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Heme oxygenase-1 (HO-1) catalyzes the first and rate-limiting enzymatic step of heme degradation, producing carbon monoxide, biliverdin, and free iron. Most iron is derived from aged erythrocytes by the decomposition of heme, which happened mainly in macrophages. However, the role of HO-1 on iron metabolism and function of macrophage is unclear. The present study investigated the effect of HO-1 on iron metabolism in macrophages, and explored the role of HO-1 on inflammatory response, polarization, and migration of macrophages. HO-1 inducer Hemin or HO-1 inhibitor zinc protoporphyrin was intravenously injected to C57BL/6 J mice every 4 d for 28 d. We found that HO-1 was mainly located in the cytoplasm of splenic macrophages of mice. Activation of HO-1 by Hemin significantly increased iron deposition in the spleen, up-regulated the gene expression of ferritin and ferroportin, and down-regulated gene expression of divalent metal transporter 1 and hepcidin. Induced HO-1 by Hemin treatment increased intracellular iron levels of macrophages, slowed down the absorption of extracellular iron, and accelerated the excretion of intracellular iron. In addition, activation of HO-1 significantly decreased the expression of pro-inflammatory cytokines including interleukin (IL)-6, IL-1β, and inducible nitric oxide synthase, but increased the expression of anti-inflammatory cytokines such as IL-10. Furthermore, activation of HO-1 inhibited macrophages to M1-type polarization, and increased the migration rate of macrophages. This study demonstrated that HO-1 was able to regulate iron metabolism, exert anti-inflammatory effects, and inhibit macrophages polarization to M1 type., (© The Author(s) 2023. Published by Oxford University Press.)

Heme b (iron protoporphyrin IX) plays important roles in biology as a metallocofactor and signaling molecule. However, the targets of heme signaling and the network of proteins that mediate the exchange of heme from sites of synthesis or uptake to heme dependent or regulated proteins are poorly understood. Herein, we describe a quantitative mass spectrometry (MS)-based chemoproteomics strategy to identify exchange labile hemoproteins in human embryonic kidney HEK293 cells that may be relevant to heme signaling and trafficking. The strategy involves depleting endogenous heme with the heme biosynthetic inhibitor succinylacetone (SA), leaving putative heme-binding proteins in their apo-state, followed by the capture of those proteins using hemin-agarose resin, and finally elution and identification by MS. By identifying only those proteins that interact with high specificity to hemin-agarose relative to control beaded agarose in an SA-dependent manner, we have expanded the number of proteins and ontologies that may be involved in binding and buffering labile heme or are targets of heme signaling. Notably, these include proteins involved in chromatin remodeling, DNA damage response, RNA splicing, cytoskeletal organization, and vesicular trafficking, many of which have been associated with heme through complementary studies published recently. Taken together, these results provide support for the emerging role of heme in an expanded set of cellular processes from genome integrity to protein trafficking and beyond., (© The Author(s) 2023. Published by Oxford University Press.)

Hemin, a substrate of heme oxygenase (HO)-1, induces HO-1 expression on a variety of cells to exert anti-oxidant and anti-inflammatory roles. However, the role of HO-1 in allergic diseases for dendritic cells (DCs) is not fully understood. Here, we report that HO-1 modulates asthmatic airway inflammation by hemin-treated DC-released extracellular vesicles (DCEVs). Following induction of bone marrow-derived DCs by hemin and then by house dust mite (HDM) in vitro, mouse CD4+ naïve T cells were cocultured with DCEVs to determine T helper (h) cell differentiation. C57BL/6 mice were sensitized by different stimuli-induced DCEVs and challenged with HDM to analyze the changes of inflammatory cells and cytokines in the lung and bronchoalveolar lavage fluid. The results showed that hemin-treated DCEVs (hemin-DCEVs) express phosphatidylserine (PS), CD81, heat shock protein 70, and HO-1, which facilitates regulatory T (Treg) cells differentiation in vitro and in vivo. In HDM-induced asthmatic mouse model, hemin-DCEVs inhalation reduced eosinophils infiltration and mucus secretion in the airway, decreased the levels of IL-4, IL-5, and IL-13 in the lung and the number of Th2 cells in mediastinal lymph nodes (MLNs), and increased the number of Treg cells in MLNs. Thus, our study demonstrated, for the first time, that EVs from HO-1-overexpressing DCs alleviate allergic airway inflammation of eosinophilic asthma by potentiating Treg cells differentiation and limiting proinflammatory cytokine secretion, which expands our understanding of HO-1 function, opening the door for HO-1 inducer-like hemin as a novel therapeutic strategy for asthma or other allergic diseases., (© 2021 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals LLC on behalf of Society for Leukocyte Biology.)

G-quadruplex (G4)/hemin DNAzyme is promising horseradish peroxidase (HRP)-mimic candidate in the biological field. However, its relatively unsatisfactory catalytic capacity limits the potential applications. Inspired by nature protease, we conducted a proximity-enhanced cofactor assembly strategy (PECA) to form an exceptional HRP mimic, namely zippered G4/hemin DNAzyme (Z-G4/H). The hybridization of short oligonucleotides induced proximity assembly of the DNA-grafted hemin (DGH) with the complementary G4 sequences (cG4s), mimicking the tight configuration of protease cofactor and apoenzyme. The detailed investigations of catalytic efficiency and mechanism verified the higher activity, more rapid catalytic rate and high environmental tolerance of the Z-G4/H than the classical G4/hemin DNAzymes (C-G4/H). Furthermore, a proximity recognition transducer has been developed based on the PECA for sensitive detection of gene rearrangement and imaging human epidermal growth factor receptor 2 protein (HER2) dimerization on cell surfaces. Our studies demonstrate the high efficiency of Z-G4/H and its universal application potential in clinical diagnostics and biomolecule interaction research. It also may offer significant opportunities and inspiration for the engineering of the protease-free mimic enzyme., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Heme is a ubiquitous compound of human tissues, and it is involved in cellular physiology and metabolism. Once released from the cell, free heme oxidizes to the ferric state (hemin). High levels of hemin can cause oxidative stress and inflammation if not neutralized immediately by specialized scavenger proteins. Human alpha1-antitrypsin (A1AT), an acute-phase glycoprotein and important inhibitor of neutrophil proteases, is also a hemin-binding protein. A short-term exposure of freshly isolated human blood neutrophils to 4 µM hemin results in cell spreading, surface expression of filament protein, vimentin, free radical production, expression of heme oxygenase-1 (HO-1), release of IL-8, and enhanced neutrophil adhesion to human endothelial cells. Consequently, the phosphorylation of protein kinase C (PKC) occurs after 25 min. Under the same experimental conditions, addition of 1 mg/ml A1AT markedly reduces or abolishes neutrophil-activating effects of hemin and prevents PKC phosphorylation. In a mouse model of acute kidney injury (AKI) plus injection of hemin, monotherapy with 4 mg/mouse A1AT significantly lowered serum levels of free hemin at 2 h after surgery. Moreover, a tendency toward lower AKI scores, reduced infiltration of neutrophils, and lower levels of serum chemokine [CXCL1/keratinocyte-derived chemokine (KC)] was observed. Our findings highlight A1AT as a potential serum scavenger of hemin and suggest that the commercial preparations of human plasma A1AT might prove to be useful therapeutics in conditions associated with hemolysis., (© Society for Leukocyte Biology.)

Hemolytic anemia is one of the hallmarks of malaria and leads to an increase in oxidized heme (hemin) within the plasma of infected individuals. While scavenger proteins sequester much of the circulating heme, it has been hypothesized that extracellular heme may play a central role in malaria pathogenesis. We have previously developed the multiplex fluidic plunger (MFP) device for the measurement of red blood cell (RBC) deformability. Here, we demonstrate that the measurement of changes in RBC deformability is a sensitive method for inferring heme-induced oxidative stress. We further show that extracellular hemin concentration correlates closely with changes in RBC deformability and we confirm that this biophysical change correlates with other indicators of cell stress. Finally, we show that reduced erythrocyte deformability corresponds with both erythrophagocytosis and RBC osmotic fragility. The MFP microfluidic device presents a simple and potentially inexpensive alternative to existing methods for measuring hemolytic cell stress that could ultimately be used to perform clinical assessment of disease progression in severe malaria.

G-quadruplex (G4) with stacked G-tetrads structure is able to bind hemin (iron (III)-protoporphyrin IX) to form a unique type of DNAzyme/RNAzyme with peroxidase-mimicking activity, which has been widely employed in multidisciplinary fields. However, its further applications are hampered by its relatively weak activity compared with protein enzymes. Herein, we report a unique intramolecular enhancement effect of the adjacent adenine (EnEAA) at 3' end of G4 core sequences that significantly improves the activity of G4 DNAzymes. Through detailed investigations of the EnEAA, the added 3' adenine was proved to accelerate the compound I formation in catalytic cycle and thus improve the G4 DNAzyme activity. EnEAA was found to be highly dependent on the unprotonated state of the N1 of adenine, substantiating that adenine might function as a general acid-base catalyst. Further adenine analogs analysis supported that both N1 and exocyclic 6-amino groups in adenine played key role in the catalysis. Moreover, we proved that EnEAA was generally applicable for various parallel G-quadruplex structures and even G4 RNAzyme. Our studies implied that adenine might act analogously as the distal histidine in protein peroxidases, which shed light on the fundamental understanding and rational design of G4 DNAzyme/RNAzyme catalysts with enhanced functions., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

There is a significant body of work suggesting that sRNA-mediated post-transcriptional regulation is a conserved mechanism among pathogenic bacteria to modulate bacterial virulence and survival. Porphyromonas gingivalis is recognized as an etiological agent of periodontitis and implicated in contributing to the development of multiple inflammatory diseases including cardiovascular disease. Using NimbleGen microarray analysis and a strand-specific method to sequence cDNA libraries of small RNA-enriched P. gingivalis transcripts using Illumina's high-throughput sequencing technology, we identified putative sRNA and generated sRNA expression profiles in response to growth phase, hemin availability after hemin starvation, or both. We identified transcripts that mapped to intergenic sequences as well as antisense transcripts that mapped to open reading frames of the annotated genome. Overall, this approach provided a comprehensive way to survey transcriptional activity to discover functionally linked RNA transcripts, responding to specific environmental cues, that merit further investigation., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Objectives: The absorption of commonly used ferrous iron salts from intestinal segments at neutral to slightly alkaline pH is low, mainly because soluble ferrous iron is easily oxidized to poorly soluble ferric iron and ferrous iron but not ferric iron is carried by the divalent metal transporter DMT-1. Moreover, ferrous iron frequently causes gastrointestinal side effects. In iron(III)-hydroxide nanoparticles hundreds of ferric iron atoms are safely packed in nanoscaled cores surrounded by a solubilising carbohydrate shell, yet bioavailability from such particles is insufficient when compared with ferrous salts. To increase their intestinal uptake iron(III)-hydroxide nanoparticles were coupled in this study with the protoporphyrin hemin, which undergoes carrier-mediated uptake in the intestine., Methods: Uptake of iron(III)-hydroxide nanoparticles with hemin covalently coupled by DCC reaction was measured in Caco-2 cells with a colorimetric assay and visualized by transmission electron microscopy., Key Findings: Nanoparticles were taken up by carrier-mediated transport, since uptake was temperature-dependent and increased with an increasing hemin substitution grade. Furthermore, uptake decreased with an increasing concentration of free hemin, due to competition for carrier-mediated uptake., Conclusions: Hemin-coupled iron(III)-hydroxide nanoparticles were carried by a heme specific transport system, probably via receptor mediated endocytosis. It can be expected that this system shows improved absorption of iron compared with uncoupled iron(III)-hydroxide nanoparticles, which exist on the market today., (© 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.)

Deferoxamine (DFO), an FDA-approved iron chelator used for treatment of iron poisoning, affects bacteria as iron availability is intimately connected with growth and several virulence determinants. However, little is known about the effect on oral pathogens. In this study, the effect of DFO on Porphyromonas gingivalis, a major periodontopathogen which has an essential growth requirement for hemin (Fe(3+)-protoporphyrin IX), was evaluated. The viability of P. gingivalis W83 was not affected by 0.06-0.24 mM DFO, whereas the doubling time of the bacterium was considerably prolonged by DFO. The inhibitory effect was evident at earlier stages of growth and reduced by supplemental iron. UV-visible spectra using the pigments from P. gingivalis cells grown on blood agar showed that DFO inhibited μ-oxo bisheme formation by the bacterium. DFO decreased accumulation and energy-driven uptake of hemin by P. gingivalis. Antibacterial effect of H(2)O(2) and metronidazole against P. gingivalis increased in the presence of DFO. Collectively, DFO is effective for hemin deprivation in P. gingivalis suppressing the growth and increasing the susceptibility of the bacterium to other antimicrobial agents such as H(2)O(2) and metronidazole. Further experiments are necessary to show that DFO may be used as a therapeutic agent for periodontal disease., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Exogenous δ-aminolevulinic acid (ALA)-induced photodynamic therapy (PDT) has been used in the treatment of cancer. To obtain a high efficacy of ALA-PDT, we have screened various chemicals affecting ALA-induced accumulation of protoporphyrin in cancerous cells. When HeLa cells were treated with quinolone chemicals including enoxacin, ciprofloxacin or norfloxacin, the ALA-induced photodamage accompanied by the accumulation of protoporphyrin was stronger than that with ALA alone. Thus, quinolone compounds such as enoxacin, ciprofloxacin and norfloxacin enhanced ALA-induced photodamage. The increased ALA-induced photodamage in enoxacin-treated HeLa cells was decreased by haemin or ferric-nitrilotriacetate (Fe-NTA), suggesting that an increase in iron supply cancels the accumulation of protoporphyrin. On the other hand, the treatment of the cells with ALA plus an inhibitor of haem oxygenase, Sn-protoporphyrin, led to an increase in the photodamage and the accumulation of protoporphyrin compared with those upon treatment with ALA alone, indicating that the cessation of recycling of iron from haem augments the accumulation. The use of quinolones plus Sn-protoporphyrin strongly enhances ALA-induced photodamage. To examine the mechanisms involved in the increased accumulation of protoporphyrin, we incubated ferric chloride with an equivalent amount of quinolones. Iron-quinolone complexes with visible colours with a maximum at 450 nm were formed. The levels of iron-metabolizing proteins in enoxacin- or ciprofloxacin-treated cells changed, indicating that quinolones decrease iron utilization for haem biosynthesis. Hence, we now propose that the use of quinolones in combination with ALA may be an extremely effective approach for the treatment modalities for PDT of various tumour tissues in clinical practice.

The aim of this study is to investigate the effect of heme oxygenase-1 (HO-1)/carbon monoxide (CO) system in pulmonary ischemia-reperfusion injury (PIRI) in rabbits. The rabbits were randomly assigned to three groups (n=10, in each): control group (C), PIR group (I-R), PIR+Hemin group (H) and PIR+zinc protoporphyrin IX (ZnPP) group (Z). There were changes to several parameters which included plasma carboxyhemoglobin (COHb), wet to dry ratio of lung tissue weight (W/D), the injured alveoli rate (IAR) and the HO-1 enzymatic activity. Immunohistochemistry and in situ hybridization for HO-1 was detected in lung. The electron microscopic observation for lung tissue injury was done after PIRI. The plasma content of COHb increased by reperfusion was strengthened by hemin but weakened by ZnPP. The HO-1 activity in lung tissue was upregulated by PIRI, further enhanced by hemin and abolished by ZnPP. Except for the C group, HO-1 was upregulated in all other groups in the pulmonary endothelial cells, some pulmonary vascular smooth muscle cells, extima of vessels and epithelial cells of airway. The injury parameters were highest in the Z group, the second was in the IR group, then the H group and the C group. HO-1/CO system was activated and may be one of the protective signal pathway during PIRI in rabbits.

A new RNA aptamer-binding hemin was synthesized by the in vitro selection (SELEX) method. A pool of 103 bases single strand DNAs containing a randomized sequence of 59 bases was synthesized. The pool was incubated with hemin on hemin-immobilized affinity column. Bound RNAs were eluted off with hemin solution and amplified by PCR. After 3 rounds of selection process, the selected RNAs were cloned and sequenced. Some RNA aptamers, which have affinity to hemin, was selected.

Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) is the most important vector of Leishmania major, and previous experiments revealed that Leishmania development in the sand fly midgut is significantly affected by temperature. Therefore, we maintained blood-fed P. papatasi females at 23 or 28 degrees C to understand the effect of temperature on bloodmeal digestion and developmental times of this sand fly. At the lower temperature, the metabolic processes were slower and developmental times were longer: defecation, oviposition, and egg hatch started later and took longer to complete. Also, the mortality of blood-fed females was significantly lower. The defecation of bloodmeal remains was delayed for 12-36 h at 23 degrees C compared with the group maintained at 28 degrees C. Such delay would provide more time for Leishmania to establish the midgut infection and could partially explain the increased susceptibility of P. papatasi to Leishmania major at 23 degrees C. In both experimental groups, blood-fed females laid similar numbers of eggs (mean 60 and 70, maximum 104 and 115 per female). Egg numbers were positively correlated with the amount of hematin excreted in feces of ovipositing females. In parallel experiments, autogeny was recorded in 8% of females. The autogenous egg batches were smaller (mean, 12; range, 1-39), but they all produced viable larvae.

Some enteropathogenic Escherichia coli (EPEC) strains, which are an important cause of diarrhea among infants, secrete a serine protease autotransporter protein called EspC. The pathogenic role of EspC upon EPEC infection is unknown. In this study, we demonstrated that purified EspC protein, obtained from supernatants of EPEC cultures, interacted with hemoglobin and degraded it. Moreover, we have shown that EspC is a hemin-binding protein. We hypothesized that hemoglobin proteolysis by EspC may contribute to the utilization of heme and hemoglobin iron for bacterial growth.

Using DNA microarray analysis, mRNA levels from wild-type Yersinia pestis cells treated with the iron chelator 2,2'-dipyridyl were compared with those supplemented with excessive iron, and subsequent to this, gene expression in the fur mutant was compared with that in the wild-type strain under iron rich conditions. The microarray analysis revealed many iron transport or storage systems that had been induced in response to the iron starvation, which is mediated by the Fur protein, using the iron as a co-repressor. The iron-Fur complex also affected some genes involved in various non-iron functions (ribonucleoside-diphosphate reductase, membrane proteins, electron transport and oxidative defense, etc.). The Fur protein still participated in the regulation of genes involved in broad cellular processes (virulence factors, pesticin activity, haemin storage and many proteins with unknown functions) that were not affected by iron depletion conditions. In addition to its classical negative regulatory activities, the Fur protein activates gene transcription. Using bioinformatics tools, we were able to predict the Y. pestis Fur box sequence that was clearly the over-presented motif in the promoter regions of members of the iron-Fur modulon.

Objectives: There is an urgent need to discover new antimalarials, due to the spread of chloroquine resistance and the limited number of available drugs. Chalcones are one of the classes of natural products that are known to possess antiplasmodial properties. Therefore, the in vitro antiplasmodial activity of the main hop chalcone xanthohumol and seven derivatives was evaluated. In addition, the influence of the compounds on glutathione (GSH)-dependent haemin degradation was analysed to determine its contribution to the antimalarial effect of chalcones., Methods: In vitro antiplasmodial activity was evaluated against the chloroquine-sensitive strain poW and the multiresistant clone Dd2 using a [(3)H]hypoxanthine-incorporation assay. Inhibition of GSH-dependent haemin degradation was analysed by a multiwell plate assay at 11 microM., Results: Of the eight compounds tested, four possessed activity with IC(50) values<25 microM against at least one of the two strains of Plasmodium falciparum. The main hop chalcone, xanthohumol, was most active with IC(50) values of 8.2+/-0.3 (poW) and 24.0 +/- 0.8 microM (Dd2). Three of these compounds were additionally active in the haemin-degradation assay., Conclusions: The results demonstrate for the first time the ability of chalcone derivatives to interfere with the haemin-degradation process of P. falciparum. This effect might contribute to their antiplasmodial activity. Nevertheless, as one compound showed inhibition of P. falciparum without being able to interact with GSH-dependent haemin degradation, other modes of action must add to the observed antiparasitic activity of hop chalcones.

Methemoglobin (MetHb), oxyhemoglobin (oxyHb), metmyoglobin (metMb), and oxymyoglobin (oxyMb) catalyze formation of the 7-carboxyheptyl and pentyl radicals from 13-hydroperoxy-9,11-octadecadienoic acid. The relative HPLC-ESR peak height of the pentyl radical to the 7-carboxyheptyl radical was found to depend on the oxygen concentration in the reaction mixture. Under aerobic conditions, the 7-carboxyheptyl radical was predominant for the reaction mixture with ferrous ions (or cytochrome c, metHb, or metMb). On the other hand, under anaerobic conditions, the pentyl radical was predominant for the reaction mixture with ferrous ions (or cytochrome c), but the 7-carboxyheptyl radical was still predominant for the reaction mixture with metHb (or metMb), suggesting that metHb (or metMb) catalyzes the reaction through a mechanism different from that in the case of ferrous ions (or cytochrome c). In order to explain the above results, a mechanism, in which molecular oxygen is not involved, is proposed for the formation of the 7-carboxyheptyl radical in the reaction mixture of 13-HPODE with metHb (or metMb) under anaerobic conditions.

Streptococcus pneumoniae causes considerable morbidity and mortality throughout the world. Iron acquisition is an important virulence factor for bacterial pathogens. Two loci, piu and pia, were identified as responsible for the hemoglobin utilization of S. pneumoniae. The binding activity and surface accessibility of the solute binding protein of PiuA were studied. PiuA is a lipoprotein, binds hemin and hemoglobin, resides on the cytoplasmic membrane, and is not exposed on the surface of S. pneumoniae. The localization of PiuA has implications in its role in hemoglobin utilization and possible use as a pneumococcal vaccine.

Forty-two compounds isolated from nine plants used within South America for the treatment of malaria were tested for haemin binding using two novel, rapid screening methods. The data obtained were analysed with respect to IC(50) values for in vitro toxicity to Plasmodium falciparum trophozoites. One method, a multiwell assay based on the inhibition of the interaction of haemin with glutathione (GSH), is sensitive in the 10 microM range, takes c. 1 h and is suitable for either a high throughput screen or rapid assay during natural product isolation. Of 19 compounds showing antiplasmodial activity (IC(50) < 40 microM), 16 (84%) showed >40% inhibition of GSH-haemin reaction. The sensitivity and specificity of the assay were 0.85 and 0.82, respectively. The positive predictive value was 0.81 and the negative predictive value 0.86. A more sensitive assay (0.1 microM range) is based on the reversal by haemin-binding compounds of the haemin inhibition of the L-dopachrome-methyl ester tautomerase activity of human macrophage migration inhibitory factor. This assay gives a better idea of the affinity of interaction and uses very small amounts of test compound. The log[RI(50)] of eight of the compounds that tested positive in the above assays together with those of quinine and chloroquine showed a positive correlation with log[antiplasmodial IC(50)] for strain T9-96 (r = 0.824) and strain K1 (r = 0.904). Several of the antimalarial compounds that bind haemin are isoquinolines, a class not shown previously to interact with haemin.

Bartonella henselae causes cat-scratch disease and bacillary angiomatosis peliosis. The bacteria reside in erythrocytes of asymptomatic cats, which represent the natural reservoir for this pathogen. B. henselae is usually grown on blood-enriched media. Growth experiments on Brucella medium without blood demonstrated that heme compounds are essential for the growth of B. henselae and can completely substitute the addition of blood components. The heme precursor protoporphyrin IX alone, or in combination with FeCl(2) or FeCl(3), as well as transferrin or lactoferrin did not support growth, indicating that B. henselae cannot synthesize heme itself. Hemin supported growth even when free iron was chelated, indicating that hemin is also used as an iron source. Binding assays showed that hemin starvation increased the binding capacity of B. henselae for hemin, providing evidence that the bacteria carry a specific hemin uptake system, which might be regulated by hemin.

In eukaryotic cells, HSP90 is associated with several protein kinases and regulates their activities. HSP90 was also reported to possess an autophosphorylase activity. In this study, we examined in vitro autophosphorylation of HSP90, which was purified from chick muscle. We show that HSP90 was not phosphorylated in vitro, but an 84-kDa protein (p84) was highly phosphorylated. P84 was neither HSP90 nor its degradative product, as it was not detected by an antibody (BF4) specific to HSP90 in denaturing immunoprecipitation and Western blot analysis. Phosphorylation of a protein similar to p84 was also detected with purified human brain and HeLa HSP90, indicating that p84 is present in many different types of cells. P84 appeared to exist as large complexes, as determined by HPLC and native gel electrophoresis. Native immunoprecipitation using anti-HSP90 (BF4)-conjugated Affi-gel revealed that this phosphoprotein is specifically associated with HSP90. The interaction of p84 and HSP90 was not affected by p84 phosphorylation. In addition, p84 phosphorylation was prevented by the presence of divalent cations such as Mg(2+) and Mn(2+). In contrast, p84 phosphorylation was significantly activated by addition of exogenous Ca(2+) between 100 and 500 microM, although it was blocked by higher concentrations (>1 mM) of Ca(2+). HSP90, but not p84, could be phosphorylated by casein kinase II. Finally, p84 phosphorylation was specifically prevented by hemin, but not by other kinase inhibitors, indicating that p84 phosphorylation may be regulated by heme-regulated protein kinase.

The effect of environmental haemin on the physiology and biochemistry of Prevotella intermedia R78 grown in batch culture was assessed. Extent and rate of growth increased as the environmental haemin concentration was raised. In addition, cell morphology was predominantly cocco-bacillary when cultured in high haemin environments, while bacillary forms were prevalent in low haemin conditions (< 2.5 mumol l-1). Cells harvested from low haemin environments produced greater numbers of extracellular vesicles and greater amounts of peptidolytic activity, haemagglutinating potential and haemin binding activity when compared with cells harvested from high haemin conditions. The results of the present study indicate that aspects of the biochemistry and physiology of P. intermedia are influenced by changes in environmental haemin levels.

Vibrio vulnificus, an opportunistic human pathogen, can obtain iron from a variety of heme proteins. This process involves the digestion of heme proteins by an exoprotease to liberate protoheme (iron-protoporphyrin IX). In the present study, we tested whether this pathogen also uses a synthetic heme compound, Fe-alpha,beta,gamma,delta-tetraphenylporphine tetrasulfonic acid (Fe-TPPS), as an iron source. When inoculated into a medium containing Fe-TPPS, V. vulnificus L-180 multiplication was seen to be dependent on the concentration of the synthetic heme compound; a mutant lacking the ability to utilize protoheme did not multiply. Cells of the strain grown under the iron-restricted condition showed time-dependent uptake of Fe-TPPS. The ability to use either protoheme or Fe-TPPS was significantly reduced by the addition of an excess amount of free TPPS or Cu-TPPS. The data suggest that, V. vulnificus may assimilate Fe-TPPS, at least partially, through the same system as that for protoheme.

(Alpha-bromoiso-valeryl) urea, a sedative or hypnotic, is metabolized to (3-methylbutyryl)urea by reductive debromination. This study was designed to evaluate the role of blood in the debromination of (alpha-bromoiso-valeryl) urea. Rat blood containing an electron donor had significant debrominating activity toward (alpha-bromoiso-valeryl)urea. This debromination proceeded by enzymatic and non-enzymatic processes which required both NADH (or NADPH) and flavin mononucleotide (FMN), under anaerobic conditions. The debrominating activity was sensitive to inhibition by carbon monoxide, and the pH optimum was 8.5. When FMN was replaced by flavin adenine dinucleotide (FAD) or riboflavin, similar results were obtained. The optimum concentration of flavins was 10(-4) M. The reductive debromination was also mediated by rat erythrocytes, but not by plasma. When the blood or erythrocytes were boiled, the debrominating activity was not abolished, but was enhanced, suggesting that the activity arises from the haemoglobin in erythrocytes, and haemoglobin had debrominating activity when supplemented with both a reduced pyridine nucleotide and a flavin. Furthermore, haematin had significant debrominating activity in the presence of these cofactors. The activity of haematin was also observed with the photochemically reduced form of FMN. The results imply that the debromination proceeds in two steps--enzymatic or non-enzymatic reduction of a flavin such as FAD, FMN or riboflavin by NADPH or NADH, then non-enzymatic reductive debromination of (alpha-bromoiso-valeryl)urea to (3-methylbutyryl)urea catalysed by the haem group of rat haemoglobin in the presence of the reduced flavin.

The effect of iron deprivation on the expression of outer membrane proteins and the ability to use heme as an iron source by uropathogenic Proteus mirabilis, Pr 6515, was studied. Examination of iron-restricted bacteria showed three outer membrane proteins ranging from 66 to 75 kDa to be affected by iron restriction, as well as a newly expressed 64-kDa protein. These proteins were induced within 15 minutes of iron-deprivation. The strain grew in the presence of ferric citrate, hemin and hemoglobin as iron sources, but could not use transferrin, lactoferrin or siderophores from exogenous sources. The 64- and 66-kDa proteins showed hemin-binding activity by affinity chromatography, and both reacted in Western blots with sera from mice transurethrally infected with the same strain. We suggest that P. mirabilis expresses iron-regulated outer membrane proteins that could be involved in heme uptake and may have a role in pathogenesis.

Actinobacillus actinomycetemcomitans was found to express a polypeptide immunologically related to the Neisseria gonorrhoeae FbpA iron binding protein. In addition, the expression of hitB and hitC homologs was detected by Northern blot analysis. This periodontal pathogen also expresses a polypeptide homologous to the 31-kDa Haemophilus influenzae protein, which shows amino acid sequence homology with the FimA and YfeA proteins from Streptococcus parasanguis and Yersinia pestis, respectively. Both A. actinomycetemcomitans protein homologs were located within the periplasmic space, and their synthesis was regulated by the iron and hemin concentration of the culture medium. Southern and Western blot analysis together with molecular cloning revealed the presence of a Fur-like repressor, which may control the iron regulation of gene expression in this bacterium. Cultivation in the presence of hemin or Congo red revealed the ability of this organism to bind hemin. This binding activity was further confirmed by isolating Escherichia coli DH5 alpha clones that produced red and brown colonies on agar plates containing Congo red and hemin, respectively, after transformation with an A. actinomycetemcomitans gene library.

Prevotella intermedia, a putative periodontopathic microorganism, requires iron for growth. Hemoglobin can be a major source of iron for bacterial growth in vivo since it is present in the crevicular fluid collected from periodontitis sites. Experiments studying the growth of P. intermedia in iron-depleted Todd-Hewitt broth supplemented with human hemoglobin showed that the bacteria were able to utilize human hemoglobin as a source of iron. The uptake of iron from hemoglobin by P. intermedia appears to be initiated by the binding of hemoglobin to the bacteria as shown by direct binding studies using 125I-labeled human hemoglobin. Scatchard analysis of saturation binding data revealed that 125I-labeled human hemoglobin had a dissociation constant (Kd) of 2.53 x 10(-8) M for the receptor on P. intermedia. Binding of labeled hemoglobin to P. intermedia was competitively inhibited by unlabeled human hemoglobin showing that the binding was specific. The ability of bovine hemoglobin, but not hemin or non-hemoglobin heme-containing compounds, to inhibit binding competitively suggested that the globin moiety of the hemoglobin molecule is recognized by the hemoglobin binding receptors.

The eel pathogen Vibrio vulnificus biotype 2 (serogroup E) is able to use hemin (Hm) or hemoglobin (Hb) as the sole iron source for growth in vitro and in vivo. The mechanism of heme-iron acquisition in this bacterium requires a direct interaction through binding sites on the bacterial surface (constitutive outer membrane proteins). Using affinity chromatography techniques, a unique protein of around 36.5 kDa was isolated from cell envelopes of E86 strain regardless of the affinity ligand used, hemoglobin or hemin. This protein was purified from both iron-enriched and iron-restricted grown cells. These results support the hypothesis that in this pathogen Hm- and Hb-iron acquisition is mediated by a common protein receptor which recognizes the heme prosthetic group of Hb.

Vibrio vulnificus, an opportunistic human pathogen, can obtain iron from a variety of heme proteins. This process involves the digestion of heme proteins by an exoprotease to liberate protoheme (iron-protoporphyrin IX). In the present study, we isolated and characterized a mutant for protoheme utilization. One mutant isolated by treatment with a chemical mutagen was shown to be unable to use either protoheme or heme proteins, but multiplied in a medium supplemented with an iron siderophore, such as iron-vulnibactin. Like a wild-type strain, the mutant sensed iron depletion, so that the 74- and 79-kDa outer membrane proteins were expressed under iron-regulated conditions. Both the parent and mutant strains secreted hemolysin independent of the iron concentration of the medium. Whole cells of either of the strains were equally capable of binding of hematin. Taken together, the data suggest that the mutant may have a mutation in a gene encoding an inner membrane or a periplasmic protein which transports protoheme or iron dissociated from protoporphyrin IX into the cell.

Haemin (iron protoporphyrin IX) is an essential growth factor for the periodontal pathogen. Porphyromonas gingivalis. Iron protoporphyrin IX (IPP IX) binding to the avirulent P. gingivalis beige variant (W50/BE1) and the black-pigmenting parent wild-type strain W50 was quantified. W50/BE1 grown in a chemostat under haemin excess-bound IPP IX under both oxidising and reducing conditions but with both lower capacity and avidity than either the haemin-limited- and haemin-excess-grown parent strain W50. Rosenthal plots for W50/BE1 indicated cooperative binding. W50/BE1 cells expressed a 32 kDa outer membrane haemin-binding protein when grown under conditions of haemin excess, and this strain might serve as a useful source from which to isolate this protein. The reduced IPP IX binding ability of W50/BE1 may be the rate-limiting factor for haem uptake and explain the reduced virulence and slower rate of pigmentation of this strain.

Several clinical isolates of Vibrio parahaemolyticus were examined for their ability to utilize either hemin or hemoglobin as a sole source of iron. Both compounds appeared to be equally good iron sources. Maximum growth was obtained at 5 microM hemin or 1.25 microM hemoglobin under the conditions tested. Using a hemin-agarose batch affinity method, the hemin-binding protein was isolated from crude total membranes of a hemin-utilizing strain, WP1, grown under iron-deficient but not under iron-sufficient conditions. This protein was identical to the 83 kDa outer membrane protein which was expressed in response to iron limitation. The protein was susceptible to proteinase K cleavage in whole cells, indicating its exposure at the cell surface. Hemin and hemoglobin, but not protoporphyrin IX, inhibited binding of the protein to hemin-agarose.

To investigate the involvement of peripheral-type benzodiazepine receptors (PBR) in heme metabolism, we examined the interaction of [55Fe]heme with PBR. Transfection of the cloned mouse PBR-isoquinoline carboxamide-binding protein (PBR/IBP) cDNA into monkey kidney Cos-1 cells resulted in a 2.5-fold increase in [55Fe]hemin binding sites, concomitant with the increase in [3H]PK11195 binding sites, as compared with those seen in antisense PBR/IBP cDNA-transfected cells. The binding of hemin to the transfected receptors exhibited a relatively high affinity with a Kd of 12 nM, and was inhibited by several benzodiazepine ligands, including PK11195, Ro 5-4864, diazepam and protoporphyrin IX. When mouse liver mitochondria were incubated with [55Fe]hemin, the binding to PBR had a Kd of 15 +/- 1.8 nM. The Bmax of [55Fe]hemin binding to the mitochondria was 6.88 +/- 0.76 pmol/mg of protein, a value consistent with that of [3H]PK11195 binding, with a lower affinity. Coproporphyrinogen III, a precursor porphyrin produced in the cytosol, is translocated into mitochondria, then is converted to protoporphyrinogen IX; this conversion decreased in the presence of benzodiazepine ligands. To examine whether this decrease was related to a decrease in the binding of coproporphyrinogen to the mitochondria, the effects of benzodiazepines on the binding of coproporphyrinogen were examined. As the binding was dose-dependently inhibited by PK11195, Ro 5-4864, and diazepam, porphyrins are likely to be endogenous ligands for PBR. We propose that PBR play a role in the intracellular transport of porphyrins and heme.

Staphylococcus aureus menadione and hemin auxotrophs, generated by in vitro gentamicin selection, demonstrated reduced hemolytic activity and enhanced intracellular survival within cultured bovine aortic endothelial cells relative to their hemolytic parent. Supplementation of the auxotrophs with exogenous menadione or hemin resulted in rapid growth, increased hemolytic activity, and reduced intracellular persistence to the level found for the hemolytic clinical parent. Aminoglycoside selection of staphylococcal menadione and hemin auxotrophs and subsequent persistence of these variants in the intracellular milieu may adapt S. aureus for evasion of host defenses and resistance to antimicrobial therapy.

Porphyromonas gingivalis strain W50 was grown in a chemostat either under haemin limitation or haemin excess at pH 7.3. Cells and the extracellular vesicle (ECV) and extracellular protein (EP) fractions were separated, quantified, and assayed for haemagglutination, protease activity and haemin binding. Under haemin-limitation, despite a reduction in cell yield, there was a 2.5-fold increase in the gravimetric yield of extracellular vesicles. Cells and vesicles from haemin-limited cultures, haemagglutinated sheep red blood cells to higher titres than their haemin-excess counterparts. Growth in haemin-excess conditions resulted in increased haemin-binding capacities of ECV, cells and EDTA-extracted outer membrane. Cells grown under haemin-excess showed a 2-fold elevation in specific activity towards the substrate N-alpha-benzoyl-L-arginine-p-nitroanilide (L-BAPNA) compared to haemin-limited cells. The specific activities against L-BAPNA for haemin-limited ECV were 3-fold greater than their haemin-excess counterparts. These vesicle activities represented 25% and 3% of the total culture protease activity under haemin limited and haemin excess conditions respectively.

The present study indicates that the outer membranes of Porphyromonas gingivalis have a high affinity for hemin, an essential growth factor for this suspected periodontal pathogen. The hemin-binding property appears to be mediated by the lipopolysaccharides, particularly the lipid A region. It is suggested that this binding mechanism may be important for the growth and establishment of P. gingivalis in environments with a low hemin content such as periodontal pockets.

Binding of hemin to globin was studied in the presence of 25 mM caffeine by measuring CD and optical absorption changes in the Soret region. CD and optical absorption spectra after mixing equimolar amounts of hemin and globin were the same as those of ferric hemoglobin. In contrast, addition of excess globin to hemin formed a complex that was distinguishable from ferric hemoglobin in terms of the CD and optical absorption spectra. By comparing the spectra of the complex with those of various hemoglobin derivatives, it was concluded that the complex was globin which carried a hemin exclusively on the alpha chain. This means that the alpha chain of the globin molecule has a greater affinity for hemin than the beta chain, as observed by other investigators using hemin-cyanide. The rate of binding of hemin to globin was estimated by the use of CD and optical absorption stopped-flow apparatus. The rate of hemin binding to the alpha chain of globin was obtained by mixing hemin and excess globin, and that to the beta chain was obtained by mixing equimolar concentrations of hemin and globin. The results showed that hemin was bound to the alpha chain in the globin molecule to form a transient intermediate, followed by its transformation into another intermediate, the transformation was the rate-limiting step, and the beta chain in the globin molecule had a greater affinity for hemin after hemin binding to the alpha chain than before.

A cell-free protein-synthesizing system active in initiation of translation of both endogenous mRNA and exogenous mRNA has been obtained from postmitochondrial supernatant (S-12) of the liver of ethionine-treated rats by adding reticulocyte ribosomal extract as a source of initiation factor. Formation of polysomes in the course of protein synthesis in vitro has also been demonstrated. Homogenization of the liver in the presence of 50 microM hemin stabilizes the initiation activity of S-12 fraction, which otherwise decays rapidly even at 0 degrees C. The mechanism of inhibition of protein synthesis by ethionine is discussed in view of these results.

In heme degradation catalyzed by the reconstituted heme oxygenase system, 8 to 9 mol of dioxygen and 11 to 12 mol of NADPH were consumed per mol of hemin lost, and about half the amount of dioxygen consumed could be accounted for by the production of hydrogen peroxide, which accumulated in the reaction mixture. Production of hydrogen peroxide in the heme oxygenase reaction did not appear to be due to the bimolecular dismutation of superoxide anions but rather seemed to be due to dissociation of a "peroxo" species formed on heme or intermediates of heme degradation. The hydrogen peroxide produced appeared to cause a considerable degree of non-specific degradation of heme (not leading to the formation of biliverdin) and also caused an inactivation of heme oxygenase. By taking into account the amount of dioxygen incorporated into hydrogen peroxide and some other factors, it could be deduced that 3 mol of dioxygen is consumed for the formation of 1 mol of biliverdin in the heme oxygenase reaction.

The effect of various concentrations of potassium salts on the activity of the N-ethylmaleimide(NEM)-treated hemin-controlled translational inhibitor (HCI) was investigated using the rabbit reticulocyte lysate system. The NEM-treated HCI was found to be less active for the inhibition of globin synthesis at higher concentrations of potassium salts. In addition, the binding of [35S]Met-tRNAf to the 40S ribosomal subunits was inhibited more profoundly at 100 mM than at 240 mM KOAc. The phosphorylation of the NEM-treated HCI and eukaryotic initiation factor, eIF-2, was studied at various concentrations of KOAc. The NEM-treated HCI was phosphorylated to an almost constant extent at 20 to 300 mM K+, but the phosphorylation of eIF-2 alpha by the inhibitor decreased with increasing concentration of KOAc. It was also found that the incubation of the 32P-labeled inhibitor with eIF-2 did not produce the 32P-labeled eIF-2. In the light of these data, a possible relationship between the phosphorylation of the inhibitor or eIF-2 alpha and the inhibition of globin synthesis by the inhibitor is discussed.

Seventeen strains of Yersinia enterocolitica and related species were examined for their susceptibility to a variety of antimicrobial agents. Twelve of the strains were clinical isolates which carried a virulence plasmid; five were food isolates which lacked plasmids. When grown on agar containing Congo red or haemin, each strain yielded pigmented (CR+) and non-pigmented (CR-) variants. CR+ bacteria of clinical origin were virulent, whereas CR+ food isolates and all CR- bacteria were avirulent. The susceptibility of CR+ and CR- bacteria to the following agents was compared: ampicillin, chloramphenicol, latamoxef, nalidixic acid, novobiocin, polymyxin B, rifampicin, streptomycin, tetracycline and tobramycin. The results showed that bacteria which bound Congo red were more susceptible to each drug examined than their CR- counterparts. This relationship held regardless of whether the CR+ bacteria possessed a virulence plasmid or not. The findings suggest that binding of antimicrobial agents by yersiniae may parallel binding of haemin and Congo red.