Immunologic evidence for structural homology between the release factors of Escherichia coli.

A cell-free chloroplast preparation obtained from greening cucumber cotyledons was tested for its ability to synthesize protoporphyrin IX from compounds previously postulated to be precursors of δ-aminolevulinic acid in plants, namely, glutamate, glutamine, α-ketoglutarate, glycine, and succinate. Of these, only glutamate caused a marked stimulation of protoporphyrin biosynthesis. A mixture of cofactors (ATP, KH₂PO₄, glutathione, coenzyme A, and NAD⁺), which was previously shown to be necessary for the incorporation of δ-aminolevulinic acid into protochlorophyll and for the maintenance of etioplasts in vitro also proved to be necessary for the conversion of glutamate to protoporphyrin IX.     

Formation of Mg-Containing Chlorophyll Precursors from Protoporphyrin IX, delta-Aminolevulinic Acid, and Glutamate in Isolated, Photosynthetically Competent, Developing Chloroplasts

Intact developing chloroplasts isolated from greening cucumber (Cucumis sativus L. var Beit Alpha) cotyledons were found to contain all the enzymes necessary for the synthesis of chlorophyllide. Glutamate was converted to Mg-protoporphyrin IX (monomethyl ester) and protoclorophyllide. δ-Aminolevulinic acid and protoporphyrin IX were converted to Mg-protoporphyrin IX, Mg-protoporphyrin IX monomethyl ester, protochlorophyllide and chlorophyllide a. The conversion of δ-aminolevulinic acid or protoporphyrin IX to Mg-protoporphyrin IX (monomethyl ester) was inhibited by AMP and p-chloromercuribenzene sulfonate. Light stimulated the formation of Mg-protoporphyrin IX from all three substrates. In the case of δ-aminolevulinic acid and protoporphyrin IX, light could be replaced by exogenous ATP. In the case of glutamate, both ATP and reducing power were necessary to replace light. With all three substrates, glutamate, δ-aminolevulinic acid, and protoporphyrin IX, the stimulation of Mg-protoporphyrin IX accumulation in the light was abolished by DCMU, and this DCMU block was overcome by added ATP and reducing power.     

Regulation of protoporphyrin IX biosynthesis by intraplastidic compartmentalization and adenosine triphosphate

Subplastidic preparations from cotyledons of cucumber (Cucumis sativus L.) were tested for their ability to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Envelope or thylakoid membranes failed to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Stromal preparations synthesized a very low amount of protoporphyrin IX. In a reconstitution experiment using stroma + envelope membranes, protoporphyrin IX synthesis from 5-aminolevulinic acid was enhanced by 660% over that of stroma alone. However, when thylakoids were added to the stroma + envelope mixture, protoporphyrin IX synthesis from 5-aminolevulinic acid was completely inhibited. In the reconstituted stroma + envelope membrane mixture, the reducing agent dithiothreitol enhanced the protoporphyrin IX-synthesizing ability and completely abolished the inhibition of protoporphyrin IX synthesis by thylakoids. This suggested that the oxidizing agents usually associated with the thylakoid membranes inhibited protoporphyrin IX biosynthesis and the inhibition was alleviated by the reducing power of dithiothreitol. This study exposes the weakness of in vitro reconstitution experiments in mimicking the in vivo-conditions. Addition of ATP stimulated protoporphyrin IX synthesis by 50% in the supernatant fraction of chloroplast lysate. This ATP-induced stimulation of protoporphyrin IX synthesis was due to the enhancement of the activities of uroporphyrinogen decarboxylase and protoporphyrinogen oxidase, involved in tetrapyrrole biosynthesis. The ATP-induced stimulation of porphyrinogen oxidase activity was an energy-dependent reaction. Received: 21 March 2000 / Accepted: 9 May 2000     

Growth Cycle-Dependent Overproduction and Accumulation of Protoporphyrin IX in Tetrahymena: Effect of Heavy Metals1

Cells of the ciliate Tetrahymena pyriformis GL overproduce and accumulate massive quantities of the heme intermediate, protoporphyrin IX. Protoporphyrin is localized intracellularly in discrete membranous compartments. The amount of porphyrin stored in the cell changes dramatically as cells progress through the growth cycle. Porphyrin overproduction is stimulated by δ-aminolevulinic acid, but only during the mid-stationary phase. Overproduction of protoporphyrin IX apparently results from an increase, late in the growth cycle, of activities subsequent to δ-aminolevulinic acid synthetase. Feedback inhibition in the pathway by accumulated protoporphyrin IX does not occur. The presence of Co²⁺ completely inhibits accumulation of protoporphyrin IX in a manner reversed by δ-aminolevulinic acid. Sn⁴⁺ stimulates protoporphyrin IX accumulation in the culture.     

Specific Binding of Protoporphyrin IX to a Membrane-Bound 63 Kilodalton Polypeptide in Cucumber Cotyledons Treated with Diphenyl Ether-Type Herbicides

Porphyrin accumulation in excised cucumber cotyledons (Cucumis sativus L.) treated with a N-phenylimide S-23142 (N-[4-chloro-2-fluoro-5-propargyloxyphenyl]-3,4,5,6- tetrahydrophthalimide) and a diphenylether acifluorfen-ethyl (ethyl-5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitro benzoic acid) was studied. Most of the accumulated porphyrins were found in the membrane fractions of 6,000g and 30,000g pellets, forming a complex with a membrane polypeptide. The complex was solubilized with 1% n-dodecyl β-d-maltoside and its molecular mass was estimated to be 63,000 and 66,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation high performance liquid chromatography (HPLC), respectively. The polypeptide also existed in untreated cotyledons but had little protoporphyrin IX. The complex was also formed in vitro by mixing the 30,000g pellets from untreated cotyledons and authentic protoporphyrin IX. However, protoporphyrin IX formed the complex specifically with the 63,000 dalton polypeptide and not with the other proteins both in vivo and in vitro. At least four fluorescent porphyrins, including protoporphyrin IX, were found in the acetone extract of the cotyledons by HPLC using a reversed phase column. Protoporphyrin IX was one of the two porphyrins that formed the complex. These results suggest that S-23142 and acifluorfenethyl enhance the accumulation of protoporphyrin IX, which forms the complex with the membrane protein.     

Regulation of Magnesium Chelatase Activity during Excessive Accumulation of Porphyrins in Green Barley Leaves

Activity of magnesium chelatase was studied in green barley leaves treated with 5-aminolevulinic acid (ALA). After this treatment, leaves accumulated excessive amounts of porphyrinic precursors of chlorophyll : protoporphyrin IX (PP), magnesium-protoporphyrin IX (MgPP), its monomethyl ester (MgPPE), and protochlorophyllide. The enzyme activity was found to be inversely dependent on the amount of MgPP formed from exogenous ALA. A conclusion was drawn about the existence of a mechanism for the regulation of the enzyme activity in vivo via its inhibition by the reaction product.     

Influence of photodynamic processes induced by 2,2′-dipyridyl on the enzymatic system of chlorophyll biosynthesis

The influence of 2,2′-dipyridyl (2,2′-DP) on the activity of one of the enzymes at the initial stages of chlorophyll (Chl) biosynthesis, δ-aminolevulinic acid dehydratase (ALAD; δ-aminolevulinate hydro-lyase, EC 4.2.1.24), as well as on δ-aminolevulinic acid (ALA) accumulation was investigated in green barley (Hordeum vulgare L.) leaves. In seven-day-old green leaves treated with 3 mM 2,2′-DP for 17 h in darkness and subsequently irradiated with "white light" (15 W m-2) for 4, 8, and 24 h the ALAD activity was 51 % as compared to that in untreated leaves. At the same time, the ALA forming system was most sensitive to the photodynamic processes caused by 2,2′-DP. After 8 h of irradiation, ALA synthesis was entirely inhibited. After the treatment the leaves accumulated exceptionally high amounts of Chl precursors such as protoporphyrin IX (Proto), Mg-protoporphyrin IX (Mg-Proto), its monomethyl ester, and protochlorophyllide (Pchlide) that are photosensitizers of photodynamic processes in plants. A comparatively low Chl and carotenoid (Car) destruction was registered during the subsequent 4 and 8 h of irradiation. At the same time, the content of Chl precursors was negligible. The low photodestruction of Chl and Car included in pigment-protein complexes, against the background of fast porphyrin disappearance, and fast decrease of enzymatic activities at the initial stages of Chl production could mean that the photodynamic effect induced by porphyrins accumulated in the presence of 2,2′-DP affected first the Chl enzymatic system and did not change the pool of already synthesized photosynthetic pigments.     

Photodynamic antimicrobial activity of new porphyrin derivatives against methicillin resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Methicillin resistant Staphylococcus aureus (MRSA) with multiple drug resistance patterns is frequently isolated from skin and soft tissue infections that are involved in chronic wounds. Today, difficulties in the treatment of MRSA associated infections have led to the development of alternative approaches such as antimicrobial photodynamic therapy. This study aimed to investigate photoinactivation with cationic porphyrin derivative compounds against MRSA in in-vitro conditions. In the study, MRSA clinical isolates with different antibiotic resistance profiles were used. The newly synthesized cationic porphyrin derivatives (P_M, P_E, P_PN, and P_PL) were used as photosensitizer, and 655 nm diode laser was used as light source. Photoinactivation experiments were performed by optimizing energy doses and photosensitizer concentrations. In photoinactivation experiments with different energy densities and photosensitizer concentrations, more than 99% reduction was achieved in bacterial cell viability. No decrease in bacterial survival was observed in control groups. It was determined that there was an increase in photoinactivation efficiency by increasing the energy dose. At the energy dose of 150 J/cm² a survival reduction of over 6.33 log₁₀ was observed in each photosensitizer type. While 200 μM P_M concentration was required for this photoinactivation, 12.50 μM was sufficient for P_E, P_PN, and P_PL. In our study, antimicrobial photodynamic therapy performed with cationic porphyrin derivatives was found to have potent antimicrobial efficacy against multidrug resistant S. aureus which is frequently isolated from wound infections.     

Shock Wave Treatment Enhances Cell Proliferation and Improves Wound Healing by ATP Release-coupled Extracellular Signal-regulated Kinase (ERK) Activation

Shock wave treatment accelerates impaired wound healing in diverse clinical situations. However, the mechanisms underlying the beneficial effects of shock waves have not yet been fully revealed. Because cell proliferation is a major requirement in the wound healing cascade, we used in vitro studies and an in vivo wound healing model to study whether shock wave treatment influences proliferation by altering major extracellular factors and signaling pathways involved in cell proliferation. We identified extracellular ATP, released in an energy- and pulse number-dependent manner, as a trigger of the biological effects of shock wave treatment. Shock wave treatment induced ATP release, increased Erk1/2 and p38 MAPK activation, and enhanced proliferation in three different cell types (C3H10T1/2 murine mesenchymal progenitor cells, primary human adipose tissue-derived stem cells, and a human Jurkat T cell line) in vitro. Purinergic signaling-induced Erk1/2 activation was found to be essential for this proliferative effect, which was further confirmed by in vivo studies in a rat wound healing model where shock wave treatment induced proliferation and increased wound healing in an Erk1/2-dependent fashion. In summary, this report demonstrates that shock wave treatment triggers release of cellular ATP, which subsequently activates purinergic receptors and finally enhances proliferation in vitro and in vivo via downstream Erk1/2 signaling. In conclusion, our findings shed further light on the molecular mechanisms by which shock wave treatment exerts its beneficial effects. These findings could help to improve the clinical use of shock wave treatment for wound healing.     

Cold Atmospheric Plasma (CAP) Changes Gene Expression of Key Molecules of the Wound Healing Machinery and Improves Wound Healing In Vitro and In Vivo

Cold atmospheric plasma (CAP) has the potential to interact with tissue or cells leading to fast, painless and efficient disinfection and furthermore has positive effects on wound healing and tissue regeneration. For clinical implementation it is necessary to examine how CAP improves wound healing and which molecular changes occur after the CAP treatment. In the present study we used the second generation MicroPlaSter ß® in analogy to the current clinical standard (2 min treatment time) in order to determine molecular changes induced by CAP using in vitro cell culture studies with human fibroblasts and an in vivo mouse skin wound healing model. Our in vitro analysis revealed that the CAP treatment induces the expression of important key genes crucial for the wound healing response like IL-6, IL-8, MCP-1, TGF-ß1, TGF-ß2, and promotes the production of collagen type I and alpha-SMA. Scratch wound healing assays showed improved cell migration, whereas cell proliferation analyzed by XTT method, and the apoptotic machinery analyzed by protein array technology, was not altered by CAP in dermal fibroblasts. An in vivo wound healing model confirmed that the CAP treatment affects above mentioned genes involved in wound healing, tissue injury and repair. Additionally, we observed that the CAP treatment improves wound healing in mice, no relevant side effects were detected. We suggest that improved wound healing might be due to the activation of a specified panel of cytokines and growth factors by CAP. In summary, our in vitro human and in vivo animal data suggest that the 2 min treatment with the MicroPlaSter ß® is an effective technique for activating wound healing relevant molecules in dermal fibroblasts leading to improved wound healing, whereas the mechanisms which contribute to these observed effects have to be further investigated.     

Temperature-Stress-Induced Impairment of Chlorophyll Biosynthetic Reactions in Cucumber and Wheat

Chlorophyll (Chl) biosynthesis in chill (7°C)- and heat (42°C)-stressed cucumber (Cucumis sativus L. cv poinsette) seedlings was affected by 90 and 60%, respectively. Inhibition of Chl biosynthesis was partly due to impairment of 5-aminolevulinic acid biosynthesis both in chill- (78%) and heat-stress (70%) conditions. Protochlorophyllide (Pchlide) synthesis in chill- and heat-stressed seedlings was inhibited by 90 and 70%, respectively. Severe inhibition of Pchlide biosynthesis in chill-stressed seedlings was caused by inactivations of all of the enzymes involved in protoporphyrin IX (Proto IX) synthesis, Mg-chelatase, and Mg-protoporphyrin IX monoester cyclase. In heat-stressed seedlings, although 5-aminolevulinic acid dehydratase and porphobilinogen deaminase were partially inhibited, one of the porphyrinogen-oxidizing enzymes, uroporphyrinogen decarboxylase, was stimulated and coproporphyrinogen oxidase and protoporphyrinogen oxidase were not substantially affected, which demonstrated that protoporphyrin IX synthesis was relatively more resistant to heat stress. Pchlide oxidoreductase, which is responsible for phototransformation of Pchlide to chlorophyllide, increased in heat-stress conditions by 46% over that of the control seedlings, whereas it was not affected in chill-stressed seedlings. In wheat (Triticum aestivum L. cv HD2329) seedlings porphobilinogen deaminase, Pchlide synthesis, and Pchlide oxidoreductase were affected in a manner similar to that of cucumber, suggesting that temperature stress has a broadly similar effect on Chl biosynthetic enzymes in both cucumber and wheat.     

ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and “pump” functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.     

Chloroplast biogenesis: Biosynthesis and accumulation of Mg-protoporphyrin IX monoester and other metalloporphyrins by isolated etioplasts and developing chloroplasts

Developing chloroplasts isolated from greening cotyledons and isolated etioplasts were capable of synthesizing and accumulating Mg-protoporphyrin IX monoester as well as longer wavelength metalloporphyrins when incubated in the dark, in the presence of air, δ-aminolevulinic acid, and cofactors (coenzyme A, glutathione, adenosine triphosphate, nicotinamide adenine dinucleotide, methyl alcohol, magnesium, potassium, and phosphate). The putative metalloporphyrins exhibited distinct fluorescence emission and excitation properties and were detected by spectrofluorometry in situ and after extraction in organic solvents. The cofactors were previously shown to be required for protochlorophyll, and chlorophyll biosynthesis and grana assembly in vitro. The putative long wavelength metalloporphyrins were suggested earlier to represent intermediates between Mg-protoporphyrin IX monomethyl ester and protochlorophyllide. The isolated plastids were similar in this aspect of their biosynthetic activity to etiolated cotyledons greening in distilled H₂O. In contrast to greening cotyledons, however, the biosynthetic activity of the isolated plastids depended on the addition of exogenous cofactors and δ-aminolevulinic acid. This was interpreted as an indication that the isolated plastids were not capable of generating their own δ-aminolevulinic acid and cofactors under the present incubation conditions. Light was not required for the conversion of added ALA to metalloporphyrins in vitro. The metalloporphyrins synthesized in vitro were more highly fluorescent in situ than those of greening cotyledons. In addition to Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins, isolated etioplasts synthesized and accumulated Zn-protoporphyrin and Zn-protoporphyrin IX monoesterlike compounds.     

Luminol as the light source for in situ photodynamic therapy

Presently, the light sources used in photodynamic therapy are high intensity lasers or light emitting diodes, making it unsuitable for large-volume tumors and those located deep inside the body. To overcome this limitation, we propose an in situ light source to excite the photosensitizer to generate toxic singlet oxygen and kill tumor cells directly. In this research, luminol served as the in situ light source in 5-aminolevulinic acid-mediated photodynamic treatment of Caco-2 cell cultures. 72 h after luminol excitation the viability of the treated cells significantly decreased compared to the control cells in assays including cell viability, cytotoxicity, flow cytometry and fluorescence confocal microscopy. According to the results, we suggested luminol could be used as an in situ light source for 5-aminolevulinic acid-mediated photodynamic therapy. This method would have great potential to extend the application of photodynamic therapy to tumors located deep inside the body.     

Interactions of Methicillin Resistant Staphylococcus aureus USA300 and Pseudomonas aeruginosa in Polymicrobial Wound Infection

Understanding the pathology resulting from Staphylococcus aureus and Pseudomonas aeruginosa polymicrobial wound infections is of great importance due to their ubiquitous nature, increasing prevalence, growing resistance to antimicrobial agents, and ability to delay healing. Methicillin-resistant S. aureus USA300 is the leading cause of community-associated bacterial infections resulting in increased morbidity and mortality. We utilized a well-established porcine partial thickness wound healing model to study the synergistic effects of USA300 and P. aeruginosa on wound healing. Wound re-epithelialization was significantly delayed by mixed-species biofilms through suppression of keratinocyte growth factor 1. Pseudomonas showed an inhibitory effect on USA300 growth in vitro while both species co-existed in cutaneous wounds in vivo. Polymicrobial wound infection in the presence of P. aeruginosa resulted in induced expression of USA300 virulence factors Panton-Valentine leukocidin and α-hemolysin. These results provide evidence for the interaction of bacterial species within mixed-species biofilms in vivo and for the first time, the contribution of virulence factors to the severity of polymicrobial wound infections.     

Accumulation of protoporphyrin-IX (PpIX) in leukemic cell lines following induction by 5-aminolevulinic acid (ALA)

We investigated the amounts of protoporphyrin IX (PpIX) accumulated in noninduced cells and following 5-aminolevulinic acid (ALA)-induction. Following ALA administration PpIX increased in all leukemic cell lines under investigation (HEL 26-fold, HL60 6-fold, Jurkat 3-fold, ML2 2-fold) but not in lymphocytes. Compared to other cell lines studied, HEL cells showed the lowest basal level of PpIX and the largest relative increase in PpIX. Despite a high increase following ALA treatment, the PpIX level in HEL cells is almost as low as in lymphocytes. It is in agreement with their relatively low sensitivities of ALA-induced photodynamic therapy (ALA-PDT) shown previously [(Grebenová, D., Cajthamlová, H., Bartosová, J., Marinov, J., Klamová, H., Fuchs, O., Hrkal, Z., 1998. Selective destruction of leukemic cells by photo-activation of 5-aminolevulinic acid induced protoporphyrin IX. J. Photochem. Photobiol. B: Biol. 47, 74-81)]. The ferrochelatase activities in the individual cell lines are in good inverse correlation with PpIX amounts accumulated in the ALA-induced cells, but not with the relative increase (ratio) of PpIX levels from basal to ALA-induced ones. This is most apparent in HEL cells and lymphocytes. There is probably different regulation of heme biosynthesis in erythroid cells, which are therefore not suitable for the studies of ALA-PDT mechanism. PpIX was accumulated more extensively in absence of fetal calf serum than in its presence. The amounts of PpIX accumulated in cells decreased exponentially with increasing fetal calf serum concentration.     

Succinylacetone pyrrole,a powerful inhibitor of vitamin B12 biosynthesis: Effect on δ-aminolevulinic acid dehydratase

Succinylacetone, a competitive inhibitor (K_I = 400 μM) of δ-aminolevulinic acid dehydratase of $\text{Clostridium}\text{tetanomorphum}$, is converted non-enzymatically upon incubation with δ-aminolevulinic acid to succinylacetone pyrrole, a much stronger competitive inhibitor (K_I = 5 μM) of the enzyme. A similar effect is seen in vivo: when present in the growth medium at concentrations of about 1 μM, the pyrrole decreases the level of corrinoids produced by this organism by half, while succinylacetone at 200 μM causes only 19 per cent inhibition of corrinoid formation. Levulinic acid is a much weaker inhibitor in vitro and in vivo. The inhibition by succinylacetone pyrrole is considered to be due to its structural resemblance to δ-aminolevulinic acid rather than to porphobilinogen, the reaction product of δ-aminolevulinic acid dehydratase: succinylacetone, succinylacetone pyrrole, and levulinic acid all contain a succinyl group.     

Relation between intracellular location and photodynamic efficacy of 5-aminolevulinic acid-induced protoporphyrin IX in vitro. Comparison between human glioblastoma cells and other cancer cell lines.

A promising clinical application of 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PP IX) is fluorescence detection and photodynamic treatment of residual tumour tissue during surgical resection of high grade malignant glioma. U373 MG human glioblastoma cells were used as a model system to study the relation between intracellular location and photodynamic efficacy of 5-ALA-induced PP IX in more detail. Therefore, ultra-sensitive fluorescence microscopy, using either optical excitation of whole cells or selective excitation of the plasma membrane by an evanescent electromagnetic field, was combined with quantitative measurements of intracellular porphyrin amount and phototoxicity. Glioblastoma cells accumulated PP IX to a moderate extent as compared to T47D breast cancer cells (high accumulation) or OV2774 ovarian cancer cells (low accumulation). Although photodynamic inactivation of the different cell lines (decreasing in the order T47D > U373 MG > OV2774) seemed to be directly related to PP IX accumulation, examination of the data in more detail revealed that photodynamic efficacy per photosensitizer molecule (PE) was about two times higher in glioblastoma and ovarian cancer cells as compared to breast cancer cells. The different photodynamic efficacy of PP IX was related to the different intracellular location. In contrast to breast cancer cells where PP IX fluorescence was localized in small granules, PP IX fluorescence in glioblastoma cells and ovarian cancer cells originated mainly from cellular membranes. Thus, the intracellular location of PP IX in a predominantly lipophilic environment, characterized by a comparably high photostability (probed by photobleaching and photoproduct formation) and a lower degree of porphyrin aggregation (probed previously by fluorescence decay kinetics), seems to be the key factor for high photodynamic efficacy of 5-ALA-induced PP IX. In the case of OV2774 ovarian cancer cells, however, a low PP IX accumulation limited cell inactivation upon irradiation, whereas the results obtained for glioblastoma cells are encouraging to develop PDT to an additional therapeutic option for the treatment of tumour margins in patients who underwent fluorescence-guided resection of high grade malignant glioma after 5-ALA administration.     

Induction of delta-Aminolevulinic Acid Synthase Activity and Inhibition of Heme Synthesis in Euglena gracilis by N-Methyl Mesoporphyrin IX

N-Methyl mesoporphyrin IX, an inhibitor of heme synthesis, increases extractable δ-aminolevulinic acid (ALA) synthase activity when administered to growing cultures of Euglena gracilis Klebs strain Z Pringsheim in micromolar concentrations. Wild-type light-grown green cells and white aplastidic cells exhibited 2.8-fold and 1.8-fold increases, respectively, in ALA synthase activity within five to six hours after incubation with 4 × 10⁻⁶ molar N-methyl mesoporphyrin IX. Protoheme levels were decreased and ⁵⁹Fe incorporation into heme was inhibited by N-methyl mesoporphyrin IX, indicating that, as in animal cells, N-methyl mesoporphyrin IX acts specifically to block iron insertion into protoporphyrin IX. Chlorophyll synthesis in wild-type cells was not affected within the first 6 hours after administration of N-methyl mesoporphyrin IX.     

Embelin (2,5-dihydroxy-3-undecyl-p-benzoquinone): A bioactive molecule isolated from Embelia ribes as an effective photodynamic therapeutic candidate against tumor in vivo

The present study was carried out to assess the photosensitizing potential of embelin, the biologically active natural product isolated from Embelia ribes in photodynamic therapy (PDT) experiments in vivo. In vitro PDT clearly indicated that embelin recorded significant cytotoxicity in Ehrlich's Ascites Carcinoma (EAC) cells, which is superior to 5-aminolevulinic acid, a known photodynamic compound. For in vivo experiments solid tumor was induced using EAC cells in the male Swiss albino mice of groups I, II, III and IV. Group I served as the control (without solid tumor), group II served as tumor bearing mice without treatment and groups III and IV served as treatments. At the completion of 4 weeks of induction, the tumor bearing mice from group III and IV were given an intraperitoneal injection with embelin (12.5 mg/kg body weight). After 24 h, tumor area in the Group III and IV animals was exposed to visible light from a 1000 W halogen lamp. The mice from groups I to III were sacrificed 2 weeks after the PDT treatment and the marker enzymes (myeloperoxidase [MPO], β-d-glucuronidase, and rhodanese) were assayed and expression of Bcl-2 and Bax were analyzed in normal and tumor tissues. Animals from group IV were sacrificed after 90 days of PDT treatment and the above mentioned parameters were recorded. Reduction in tumor volume and reversal of biochemical markers to near normal levels were observed in the treated groups. This is the first report on PDT using a natural compound for solid tumor control in vivo. The uniqueness of the mode of treatment lies in the selective uptake of the nontoxic natural compound, embelin from the medicinal plant E. ribes used in Indian system of medicine, by the solid tumor cells and their selective destruction using PDT without affecting the neighboring normal cells, which is much advantageous over radiation therapy now frequently used.