Expression of peptide transporter 1 has a positive correlation in protoporphyrin IX accumulation induced by 5-aminolevulinic acid with photodynamic detection of non-small cell lung cancer and metastatic brain tumor specimens originating from non-small cell lung cancer.

BACKGROUND: Recently, 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX fluorescence was reported to be a useful tool during total surgical resection of high-grade gliomas. However, the labeling efficacy of protoporphyrin IX fluorescence is lower in metastatic brain tumors compared to that in high-grade gliomas, and the mechanism underlying protoporphyrin IX fluorescence in metastatic brain tumors remains unclear. Lung cancer, particularly non-small cell lung cancer (NSCLC), is the most common origin for metastatic brain tumor. Therefore, we investigated the mechanism of protoporphyrin IX fluorescence in NSCLC and associated metastatic brain tumors. METHODS: Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) was employed to evaluate the protein and mRNA levels of five transporters and enzymes involved in the porphyrin biosynthesis pathway: peptide transporter 1 (PEPT1), hydroxymethylbilane synthase (HMBS), ferrochelatase (FECH), ATP-binding cassette 2 (ABCG2), and heme oxygenase 1 (HO-1). The correlation between protein, mRNA, and protoporphyrin IX levels in NSCLC cells were evaluated in vitro. Immunohistochemistry was used to determine proteins that played a key role in intraoperative protoporphyrin IX fluorescence in clinical samples from patients with NSCLC and pathologically confirmed metastatic brain tumors. RESULTS: A significant correlation between PEPT1 expression and protoporphyrin IX accumulation in vitro was identified by western blotting (P = 0.003) and qRT-PCR (P = 0.04). Immunohistochemistry results indicated that there was a significant difference in PEPT1 between the intraoperative protoporphyrin IX fluorescence-positive and protoporphyrin IX fluorescence-negative groups (P = 0.009). CONCLUSION: Expression of PEPT1 was found to be positively correlated with 5-ALA-induced protoporphyrin IX accumulation detected by photodynamic reaction in metastatic brain tumors originating from NSCLC.

Cloning, purification and characterization of Geobacillus stearothermophilus V uroporphyrinogen-III C-methyltransferase: evaluation of its role in resistance to potassium tellurite in Escherichia coli.

The Geobacillus stearothermophilus V cobA gene encoding uroporphyrinogen-III C-methyltransferase (also referred to as SUMT) was cloned into Escherichia coli and the recombinant enzyme was overexpressed and purified to homogeneity. The enzyme binds S-adenosyl-L-methionine and catalyzes the production of III methyl uroporphyrinogen in vitro. E. coli cells expressing the G. stearothermophilus V cobA gene exhibited increased resistance to potassium tellurite and potassium tellurate. Site-directed mutagenesis of cobA abolished tellurite resistance of the mesophilic, heterologous host and SUMT activity in vitro. No methylated, volatile derivatives of tellurium were found in the headspace of tellurite-exposed cobA-expressing E. coli, suggesting that the role of SUMT methyltransferase in tellurite(ate) detoxification is not related to tellurium volatilization.

The biosynthesis of adenosylcobalamin (vitamin B12).

Vitamin B12, or cobalamin, is one of the most structurally complex small molecules made in Nature. Major progress has been made over the past decade in understanding how this synthesis is accomplished. This review covers some of the most important findings that have been made and provides the reader with a complete description of the transformation of uroporphyrinogen III into adenosylcobalamin (AdoCbl). 183 references are cited.

Biosynthesis of cobalamin (vitamin B(12)).

The biosynthesis of vitamin B(12) is summarized, emphasizing the differences observed between the aerobic and anaerobic pathways. The biosynthetic route to adenosylcobalamin from its five-carbon precursor, 5-aminolaevulinic acid, can be divided into three sections: (1) the biosynthesis of uroporphyrinogen III from 5-aminolaevulinic acid, which is common to both pathways; (2) the conversion of uroporphyrinogen III into the ring-contracted, deacylated intermediate precorrin 6 or cobalt-precorrin 6, which includes the primary differences between the two pathways; and (3) the transformation of this intermediate to form adenosylcobalamin.

Effect of arsenite on the induction of CYP1A4 and CYP1A5 in cultured chick embryo hepatocytes.

We had reported previously that 2.5-5 microM sodium arsenite decreased the phenobarbital-mediated induction of CYP2H activity and protein but not CYP2H1 mRNA in chick-embryo hepatocyte cultures. Induction of a CYP1A activity and protein by 3-methylcholanthrene was also decreased by low arsenite concentrations; however, CYP1A mRNAs were not measured in those studies. We report here that low concentrations of arsenite decreased induction of activities and mRNAs of two chicken cytochromes P450, CYP1A (1A4 and 1A5), by 3-methylcholanthrene in chick-embryo hepatocyte cultures. Arsenite treatment did not affect the turnover of either mRNA, nor did it decrease the superinduction of each mRNA caused by treatment with cycloheximide in addition to 3-methylcholanthrene. Glutathione depletion enhanced the effect of arsenite to decrease induction of CYP1A4. These results indicate the induction of CYP1A4 and 1A5 is inhibited by sodium arsenite at the level of transcription, suggesting that the Ah receptor complex may be involved.

Rat harderian gland porphobilinogen deaminase: characterization studies and regulatory action of protoporphyrin IX.

Properties of purified porphobilinogen deaminase (PBG-D; EC 4.3.1.8) from rat harderian gland are here presented. The enzyme behaves as a monomer of Mr 38 +/- 2 kDa and is optimally active at pH 8.0-8.2. Its activation energy, determined by an Arrhenius plot, is 76.1 kJ/mol. Initial velocity studies showed a linear progress curve for uroporphyringen I formation and a hyperbolic dependence of the initial rate on substrate concentration, indicating the existence of a sequential displacement mechanism. Apparent kinetic constants, Km and Vm, calculated at 37 degrees C and pH 8.0 were 1.1 microM and 170 pmol/min mg, respectively. The pH dependence of the apparent kinetic parameters revealed the ionization of residues with pKAES and pKBES of 7.4 +/- 0.1 and 8.6 +/- 0.1, respectively, and a pKE value of 8.0 +/- 0.1. Incubation of PBG-D with 5.0 mM N-ethylmaleimide and 5.0 mM 5,5'-dithiobis(2-nitrobenzoic acid) at pH 8.0 led to inhibitions of 70 and 50%, respectively. The effect of pH, as well as the effect of thiol reagents, on enzyme activity strongly suggests the involvement of cysteine residue(s) in the mechanism of uroporphyrinogen I biosynthesis, in both the catalytic reaction and the substrate binding. Rat harderian gland PBG-D activity decreased with increasing concentrations of protoporphyrin IX, reaching a 40% inhibition at the in vivo concentration of the porphyrin and 7 microM PBG. Even at saturating concentrations of substrate, inhibition by protoporphyrin was not completely reversed. So, accumulated porphyrin may act as an regulator of PBG-D activity in rat harderian gland.

The three-dimensional structure of Escherichia coli porphobilinogen deaminase at 1.76-A resolution.

Porphobilinogen deaminase (PBGD) catalyses the polymerization of four molecules of porphobilinogen to form the 1-hydroxymethylbilane, preuroporphyrinogen, a key intermediate in the biosynthesis of tetrapyrroles. The three-dimensional structure of wild-type PBGD from Escherichia coli has been determined by multiple isomorphous replacement and refined to a crystallographic R-factor of 0.188 at 1.76 A resolution. the polypeptide chain of PBGD is folded into three alpha/beta domains. Domains 1 and 2 have a similar overall topology, based on a five-stranded, mixed beta-sheet. These two domains, which are linked by two hinge segments but otherwise make few direct interactions, form an extensive active site cleft at their interface. Domain 3, an open-faced, anti-parallel sheet of three strands, interacts approximately equally with the other two domains. The dipyrromethane cofactor is covalently attached to a cysteine side-chain borne on a flexible loop of domain 3. The cofactor serves as a primer for the assembly of the tetrapyrrole product and is held within the active site cleft by hydrogen-bonds and salt-bridges that are formed between its acetate and propionate side-groups and the polypeptide chain. The structure of a variant of PBGD, in which the methionines have been replaced with selenomethionines, has also been determined. The cofactor, in the native and functional form of the enzyme, adopts a conformation in which the second pyrrole ring (C2) occupies an internal position in the active site cleft. On oxidation, however, this C2 ring of the cofactor adopts a more external position that may correspond approximately to the site of substrate binding and polypyrrole chain elongation. The side-chain of Asp84 hydrogen-bonds the hydrogen atoms of both cofactor pyrrole nitrogens and also potentially the hydrogen atom of the pyrrole nitrogen of the porphobilinogen molecule bound to the proposed substrate binding site. This group has a key catalytic role, possibly in stabilizing the positive charges that develop on the pyrrole nitrogens during the ring-coupling reactions. Possible mechanisms for the processive elongation of the polypyrrole chain involve: accommodation of the elongating chain within the active site cleft, coupled with shifts in the relative positions of domains 1 and 2 to carry the terminal ring into the appropriate position at the catalytic site; or sequential translocation of the elongating polypyrrole chain, attached to the cofactor on domain 3, through the active site cleft by the progressive movement of domain 3 with respect to domains 1 and 2. Other mechanisms are considered although the amino acid sequence comparisons between PBGDs from all species suggest they share the same three-dimensional structure and mechanism of activity.

Evidence for pteridine regulation of lead-mediated inhibition of uroporphyrinogen and heme formation in rat bone marrow.

Uroporphyrin I (URO I) accumulation has been reported in the bone marrow of rats exposed to lead, suggesting a sensitivity of uroporphyrinogen III cosynthase (COSYN) to this heavy metal. Furthermore, it has been reported that a polyglutamated folate derivative may serve as a coenzyme for the catalytic action of hepatic uroporphyrinogen III cosynthase. These findings raised the question of whether depletion of polyglutamated folate could enhance the susceptibility of bone marrow COSYN to lead and potentially interfere with the formation of heme. Nitrous oxide, an anesthetic agent capable of causing bone marrow tetrahydrofolate deficiency, depressed total bone marrow polyglutamated folate content by 42% with significant reductions in all three chain lengths (5-7) identified in the bone marrow during an exposure period of 7 days at 4 hr/day. Lead acetate (15 mg/kg) administered by ip injection at Days 0 and 2 during a 7-day exposure to nitrous oxide resulted in an 84% increase of bone marrow URO I content, which was markedly higher than the increases of 22 and 38% seen with sole administration of lead or nitrous oxide, respectively. The combination of agents also produced a 48% rise in COPRO I, a 39 and 43% decrease in COPRO III and protoporphyrin, respectively, and a 42% decline in the activity of microsomal 7-ethoxycoumarin O-deethylase, which is hemoprotein, cytochrome P-450 mediated. Heme oxygenase activity was not altered by nitrous oxide, lead, or their combination. These results suggest that bone marrow folate deficiency may render COSYN more sensitive to lead as characterized by increased uroporphyrin I and coproporphyrin I isomer content, decreased coproporphyrin III and protoporphyrin content, and depressed microsomal hemoprotein, cytochrome P-450-mediated drug-metabolizing capability.

Pteridine modulation of lead inhibition of uroporphyrinogen synthesis in erythroid precursor cells.

The role of nutritional factors on heme synthesis and their influence on the development of anemia in the bone marrow during lead exposure is unclear. Previous reports suggested that pteridines could regulate the formation of uroporphyrinogen, a step midway along the heme synthetic pathway. Studies were undertaken to determine if pteridines could modulate lead inhibition of uroporphyrinogen synthesis in erythroid precursor cells. Pteroylpolyglutamates of various glutamate chain lengths were tested for the ability to protect against lead inhibition of uroporphyrinogen I (URO) synthase prepared from murine erythroleukemia cells (MELC). Pteroylpentaglutamate, the major endogenous polyglutamate form by chain length found to be present in MELC, afforded rapid and specific protection of URO synthase against lead inhibition. MELC are expected to be a useful in vitro model for studying the role of endogenous folates on uroporphyrinogen synthesis and heme formation in erythroid precursor cells following lead exposure.

Destruction of erythroleukaemic cells by photoactivation of endogenous porphyrins.

Selective destruction of Friend erythroleukaemic cells (FELC) was potentiated by stimulation of endogenous porphyrin synthesis followed by light sensitization. Endogenous porphyrin biosynthesis in FELC was induced by supplementation of 5-amino levulinic acid (5-ALA) at a concentration of 5 X 10(-4) M. The main accumulated product, after 4 days culture, was uroporphyrin, while after 8 days culture the cells were loaded with protoporphyrin, up to 1.5 micrograms 10(-7) cells. Photoirradiation of the cells for 2 min, accumulating endogenous porphyrins, induced cardinal deformations and cell disintegration in greater than 95% of the cells, as examined by scanning electron microscopy (SEM). The photodynamic destruction effects were dependent on cultivation time with 5-ALA. Flow cytometry analysis showed an immediate expansion of cell volume subsequent to irradiation, presumably a consequence of water influx. Transmission electron microscopy (TEM) of photosensitized cells after different time intervals of culture in 5-ALA medium, revealed initial damage to mitochondria and water influx into the nuclear envelope, after 2 days. After 3-4 days in culture the water influx phenomenon was pronounced, chromatin condensation took place and slight rupture of the outer membrane was detected. Cells photosensitized after 5-6 days of culture were completely disintegrated leaving a nuclear remnant and an enormously swollen nuclear envelope. The culture time dependence of the process, showed an interrelationship between the photodynamic effect and porphyrin accumulation sites in cellular compartments. The study presents a specific method for erythroleukaemic cell inactivation.