Presence Of Haemin Research Articles

WS19.3 Prevalence of small colony variants of Staphylococcus aureus from lower respiratory tract specimens

Background Staphylococcus aureus , one of the first bacteria isolated from cystic fibrosis (CF) airways, is the most common cause of respiratory infections. Small-colony phenotypic variants (SCVs) of S. aureus are selected by long-term exposure to antibiotics or co-culture with Pseudomonas aeruginosa and are often difficult to detect. The prevalence of this pathogen is largely unknown. Aim To establish the prevalence of SCV S. aureus by screening lower airway specimens from children with CF. Methods SCV S. aureus were obtained from the hospital clinical laboratory from lower airway specimens of children with CF. Each visible morphotype was subcultured onto Horse blood agar (HBA) to enable identification of SCVs. If identified, isolates required confirmation by PCR amplification of the S. aureus -specific gene, nuc and metabolic defects determined by growing isolates in the presence of haemin, menadione, thymidine and CO 2 . Paired normal and SCV S. aureus isolates were to be genotyped using pulse-field gel electrophoresis. Results 26 children (mean age 11.35(5.05) years) from the AREST CF study (Melbourne) were identified with a positive S. aureus culture from lower respiratory tract specimens collected in 2014. All children were on at least one or multiple antibiotics prior to or at time of collection, namely Augmentin (46%), Tobramycin (15%), Bactrim (11%), or other (27%). 57% of samples were visible on large MSA plates and subcultured onto wild-type HBA plates, but no SCVs of S. aureus were isolated. Conclusion SCV's were not isolated from the lower airways specimens and therefore not identified as contributing to lung infections in our cohort of young subjects with CF.

Investigation of the multifaceted iron acquisition strategies of Burkholderia cenocepacia

Burkholderia cenocepacia is a bacterial pathogen which causes severe respiratory infections in cystic fibrosis (CF). These studies were aimed at gaining an insight into the iron acquisition strategies of B. cenocepacia. In iron restricted conditions, genes associated with the synthesis and utilisation of ornibactin (pvdA, orbA, orb F) were significantly upregulated compared to the expression of pyochelin associated genes (pchD, fptA). In the absence of alternative iron sources, B. cenocepacia J2315 and 715j utilised ferritin and haemin, but not transferrin or lactoferrin for growth. Significantly, mutants unable to produce ornibactin, (715j-orbI) or ornibactin and pyochelin, (715j-pobA), utilised haemin and ferritin more efficiently than the wild-type. Moreover, both mutants were also able to utilise lactoferrin for growth (P ≤ 0.01) and additionally 715j-pobA utilised transferrin (P ≤ 0.01), potentially facilitating adaptation to the host environment. Furthermore, B. cenocepacia increased ornibactin gene expression in response to pyoverdine from Pseudomonas aeruginosa (P ≤ 0.01), demonstrating the capacity to compete for iron in co-colonised niches. Pyoverdine also significantly diminished the growth of B. cenocepacia (P < 0.001) which was related to its iron chelating activity. In a study of three B. cenocepacia sequential clonal isolates obtained from a CF patient over a 3.5 year period, ornibactin upregulation in response to pyoverdine was less pronounced in the last isolate compared to the earlier isolates, as was growth in the presence of haemin and ferritin, indicating alternative iron acquisition mechanism(s) may dominate as chronic infection progresses. These data demonstrate the multifaceted iron acquisition strategies of B. cenocepacia and their capacity to be differentially activated in the presence of P. aeruginosa and during chronic infection.

Efficacy of Synriam™, a new antimalarial combination of OZ277 and piperaquine, against different developmental stages of Schistosoma mansoni

Control of schistosomiasis relies on a single drug, praziquantel (PZQ). Given the rising concerns about the potential emergence of PZQ-resistant strains, it has now become necessary to search for novel therapeutics. However, the current pace for anti-schistosomal drug discovery is slow; hence, repositioning of existing approved drugs can offer a safe, rapid and cost-effective solution. The anti-malarial synthetic artemisinin-derivatives trioxolanes demonstrated anti-schistosomal efficacies against the three major species infecting humans and, unlike PZQ, showed activities against both juvenile and adult worm stages. The 1,2,4-trioxolane/OZ277 (arterolane maleate) in combination with a partner drug: piperaquine phosphate was recently developed as an anti-malarial drug and manufactured by Ranbaxy (India) as Synriam™ (SYN). Herein, the in vivo activities of SYN were investigated in a mouse model of Schistosoma mansoni (S. mansoni), compared to PZQ. We show that a single fixed dose of 240mg/kg SYN (40mg/kg arterolane and 200mg/kg piperaqine) induced significant protective effects in mice, in terms of reduction in worm and tissue egg burdens, which were evident against all schistosome developmental stages. Extensive alterations in the tegument and subtegumental tissues of SYN-exposed worms were revealed by both scanning and transmission electron microscopes. Progressive decrease in worm activity and occurrence of death were noticed in vitro upon exposure to the drug − more pronounced in the presence of haemin. This report provides the first evidence of the efficacy of a combination of 1,2,4-trioxolane and piperaquine against S. mansoni in mice. Being effective against young stages, SYN could be used to prevent early Schistosoma infection.

Haemin represses the haemolytic activity of Staphylococcus aureus in an Sae-dependent manner.

Staphylococcus aureus is a major human pathogen and a common cause of nosocomial infections. This facultative pathogen produces a large arsenal of virulence factors, including the haemolysins, which allow the bacterium to lyse erythrocytes and thereby release large amounts of the haem-containing haemoglobin. The released haem is thought to be the main iron source of this organism during the course of infection, and is considered to be crucial for bacterial proliferation in vivo. High concentrations of haem and its degradation products, on the other hand, are known to be toxic for S. aureus, making it essential for the pathogen to tightly control haem release from red blood cells. Here we show that S. aureus responds to haemin by downregulating the expression of haemolysins. Subinhibitory concentrations of haemin were found to significantly reduce transcription of the haemolysin genes hlb (encoding β-haemolysin) and hlgA (encoding the S-class component of γ-haemolysin), while hla (encoding α-haemolysin) and RNAIII (encoding δ-haemolysin) transcription did not appear to be affected. The presence of haemin also reduced the haemolytic potential of the supernatants of S. aureus LS1 cultures. Inactivation of the sae locus in LS1 abolished the haemin effect on the transcription of haemolysin genes, indicating that the two-component regulatory system is required for this regulatory effect. Iron limitation, on the other hand, was found to induce the expression of haemolysins, and this effect was again abolished in the sae mutant, indicating that S. aureus modulates its haemolysin production in response to iron and haem availability in an Sae-dependent manner.

Schistosoma mansoni: Antischistosomal activity of the four optical isomers and the two racemates of mefloquine on schistosomula and adult worms in vitro and in vivo

Recent studies have shown that mefloquine (MQ) reveals interesting antischistosomal properties. We examined the antischistosomal activities of the erythro and threo isomers and racemates of MQ on newly transformed schistosomula (NTS) and adult Schistosoma mansoni in vitro and in mice harbouring adult S. mansoni. The in vitro effects in the presence and absence of haemin were monitored by means of microcalorimetry, scanning electron microscopy and phenotypic evaluation. Incubation of NTS with the erythro derivatives at concentrations of 3 μg/ml and above resulted in convulsions, granularity, decrease in heat flow, and death while NTS incubated with the threo derivatives were only affected at high concentrations (100 μg/ml). Extensive tegumental alterations, decrease in metabolic activity, viability, and death were observed when adult schistosomes had been exposed to 10 μg/ml of the erythro compounds. Moderate tegumental and viability changes but reduced heat production rates were observed with the threo derivatives at 10 μg/ml. In the presence of haemin, all MQ derivatives showed pronounced antischistosomal properties against adult S. mansoni in vitro. In vivo, MQ derivatives achieved statistically significant total and female worm burden reductions ranging between 65.4% and 100%. The highest total worm burden reductions of 93.4% and 90.2% were observed following treatment with the erythro and threo racemates, respectively. In conclusion, the optical isomers and racemates of MQ show only moderate stereoselectivity, in particular in vivo. Our results may enhance our understanding of the mechanism of action and therapeutic profile of MQ derivates on schistosomes.

Bacteroides graminisolvens sp. nov., a xylanolytic anaerobe isolated from a methanogenic reactor treating cattle waste

A strictly anaerobic bacterial strain, designated XDT-1T, was isolated from plant residue from a methanogenic reactor treating waste from cattle farms. Cells of the strain were Gram-negative, non-motile, non-spore-forming rods. Haemin was required for growth. The strain utilized xylan as well as various sugars including arabinose, xylose, glucose, mannose, cellobiose, raffinose, starch and pectin. The strain produced acetate, propionate and succinate from saccharides in the presence of haemin. The optimum pH for growth was approximately 7.2 and the optimum growth temperature was 30-35 degrees C. The strain was sensitive to bile. The major cellular fatty acids of the strain were anteiso-C15:0 and iso-C17:0 3-OH, MK-10(H0) was the major respiratory quinone and the genomic DNA G+C content was 38.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences placed the strain in the phylum Bacteroidetes. The closest phylogenetic neighbour of strain XDT-1T was Bacteroides ovatus NCTC 11153T, with a 16S rRNA gene sequence similarity of 94.2%. On the basis of data from the phylogenetic, physiological and chemotaxonomic analyses, strain XDT-1T represents a novel species of the genus Bacteroides, for which the name Bacteroides graminisolvens sp. nov. is proposed. The type strain is XDT-1T (=JCM 15093T=DSM 19988T).

Increased biomass yield of Lactococcus lactis during energetically limited growth and respiratory conditions

Lactococcus lactis is known to be capable of respiration under aerobic conditions in the presence of haemin. In the present study the effect of respiration on ATP production during growth on different sugars was examined. With glucose as the sole carbon source, respiratory conditions in L. lactis MG1363 resulted in only a minor increase, 21%, in biomass yield. Since ATP production through substrate-level phosphorylation was essentially identical with and without respiration, the increased biomass yield was a result of energy-saving under respiratory conditions estimated to be 0.4 mol of ATP/mol of glucose. With maltose as the energy source, the increase in biomass yield amounted to 51% compared with an aerobic culture that lacked haemin. This higher ATP yield was obtained by redirecting pyruvate metabolism from lactate to acetate production, and from savings through respiration. However, even after subtracting these contributions, approx. 0.3 mol of ATP/mol of glucose remained unaccounted for. A similar response to respiratory conditions (0.2 mol of ATP/mol of glucose) was observed in a mutant that had a decreased glucose uptake rate during growth on glucose caused by disruption of the PTS(mannose) (glucose/mannose-specific phosphotransferase system). Amino acid catabolism could be excluded as the source of the additional ATP. Since mutants without a functional H+-ATPase produced less ATP under sugar starvation and respiratory conditions, the additional ATP yield appears to come partly from energy saved on proton pumping through the H+-ATPase due to respiration and partly from a reversed function of the H+-ATPase towards oxidative phosphorylation. These results may contribute to the design and implementation of carbon-efficient high-cell-density cultures of this industrially important species of bacterium.

Bacteroides propionicifaciens sp. nov., isolated from rice-straw residue in a methanogenic reactor treating waste from cattle farms

Two strictly anaerobic bacterial strains (SV434(T) and S562) were isolated from rice-straw residue in a methanogenic reactor treating waste from cattle farms in Japan. They had identical 16S rRNA gene sequences and showed almost the same phenotypic properties. The cells of both strains were Gram-negative, non-motile, non-spore-forming rods; extraordinarily long rods often occurred. Remarkable stimulation of growth occurred with the addition of haemin and cobalamin (vitamin B(12)) to the medium. The supplementary cobalamin and haemin could be replaced if autoclaved and clarified sludge fluid obtained from the reactor was added. Both strains utilized a range of growth substrates, including arabinose, fructose, galactose, glucose, mannose, cellobiose, maltose, glycogen, starch, dextrin, amygdalin, lactate and pyruvate. Both strains produced acetate and propionate with a small amount of succinate from these substrates in the presence of haemin and cobalamin. Both strains were slightly alkaliphilic, having a pH optimum at about 7.9. The temperature range for growth was 5-35 degrees C, the optimum being 30 degrees C. The NaCl concentration range for growth was 0-4 % (w/v). Catalase activity was not detected in cells cultivated without haemin, whereas cells cultivated with haemin usually had the enzyme activity. Oxidase and nitrate-reducing activities were not detected. Aesculin was hydrolysed, but gelatin was not hydrolysed. Both strains were sensitive to bile acids. The major cellular fatty acids of both strains were anteiso-C(15 : 0) and iso-C(15 : 0). Menaquinones MK-8(H(0)) and MK-9(H(0)) were the major respiratory quinones and the genomic DNA G+C contents were 46.2-47.5 mol%. A phylogenetic analysis based on 16S rRNA gene sequences placed both strains in the phylum Bacteroidetes. Bacteroides coprosuis (isolated from swine-manure storage pits) was the species most closely related to both strains (95.9 % 16S rRNA gene sequence similarity to the type strain). On the basis of the phylogenetic, physiological and chemotaxonomic analyses, strains SV434(T) and S562 represent a novel species of the genus Bacteroides, for which the name Bacteroides propionicifaciens sp. nov. is proposed. The type strain is SV434(T) (=JCM 14649(T) =DSM 19291(T)).

Regulation of Protein Synthesis in Reticulocyte Lysates

Heating of a rabbit reticulocyte lysate at 44 degrees C, in the presence of optimal concentrations of haemin, results in an inhibition of protein synthesis and in the appearance of an inhibitory activity in the postribosomal supernatant. These effects of supraoptimal heating are similar to those observed in lysates and supernatants incubated at physiological temperatures in the absence of haemin. In this paper we examined whether the haem-regulated inhibitor produced by treating postribosomal supernatant with N-ethylmaleimide and the inhibitor generated by heating at 44 degrees C in the presence of haemin are the same molecular entity. Both inhibitors behaved similarly through a partial purification consisting of 50% (NH4)2SO4 precipitation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography, and had the same pattern of polypeptides after polyacrylamide gel electrophoresis. However, when incubated with an antiserum to the haem-regulated inhibitor the activity of the 44 degrees C heated supernatant was not neutralized, whereas that of its more purified fractions was. This apparent contradiction was shown to be due to an interference of the immune serum assay by the levels of proinhibitor and haemoglobin present in the crude supernatant. Further experiments, with extensively diluted 44 degrees C supernatants or with isolated proinhibitor subsequently heated, are consistent with the conclusion that both heating at supraoptimal temperatures and incubating in the absence of haem finally activate the same inhibitor.

Enhancement of glutathione-dependent haemin degradation by ascorbic acid

In the current work, we investigated the effect of ascorbic acid on GSH-mediated haemin degradation. GSH-mediated haemin degradation in the presence of ascorbic acid in phosphate-buffered saline and in erythrocyte ghosts was determined by recording absorbance at 365 and 399 nm, respectively. Generation of intracellular H 2O 2 was measured indirectly in terms of the inactivation of endogenous catalase in erythrocytes in the presence of 3-amino-1,2,4-triazole. Although ascorbic acid itself did not induce haemin degradation, it enhanced GSH-mediated haemin degradation. Experiments with catalase showed that H 2O 2 was essential in this process. The oxidation of ascorbic acid in the presence of haemin was stimulated by GSH, suggesting that ascorbic acid can alter the mechanism of H 2O 2 generation observed with GSH and haemin alone. These results suggest that enhancement of GSH-mediated haemin degradation by ascorbic acid may be due to an increase in the production of H 2O 2 generated by GSH and haemin in the absence of ascorbic acid.

Characterization of a Vibrio vulnificus LysR homologue, HupR, which regulates expression of the haem uptake outer membrane protein, HupA

In Vibrio vulnificus, the ability to acquire iron from the host has been shown to correlate with virulence. Here, we show that the DNA upstream of hupA (haem uptake receptor) in V. vulnificus encodes a protein in the inverse orientation to hupA (named hupR). HupR shares homology with the LysR family of positive transcriptional activators. A hupA–lacZ fusion contained on a plasmid was transformed into Fur−, Fur+and HupR−strains of V. vulnificus. The β-galactosidase assays and Northern blot analysis showed that transcription of hupA is negatively regulated by iron and the Fur repressor in V. vulnificus. Under low-iron conditions with added haemin, the expression of hupA in the hupR mutant was significantly lower than in the wild-type. This diminished response to haem was detected by both Northern blot andhupA–lacZ fusion analysis. The haem response of hupA in the hupR mutant was restored to wild-type levels when complemented with hupR in trans. These studies suggest that HupR may act as a positive regulator ofhupA transcription under low-iron conditions in the presence of haemin.

Enhanced in vitro neurotoxicity of artemisinin derivatives in the presence of haemin

The role of haem in the neurotoxicity of artemisinin derivatives has been studied in vitro by examining neurite outgrowth measured by image analysis and cellular metabolism of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) measured spectrophotometrically in the neuroblastoma cell line NB2a, and by examining binding of radiolabelled dihydroartemisinin to NB2a cell and rat brain proteins. In the cases of artemether, dihydroartemisinin, and arteether, haemin (ferriprotoporphyrin IX) significantly increased the dose-related inhibition of neurite outgrowth from differentiating NB2a cells and significantly increased the dose-dependent inhibition of MTT metabolism. Inhibition of neurite outgrowth and metabolism of MTT in the presence or absence of haemin ranged from 72% to 93% and from 27% to 49% at a drug concentration of 300 nM. Haemin also significantly increased the dose-related binding of radiolabelled dihydroartemisinin to proteins from NB2a cells approximately twofold and to rat brain between three- and sixfold. Haemin did not enhance the neurotoxicity of desoxyarteether, a structural analogue of arteether with an ether linkage in the place of the endoperoxide bridge. It is suggested that haemin may catalyse the transformation of these derivatives via an interaction with the endoperoxide bridge of the artemisinin derivative to produce free radicals or electrophilic intermediates that are toxic to neuronal cells.

Erythroid development of the FDCP-Mix A4 multipotent cell line is governed by the relative concentrations of erythropoietin and interleukin 3.

Conditions are described which promote the erythroid development of the FDCP-Mix A4 (A4) cell line with accompanying proliferation of the cells. The requirements for this development are low concentrations of interleukin 3 (IL-3) plus the presence of erythropoietin (epo) and haemin. When high concentrations of IL-3 are added with erythropoietin and haemin the cells do not differentiate and maintain their blast cell morphology. Addition of haemin, in the absence of erythropoietin, does not promote erythroid development, but the presence of haemin with erythropoietin promotes increased proliferation and maturation. The morphological maturation of A4 cells along the erythroid lineage is accompanied by a gradual loss of clonogenic potential, loss of A4 cell multipotency, increased erythropoietin receptor expression, and an increased expression of the beta-globin gene. An initial increase in mitogenic responsiveness to erythropoietin is followed by a decrease as the cells become refractory to all mitogenic stimuli with the acquisition of a postmitotic, mature erythroid cell phenotype.

Inhibition of lipid autoxidation by bovine superoxide dismutase

For autoxidation, the initiation reactions, which are believed to be responsible for the formation of hydroxyl radicals, are inhibited by superoxide dismutase (SOD). Soybean lipoxygenase types I, IV and V were not inhibited by SOD in model systems containing linoleic acid. Autoxidation in a low-iron model system was further reduced by SOD. Autoxidation in the presence of haemin was rapid but also inhibited by SOD. These observations indicate that SOD mainly prevents the formation of hydroxyl radicals via the Haber-Weiss reaction during the iron-catalysed oxidation of linoleic acid, although it is possible that the enzyme might also inhibit haemin-catalysed oxidation by scavenging an oxy-haem complex.

Stromal cell-dependent terminal maturation of K562 erythroleukaemia cells

K562 cells were cultured over monolayers of various cell types for four days and the characteristics of those cells which became firmly adherent to such monolayers studied. The monolayers used were composed of stromal cells derived from human bone marrow, two human fibroblast lines, human umbilical cord-derived endothelial cells, bone marrow macrophages, blood-monocyte-derived macrophages, a neuroglial cell line (U-251 MG) or mixtures of some of the above. The data showed that K562 cells adhered much more frequently to fibroblasts than to other cell types. In the case of some marrow-derived stromal cell layers grown in human group AB serum, the firmly adherent K562 cells showed morphological evidence of nuclear and cytoplasmic maturation and, especially in the presence of haemin, sometimes developed into erythrocyte-like cells. The maturing cells were weakly or moderately benzidine-positive. By contrast, K562 cells which adhered to monolayers of fibroblasts, endothelial cells or macrophages showed little or no maturation and were either benzidine-negative or only very weakly positive. In experiments employing monolayers made up by mixing skin fibroblasts and endothelial cells or skin fibroblasts, endothelial cells and macrophages, some adherent K562 cells developed into cells resembling early and late polychromatic normoblasts and erythrocytes. The maturing cells were moderately or strongly benzidine-positive. Maturation was most marked in K562 cells adherent to regions of the monolayer containing all three of the stromal cell types. The results indicate that the optimal microenvironmental niche for the development of K562 cells into erythroblasts and erythrocytes may consist of a cluster of closely associated fibroblasts, macrophages and endothelial cells.

The effect of haemin on RNA synthesis and stability in differentiating rabbit erythroblasts.

Haemin accelerates the maturation of erythroid cells but whether this is the result of increased globin gene transcription or processing and stabilization of globin mRNA is not clear. The effect of haemin on the synthesis and stability of non-globin messengers is also unknown. We examined the changes that occur in RNA metabolism when anaemic rabbit bone marrow erythroblasts, fractionated into immature and mature fractions, are cultured with 20 microM or 50 microM haemin for brief periods (5-8 h). With both cell types haemin increases the incorporation of [3H]uridine into newly synthesized RNA, particularly into the poly(A)-rich fraction which can increase threefold. Haemin also increases the synthesis of globin mRNA (up to 500% absolutely and 50% relative to the synthesis of total RNA) in the immature, but not in the mature, cells. These results suggest that haemin increases the transcription of both globin and non-globin mRNAs and that the relative increase of each depends on the stage of erythroid cell development. When the [3H]RNA from prelabelled cells was chased in the presence of haemin (with or without actinomycin D) the proportion of 3H remaining in globin mRNA increased in the immature, but not in the mature, cells. These changes in the relative concentration of globin mRNA were also shown by the translation of extracted bone marrow RNAs in a nuclease-treated reticulocyte lysate. We conclude that a secondary effect of haemin is on RNA stability and that it enhances the accumulation of globin mRNA by both molecular and cellular mechanisms.

Partial purification of prostaglandin synthetase from silkmoth Bombyx mori L.

Prostaglandin synthetase has been isolated and purified from the post mitochondrial supernatant of the testis of male silkmoth Bombyx mori L. A 3-fold purification was achieved compared to that of post mitochondrial supernatant. The partially purified enzyme converted arachidonic acid to PGE 2 in vitro in the presence of haemin and hydroquinone.

Biogenesis of mitochondrial proteins. Regulation of production of delta-aminolaevulinate synthase by haemin in embryonic-chick liver.

The effect of haemin on the biogenesis of delta-aminolaevulinate synthase (ALA synthase) was investigated in primary cultures of embryonic-chick liver. The activity of the enzyme and the amount of the enzyme detected by 'immune-blotting' were determined in hepatocytes incubated with the porphyrogenic agent allylisopropylacetamide. The results of these studies indicated that the loss in ALA synthase activity in cells incubated in the presence of haemin (10 microM) was roughly proportional to a loss in the immune-reactive mass of the enzyme. Haemin was as effective as cycloheximide in causing depletion of ALA synthase in hepatocytes. We had previously established that haemin blocked the maturation of the precursor of ALA synthase [Ades (1983) Biochem. Biophys. Res. Commun. 110, 42-47]. From results reported in the present paper on analyses of immune-precipitated ALA synthase after pulse-labelling with [35S]methionine in the presence and in the absence of haemin, we determined that the inhibition of processing of pre-ALA synthase in cells by haemin was concentration-dependent. A concentration of 2 microM in the culture medium blocked the processing of pre-ALA synthase by 50% in hepatocytes. We also determined that, after inhibition of its maturation by haemin, pre-ALA synthase turned-over with a half-time of 30 min; on the other hand, mature ALA synthase turned-over with a half-time of 120 min.

The high-temperature inactivation of rabbit reticulocyte lysates by a haemin-independent mechanism.

Exposure of rabbit reticulocytes or their lysates to 42–45 °C leads to a decrease in the rate of polypeptide chain initiation which resembles in several respects the lesion produced in reticulocyte lysates incubated in the absence of haemin. The aim of the present investigation was to establish whether the two processes were mediated by a similar mechanism, i.e. by a haemin-controlled, catalytically active, soluble inhibitor. It was found that the addition of haemin during exposure to elevated temperatures cannot prevent nor rescue the high-temperature-dependent inactivation of reticulocytes or their lysates. Assay systems which can detect the haemin-controlled repressor revealed that supraoptimally heated lysates and the postribosomal supernatants derived from them contain a soluble inhibitor. This inhibitor, although forming in the presence of haemin, resembles in several properties the haemin-controlled repressor. However, it appears to fractionate differently with ammonium sulphate and to decay after prolonged supraoptimal heating of either cells or lysates (but not of postribosomal supernatant). Moreover the 0.5 M KCl ribosomal wash obtained from heated cells or lysates was as active as preparations from control cells in supporting protein synthesis at 30–37 °C in fresh or preheated lysates and in fractionated cell-free systems synthesizing globin de novo. These results suggest that the lesion in polypeptide chain initiation produced in reticulocyte lysates by elevated temperatures or by incubation in the absence of haemin may be mediated by similar but not necessarily identical mechanisms.

Enhancement of nitro reduction in rat liver microsomes by haemin and haemoproteins.

1. Reductive metabolism of p-nitrobenzoic acid and neoprontosil in rat liver microsomes was studied in the presence of haemin, haemoglobin and myoglobin. 2. Microsomal nitro reduction is enhanced 4-fold in the presence of haemoglobin, whereas azo reduction is not affected. 3. Microsomal nitro reduction is enhanced to a similar extent by haemoglobin, haemin and boiled haemoglobin, whereas myoglobin is about half as active. 4. Maximal enhancement of microsomal nitro reductase activity by haemoglobin is achieved at high substrate concentration (6 mM) and low microsomal protein concentration (0.5--1.0 mg/ml). 5. Control microsomal nitro reduction as well as the haemoglobin-enhanced microsomal nitro reduction are inhibited completely by O2 and CO whereas potassium azide as a ligand of ferric haem iron is a less potent inhibitor.