Addition Of Hematin Research Articles

Convergence of the 5-LOX and COX-2 pathways: heme-catalyzed cleavage of the 5S-HETE-derived di-endoperoxide into aldehyde fragments

Oxygenation of the 5-lipoxygenase product 5S-hydroxyeicosatetraenoic acid by cyclooxygenase-2 yields a bicyclic di-endoperoxide. The di-endoperoxide contains two peroxides spanning from carbons 9 to 11 and 8 to 12, and two hydroxyls at carbons 5 and 15 of arachidonic acid (Schneider C., et al. 2006. Convergent oxygenation of arachidonic acid by 5-lipoxygenase and cyclooxygenase-2. J. Am. Chem. Soc. 128: 720). Here, we report that treatment of the di-endoperoxide with hematin or ferrous chloride results in cleavage of both peroxide O-O bonds and of the bonds between the carbons that carry the peroxide groups, producing the aldehydes 4-hydroxy-2E-nonenal (4-HNE), 8-oxo-5S-hydroxy-6E-octenoic acid, and malondialdehyde (MDA). The hematin- and ferrous iron-catalyzed transformation of the di-endoperoxide proceeded with a similar yield of products as the cleavage of the prostaglandin endoperoxide PGH(2) to 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid and MDA. Chiral phase HPLC analysis of the 4-HNE cleavage product showed greater than 98% 4S and thus established the S configuration of the 15-carbon of the di-endoperoxide that had not previously been assigned. This transformation of the 5-lipoxygenase/cyclooxygenase-2 derived di-endoperoxide invokes the possibility of a novel pathway to formation of the classic lipid peroxidation products 4-HNE and MDA.

Development of the AmpFISTR SEfiler PCR amplification kit: a new multiplex containing the highly discriminating ACTBP2 (SE33) locus.

The AmpFISTR SEfiler kit co-amplifies 11 short tandem repeat loci including SE33 in a single multiplex. After establishing the optimum in primer titration studies, the primer concentrations of all loci in the multiplex were chosen such that the heterozygote peak height ratios of each of the loci were balanced. The combined primer set was then tested to determine the robustness of the multiplex under various conditions. Different MgCl(2) concentrations were evaluated to establish the optimum concentration for the multiplex. The amplification of the various loci in the multiplex was tested at several annealing temperatures (55-63 degrees C). Additionally, DNA from primates, non-primates and microorganisms were amplified to investigate the specificity of the kit. The stability of the AmpFISTR SEfiler kit was determined by addition of hematin, to simulate inhibition, and the use of degraded DNA. Population studies revealed a probability of identity of 6.47x10(-15) for African Americans and 7.46x10(-14) for US Caucasians. To assess the ability of the multiplex to analyze forensic samples, testing on blood, oral swabs and mixtures was performed. Based on the various studies, it was determined that the AmpFISTR SEfiler PCR amplification kit can be used to successfully analyze a variety of forensic, databasing and paternity samples.

Effects of nitric oxide on the eosinophil survival in vitro. A role for nitrosyl-heme

In a previous paper we showed that the nitric oxide (NO) donors azide and hydroxylamine inhibited eosinophil apoptosis. Azide and hydroxylamine generate a nitrosyl-heme complex – due to endogenous catalase activity – which activates soluble guanylate cyclase. In contrast, in the present paper, we show that NO donors (SNAP, SIN-1, S-nitroso- l-cysteine, NOC-18) which spontaneously release NO in physiological solutions did not support the survival of eosinophils and induced apoptosis or necrosis. However, the addition of hematin (the ferric form of heme) together with low doses of NO (SNAP 10 μM) promoted eosinophil survival. In conclusion, we propose that NO and heme (e.g. from heme-containing enzymes such as peroxidase or catalase), both released in inflammation sites, could form nitrosyl-heme and thus promote eosinophilic inflammation.

Physiological Diversity Among Strains of Tetragenococcus halophilus

Summary Twenty one strains of Tetragenococcus halophilus were investigated for their ecological properties, e.g. growth rate, salt tolerance, pH-dependence and influence of oxygen. All strains tolerated 26% NaCl. The growth rate was optimum at pH 7.5. Anaerobically grown cultures of the type strain achieved a higher growth rate than aerobic cultures. The addition of hematin to aerobic cultures resulted in a reduction in the lag phase and in generation time and a twofold increase in growth yield. Under these growth conditions 12 strains exhibited catalase activity. The composition of the metabolic end products was strongly affected by the growth conditions. Anaerobically grown cultures produced lactate, acetate and formate, whereas acetate was the main product of aerobic cultures.

Molecular analysis of mutated thyroid peroxidase detected in patients with total iodide organification defects.

Wild-type and mutant thyroid peroxidase (TPO) was expressed in a Semliki Forest Virus (SFV)-based transient expression system in Chinese hamster ovary-K1 cells. Twenty four hours after transfection proteins immunoreactive with TPO antibodies could be detected on a Western blot. Peroxidase activity was assayed using both the guaiacol and the I3- assay. Addition of hematin was necessary to obtain enzymatic active TPO. Thyroid peroxidase complementary DNA constructs containing mutations originally found in patients with hereditary congenital hypothyroidism caused by total iodide organification defects were analyzed using these techniques. In all cases TPO was expressed as shown by Western blotting and immunostaining. Enzymatic activity (measured by guaiacol and iodide oxidation assay) was below the detection level in four out of five mutants. The only mutant yielding TPO with enzymatic activity was G 1858 A (Gly 590 Ser). However, the mutation could affect splicing of TPO messenger RNA, leading to inactive TPO, because it is located at the exon 10/intron 10 border.

Design, synthesis, and biochemical evaluation of N-substituted maleimides as inhibitors of prostaglandin endoperoxide synthases.

N-(Carboxyalkyl)maleimides are rapid as well as time-dependent inhibitors of prostaglandin endoperoxide synthase (PGHS). The corresponding N-alkylmaleimides were only time-dependent inactivators of PGHS, suggesting that the carboxylate is critical for rapid inhibition. Several N-substituted maleimide analogs containing structural features similar to those of the nonsteroidal anti-inflammatory drug aspirin were synthesized and evaluated as inhibitors of PGHS. Most of the aspirin-like maleimides inactivated the cyclooxygenase activity of purified ovine PGHS-1 in a time- and concentration-dependent manner similar to that of aspirin. The peroxidase activity of PGHS was also inactivated by the maleimide analogs. The cyclooxygenase activity of the inducible isozyme, i.e., PGHS-2, was also inhibited by these compounds. The corresponding succinimide analog of N-5-maleimido-2-acetoxy-1-benzoic acid did not inhibit either enzyme activity, suggesting that inactivation was due to covalent modification of the protein. The mechanism of inhibition of PGHS-1 by N-(carboxyheptyl)maleimide was investigated. Incubation of apoPGHS-1 with 2 equiv of N-(carboxyheptyl)[3,4-14C]maleimide led to the incorporation of radioactivity in the protein, but no adduct was detected by reversed-phase HPLC, suggesting that it was unstable to the chromatographic conditions. Furthermore, hematin-reconstituted PGHS-1, which was rapidly inhibited by N-(carboxyheptyl)maleimide, displayed spontaneous regeneration of about 50% of the cyclooxygenase and peroxidase activities, suggesting that the adduct responsible for the inhibition breaks down to regenerate active enzyme. ApoPGHS-1, inhibited by N-(carboxyheptyl)maleimide, did not display regeneration of enzyme activity, but addition of hematin to the inhibited apoenzyme led to spontaneous recovery of about 50% of cyclooxygenase activity. These results suggest that addition of heme leads to a conformational change in the protein which increases the susceptibility of the adduct toward hydrolytic cleavage. ApoPGHS-1, pretreated with N-(carboxyheptyl)maleimide, was resistant to trypsin cleavage, suggesting that the carboxylate functionality of the maleimide binds in the cyclooxygenase channel. A model for the interaction of N-(carboxyheptyl)maleimide in the cyclooxygenase active site is proposed.

Heme-Dependent Cytochrome Formation in Lactobacillus maltaromicus

Summary Functional cytochromes of the b and d types were detected in crude extracts of Lactobacillus maltaromicus grown aerobically in the presence of hematin. With the addition of hematin to the medium the growth yield under aerobic growth conditions was tenfold higher than observed with anaerobically grown cultures. In addition, an increase was observed of acetoin formation. Our results are consistent with the phylogenetic and metabolic relationship between enterococci and carnobacteria including L. maltaromicus as a species to be transferred to the genus Carnobacterium .

Inhibition of human lymphoproliferative responses and altered lymphocyte membrane phenotype by succinylacetone

Succinylacetone (SA) proved to be a potent inhibitor of in vitro lymphoproliferative responses. This compound (3.0 mM) reduced the incorporation of 3HTdr by > 90% in mononuclear cell cultures stimulated with PHA, anti-CD3, IL-2 or phorbol dibutyrate-Ca2+ ionomycin. Furthermore, SA caused profound reduction in isotope uptake even if added to 3-day PHA-stimulated cultures as late as 6 h prior to harvest. Cells exposed to SA prior to mitogenic challenge and washed were not impaired in their proliferative activities. The addition of hematin to SA-containing cultures did not reverse the proliferative block. Phenotypic studies of stimulated cells suggested that SA does not preferentially affect one functional group of lymphocytes. However, it appeared that SA may act selectively to inhibit expression of transferrin receptors (CD71), a T-cell activation antigen. These data suggest that SA acts as a noncytotoxic immune inhibitor; this activity may be mediated, in part, by blocking cell activation and subsequent progress through the mitotic cycle.

Gamma-rays from Mössbauer sources: a low-dose approach to cancer therapy

A new approach to cancer treatment using low-energy gamma-rays is discussed. Cultures of bone marrow mononuclear cells collected from normal donors and patients with chronic myelogenous leukaemia have been studied after the addition of hematin, at different concentrations, and irradiation by a Mossbauer gamma-ray source. Growth stimulation has been observed when hematin is added to normal bone marrow cultures, while growth inhibition is observed when hematin is added to leukaemic cultures. The effect becomes then more pronounced when hematin is used in combination with Mossbauer gamma-rays.

A simplified, defined medium for growth of Entomophaga aulicae protoplasts

A defined medium, which allowed growth in both stationary and shaken cultures in the absence of fetal calf serum, was developed for protoplasts of the fungus Entomophaga aulicae. The protein "requirement" was obviated and growth was enhanced by the addition of hematin (0.5 μg/mL) and oleic acid (1.0 μg/mL). The utilization and production of ninhydrin-positive compounds was monitored at various stages of medium simplification. The protoplasts were autotrophic for all vitamins and organic acids. Fructose was not required for protoplast growth; however, it could function as a carbon source. The optimum nitrogen level was 635.08 mg N/L, and the optimum carbon level was 280 mg C/L under the conditions utilized. The protoplasts did not utilize either inorganic nitrogen (ammonium ion) or sulfur (sulfate ion) at the levels tested. No amino acid could function as a sole nitrogen source. Analysis of the amino acid composition of a general (> 14 000 molecular weight) protein extract gave no evidence of a skewed amino acid content. The final simplified medium included 8 amino acids (aspartic acid, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, and threonine) of the 21 in the original medium. This medium is useful in isolating vitamin auxotrophs for use as markers in virulence and in protoplast fusion studies, and in studies on the chemical nature of the limiting membrane and its outer surface.

Effect of hematin on the activities of nitrite reductase and catalase in lactobacilli

The effect of the addition of hematin on the activities of nitrite reductase and catalase was studied with cell suspensions of strains of lactobacillus species. In cells of Lactobacillus plantarum grown under aerobic or anaerobic conditions nitrite reductase was present. This activity was not exhibited by cells of L. curvatus and only rarely by L. sake. In addition, catalase activity was detected in aerobically grown cells only, with all strains of L. plantarum and L. sake; again L. curvatus was devoid of this activity. Ammonia was formed as the main product of nitrite reduction by L. plantarum. With lactate as the electron donor, the end products of carbohydrate catabolism were carbon dioxide, acetoin and acetate. The activities of nitrite reductase and catalase in strains of lactobacillus species may be used for optimizing the quality of starter cultures applied for the production of raw sausages.

Arachidonic acid-dependent activation of benzo[a]pyrene to bind to proteins with cytosolic and microsomal fractions from rat liver and lung.

Activation of benzo[a]pyrene to bind to proteins by co-oxidation with prostaglandin synthesis was studied in rat liver and lung microsomes and cytosols. The kinetics of this activation showed a two-phase reaction; a rapid initial reaction for 2-4 min after addition of arachidonic acid and then a slow reaction or a plateau state. The reaction was linear only with low contents of the enzyme proteins, and was slower with more than 1 mg of enzyme per ml of incubation mixture. Other unsaturated fatty acids were also effective in activating benzo[a]pyrene with both microsomes and cytosols. With microsomal proteins linoleic acid was more effective than arachidonic acid, whereas with cytosolic proteins arachidonic acid was the best cofactor. Linolenic acid could also activate benzo[a]pyrene, though less efficiently, but oleic acid had no influence on the binding. Indomethacin did not inhibit the activation, but nordihydroguaiaretic acid and quercetin significantly reduced the binding. Addition of hematin significantly increased the binding. The NADPH-dependent bindings of benzo[a]pyrene to proteins with liver and lung microsomes were one-third and one-twelfth the values after incubation with arachidonic acid. Addition of glutathione or Ca2+ ion reduced the binding significantly. The present results suggest the importance of co-oxidation with lipoxygenase for activation of benzo[a]pyrene and the possible role of both the arachidonic acid cascade system and the NADPH-dependent cytochrome P-450 system in metabolic activation of chemical carcinogens.

Synthesis of catalase by ?Streptococcus faecalis subsp. zymogenes?

"Streptococcus faecalis subsp. zymogenes" was grown aerobically and anaerobically with glycerol as the source of carbon, and in the presence and absence of haematin. Catalase activity was found only during aerobic growth in the presence of haematin. The rate of appearance of catalase activity was measured on (a) addition of haematin to haematin-less aerobic cultures, and (b) aeration of haematin-containing anaerobic cultures in the presence or absence of chloramphenicol. These experiments suggested that apocatalase synthesis was induced by aeration and was not dependent on the presence of haematin in the growth medium. The binding of haem to apocatalase was oxygen dependent.

Requirement for heme in the activation of purified guanylate cyclase by nitric oxide

Guanylate cyclase activity was purified to apparent homogeneity from rat liver (7700-fold) and bovine lung (8600-fold) soluble fractions by ammonium sulfate precipitation, DEAE-cellulose chromatography, agarose gel filtration and isoelectric focussing. The purified enzymes did not contain heme and did not respond to NO, nitroprusside or NO-cysteine in the absence of exogenous hematin. By contrast, preformed NO-hemoglobin increased enzyme activity 10–12-fold or 60–80-fold when 4 mM MnCl 2 or 4 mM MgCl 2, respectively, were employed as the metal ion co-factor. Addition of hematin to the enzyme preparations restored responsiveness to NO, nitroprusside or NO-cysteine to levels seen with NO-hemoglobin. Partial purification of guanylate cyclase from the soluble fraction of bovine lung (2400-fold) by ammonium sulfate precipitation, DEAE-cellulose chromatography, agarose gel filtration and high pressure liquid chromatography (HPLC) resulted in a preparation which contained endogenous heme as indicated by absorbance at 436 nm and responded to NO, nitroprusside and NO-cysteine in the absence of added hematin. By contrast, guanylate cyclase purified from the hepatic supernatant by the identical procedure, did not contain detectable absorption due to heme and did not respond or responded poorly to NO, nitroprusside or NO-cysteine in the absence of exogenous hematin. Analogous to hepatic guanylate cyclase purified by isoelectric focussing, the HPLC purified hepatic enzyme was activated 14-fold by NO-hemoglobin in assays which contained 4mM MnCl 2 and 60-fold in assays with 4 mM MgCl 2. Further, addition of hematin to the HPLC purified enzyme restored responsiveness to NO, nitroprusside and NO-cysteine to levels seen with NO-hemoglobin. These effects of hematin were specific for hematin and were not mimicked by albumin, sucrose or dithiothreitol. Moreover, the failure to observe stimulation of purified hepatic guanylate cyclase was not explained by a shift in the concentration response relationship between NO and guanylate cyclase activity. Several observations indicated that neither NO-thiol complexes nor [Fe(CN) 5NO] −3 were the proximate moieties responsible for activation of guanylate cyclase by nitroprusside and related agents, as has been previously suggested. These results strongly support the proposal that activation of guanylate cyclase by NO and related agents specifically requires formation of an NO-heme complex.

Growth inhibitory activity of succinylacetone: studies with Walker 256 carcinosarcoma, Novikoff hepatoma and L1210 leukemia.

Succinylacetone (SA, 4,6-dioxoheptanoic acid) inhibits d-aminolevulinic acid dehydrase, the second enzyme of the heme biosynthetic pathway and thereby inhibits heme biosynthesis. In the present study SA is shown to inhibit the growth of the Walker carcinosarcoma (W256) in vitro and in vivo, the Novikoff hepatoma in vivo, and L1210 leukemia in vitro, but only slightly in vivo. Rats can tolerate significantly larger doses of SA for at least twice as long as were administered in the present study without gross evidence of toxicity. In contrast to findings in previously published studies with murine erythroleukemia cells, the inhibition of growth of L1210 and W256 cells by SA in vitro is not accompanied by a decrease in cellular heme and is not reversed by addition of hematin to the medium. This suggests a second mechanism of growth inhibition of tumor cells unrelated to heme biosynthesis. Although the growth of both W256 and L1210 cells was markedly inhibited by the same concentration of SA in culture, there was a great difference in responsiveness in vivo, in that much greater inhibition of the growth of the Walker tumor was produced by SA.

Interactions de la chloroquine avec la ferriprotoporphyrine IX: Étude par résonance magnétique nucléaire

Chloroquine is still the antimalarial drug which is the most utilized. Nevertheless the molecular mode of action of this drug is not very well understood. When mouse erythrocytes injected with Plasmodium berghei are exposed to chloroquine, the first biochemical event is rapid accumulation of the drug. This process is energy dependent, saturable and competitively inhibited by drugs of the same therapeutic class (Quinine, Amodiaquine, Mefloquine). Receptors for chloroquine have been proposed for the process of accumulation. The nature of the chloroquine receptor is presently the subject of debates. The latest hypothesis proposed by Chou and coll. [12], is that ferriprotoporphyrin IX, formed by the degradation of hemoglobin by the parasite, binds to chloroquine with a dissociation constant of 3.5.10(-9) M. We studied here the molecular interactions between these two species by Proton Nuclear Magnetic Resonance in order to elucidate the nature and the geometry of were undertaken. The perturbations of the NMR spectra of chloroquine (10(-2) M) induced by addition of hematin or hemin were measured. Two types of measures were undertaken. The first study carried out in organic solvent (DMSO) has shown that the interaction occurred between the acidic functions of hemin and the side-chain nitrogen of chloroquine. The iron atom was not implicated in this process. The second study carried out in aqueous medium (phosphate buffer; 0.1 M; pH = 7) allowed us to demonstrate that chloroquine is able to intercalate into a polymer of hematin. The quinoleic nucleus of chloroquine was intercalated between two dimers of hematin as shown by the broadening of the signal of the quinoleic protons due to very large increase in the correlation time. Finally it was shown that chloroquine is associated as a dimer in aqueous medium by hydrophobic interactions. The association constant is 5.5 M-1.

Purification and properties of heme-deficient hepatic soluble guanylate cyclase: Effects of heme and other factors on enzyme activation by NO, NO-heme, and protoporphyrin IX

Purified hepatic soluble guanylate cyclase (EC 4.6.1.2) had maximal specific activities in the unactivated state of 0.4 and 1 μmol cyclic GMP min −1 mg protein −1, when MgGTP and MnGTP, respectively, were used as substrates. The apparent K m for GTP was 85 or 10 μ m in the presence of excess Mg 2+ or Mn 2+, respectively. Guanylate cyclase purified as described was deficient in heme but could be readily reconstituted with heme by reacting enzyme with hematin and excess dithiothreitol at 4 °C and pH 7.8. Unpurified guanylate cyclase was activated 20- to 84-fold by NO, nitroso compounds, NO-heme, and protoporphyrin IX. The purified enzyme was only slightly (2- to 3-fold) activated by NO and nitroso compounds but was markedly (50-fold) activated by NO-heme and protoporphyrin IX, achieving maximal specific activities of 10 μmol cyclic GMP min −1 mg protein −1. Enzyme activation by NO and nitroso compounds was restored by addition of hematin or by reconstitution of guanylate cyclase with heme. Excess hematin, however, inhibited enzyme activity. A partially purified heat-stable factor (activation-enhancing factor) was found to enhance (2- to 35-fold) enzyme activation without directly stimulating guanylate cyclase. In the presence of optimal concentrations of hematin, enzyme activation was still increased (2-fold) by the activation-enhancing factor but not by bovine serum albumin. Guanylate cyclase was markedly inhibited by SH reactive agents such as cystine, o-iodosobenzoic acid, periodate, and 5,5′-dithiobis (2-nitrobenzoic acid). In addition, CN − and FMN inhibited enzyme activation by NO-heme, but not by protoporphyrin IX, and did not affect basal enzymatic activity. Hepatic soluble guanylate cyclase appears to possess SH groups required for catalysis and to require heme and/or other unknown factors for the full expression of enzyme activation by NO and nitroso compounds.

Selective destruction and removal of heme from prostaglandin H synthase

Treatment of the holoenzyme form of prostaglandin H synthase with oxygen gas in the presence of excess dithionite has been found to selectively oxidize the enzyme's heme cofactor. Both the cyclooxygenase and peroxidase activities of the PGH synthase were restored upon addition of hematin. A convenient procedure has been developed to prepare milligram amounts of apo-PGH synthase from the holoenzyme. This procedure appears to involve a reactive species generated during cooxidation of dithionite and heme. The reactive species differs from that generated during the cyclooxgenase catalytic reactions which inactivates the enzyme. The heme in hemoglobin and hematin is destroyed by the same treatment. Direct addition of hydrogen peroxide converted holo-PGH synthase to the apoenzyme, but with extensive loss of enzymatic activity.

Immunosuppressive activity of succinylacetone.

SA, an inhibitor of ALA dehydrase, the second enzyme of the heme biosynthetic pathway, has been shown to exert immunosuppressive activity in several systems. When the Walker 256 tumor was grown subcutaneously in outbred Sprague-Dawley rats, SA prevented rejection of the tumor by the host. Human peripheral blood lymphocyte transformation in response to mitogens and antigens in vitro was markedly impaired by addition of SA to the medium. Addition of hematin to the medium did not reverse the SA-mediated inhibition of transformation. This finding suggests that SA may exert immunosuppressive activity by a mechanism separate from inhibition of heme biosynthesis. Continuous administration of SA to Fisher 344 rats profoundly suppressed hemolytic antibody production after immunization with SRBC. Administration of large doses (equivalent to the highest dose of Sa given to tumor-bearing animals) for a month produced a 20% decrease of hemoglobin concentration, a 12% decrease in hematocrit, and no significant effect on leukocyte or erythrocyte concentration. There were no significant changes in tissue histology, including marrow cellularity.

The effect of haematin and catalase on Streptococcus faecalis var. zymogenes growing on glycerol.

Streptococcus faecalis var. zymogenes was grown aerobically on a complex medium containing glycerol as the carbon source. Addition of hematin or bovine liver catalase to the growth medium resulted in a small increment in growth yield. Suspensions of bacteria that had been grown in the presence of haematin or catalase, respectively, translocated 0.83 to 1.98 and 1.33 to 2.53 protons per oxygen atom consumed in glycerol oxidation. Bacterial grown without haematin or catalase had nil or little respiratory-induced proton translocation during glycerol oxidation. Inclusion of haematin in the growth medium caused the bacterium to form a cyanide- and azide-sensitive catalase. Superoxide dismutase activity was similar whether or not haematin was added to the growth medium.