Rapid in vivo inactivation by acivicin of CTP synthetase,carbamoyl-phosphate synthetase II,and amidophosphoribosyltransferase in hepatoma

A single injection of the anti-glutamine drug, acivicin (NSC 163501), in tumor-bearing rats in 30 min decreased the activities of amidophosphoribosyltransferase, carbamoyl-phosphate synthetase II and CTP synthetase to 56, 50 and 7% of those of the controls. By 1 hr the activities were down to 32, 13 and 3% and they remained low for 12 hr, after which they slowly returned towards normal range in 72 hr. The decline of the activity of CTP synthetase (a loss of 80% in 10 min) was the most rapid, and the activity only returned to 60% of the controls by 3 days after the acivicin injection. In the hepatoma the concentrations of ATP and UTP changed little, but those of GTP and CTP rapidly decreased, reaching at the lowest point 32 and 2%, respectively, of control values 2 hr after acivicin; concentrations started to rise at 12 hr, reaching normal levels by 48 hr. The drop in enzyme activities preceded the decline in the pools of GTP and CTP. The behavior of enzyme activities and nucleotide concentrations in the host liver had a pattern similar to that in the hepatoma; however, the changes were less extensive than those in the tumor. The differential response between tumor and liver is attributed, in part at least, to the tissue L-glutamine concentration which in the hepatoma (0.5 mM) was 9 times lower than in the liver (4.5 mM). The selectivity of acivicin action in inhibiting glutamine-utilizing enzymes is also demonstrated by the lack of effect on aspartate carbamoyltransferase, a an enzymic activity which resides in the same complex as that of carbamoyl-phosphate synthetase II. The rapid decline in the activities of glutamine-utilizing enzymes is attributed to an in-activation of the enzymes by acivicin which functions as an active site-directed affinity analog of L-glutamine. The rapid modulation of the enzymic phenotype and ribonucleotide concentrations by acivicin provides a useful tool for elucidating the role of enzymic and nucleotide imbalance in the commitment of cancer cells to replication and in the targeting of anticancer chemotherapy.     

The in vitro and in vivo effects of mefloquine on Trypanosoma brucei brucei.

Blood stream forms of Trypanosoma brucei brucei were grown over mouse kidney (MK) cells in minimum essential medium with various concentrations of mefloquine. The drug was observed to inhibit multiplication of the parasites in vitro. Groups of male albino mice were treated with mefloquine at 24, 48 and hours after T. b. brucei infection. Mefloquine at 0.03 mg/kg body weight administered for 4 consecutive days cleared the infection. No trypanosomes were detected in the blood of these mice for 90 days and over after the clearance of parasite from the blood. The doses for both the in vitro and in vivo therapy, were well below those prescribed for humans.     

Nitric oxide-mediated cytostatic activity on Trypanosoma brucei gambiense and Trypanosoma brucei brucei.

Macrophages collected from BCG-infected mice or exposed in vitro to interferon-gamma plus lipopolysaccharide developed a cytostatic activity on Trypanosoma brucei gambiense and Trypanosoma brucei brucei. This trypanostatic activity of activated macrophages was inhibited by addition of N-monomethyl-L-arginine, an inhibitor of the L-arginine-nitric oxide (NO) metabolic pathway, indicating a role for NO as the effector molecule. Contrary to trypanosomes treated with N2gas, trypanosomes treated with NO gas did not proliferate in vitro on normal macrophages. Compared to mice infected with control parasites, mice infected with NO-treated parasites had decreased parasitemias in the first days postinfection and had a prolonged survival. Addition of excess iron reversed the trypanostatic effect of both activated macrophages and NO gas. These data show that activated macrophages exert an antimicrobial effect on T.b. gambiense and T.b. brucei through the L-arginine-NO metabolic pathway. In trypanosomes, NO could trigger iron loss from critical targets involved in parasite division. The participation of this effector mechanism among the other immune elements involved in the control of African trypanosomes (antibodies, complement, phagocytic events) remains to be defined.     

Kinetics of S-adenosylmethionine cellular transport and protein methylation in Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

African trypanosomes of the Trypanosoma brucei group are agents of disease in man and animals. They present unique biochemical characteristics such as the need for preformed purines and have extensive salvage mechanisms for nucleoside recovery. In this regard we have shown that trypanosomes have a dedicated transporter for S-adenosylmethionine (AdoMet), a key metabolite in transmethylation reactions and polyamine synthesis. In this study we compared the apparent kinetics of AdoMet transport, cytosolic AdoMet pool formation, and utilization of AdoMet in protein methylation reactions using two isolates: Trypanosoma brucei brucei, a veterinary parasite, and Trypanosoma brucei rhodesiense, a human pathogen that is highly refractory and has greatly reduced susceptibility to standard trypanocidal agents active against T. b. brucei. The apparent Km values for [methyl-3H]AdoMet transport, derived by Hanes-Woolf analysis, for T. b. brucei was 4.2 and 10 mM for T. b. rhodesiense, and the Vmax values were 124 and 400 micromol/liter/min, respectively. Both strains formed substantial cytosolic pools of AdoMet, 1600 nmol/10(9) T. b. brucei and 3500 nmol/10(9) T. b. rhodesiense after 10 min incubation with 25 mM exogenous AdoMet. Data obtained from washed trichloroacetic acid precipitates of cells incubated with [methyl-3H]AdoMet indicated that the rate of protein methylation in T. b. brucei was fourfold greater than in T. b. rhodesiense. These results demonstrate that the unique rapid uptake and utilization of AdoMet by African trypanosomes is an important consideration in the design and development of new agents of potential use in chemotherapy.     

Experimental therapy of African trypanosomiasis with a nanobody-conjugated human trypanolytic factor

High systemic drug toxicity and increasing prevalence of drug resistance hampers efficient treatment of human African trypanosomiasis (HAT). Hence, development of new highly specific trypanocidal drugs is necessary. Normal human serum (NHS) contains apolipoprotein L-I (apoL-I), which lyses African trypanosomes except resistant forms such as Trypanosoma brucei rhodesiense. T. b. rhodesiense expresses the apoL-I-neutralizing serum resistance-associated (SRA) protein, endowing this parasite with the ability to infect humans and cause HAT. A truncated apoL-I (Tr-apoL-I) has been engineered by deleting its SRA-interacting domain, which makes it lytic for T. b. rhodesiense. Here, we conjugated Tr-apoL-I with a single-domain antibody (nanobody) that efficiently targets conserved cryptic epitopes of the variant surface glycoprotein (VSG) of trypanosomes to generate a new manmade type of immunotoxin with potential for trypanosomiasis therapy. Treatment with this engineered conjugate resulted in clear curative and alleviating effects on acute and chronic infections of mice with both NHS-resistant and NHS-sensitive trypanosomes.     

In situ regulation of mammalian CTP synthetase by allosteric inhibition

The regulatory role of the allosteric site of CTP synthetase on flux through the enzyme in situ and on pyrimidine nucleotide triphosphate (NTP) pool balance was investigated using a mutant mouse T lymphoblast (S49) cell line which contains a CTP synthetase refractory to complete inhibition by CTP. Measurements of [3H]uridine incorporation into cellular pyrimidine NTP pools as a function of time indicated that CTP synthesis in intact wild type cells was markedly inhibited in a cooperative fashion by small increases in CTP pools, whereas flux across the enzyme in mutant cells was much less affected by changes in CTP levels. The cooperativity of the allosteric inhibition of the enzyme was greater in situ than in vitro. Exogenous manipulation of levels of GTP, an activator of the enzyme, indicated that GTP had a moderate effect on enzyme activity in situ, and changes in pools of ATP, a substrate of the enzyme, had small effects on CTP synthetase activity. The consequences of incubation with actinomycin D, cycloheximide, dibutyryl cyclic AMP, and 6-azauridine on the flux across CTP synthetase and on NTP pools differed considerably between wild type and mutant cells. Under conditions of growth arrest, an intact binding site for CTP on CTP synthetase was required to maintain a balance between the CTP and UTP pools in wild type cells. Moreover, wild type cells failed to incorporate H14CO3- into pyrimidine pools following growth arrest. In contrast, mutant cells incorporated the radiolabel at a high rate indicating loss of a regulatory function. These results indicated that uridine nucleotides are important regulators of pyrimidine nucleotide synthesis in mouse S49 cells, and CTP regulates the balance between UTP and CTP pools.     

Crystal structures of CTP synthetase reveal ATP, UTP, and glutamine binding sites

CTP synthetase (CTPs) catalyzes the last step in CTP biosynthesis, in which ammonia generated at the glutaminase domain reacts with the ATP-phosphorylated UTP at the synthetase domain to give CTP. Glutamine hydrolysis is active in the presence of ATP and UTP and is stimulated by the addition of GTP. We report the crystal structures of Thermus thermophilus HB8 CTPs alone, CTPs with 3SO4(2-), and CTPs with glutamine. The enzyme is folded into a homotetramer with a cross-shaped structure. Based on the binding mode of sulfate anions to the synthetase site, ATP and UTP are computer modeled into CTPs with a geometry favorable for the reaction. Glutamine bound to the glutaminase domain is situated next to the triad of Glu-His-Cys as a catalyst and a water molecule. Structural information provides an insight into the conformational changes associated with the binding of ATP and UTP and the formation of the GTP binding site.     

Substrate specificity of CTP synthetase from Escherichia coli

The stoichiometry of the enzymatic reaction catalyzed by CTP synthetase from Escherichia coli was analyzed by high-performance liquid chromatography. The results revealed that for every mole of UTP transformed to CTP, one mole of ATP was converted to ADP. The substrate specificity of CTP synthetase from E. coli was investigated by means of UTP analogs. Chemical modification of UTP involved either the uracil, ribose or 5'-triphosphate part. None of the UTP analogs studied proved to be a substrate. The capacity of the UTP analogs to inhibit CTP synthetase was investigated. From the UTP derivatives employed only 2-thiouridine 5'-triphosphate was found to inhibit the enzyme competitively with reasonable affinity: Ki/Km(UTP) = 1. This study indicated that the three main structural elements of the UTP molecule: uracil, ribose and 5'-triphosphate moiety, contribute to substrate specificity. The behaviour of a limited number of CTP analogs as product-like inhibitors supported this view.     

The flagellum of Trypanosoma brucei: new tricks from an old dog

African trypanosomes, i.e. Trypanosoma brucei and related sub-species, are devastating human and animal pathogens that cause significant human mortality and limit sustained economic development in sub-Saharan Africa. T. brucei is a highly motile protozoan parasite and coordinated motility is central to both disease pathogenesis in the mammalian host and parasite development in the tsetse fly vector. Therefore, understanding unique aspects of the T. brucei flagellum may uncover novel targets for therapeutic intervention in African sleeping sickness. Moreover, studies of conserved features of the T. brucei flagellum are directly relevant to understanding fundamental aspects of flagellum and cilium function in other eukaryotes, making T. brucei an important model system. The T. brucei flagellum contains a canonical 9+2 axoneme, together with additional features that are unique to kinetoplastids and a few closely-related organisms. Until recently, much of our knowledge of the structure and function of the trypanosome flagellum was based on analogy and inference from other organisms. There has been an explosion in functional studies in T. brucei in recent years, revealing conserved as well as novel and unexpected structural and functional features of the flagellum. Most notably, the flagellum has been found to be an essential organelle, with critical roles in parasite motility, morphogenesis, cell division and immune evasion. This review highlights recent discoveries on the T. brucei flagellum.     

Development of metacyclic forms of Trypanosoma brucei sspp. in cultures containing explants of Phormia regina Meigen

When procyclic trypanosomes of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense were cultivated in Nunclon 25 cm2 flasks at 27 C in a liquid medium containing various tissue explants of Phormia regina Meigen, some of them developed into forms infective for mice. The infective stages were present at various periods of up to 29 days when the cultures were terminated. Larger numbers of explants of head-salivary glands than the other tissues used were required to produce infections. Infectivity titrations on trypanosome suspensions of T. b. brucei TRUM 252 and T. b. rhodesiense TRUM 497 indicated that only a small proportion of the populations was infective. Mice were rarely infected with trypanosomes grown in medium without explants. Only 1 mouse of the 11 inoculated developed a parasitemia from a control culture of T. b. rhodesiense TRUM 545. A few trypanosomes resembling epimastigotes and metacyclic forms were seen in stained samples of infective inocula.     

Identification of Ser424 as the protein kinase A phosphorylation site in CTP synthetase from Saccharomyces cerevisiae.

The URA7-encoded CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] in the yeast Saccharomyces cerevisiae is phosphorylated on a serine residue and stimulated by cAMP-dependent protein kinase (protein kinase A) in vitro. In vivo, the phosphorylation of CTP synthetase is mediated by the RAS/cAMP pathway. In this work, we examined the hypothesis that amino acid residue Ser424 contained in a protein kinase A sequence motif in the URA7-encoded CTP synthetase is the target site for protein kinase A. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing the protein kinase A motif was a substrate (Km = 30 microM) for protein kinase A. This peptide also inhibited (IC50 = 45 microM) the phosphorylation of purified wild-type CTP synthetase by protein kinase A. CTP synthetase with a Ser424 --> Ala (S424A) mutation was constructed by site-directed mutagenesis. The mutated enzyme was not phosphorylated in response to the activation of protein kinase A activity in vivo. Purified S424A mutant CTP synthetase was not phosphorylated and stimulated by protein kinase A. The S424A mutant CTP synthetase had reduced Vmax and elevated Km values for ATP and UTP when compared with the protein kinase A-phosphorylated wild-type enzyme. The specificity constants for ATP and UTP for the S424A mutant CTP synthetase were 4.2- and 2.9-fold lower, respectively, when compared with that of the phosphorylated enzyme. In addition, the S424A mutant enzyme was 2.7-fold more sensitive to CTP product inhibition when compared with the phosphorylated wild-type enzyme. These data indicated that the protein kinase A target site in CTP synthetase was Ser424 and that the phosphorylation of this site played a role in the regulation of CTP synthetase activity.     

Early invasion of brain parenchyma by african trypanosomes

Human African trypanosomiasis or sleeping sickness is a vector-borne parasitic disease that has a major impact on human health and welfare in sub-Saharan countries. Based mostly on data from animal models, it is currently thought that trypanosome entry into the brain occurs by initial infection of the choroid plexus and the circumventricular organs followed days to weeks later by entry into the brain parenchyma. However, Trypanosoma brucei bloodstream forms rapidly cross human brain microvascular endothelial cells in vitro and appear to be able to enter the murine brain without inflicting cerebral injury. Using a murine model and intravital brain imaging, we show that bloodstream forms of T. b. brucei and T. b. rhodesiense enter the brain parenchyma within hours, before a significant level of microvascular inflammation is detectable. Extravascular bloodstream forms were viable as indicated by motility and cell division, and remained detectable for at least 3 days post infection suggesting the potential for parasite survival in the brain parenchyma. Vascular inflammation, as reflected by leukocyte recruitment and emigration from cortical microvessels, became apparent only with increasing parasitemia at later stages of the infection, but was not associated with neurological signs. Extravascular trypanosomes were predominantly associated with postcapillary venules suggesting that early brain infection occurs by parasite passage across the neuroimmunological blood brain barrier. Thus, trypanosomes can invade the murine brain parenchyma during the early stages of the disease before meningoencephalitis is fully established. Whether individual trypanosomes can act alone or require the interaction from a quorum of parasites remains to be shown. The significance of these findings for disease development is now testable.     

Characterization of a novel dUTP-dependent activity of CTP synthetase from Saccharomyces cerevisiae

CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] from the yeast Saccharomyces cerevisiae catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In this work, we demonstrated that CTP synthetase utilized dUTP as a substrate to synthesize dCTP. The dUTP-dependent activity was linear with time and with enzyme concentration. Maximum dUTP-dependent activity was dependent on MgCl(2) (4 mM) and GTP (K(a) = 14 microM) at a pH optimum of 8.0. The apparent K(m) values for dUTP, ATP, and glutamine were 0.18, 0.25, and 0.41 mM, respectively. dUTP promoted the tetramerization of CTP synthetase, and the extent of enzyme tetramerization correlated with dUTP-dependent activity. dCTP was a poor inhibitor of dUTP-dependent activity, whereas CTP was a potent inhibitor of this activity. The enzyme catalyzed the synthesis of dCTP and CTP when dUTP and UTP were used as substrates together. CTP was the major product synthesized when dUTP and UTP were present at saturating concentrations. When dUTP and UTP were present at concentrations near their K(m) values, the synthesis of dCTP increased relative to that of CTP. The synthesis of dCTP was favored over the synthesis of CTP when UTP was present at a concentration near its K(m) value and dUTP was varied from subsaturating to saturating concentrations. These data suggested that the dUTP-dependent synthesis of dCTP by CTP synthetase activity may be physiologically relevant.     

Infectivity of Trypanosoma brucei cultivated at 28 C with tsetse fly salivary glands

When transformed procyclic noninfective trypanosomes of several unrelated stocks of Trypanosoma brucei were cultivated in T-30 Falcon flasks at 28 C in a liquid medium containing head-salivary gland explants of Glossina morsitans morsitans some of the organisms developed into forms infective for mice. Infective trypanosomes were detected 7 to 14 days after the cultures were prepared and they persisted for varying periods of up to 88 days when the cultures were terminated. A few of the salivary glands became invaded with parasites about the time infective organisms appeared in the cultures. Using T. brucei TREU 929, it was shown that trypanosomes grown with between 2m and 50 explants were capable of producing infections consistently for prolonged periods. On the other hand, trypanosomes cultivated with 25 or fewer explants rarely infected mice. Infectivity titrations on trypanosome suspensions from cultures of stocks TREU 1275 and TREU 929 revealed that the maximum number of infective organisms was present 26 to 50 days after initiation of the cultures. Control cultures of trypanosomes grown in medium alone were generally not infective but 2 of the 6 stocks gave rise to a few sporadic infections. A few epimastigote-like and metacyclic-like trypanosomes were seen in stained preparations of infective inocula.     

Effect of in vitro cultivation on the stability of resistance of Trypanosoma brucei brucei to diminazene, isometamidium, quinapyramine, and Mel B

A Trypanosoma brucei brucei stock resistant to diminazene aceturate, isometamidium chloride, quinapyramine sulfate, and Mel B was grown in vitro and its response to these drugs compared to that of a drug-sensitive trypanosome stock. There was little if any change of drug sensitivity after in vitro propagation as bloodstream forms for 120, 177, and 275 days and after in vitro transformation of bloodstream forms into procyclic, epimastigote, and finally metacyclic forms. Drug resistance was stable during in vitro maintenance in the absence of drugs in both culture systems. The response of resistant and sensitive T. b. brucei to diminazene in vitro correlated with their sensitivity pattern in vivo. Thus, in vitro techniques can be used to study drug resistance in trypanosomes.     

Utilization of L-cell nucleoside triphosphates by Chlamydia psittaci for ribonucleic acid synthesis.

Long-term, 32-P-labeled L cells were infected with the obligately intracellular parasite Chlamydia psittaci (strain 6 BC). At 20 h postinfection, [3-H]uridine was added, and the infected cells were sampled at intervals for incorporation of the labels into the uridine triphosphate (UTP) and cytidine triphosphate (CTP) pools of the host L cell and the uridine monophosphate (UMP) and cytidine monophosphate (CMP) in 16S ribosomal ribonucleic acid (RNA) of the parasite. The specific activity of the nucleotides was calculated from the ratio of 3-H to 32-P counts in the nucleotides. The rate of approach to equilibrium labeling of UTP and CTP in L-cell pools and UMP and CMP in 16S RNA from the exogenous uridine label was determined from the increase in the ratios of the specific activities of CTP to UTP and CMP to UMP with time. The rate of approach to equilibrium CMP:UMP labeling of the 16S RNA of C. psittaci was consistent with the rate predicted from the kinetics of labeling of the CTP and UTP pools of the host L cell. In analogous experiments, the rate of approach to equilibrium guanosine monophosphate:adenosine monophosphate labeling of 16S RNA from an exogenous [14-C]adenine label was consistent with the rate predicted from the kinetics of labeling of the purine nucleoside triphosphate pool of the host cell. These results support the concept that members of the genus Chlamydia owe their obligate intracellular mode of reproduction to a requirement for energy intermediates which is fulfilled by the host cell. In addition, evidence was obtained that the total acid-soluble purine nucleoside triphosphate pool of L cells accurately represents the precursors of L-cell 18S ribosomal RNA.     

Antipyrimidine effects of five different pyrimidine de novo synthesis inhibitors in three head and neck cancer cell lines

The pyrimidine de novo nucleotide synthesis consists of 6 sequential steps. Various inhibitors against these enzymes have been developed and evaluated in the clinic for their potential anticancer activity: acivicin inhibits carbamoyl-phosphate-synthase-II, N-(phosphonacetyl)-L- aspartate (PALA) inhibits aspartate-transcarbamylase, Brequinar sodium and dichloroallyl-lawsone (DCL) inhibit dihydroorotate-dehydrogenase, and pyrazofurin (PF) inhibits orotate-phosphoribosyltransferase. We compared their growth inhibition against 3 cell lines from head-and-neck-cancer (HEP-2, UMSCC-14B and UMSCC-14C) and related the sensitivity to their effects on nucleotide pools. In all cell lines Brequinar and PF were the most active compounds with IC50 (50% growth inhibition) values between 0.06–0.37 µM, Acivicin was as potent (IC50s 0.26-1 µM), but DCL was 20-31-fold less active. PALA was most inactive (24–128 µM). At equitoxic concentrations, all pure antipyrimidine de novo inhibitors depleted UTP and CTP after 24 hr exposure, which was most pronounced for Brequinar (between 6–10% of UTP left, and 12–36% CTP), followed by DCL and PF, which were almost similar (6–16% UTP and 12–27% CTP), while PALA was the least active compound (10–70% UTP and 13–68% CTP). Acivicin is a multi-target inhibitor of more glutamine requiring enzymes (including GMP synthetase) and no decrease of UTP was found, but a pronounced decrease in GTP (31–72% left). In conclusion, these 5 inhibitors of the pyrimidine de novo nucleotide synthesis varied considerably in their efficacy and effect on pyrimidine nucleotide pools. Inhibitors of DHO-DH were most effective suggesting a primary role of this enzyme in controlling pyrimidine nucleotide pools