Midpoint potentials of the mitochondrial cytochromes of Crithidia fasciculata

The midpoint potentials of the mitochondrial respiratory chain cytochromes of the protozoan Crithidia fasciculata at pH 7.2, Em7.2, show great similarity to those measured in higher organisms. Values of Em7.2 for cytochromes a and a3 are +165 and +340 mV. Both c cytochromes have Em7.2 = +230 mV. There are two b cytochromes with the same spectral characteristics with Em7.2 = -20 and -135 mV. These values are compatible with two sites of energy conservation for oxidative phosphorylation in these mitochondria. All cytochrome components show potentiometric titrations with n = 1. There is a fluorescent flavoprotein in these mitochondria with Em7.2 = -40 mV and n =2, whose function is not known.     

Citrate synthase from Crithidia fasciculata: inhibition by adenine nucleotides and suramin

Citrate synthase (EC 4.1.3.7) was purified to electrophoretic homogeneity from Crithidia fasciculata ATCC 11745. 2. The purified enzyme had an optimal pH of 8.0-8.5, apparent Km values for acetyl-CoA and oxaloacetate of 5.5 and 3.5 microM, respectively, and was not activated by NH4Cl or KCl, nor inhibited by NADH or alpha-oxoglutarate. 3. Adenine nucleotides inhibited the enzyme, ATP being the most effective. The inhibition was strictly competitive towards acetyl-CoA and of the mixed type with respect to oxaloacetate. 4. The trypanocidal drug suramin inhibited both the C. fasciculata and the pig liver citrate synthases, being strictly competitive with respect to oxaloacetate, and non-competitive towards acetyl-CoA. The competitive inhibition with respect to the divalent anion oxaloacetate might be due to the strongly anionic nature of suramin, which has six sulfonic groups in its molecule.     

A structure-specific DNA endonuclease is enriched in kinetoplasts purified from Crithidia fasciculata

The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata consists of minicircles and maxicircles topologically interlocked in a single network per cell. Individual minicircles replicate unidirectionally from either of two replication origins located 180 degrees apart on the minicircle DNA. Initiation of minicircle leading-strand synthesis involves the synthesis of an RNA primer which is removed in the last stage of replication. We report here the purification to near homogeneity of a structure-specific DNA endo-nuclease based on the RNase H activity of the enzyme on a poly(rA).poly(dT) substrate. RNase H activity gel analysis of whole cell and kinetoplast extracts shows that the enzyme is enriched in kinetoplast fractions. The DNA endonuclease activity of the enzyme is specific for DNA primers annealed to a template strand and requires an unannealed 5' tail. The enzyme cleaves 3' of the first base paired nucleotide releasing the intact tail. The purified enzyme migrates as a 32 kDa protein on SDS gels and has a Stoke's radius of 21.5 A and a sedimentation coefficient of 3.7 s, indicating that the protein is a monomer in solution with a native molecular mass of 32.4 kDa. These results suggest that the enzyme may be involved in RNA primer removal during minicircle replication.     

Entamoeba dispar: cultivation with sterilized Crithidia fasciculata

Four isolates of Entamoeba dispar identified by their hexokinase and phosphoglucomutase isoenzyme profile and by their failure to react with Entamoeba histolytica-specific monoclonal antibody (4G6) could be grown in either Diamond's BI-S-33 medium, newly developed BCSI-S (Biosate cysteine starch iron-serum) medium, or casein-free YI-S medium in the presence of Crithidia fasciculata (ReF-1:PRR) sterilized by heating 56 degrees C for 30 min and subsequent incubation with 1% hydrogen peroxide for 24 hours at 4 degrees C. After the cultures were maintained for over 50 passages, the amebae were identified as E. dispar by isoenzyme analysis, polymerase chain reaction with E. histolytica- and E. dispar-specific primers, i.e. p11 plus p12 and p13 plus p14, respectively, and by negative reactivity with monoclonal antibody 4G6. The flagellates added to the culture were judged to be metabolically inactive based on the results of nuclear magnetic resonance spectroscopy, electron microscopy, and polarographic analysis. All of these findings suggest that E. dispar can grow in vitro with metabolically inactive C. fasciculata as a culture associate.     

Sequence, heterologous expression and functional characterization of a novel tryparedoxin from Crithidia fasciculata

Tryparedoxin has recently been discovered as a constituent of the trypanosomal peroxidase system catalysing the reduction of a peroxiredoxin-type peroxidase by trypanothione [Nogoceke et al. (1997) Biol. Chem. 378, 827-836] and has attracted interest as a potential molecular target for the development of trypanocidal agents. Here we describe the first isolation of a novel gene from Crithidia fasciculata encoding a different tryparedoxin designated tryparedoxin II. The deduced amino acid sequence of tryparedoxin II (accession number AF055986) differs substantially from the partial sequence reported for the tryparedoxin described previously and now renamed tryparedoxin I. It shares the sequence motif Vx3FSAxWCPPCR shown to represent the catalytic site in tryparedoxin I [Gommel et al. (1997) Eur. J. Biochem. 248, 913-918] with mouse nucleoredoxin (accession number X92750), and a thioredoxin-like gene product of Caenorhabditis elegans (accession number U23511). Depending on which ATG is considered functional as translation start codon, tryparedoxin II, with 150 or 165 amino acid residues, is 50% larger than the typical thioredoxins. The tryparedoxins appear phylogenetically related to the thioredoxins, but sequence similarities are restricted to the active site motifs and their intimate neighbourhood. His-tagged tryparedoxin II expressed in E. coli exhibited ping-pong kinetics in the trypanothione:peroxiredoxin assay with kinetic parameters (KM peroxiredoxin = 4.2 microM, KM trypanothione = 33 microM, Vmax/[E] = 952 min(-1)) similar to those reported for tryparedoxin I [Gommel et al. (1997) Eur. J. Biochem. 248, 913-918]. The co-existence of two distinct tryparedoxins in C. fasciculata suggests diversified biological roles of this novel type of protein, which in trypanosomatids may substitute for the pleiotropic redox catalyst thioredoxin.     

Sequence analysis of the tryparedoxin peroxidase gene from Crithidia fasciculata and its functional expression in Escherichia coli

Tryparedoxin peroxidase from Crithidia fasciculata is an essential component of the trypanothione-dependent hydroperoxide metabolism in the trypanosomatids (Nogoceke, E., Gommel, D. U., Kiebeta, M., Kalisz, H. M., and Flohé, L. (1997) Biol. Chem. 378, 827-836). The tryparedoxin peroxidase gene and its flanking regions have been isolated and sequenced from a C. fasciculata genomic DNA library. It consists of an open reading frame of 564 base pairs encoding a protein of 188 amino acid residues. The gene, modified to encode 6 additional histidine residues, was expressed in Escherichia coli and the recombinant protein was purified to homogeneity by metal chelating chromatography. Recombinant tryparedoxin peroxidase has a subunit molecular mass of 21884 +/- 22 and contains two isoforms of pI 6.2 and 6.3. It exhibits a kinetic pattern identical to that of the authentic tryparedoxin peroxidase and has a similar specific activity of 2.51 units mg-1. The enzyme unequivocally belongs to the peroxiredoxin family of proteins, whose members have been found in all phyla. A phylogenetic tree comprising 47 protein and DNA sequences showed tryparedoxin peroxidase and a homologous Trypanosoma brucei sequence to form a distinct molecular clade. The consensus sequence: xnAx5-6Fx9Gx3Vx2Fx1Px2Fx1FVCPTEx21Sx1Dx7Wx16-19Dx15- 16Gx3Rx2Fx2Dx27Ax 1Qx4-11Cx1-3Wxn was demonstrated by alignment of the sequences of tryparedoxin peroxidase and 8 other peroxiredoxins with established peroxidase function.     

Cloning, expression and reconstitution of the trypanothione-dependent peroxidase system of Crithidia fasciculata

Tyrosine phosphoproteins of size 115-120 kDa were purified from membranes of chicken embryo fibroblasts (CEF) infected with Rous sarcoma virus (RSV). A mouse was immunized with these proteins, and the immune serum was used to screen a CEF cDNA expression library. A highly immunoreactive clone (KS5) was identified and characterized. The cDNA of this clone is 2.3 kb in length with a short 5' UTR and a single major open reading frame (ORF) encoding a polypeptide of 719 amino acids, with a calculated molecular weight of 81.1 kDa. The encoded protein contains an amino terminal PDZ domain, followed by a predicted coiled-coil region, a PEST domain, and a carboxy-terminal SAM domain. Consensus sequence motifs for tyrosine phosphorylation are also present, as are consensus sequences for the binding of SH2 and PDZ domains. Antisera from mice immunized with bacterially expressed fragments of the KS5 protein recognized proteins of size 230, 116, and 65 kDa in CEF. In other chicken embryo tissues, a 116-kDa species was the predominant protein recognized. The 116-kDa species is tyrosine-phosphorylated in RSV-CEF. The presence of PDZ and SAM domains in the KS5 protein suggests that it may act as a molecular adaptor, promoting and relaying information in a signal transduction pathway. It is a member of a family of related proteins, all of which have a highly conserved PDZ domain adjacent to a coiled-coil region. Two other members of this family are the neuronal proteins spinophilin (Allen, P.B., Ouimet, C.C., Greengard, P., 1997. Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines. Proc. Natl. Acad. Sci. USA 94, 9956-9961) and neurabin (Nakanishi, H., Obaishi, H., Satoh, A., Wada, M., Mandai, K., Satoh, K., Nishioka, H., Matsuura, Y., Mizoguchi, A. , Takai, Y., 1997. Neurabin: A novel neural tissue-specific actin filament-binding protein involved in neurite formation. J. Cell Biol. 139, 951-961).     

A unique cascade of oxidoreductases catalyses trypanothione-mediated peroxide metabolism in Crithidia fasciculata

Parasitic trypanosomatids comprise causative agents of debilitating or life-threatening tropical diseases. The limited capacity of these parasites to cope with oxidative stress has been discussed as a target area for therapeutic approaches but success has been hampered by a lack of comprehension of their peculiar oxidant defense system depending on the unique redox metabolite trypanothione. Here we report that trypanothione-dependent hydroperoxide metabolism in Crithidia fasciculata is catalysed by two distinct proteins working in concert. One is Cf16, a unique protein which, apart from a WCPPC sequence that resembles the thioredoxin-type WCG(A)PC motif, only shows low similarity to thioredoxin-like proteins of bacteria and invertebrates. The second component is Cf21, which can be classified as a member of the peroxiredoxin family of proteins. The two proteins have been purified to homogeneity and shown to be essential for the trypanothione-dependent removal of hydroperoxides. By means of selective derivatisation of the substrate-reduced proteins the flux of reduction equivalents from trypanothione to Cf16, Cf21 and finally to the hydroperoxide was elucidated. Cf21 proved to be a moderately efficient peroxidase with broad specificity. The rate constants for the reaction of the reduced protein with H2O2, t-butyl hydroperoxide, linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide were 1.0 x 10(5), 1.2 x 10(5), 1.0 x 10(5) and 0.4 x 10(5) M-1S-1, respectively. The apparent rate constant for the regeneration of reduced Cf21 by Cf16 was in the range of 1.5-3.5 x 10(6) M-1S-1. This newly discovered metabolic pathway adds two further candidates to the list of potential targets for trypanocidal drugs.     

Isolation and characterization of mitochondria from turkey spermatozoa

The purpose of the present investigation was to evaluate 99Tcm-labelled alpha-D-glucose 1-phosphate (GP) aerosols for single photon emission computed tomographic (SPECT) ventilation lung imaging in comparison to 99Tcm-diethylenetriaminepentaacetate (DTPA) aerosols. Ten normal nonsmoking male volunteers (aged 20-30 years) were included in this study after obtaining their informed consent. 99Tcm-GP, 30 mCi, in 2 ml was placed in the nebulizer (Venticis II) and inhalation continued for 5 min of normal breathing with oxygen flowing through. In 10 subjects dynamic images were obtained from the posterior position for 90 min with 45 frames on a 64 x 64 matrix by the use of a gamma camera. At the end of the dynamic study planar images of the lung (anterior, posterior and laterals) were recorded. Decay corrected clearance curves and kep values were obtained by the pulmonary epithelial programme and T1/2 values were calculated. The same procedure was followed by the use of 99Tcm-DTPA in the same subjects 2 weeks later. SPECT studies of the lung were performed in five subjects after inhalation of 99Tcm-GP aerosols. Clearance curves were monoexponential. The difference in T1/2 values between the right and left lungs was statistically insignificant (P > 0.10). The mean T1/2 values were 316.5 +/- 44.7 and 80.8 +/- 13.4 min for 99Tcm-GP and 99Tcm-DTPA, respectively. The difference was significant (P < 0.0005). On scintigraphic images 99Tcm-GP showed high alveolar deposition and low adhesion to major airways like 99Tcm-DTPA. However, it is preferred to 99Tcm-DTPA for SPECT studies because of its prolonged pulmonary clearance.     

Isolation and characterization of 6-hydroxymethylpterin as the Crithidia growth-promoting factor from spinach chloroplasts

2-Amino-4-hydroxypteridine compounds were isolated as the Crithidia factor from spinach chloroplasts by DEAE-Sephadex A-50, DEAE-cellulose, Sephadex G-25, and thin-layer chromatography. One of the compounds was characterized as 6-hydroxymethylpterin by gas-liquid chromatography-mass spectrometry and by comparison with authentic specimen.     

Presence of mitochondrial large ribosomal RNA outside mitochondria in germ plasm of Drosophila melanogaster

Mitochondrial large ribosomal RNA (mtlrRNA) has been identified as a cytoplasmic factor that induces pole cell formation in embryos whose ability to form a germ line has been abolished by treatment with ultraviolet light. In situ hybridization analyses reveal that mtlrRNA is enriched in germ plasm and is tightly associated with polar granules, the distinctive organelles of germ plasm, which supports the idea that mtlrRNA functions in pole cell formation. This suggests that a product from the mitochondrial genome, along with nuclear products, participates in a key event in embryonic development: determination of the germ line.     

Lability of RNA from the large cytoplasmic ribosomal subunit of the protozoon Crithidia oncopelti

Cytoplasmic ribosomalRNA extracted from Crithidia oncopelti and analysed by gel electrophoresis at 4 degrees C consisted of two components, with molecular weights (relative to E. coli rRNA) of 1-30 X 10(6) and 0-83 X 10(6) daltons, present in equimolar amounts. On heating briefly at 51 degrees C followed by rapid cooling, the 1-30 X 10(6) RNA completely dissociated into two components of molecular weights 0-70 X 10(6) and 0-56 X 10(6) (present in equimolar amounts). Fifty per cent dissociation of the molecule occurred at 28 degrees C. That the integrity of the RNA molecule at low temperatures is maintained by its secondary structure was confirmed by electrophoresis under denaturing conditions (98%, v/v, formamide). To account for these phenomena, latent cleavage of the molecule in vivo is proposed.     

Further evidence for the presence of mitochondrially encoded subunits in cytochrome c oxidase of the trypanosomatid Crithidia fasciculata

Trimethylaminuria is an autosomal recessive human disorder affecting a small part of the population as an inherited polymorphism. Individuals diagnosed with trimethylaminuria excrete relatively large amounts of trimethylamine in their urine, sweat, and breath, and this results in a fishy odor characteristic of trimethylamine. Activity of the human flavin-containing monooxygenase (FMO) has been proposed to be deficient in trimethylaminuria patients causing a decrease in the metabolism of trimethylamine that results in a fishy body odor. Cohorts of Australian, American, and British individuals suffering from trimethylaminuria have been identified. The human FMO3 cDNA was amplified from lymphocytes of affected patients. We report preliminary evidence of substitutions detected by screening of the cDNA and genomic DNA. The variant human FMO3 cDNA was constructed from wild type human FMO3 cDNA by site-directed mutagenesis as maltose-binding protein fusions. Five distinct human FMO3 mutants were expressed as fusion proteins in Escherichia coli and compared with wild type human FMO3 maltose-binding proteins (FMO3-MBP) for the N-oxygenation of 10-[(N,N-dimethylamino)pentyl]-2-(trifluoromethyl)phenothiazine, tyramine, and trimethylamine. Human Lys158 FMO3-MBP and, to a greater extent, human Glu158 FMO3-MBP efficiently N-oxygenated the three amine substrates. Human Lys158 Ile66 FMO3-MBP, Glu158 Ile66 FMO3-MBP, Lys158 Leu153 FMO3-MBP, and Glu158 Leu153 FMO3-MBP were all constructed as mutants identified as possible FMO3 variants responsible for trimethylaminuria and were found to be inactive as N-oxygenases. The results suggest that mutations at codons 66 and 153 of FMO3 can cause trimethylaminuria in humans. We observed a common polymorphism of Lys to Glu at codon 158 of FMO3 that segregated with almost equal allele frequencies in a number of control Australian and North American samples studied. The Lys158 to Glu158 human FMO3 polymorphism does not decrease trimethylamine N-oxygenation for the cDNA-expressed enzyme and thus does not appear to be causative of trimethyaminuria. The data show that the functional activity of human FMO3 can be significantly altered by amino acid changes that have been observed in individuals with clinically diagnosed trimethylaminuria.     

Isolation and translation of non-crystallin messenger RNA from calf lens

Affinith chromatography of lens polyribosomal RNA on oligo(dT)-cellulose yields three fractions. As arule the second fraction has been neglected in other studies reported in the literature. According to our investigations this fraction in particular contains the messengers for the non-crystallin lens proteins.     

Isolation of polyadenylated RNA from cultured cells and intact tissues

Burns in children carry many age-related problems. Maintenance of immobility and cleanness of absorbent dressings are very difficult, especially in infancy and large wounds. These problems are very important when meshed skin grafts are used. Graft survival is disturbed by patient movements and septic colonization, especially in the early postoperative period. Over a two years period we have been applying Nobecutan as meshed skin graft fixation procedure, antiseptic and protecting wound coat. The use of Nobecutan is a time-saving technique. No sutures nor staples are employed, which is very important when working with burned children. No local adverse effects, nor prolongation of wound healing had been observed.     

Isolation and characterization of a multienzyme complex containing DNA replicative enzymes from mitochondria of S. cerevisiae. Multienzyme complex from yeast mitochondria

A 40 S multienzyme complex containing mtDNA polymerase was isolated from mitochondria of S. cerevisiae by density gradient centrifugation and by gel filtration chromatography. Besides DNA polymerase, RNA polymerase, primase, 3'-->5' exonuclease and an ATPase activities were found to be associated with it. The presence of some of these enzymes were confirmed by Western blot. This high molecular weight multienzyme complex containing DNA has most of the attributes of a putative replisome.