Presence Of Haematin Research Articles

Biliverdin reductase is a transporter of haem into the nucleus and is essential for regulation of HO-1 gene expression by haematin

hBVR (human biliverdin reductase) is an enzyme that reduces biliverdin (the product of haem oxygenases HO-1 and HO-2 activity) to the antioxidant bilirubin. It also functions as a kinase and as a transcription factor in the MAPK (mitogen-activated protein kinase) signalling cascade. Fluorescence correlation spectroscopy was used to investigate the mobility of hBVR in living cells and its function in the nuclear transport of haematin for induction of HO-1. In transiently transfected HeLa cells only kinase-competent hBVR translocates to the nucleus. A reduced mobility in the nucleus of haematin-treated cells suggests formation of an hBVR-haematin complex and its further association with large nuclear components. The binding of haematin is specific, with the formation of a 1:1 molar complex, and the C-terminal 7-residue fragment KYCCSRK(296) of hBVR contributes to the binding. The following data suggest formation of dynamic complexes of hBVR-haematin with chromatin: (i) the reduction of hBVR mobility in the presence of haematin is greater in heterochromatic regions than in euchromatic domains and (ii) hBVR mobility is not retarded by haematin in nuclear lysates that contain only soluble factors. Moreover, hBVR kinase activity is stimulated in the presence of double-stranded DNA fragments corresponding to HO-1 antioxidant and HREs (hypoxia response elements), as well as by haematin. Experiments with nuclear localization, export signal mutants and si-hBVR [siRNA (small interfering RNA) specific to hBVR] indicate that nuclear localization of hBVR is required for induction of HO-1 by haematin. Because gene regulation is energy-dependent and haematin regulates gene expression, our data suggest that hBVR functions as an essential component of the regulatory mechanisms for haem-responsive transcriptional activation.

Photochemical method for the determination of hydrogen peroxide and glucose

Reduction of hydrogen peroxide was achieved with leuco-phloxin in the presence of haematin. The leuco dye was generated through the photochemical reaction between phloxin and ethylenediaminetetraacetic acid. The method involves measuring the time to reach the end-point of the photochemical titration, i.e., the photolysis time necessary for the total reduction of the peroxide. The method can be extended to the determination of substrate–enzyme systems that produce hydrogen peroxide, e.g., glucose–glucose oxidase. The assay is linear between 0.12 and 4.61 µg cm–3 for hydrogen peroxide (r= 0.9992) and between 3.99 and 43.2 µg cm–3 for glucose (r= 0.9989). The detection limit, defined as three times the standard deviation of the reagent blank, was 0.03 µg cm–3 for hydrogen peroxide and 1.0 µg cm–3 for glucose.

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.

Growth of Streptococcus faecalis var. zymogenes on glycerol: the effect of aerobic and anaerobic growth in the presence and absence of haematin on enzyme synthesis.

Streptococcus faecalis var. zymogenes was grown aerobically and anaerobically in the presence and absence of haematin, with glycerol as the carbon and energy source. Aerobic growth was stimulated by the inclusion of haematin in the medium but fumarate had no effect on growth. The bacterium was unable to grow anaerobically on glycerol unless fumarate was present; haematin had no effect on growth. NADH oxidase activity, which catalysed the oxidation of NADH + H+ to form H2O rather than H2O2, was found in the soluble fraction and was induced by aerobic growth but partially repressed when haematin was present in the medium. In contrast, a particulate NADH oxidase, which was sensitive to inhibition by antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide, was induced by aerobic growth in the presence of haematin. NADH peroxidase was massively induced by aerobic growth, whereas more lactate dehydrogenase activity was found in anaerobically grown bacteria. Catalase was formed only during aerobic growth in the presence of haematin.

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.

Cytochrome Formation, Oxygen-induced Proton Extrusion and Respiratory Activity in Streptococcus faecalis var. zymogenes Grown in the Presence of Haematin

Streptococcus faecalis var. zyrnogenes was grown in oxygen-limited continuous culture in a lactate/tryptone/yeast extract medium containing 7·5 μg haematin ml-l. Low temperature difference spectra of whole organisms and washed membranes showed two peaks in the cytochrome b region, at 558 nm and 562 nm, and a peak at 627 nm indicative of a d-type cytochrome. A CO-binding cytochrome was also present in the haematin-grown bacteria. These cytochromes were not detected in bacteria grown without haematin. The haematin- grown bacteria produced proton pulses in a weakly buffered medium when pulsed with oxygen-saturated buffer. The average →H+/O ratio of such pulses was 1·4 and they were abolished by carbonyl cyanide m-chlorophenylhydrazone (CCCP). The haematin-grown bacteria had a high activity of particulate NADH oxidase, at least 10 times that of bacteria grown without haematin. They also had a high lactate Q 02 which was strongly inhibited by CCCP and gramicidin whereas bacteria grown without haematin had a very low lactate Q 02 largely insensitive to the uncoupling agents. The glucose Q 02 was similar in bacteria grown in the presence or absence of haematin but was stimulated by uncoupling agents in haematin- grown organisms and slightly inhibited by these agents in organisms grown without haematin. These results confirm earlier findings of the ability of S. faecalis to form a functional cytochrome system in the cell membrane when supplied with haematin and show that electron transport to oxygen by such a system is coupled to proton translocation.

Distribution of Cytochrome-like Respiration in Streptococci

The electron transport systems of 134 strains of streptococci were studied after aerobic growth on glucose in the presence of haematin, by examining the inhibition of electron transport as well as the cellular site of NADH oxidation. Each strain was placed into one of three possible groups: cytochrome-like NADH oxidase; flavin-like NADH oxidase; or no NADH oxidase. Most (88%) of the strains of Streptococcus faecalis and its variants liquefaciens and zymogenes and a few strains of S. lactis and its variant diacetylactis contained cytochrome-like respiratory systems. Other streptococci including S. faecium fell into one of the other groups but did not contain cytochrome-like NADH oxidases.

Mechanism of action of specific antiserum on Pasteurella septica. Selective inhibition of net macromolecular synthesis and its reversal by iron compounds.

Circumstantial evidence suggests that the ability of the host to prevent the uptake of iron by pathogenic bacteria may constitute an important means of defence. A detailed biochemical study of the way antisera exert their antibacterial effects and the possible involvement of iron has now been initiated. Work with Pasteurella septica antiserum has revealed a totally unsuspected way in which antiserum and complement exert their antibacterial effects. The process is very quick and specific and results in the cessation of bacterial multiplication; cells in stasis then appear to be killed by the operation of a secondary event. The inhibitory process operates by affecting the biochemistry of the bacterial cell leading first to an inhibition of net RNA synthesis and then to an inhibition of all macromolecular synthesis. The presence of free iron in the serum did not prevent the initiation of the inhibition but it did allow macromolecular synthesis, cell multiplication and rapid growth to be resumed later. In contrast, the presence of haematin in the serum allowed the cells to continue multiplying in the presence of antiserum without delay. Addition of haematin to bacteria which had been inhibited by antiserum restored net RNA synthesis immediately. Net protein synthesis and multiplication were restarted within 15–20 min and DNA synthesis in 40 min.

A technique for the detection of Polystoma integerrimum in the common frog (Rana temporaria).

An infection of the frog bladder by the Monogenetic Trematode Polystoma integerrimum can be detected by testing the urine for the presence of the pigment haematin. This substance is an end product of the digestion of blood, which the parasite sucks from the capillaries of the bladder wall, and it is voided from the flatworm's gut into the bladder to pass eventually to the exterior with the urine (Jennings—unpublished work).An alkaline solution of luminol (3-amino pthallic cyclic hydrazide hydrochloride) and hydrogen peroxide exhibits an intense blue luminescence in the presence of haematin (Proescher & Moody, 1938) and this reaction, which is very sensitive, can be used to detect the pigment in the urine of infected frogs.