Heme is a positive regulator of cytochrome P-450 gene transcription

Inhibitors of heme biosynthesis such as CoCl2, 3-amino-1,2,4-triazole, and thioacetamide block the 3-methylcholanthrene-mediated induction of cytochrome P-450 (c + d) messenger RNAs and their transcription in rat liver. This effect is specific, since the messenger RNA levels for albumin and glutathione transferase (Ya + Yc) and their transcription are not significantly influenced under conditions of heme depletion. Exogenous administration of heme at very low doses (50 μg/100 g body wt) is able to completely counteract the effects of the heme biosynthetic inhibitors on cytochrome P-450 (c + d) messenger RNA levels and their transcription. This constitutes a direct proof for the role of heme as a positive regulator of cytochrome P-450 gene transcription.

Heme regulates cytochrome P-450 gene transcription elongation

Administration of 3-methylcholanthrene (MC) to rats results in a striking increase in the transcription of cytochrome P-450 (c+d) messenger RNA with isolated nuclei, which is blocked by the simultaneous administration of cobalt chloride, an inhibitor of heme biosynthesis. Transcription of cytochrome P-450 (c+d) mRNAs with nuclei isolated from MC treated rats shows a linear increase with time of incubation, whereas it shows a progressive decrease with incubation time in the case of nuclei isolated from MC+CoCl2 treated rats. Addition of heme in vitro (10−6M) to the latter nuclei results in a significant counteraction of the decreased cytochrome P-450 (c+d) mRNA transcription. The inhibition in transcription rates observed in MC+CoCl2 treated rat liver nuclei is more pronounced with the seventh exon probe than with the second exon probe. Once again, in vitro heme addition can counteract the inhibition observed with both the probes. Since run off transcription with isolated nuclei represents essentially elongation of the initiated transcripts, the data obtained can be interpreted on the basis that heme regulates cytochrome P-450 gene transcription elongation.

Chloroquine Inhibits Heme-Dependent Protein Synthesis in Plasmodium falciparum

A cell-free protein-synthesizing system has been reconstituted using the S-30 fraction or ribosomes and the S-100 fraction from Plasmodium falciparum. Addition of heme in vitro stimulates cell-free protein synthesis strikingly. Chloroquine inhibits the heme-dependent protein synthesis in the parasite lysate. The drug has also been found to inhibit parasite protein synthesis in situ at therapeutic concentrations soon after addition to parasite cultures. Ribosomes as well as the S-100 fraction isolated from such chloroquine-treated cultures are defective in protein synthesis. Addition of hemin plus glucose 6-phosphate or high concentrations of GTP, cAMP, and an active preparation of eIF-2 to the parasite cell-free system restores protein synthesis to a significant extent in chloroquine-treated cultures. Under conditions of inhibition of protein synthesis in situ by chloroquine in the culture, the parasite eukaryotic initiation factor 2-alpha- (eIF-2-alpha) is phosphorylated in the parasite lysate to a greater extent than that observed in the control culture. Addition of hemin in vitro suppresses this phosphorylation. eIF-2-alpha kinase activity is present in the parasite lysate and is not a contaminant derived from the human erythrocytes used to culture the parasite. The heme-chloroquine interactive effects can also be demonstrated with purified eIF-2-alpha kinase from rabbit reticulocyte lysate. It is proposed that chloroquine inhibits heme-dependent protein synthesis in the parasite and this is an early event mediating the growth-inhibitory effects of the drug.

Indole oxygenase from the leaves of Jasminum grandiflorum

An indole oxygenase from the leaves of Jasminum grandiflorum was isolated and purified to near homogeneity. The purified enzyme system catalyses the conversion of indole to anthranilic acid. It is optimally active at pH 4.8 and at 30°C. Apart from indole, the oxygenase also attacks 5-hydroxy indole and 5-bromoindole. Both sulfhydryl reagents and sulfhydryl compounds inhibited the enzyme activity. Copper specific metal chelators such as salicylaldoxime, diethyl dithiocarbamate and neocuproine, inhibited the enzyme activity drastically. Inhibition caused by atebrine, could be reversed by FAD. Dialysis resulted in complete loss of enzyme activity. Inactive enzyme could be reactivated only by the addition of both FAD and Cu2+, suggesting that indole oxygenase is a cuproflavoprotein.

devovo biosynthesis of heme offers a new chemotherapeutic target in the human malarial parasite

The human malarial parasite, Image , has been found to synthesize heme Image , despite the accumulation of large quantities of polymeric heme derived from the hemoglobin of the red cell host. The parasite δ-aminolevulinate dehydrase level is significantly lower than that of the host and its inhibition by succinylacetone leads to inhibition of parasite protein synthesis and viability.

Malaria Parasite-Synthesized Heme Is Essential in the Mosquito and Liver Stages and Complements Host Heme in the Blood Stages of Infection

Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, d-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of Plasmodium berghei (Pb). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using 4-(14) C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.

X-ray Structure of Gelonin at 1.8 Å Resolution

Gelonin is a single chain ribosome inactivating protein (RIP) with potential application in the treatment of cancer and AIDS. Diffraction quality crystals grown using PEG3350, belong to the space group P2(1), with it a = 49.4 Angstrom b = 44.9 Angstrom, c = 137.4 Angstrom and beta = 98.4 degrees, and contain two molecules in the asymmetric unit. Diffraction data collected to 1.8 Angstrom resolution has a R(m) value of 7.3%. Structure of gelonin has been solved by the molecular replacement method, using ricin A chain as the search model. Crystallographic refinement using X-PLOR resulted in a model for which the r.m.s deviations from ideal bond lengths and bond angles are 0.012 Angstrom and 2.7 degrees, respectively The final R-factor is 18.4% for 39,806 reflections for which I > 1.0 sigma(I).The C-alpha atoms of the two molecules in the asymmetric unit superpose to within 0.38 Angstrom for 247 atom pairs. The overall fold of gelonin is similar to that of other RIPs such as ricin A chain and alpha-momorcharin, the r.m.s.d. for C-alpha superpositions being 1.3 and 1.4 Angstrom, respectively The-catalytic residues (Glu166, Arg169 and Tyr113) in the active site form a hydrogen bond scheme similar to that observed in other RIPs. The conformation of Tyr74 in the active site, however, is significantly different from that in alpha-momorcharin. Three well defined water molecules are located in the active site cavity and one of them, X319, superposes to within 0.2 Angstrom of a corresponding water molecule in the structure of alpha-momorcharin. Any of the three could be the substrate water molecule in the hydrolysis reaction catalysed by gelonin.Difference electron density for a N-linked sugar moiety has been observed near only one of the two potential glycosylation sites in the sequence. The amino acid at position 239 has been established as Lys by calculation of omit electron density maps.The two cysteine residues in the sequence, Cys44 and Cys50, form a disulphide bond, and are therefore not available for disulphide conjugation with antibodies. Based on the structure, the region of the molecule that is involved in intradimer interactions is suggested to be suitable for introducing a Cys residue for purposes of conjugation with an antibody to produce useful immunotoxins.

Ethyl 1-(2-cyanoethyl)-3,5-dimethyl-1H-indole-2-carboxylate

The title compound, C16H18N2O2, is an important precursor in the synthesis of 1,2,3,4-tetrahydropyrazinoindoles, which show excellent antihistamine, antihypertensive and central nervous system depressant properties. The carbethoxy group attached to C2 and the planar cyanoethyl group attached to N1 make dihedral angles of 11.0(4) and 75.0(3)degrees, respectively, with the mean plane of the indole ring, The C-C=N chain is linear with a bond angle of 179.3 (4)degrees.

3-Phenylisoquinolin-1(2H)-one

The title compound, C15H11NO, consists of a planar isoquinolinone group to which a phenyl ring is attached in a twisted fashion [dihedral angle = 39.44 (4)degrees]. The crystal packing is dominated by intermolecular N-H center dot center dot center dot O and C-H center dot center dot center dot O hydrogen bonds which define centrosymmetric dimeric entitities.

Methyl 1H-1,2,3-triazole-4-carboxylate

The title compound, C4H5N3O2, features an essentially planar molecule (r.m.s. deviation for all non-H atoms = 0.013 angstrom). The crystal structure is stabilized by intermolecular N-H center dot center dot center dot O hydrogen bonds and pi-pi stacking interactions (centroid centroid distance 3.882 angstrom).

1-(4-Chloro-3-fluorophenyl)-2-[(3-phenylisoquinolin-1-yl)sulfanyl]ethanone

In the title compound, C23H15ClFNOS, the isoquinoline system and the 4-chloro-3-fluorophenyl ring are aligned at 80.4 (1)degrees. The dihedral angle between the isoquinoline system and the pendant (unsubstituted) phenyl ring is 19.91 (1)degrees.

2-[2-(Hydroxymethyl)phenyl]-1-phenylethanol

The title compound, C15H16O2, has a dihedral angle of 19.10 (5)degrees between the mean planes of the two benzene rings. There is an intramolecular O-H center dot center dot center dot O hydrogen bond and the C-C-C-C torsion angle across the bridge between the two rings is 173.13 (14)degrees. The molecules form intermolecular O-H center dot center dot center dot O hydrogen-bonded chains extending along the a axis. C-H center dot center dot center dot pi contacts are also observed between molecules within the chains.