Absence Of Haemin Research Articles

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.

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.

Overexpression of neuronal nitric oxide synthase in insect cells reveals requirement of haem for tetrahydrobiopterin binding.

Nitric oxide synthase (NOS) catalyses the conversion of L-arginine into L-citrulline and nitric oxide. Recently we have developed a method for expression of recombinant rat brain NOS in baculovirus-infected Sf9 cells and purification of the enzymically active enzyme [Harteneck, Klatt, Schmidt and Mayer (1994) Biochem J. 304, 683-686]. To study how biosynthetic manipulation of the NOS cofactors haem, FAD/FMN, and tetrahydrobiopterin (H4biopterin) affects the properties of the isolated enzyme, Sf9 cells were infected in the absence and presence of haemin chloride (4 microg/ml), riboflavin (0.1.mM), and the inhibitor of H4biopterin biosynthesis 2,4-diamino-6-hydroxypyrimidine (10 mM). In the absence of haemin, NOS was expressed to a very high level but remained predominantly insoluble. Purification of the soluble fraction of the expressed protein showed that it had poor activity (0.35 micromol of citrulline x mg(-1) x min(-1)) and was haem-deficient (0.37 equiv. per monomer). Supplementing the culture medium with haemin resulted in pronounced solubilization of the expressed enzyme, which had a specific activity of approximately 1 micromol of citrulline x mg(-1) x min(-1) and contained 0.95 equiv. of haem per monomer under these conditions. Unexpectedly, the amount of H(4) biopterin endogenously present in the different NOS preparations positively correlated with the amount of enzyme-bound haem (y = 0.066+0.430x; r = 0.998). Radioligand binding experiments demonstrated that haem-deficient enzyme preparations containing 30-40% of the holoenzyme bound only approximately 40% of H4biopterin as compared with haem-saturated controls. These results suggest that the prosthetic haem group is essentially involved in the correct folding of NOS that is a requisite for solubilization of the protein and tight binding of H4biopterin.

Effect of ferric and ferrous iron chelators on growth of Bacteroides fragilis under anaerobic conditions

Growth of Bacteroides fragilis under anaerobic conditions in the presence of either haemin or protoporphyrin IX was inhibited by the ferrous iron chelator bipyridyl. The ferric-iron chelator desferrioxamine inhibited growth in the presence of protoporphyrin but not haemin, suggesting that even under anaerobic conditions Fe3+ is involved in uptake of non-haem iron, which is required in the absence of haemin. However, the ferric iron chelators 1,2-dimethyl-3-hydroxy-pyrid-4-one (L1) and pyridoxal isonicotinoyl hydrazone (PIH) were only weakly inhibitory. Apotransferrin, which also binds Fe3+, inhibited growth, but this was not simply due to binding of iron in the medium, as under the reducing conditions present, transferrin was unable to bind iron. This study suggests that even under anaerobic conditions, uptake of non-haem iron by B. fragilis may involve conversion of Fe2+ to Fe3+.

Effect of ferric and ferrous iron chelators on growth ofBacteroides fragilisunder anaerobic conditions

Growth of Bacteroides fragilis under anaerobic conditions in the presence of either haemin or protoporphyrin IX was inhibited by the ferrous iron chelator bipyridyl. The ferric-iron chelator desferrioxamine inhibited growth in the presence of protoporphyrin but not haemin, suggesting that even under anaerobic conditions Fe3+ is involved in uptake of non-haem iron, which is required in the absence of haemin. However, the ferric iron chelators 1,2-dimethyl-3-hydroxy-pyrid-4-one (L1) and pyridoxal isonicotinoyl hydrazone (PIH) were only weakly inhibitory. Apotransferrin, which also binds Fe3+, inhibited growth, but this was not simply due to binding of iron in the medium, as under the reducing conditions present, transferrin was unable to bind iron. This study suggests that even under anaerobic conditions, uptake of non-haem iron by B. fragilis may involve conversion of Fe2+ to Fe3+.

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.

Control of protein synthesis in reticulocyte lysates: The effect of nucleotide triphosphates on formation of the translational repressor

High concentrations (1–2mM) of GTP completely prevented the decline in protein synthesis which occurs in a reticulocyte lysate incubated in the absence of haemin. The effect of GTP was diminished by raising the concentration of ATP. When formation of the haemin-controlled translational repressor was studied as a partial reaction using post-ribosomal supernatant, it was found that GTP acted by blocking repressor formation, whilst ATP was found to stimulate this process.