Metabolic evolutionary roots of the macrophage immune response in amoeba-bacteria interactions: The conserved role of hypoxia-induced Factor and AMP kinase.

The bacteria Legionella, being able to infect both macrophages and protozoans, reduce oxidative phosphorylation and induce glycolysis, which allows pathogens to grow and replicate in these cells. In amoeba-like inflammatory macrophages (M1), the phagocytizing cells of the primary immune defense, an increase in the rate of glycolysis is followed by a decrease of oxidative phosphorylation. The opposite takes place in anti-inflammatory macrophages (M2). They change from glycolysis to oxidative metabolism when AMP-dependent kinase (AMPK) is activated by a high ratio of AMP/ATP. Stimulation of macrophages with anti-inflammatory cytokines causes activation of AMPK. Infection of macrophages with the parasitic flagellate Leishmania infantum induces a switch from an initial glycolytic phase to oxidative phase with the essential role of AMPK in this change. Activated AMPK induces catabolic pathways effectively producing ATP as well as processes requiring the energy supply. AMPK regulates the migration of cells and enhances the phagocytic activity of macrophages. In macrophages, bacterial products activate TLRs and NF-κB signaling, causing an increase of transcription of hypoxia-induced factor HIF-1α (a subunit of HIF-1). This brings about induction of the enzyme and transporter expression essential for glycolysis and the pentose phosphate pathway to proceed and makes biosynthetic processes and ROS production in macrophages possible. Hypoxia augments macrophage phagocytosis in a HIF-1α-dependent manner. Multicellular parasites experience changes in the availability of oxygen in their life cycle. In the nematode Ascaris suum, HIF participates in the pre-adaptation to hypoxic conditions after infection of their hosts. Also, the freshwater and marine invertebrates meet changes of oxygen concentrations. In the anaerobic branch of the respiratory chain of these invertebrates, fumarate serves as the terminal electron acceptor that is reduced to succinate in complex II of the ETC. In mammalian cells, accumulation of succinate under hypoxic conditions suggests that the mammalian complex II may reduce fumarate to succinate, too. The data reviewed here show that the ability to shift the cell metabolism towards glycolysis observed in activated macrophages can be traced back in evolution to metabolic changes characterizing protozoans infected with bacteria. Anabolic needs of multiplying bacteria direct host metabolism to glycolysis that produces, aside from ATP, precursors of the amino acids used by the pathogen for its protein synthesis. M1-activated mammalian macrophages behave in the same way. Regulation of metabolism in M1 and M2 macrophages is further enhanced by HIF-1 and AMPK, respectively. These archaic functions of AMPK and HIF, important also to control phagocytosis and cell migration were extended to embryonic development in multicellular organisms.

Dielectric and Electrical Properties of BLT Ceramics Modified by Fe Ions.

The solid solution of the perovskite type structure Ba0.996La0.004Ti1-yFeyO3 (BLTF) for varying iron content (y = 0.1-0.4 mol.%) was obtained as a result of a solid state reaction using the conventional method. At room temperature (Tr < TC), the as-received ceramics reveals a single-phase, tetragonal structure and a P4mm space group. An increase in the iron content causes a slight decrease in the volume of the elementary cell. In addition, this admixture significantly reduces the maximum permittivity value (εm) and the shift of the phase transition temperature (TC) towards lower temperatures. The BLTF solid solution shows a classical phase transition and low values of dielectric loss tangent (tgδ), both at room temperature and in the phase transition area. The Curie-Weiss temperature (T0) and Curie constant (C) were also determined on the basis of the dielectric measurements results. The analysis of temperature changes in DC conductivity revealed presence of the positive temperature coefficient of resistivity (PTCR) effect in the phase transition area.

Preparation and Dielectric Properties of K1/2Na1/2NbO3 Ceramics Obtained from Mechanically Activated Powders.

Alkaline based materials have been considered as a replacement for environmentally harmful Pb(Zr,Ti)O3 (PZT) electro-ceramics. In this paper, the K1/2Na1/2NbO3 (KNN) ceramics were prepared in a three stage process: first Nb2O5, Na2CO3, and K2CO3 were milled in a high energy mill (shaker type) for different periods, between 25 h and 100 h, consecutively a solid state reaction was carried out at 550 °C. Finally, the uniaxially pressed samples were sintered at 1000 °C. The reaction temperature is lower for mechanically activated powders than in the case of the conventional solid-state method. The ceramic samples, prepared from the mechanically activated powders, were investigated by dielectric spectroscopy. The influence of the duration of the mechanical activation on the properties of the ceramic materials, e.g., ceramic microstructures, phase transition temperatures, character of the temperature dependences of dielectric permittivity, are discussed.

Systemic changes in photosynthesis and reactive oxygen species homeostasis in shoots of Arabidopsis thaliana infected with the beet cyst nematode Heterodera schachtii.

Photosynthetic efficiency and redox homeostasis are important for plant physiological processes during regular development as well as defence responses. The second-stage juveniles of Heterodera schachtii induce syncytial feeding sites in host roots. To ascertain whether the development of syncytia alters photosynthesis and the metabolism of reactive oxygen species (ROS), chlorophyll a fluorescence measurements and antioxidant responses were studied in Arabidopsis thaliana shoots on the day of inoculation and at 3, 7 and 15 days post-inoculation (dpi). Nematode parasitism caused an accumulation of superoxide and hydrogen peroxide molecules in the shoots of infected plants at 3 dpi, probably as a result of the observed down-regulation of antioxidant enzymes. These changes were accompanied by an increase in RNA and lipid oxidation markers. The activities of antioxidant enzymes were found to be enhanced on infection at 7 and 15 dpi, and the content of anthocyanins was elevated from 3 dpi. The fluorescence parameter Rfd , defining plant vitality and the photosynthetic capacity of leaves, decreased by 11% only at 7 dpi, and non-photochemical quenching (NPQ), indicating the effectiveness of photoprotection mechanisms, was about 16% lower at 3 and 7 dpi. As a result of infection, the ultrastructure of chloroplasts was changed (large starch grains and plastoglobules), and more numerous and larger peroxisomes were observed in the mesophyll cells of leaves. We postulate that the joint action of antioxidant enzymes/molecules and photochemical mechanisms leading to the maintenance of photosynthetic efficiency promotes the fine-tuning of the infected plants to oxidative stress induced by parasitic cyst nematodes.

Monocyte/Macrophage: NK Cell Cooperation-Old Tools for New Functions.

Monocyte/macrophage and natural killer (NK) cells are partners from a phylogenetic standpoint of innate immune system development and its evolutionary progressive interaction with adaptive immunity. The equally conservative ways of development and differentiation of both invertebrate hemocytes and vertebrate macrophages are reviewed. Evolutionary conserved molecules occurring in macrophage receptors and effectors have been inherited by vertebrates after their common ancestor with invertebrates. Cytolytic functions of mammalian NK cells, which are rooted in immune cells of invertebrates, although certain NK cell receptors (NKRs) are mammalian new events, are characterized. Broad heterogeneity of macrophage and NK cell phenotypes that depends on surrounding microenvironment conditions and expression profiles of specific receptors and activation mechanisms of both cell types are discussed. The particular tissue specificity of macrophages and NK cells, as well as their plasticity and mechanisms of their polarization to different functional subtypes have been underlined. The chapter summarized studies revealing the specific molecular mechanisms and regulation of NK cells and macrophages that enable their highly specific cross-cooperation. Attention is given to the evolving role of human monocyte/macrophage and NK cell interaction in pathogenesis of hypersensitivity reaction-based disorders, including autoimmunity, as well as in cancer surveillance and progression.

Protease activity and phytocystatin expression in Arabidopsis thaliana upon Heterodera schachtii infection.

The activity of plant proteases is important for amino acids recycling, removal of damaged proteins as well as defence responses. The second-stage juvenile of the beet cyst nematode Heterodera schachtii penetrates host roots and induces the feeding site called a syncytium. To determine whether infection by H. schachtii affects proteolysis, the protease activity was studied in Arabidopsis roots and shoots at the day of inoculation and 3, 7 and 15 days post inoculation (dpi). Nematode infection caused a decrease of protease activities in infected roots over the entire examination period at all studied pH values. In contrast, the activities of the low molecular mass as well as Ca2+-dependent cysteine proteases were found to be stimulated. In shoots of infected plants, the protease activity was diminished only at 15 dpi at all tested pH values. It was accompanied by changes in total soluble protein content, a higher protein carbonylation and a total polyphenol content. To go deeper into proteolysis regulation, the expression of phytocystatin genes, endogenous inhibitors of cysteine proteases, was examined in syncytia, roots and shoots. Expression of AtCYS1, AtCYS5 and AtCYS6 genes was enhanced upon cyst nematode infection. Our results suggest that changes in protease activities in roots and shoots and altered cystatin expression patterns in syncytia, roots and shoots are important for protein metabolism during cyst nematode infection.

Magnetoelectric Effect in Ceramics Based on Bismuth Ferrite.

Solid-state sintering method was used to prepare ceramic materials based on bismuth ferrite, i.e., (BiFeO3)1 - x -(BaTiO3) x and Bi1 - x Nd x FeO3 solid solutions and the Aurivillius Bi5Ti3FeO15 compound. The structure of the materials was examined using X-ray diffraction, and the Rietveld method was applied to phase analysis and structure refinement. Magnetoelectric coupling was registered in all the materials using dynamic lock-in technique. The highest value of magnetoelectric coupling coefficient α ME was obtained for the Bi5Ti3FeO15 compound (α ME ~ 10 mVcm(-1) Oe(-1)). In the case of (BiFeO3)1 - x -(BaTiO3) x and Bi1 - x Nd x FeO3 solid solutions, the maximum α ME is of the order of 1 and 2.7 mVcm(-1) Oe(-1), respectively. The magnitude of magnetoelectric coupling is accompanied with structural transformation in the studied solid solutions. The relatively high magnetoelectric effect in the Aurivillius Bi5Ti3FeO15 compound is surprising, especially since the material is paramagnetic at room temperature. When the materials were subjected to a preliminary electrical poling, the magnitude of the magnetoelectric coupling increased 2-3 times.

Melatonin nephroprotective action in Zucker diabetic fatty rats involves its inhibitory effect on NADPH oxidase.

Excessive activity of NADPH oxidase (Nox) is considered to be of importance for the progress of diabetic nephropathy. The aim of the study was to elucidate the effect of melatonin, known for its nephroprotective properties, on Nox activity under diabetic conditions. The experiments were performed on three groups of animals: (i) untreated lean (?/+) Zucker diabetic fatty (ZDF) rats; (ii) untreated obese diabetic (fa/fa) ZDF rats; and (iii) ZDF fa/fa rats treated with melatonin (20 mg/L) in drinking water. Urinary albumin excretion was measured weekly. After 4 wk of the treatment, the following parameters were determined in kidney cortex: Nox activity, expression of subunits of the enzyme, their phosphorylation and subcellular distribution. Histological studies were also performed. Compared to ?/+ controls, ZDF fa/fa rats exhibited increased renal Nox activity, augmented expression of Nox4 and p47(phox) subunits, elevated level of p47(phox) phosphorylation, and enlarged phospho-p47(phox) and p67(phox) content in membrane. Melatonin administration to ZDF fa/fa rats resulted in the improvement of renal functions, as manifested by considerable attenuation of albuminuria and some amelioration of structural abnormalities. The treatment turned out to nearly normalize Nox activity, which was accompanied by considerably lowered expression and diminished membrane distribution of regulatory subunits, that is, phospho-p47(phox) and p67(phox) . Thus, it is concluded that: (i) melatonin beneficial action against diabetic nephropathy involves attenuation of the excessive activity of Nox; and (ii) the mechanism of melatonin inhibitory effect on Nox is based on the mitigation of expression and membrane translocation of its regulatory subunits.

ERK1/2 pathway is involved in renal gluconeogenesis inhibition under conditions of lowered NADPH oxidase activity.

The aim of this study was to elucidate the mechanisms involved in the inhibition of renal gluconeogenesis occurring under conditions of lowered activity of NADPH oxidase (Nox), the enzyme considered to be one of the main sources of reactive oxygen species in kidneys. The in vitro experiments were performed on primary cultures of rat renal proximal tubules, with the use of apocynin, a selective Nox inhibitor, and TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a potent superoxide radical scavenger. In the in vivo experiments, Zucker diabetic fatty (ZDF) rats, a well established model of diabetes type 2, were treated with apocynin solution in drinking water. The main in vitro findings are the following: (1) both apocynin and TEMPOL attenuate the rate of gluconeogenesis, inhibiting the step catalyzed by phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of the process; (2) in the presence of the above-noted compounds the expression of PEPCK and the phosphorylation of transcription factor CREB and ERK1/2 kinases are lowered; (3) both U0126 (MEK inhibitor) and 3-(2-aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione (ERK inhibitor) diminish the rate of glucose synthesis via mechanisms similar to those of apocynin and TEMPOL. The observed apocynin in vivo effects include: (1) slight attenuation of hyperglycemia; (2) inhibition of renal gluconeogenesis; (3) a decrease in renal PEPCK activity and content. In view of the results summarized above, it can be concluded that: (1) the lowered activity of the ERK1/2 pathway is of importance for the inhibition of renal gluconeogenesis found under conditions of lowered superoxide radical production by Nox; (2) the mechanism of this phenomenon includes decreased PEPCK expression, resulting from diminished activity of transcription factor CREB; (3) apocynin-evoked inhibition of renal gluconeogenesis contributes to the hypoglycemic action of this compound observed in diabetic animals. Thus, the study has delivered some new insights into the recently discussed issue of the usefulness of Nox inhibition as a potential antidiabetic strategy.

Evolutionary roots of arginase expression and regulation.

TWO MAIN TYPES OF MACROPHAGE FUNCTIONS ARE KNOWN: classical (M1), producing nitric oxide, NO, and M2, in which arginase activity is primarily expressed. Ornithine, the product of arginase, is a substrate for synthesis of polyamines and collagen, important for growth and ontogeny of animals. M2 macrophages, expressing high level of mitochondrial arginase, have been implicated in promoting cell division and deposition of collagen during ontogeny and wound repair. Arginase expression is the default mode of tissue macrophages, but can also be amplified by signals, such as IL-4/13 or transforming growth factor-ß (TGF-ß) that accelerates wound healing and tissue repair. In worms, the induction of collagen gene is coupled with induction of immune response genes, both depending on the same TGF-ß-like pathway. This suggests that the main function of M2 "heal" type macrophages is originally connected with the TGF-ß superfamily of proteins, which are involved in regulation of tissue and organ differentiation in embryogenesis. Excretory-secretory products of metazoan parasites are able to induce M2-type of macrophage responses promoting wound healing without participation of Th2 cytokines IL-4/IL-13. The expression of arginase in lower animals can be induced by the presence of parasite antigens and TGF-ß signals leading to collagen synthesis. This also means that the main proteins, which, in primitive metazoans, are involved in regulation of tissue and organ differentiation in embryogenesis are produced by innate immunity. The signaling function of NO is known already from the sponge stage of animal evolution. The cytotoxic role of NO molecule appeared later, as documented in immunity of marine mollusks and some insects. This implies that the M2-wound healing promoting function predates the defensive role of NO, a characteristic of M1 macrophages. Understanding when and how the M1 and M2 activities came to be in animals is useful for understanding how macrophage immunity, and immune responses operate.

The ancestry and cumulative evolution of immune reactions.

The last two decades of study enriched greatly our knowledge of how the immune system originated and the sophisticated immune mechanisms of today's vertebrates and invertebrates developed. Even unicellular organisms possess mechanisms for pathogen destruction and self recognition. The ability to distinguish self from non-self is a prerequisite for recognition of sexual compatibility and ensuring survival. Molecules involved in these processes resemble those found in the phagocytic cells of higher organisms. Recognition of bacteria by scavenger receptors induces phagocytosis or endocytosis. The phagocytic mechanisms characterizing the amoeboid protozoans developed further during the evolution towards innate immunity. The scavenger receptor cysteine-rich domain SRCR is encoded in the genomes from the most primitive sponges to mammals. The immune system of sponges comprises signal transduction molecules which occur in higher metazoans as well. Sponges already possess recognition systems for pathogenic bacteria and fungi, based on membrane receptors (a lipopolysaccharide-interacting protein, a cell surface receptor recognizing ß(1 → 3)-d-glucans of fungi). Perforin-like molecules and lysozymes are involved, among others, in defense in sponges. Reactive oxygen and nitrogen species function in the immunity of early metazoan. Genes encoding the family of reactive oxygen-generating NADPH oxidases (Noxes) are found in a variety of protists and plants. The NO synthases of cnidarians, mollusks, and chordates are conserved with respect to the mammalian NOS. The antimicrobial peptides of protozoans, amoebapores, are structural and functional analogs of the natural killer cell peptide, NK-lysin, of vertebrates. An ancestral S-type lectin has been found in sponges. Opsonizing properties of lectins and the ability to agglutinate cells justify their classification as primitive recognition molecules. Invertebrate cytokines are not homologous to those of vertebrate, and their functional convergence was presumably enabled by the general similarity of the lectin-like recognition domain three-dimensional structure. Sponges contain molecules with SCR/CCP domains that show high homology to the mammalian regulators of complement activation (RCA family). A multi-component complement system comprising at least the central molecule of the complement system, C3, Factor B, and MASP developed in the cnidarians and evolved into the multilevel cascade engaged in innate and acquired immunity of vertebrates. The adaptive immune system of mammals is also deeply rooted in the metazoan evolution. Some its precursors have been traced as deep as in sponges, namely, two classes of receptors that comprise Ig-like domains, the receptor tyrosine kinases (RTK), and the non-enzymic sponge adhesion molecules (SAM). The antibody-based immune system defined by the presence of the major histocompatibility complex (MHC), T-cell receptor (TCR), B-cell receptor (BCR) or recombination activating genes (RAGs) is known beginning from jawed fishes. However, genes closely resembling RAG1 and RAG2 have been uncovered in the genome of a see urchin. The ancestry of MHC gene remains unknown. Similarly, no homologue of the protein binding domain (PBD) in MHC molecules has been found in invertebrates. The pathway by which endogenous peptides are degraded for presentation with class I MHC molecules utilizes mechanisms similar to those involved in the normal turnover of intracellular proteins, apparently recruited to work also for the immune system. Several cDNAs coding for lysosomal enzymes, e.g., cathepsin, have been isolated from sponges. All chromosomal duplication events in the MHC region occurred after the origin of the agnathans but before the gnathostomes split from them. The V-domains of the subtype found in the receptors of T and B-cells are known from both agnathans and cephalochordates, although they do not rearrange. The rearrangement mechanism of the lymphocyte V-domains suggests its origin from a common ancestral domain existing before the divergence of the extant gnathostome classes. Activation-induced deaminase (AID) - homologous proteins have been found only in the gnathostomes. It appears thus that the adaptive immunity of vertebrates is a result of stepwise accumulation of small changes in molecules, cells and organs over almost half a billion years.

Molecules released by helminth parasites involved in host colonization.

Parasites are designed by evolution to invade the host and survive in its organism until they are ready to reproduce. Parasites release a variety of molecules that help them to penetrate the defensive barriers and avoid the immune attack of the host. In this respect, particularly interesting are enzymes and their inhibitors secreted by the parasites. Serine-, aspartic-, cysteine-, and metalloproteinases are involved in tissue invasion and extracellular protein digestion. Helminths secrete inhibitors of these enzymes (serpins, aspins, and cystatins) to inhibit proteinases, both of the host and their own. Proteinases and their inhibitors, as well as helminth homologues of cytokines and molecules containing phosphorylcholine, influence the immune response of the host biasing it towards the anti-inflammatory Th2 type. Nucleotide-metabolizing enzymes and cholinesterase are secreted by worms to reduce inflammation and expel the parasites from the gastrointestinal tract. An intracellular metazoan parasite, Trichinella spiralis, secretes, among others, protein kinases and phosphatases, endonucleases, and DNA-binding proteins, which are all thought to interfere with the host cellular signals for muscle cell differentiation. Secretion of antioxidant enzymes is believed to protect the parasite from reactive oxygen species which arise from the infection-stimulated host phagocytes. Aside from superoxide dismutase, catalase (rarely found in helminths), and glutathione peroxidase (selenium-independent, thus having a poor activity with H(2)O(2)), peroxiredoxins are probably the major H(2)O(2)-detoxifying enzymes in helminths. Secretion of antioxidant enzymes is stage-specific and there are examples of regulation of their expression by the concentration of reactive oxygen species surrounding the parasite. The majority of parasite-secreted molecules are commonly found in free-living organisms, thus parasites have only adapted them to use in their way of life.

Trichinella spiralis infection affects p47(phox) protein expression in guinea-pig alveolar macrophages.

To establish whether NADPH oxidase activation, responsible for previously demonstrated Trichinella spiralis-induced respiratory burst, results from assembling of membrane and cytosolic NADPH oxidase components and/or increased expression of the oxidase complex proteins, the superoxide anion production and expression of the regulatory p47(phox) subunit were measured in cultured alveolar macrophages obtained during T. spiralis infection of guinea pigs. The results demonstrate for the first time helminth parasite-infection-induced stimulation of NADPH oxidase p47(phox) subunit protein expression, with the effect being decreased by in vivo treatment with cyclosporin A, previously shown to inhibit T. spiralis infection-induced respiratory burst in guinea-pig alveolar macrophages. However, although the expression of the p47(phox) subunit protein remained induced during secondary infection, it was accompanied by superoxide anion production that was significantly suppressed in comparison with that observed during primary infection, suggesting suppressive action of T. spiralis on host's alveolar macrophage immune response, presumably connected with NADPH oxidase complex activity attenuation.

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.

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 O(2)(-) 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)-gamma-like activity in sera of infected animals, macrophage nitric oxide production was not affected by infection, even after additional stimulation in vitro (lipopolisaccharide + hrIFN-gamma) 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.

Synthesis and biological activity of N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid.

2-Deamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) is a potent inhibitor of thymidylate synthase. Its analogue, N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid, containing p-aminophenylacetic acid residue substituting p-aminobenzoic acid residue, was synthesized. The new analogue exhibited a moderately potent thymidylate synthase inhibition, of linear mixed type vs. the cofactor, N(5,10)-methylenetetrahydrofolate. The Ki value of 0.34 microM, determined with a purified recombinant rat hepatoma enzyme, was about 30-fold higher than that reported for inhibition of thymidylate synthase from mouse leukemia L1210 cells by ICI 198583 (Hughes et al., 1990, J. Med. Chem. 33, 3060). Growth of mouse leukemia L5178Y cells was inhibited by the analogue (IC50 = 1.26 mM) 180-fold weaker than by ICI 198583 (IC50 = 6.9 microM).

Effect of cyclosporin A on immunological response in lungs of guinea pigs infected with Trichinella spiralis.

The effects of cyclosporin A (CsA), a potent immunosuppressive drug with antiparasitic activity, on the innate immunological response in guinea pig lungs during an early period (6th and 14th days) after T. spiralis infection were studied. CsA treatment of T. spiralis-infected guinea pigs caused a significant attenuation of immunological response in lungs by decreasing lymphocyte infiltration into pulmonary alveolar space, inhibiting alveolar macrophage superoxide anion production and lowering both the production of NO metabolites measured in bronchoalveolar lavage fluid and expression of the iNOS protein in lung homogenates, allowing us to speculate that the T. spiralis-dependent immunological response is dependent on lymphocyte T function. Interestingly, CsA itself had a pro-inflammatory effect, promoting leucocyte accumulation and macrophage superoxide production in guinea pig lungs. This observation may have a relevance to the situation in patients undergoing CsA therapy. Macrophage expression of the iNOS protein, evaluated by immunoblotting was not influenced by treatment of animals with CsA or anti-TGF-antibody, indicating different regulation of the guinea pig and murine enzymes.

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. METHODS: Urine nitrites and nitrates were determined by means of the Griess reaction, after reduction of nitrates to nitrites with nitrate reductase. RESULTS: The NOx level was higher in preterm (278 nmol/mL) than full-term (176 nmol/mL) infants. Low NOx (115 nmol/mL) levels accompanied generalized infections, while its high contents (650 nmol/mL) was found in cytomegalovirus and one case of Pneumocystis carinii infection. Moderate increase of NOx production was observed in infants with local pulmonary infections and encephalopathies. CONCLUSIONS: The results indicate urinary NOx level is lowered in infants with life-threatening generalized infection. A possibility of a rapid test based on newborn urinary NOx level determination is considered.