Elucidation of the biosynthesis of the methane catalyst coenzyme F430.

Methane biogenesis in methanogens is mediated by methyl-coenzyme M reductase, an enzyme that is also responsible for the utilization of methane through anaerobic methane oxidation. The enzyme uses an ancillary factor called coenzyme F430, a nickel-containing modified tetrapyrrole that promotes catalysis through a methyl radical/Ni(ii)-thiolate intermediate. However, it is unclear how coenzyme F430 is synthesized from the common primogenitor uroporphyrinogen iii, incorporating 11 steric centres into the macrocycle, although the pathway must involve chelation, amidation, macrocyclic ring reduction, lactamization and carbocyclic ring formation. Here we identify the proteins that catalyse the biosynthesis of coenzyme F430 from sirohydrochlorin, termed CfbA-CfbE, and demonstrate their activity. The research completes our understanding of how the repertoire of tetrapyrrole-based pigments are constructed, permitting the development of recombinant systems to use these metalloprosthetic groups more widely.

Accumulation of Uroporphyrin I in Necrotic Tissues of Squamous Cell Carcinoma after Administration of 5-Aminolevulinic Acid.

5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is widely used for the intraoperative detection of malignant tumors. However, the fluorescence emission profiles of the accompanying necrotic regions of these tumors have yet to be determined. To address this, we performed fluorescence and high-performance liquid chromatography (HPLC) analyses of necrotic tissues of squamous cancer after 5-ALA administration. In resected human lymph nodes of metastatic squamous cell carcinoma, we found a fluorescence peak at approximately 620 nm in necrotic lesions, which was distinct from the PpIX fluorescence peak at 635 nm for viable cancer lesions. Necrotic lesions obtained from a subcutaneous xenograft model of human B88 oral squamous cancer also emitted the characteristic fluorescence peak at 620 nm after light irradiation: the fluorescence intensity ratio (620 nm/635 nm) increased with the energy of the irradiation light. HPLC analysis revealed a high content ratio of uroporphyrin I (UPI)/total porphyrins in the necrotic cores of murine tumors, indicating that UPI is responsible for the 620 nm peak. UPI accumulation in necrotic tissues after 5-ALA administration was possibly due to the failure of the heme biosynthetic pathway. Taken together, fluorescence imaging of UPI after 5-ALA administration may be applicable for the evaluation of tumor necrosis.

The Hydrogenobyric Acid Structure Reveals the Corrin Ligand as an Entatic State Module Empowering B12 Cofactors for Catalysis.

The B12 cofactors instill a natural curiosity regarding the primordial selection and evolution of their corrin ligand. Surprisingly, this important natural macrocycle has evaded molecular scrutiny, and its specific role in predisposing the incarcerated cobalt ion for organometallic catalysis has remained obscure. Herein, we report the biosynthesis of the cobalt-free B12 corrin moiety, hydrogenobyric acid (Hby), a compound crafted through pathway redesign. Detailed insights from single-crystal X-ray and solution structures of Hby have revealed a distorted helical cavity, redefining the pattern for binding cobalt ions. Consequently, the corrin ligand coordinates cobalt ions in desymmetrized "entatic" states, thereby promoting the activation of B12 -cofactors for their challenging chemical transitions. The availability of Hby also provides a route to the synthesis of transition metal analogues of B12 .

Desulfovibrio vulgaris CbiKP cobaltochelatase: evolution of a haem binding protein orchestrated by the incorporation of two histidine residues.

The sulfate-reducing bacteria of the Desulfovibrio genus make three distinct modified tetrapyrroles, haem, sirohaem and adenosylcobamide, where sirohydrochlorin acts as the last common biosynthetic intermediate along the branched tetrapyrrole pathway. Intriguingly, D. vulgaris encodes two sirohydrochlorin chelatases, CbiKP and CbiKC , that insert cobalt/iron into the tetrapyrrole macrocycle but are thought to be distinctly located in the periplasm and cytoplasm respectively. Fusing GFP onto the C-terminus of CbiKP confirmed that the protein is transported to the periplasm. The structure-function relationship of CbiKP was studied by constructing eleven site-directed mutants and determining their chelatase activities, oligomeric status and haem binding abilities. Residues His154 and His216 were identified as essential for metal-chelation of sirohydrochlorin. The tetrameric form of the protein is stabilized by Arg54 and Glu76, which form hydrogen bonds between two subunits. His96 is responsible for the binding of two haem groups within the main central cavity of the tetramer. Unexpectedly, CbiKP is shown to bind two additional haem groups through interaction with His103. Thus, although still retaining cobaltochelatase activity, the presence of His96 and His103 in CbiKP , which are absent from all other known bacterial cobaltochelatases, has evolved CbiKP a new function as a haem binding protein permitting it to act as a potential haem chaperone or transporter.

Liver Transplantation for Acute Intermittent Porphyria: Biochemical and Pathologic Studies of the Explanted Liver.

Acute intermittent porphyria (AIP) is an autosomal-dominant hepatic disorder caused by the half-normal activity of hydroxymethylbilane (HMB) synthase. Symptomatic individuals experience life-threatening acute neurovisceral attacks that are precipitated by factors that induce the hepatic expression of 5-aminolevulinic acid synthase 1 (ALAS1), resulting in the marked accumulation of the putative neurotoxic porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG). Here, we provide the first detailed description of the biochemical and pathologic alterations in the explanted liver of an AIP patient who underwent orthotopic liver transplantation (OLT) due to untreatable and debilitating chronic attacks. After OLT, the recipient's plasma and urinary ALA and PBG rapidly normalized, and her attacks immediately stopped. In the explanted liver, (a) ALAS1 mRNA and activity were elevated approximately ~3- and 5-fold, and ALA and PBG concentrations were increased ~3- and 1,760-fold, respectively; (b) uroporphyrin III concentration was elevated; (c) microsomal heme content was sufficient, and representative cytochrome P450 activities were essentially normal; (d) HMB synthase activity was approximately half-normal (~42%); (e) iron concentration was slightly elevated; and (f) heme oxygenase I mRNA was increased approximately three-fold. Notable pathologic findings included nodular regenerative hyperplasia, previously not reported in AIP livers, and minimal iron deposition, despite the large number of hemin infusions received before OLT. These findings suggest that the neurovisceral symptoms of AIP are not associated with generalized hepatic heme deficiency and support the neurotoxicity of ALA and/or PBG. Additionally, they indicate that substrate inhibition of hepatic HMB synthase activity by PBG is not a pathogenic mechanism in acute attacks.

An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis.

The biosynthesis of many vitamins and coenzymes has often proven difficult to elucidate owing to a combination of low abundance and kinetic lability of the pathway intermediates. Through a serial reconstruction of the cobalamin (vitamin B(12)) pathway in Escherichia coli and by His tagging the terminal enzyme in the reaction sequence, we have observed that many unstable intermediates can be isolated as tightly bound enzyme-product complexes. Together, these approaches have been used to extract intermediates between precorrin-4 and hydrogenobyrinic acid in their free acid form and permitted the delineation of the overall reaction catalyzed by CobL, including the formal elucidation of precorrin-7 as a metabolite. Furthermore, a substrate-carrier protein, CobE, that can also be used to stabilize some of the transient metabolic intermediates and enhance their onward transformation, has been identified. The tight association of pathway intermediates with enzymes provides evidence for a form of metabolite channeling.

Evolution in a family of chelatases facilitated by the introduction of active site asymmetry and protein oligomerization.

The class II chelatases associated with heme, siroheme, and cobalamin biosynthesis are structurally related enzymes that insert a specific metal ion (Fe(2+) or Co(2+)) into the center of a modified tetrapyrrole (protoporphyrin or sirohydrochlorin). The structures of two related class II enzymes, CbiX(S) from Archaeoglobus fulgidus and CbiK from Salmonella enterica, that are responsible for the insertion of cobalt along the cobalamin biosynthesis pathway are presented in complex with their metallated product. A further structure of a CbiK from Desulfovibrio vulgaris Hildenborough reveals how cobalt is bound at the active site. The crystal structures show that the binding of sirohydrochlorin is distinctly different to porphyrin binding in the protoporphyrin ferrochelatases and provide a molecular overview of the mechanism of chelation. The structures also give insights into the evolution of chelatase form and function. Finally, the structure of a periplasmic form of Desulfovibrio vulgaris Hildenborough CbiK reveals a novel tetrameric arrangement of its subunits that are stabilized by the presence of a heme b cofactor. Whereas retaining colbaltochelatase activity, this protein has acquired a central cavity with the potential to chaperone or transport metals across the periplasmic space, thereby evolving a new use for an ancient protein subunit.

Crystal structure of putative CbiT from Methanocaldococcus jannaschii: an intermediate enzyme activity in cobalamin (vitamin B12) biosynthesis.

BACKGROUND: In the anaerobic pathway of cobalamin (vitamin B12) synthesis, the CbiT enzyme plays two roles, as a cobalt-precorrin-7 C15-methyltransferase and a C12-decarboxylase, to produce the intermediate, cobalt-precorrin 8. RESULTS: The primary structure of the hypothetical protein MJ0391, from Methanocaldococcus jannaschii, suggested that MJ0391 is a putative CbiT. Here, we report the crystal structure of MJ0391, solved by the MAD procedure and refined to final R-factor and R-free values of 19.8 & 27.3%, respectively, at 2.3 Å resolution. The asymmetric unit contains two NCS molecules, and the intact tetramer generated by crystallographic symmetry may be functionally important. The overall tertiary structure and the tetrameric arrangements are highly homologous to those found in MT0146/CbiT from Methanobacterium thermoautotrophicum. CONCLUSIONS: The conservation of functional residues in the binding site for the co-factor, AdoMet, and in the putative precorrin-7 binding pocket suggested that MJ0391 may also possess CbiT activity. The putative function of MJ0391 is discussed, based on structural homology.

Immediate pigment darkening: its evolutionary roles may include protection against folate photosensitization.

The evolution of dark human skin colors in tropical areas is possibly related to photoprotection of folates. However, natural folates absorb mainly UVB radiation, and too little UVB can penetrate down to folates in dermal vessels to cause serious damage. However, endogenous photosensitizers, like riboflavin and uroporphyrin, absorbing UVA and visible light, can cause photosensitization of folates. Immediate pigment darkening (IPD), generated by UVA, has an absorption spectrum covering those of the endogenous photosensitizers. IPD is most prominent for darker skin types, which were typical for populations living under tropical solar fluences. We here propose that the biological role of IPD is protection of folates against photodegradation, which would be of large evolutionary importance for early hominids.

Acitretin mitigates uroporphyrin-induced bone defects in congenital erythropoietic porphyria models.

Congenital erythropoietic porphyria (CEP) is a rare genetic disorder leading to accumulation of uro/coproporphyrin-I in tissues due to inhibition of uroporphyrinogen-III synthase. Clinical manifestations of CEP include bone fragility, severe photosensitivity and photomutilation. Currently there is no specific treatment for CEP, except bone marrow transplantation, and there is an unmet need for treating this orphan disease. Fluorescent porphyrins cause protein aggregation, which led us to hypothesize that uroporphyrin-I accumulation leads to protein aggregation and CEP-related bone phenotype. We developed a zebrafish model that phenocopies features of CEP. As in human patients, uroporphyrin-I accumulated in the bones of zebrafish, leading to impaired bone development. Furthermore, in an osteoblast-like cell line, uroporphyrin-I decreased mineralization, aggregated bone matrix proteins, activated endoplasmic reticulum stress and disrupted autophagy. Using high-throughput drug screening, we identified acitretin, a second-generation retinoid, and showed that it reduced uroporphyrin-I accumulation and its deleterious effects on bones. Our findings provide a new CEP experimental model and a potential repurposed therapeutic.

Functional characterization of the early steps of tetrapyrrole biosynthesis and modification in Desulfovibrio vulgaris Hildenborough.

The biosynthesis of the tetrapyrrole framework has been investigated in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough by characterization of the enzymes required for the transformation of aminolaevulinic acid into sirohydrochlorin. PBG (porphobilinogen) synthase (HemB) was found to be a zinc-dependent enzyme that exists in its native state as a homohexamer. PBG deaminase (HemC) was shown to contain the dipyrromethane cofactor. Uroporphyrinogen III synthase is found fused with a uroporphyrinogen III methyltransferase (HemD-CobA). Both activities could be demonstrated in this amalgamated protein and the individual enzyme activities were separated by dissecting the relevant gene to allow the production of two distinct proteins. A gene annotated in the genome as a bifunctional precorrin-2 dehydrogenase/sirohydrochlorin ferrochelatase was in fact shown to act only as a dehydrogenase and is simply capable of synthesizing sirohydrochlorin rather than sirohaem. Genome analysis also reveals a lack of any uroporphyrinogen III decarboxylase, an enzyme necessary for the classical route to haem synthesis. However, the genome does encode some predicted haem d1 biosynthetic enzymes even though the bacterium does not contain the cd1 nitrite reductase. We suggest that sirohydrochlorin acts as a substrate for haem synthesis using a novel pathway that involves homologues of the d1 biogenesis system. This explains why the uroporphyrinogen III synthase is found fused with the methyltransferase, bypassing the need for uroporphyrinogen III decarboxylase activity.

Siroheme synthase orients substrates for dehydrogenase and chelatase activities in a common active site.

Siroheme is the central cofactor in a conserved class of sulfite and nitrite reductases that catalyze the six-electron reduction of sulfite to sulfide and nitrite to ammonia. In Salmonella enterica serovar Typhimurium, siroheme is produced by a trifunctional enzyme, siroheme synthase (CysG). A bifunctional active site that is distinct from its methyltransferase activity catalyzes the final two steps, NAD+-dependent dehydrogenation and iron chelation. How this active site performs such different chemistries is unknown. Here, we report the structures of CysG bound to precorrin-2, the initial substrate; sirohydrochlorin, the dehydrogenation product/chelation substrate; and a cobalt-sirohydrochlorin product. We identified binding poses for all three tetrapyrroles and tested the roles of specific amino acids in both activities to give insights into how a bifunctional active site catalyzes two different chemistries and acts as an iron-specific chelatase in the final step of siroheme synthesis.

How nature builds the pigments of life: the conquest of vitamin B12.

In part because humans cannot synthesize vitamin B12 and must obtain it from organisms that produce it and because B12 deficiency leads to pernicious anemia, it has been important to understand how microorganisms build this quite complex substance. As shown here, an interdisciplinary attack was needed, which combined the strengths of genetics, molecular biology, enzymology, chemistry, and spectroscopy. This allowed the step-by-step synthetic pathway of B12 to be elucidated, and this approach has acted as a model for future research on the synthesis of substances in living organisms. One practical outcome of such an approach has been the improved availability of B12 for animal feedstuffs and human health.

Absolute requirement for iron in the development of chemically induced uroporphyria in mice treated with 3-methylcholanthrene and 5-aminolevulinate.

Accumulating evidence, including experiments using cytochrome P450 1a2 (Cyp1a2) gene knock-out mice (Cyp1a2(-/-)), indicates that the development of chemically induced porphyria requires the expression of CYP1A2. It has also been demonstrated that iron enhances and expedites the development of experimental uroporphyria, but that iron alone without CYP1A2 expression, as in Cyp1a2(-/-) mice, does not cause uroporphyria. The role of iron in the development of porphyria has not been elucidated. We examined the in vivo effect of iron deficiency on hepatic URO accumulation in experimental porphyria. Mice were fed diets containing low (iron-deficient diet (IDD), 8.5 mg iron/kg) or normal (normal diet (ND), 213.7 mg iron/kg) levels of iron. They were treated with 3-methylcholanthrene (MC), an archetypal inducer of CYP1A, and 5-aminolevulinate (ALA), precursors of porphyrin and heme. We found that uroporphyrin (URO) levels and uroporphyrinogen oxidation (UROX) activity were markedly increased in ND mice treated with MC and ALA, while the levels were not raised in IDD mice with the same treatments. CYP1A2 levels and methoxyresorufin O-demethylase (MROD) activities, the CYP1A2-mediated reaction, were markedly induced in the livers of both ND and IDD mice treated with MC and ALA. UROX activity, supposedly a CYP1A2-dependent activity, was not enhanced in iron-deficient mice in spite of the fact of induction of CYP1A2. We showed that a sufficient level of iron is essential for the development of porphyria and UROX activity.

Transgenic Leishmania model for delta-aminolevulinate-inducible monospecific uroporphyria: cytolytic phototoxicity initiated by singlet oxygen-mediated inactivation of proteins and its ablation by endosomal mobilization of cytosolic uroporphyrin.

Inherent deficiencies of Leishmania in heme biosynthesis were genetically complemented for delta-aminolevulinate-inducible biosynthesis and accumulation of light-excitable uroporphyrin. The phototoxic flagellar immobilization and cytolysis phenotypes and porphyrin mobilization noted previously were further analyzed biochemically and cytologically to delineate the mechanism of phototoxicity and detoxification in this monoporphyric model. Under optimal conditions of induction for approximately 3 days, cells remained viable but became increasingly uroporphyric, peaking at > or =90% of the population by approximately day 2; thereafter, a small population of less porphyric or aporphyric cells emerged. On exposure to light, the flagella of porphyric cells were immobilized in milliseconds, and singlet oxygen became detectable in their lysates. Both photosensitive phenotypes increased proportionally with the cellular uroporphyric levels and were susceptible to inhibition by azide, but not by D-mannitol. Brief irradiation of the uroporphyric cells produced no appreciable protein degradation but inactivated cytosolic neomycin phosphotransferase and significantly bleached cytosolic green fluorescent protein, which was azide reversible. These cells were irreparably photodamaged, as indicated by their subsequent loss of membrane permeability and viability. This is the first in situ demonstration that early inactivation of functional proteins by singlet oxygen initiates the cytolytic phototoxicity in uroporphyria. Detoxification appears to involve endocytic/exocytic mobilization of uroporphyrin from cytosol to "porphyrinosomes" for its eventual extracellular expulsion. This is proposed as the sole mechanism of detoxification, since it is attributable to the reversion of porphyric to aporphyric cells during uroporphyrinogenesis and repeated cycles of this event plus photolysis selected no resistant mutants, only aporphyric clones of the parental phenotypes. Further characterization of the transport system for uroporphyrin in this model is expected to benefit not only our understanding of the cellular mechanism for disposal of toxic soluble wastes but also potentially the effective management of human uroporphyria and the use of uroporphyric Leishmania for vaccine/drug delivery.

Structure and function of SirC from Bacillus megaterium: a metal-binding precorrin-2 dehydrogenase.

In Bacillus megaterium, the synthesis of vitamin B(12) (cobalamin) and sirohaem diverges at sirohydrochlorin along the branched modified tetrapyrrole biosynthetic pathway. This key intermediate is made by the action of SirC, a precorrin-2 dehydrogenase that requires NAD(+) as a cofactor. The structure of SirC has now been solved by X-ray crystallography to 2.8 A (1 A = 0.1 nm) resolution. The protein is shown to consist of three domains and has a similar topology to the multifunctional sirohaem synthases Met8p and the N-terminal region of CysG, both of which catalyse not only the dehydrogenation of precorrin-2 but also the ferrochelation of sirohydrochlorin to give sirohaem. Guided by the structure, in the present study a number of active-site residues within SirC were investigated by site-directed mutagenesis. No active-site general base was identified, although surprisingly some of the resulting protein variants were found to have significantly enhanced catalytic activity. Unexpectedly, SirC was found to bind metal ions such as cobalt and copper, and to bind them in an identical fashion with that observed in Met8p. It is suggested that SirC may have evolved from a Met8p-like protein by loss of its chelatase activity. It is proposed that the ability of SirC to act as a single monofunctional enzyme, in conjunction with an independent chelatase, may provide greater control over the intermediate at this branchpoint in the synthesis of sirohaem and cobalamin.

Porphyrias and photosensitivity: pathophysiology for the clinician.

Porphyrias are disorders caused by defects in the biosynthetic pathway of heme. Their manifestations can be divided into three distinct syndromes, each attributable to the accumulation of three distinct classes of molecules. The acute neurovisceral syndrome is caused by the accumulation of the neurotoxic porphyrin precursors, delta aminolevulinic acid, and porphobilinogen; the syndrome of immediate painful photosensitivity is caused by the lipid-soluble protoporphyrin IX and, the syndrome of delayed blistering photosensitivity, caused by the water-soluble porphyrins, uroporphyrin, and coproporphyrin. Porphyrias can manifest with one, or with a combination, of these syndromes, depending on whether one or more types of molecules are being accumulated. Iron plays a significant role in some of these conditions, as evidenced by improvements in both clinical manifestations and laboratory parameters, following iron depletion in porphyria cutanea tarda, or iron administration in some cases of X-linked erythropoietic protoporphyria. While the pathophysiology of a specific type of porphyrias, the protoporphyrias, appears to favor the administration of zinc, results so far have been conflicting, necessitating further studies in order to assess its potential benefit. The pathways involved in each disease, as well as insights into their pathobiological processes are presented, with an emphasis on the development of photosensitivity reactions.

Preliminary field study of hepatic porphyrin profiles of Astyanax fasciatus (Teleostei, Characiformes) to define anthropogenic pollution.

The implementation of eco-toxicological assessment in South America is presently limited due to significant scientific information gaps concerning native species and their potential use as biomarkers. Recently, a common southern hemisphere fish species, Astyanax fasciatus, has been pointed out as a potential bio-indicator to anthropogenic pollution. This is a small, abundant, Neotropical characid, which is widely distributed from Central America south, to the Rio de la Plata Basin of western Uruguay. Our study found a statistically significant increase of coproporphyrin, uroporphyrin and protoporphyrin concentrations in hepatic tissues of A. fasciatus collected from a stream segment with high anthropogenic disturbance (due mainly to agricultural derivatives and motor vehicle transportation activities). Although the area studied showed differences in up and downstream limno-chemical parameters, these differences were not related to the increase of hepatic porphyrin concentrations. Based on the results of our study, we conclude that A. fasciatus is a good bio-indicator of exposure to environmental contaminants, and we propose that this abundant fish species be considered as a sentinel organism for monitoring potential disturbances to freshwater ecosystems.

Crystal structure of precorrin-8x methyl mutase.

BACKGROUND: The crystal structure of precorrin-8x methyl mutase (CobH), an enzyme of the aerobic pathway to vitamin B12, provides evidence that the mechanism for methyl migration can plausibly be regarded as an allowed [1,5]-sigmatropic shift of a methyl group from C-11 to C-12 at the C ring of precorrin-8x to afford hydrogenobyrinic acid. RESULTS: The dimeric structure of CobH creates a set of shared active sites that readily discriminate between different tautomers of precorrin-8x and select a discrete tautomer for sigmatropic rearrangement. The active site contains a strictly conserved histidine residue close to the site of methyl migration in ring C of the substrate. CONCLUSION: Analysis of the structure with bound product suggests that the [1,5]-sigmatropic shift proceeds by protonation of the ring C nitrogen, leading to subsequent methyl migration.

Selective in vivo tumor localization of uroporphyrin isomer I in mouse mammary carcinoma: superiority over other porphyrins in a comparative study.

Twenty-eight porphyrins were evaluated for tumor localization as delineated by fluorescence using a transplantable KHJJ mammary carcinoma in BALB/c mice as the tumor model. Five of the 28 porphyrins were found to localize and of these, one, i.e., uroporphyrin I (UROP I), showed a higher tumor:skin ratio than any of the others; moreover, as no measurable UROP I was present in the gut, the tumor:intestinal porphyrin ratio under the conditions of assay was infinity. Because hematoporphyrin derivative (HPD), a complex mixture of porphyrins has been studied extensively as a tumor localizer, we compared HPD with UROP I at differing doses (2-40 mg/kg) and at different times (3-96 h) following i.v. administration. Dose response curves showed tissue levels of porphyrin to plateau out at doses above 20 mg/kg. Peak tumor HPD and UROP I levels attained 6-18 h after i.v. administration (40 mg porphyrin/kg) were comparable, but tumor retention of HPD over the ensuing 96 h was higher. The ratio of UROP I in tumor compared to skin was significantly greater throughout the period of observation. At all times, no UROP I was detectable in gastrointestinal mucosa. At differing doses (10-40 mg/kg), the tumor:skin ratio for HPD ranged from 1.47-1.85, and for UROP I from 6.06-12.33. As a function of time (6-72 h), the tumor:skin ratios respectively were 1.03-2.38, and 11.9 to infinity. At all times, the tumor:colon mucosa ratio at different doses for HPD approached 1 and for UROP I was infinity. We conclude that the greater specificity of tumor uptake by UROP I and its lack of retention by gut mucosa warrants further study to determine its potential clinical application as a diagnostic marker, particularly for early mucosal cancer, and in photoradiation therapy.