Impact of thiosemicarbazones on the accumulation of PpIX and the expression of the associated genes.

Thiosemicarbazone derivatives are known for their broad biological activity including their antitumor potency. The aim of the current study was to examine the effect of a novel series of non-toxic iron chelators on the accumulation of protoporphyrin IX after external 5-aminolevulonic acid administration. From this series we selected one the most promising derivative which causes a pronounced increase in the concentration of protoporphyrin IX. The increase of the photosensitizer concentration is necessary for the trigger the efficient therapeutic effect of the photodynamic reaction. For selected compound 2 we performed an examination of a panel of the genes that are involved in the heme biosynthesis and degradation. Results indicated the crucial roles of ferrochelatase and heme oxygenase in the described processes. Surprisingly, there was a strict dependence on the type of the tested cell line. A decrease in the expression of the two aforementioned enzymes after incubation with compound 2 and 5-aminolevulonic acid is a commonly known fact and we detected this trend for the MCF-7 and HCT 116 cell lines. However, we noticed the upregulation of the tested targets for the Hs683 cells. These unconventional results prompted us to do a more in-depth analysis of the described processes. In conclusion, we found that compound 2 is a novel, highly effective booster of photodynamic therapy that has prospective applications.

Differential gene content and gene expression for bacterial evolution and speciation of Shewanella in terms of biosynthesis of heme and heme-requiring proteins.

BACKGROUND: Most species of Shewanella harbor two ferrochelatase paralogues for the biosynthesis of c-type cytochromes, which are crucial for their respiratory versatility. In our previous study of the Shewanella loihica PV-4 strain, we found that the disruption of hemH1 but not hemH2 resulted in a significant accumulation of extracellular protoporphyrin IX (PPIX), but it is different in Shewanella oneidensis MR-1. Hence, the function and transcriptional regulation of two ferrochelatase genes, hemH1 and hemH2, are investigated in S. oneidensis MR-1. RESULT: In the present study, deletion of either hemH1 or hemH2 in S. oneidensis MR-1 did not lead to overproduction of extracellular protoporphyrin IX (PPIX) as previously described in the hemH1 mutants of S. loihica PV-4. Moreover, supplement of exogenous hemins made it possible to generate the hemH1 and hemH2 double mutant in MR-1, but not in PV-4. Under aerobic condition, exogenous hemins were required for the growth of MR-1ΔhemH1ΔhemH2, which also overproduced extracellular PPIX. These results suggest that heme is essential for aerobic growth of Shewanella species and MR-1 could also uptake hemin for biosynthesis of essential cytochrome(s) and respiration. Besides, the exogenous hemin mediated CymA cytochrome maturation and the cellular KatB catalase activity. Both hemH paralogues were transcribed in wild-type MR-1, and the hemH2 transcription was remarkably up-regulated in MR-1ΔhemH1 mutant to compensate for the loss of hemH1. The periplasmic glutathione peroxidase gene pgpD, located in the same operon with hemH2, and a large gene cluster coding for iron, heme (hemin) uptake systems are absent in the PV-4 genome. CONCLUSION: Our results indicate that the genetic divergence in gene content and gene expression between these Shewanella species, accounting for the phenotypic difference described here, might be due to their speciation and adaptation to the specific habitats (iron-rich deep-sea vent versus iron-poor freshwater) in which they evolved and the generated mutants could potentially be utilized for commercial production of PPIX.

Effects of 5-Aminolevulinic Acid on Gene Expression, Immunity, and ATP Levels in Pacific White Shrimp, Litopenaeus vannamei.

With the emergence of several infectious diseases in shrimp aquaculture, there is a growing interest in the use of feed additives to enhance shrimp immunity. Recently, the use of 5-aminolevulinic acid (5-ALA), a non-protein amino acid that plays a rate-limiting role in heme biosynthesis, has received attention for its positive effect on immunity in livestock animals. To evaluate the effect of 5-ALA in the Pacific white shrimp, Litopenaeus vannamei, we conducted microarray analysis, a Vibrio parahaemolyticus immersion challenge test, an ATP level assay, and gene expression analysis of some hemoproteins and genes associated with heme synthesis and degradation. Out of 15,745 L. vannamei putative genes on the microarray, 101 genes were differentially expressed by more than fourfold (p < 0.05) between 5-ALA-supplemented and control shrimp hepatopancreas. 5-ALA upregulated 99 of the 101 genes, 41 of which were immune- and defense-related genes based on sequence homology. Compared to the control, the 5-ALA-supplemented group had a higher survival rate in the challenge test, higher transcript levels of porphobilinogen synthase, ferrochelatase, catalase, nuclear receptor E75, and heme oxygenase-1 and higher levels of ATP. These findings suggest that dietary 5-ALA enhanced the immune response of L. vannamei to V. parahaemolyticus, upregulated immune- and defense-related genes, and enhanced aerobic energy metabolism, respectively. Further studies are needed to elucidate the extent of 5-ALA use in shrimp culture.

The Plasma Membrane Protein Nce102 Implicated in Eisosome Formation Rescues a Heme Defect in Mitochondria.

The cellular transport of the cofactor heme and its biosynthetic intermediates such as protoporphyrin IX is a complex and highly coordinated process. To investigate the molecular details of this trafficking pathway, we created a synthetic lesion in the heme biosynthetic pathway by deleting the gene HEM15 encoding the enzyme ferrochelatase in S. cerevisiae and performed a genetic suppressor screen. Cells lacking Hem15 are respiratory-defective because of an inefficient heme delivery to the mitochondria. Thus, the biogenesis of mitochondrial cytochromes is negatively affected. The suppressor screen resulted in the isolation of respiratory-competent colonies containing two distinct missense mutations in Nce102, a protein that localizes to plasma membrane invaginations designated as eisosomes. The presence of the Nce102 mutant alleles enabled formation of the mitochondrial respiratory complexes and respiratory growth in hem15Δ cells cultured in supplemental hemin. Respiratory function in hem15Δ cells can also be restored by the presence of a heterologous plasma membrane heme permease (HRG-4), but the mode of suppression mediated by the Nce102 mutant is more efficient. Attenuation of the endocytic pathway through deletion of the gene END3 impaired the Nce102-mediated rescue, suggesting that the Nce102 mutants lead to suppression through the yeast endocytic pathway.

N-alkylprotoporphyrin formation and hepatic porphyria in dogs after administration of a new antiepileptic drug candidate: mechanism and species specificity.

A new antiepileptic synaptic vesicle 2a (SV2a) ligand drug candidate was tested in 4-week oral toxicity studies in rat and dog. Brown pigment inclusions were found in the liver of high-dose dogs. The morphology of the deposits and the accompanying liver changes (increased plasma liver enzymes, increased total hepatic porphyrin level, decreased liver ferrochelatase activity, combined induction, and inactivation of cytochrome P-450 CYP2B11) suggested disruption of the heme biosynthetic cascade. None of these changes was seen in rat although this species was exposed to higher parent drug levels. Toxicokinetic analysis and in vitro metabolism assays in hepatocytes showed that dog is more prone to oxidize the drug candidate than rat. Mass spectrometry analysis of liver samples from treated dogs revealed an N-alkylprotoporphyrin adduct. The elucidation of its chemical structure suggested that the drug transforms into a reactive metabolite which is structurally related to a known reference porphyrogenic agent allylisopropylacetamide. That particular metabolite, primarily produced in dog but neither in rat nor in human, has the potential to alkylate the prosthetic heme of CYP. Overall, the data suggested that the drug candidate should not be porphyrogenic in human. This case study further exemplifies the species variability in the susceptibility to drug-induced porphyria.

The role of coproporphyrinogen III oxidase and ferrochelatase genes in heme biosynthesis and regulation in Aspergillus niger.

Heme is a suggested limiting factor in peroxidase production by Aspergillus spp., which are well-known suitable hosts for heterologous protein production. In this study, the role of genes coding for coproporphyrinogen III oxidase (hemF) and ferrochelatase (hemH) was analyzed by means of deletion and overexpression to obtain more insight in fungal heme biosynthesis and regulation. These enzymes represent steps in the heme biosynthetic pathway downstream of the siroheme branch and are suggested to play a role in regulation of the pathway. Based on genome mining, both enzymes deviate in cellular localization and protein domain structure from their Saccharomyces cerevisiae counterparts. The lethal phenotype of deletion of hemF or hemH could be remediated by heme supplementation confirming that Aspergillus niger is capable of hemin uptake. Nevertheless, both gene deletion mutants showed an extremely impaired growth even with hemin supplementation which could be slightly improved by media modifications and the use of hemoglobin as heme source. The hyphae of the mutant strains displayed pinkish coloration and red autofluorescence under UV indicative of cellular porphyrin accumulation. HPLC analysis confirmed accumulation of specific porphyrins, thereby confirming the function of the two proteins in heme biosynthesis. Overexpression of hemH, but not hemF or the aminolevulinic acid synthase encoding hemA, modestly increased the cellular heme content, which was apparently insufficient to increase activity of endogenous peroxidase and cytochrome P450 enzyme activities. Overexpression of all three genes increased the cellular accumulation of porphyrin intermediates suggesting regulatory mechanisms operating in the final steps of the fungal heme biosynthesis pathway.

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, δ-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.

Photodynamic therapy involves an antiangiogenic mechanism and is enhanced by ferrochelatase inhibitor in urothelial carcinoma.

The purpose of the present study was to investigate the mechanism of photodynamic therapy (PDT) supplemented with exogenously added 5-aminolevulinic acid (ALA) on human urothelial cancer (UC). Moreover, we aimed to determine whether the therapeutic effects of ALA-based PDT (ALA-PDT) for UC could be enhanced by deferoxamine (DFX), an inhibitor of ferrochelatase. The efficiency of ALA-PDT on these cells was analyzed using flow cytometry and the type of cell death was also assessed. The ALA-PDT promoting effect of DFX was examined on both UC cells and human umbilical vein endothelial cells (HUVEC). The ALA-PDT decreased levels of mitochondrial membrane potential and induced cell death mainly via apoptosis in these cells. Moreover, inhibition of ferrochelatase by DFX led to an increase of protoporphyrin IX (PpIX) accumulation and enhanced the effect of ALA-PDT on UC cells. We further investigated the effect of DFX on in vivo PDT with a tumor-bearing animal model and found that DFX efficiently enhanced tumor cell apoptosis. ALA-PDT induced death of neovascular endothelial cells in tumors but did not affect small vessel endothelial cells in normal tissues surrounding the tumor. Furthermore, DFX enhanced inhibition of neovascularization. These results demonstrated ALA-PDT dominantly induced apoptosis over necrosis by direct action on UC as well as via antiangiogenic action on neovacular endothelial cells, suggesting that the therapeutic damage by ALA-PDT could be kept to a minimum in the surrounding normal tissues. In addition, increased accumulation of PpIX by DFX could enhance this effectiveness of ALA-PDT.

Characterization of the evolutionarily conserved iron-sulfur cluster of sirohydrochlorin ferrochelatase from Arabidopsis thaliana.

Sirohaem is a cofactor of nitrite and sulfite reductases, essential for assimilation of nitrogen and sulfur. Sirohaem is synthesized from the central tetrapyrrole intermediate uroporphyrinogen III by methylation, oxidation and ferrochelation reactions. In Arabidopsis thaliana, the ferrochelation step is catalysed by sirohydrochlorin ferrochelatase (SirB), which, unlike its counterparts in bacteria, contains an [Fe-S] cluster. We determined the cluster to be a [4Fe-4S] type, which quickly oxidizes to a [2Fe-2S] form in the presence of oxygen. We also identified the cluster ligands as four conserved cysteine residues located at the C-terminus. A fifth conserved cysteine residue, Cys(135), is not involved in ligating the cluster directly, but influences the oxygen-sensitivity of the [4Fe-4S] form, and possibly the affinity for the substrate metal. Substitution mutants of the enzyme lacking the Fe-S cluster or Cys(135) retain the same specific activity in vitro and dimeric quaternary structure as the wild-type enzyme. The mutant variants also rescue a defined Escherichia coli sirohaem-deficient mutant. However, the mutant enzymes cannot complement Arabidopsis plants with a null AtSirB mutation, which exhibits post-germination arrest. These observations suggest an important physiological role for the Fe-S cluster in Planta, highlighting the close association of iron, sulfur and tetrapyrrole metabolism.

Abnormal mitoferrin-1 expression in patients with erythropoietic protoporphyria.

OBJECTIVE: Most patients with erythropoietic protoporphyria have deficient ferrochelatase (FECH) activity due to changes in FECH DNA. We evaluated seven patients with erythropoietic protoporphyria phenotype in whom abnormalities of FECH DNA were not found by conventional analysis. The major focus was mitoferrin-1 (MFRN1), the mitochondrial transporter of Fe used for heme formation by FECH and for 2Fe2S cluster synthesis, which is critical to FECH activity/stability. MATERIALS AND METHODS: Four patients had a deletion in ALAS2 that causes enzyme gain-of-function, resulting in increased formation of protoporphyrin; one had a heterozygous major deletion in FECH DNA. All had an abnormal transcript of MFRN1 in messenger RNA extracted from blood leukocytes and/or liver tissue. The abnormal transcript contained an insert of intron 2 that had a stop codon. The consequences of abnormal MFRN1 expression were examined using zebrafish and yeast MFRN-deficient strains and cultured lymphoblasts from the patients. RESULTS: Abnormal human MFRN1 complementary DNA showed loss-of-function in zebrafish and yeast mutants, whereas normal human MFRN1 complementary DNA rescued both. Using cultured lymphoblasts, quantitative reverse transcription polymerase chain reaction showed increased formation of abnormal transcript that was accompanied by decreased formation of normal transcript and reduced FECH activity in patients compared to normal lines. A positive correlation coefficient (0.75) was found between FECH activity and normal MFRN1 messenger RNA in lymphoblasts. However, no obvious cause for increased formation of abnormal transcript was identified in MFRN1 exons and splice junctions. CONCLUSIONS: Abnormal MFRN1 expression can contribute to erythropoietic protoporphyria phenotype in some patients, probably by causing a reduction in FECH activity.

Erythropoietic protoporphyria in Sweden: demographic, clinical, biochemical and genetic characteristics.

OBJECTIVE: To investigate the demographic, clinical, biochemical and genotypic features of patients with erythropoietic protoporphyria (EPP) in a Swedish cohort. DESIGN: Cross-sectional questionnaire, biochemical and genetic study. SETTING: Sweden. SUBJECTS: Fifty-one Swedish individuals known in 2008 to have EPP confirmed by molecular diagnosis. There were no exclusion criteria; all patients were included in the demographic and genetic study. A total of 92% participants completed the questionnaire study and 82% the biochemical study. RESULTS: The prevalence of EPP was 1 : 180,000. Nine novel ferrochelatase gene mutations were found. The most commonly reported age at onset of symptoms was the first year of life and the mean age at diagnosis was 22 years. Painful photosensitivity was the main symptom. Exogenous factors other than sunlight were frequently reported to cause cutaneous symptoms. One in five patients reported a positive effect of beta-carotene therapy. A marked impact of EPP on quality of life was reported. Women had a significantly lower mean erythrocyte protoporphyrin concentration than men. Of all participants, 84% had insufficient vitamin D concentrations, 44% had below normal serum ferritin or transferrin saturation levels and red cell abnormalities were common. CONCLUSIONS: The notably delayed diagnosis suggests the need for an increased awareness of EPP. Disturbed erythropoiesis, biochemical signs of iron deficiency and low vitamin D levels are frequent findings in this disease. New and better treatments are needed as current treatment options for symptom amelioration are limited. Vitamin D supplementation should be considered.

Porcine ferrochelatase: the relationship between iron-removal reaction and the conversion of heme to Zn-protoporphyrin.

At the terminal step of heme biosynthesis, ferrochelatase (FECH) catalyzes the insertion of Fe2+ into protoporphyrin to form heme. It is located on the inner membrane of the mitochondria of animals. The enzyme inserts divalent metal ions, including Fe2+, Co2+, and Zn2+, into porphyrins in vitro. We have reported that it can remove Fe2+ from heme. To characterize the iron-removal reverse activity of FECH, we examined its properties in porcine liver and muscle mitochondria, and isolated porcine FECH cDNA. The amino acid sequence of porcine FECH showed high homology with bovine (91%), human (85%), mouse (87%), and rat (76%) equivalents. It was expressed in Escherichia coli, and purified, and the kinetic properties of the zinc-chelating and iron-removal activities were examined. Both activities peaked at 45 degrees C, but different optimal pH values, of 7.5-8.0 for zinc-ion insertion and 5.5-6.0 for the reverse reaction were found. The K(m) values for mesoporphyrin IX and Zn2+ were 6.6 and 1.1 microM, respectively, and the K(m) for heme was 5.7 microM. The k(cat) value of the forward reaction was about 11-fold higher than that of the reverse reaction, indicating that the enzyme preferably catalyzes the forward reaction rather than the iron-removal reaction. Reverse activity was stimulated by fatty acids and phospholipids, similarly to the case of the forward reaction, indicating that lipids play a role in regulating both enzyme activities.

Pure red cell precursor toxicity by linezolid in a pediatric case.

Linezolid (LZD)-induced myelosuppression has been reported in adults; however, LZD-induced pure red cell precursor toxicity rarely occurs. A 2-year-old boy diagnosed with infective endocarditis by Streptococcus mitis received LZD after developing resistance to multiple antibiotics. Although his infective symptoms were improved by LZD, progressive anemia was noticed 2 weeks after LZD therapy. Four weeks after LZD administration, his hemoglobin level was 6.5 g/dL and reticulocytes less than 0.1%. Bone marrow examination revealed markedly decreased erythropoiesis with cytoplasmic vacuolation of erythroblasts. Anemia recovered 19 days after cessation of LZD. Elevated protoporphyrin and a high LZD level in the blood suggested that mitochondrial disturbance by high-dose and long-term treatment with LZD may have been responsible for LZD-induced pure red cell precursor toxicity.

[Effect of treadmill exercise and nutrition supplement on activity and gene expression of rate-limiting enzyme of heme metabolism and globin].

AIM: To investigate the possible role of rate-limiting enzyme of heme metabolism and globin in the development of the low hemoglobin (Hb), red blood (cell) count (RBC) and hematocrit (Hct) after long-term exercise, and effect of nutrition supplement on sports anemia. METHODS: Male Wistar rats were randomly assigned to three groups (n = 10): control (C), exercise (P) and exercise + nutrition (G). Animals in the P and G groups started treadmill running at 30 m/min, 0% grade, 1 min/time. Running time was gradually increased with 2 min/time during initial 5 weeks and final 4 weeks. In addition, running frequency was 2 times/day except initial 2 weeks. At the end of eleventh week, gene expression of 5-aminolevulinate synthase (ALAS), ferrochelatase, alpha-globin and beta-globin in bone marrow were measured with RT-PCR. Mean-while heme oxygenase 1 (HO-1) activity in liver was measured with immunohistochemical method. RESULTS: Eleven weeks of exercise induced a significant increase in HO-1 and a significant increase in gene expression of beta-globin (P < 0.01, P < 0.05, respectively). Treatment with anti-sports anemia compound dosage led to no significant differences in rate-limiting enzyme of heme metabolism and globin in the exercised rats. The G group had a significantly higher HO-1 level in liver than the C group (P < 0.01). These finds showed that exercise was associated with no significant difference in heme synthetase and alpha-globin gene expression, and significant difference in heme catabolic enzyme and beta-globin gene expression. CONCLUSION: The increase of HO-1 activity in liver might be one of the causes of the lower Hb, RBC and Hct status in exercised rats.

Effect of treadmill exercise and nutrition supplement on activity and gene expression of rate-limiting enzyme of heme metabolism and globin / 中国应用生理学杂志

<p><b>AIM</b>To investigate the possible role of rate-limiting enzyme of heme metabolism and globin in the development of the low hemoglobin (Hb), red blood (cell) count (RBC) and hematocrit (Hct) after long-term exercise, and effect of nutrition supplement on sports anemia.</p><p><b>METHODS</b>Male Wistar rats were randomly assigned to three groups (n = 10): control (C), exercise (P) and exercise + nutrition (G). Animals in the P and G groups started treadmill running at 30 m/min, 0% grade, 1 min/time. Running time was gradually increased with 2 min/time during initial 5 weeks and final 4 weeks. In addition, running frequency was 2 times/day except initial 2 weeks. At the end of eleventh week, gene expression of 5-aminolevulinate synthase (ALAS), ferrochelatase, alpha-globin and beta-globin in bone marrow were measured with RT-PCR. Mean-while heme oxygenase 1 (HO-1) activity in liver was measured with immunohistochemical method.</p><p><b>RESULTS</b>Eleven weeks of exercise induced a significant increase in HO-1 and a significant increase in gene expression of beta-globin (P < 0.01, P < 0.05, respectively). Treatment with anti-sports anemia compound dosage led to no significant differences in rate-limiting enzyme of heme metabolism and globin in the exercised rats. The G group had a significantly higher HO-1 level in liver than the C group (P < 0.01). These finds showed that exercise was associated with no significant difference in heme synthetase and alpha-globin gene expression, and significant difference in heme catabolic enzyme and beta-globin gene expression.</p><p><b>CONCLUSION</b>The increase of HO-1 activity in liver might be one of the causes of the lower Hb, RBC and Hct status in exercised rats.</p>

The influence of sex steroid hormones on ferrochelatase gene expression in Harderian gland of hamster (Mesocricetus auratus).

Ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the insertion of ferrous iron into protoporphyrin IX to form protohaem. The Syrian hamster Harderian gland (HG) is known for its ability to produce and accumulate large amounts of protoporphyrins. In this species, the female gland contains up to 120 times more porphyrin than the male gland. Data from biochemical studies suggest that this gland possesses the enzymatic complex for haem biosynthesis but lacks ferrochelatase activity. The abundance of intraglandular haem proteins does not support this idea. To gain more insight into this process, we isolated cDNA for ferrochelatase from hamster liver, using the 5'- and 3'- rapid amplification of complementary DNA ends (RACE), and investigated its expression in HG from males and females. The full-length cDNA comprises an open reading frame of 1269 bp encoding a polypeptide of 422 amino-acid residues. Hamster DNA sequence exhibits 92% identity to mouse and 87% identity to human sequences. The predicted hamster enzyme was shown to have structural features of mammalian ferrochelatase, including a putative NH2- terminal presequence, a central core of about 330 amino-acid residues and an extra 30-50-amino-acid stretch at the carboxyl-terminus. RNA blotting experiments indicated that this cDNA hybridized to a liver mRNA of about 2.1 kb, while a weak hybridization signal was observed with mRNA from HG preparations. RT-PCR assays confirmed the expression of specific transcripts in both tissues. Male glands contained approximately twofold more enzyme mRNA than female glands. Likewise, the intraglandular content of mRNA varied during the oestrous cycle, with the highest levels found in the oestrous phase. These cyclic variations were less evident in liver. Ovariectomy plus treatment with progesterone or 17beta-oestradiol plus progesterone increased ferrochelatase mRNA of the gland. In HG of short- or long-term castrated males, the administration of testosterone did not affect the ferrochelatase mRNA concentration. Based on mRNA expression levels, we conclude that Harderian ferrochelatase may play an active role in maintaining the physiological pool of haem required for processing cytochromes and other glandular haem proteins. Likewise, the sex-steroid hormones appear to have only a modest influence upon Harderian ferrochelatase.

Terapia fotodinámica en dermatología / Photodynamic therapy in dermatology

La terapia fotodinámica (TF) es una modalidad terapéutica basada en la fotooxidación de materiales biológicos inducida por un fotosensibilizante, el cual se localiza selectivamente en determinadas células o tejidos tumorales, de forma que al ser iluminadas con una luz de adecuada longitud y en dosis suficiente, dichas células resultan destruidas. En dermatología, la TF con ácido 5-aminolevulínico o 5-metilo aminolevulinato tópicos es muy efectiva en el tratamiento de queratosis actínicas, carcinomas basocelulares y enfermedad de Bowen. Además, se han obtenido resultados muy prometedores en patología inflamatoria como la morfea o la sarcoidosis, infecciones como las verrugas y procesos cosméticos, como el fotoenvejecimiento, entre otras. El presente artículo revisa los aspectos más relevantes de la TF en dermatología. En primer lugar se hará una revisión de los fundamentos básicos del tratamiento fotodinámico; posteriormente se expondrán sus aplicaciones clínicas en dermatología, tanto las oncológicas como todos aquellos procesos dermatológicos en los que la TF puede desempeñar un papel en su manejo, sin olvidar su prometedora aplicación cosmética en el tratamiento del fotoenvejecimiento. Finalizaremos la revisión con el fotodiagnóstico y las diferentes formas de monitorización no invasiva de la efectividad de la TF

Photodynamic therapy (PDT) is a therapeutic modality based on the photooxidation of biological materials induced by a photosensitizer, which selectively locates itself in certain tumorous cells or tissues, so that when illuminated by a light of the right length and at a sufficient dose, these cells are destroyed. In dermatology, PDT with topical 5-aminolevulinic acid or 5-methyl aminolevulinate is very effective in the treatment of actinic keratoses, basal cell carcinomas and Bowen's disease. In addition, very promising results have been obtained in inflammatory pathologies like morphea or sarcoidosis, infections like warts, and cosmetic processes such as photoaging, among others. This article reviews the most significant aspects of PDT in dermatology. First of all, we will review the basic fundamentals of photodynamic treatment. Next, we will outline its clinical applications in dermatology, both in oncological applications and all those dermatological processes in which PDT may play a role in their management. We will also discuss its promising cosmetic application in the treatment of photoaging. We will complete the review with photodiagnosis and the different non-invasive ways to monitor the effectiveness of PDT

Enzymes of the heme biosynthetic pathway in the nonphotosynthetic alga Polytomella sp.

Heme biosynthesis involves a number of enzymatic steps which in eukaryotes take place in different cell compartments. Enzyme compartmentalization differs between photosynthetic and nonphotosynthetic eukaryotes. Here we investigated the structures and subcellular localizations of three enzymes involved in the heme pathway in Polytomella sp., a colorless alga evolutionarily related to the green alga Chlamydomonas reinhardtii. Functional complementation of Escherichia coli mutant strains was used to isolate cDNAs encoding three heme biosynthetic enzymes, glutamate-1-semialdehyde aminotransferase, protoporphyrinogen IX oxidase, and ferrochelatase. All three proteins show highest similarity to their counterparts in photosynthetic organisms, including C. reinhardtii. All three proteins have N-terminal extensions suggestive of intracellular targeting, and immunoblot studies indicate their enrichment in a dense cell fraction that is enriched in amyloplasts. These results suggest that even though the plastids of Polytomella sp. are not photosynthetically active, they are the major site of heme biosynthesis. The presence of a gene for glutamate-1-semialdehyde aminotransferase suggests that Polytomella sp. uses the five-carbon pathway for synthesis of the heme precursor 5-aminolevulinic acid.

Subcellular localization and light-regulated expression of protoporphyrinogen IX oxidase and ferrochelatase in Chlamydomonas reinhardtii.

Protoporphyrinogen IX oxidase (PPO) catalyzes the last common step in chlorophyll and heme synthesis, and ferrochelatase (FeC) catalyzes the last step of the heme synthesis pathway. In plants, each of these two enzymes is encoded by two or more genes, and the enzymes have been reported to be located in the chloroplasts or in the mitochondria. We report that in the green alga Chlamydomonas reinhardtii, PPO and FeC are each encoded by a single gene. Phylogenetic analysis indicates that C. reinhardtii PPO and FeC are most closely related to plant counterparts that are located only in chloroplasts. Immunoblotting results suggest that C. reinhardtii PPO and FeC are targeted exclusively to the chloroplast, where they are associated with membranes. These results indicate that cellular needs for heme in this photosynthetic eukaryote can be met by heme that is synthesized in the chloroplast. It is proposed that the multiplicity of genes for PPO and FeC in higher plants could be related to differential expression in differently developing tissues rather than to targeting of different gene products to different organelles. The FeC content is higher in C. reinhardtii cells growing in continuous light than in cells growing in the dark, whereas the content of PPO does not significantly differ in light- and dark-grown cells. In cells synchronized to a light/dark cycle, the level of neither enzyme varied significantly with the phase of the cycle. These results indicate that heme synthesis is not directly regulated by the levels of PPO and FeC in C. reinhardtii.

Iron-dependent cytochrome c1 expression is mediated by the status of heme in Bradyrhizobium japonicum.

The heme prosthetic group of heme proteins contains iron, which can be a limiting nutrient. Here, we show that cytochrome c1 protein from Bradyrhizobium japonicum was strongly affected by the iron status, with low expression in cells grown under iron limitation. This control was not affected in mutants encoding the iron regulator Irr or Fur. Furthermore, cytochrome c1 mRNA was not influenced by the iron status, suggesting control at a posttranscriptional step. Cytochrome c1 protein levels were very low in mutants defective in the genes encoding delta-aminolevulinic acid (ALA) synthase and ferrochelatase, enzymes that catalyze the first and final steps of the heme biosynthetic pathway, respectively. Iron-dependent cytochrome c1 expression was restored in the ALA synthase mutant by supplementation of the medium with the heme precursor ALA. Supplementation with heme resulted in high levels of cytochrome c1 protein in the wild type and in both mutants, but expression was no longer iron dependent. Cytochrome c1 is synthesized as a protein precursor fused with cytochrome b. A plasmid-borne construct encoding only cytochrome c1 was expressed in an iron- and heme-dependent manner similar to that of the wild-type gene, indicating that control by those effectors is not linked to posttranslational processing of the fusion protein. Mutation of the cytochrome c1 cysteines involved in covalent binding to heme nearly abolished immunodetectable protein. Thus, defects in heme synthesis or heme binding abrogate cytochrome c1 accumulation, apparently due to protein degradation. We suggest that iron-dependent cytochrome c1 expression is mediated by heme availability for heme protein formation.