Jolanta M. Dzik

A non-classical type of alveolar macrophage response to Trichinella spiralis infection

Studies of arginase expression and activity in guinea pig alveolar macrophages during Trichinella spiralis infection, prompted by earlier observation of innate lung response to the parasite, showed the macrophages to express both activity and protein of arginase type I. In cultured macrophages part of the enzyme was found to be always released to the extracellular medium. Whereas BCG in vivo treatment, alone or preceded by T. spiralis infection, stimulated arginase activity, T. spiralis infection alone affected the enzyme distribution between intracellular and extracellular fractions, and properties (Km and Vmax), rather than total (intracellular + extracellular) activity, with TGF‐β apparently responsible for a part of the effect. Anti‐TGF‐β antibody treatment of the animals influenced both arginase activation by Mn2+ and dependence of the enzyme‐catalysed reaction on pH. Whereas T. spiralis infection activated guinea pig alveolar macrophages by the type II macrophage activation, as indicated by constant arginase expression, associated with previously demonstrated lack of stimulation of nitric oxide production, BCG treatment invoked an alternative type of activation mechanism, reflected by stimulation of macrophage arginase, but not iNOS, activity.

Early response of guinea-pig lungs to Trichinella spiralis infection.

In order to assess immunological response, induced in guinea‐pig lungs by Trichinella spiralis, cellular infiltration into pulmonary alveolar space and production of and NO in alveolar macrophages obtained from bronchoalveolar lavage fluid (BALF), as well as accumulation of nitric oxide (NO) metabolites in BALF and serum, were evaluated during the early period of primary T. spiralis infection (from 4th to 8th and on 14th day after oral administration of larvae) and on 6th day after secondary infection. Primary infection caused increased infiltration of lymphocytes, macrophages, neutrophils and eosinophils, while secondary infection resulted in raised lymphocyte and eosinophil numbers. In spite of marked cellular infiltration of alveolar space, only very limited activation of effector cells, pointing to a suppressed innate response, was apparent, as (i) BALF supernatant phospholipid/protein concentration ratio, and lung levels of phospholipid peroxidation markers, conjugated dienes and malondialdehyde, did not change during 7 days following infection; (ii) primary, but not secondary, infection caused only a transient increase of superoxide anion production by alveolar macrophages; (iii) despite expression of inducible nitric oxide synthase in macrophages of control, infected and BCG‐treated animals, and of interferon (IFN)‐γ‐like activity in sera of infected animals, macrophage nitric oxide production was not affected by infection, even after additional stimulation in vitro (lipopolisaccharide + hrIFN‐γ) or in vivo (BCG or secondary T. spiralis infection); and (iv) increased nitrate concentrations were found in BALF supernatant and serum, but not in lung homogenates, of infected animals.

Interaction of 5-fluoro-4-thio-2′-deoxyuridine 5′-phosphate with mammalian tumour thymidylate synthase: Role of the pyrimidine N(3)-H dissociation

The role of the pyrimidine N(3)-H in binding of dUMP derivatives to thymidylate synthase was evaluated with the aid of a new dUMP analogue, 5-fluoro-4-thio-dUMP, synthesized by an improved thiation and enzymatic phosphorylation. The interaction of this analogue, and of 5-FdUMP, with the enzyme, and the pH-dependence of these interactions, were compared. Both were slow-binding competitive inhibitors of the enzyme from Ehrlich carcinoma, L1210 and CCRF-CEM cells, with Ki an order of magnitude higher for 5-fluoro-4-thio-dUMP than for 5-FdUMP. With both nucleotides, as well as the parent nucleosides, enzyme inactivation increased as the pH was lowered from 8 to 6. Maximum inactivation with 5-FdUrd was at pH 7.0, and with 5-fluoro-4-thio-dUrd at pH 6.0, in agreement with the higher pKa for the N(3)-H dissociation of the former, and pointing to participation of the N(3)-H as a hydrogen donor in binding to the enzyme.

Nitric oxide metabolites in the urine of full-term and preterm infants.

Background : In newborn full‐term and preterm infants the urine nitrites and nitrates (NOx) were measured, in order to investigate the effects of different pathological conditions (infection, hypoxia) on systemic nitric oxide production.

Trichinella spiralis infection enhances protein kinase C phosphorylation in guinea pig alveolar macrophages

To learn more about the signalling pathways involved in superoxide anion production in guinea pig alveolar macrophages, triggered by Trichinella spiralis infection, protein level and phosphorylation of mitogen activated protein (MAP) kinases and protein kinase C (PKC) were investigated. Infection with T. spiralis, the nematode having ‘lung phase’ during colonization of the host, enhances PKC phosphorylation in guinea pig alveolar macrophages. Isoenzymes β and δ of PKC have been found significantly phosphorylated, although their location was not changed as a consequence of T. spiralis infection. Neither in macrophages from T. spiralis‐infected guinea pig nor in platelet‐activating factor (PAF)‐stimulated macrophages from uninfected animals, participation of MAP kinases in respiratory burst activation was statistically significant. The parasite antigens seem to act through macrophage PAF receptors, transducing a signal for enhanced NADPH oxidase activity, as stimulating effect of newborn larvae homogenate on respiratory burst was abolished by specific PAF receptor antagonist CV 6209. A suppressive action of T. spiralis larvae on host alveolar macrophage innate immunological response was reflected by diminished protein level of ERK2 kinase and suppressed superoxide anion production, in spite of high level of PKC phosphorylation.

Sulfamide antifolates inhibiting thymidylate synthase: synthesis, enzyme inhibition and cytotoxicity

Synthesis and biological evaluation are described of seven new analogues (3-9) of two potent thymidylate synthase inhibitors, 10-propargyl-5,8-dideazafolate (1) and its 2-methyl-2-deamino congener ICI 198583 (2). While the new compunds 3 and 4 were analogues of 1 and 2, respectively, containing a p-aminobenzenesulfonyl residue in place of the p-aminobenzoic acid residue, the remaining 5 new compounds were analogues of 4 with the L-glutamic acid residue replaced by glycine (5), L-valine (6), L-alanine (7), L-phenylglycine (8) or L-norvaline (9). The new analogues were tested as inhibitors of thymidylate synthases isolated from tumour (Ehrlich carcinoma), parasite (Hymenolepis diminuta) and normal tissue (regenerating rat liver) and found to be weaker inhibitors than the parent 10-propargyl-5,8-dideazafolic acid. Selected new analogues, tested as inhibitors of growth of mouse leukemia L 5178Y cells, were less potent than the parent 10-propargyl-5,8-dideazafolic acid. Substitution of the glutamyl residue in compound 4 with L-norvaline (9) resulted in only a 5-fold stronger thymidylate synthase inhibitor, but a 40-fold weaker cell growth inhibitor.

Utilization of glutamate for phosphoenolpyruvate and aspartate synthesis in kidney cortex mitochondria of rabbit

1. 1. Utilization of glutamate for both phosphoenolpyruvate and aspartate synthesis in kidney cortex mitochondria of the rabbit was studied under three metabolic conditions (state 3, uncoupled state and in the presence of uncoupler and oligomycin) in order to induce a high respiratory activity with an oxidized state of the nicotinamide dinucleotides but with varied intramitochondrial ATP levels. 2. 2. The highest rates of glutamate deamination and thus of phosphoenolpyruvate formation were observed in uncoupled mitochondria incubated without oligomycin, i.e. under conditions of low intramitochondrial ATP content. Aspartate synthesis was high in both State 3 and the uncoupled state. 3. 3. In contrast to liver, addition of oligomycin to uncoupled kidney cortex mitochondria in order to increase the mitochondrial ATP/ADP ratio resulted in an inhibition of glutamate deamination, phosphoenolpyruvate formation and aspartate synthesis. Atractyloside augmented an inhibitory effect of oligomycin. 4. 4. Addition of either ATP trapping system or ADP to mitochondria incubated in uncoupled-plus-oligomycin state resulted in the abolishing of the inhibition of both phosphoenolpyruvate and aspartate synthesis, due to a depletion of intramitochondrial ATP content. 5. 5. In contrast to liver, leucine, which activates glutamate dehydrogenase, caused an acceleration of glutamate deamination in kidney cortex mitochondria, accompanied by an enhancement of phosphoenolpyruvate formation. On the other hand, aspartate synthesis was not affected by leucine. 6. 6. In the uncoupled-plus-oligomycin state, leucine abolished an inhibitory effect of ATP and/or GTP on both glutamate deamination and phosphoenolpyruvate synthesis. However, it did not relieve the inhibition of aspartate formation. 7. 7. In the presence of malate in addition to glutamate, oligomycin inhibited neither aspartate formation nor phosphoenolpyruvate synthesis, since under these conditions an accumulation of energy did not occur in mitochondria due to an efficient phosphoenolpyruvate formation. 8. 8. The data indicate that in contrast to liver, in kidney cortex mitochondria, utilizing glutamate as a substrate, the rates of both phosphoenolpyruvate and aspartate synthesis depend greatly upon the mitochondrial energy level and the availability of both oxaloacetate and leucine. d

Thiated pyrimidine deoxynucleoside analogues, potential chemotherapeutic agents, and substrates/inhibitors in various enzyme systems

Abstract The synthesis of thated nucleoside and nucleotide analogues, and determination of their structures, conformations, potential chemotherapeutic activities, and substratehnhibitor properties in various enzyme systems, with emphasis on enzymes related to chemotherapeutic activities, are reported.

Thymidylate synthases from Hymenolepis diminuta and regenerating rat liver: purification, properties, and inhibition by substrate and cofactor analogues

Comparative studies of thymidylate synthases, isolated from the tapeworm, Hymenolepis diminuta, and regenerating liver of its host, rat, aimed at a possibility of specific inhibition of the helminthic enzyme, are presented. While similar in structure (dimers with monomer molecular masses of 33.7 kDa and 34.9 kDa, respectively) and parameters describing interactions with substrates and products, the tapeworm and rat enzymes differed in the dependences of reaction velocity on temperature (Arrhenius plots biphasic and linear, respectively). The tapeworm, compared with the host, enzyme was less sensitive to the competitive slow-binding inhibition by 5-fluoro-dUMP and its 2-thio congener, but equally sensitive to inhibition by 4-thio-5-fluoro-dUMP, N4-hydroxy-dCMP and N4-hydroxy-5-fluoro-dCMP, the latter being more potent inhibitor of the parasite enzyme than 5-fluoro-dUMP. alpha-Anomer of 5-fluoro-dUMP behaved as a very weak competitive slow-binding inhibitor of both enzymes. Both enzymes differed markedly in sensitivity to inhibition by 10-propargyl-5,8-dideazafolate and its di- and triglutamates (pddPteGlu1-3), with pddPteGlu1 being stronger inhibitor of the mammalian enzyme, but pddPteGlu3 showing opposite specificity. Sulfonamidobenzoylglutamate analogue of pddPteGlu (pddPteSO2Glu) and 2-desamino-2-methyl derivative of this analogue (CH3pddPteSO2Glu) were weaker inhibitors of both enzymes than the parent compound. Substitution of the glutamyl residue in CH3pddPteSO2Glu with either norvaline or alanine increased inhibition potency, whereas similar substitutions with glycine, valine or phenylglycine were without a distinct effect with the host enzyme but weakened inhibition of the tapeworm enzyme.

Phosphoenolpyruvate efflux from kidney cortex mitochondria of rabbit

(1) The relationship between phosphoenolpyruvate formation and its accumulation in kidney cortex mitochondria of rabbit was studied in the presence of glutamate as substrate. (2) In mitochondria incubated in either State 4 or under uncoupled conditions, both 1,2,3-benzenetricarboxylate and atractyloside resulted in a marked elevation of the intramitochondrial phosphoenolpyruvate accompanied by a 2-4-fold decline in production of this compound. The same effect was induced by n-butylmalonate in uncoupled mitochondria, while both phosphoenolpyruvate efflux and its production were inhibited to a smaller extent in mitochondria incubated with 1,2,3-benzenetricarboxylate in State 3. (3) Citrate, malate or 2-phosphoglycerate caused a fast displacement of phosphoenolpyruvate from atractyloside-inhibited mitochondria to the reaction medium. In contrast, on the addition of ATP to mitochondria incubated with 1,2,3-benzenetricarboxylate, the rate of phosphoenolpyruvate efflux was lower than that induced by either malate or citrate. (4) Despite the presence of both 1,2,3-benzenetricarboxylate and atractyloside, arsenite and rotenone plus antimycin resulted in a leakage of phosphoenolpyruvate from the mitochondria, probably via a carrier-independent mechanism. (5) Based on the present results it seems that depending on the metabolic condition, the tricarboxylate carrier and the adenine nucleotide translocase are functioning to different extents in the efflux of phosphoenolpyruvate from rabbit renal mitochondria to the surrounding medium.