located about -310 bp upstream of the human c-fos gene promoter, may not contribute to the heme-dependent induction. With in-gel assay of protein kinases, the activity of the mitogen-activated protein (MAP) kinases such as extracellular signal-regulated kinase 12 or p38 MAP kinase in hemin-treated HeLa cells was not stimulated. Stimulation of c-Jun N-terminal kinase by hemin was nil. Furthermore, PD58059 and SB203580, inhibitors for MAP kinases, did not affect the hemin-dependent c-fos induction. Of the inhibitors for protein kinases so far tested, KN-62, a specific inhibitor for calmodulin-dependent protein kinase II (CaMK II), inhibited the induction of c-fos mRNA by hemin. Phosphorylation of CaMK II in hemin-treated cells increased. With gel mobility assay, the DNA AP-1 binding activity transiently increased when treating HeLa cells with hemin. Therefore, induction of c-fos led to an activation of AP-1 in the presence of hemin. We suggest that calmodulin-dependent protein kinase II rather than the MAP kinase family regulates the induction of the human c-fos gene expression by hemin., (Copyright 1999 Academic Press.)

Heat shock transcription factor 2 (HSF-2) activates transcription of heat shock proteins in response to hemin in the human erythroleukemia cell line, K562. To understand the regulation of HSF-2 activation, a series of deletion mutants of HSF-2 fused to the GAL-4 DNA binding domain were generated. We have found that human HSF-2 has a regulatory domain located in the carboxyl-terminal portion of the protein which represses the activity of its activation domain under normal physiological conditions. The repressive effects of this domain can be eliminated by its deletion in GAL4-HSF-2 fusion constructs. The regulatory domain of HSF-2 can also repress a heterologous chimeric activator that contains a portion of the VP16 activation domain. The activation domain of HSF-2 is a segment of approximately 77 amino acids located proximal to the carboxyl-terminal hydrophobic heptad repeat (leucine zipper 4) of the molecule. Interestingly, the GAL4-HSF-2 fusion protein and the 77 amino acids activation domain are inactive and are not activated by pretreatment of cells with either hemin or elevated temperature. Our data suggest that regulation of HSF-2 differs from HSF-1 in that its regulatory domain is not responsive to hemin or heat directly.

The heme-regulated eukaryotic initiation factor 2alpha (eIF2alpha) kinase (heme-regulated inhibitor (HRI)) is activated by heme deficiency in reticulocytes and plays an important role in translational control in these cells. Previously, HRI was cloned from rabbit reticulocytes and rat brain, but a heme-regulated eIF2alpha kinase activity has only been purified from erythroid cells. In this study, we report the purification of a heme-sensitive eIF2alpha kinase activity from both mouse liver and NIH 3T3 cell extracts. Furthermore, we have cloned and characterized this mouse liver eIF2alpha kinase (mHRI), which exhibits 83 and 94% identities to rabbit and rat HRIs, respectively. Both the purified enzyme and recombinant mHRI exhibited an autokinase and an eIF2alpha kinase activity, and both activities were inhibited in vitro by hemin. In addition, wild-type mHRI, but not the inactive mHRI-K196R mutant, was autophosphorylated in vivo when it was expressed in 293 cells. Quantitation of mHRI mRNA expression in various mouse tissues by reverse transcription-polymerase chain reaction revealed relatively high levels in liver, kidney, and testis. These results provide strong evidence that mHRI is a ubiquitous eIF2alpha kinase of mammalian cells, suggesting that it could play important roles in the translational regulation of nonerythroid tissues.

Transcriptional induction of the hsp70 gene is mediated by heat shock factor 1 (HSF1) rapidly activated upon heat and other stresses. HSF2 has been thought to be responsible for accumulation of HSP70 during hemin-induced differentiation of human K562 erythroleukemia cells because of accompanying acquisition of HSF2 DNA binding activity. However, there has not been any direct evidence for such a functional role of HSF2. The purpose of this study is to clarify the roles of HSF1 and HSF2 in HSP70 induction in hemin-treated K562 cells. We show here that a chimeric polypeptide of HSF2 and GAL4 DNA binding domain (GAL4-BD-HSF2) was unable to induce a GAL4 binding site-containing luciferase reporter gene in response to hemin and that exogenously overproduced HSF2 also failed to increase expression of a heat shock element-containing reporter. On the contrary, expression of a GAL4-BD-HSF1 chimeric protein responded to hemin treatment as well as to heat shock, and transiently overexpressed HSF1 caused hemin-responsive induction of the reporter gene in a dose-dependent manner. These results indicate that HSF1, rather than HSF2, primarily mediates the hemin-induced transcription of the hsp70 gene.

P. gingivalis is considered to be a major pathogen of adult periodontitis. Among its cadre of putative virulence factors are hemagglutinins (adhesins) and proteases. We here report the cloning, sequencing and characterization of two genes, designated kgp(381) and hagD. Kgp(381), an open reading frame (ORF) of 1095 bp encoding a 40.1 kda protein, has high homology to the proteolytic domain of cysteine protease/hemagglutinin genes. HagD, an ORF of 4077 bp encoding a 147.1 kda protein, contains one HArep sequence which establishes it as an additional member of the HArep multigene family. Although similar in sequence to kgp and prtP which were identified from strains HG66 and W12, respectively, the kgp(381)-hagD genes have several characteristics which distinguish them from kgp and prtP. Foremost among these is a single base difference which produces a termination codon and an immediate frame shift resulting in two ORFs in strain 381 as compared to one ORF in strains HG66 and W12. In addition, a 172 amino acid sequence near the C-terminal end of hagD has very low identity (20.5-27.8%) to the corresponding region of kgp and prtP. These demonstrate that the homologue of kgp and prtP in strain 381 occurs as two separate genes which may genetically separate the adhesive and enzymatic domains of Kgp and PrtP proteins. Reverse polymerase chain reaction (PCR) analysis indicates that hagD expression is regulated by hemin concentration.

We are interested in the genetic mechanisms whereby several classes of drugs increase fetal hemoglobin (HbF) in patients with sickle-cell anemia or beta-thalassemia. Recently, we have shown (Kollia et al., Proc. Natl. Acad. Sci. U.S.A. 93: 5693, 1996) that cultured primary human adult erythroid cells (hAEC) offer a useful model for the study of transcriptional and posttranscriptional regulation of globin gene expression. We have found also that hemin markedly increases HbF levels in these cells. We report here the effect of hemin on globin gene transcription and RNA processing in hAEC. Quantitative reverse transcriptase-polymerase chain reaction analysis showed that the gamma-globin message levels in the cytoplasm and nucleus were increased two-fold by hemin. In the untreated cells, only spliced gamma-transcripts were detected in the cytoplasm, indicating that only completely processed gamma-RNA is transported to the cytoplasm, whereas approximately half of the nuclear gamma-globin RNA transcripts were unspliced. After treatment with hemin, correctly spliced gamma-transcripts increased in the cytoplasm and nucleus, while the unprocessed gamma-transcripts decreased in number in the nucleus. We also studied epsilon-globin RNA transcripts; in the cytoplasm of untreated cells, only correctly processed transcripts were present, whereas the nuclear epsilon-globin RNA transcripts were unspliced. In hemin-induced cells, unspliced nuclear epsilon-transcripts decreased in number. In contrast to the gamma- and epsilon-globin genes, the levels of full-length, correctly spliced beta-globin message are not affected by hemin. Nuclear run-on transcription assays confirmed the increase in the rate of transcription of gamma- and epsilon-globin genes in hemin-treated versus untreated hAEC. These results indicate that hemin affects the expression of embryonic and fetal globin genes by acting both at the transcriptional and posttranscriptional levels. These results may be relevant to the action of other agents that affect the hemoglobin phenotype of human erythroid cells.

Acquisition of heat shock factor 2 (HSF2) DNA binding activity is accompanied by induced transcription of heat shock genes in hemin-treated K562 cells undergoing erythroid differentiation. Previous studies revealed that HSF2 consists of two alternatively spliced isoforms, HSF2-alpha and HSF2-beta, whose relative abundance is developmentally regulated and varies between different tissues. To investigate whether the molar ratio of HSF2-alpha and HSF2-beta isoforms is crucial for the activation of HSF2 and whether the HSF2 isoforms play functionally distinct roles during the hemin-mediated erythroid differentiation, we generated cell clones expressing different levels of HSF2-alpha and HSF2-beta. We show that in parental K562 cells, the HSF2-alpha isoform is predominantly expressed and HSF2 can be activated upon hemin treatment. In contrast, when HSF2-beta is expressed at levels exceeding those of endogenous HSF2-alpha, the hemin-induced DNA binding activity and transcription of heat shock genes are repressed, whereas overexpression of HSF2-alpha results in an enhanced hemin response. Furthermore, the hemin-induced accumulation of globin, known as a marker of erythroid differentiation, is decreased in cells overexpressing HSF2-beta. We suggest that HSF2-beta acts as a negative regulator of HSF2 activity during hemin-mediated erythroid differentiation of K562 cells.

One way prokaryotes respond to environmental stresses is by modifying selected outer membrane components. Iron, in the form of hemin, has been shown to be a significant regulator of Porphyromonas gingivalis growth and virulence and of the expression of outer membrane proteins and lipopoly saccharide. Since lipopoly saccharide has profound effects on host immune cells, this study compared the effect of hemin-restricted and hemin-normal P. gingivalis growth conditions on lipopolysaccharide priming of N-formylmethionyl-leucyl-phenylalanine-induced superoxide generation by human neutrophils. P. gingivalis was grown in a chemostat under normal (5 micrograms hemin/ml) and hemin-restricted (0.08 microgram hemin/ml) conditions. Purified lipopolysaccharide from both P. gingivalis normal and hemin-limited environments increased N-formylmethionyl-leucyl-phenylalanine-induced superoxide release by neutrophils in a dose-dependent manner. Lipopolysaccharide isolated from the hemin-normal conditions was a significantly more potent neutrophil priming agent than the lipopolysaccharide isolated from hemin-restricted conditions. Addition of normal human serum enhanced the priming effect of both lipopolysaccharide preparations; this effect, however, was more evident with the hemin-normal lipopolysaccharide. Further, this enhancing effect of serum was partly reduced in the presence of antibodies raised against the serum lipopolysaccharide-binding protein. The differences in the biological activity of the two lipopolysaccharide preparations could be associated with structural differences detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These results indicate that hemin availability affects regulation of an aspect of P. gingivalis virulence, lipopolysaccharide-human neutrophils priming. The reduced capacity for neutrophil priming by hemin-restricted lipopolysaccharide appears to be related to lipopolysaccharide-neutrophil interactions and not to serum factors Targeting bacterial cell-surface components involved in hemin transport might be effective therapy for P. gingivalis-associated periodontal diseases.

Background: We previously reported that hemin-induced mitogenicity in mouse splenocytes was potentiated up to two-fold by interleukin (IL)-2, and the combination of hemin and IL-2 was also effective in inducing cytotoxicity for NK-resistant target cells. The purpose of this study was to investigate the effects of hemin both on expression of membrane surface receptors and on production of cytokine in human peripheral blood mononuclear cells (PBMC)., Methods: Human PBMC were obtained from 16 healthy volunteers. We analyzed hemin-mediated surface phenotypes of cells using flow cytometry, and levels of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma in the culture-supernatant of cells by ELISA system., Results: Hemin induced increased expression of both transferrin receptor and IL-2 receptor (CD25) on PBMC in a dose-dependent manner. When the combination of hemin (30 mumol/L) and IL-2 (100 U/mL) was added to the culture of PBMC, the population of double positive cells was increased up to 70.4%. These effects of hemin were enhanced by the addition of catalase in the culture. Treatment with hemin plus IL-2 effectively enhanced the production of TNF-alpha and IFN-gamma in PBMC., Conclusion: Hemin upregulated both IL-2 receptor and transferrin receptor expression, and stimulated TNF-alpha and IFN-gamma production in PBMC. IL-2 cooperated with hemin in eliciting these effects.

In this study, we investigated whether Porphyromonas gingivalis can bind hemoglobin as an initial step in the acquisition of heme from hemoglobin. The binding of human hemoglobin by P. gingivalis cells was determined using [3H]hemoglobin. Hemoglobin binding occurred rapidly, reversibly and specifically. A Scatchard analysis of the binding data generated a linear plot, indicating a single population of binding proteins. The apparent Kd was 1.0 +/- 0.19 x 10(-6) M and there were 3.2 +/- 0.76 x 10(4) binding sites per cell. Hemoglobin binding was inhibited by unlabeled human hemoglobin but not by hemin and protoporphyrin IX. The binding was only partially inhibited by human serum albumin, transferrin, lactoferrin, catalase and cytochrome c. These results suggest that the ligand recognized by the binding protein may not be the heme moiety. The binding of hemoglobin considerably increased when the organisms were grown under hemin-limited conditions. Hemoglobin bound to outer membrane proteins extracted from P. gingivalis cells on a dot blot binding assay and binding ability was lost after heating bacterial proteins. These results suggest that P. gingivalis cells interact with human hemoglobin through specific binding sites on their surfaces as a preliminary step in iron acquisition.

Hemin is a prominent breakdown product of hemoglobin, and high levels of hemin are found in the cerebrospinal fluid during subarachnoid hemorrhage-induced vasospasm. The possible role of hemin in modifying vascular function was examined in the present study by testing its effects on nitric oxide synthase (NOS) activity in cultured rat aortic smooth-muscle cells. Nitric oxide synthase activity was estimated from the amounts of accumulated nitrite and nitrate, which are oxidative products of nitric oxide (NO). Hemin (1-100 microM) increased the levels of nitrite and nitrate in culture medium in a dose- and time-dependent manner. The hemin-induced elevation of nitrite and nitrate was inhibited significantly by the NOS inhibitor, N omega-nitro-L-arginine (300 microM), and by the protein synthesis inhibitor, cycloheximide (5 micrograms/ml). These results indicate that hemin is capable of stimulating the expression of an inducible isoform of NOS (iNOS) in vascular smooth muscle. Transcriptional expression of iNOS is known to cause injurious effects on the maintenance of cellular homeostasis by generating extremely high levels of NO. The generation of hemin from methemoglobin during hemolysis of a subarachnoid blood clot could therefore stimulate an excessive production of NO in vascular smooth-muscle cells. It is postulated that this series of events contributes to the development of vascular injury associated with cerebral vasospasm after aneurysmal subarachnoid hemorrhage.

Cerebral arteries in spasm have been found to contain low levels of adenosine triphosphate (ATP), and it has been postulated that this change in levels results from hypoxia produced by arterial encasement in clotted material. This study was undertaken to determine whether any of four blood-derived agents, ferrous hemoglobin, methemoglobin, hemin, or bilirubin, is capable of reducing energy levels in cerebral artery smooth-muscle cells. Twenty-four-hour exposure of cultured canine basilar artery cells to ferrous hemoglobin and bilirubin led to a significant decline in ATP levels (to 8.9 nmol/mg protein and 2.8 nmol/mg protein, respectively) versus control (16.6 nmol/mg protein); methemoglobin and hemin showed no effect. Bilirubin but not hemoglobin was found to interfere with electron transport and with creatine phosphokinase activity in intact cells; however, bilirubin showed no inhibitory effect on this enzyme in cell-free conditions. The findings indicate that hemoglobin and bilirubin may be responsible for diminished energy levels in cerebral arteries. These observations also suggest that bilirubin may exert its effect on ATP by impairing mitochondrial function.

We have purified a soluble rabbit reticulocyte protein, previously termed the supernatant factor, that reverses the inhibition of protein synthesis in hemin-deficient lysate by promoting the inactivation of the hemin-controlled eIF-2 alpha kinase (HCR) mediating the effect of hemin deficiency. We have identified the supernatant factor as a member of the heat shock protein 70 family, denoted hsp 70(R), based upon its size (72 kDa), specific reaction to a monoclonal antibody against eukaryotic hsp 70, strong binding affinity for ATP, and endogenous ATPase activity. We have investigated the role of hsp 70(R) and hemin in the regulation of the activation of HCR from its latent precursor (ProHCR) and the translational control of protein synthesis in rabbit reticulocyte lysate. We find that autophosphorylation of Pro-HCR is reduced by about 75% by adding saturating hsp 70(R) and almost completely reduced by adding either saturating hemin or limiting hemin plus limiting hsp 70(R). In contrast, autophosphorylation of HCR, which is similar in magnitude to that of ProHCR, is unaffected by adding either saturating hsp 70(R), saturating hemin, or limiting amounts of both. The activation of HCR (measured by inhibition of protein synthesis) from isolated ProHCR is completely prevented by hsp 70(R) in the presence, but not absence, of dithiothreitol. This suppression appears to be due to the association of hsp 70(R) with ProHCR, since hsp 70(R) action is prevented by ATP/Mg2+ and because activation of HCR from less purified ProHCR, that has associated hsp 70(R), is suppressed by dithiothreitol alone. This association is confirmed by sucrose gradient centrifugation, which shows co-sedimentation of some hsp 70(R) with ProHCR following preincubation together that is prevented by ATP/Mg2+ and does not occur after conversion of ProHCR to HCR. Limiting hsp 70(R) reduces the concentration of hemin required to prevent activation of HCR from isolated ProHCR from 0.75 to 0.15 microM and the optimal hemin concentration needed to maintain protein synthesis in reticulocyte lysate from 25 to 10 microM. Limiting hsp 70(R) also allows the delayed addition of hemin to suppress activation of HCR from ProHCR and to reverse inhibition of protein synthesis in hemin deficient lysate. The association of hsp 70(R) with ProHCR also underlies the observation that much more protein is synthesized in reticulocyte lysate in the absence of hemin at 25 degrees C than at temperatures of 30 degrees C or greater. These observed effects may be specific to hsp 70(R), since they are not observed with rabbit reticulocyte eIF-2 or eIF-2B, and since the comparable hsp 70 from bovine brain is incapable of maintaining or restoring protein synthesis in hemin-deficient lysate.

Hemin hemolysis of human erythrocytes at 37 degrees C has been investigated by the kinetic method. It has been observed that hemin affects erythrocytes by two mechanisms, inducing "quick" and "slow" hemolysis. The "quick" hemolysis is completed after the first few minutes. The final level of "quick" hemolysis is a function of relative hemin concentration. It is equal to zero when the latter is smaller than the minimum concentration Cmin = 10(8) molecules of added hemin per cell. The final level of "slow" hemolysis is always 100%. It is suggested that the limiting step of autohemolysis is the membrane proteins oxidation catalyzed by endogenous hemin originating from methaemoglobin during erythrocyte incubation at 37 degrees C.

Although hemin is known to exert toxic effects on a variety of cell types, its possible participation in the genesis of cerebral vasospasm has received little attention. The authors measured the concentration of hemin in experimental subarachnoid clot and studied its effects on the morphology and 45Ca++ uptake of vascular smooth-muscle cells dissociated from canine carotid artery. Craniectomies were performed in five dogs under general anesthesia, and 3 to 5 ml of autologous whole blood was deposited in the supraclinoid subarachnoid compartment. The concentration of hemin recovered by Folch extraction from clotted material removed 7 days after surgery was 390 +/- 247 microM (mean +/- standard error of the mean). Mean vascular smooth-muscle cell length after 40 minutes of exposure to 50 microM hemin was 37.3 +/- 1.2 microns (control 51.6 +/- 1.6 microns) (p < 0.01). The mean percent permeation of 45Ca++, measured by a dual label technique, of cells exposed to hemin was 200.9% +/- 23% (control 102.9% +/- 4.3%) (p < 0.01). These findings indicate that hemin accrues in subarachnoid hematoma, that it exerts a constrictive effect on vascular smooth-muscle cells, and that this effect is associated with an increased uptake of Ca++. This study demonstrates that hemin should be included in the list of potential agents that participate in the development of cerebral vasospasm.

Based on the unusually high and stage-dependent susceptibility of Plasmodia to oxidant stress it has been proposed that during parasite development, increasing levels of redox-active forms of iron are gradually released. The purpose of this study was to examine this proposal by using an assay monitoring the levels of available forms of iron for redox reactions. Ascorbate-driven and iron-mediated degradation of adventitious DNA served as the basis for this functional assay. Incubation of DNA with lysate from infected RBC caused massive degradation, which was dose, time- and parasite-stage dependent. In contrast, lysate from non-infected RBC did not induce DNA degradation. Likewise, lysate only from infected RBC enhanced the aerobic oxidation of ascorbate. These effects on both reaction, DNA degradation and ascorbate oxidation, could be reconstructed with hemin, instead of lysate. Also, chelators exerted similar effects on both reactions. The results suggest that increased levels of redox-active forms of iron are liberated during parasite development. We propose that hemin or hemin-like structures are the appropriate candidates which could catalyze oxidative stress and deregulate the delicate redox balance of the host-parasite system.

1. Heme synthesis from delta-aminolevulinic acid (delta-ALA) in freshly isolated rat hepatocytes was maximal at 100 microM with a rate of approx. 7 nmol being synthesized per g wet weight cells. 2. Approximately 8% of synthesized heme was converted to bilirubin and 50% of the newly synthesized bilirubin was conjugated. 3. The ratio of di to monoconjugate was approx. 2.5. Incorporation of delta-ALA into bilirubin was increased by additional delta-ALA, heme and was also doubled in cells isolated from animals treated with CoCl2. 4. Bilirubin formation was inhibited approx. 90% by in vitro treatment with heme oxygenase inhibitors zinc and tin protoporphyrin.

Hemin induces nonterminal differentiation of human K562 erythroleukemia cells, which is accompanied by the expression of certain erythroid cell-specific genes, such as the embryonic and fetal globins, and elevated expression of the stress genes hsp70, hsp90, and grp78/BiP. Previous studies revealed that, as during heat shock, transcriptional induction of hsp70 in hemin-treated cells is mediated by activation of heat shock transcription factor (HSF), which binds to the heat shock element (HSE). We report here that hemin activates the DNA-binding activity of HSF2, whereas heat shock induces predominantly the DNA-binding activity of a distinct factor, HSF1. This constitutes the first example of HSF2 activation in vivo. Both hemin and heat shock treatments resulted in equivalent levels of HSF-HSE complexes as analyzed in vitro by gel mobility shift assay, yet transcription of the hsp70 gene was stimulated much less by hemin-induced HSF than by heat shock-induced HSF. Genomic footprinting experiments revealed that hemin-induced HSF and heat shock-induced HSF, HSF2, and HSF1, respectively, occupy the HSE of the human hsp70 promoter in a similar yet not identical manner. We speculate that the difference in occupancy and/or in the transcriptional abilities of HSF1 and HSF2 accounts for the observed differences in the stimulation of hsp70 gene transcription.

Nuclear extract of human erythroleukemic cell line K562 contains a 70 kDa protein which is gradually reduced when cells are induced to express globin genes by 25 microM hemin. When globin synthesis was inhibited by cycloheximide (100 micrograms/ml) or Actinomycin D (1 microgram/ml), the disappearance of this protein was prevented. The 70 kDa nuclear protein exhibited strong binding to G gamma and A gamma globin promoters but not to beta-globin promoter. This suggests that this 70 kDa nuclear protein may be involved in the regulation of fetal globin gene expression.

Hemin stimulates cAMP production in human peripheral blood mononuclear cells (PBMC). The kinetics are similar to that of hormone-induced cAMP generation, namely a rapid effect followed by a desensitization phase. Several experimental findings suggest that prostaglandins do not mediate this effect. First, macrophage depleted T and B cells purified by erythrocyte-rosetting were as responsive as unfractionated PBMC to hemin. Second, indomethacin, an inhibitor of prostaglandin synthesis, and meclofenamate, a prostaglandin E2 receptor antagonist, had no effect on hemin stimulated cAMP production. In addition, propranolol, a beta-adrenergic receptor antagonist, had no effect on hemin-stimulated cAMP production. We also examined structural analogues of hemin. Among the metalloporphyrins (Fe, Ni, Co, Zn and Sn) and protoporphyrin IX tested only hemin (Fe-protoporphyrin) was active in stimulating cAMP production. No correlation was found between the ability of metalloporphyrins to stimulate cAMP production and their ability to generate H2O2. The data indicate that hemin stimulates cAMP production by directly affecting lymphocytes and that prostaglandins do not mediate this effect.

The antigens from outer membrane protein extracts of Porphyromonas gingivalis (W50), grown under different haemin concentrations, were examined for binding with serum antibodies from patients with severe progressive periodontitis or from periodontally healthy control subjects. P. gingivalis was grown under haemin limitation (0.33 micrograms/ml) and haemin excess (2.5 micrograms/ml) conditions in a chemostat at a mean generation time of 6.9 h, at pH 7.5. Sarkosyl-insoluble fractions of outer membrane proteins from P. gingivalis were prepared, and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot techniques. The SDS-PAGE analysis of the outer membrane of haemin-limited P. gingivalis identified several new protein components, or changed expression of bands compared with cells grown under haemin excess. Immunoblot analysis showed IgG antibodies to 2 haemin deprivation-induced proteins in patients with severe progressive periodontitis, but not in the control sera. These results confirm the immunogenicity of some of the haemin-regulated outer membrane proteins of P. gingivalis in severe progressive periodontitis.

Oxidized low density lipoprotein (LDL), formed in vivo from presently unknown reactions, may play a role in atherogenesis. In vitro, transition metals such as iron and copper will facilitate LDL oxidation, but these metals are unlikely to exist in free form in normal body fluids. We have explored the possibility that LDL oxidation may be promoted by heme, a physiologically ubiquitous, hydrophobic, iron-containing compound. Indeed, during several-hour incubation, heme caused extensive oxidative modification of LDL; however, such modification requires only minutes in the presence of small amounts of H2O2 or preformed lipid hydroperoxides within the LDL. Oxidative interactions between heme, LDL, and peroxides lead to degradation of the heme ring and consequent release of heme iron, which further accelerates heme degradation. Coupled (evidently iron-catalyzed) heme degradation and LDL oxidation are both effectively inhibited by hydrophobic antioxidants and iron chelators. That such hemin-induced LDL oxidation may be involved in atherogenesis is supported by the finding that LDL oxidized by hemin is extremely cytotoxic to cultured aortic endothelial cells. Overall, these investigations not only lend support to the idea that LDL oxidation by physiological substances such as heme may play a role in the process of atherogenesis but also may have broader implications, as similar oxidative reactions between heme and unsaturated fatty acids may occur consequent to hemorrhagic injury.

Erythropoietin (Ep) regulates the proliferation and differentiation of erythroid progenitor cells, but whether it functions solely as a survival factor or also acts as a mitogen is unresolved. Because late erythroid progenitor cells (CFU-E) are largely in cell cycle, we examined this issue by using an Ep-dependent, murine erythroleukemia cell line, HCD-57. In the presence of human Ep and fetal calf serum, HCD-57 cells had a doubling time of approximately 24 hours, and during log-phase growth approximately 36% of the cells were in G1, 45% in S, and 19% in G2/M. With Ep deprivation, there was a gradual loss of viability and an arrest of proliferation with a 44% increase in the G0/G1 population, which could be reversed by reexposure to Ep even after 72 hours of hormone withdrawal. As little as 2 hours of exposure to Ep was sufficient to stimulate DNA synthesis, and the lag time for initiation of DNA synthesis after exposure to the hormone was approximately 10 hours as measured by either incorporation of labeled thymidine into DNA or cell cycle analysis by flow cytometry. RNA synthesis, by contrast, was initiated within 2 hours after exposure to Ep and did not require DNA synthesis. Total cell DNA content increased after exposure to Ep, indicating that it was acting as mitogen in HCD-57 cells. Ep was also able to stimulate DNA synthesis in the absence of serum as well as in its presence, indicating that the hormone could act as both a competence and a progression factor. Qualitative analysis of the integrity of HCD-57 DNA by electrophoresis in agar as well as direct measurement of DNA fragmentation after metabolic labeling with radioactive thymidine indicated that programmed cell death was occurring that could be reduced but not completely prevented by Ep. These data indicate that Ep acts as both a mitogen and a survival factor for HCD-57 cells.