Elżbieta Wałajtys-Rode

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.

Developmental arrest in Caenorhabditis elegans dauer larvae causes high expression of enzymes involved in thymidylate biosynthesis, similar to that found in Trichinella muscle larvae

Crude extract specific activities of thymidylate synthase, dUTPase, thymidine kinase and dihydrofolate reductase were high during the development of Caenorhabditis elegans, the dauer larva activities being similar to those previously determined in Trichinella spiralis and T. pseudospiralis muscle larvae (with the exception of thymidine kinase, not detected in Trichinella). High thymidylate synthase expression in developmentally arrested larvae, demonstrated also at the mRNA and protein levels, is in agreement with a global cell cycle arrest of dauer larvae and indicates this unusual cell cycle regulation pattern can be shared by developmentally arrested larvae of C. elegans and the two Trichnella species. Hence, the phenomenon may be characteristic for developmentally arrested larvae of different nematodes, rather than specific for the parasitic Trichinella muscle larvae. Endogenous C. elegans thymidylate synthase was purified and its molecular properties compared with those of the recombinant protein, expression of the latter in E. coli cells confirming the NCBI database sequence identity.

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.

Different patterns of nuclear and mitochondrial penetration by the G3 PAMAM dendrimer and its biotin–pyridoxal bioconjugate BC-PAMAM in normal and cancer cells in vitro

The intracellular localization and colocalization of a fluorescently labeled G3 amine-terminated cationic polyamidoamine (PAMAM) dendrimer and its biotin–pyridoxal (BC-PAMAM) bioconjugate were investigated in a concentration-dependent manner in normal human fibroblast (BJ) and squamous epithelial carcinoma (SCC-15) cell lines. After 24 hours treatment, both cell lines revealed different patterns of intracellular dendrimer accumulation depending on their cytotoxic effects. Cancer cells exhibited much higher (20-fold) tolerance for native PAMAM treatment than fibroblasts, whereas BC-PAMAM was significantly toxic only for fibroblasts at 50 µM concentration. Fibroblasts accumulated the native and bioconjugated dendrimers in a concentration-dependent manner at nontoxic range of concentration, with significantly lower bioconjugate loading. After reaching the cytotoxicity level, fluorescein isothiocyanate-PAMAM accumulation remains at high, comparable level. In cancer cells, native PAMAM loading at higher, but not cytotoxic concentrations, was kept at constant level with a sharp increase at toxic concentration. Mander’s coefficient calculated for fibroblasts and cancer cells confirmed more efficient native PAMAM penetration as compared to BC-PAMAM. Significant differences in nuclear dendrimer penetration were observed for both cell lines. In cancer cells, PAMAM signals amounted to ~25%–35% of the total nuclei area at all investigated concentrations, with lower level (15%–25%) observed for BC-PAMAM. In fibroblasts, the dendrimer nuclear signal amounted to 15% at nontoxic and up to 70% at toxic concentrations, whereas BC-PAMAM remained at a lower concentration-dependent level (0.3%–20%). Mitochondrial localization of PAMAM and BC-PAMAM revealed similar patterns in both cell lines, depending on the extracellular dendrimer concentration, and presented significantly lower signals from BC-PAMAM, which correlated well with the cytotoxicity.

In vitro cytotoxicity of the ternary PAMAM G3–pyridoxal–biotin bioconjugate

A third-generation polyamidoamine dendrimer (PAMAM G3) was used as a macromolecular carrier for pyridoxal and biotin. The binary covalent bioconjugate of G3, with nine molecules of biotin per one molecule of G3 (G39B), and the ternary covalent bioconjugate of G3, with nine biotin and ten pyridoxal molecules (G39B10P), were synthesized. The biotin and pyridoxal residues of the bioconjugate were available for carboxylase and transaminase enzymes, as demonstrated in the conversion of pyruvate to oxaloacetate and alanine to pyruvate, respectively, by in vitro monitoring of the reactions, using 1H nuclear magnetic resonance spectroscopy. The toxicity of the ternary bioconjugate (BC-PAMAM) was studied in vitro on BJ human normal skin fibroblasts and human squamous cell carcinoma (SCC-15) cell cultures in comparison with PAMAM G3, using three cytotoxicity assays (XTT, neutral red, and crystal violet) and an estimation of apoptosis by confocal microscopy detection. The tests have shown that BC-PAMAM has significantly lower cytotoxicity compared with PAMAM. Nonconjugated PAMAM was not cytotoxic at concentrations up to 5 μM (NR) and 10 μM (XTT), and BC-PAMAM was not cytotoxic up to 50 μM (both assays) for both cell lines. It has been also found that normal fibroblasts were more sensitive than SCC to both PAMAM and BC-PAMAM. The effect of PAMAM and BC-PAMAM on the initiation of apoptosis (PAMAM in fibroblasts at 5 μM and BC-PAMAM at 10 μM in both cell lines) corresponded with cytotoxicity assays for both cell lines. We concluded that normal fibroblasts are more sensitive to the cytotoxic effects of the PAMAM G3 dendrimer and that modification of its surface cationic groups by substitution with biologically active molecules significantly decreases that effect, confirming that PAMAM G3 is a useful candidate as a carrier for active biocompound delivery.

Properties of phosphorylated thymidylate synthase.

Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent.

Immunofluorescent localization of thymidylate synthase in the development of Trichinella spiralis and Caenorhabditis elegans.

Localization of thymidylate synthase protein in Trichinella spiralis and Caenorhabditis elegans development was followed with the use of confocal microscopy, revealing similar expression patterns in both nematode species. In T. spiralis premature muscle larvae and C. elegans dauer, L3 and L4 larvae, thymidylate synthase was detected in the nerve ring and gonad primordia, as well as T. spiralis stichosome and C. elegans pharyngeal glandular cells. In developmentally arrested T. spiralis muscle larvae, the enzyme was found localized to the gonad primordia and stichosome. High enzyme level was also observed in the embryos developing in uteri of T. spiralis female adult and C. elegans hermaphrodite forms. In the case of T. spiralis adult forms, thymidylate synthase was detected in stichosome, along esophagus wall, as well as in egg and sperm cells. While the enzyme protein present in the embryos remains in accord with its known association with proliferating systems, thymidylate synthase presence in the nerve ring, and reproductive and secretory (T. spiralis stichosomal and C. elegans pharyngeal glandular cells) systems, points to a state of cell cycle-arrest, also known to preserve the enzyme protein.

The effect of G3 PAMAM dendrimer conjugated with B-group vitamins on cell morphology, motility and ATP level in normal and cancer cells

&NA; In a search for the safe vitamin carrier the PAMAM G3 dendrimer covalently substituted with 9 and 10 molecules of vitamin B7 (biotin) and B6 (pyridoxal), respectively (BC‐PAMAM) was investigated. Dendrimer substitution with B‐group vitamins significantly alters its biological properties as compared to native form. Observed effects on investigated cell parameters including morphology, adhesion, migration and ATP level were different for normal human fibroblasts (BJ) and squamous cell carcinoma (SCC‐15) cell lines. BC‐PAMAM revealed significantly less pronounced effects on investigated parameters, particularly at higher concentrations (5–50 &mgr;M), which is relevant with its lower positive surface charge, as compared with native form. The bioconjugate, up to 50 &mgr;M concentration, appeared to be a safe vitamin carrier to normal fibroblasts, without significant effect on their adhesion, shape and migration as well as on intracellular ATP level. In SCC‐15 cells BC‐PAMAM, at low concentrations (0.1–0.5 &mgr;M), altered the cell shape and increase adhesion, whereas at higher concentrations opposite effects were seen. Measurements of cellular level of ATP showed that higher resistance of cancer cells to toxic effects of native PAMAM dendrimers may be due to higher energy supply of cancer cells. Graphical abstract Figure. No caption available.

Unusual Developmental Pattern of Expression of Enzymes Involved in DNA Biosynthesis in Trichinella spiralis and Trichinella pseudospiralis

All species in the genus Trichinella, between them T. spiralis and T. pseudospiralis, have been successful in colonizing striated sceletal muscle tissue and remain infective in this niche for months to years. Trichinella spiralis causes trichinellosis, a serious disease in man and other mammals. Mating of adult worms (developing from infective larvae, deriving from digested infected meat) occurs in a non membrane-bound portion of columnar epithelium of the host’s small intestine. The fertilized females enter the intestinal wall and release to the bloodstream the newborn larvae. Each of these penetrates host’s skeletal muscle cell and lives in its modified portion, the nurse cell, surrounded by a collagen capsule around which a circulatory rete develops. The nurse cell development, initiated by T. spiralis infection, is associated with a variety of changes, including cell cycle re-entry and induction of DNA synthesis, followed by the apparent G2/M arrest of the infected cell in the cell cycle. Similar changes appear to be caused by T. pseudospiralis infection, albeit the nurse cell complexes are not encapsulated by collageneous fibres and the larvae may move between muscle cells. Thymidylate (dTMP) is formed intracellularly either de novo, in a process of the C(5) methylation of 2′-deoxyuridylate (dUMP), catalyzed by the enzyme thymidylate synthase (TS), or as a product of thymidine salvage via phosphorylation, catalyzed by the enzyme thymidine kinase. The dUMP methylation reaction involves a concerted transfer and reduction of the one-carbon group of N5,10-methylenetetrahydrofolate, with concomitant production of thymidylate and dihydrofolate. The coenzyme tetrahydrofolate is regenerated via dihydrofolate reduction by the enzyme dihydrofolate reductase (DHFR). One of the sources of TS substrate, dUMP, is dUTP hydrolysis in a pyrophosphatase reaction catalyzed by the enzyme dUTPase. TS and dUTPase induction is known to be associated with cell proliferation. Thymidylate synthesis inhibition by drugs targeted at either TS or DHFR is taken advantage of in chemotherapy. TS, DHFR and dUTPase were found to be persistently expressed at a high and constant level, comparable to that found in regenerating rat liver, in crude extracts from adult worms of Trichinella spiralis, as well as from developmentally arrested muscle larvae of both Trichinella spiralis (isolated 1–24 months after infection) and Trichinella pseudospiralis (isolated 5.5–13 months after infection). The results obtained with Trichinella pseudospiralis muscle larvae isolated with the use of pepsin did not differ from those obtained when pepsin was not used. Moreover, T. spiralis muscle larvae (T. pseudospiralis larvae were not tested) contained also high level, comparable with that found in mouse leukemia L1210 cells, of DNA polymerase α, a key enzyme of the eukaryotic replication complex, its expression also known to be associated with cell proliferation. Immunofluorescent detection of TS protein was done with the use of monoclonal antibodies, developed by in vivo immunization of Balb/c mice with homogeneous recombinant rat hepatoma TS protein as an antigen. The specific anti-rat TS antibodies recognized also T. spiralis TS, as indicated by cross-reactivity on Western blot. Localization of the enzyme was based on analysis of pictures collected by confocal microscopy. Two types of T. spiralis muscle larvae preparations were studied: muscle larvae isolated from mouse muscles by a procedure destroying nurse cells and muscle larvae remaining in nurse cells, isolated as an intact nurse cell preparation. The results revealed reproducible TS localization patterns, reflected by strong fluorescence emitted by cells of both female and male gonad primordium, as well as from the regions around stichocyte nuclei. High expression in Trichnella muscle larva of thymidylate synthase, and certain other enzymes involved in DNA biosynthesis, was found also in Caenorhabditis dauer larva and appears to be connected with their cells being arrested in the cell cycle.