Effects of acriflavine on the mitochondria and kinetoplast of Crithidia fasciculata. Correlation of fine structure changes with decreased mitochondrial enzyme activity.

The effects of acriflavine on the fine structure and function of the mitochondria and the kinetoplast in Crithidia fasciculata have been investigated. A mitochondrial fraction was prepared by differential centrifugation of cells broken by grinding with neutral alumina. Isolated mitochondria or intact cells revealed by spectrophotometric measurements the presence of cytochromes a + a(3), b, c(555) and o. After cells were grown in acriflavine for 3-4 days, the fine structure of the mitochondria and their cytochrome content were affected. Cells grown in 5.0 microM acriflavine had a threefold decrease in cytochrome a + a(3) and decreased respiratory activity. The mitochondrial preparation from these cells had a fivefold decrease in cytochrome a + a(3) and a less but significant decrease of other cytochromes present. There was also a decrease in the mitochondrial enzyme activities of NADH, succinic and L-alpha-glycerophosphate oxidases, and succinic and L-alpha-glycerophosphate dehydrogenases. Dyskinetoplastic cells could be demonstrated after growth in 1.0 microM acriflavine. At 5 microM, 80-90% of the cells were dyskinetoplastic. The kinetoplastic DNA was condensed, nonfibrillar, and did not incorporate thymidine-(3)H. The mitochondria in these cells had few cristae and were shorter and more swollen than the controls. Acriflavine may induce the fine structure effects we have observed and may affect the formation of the mitochondria in C. fasciculata.

Growth of infective forms of Trypanosoma rhodesiense in vitro, the causative agent of African trypanosomiasis.

A new approach to the culture of African trypanosomes led to the growth of the infective forms of the causative agent of human African trypanosomiasis. Infective cultures of Trypanosoma rhodesiense were initiated and maintained in vitro on Chinese hamster lung cells. By changing daily one-third of the Hepes-buffered RPMI 1640 medium containing 20 percent fetal bovine serum, the trypanosome numbers increased to 3 X 10(6) to 5 X 10(6) cells per milliliter. After 80 days in vitro at 37 degrees C, the cultured trypomastigotes are infective for mice and rats and morphologically similar to bloodstream trypomastigotes in having a subterminal kinetoplast and a surface coat. In addition, they possess L-alpha-glycerophosphate oxidase, the predominant steady-state terminal oxidase of bloodstream trypomastigotes.

Characterization of a novel developmentally regulated gene from Trypanosoma brucei encoding a potential phosphoprotein.

We have isolated a cDNA clone corresponding to a single-copy nuclear gene that is upregulated at the mRNA level during in vitro differentiation of bloodstream trypomastigotes of strains of both Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense to procyclic forms. Transcript levels begin to increase within minutes of introduction of bloodstream forms into culture and peak well before cultures exhibit a procyclic morphology. This increase in transcript levels was found to occur both in the absence of protein synthesis and in a nontransforming strain blocked very early in the developmental program, both conditions under which accumulation of procyclic acidic repetitive protein (PARP) transcripts did not occur in control experiments. DNA sequence analysis reveals an open reading frame sufficient to encode a protein of approximately 50 kDa within the cDNA, but data base searches for homology at either the amino acid or nucleotide level revealed no related sequences. A high density of kinase consensus target sites in the deduced amino acid sequence suggests that the gene product may be a phosphoprotein.

Biochemical and molecular properties of the Trypanosoma brucei alternative oxidase.

The protozoal parasite Trypanosoma brucei depends on a mitochondrial non-cytochrome terminal oxidase known as the trypanosome alternative oxidase (TAO) in its mammalian host. We have recently cloned the cDNA from T. brucei bloodstream form and have characterized a 33 kDa mitochondrial protein as TAO. Here we report that the TAO is a single copy gene in T. brucei and its expression is down regulated at the level of transcript abundance during differentiation from the bloodstream to the procyclic trypanosomes. Like other alternative oxidases (AOXs) cloned from different plants and fungi, TAO possesses the conserved sequences at the centrally located predicted membrane spanning domains and the signature sequence at the C-terminal hydrophilic domain for a pair of putative iron binding motifs (E-X-X-H). Phylogenetic analysis of the deduced protein sequences of eight different alternative oxidases cloned from different plants and fungi revealed that TAO is more closely related to the alternative oxidases of the fungi clade than that of plants. TAO has been functionally expressed in Escherichia coli. In the first of the two putative iron binding motifs, site-directed mutagenesis of E215 to A, L, N and Q resulted in the loss of the ability of the TAO gene to complement the heme deficiency of the E. coli mutants (SASX41B and GE1387) by conferring on them a CN-insensitive pathway of respiration. The conservative substitution of E215 by aspartate and histidine reduced the growth of the E. coli auxotrophs by approximately 80%. The mutations apparently did not have any effect on the stability of the expressed protein as revealed by the immunoblot analysis of the bacterial protein using TAO monoclonal antibody, which we have developed. Together, these points suggest that E215 plays an important role in the function of TAO. The steady state level of TAO mRNA is down-regulated in the procyclic stage presumably accounting for the low levels of TAO protein in these forms.

Evidence for a branched electron transport chain in Trypanosoma brucei.

The flow of electrons the terminal oxidases present in the bloodstream and procyclic trypomastigotes of Trypanosoma brucei LUMP 1026 has been investigated by the use of salicylhydroxamic acid (SHAM) and cyanide. Respiration in bloodstream trypomastigotes was completely inhibited by 0.5 mM SHAM with a Ki below 10 microM. The Ki for SHAM in procyclic trypomastigotes was 70 microM. In procyclic trypomastigotes there are at least three terminal oxidases of which the two major ones are cytochrome aa3 oxidase, sensitive to cyanide inhibition, and alpha-glycerophosphate oxidase (GPO), sensitive to SHAM inhibition. These two oxidases contribute 60 and 30%, respectively, to total cell respiration. Inhibition of the cytochrome system with cyanide causes an increase in the flow of electrons through the GPO system, and inhibition of the GPO system with SHAM stimulates electron flow in the cytochrome system. Succinate oxidation in the mitochondrial fraction is partially inhibited by SHAM and this SHAM-sensitive respiration is not inhibited by antimycin A. The kinetic data of respiration by procyclic trypomastigotes fit a model proposed by Bahr and Bonner to determine the maximum rates of two competing electron transport pathways. It is concluded that the electron transport chain in T. brucei is branched.

Inhibitory effects of chloramphenicol isomers and other antibiotics on protein synthesis and respiration in procyclic Trypanosoma brucei brucei.

The effects of various antibiotics on protein synthesis systems in procyclic trypomastigotes of Trypanosoma brucei brucei LUMP 1026 have been determined in an attempt to identify a mitochondrial translation system. The function of cytoplasmic ribosomes in T. b. brucei in vivo is sensitive to inhibition not only by cycloheximide but also by high concentrations of D-chloramphenicol, L-chloramphenicol, erythromycin and tetracycline as a possible secondary consequence of a primary inhibition of mitochondrial respiration in vivo by these latter four compounds. In support of this conclusion, antimycin A inhibits mitochondrial respiration and, secondarily, cytoplasmic protein synthesis in vivo, suggesting that mitochondrial phosphorylation is necessary for cytoplasmic translation in T. b. brucei. Tetracycline inhibition of cytoplasmic protein synthesis in vivo may also be due to a direct effect on cytoplasmic ribosomes, since this drug inhibits ribosome function in vivo. Low concentrations of D-chloramphenicol, erythromycin and tetracycline do not inhibit cycloheximide-insensitive protein synthesis in vivo, implying the presence of permeability barriers to these drugs in T. b. brucei. Mitochondrial fractions isolated from T. b. brucei possess a ribosomal translation system, the function of which is insensitive to cycloheximide but sensitive to inhibition by low concentrations of D-chloramphenicol should only be used with caution in studies of mitochondrial biogenesis in T. b. brucei.

The maxicircle of Trypanosoma brucei kinetoplast DNA hybridizes with a mitochondrial gene encoding cytochrome oxidase subunit II.

A restriction endonuclease fragment of the maxicircle of Trypanosoma brucei brucei kinetoplast DNA hybridizes with a cloned mitochondrial DNA sequence which encodes cytochrome oxidase subunit II of Zea mays. A cloned mitochondrial DNA sequence encoding cytochrome oxidase subunit II of Saccharomyces cerevisiae also hybridized with kDNA, but exhibits less homology with the maxicircle than does the maize gene. The hybridizing maxicircle DNA was localized to a 2.8 kbp segment which is bounded by TaqI restriction endonuclease sites and nearby HindIII and EcoRI restriction sites. The TaqI restriction fragment is conserved between T. brucei brucei, T. brucei rhodesiense and T. brucei gambiense and hybridizes with the Zea mays probe in each case.

The maxicircle of Trypanosoma brucei kinetoplast DNA encodes apocytochrome b.

The region of the maxicircle of Trypanosoma brucei kinetoplast DNA which hybridizes at low stringency with the apocytochrome b gene of Saccharomyces cerevisiae has been identified and cloned. The nucleotide sequence of a 1.7 kb segment of this region is reported. This segment contains a single long open reading from capable of coding for a 350 amino acid protein with substantial homology to apocytochromes b of other species. The trypanosome protein is considerably more distantly related to other apocytochromes b than they are to each other. Several short unassigned open reading frames (300 nucleotides or shorter) also are described. If polypeptides are synthesized from these regions, they are more hydrophilic than known mitochondrially coded proteins.

Computer simulation of the binding of saframycin A to d (GATGCATC)2.

The binding of Saframycin A to the octanucleotide duplex d(GATGCATC)2 was investigated using molecular dynamics. For covalent binding at N2 of the central guanine, only the R configuration at the alkylating carbon (C7) was permitted for B DNA and the 3' direction in the minor groove was preferred by 50.6 kcal/mol. The dihydroquinone form of saframycin A gave stronger binding than the quinone, in agreement with the literature. Addition of solvent and counterions made no significant change in the geometry model. The proposed mechanism of DNA alkylation, involving iminium ion intermediates from the dihydroquinone or quinone, was investigated by modeling these species. They gave models with good net binding enthalpies, and C7 was in close proximity to N2 of guanine. The noncovalent binding of saframycin A and its dihydroquinone in the vicinity of guanine also was favorable in the 3' direction.

Template-directed design of a DNA-DNA cross-linker based upon a bis-tomaymycin-duplex adduct.

A template-directed approach to the design of a DNA-DNA interstrand cross-linker based upon the structure of a bis-tomaymycin-duplex adduct has been carried out. Tomaymycin is a member of the pyrrolo[1,4]benzodiazepines antitumor antibiotics. In a previous study (F.L. Boyd et al., Biochemistry 1990, 29, 2387-2403), we have shown that two tomaymycin molecules can be covalently bound to a 12-mer duplex molecule, where the drug molecules are on opposite strands six base-pairs apart, and the stereochemistry at the drug bonding site, and orientation in the minor groove, was defined by high-field NMR. This bis-tomaymycin 12-mer duplex adduct maintains the self-complementarity of the duplex and a B-type structure. In the present study we have shown using high-field NMR that this same 12-mer sequence can be truncated by two base pairs so that the two tomaymycin-modified guanines are now only four base-pairs apart, the two species of tomaymycin molecules are still bound with the same stereochemistry and orientation, and the 10-mer duplex adduct maintains its self-complementarity. In a second 10-mer duplex we have shown that changing the bonding sequence from 5'CGA to 5'AGC does not significantly affect the structure of the bis-tomaymycin-duplex adduct. However, when the sequence is rearranged so that the drugs point in a tail-to-tail orientation rather than in the previous head-to-head configuration, there are more than one species of tomaymycin bound to DNA, and, as a consequence, the bis-tomaymycin 10-mer duplex adduct loses its self-complementarity. Last, we have used the 10-mer duplex containing the 5'CGA sequence, in which the tomaymycin molecules are oriented head to head, to design an interstrand cross-linking species in which the two drug molecules are linked together with a flexible linker molecule.

Computer simulation of the binding of naphthyridinomycin and cyanocycline A to DNA.

Cyanocycline A was found to have a pKa of 6.6. Protonation of N14 was established by 1H NMR spectroscopy. In strongly acidic solution the oxazolidine ring opened irreversibly. A model was derived for the binding of naphthyridinomycin and cyanocycline A to the hexanucleotide duplex d(ATGCAT)2, by using the molecular mechanics and dynamics modules of AMBER 3.0. It involved protonation on the oxazolidine-ring nitrogen, reduction of the quinone ring to a hydroquinone, formation of an iminium ion with loss of the C7 substituent, noncovalent binding in the minor groove with the hydroquinone ring in the 3'-direction from guanine, and covalent binding to the 2-amino group of this guanine with C7 adopting the R configuration. This model is consistent with the experimental evidence on the DNA binding of these drugs. An alternative binding mode based on opening of the oxazolidine ring and alkylation at C3a also was feasible according to molecular mechanics calculations. The geometry of naphthyridinomycin does not permit interstrand cross-linking involving both C3a and C7, but formation of a cross-link to protein appears possible. When the covalent naphthyridinomycin-d(ATGCAT)2 models were refined in the presence of water and counterions, the models with the most favorable net binding enthalpies were the same as those produced by simulation in vacuum. Qualitative estimates of the relative entropy changes resulting from adduct formation were based on the number of ordered (hydrogen bonded) water molecules released from d(ATGCAT)2 and from the drug. In all cases but one, d(ATGCAT)2 loses five water molecules. It loses six in the C3a covalent model with 5',S geometry. Naphthyridinomycin hydroquinone loses up to two water molecules, depending on the particular adduct. The 3',R model was again favored for the C7 covalent adduct. Among the C3a covalent models, the one with 5',R geometry lost the second most water molecules, but it had the best binding enthalpy.

DNA binding by antitumor anthracene derivatives.

The relative DNA binding strengths of bisantrene and nine new analogues were measured by spectrophotometric titration and melt transition temperature (Tm) techniques. Data from the spectrophotometric titrations could not be fit by simple Scatchard plots. However, they were fit by a McGhee-von Hippel equation over part of the binding range. The entire range of data was fit by a smoothing cubic spline function. The first derivative of this function gave, for each compound, a curve whose intercept provided a measure of relative binding strength. The delta Tm values agreed qualitatively with the spectrophotometric titration results, although there was not a precise linear relationship. Determinations of macroscopic pKas revealed that most of the compounds were dications at pH 7.0, but a few were mixtures of monocations and dications. No correlation was found between these binding studies and antitumor potencies in a clonogenic assay, which suggests that factors other than DNA binding can determine cytotoxicity for some of the analogues.

Growth of infective forms of Trypanosoma (T.) brucei on buffalo lung and Chinese hamster lung tissue culture cells.

Infective cultures of Trypanosoma (T.) brucei (strain 427) have been initiated and maintained on Chinese hamster lung tissue culture cells and buffalo lung tissue culture cells. By changing daily one-third of the RPMI-1640 plus 20% fetal bovine serum medium, the cell numbers can be maintained at 2--4 X 10(6) cells/ml. The cultured trypanosomes on these two tissue culture cell types were infective to mice and morphologically similar to bloodstream slender trypomastigotes in having a subterminal kinetoplast and a surface coat. In addition, they possessed the L-alpha-glycerophosphate oxidase, the predominant steady state terminal oxidase identified in bloodstream trypomastigotes.

Pentamidine congeners. 3. Crystal structure and molecular modeling studies of trans-1,4-bis(4-amidinophenoxy)-2-butene.

X-ray diffraction was used to confirm the geometry of trans-1,4-bis(4-amidinophenoxy)-2-butene dihydrochloride dihydrate (trans-butenamidine). trans-Butenamidine is a semirigid analogue of pentamidine that has demonstrated good anti-Pneumocystis carinii activity in rats. Molecular modeling studies revealed that unlike pentamidine or propamidine, trans-butenamidine does not discriminate between AT and TA sequences in its binding to the minor groove of DNA. Crystal data: [C18H22N4O2(2+)][Cl(-)]2[H2O]2, triclinic space group, P1, a = 9.443(1) A, b = 11.400(1) A, c = 11.919(1) A, alpha = 62.19(1) degree, beta = 81.10(1) degree, gamma = 72.19(1) degree, V = 1080.3(3) A3, Z = 2, R = 0.054 for 1149 observed reflections with I > 3 sigma (1).

Epidermoid carcinoma of the nasal vestibule: current treatment evaluation.

Twelve cases of epidermoid carcinoma of the nasal vestibule with a median follow-up of three years were reviewed to evaluate effectiveness of current treatment modalities. Patients were initially treated by surgery, irradiation, or a combination of both therapies. The authors conclude 1. epidermoid carcinoma of the nasal vestibule is an aggressive disease; 2. once metastases have occurred, they are poorly controlled by surgery and/or irradiation; and 3. initial therapy should be directed to both control of the primary and prevention of distant metastases.

A comparison of the amino acid sequences of Crithidia fasciculata and Trypanosoma rhodesiense cytochromes c.

Apocytochrome c was isolated from procyclic trypomastigotes of Trypanosoma rhodesiense EATRO 1895 and purified on Amberlite IRC-50 ion-exchange resin. Tryptic peptides were generated from the purified apoprotein and a partial amino acid sequence was determined. A comparison of the amino acid sequence of Crithidia fasciculata with the partial amino acid sequence of T. rhodesiense reveals significant homology.

The drug insert and the physician's concern.

The package insert has achieved an increasingly important role, not only as a device to communicate prescribing information to physicians, but also as a key element in the standards used to determine the liability of physicians for adverse drug reactions. Regulations recently proposed by the FDA illustrate the significance of the package insert from the governmental perspective. This paper deals with the impact of these regulations on the medical profession.