FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1968047991> ?p ?o ?g. }

• W1968047991 endingPage "14909" @default.
• W1968047991 startingPage "14902" @default.
• W1968047991 abstract "Leishmania was found deficient in at least five and most likely seven of the eight enzymes in the heme biosynthesis pathway, accounting for their growth requirement for heme compounds. The xenotransfection of this trypanosomatid protozoan led to their expression of the mammalian genes encoding δ-aminolevulinate (ALA) dehydratase and porphobilinogen deaminase, the second and the third enzymes of the pathway, respectively. These transfectants still require hemin or protoporphyrin IX for growth but produce porphyrin when ALA was supplied exogenously. Leishmania is thus deficient in all first three enzymes of the pathway. Uroporphyrin I was produced as the sole intermediate by these transfectants, further indicating that they are also deficient in at least two porphyrinogen-metabolizing enzymes downstream of porphobilinogen deaminase, i.e. uroporphyrinogen III co-synthase and uroporphyrinogen decarboxylase. Pulsing the transfectants with ALA induced their transition from aporphyria to uroporphyria. Uroporphyrin I emerged in these cells initially as diffused throughout the cytosol, rendering them sensitive to UV irradiation. The porphyrin was subsequently sequestered in cytoplasmic vacuoles followed by its release and accumulation in the extracellular milieu, concomitant with a reduced photosensitivity of the cells. These events may represent cellular mechanisms for disposing soluble toxic waste from the cytosol. Monocytic tumor cells were rendered photosensitive by infection with uroporphyric Leishmania, suggestive of their potential application for photodynamic therapy. Leishmania was found deficient in at least five and most likely seven of the eight enzymes in the heme biosynthesis pathway, accounting for their growth requirement for heme compounds. The xenotransfection of this trypanosomatid protozoan led to their expression of the mammalian genes encoding δ-aminolevulinate (ALA) dehydratase and porphobilinogen deaminase, the second and the third enzymes of the pathway, respectively. These transfectants still require hemin or protoporphyrin IX for growth but produce porphyrin when ALA was supplied exogenously. Leishmania is thus deficient in all first three enzymes of the pathway. Uroporphyrin I was produced as the sole intermediate by these transfectants, further indicating that they are also deficient in at least two porphyrinogen-metabolizing enzymes downstream of porphobilinogen deaminase, i.e. uroporphyrinogen III co-synthase and uroporphyrinogen decarboxylase. Pulsing the transfectants with ALA induced their transition from aporphyria to uroporphyria. Uroporphyrin I emerged in these cells initially as diffused throughout the cytosol, rendering them sensitive to UV irradiation. The porphyrin was subsequently sequestered in cytoplasmic vacuoles followed by its release and accumulation in the extracellular milieu, concomitant with a reduced photosensitivity of the cells. These events may represent cellular mechanisms for disposing soluble toxic waste from the cytosol. Monocytic tumor cells were rendered photosensitive by infection with uroporphyric Leishmania, suggestive of their potential application for photodynamic therapy. Leishmania, like other trypanosomatid protozoa, are among the rare examples of aerobic organisms, which depend on oxidative phosphorylation (for Leishmania mexicana andLeishmania amazonensis, see Refs. 1.Hart D.T. Coombs G.H. Exp. Parasitol. 1982; 54: 397-409Crossref PubMed Scopus (133) Google Scholar and 2.Bermúdez R. Dagger F. D'Aquino J.A. Benaim G. Dawidowicz K. Mol. Biochem. Parasitol. 1997; 90: 43-54Crossref PubMed Scopus (18) Google Scholar), but are defective in the synthesis of heme (3.Chang C.S. Chang K.-P. Mol. Biochem. Parasitol. 1985; 16: 267-276Crossref PubMed Scopus (82) Google Scholar) required for electron transport respiratory complexes. This peculiar defect in tetrapyrrole biosynthesis is manifested as a nutritional requirement for hemin by these organisms in chemically defined medium (for review, see Ref. 3.Chang C.S. Chang K.-P. Mol. Biochem. Parasitol. 1985; 16: 267-276Crossref PubMed Scopus (82) Google Scholar). In nature, these parasitic protozoa must acquire protoporphyrin IX or heme exogenously from their hosts as a nutritional factor (3.Chang C.S. Chang K.-P. Mol. Biochem. Parasitol. 1985; 16: 267-276Crossref PubMed Scopus (82) Google Scholar). Exceptional are several entomophilic nonpathogenic Crithidiaspecies that harbor β-proteobacteria as endosymbionts presumably to help them complete the heme biosynthetic pathway, thereby sparing their nutritional requirement for hemin as an essential growth factor (4.Du Y. Maslov D.A. Chang K.-P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8437-8441Crossref PubMed Scopus (60) Google Scholar). Earlier biochemical studies of trypanosomatid protozoa have shown that they are deficient in heme biosynthesis (3.Chang C.S. Chang K.-P. Mol. Biochem. Parasitol. 1985; 16: 267-276Crossref PubMed Scopus (82) Google Scholar, 5.Chang K.-P. Chang C.S. Sassa S. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2979-2983Crossref PubMed Scopus (85) Google Scholar, 6.Salzman T.A. Stella A.M. De Xifra E.A.W. Batlle A.M.D.C. Docampo R. Stoppani A.O.M. Comp. Biochem. Physiol. 1982; 72: 663-667Crossref Scopus (4) Google Scholar, 7.Srivastava P. Sharma G.D. Kamboj K.K. Rastogi A.K. Pandey V.C. Mol. Cell. Biochem. 1997; 171: 65-68Crossref PubMed Scopus (18) Google Scholar, 8.Sagar R. Salotra P. Bhatnagar R. Datta K. Microbiol. Res. 1995; 150: 419-423Crossref PubMed Scopus (3) Google Scholar). This was examined according to the following conventional pathway: glycine + succinyl-CoA or 4,5-dioxovalerate + alanine → δ-aminolevulinate (ALA) 1The abbreviations used are: ALAδ-aminolevulinateALADδ-aminolevulinate dehydratasePBGDporphobilinogen deaminase1The abbreviations used are: ALAδ-aminolevulinateALADδ-aminolevulinate dehydratasePBGDporphobilinogen deaminase → porphobilinogen → hydroxymethylbilane (by-product = uroporphyrinogen I) →uroporphyrinogen III → co-proporphyrinogen III→ protoporphyrinogen IX→ protoporphyrin IX→ heme (9.Sassa S. Beutler E. Lichtman M.A. Coller B.S. Kipps T.J. William's Hematology. McGraw-Hill Inc., New York1995: 726-746Google Scholar). Table I lists the eight enzymes, which are known to catalyze this pathway. The activities of these enzymes are often undetectable or negligible in trypanosomatid protozoa. Reported previously in these organisms were the activities of ALA-synthase/dioxovalerate transaminase and ferrochelatase (7.Srivastava P. Sharma G.D. Kamboj K.K. Rastogi A.K. Pandey V.C. Mol. Cell. Biochem. 1997; 171: 65-68Crossref PubMed Scopus (18) Google Scholar, 8.Sagar R. Salotra P. Bhatnagar R. Datta K. Microbiol. Res. 1995; 150: 419-423Crossref PubMed Scopus (3) Google Scholar, 10.Salzman T.A. Batlle A.M. Angluster J. de Souza W. Int. J. Biochem. 1985; 17: 1343-1347Crossref PubMed Scopus (24) Google Scholar), the first and the last enzymes of the pathway normally present in mitochondria (9.Sassa S. Beutler E. Lichtman M.A. Coller B.S. Kipps T.J. William's Hematology. McGraw-Hill Inc., New York1995: 726-746Google Scholar). Much less or absent are activities of the second and the third enzymes, i.e. δ-aminolevulinate dehydratase (ALAD, EC 4.2.1.24) and porphobilinogen deaminase (PBGD, EC4.3.1.8) (3.Chang C.S. Chang K.-P. Mol. Biochem. Parasitol. 1985; 16: 267-276Crossref PubMed Scopus (82) Google Scholar, 5.Chang K.-P. Chang C.S. Sassa S. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2979-2983Crossref PubMed Scopus (85) Google Scholar, 6.Salzman T.A. Stella A.M. De Xifra E.A.W. Batlle A.M.D.C. Docampo R. Stoppani A.O.M. Comp. Biochem. Physiol. 1982; 72: 663-667Crossref Scopus (4) Google Scholar, 7.Srivastava P. Sharma G.D. Kamboj K.K. Rastogi A.K. Pandey V.C. Mol. Cell. Biochem. 1997; 171: 65-68Crossref PubMed Scopus (18) Google Scholar). The pathway thus appears to be incomplete in this group of organisms (3.Chang C.S. Chang K.-P. Mol. Biochem. Parasitol. 1985; 16: 267-276Crossref PubMed Scopus (82) Google Scholar, 6.Salzman T.A. Stella A.M. De Xifra E.A.W. Batlle A.M.D.C. Docampo R. Stoppani A.O.M. Comp. Biochem. Physiol. 1982; 72: 663-667Crossref Scopus (4) Google Scholar). Endosymbionts are thought to complement this incomplete pathway in their Crithidia host by supplying the missing enzymes, i.e. PBGD (5.Chang K.-P. Chang C.S. Sassa S. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2979-2983Crossref PubMed Scopus (85) Google Scholar).Table IEnzymatic defects of trypanosomatid protozoa in heme biosynthesisEnzymeFunctional statusPrevious (Ref.)Present1.ALAS:ALA synthase+(6–8)−2.ALAD:ALA dehydratase+/−(3, 5, 7)−3.PBGD:Porphobilinogen deaminase+/−(3, 5, 7)−4.UCS:Uroporphyrinogen cosynthase?−5.UROD:Uroporphyrinogen decarboxylase?−6.CPO:Coproporphyrinogen oxidase?−(?)7.PPO:Protoporphyrinogen oxidase?−(?)8.FeC:Ferrochelatase+(3, 6, 8)+ Open table in a new tab δ-aminolevulinate δ-aminolevulinate dehydratase porphobilinogen deaminase δ-aminolevulinate δ-aminolevulinate dehydratase porphobilinogen deaminase In the present studies, the heme biosynthetic pathway was found far more defective in trypanosomatids than expected as determined by genetic complementation of naturally symbiont-freeLeishmania. These organisms normally infect mammalian macrophages as intracellular parasites and acquire heme via the activity of their host cells (3.Chang C.S. Chang K.-P. Mol. Biochem. Parasitol. 1985; 16: 267-276Crossref PubMed Scopus (82) Google Scholar). We report here that transgenicLeishmania with alad and pbgd became highly porphyric when supplied with ALA, indicative of their deficiencies in δ-aminolevulinate synthase in addition to ALAD and PBGD. The production of uroporphyrin I as the sole intermediate under these conditions also indicates the deficiency of porphyrinogen-modifying enzymes further downstream of the pathway. Supplying the transfectants with exogenous ALA caused cellular accumulation of uroporphyrin I followed by its release. The infection of monocytic tumor cells in vitro with these porphyricLeishmania followed by UV irradiation resulted in their cytolysis, suggestive of its potential application for photodynamic therapy of various diseases. Wild type L. amazonensis (LV78) promastigotes (clone 12-1) were grown at 25 °C in Hepes-buffered Medium199 to pH 7.4 and supplemented with 10% heat-inactivated fetal bovine serum. Transfectants were grown under similar conditions with different concentrations of selective pressure, i.e.G418 and/or tunicamycin. Cells were also adapted to grow in a chemically defined medium (11.Steiger R.F. Steiger E. J. Parasitol. 1976; 62: 1010-1011Crossref PubMed Scopus (40) Google Scholar). To initiate such cultures, cells were washed twice with the defined medium by centrifugation at 3500 ×g for seeding at 2–5 × 106 cells/ml. Cells were counted using a hemacytometer. Macrophages (J774A1) were grown in RPMI 1640 medium-supplemented with 10 or 20% heat-inactivated fetal bovine serum at 35 °C. Cultures of all cells rendered porphyric were kept in the dark to avoid cytolysis because of photosensitivity. For all microscopic examinations of Leishmania, living cell suspension in 5–10 μl aliquots was placed on a glass slide and then covered with an 18 mm2 glass coverslip. For routine examinations, the preparations were viewed under phase contrast for cellular structures in conjunction with epifluorescence for porphyrins using a filter set consisting of D405/10X (405 nm exciter), 485DCXR (485 nm dichroic) and RG610LP (610 nm emitter) (Chroma Tech Co., Brattleboro, VT) in a Zeiss standard microscope with super pressure mercury lamp (HBO 50 W, Osram). Images were obtained by confocal microscopy using an Olympus FluoView confocal microscope equipped with a krypton/argon-mixed gas laser. Specimens were illuminated with the 488 nm excitation line. The specific fluorescent emission of the porphyrin was collected by a photomultiplier tube after passing through a 605 nm bandpass emission filter. Differential interference contrast images were simultaneously collected using a transmission field detector coupled to a photomultiplier tube. Detection settings were determined using a negative control by adjusting the gain and offset settings to eliminate background. Images were collected using a ×100 oil immersion objective (NA 1.40) with an electronic zoom of ×3. The confocal aperture was set to 5 mm to maximize the depth of field within the specimen. Digital image acquisition took ∼7 s/frame, resulting in movement-induced blurring of the flagella in viable specimens. Images were composed in Adobe Photoshop. Only differential interference contrast images were adjusted for brightness. The cDNA of rat pbgd(1038 bp) (GenBank™ accession number X06827) (12.Stubnicer A.C. Picat C. Grandchamp B. Nucleic Acids Res. 1988; 16: 3102Crossref PubMed Scopus (24) Google Scholar) was obtained by digesting the plasmids with BamHI. The humanalad (993 bp) (GenBank™ accession numberM13928) (13.Beaumont C. Porcher C. Picat C. Nordmann Y. Grandchamp B. J. Biol. Chem. 1989; 264: 14829-14834Abstract Full Text PDF PubMed Google Scholar) was PCR-amplified from a cDNA cloned in pGEM vector using a high fidelity Taq polymerase (Expand Hi Fi, Roche Molecular Biochemicals). The forward and reverse primers used were 5′-TGCCCACTGGATCCCCGCCATG-3′ and 5′-CACTGGGATCCATCATTCCTCC-3′. To facilitate cloning into the Leishmania expression vectors, the primer sequences were designed to include BamHl sites (underlined) flanking the PCR products. The amplified products of alad was first cloned in pGEM-T for expansion and then gel-purified afterBamHI digestion for cloning intoLeishmania-specific vector, pX-neo (14.Ha D.S. Schwarz J.K. Turco S.J. Beverley S.M. Mol. Biochem. Parasitol. 1996; 77: 57-64Crossref PubMed Scopus (221) Google Scholar). The ratpbgd was cloned into p6.5 withN-acetylglucosamine-1-phosphate transferase gene for tunicamycin-resistance (15.Liu X. Chang K.P. Mol. Cell. Biol. 1992; 12: 4112-4122Crossref PubMed Scopus (64) Google Scholar, 16.Kawazu S.-I. Lu H.G. Chang K.P. Gene (Amst.). 1997; 196: 49-59Crossref PubMed Scopus (11) Google Scholar, 17.Chen D.Q. Kolli B.K. Yadava N. Lu H.G. Gilman-Sachs A. Peterson D.A. Chang K.-P. Infect. Immun. 2000; 68: 80-86Crossref PubMed Scopus (85) Google Scholar). The clones with the inserts in correct orientation were identified by restriction mapping. Promastigotes were transfected with pX-alad and/or p6.5-pbgd (see Fig. 1) by electroporation as described earlier (18.McGwire B. Chang K.-P. J. Biol. Chem. 1996; 271: 7903-7909Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) and selected initially for resistance of up to 10 μg of tunicamycin/ml or 20 μg of G418/ml or a combination of both. Stable transfectants emerged in 8–10 days and were subsequently passaged continuously in media with appropriate drug pressures. Cells were harvested by centrifugation for 5 min at 3500 × g, resuspended in phosphate-buffered saline (pH 7.4), and lysed by three cycles of freezing-thawing in dry ice/acetone bath. Cell lysates equivalent to 20–50 × l06 cells and to 2–5 × 106 cells were used for ALAD and PBGD assays, respectively. The activity of ALAD was assayed by monitoring the absorption at 553 nm of the color salt of porphobilinogen using the modified Ehrlich reagent as described previously (19.Sassa S. Enzyme (Basel). 1982; 28: 133-145Crossref PubMed Scopus (380) Google Scholar). PBGD activities were assayed by a microfluorometric method (20.Sassa S. Granick S. Bickers D.R. Bradlow H.L. Kappas A. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 732-736Crossref PubMed Scopus (72) Google Scholar). Porphyrin levels were determined fluorometrically using 5 μl of cell suspensions (2–5 × 106 cells/ml) and 0.5 ml of 1 m perchloric acid/methanol (1:1, v/v) as described previously (21.Kappas A. Song C.S. Sassa S. Levere R.D. Granick S. Proc. Natl. Acad. Sci. U. S. A. 1969; 64: 557-564Crossref PubMed Scopus (14) Google Scholar). Samples were assayed for proteins using Coomassie Blue R-250 binding dye. The type of porphyrins produced was determined by thin-layer chromatography of relevant samples using porphyrin ester chromatographic marker kit as the standard (Porphyrin Products Co., Logan, UT). Cells were grown in porphyrin-free chemically defined medium to 3–4 × 108 cells. Porphyrins were extracted from the cell pellets, methylated, and analyzed by thin-layer chromatography as described previously (22.Anderson W.L. Shechter Y. Parikh I. Anal. Biochem. 1978; 91: 481-489Crossref PubMed Scopus (2) Google Scholar). Stable transfectants grown in Medium199 supplemented with heat-inactivated fetal bovine serum and selected with appropriate drugs were assessed for the presence of ALAD and PBGD by Western blot analysis. Protein samples each equivalent to 20 × l06 cells were subjected to SDS-PAGE using MiniProtean II (Bio-Rad) and blotted to nitrocellulose. The primary anti-PBGD and anti-ALAD antisera were generated by immunization of rabbits with purified enzymes (23.Sassa S. J. Exp. Med. 1976; 143: 305-315Crossref PubMed Scopus (215) Google Scholar). Both were used at 1:105dilution. Peroxidase-conjugated goat anti-rabbit IgG (Sigma) was used as the secondary antibody. Immunoblots were subsequently developed with the ECL reagent and exposed to x-ray films. For these experiments, transfectants with alad and pbgd and those with pbgdalone were grown in chemically defined medium supplemented with up to 1.6 mm ALA to generate different levels of porphyria. Cell suspensions in 24-well microtiter plates (107promastigotes/ml/well or 5 × 106 promastigotes + 5 × 105 J774A1 macrophages/ml/well) were irradiated after infection or immediately at room temperature under a long wave UV lamp (254–366 nm multi-bands, Mineralight Lamp, Model UVSL-58, Ultraviolet Products, Inc, San Gabriel, CA) placed ∼5 cm above the cell layers. Porphyric Leishmania prepared under other conditions and their spent media with different concentrations of released porphyrins were also examined for their effects on J774A1 cells. After illumination for variable time periods, cells were microscopically examined immediately. Cells of the monocytic tumor line were counted using a hemacytometer 1–2 days after irradiation. All experiments were repeated at least twice. Western blot analysis of various cell lysates revealed that both enzymes were undetectable in the wild type (Fig. 2, lane 1) and appeared as specific protein bands of the expected size (Fig. 2, lanes 2–5) in the transfectants. Probing the blots with anti-ALAD antiserum alone revealed a single band of ∼36 kDa in the transfectants with pX-alad (panel A, lane 2) and those with this plasmid in combination with p6.5-pbgd (panel A, lane 5) but not in those with p6.5-pbgd and p6.5-pbgd + pX (panel A, lanes 3 and 4). Reprobing the same blot with anti-PBGD antiserum showed that transfectants with pX-alad (panel B, lane 2), p6.5-pbgd (lane 3), and p6.5-pbgd + pX (lane 4) each contained a single band of the expected size,i.e. ∼36 or ∼42 kDa, respectively, whereas those with both genes (lane 5) contained both protein bands. The results thus indicate that both genes are expressed at the protein level individually in different transfectants and simultaneously in the same one using different vectors. Both ALAD and PBGD activities are absent in wild type cells (data not shown) and present only in transfectants with the genes of relevance (Table II). The specific activities in pmol products/mg protein/h fall within the range of ∼2500 to ∼9500 and ∼400 to ∼1400 for ALAD and PBGD, respectively. The variations in the specific activities among different experiments seen may be accounted for by differences introduced inadvertently in the culture and selective conditions used. Clearly, both enzymes are fully functional alone or in combination in the transgenic Leishmania cells.Table IIALAD- and PBGD-specific activities in Leishmania transfectantsALAD activityPBGD activityExpt. No.transfectants containing:2-1Grown to stationary phase in a defined medium and harvested for enzyme assays as described under “Experimental Procedures.” See Fig. 1 for the plasmid constructs used for the transfection.aladpbgdalad & pbgdaladpbgd2-150Transfectants with the pX vector alone in addition to p6.5-pbgd.alad & pbgdpmol PBG/mg protein/hpmol URO/mg protein/h19528081870138069829230065420985420326600391004916902-150 Transfectants with the pX vector alone in addition to p6.5-pbgd.2-1 Grown to stationary phase in a defined medium and harvested for enzyme assays as described under “Experimental Procedures.” See Fig. 1 for the plasmid constructs used for the transfection. Open table in a new tab Whereas both ALAD and PBGD were expressed and fully active in Leishmania transfected with the respective gene, the transfectants produced no detectable porphyrins (see Figs.3, lanes 3 and 6, and 4, panel N, 0 μm ALA) unless ALA was provided to those with both transgenes (Figs. 3, lanes 2 and 5, and 4,panel N, 125–1000 μm ALA). However, this porphyric Leishmania along with all other transfectants resembled nontransfected wild type cells in that they grew continuously only in the defined medium supplemented with either hemin or protoporphyrin IX (data not shown). Deletion of the heme compound from this medium resulted in the eventual cessation of their growth in all cases after several passages. Heme biosynthesis pathway thus remains incomplete in these transgenicLeishmania clearly because of additional enzymatic defect(s) downstream of PBGD.Figure 4Cellular localization and ALA dose-dependent release of porphyrin from porphyric L. amazonensis. Panels A–L, cells transfected with p6.5-pbgd/pX-alad and p6.5-pbgd/pX were examined by confocal microscopy for the presence of cellular porphyrins after exposure to 1 mm ALA for 2 days. See “Experimental Procedures” for the settings used for differential interference (DIC) (panels A, D,G, and J), porphyrin fluorescence (Porphyrin) (panels B, E,H, and K), and merged images of DIC and porphyrin (Merged) (panels C, F, I, and L). Panels A–F, cells transfected with the control P6.5-pbgd/pX; panels G–L, cells transfected with P6.5-pbgd/pX-alad. Note that the porphyrin signals diffused in the cytosol and localized more intensely to cytoplasmic vacuoles (panels H, I,K, and L) in some cells and in the cytosol as a diffused pattern (panel I) in others. Panels Mand N, transfectants with P6.5-pbgd/pX-alad (PBGD+ ALAD) and the control with P6.5-pbgd/pX (PBGD only) were exposed to 0–1 mm ALA for 4 days. The cultures in 200 μl aliquots were centrifuged to sediment cells for photography with (panel N) and without (panel M) long wave UV illumination. Note: porphyrin fluorescence appears only in the spent medium of PBGD+ALAD increasing with ALA concentrations (125–1000 μm) but not in the controls, i.e. cells with PBGD alone, PBGD+ALAD cells without ALA induction (panel N), and in the absence of UV illumination (panel M).View Large Image Figure ViewerDownload Hi-res image Download (PPT) This finding was originally suggested by the fluorescence emission spectra of porphyrins extracted from porphyricLeishmania observed (data not shown) and confirmed by thin-layer chromatography analysis of these samples (Fig. 3). Thin-layer chromatography of porphyrins extracted by standard procedures from porphyric Leishmania and their spent medium revealed only a single UV-fluorescent species (Fig. 3, lanes 2 and 5), which co-migrated with uroporphyrin I octamethyl esters (lanes 1, 4, and7). This finding indicates that only uroporphyrin I was produced by these cells. No porphyrin bands were visible in samples prepared simultaneously from controls, e.g. transfectants with one or the other gene and their culture supernatants (Fig. 3,lanes 3 and 6). The cells used for sample extraction were grown in porphyrin-free defined medium, eliminating the possibility that the porphyrin species detected may have derived from an exogenous source. The porphyrins emerged only in the double transfectants after the addition of ALA into their culture media. Porphyrin-specific signals were followed by epifluorescent microscopy and imaged by confocal fluorescent microcopy (see “Experimental Procedures” for the settings used). By differential interference contrast microscopy, living cells under all conditions used appeared granulated with anterior flagella (Fig. 4,panels A, D, G, and J). Under the settings for confocal microscopy used for porphyrin, fluorescence signals emerged only in the double transfectants (Fig. 4,panels H and K) but not in the control cells,e.g. the single transfectants with pbgd alone (panels B and E). When the two sets of images from the same preparations were merged, porphyrin fluorescent signals appeared to be diffused in the cytosol (Fig. 4, panel I) as well as localized in cytoplasmic vacuoles (panels I andL). Porphyric Leishmania released uroporphyrin I into the medium, independent of cytolysis. This was demonstrated under two different conditions to generate modest and high levels of uroporphyria. Cells were handled gently to avoid inadvertent cytolysis. The kinetics of uroporphyrin accumulation in and release from porphyric Leishmania was quantitatively assessed fluorometrically. Initially used were cells grown in a chemically defined medium with a modest selective pressure of 2 μg of tunicamycin and 10 μg of G418/ml in conjunction with increasing but low concentrations of ALA from 0 to 200 μm (Fig. 5). Under all these conditions, cells grew from 2.5 × 106 to ∼107/ml in a period of 3 days (Fig. 5, left panels), except the one with the highest ALA concentration of 200 μm in which case the cell density decreased on day 3 (Fig. 5, bottom left panel). In the absence of ALA, porphyrin was detected neither in cells nor in their spent media throughout the period of cell growth (Fig. 5,top middle and right panels). In the presence of ALA, the cells produced uroporphyrin in an ALA dose-dependent manner, namely an increase from ∼3 to ∼8 pmol uroporphyrin/106 cells in the presence of 25 to 200 μm ALA during the first day (Fig. 5, middle panels). The cellular levels of uroporphyrin declined in these cells from days 2 to 3, concomitant with its release also in an ALA dose-dependent manner from 5 to 28 pmol uroporphyrin/ml in the culture medium (Fig. 5, right panels). In a separate set of experiments, cells were grown in Medium199 plus heat-inactivated fetal bovine serum under the optimal conditions for uroporphyria, i.e. a 10-fold increase of the selective pressure (20 μg of tunicamycin and 100 μg of G418/ml) and a 5- to 8-fold increase of the substrate (up to 1.0–1.6 mm ALA provided exogenously). Under these conditions, both cellular and released uroporphyrin levels were considerably enhanced (Fig. 4,panels N, 125–1000 μ m ALA), the latter reaching a level as much as ∼2 μm. Cytolysis was observed in <1% of these cells that did not account for the level of porphyrin release seen. The results from both sets of the experiments indicate that uroporphyria is induced in an ALA dose-dependent fashion, which is marked by initial cellular accumulation of uroporphyrin followed by its release and accumulation in the culture medium. PorphyricLeishmania remained motile and thus viable under all culture and selective conditions used, except when they were subjected to UV irradiation. This sensitivity was indicated by the immediate cessation of the motility of the early porphyric cells after exposure to illumination under the setting for epifluorescent microscopy or with the long wave UV lamp. Late porphyric cells exposed to ALA 2 days or longer were less sensitive, whereas nonporphyric cells were totally insensitive to UV irradiation under these conditions as indicated by their motility. The monocytic tumor cells, J774A1, were also rendered sensitive to long wave UV irradiation after infection with porphyricLeishmania. Used for these experiments were double transfectants with both alad and pbgd and single transfectants with only pbgd grown under the same conditions. Uroporphyria was generated only in the double transfectants. The results (Fig. 6) showed that UV irradiation lysed only the macrophages infected with porphyric Leishmania and that the cytolysis was proportional to the porphyric levels of the latter modulated by prior exposure to different ALA concentrations (Fig. 6, PBGD/ALAD). The nonporphyricLeishmania produced no such effect (Fig. 6, PBGD) regardless of their exposure to ALA and UV irradiation under the same conditions. There was also no cytolysis of the tumor cells when irradiated immediately after mixing them with the porphyricLeishmania or in the presence of their spent media containing uroporphyrin I. The results obtained from these experiments were similar to the control in Fig. 6 (data not shown). In this study, both alad and pbgd from mammalian sources were successfully expressed in Leishmaniaby transfection (Fig. 1), yielding products of expected size (Fig. 2) with enzymatic activities (Table II). Significantly, the episomal transgenes in two different vectors can be selected appropriately to co-express both enzymes with activities. These activities are at least 10 times higher than those normally found in the mammalian cells,e.g. macrophages (3.Chang C.S. Chang K.-P. Mol. Biochem. Parasitol. 1985; 16: 267-276Crossref PubMed Scopus (82) Google Scholar), and more comparable to those in murine Friend virus-transformed leukemia cells induced for erythroid differentiation with a heightened level of heme biosynthesis (23.Sassa S. J. Exp. Med. 1976; 143: 305-315Crossref PubMed Scopus (215) Google S" @default.
• W1968047991 created "2016-06-24" @default.
• W1968047991 creator A5022961788 @default.
• W1968047991 creator A5028337949 @default.
• W1968047991 creator A5059871537 @default.
• W1968047991 creator A5068614860 @default.
• W1968047991 creator A5086841844 @default.
• W1968047991 creator A5087909694 @default.
• W1968047991 date "2002-04-01" @default.
• W1968047991 modified "2023-10-18" @default.
• W1968047991 title "Genetic Rescue of Leishmania Deficiency in Porphyrin Biosynthesis Creates Mutants Suitable for Analysis of Cellular Events in Uroporphyria and for Photodynamic Therapy" @default.
• W1968047991 cites W1489042738 @default.
• W1968047991 cites W1500528380 @default.
• W1968047991 cites W1528934273 @default.
• W1968047991 cites W1593538558 @default.
• W1968047991 cites W1605840656 @default.
• W1968047991 cites W1783140534 @default.
• W1968047991 cites W1838242598 @default.
• W1968047991 cites W1964730136 @default.
• W1968047991 cites W1967528101 @default.
• W1968047991 cites W1974894669 @default.
• W1968047991 cites W1983545930 @default.
• W1968047991 cites W1987877328 @default.
• W1968047991 cites W1993084091 @default.
• W1968047991 cites W1996043491 @default.
• W1968047991 cites W1997978756 @default.
• W1968047991 cites W2003360120 @default.
• W1968047991 cites W2011553235 @default.
• W1968047991 cites W2023298697 @default.
• W1968047991 cites W2023445327 @default.
• W1968047991 cites W2032259454 @default.
• W1968047991 cites W2037039906 @default.
• W1968047991 cites W2040775167 @default.
• W1968047991 cites W2042633669 @default.
• W1968047991 cites W2046935881 @default.
• W1968047991 cites W2047139085 @default.
• W1968047991 cites W2049349198 @default.
• W1968047991 cites W2049459769 @default.
• W1968047991 cites W2055226154 @default.
• W1968047991 cites W2056821345 @default.
• W1968047991 cites W2060790010 @default.
• W1968047991 cites W2067063027 @default.
• W1968047991 cites W2071760126 @default.
• W1968047991 cites W2074779130 @default.
• W1968047991 cites W2119332435 @default.
• W1968047991 cites W2128489920 @default.
• W1968047991 cites W2156414842 @default.
• W1968047991 cites W2295196541 @default.
• W1968047991 cites W2318797480 @default.
• W1968047991 cites W2400418602 @default.
• W1968047991 doi "https://doi.org/10.1074/jbc.m200107200" @default.
• W1968047991 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11836252" @default.
• W1968047991 hasPublicationYear "2002" @default.
• W1968047991 type Work @default.
• W1968047991 sameAs 1968047991 @default.
• W1968047991 citedByCount "71" @default.
• W1968047991 countsByYear W19680479912012 @default.
• W1968047991 countsByYear W19680479912013 @default.
• W1968047991 countsByYear W19680479912014 @default.
• W1968047991 countsByYear W19680479912015 @default.
• W1968047991 countsByYear W19680479912016 @default.
• W1968047991 countsByYear W19680479912017 @default.
• W1968047991 countsByYear W19680479912018 @default.
• W1968047991 countsByYear W19680479912019 @default.
• W1968047991 countsByYear W19680479912020 @default.
• W1968047991 countsByYear W19680479912021 @default.
• W1968047991 countsByYear W19680479912022 @default.
• W1968047991 crossrefType "journal-article" @default.
• W1968047991 hasAuthorship W1968047991A5022961788 @default.
• W1968047991 hasAuthorship W1968047991A5028337949 @default.
• W1968047991 hasAuthorship W1968047991A5059871537 @default.
• W1968047991 hasAuthorship W1968047991A5068614860 @default.
• W1968047991 hasAuthorship W1968047991A5086841844 @default.
• W1968047991 hasAuthorship W1968047991A5087909694 @default.
• W1968047991 hasBestOaLocation W19680479911 @default.
• W1968047991 hasConcept C104317684 @default.
• W1968047991 hasConcept C136764020 @default.
• W1968047991 hasConcept C143065580 @default.
• W1968047991 hasConcept C178790620 @default.
• W1968047991 hasConcept C185592680 @default.
• W1968047991 hasConcept C23085057 @default.
• W1968047991 hasConcept C2779480358 @default.
• W1968047991 hasConcept C2781092759 @default.
• W1968047991 hasConcept C2781323092 @default.
• W1968047991 hasConcept C41008148 @default.
• W1968047991 hasConcept C55493867 @default.
• W1968047991 hasConcept C70721500 @default.
• W1968047991 hasConcept C71928629 @default.
• W1968047991 hasConcept C86803240 @default.
• W1968047991 hasConcept C95444343 @default.
• W1968047991 hasConceptScore W1968047991C104317684 @default.
• W1968047991 hasConceptScore W1968047991C136764020 @default.
• W1968047991 hasConceptScore W1968047991C143065580 @default.
• W1968047991 hasConceptScore W1968047991C178790620 @default.
• W1968047991 hasConceptScore W1968047991C185592680 @default.
• W1968047991 hasConceptScore W1968047991C23085057 @default.
• W1968047991 hasConceptScore W1968047991C2779480358 @default.
• W1968047991 hasConceptScore W1968047991C2781092759 @default.

• next