Do serum and urinary concentrations of kidney injury molecule-1 in healthy newborns depend on birth weight, gestational age or gender?

OBJECTIVE: The aim of work was to establish the normal levels of serum and urinary kidney injury molecule-1 (sKIM-1 and uKIM-1) in healthy full-term newborns. STUDY DESIGN: The study included 88 healthy full-term neonates from normal, uncomplicated pregnancies. The serum and urinary concentrations of KIM-1 in the material obtained in the first or second day of life were determined with a commercially available enzyme-linked immunosorbent assay kits. In addition, uKIM-1 was normalized for urinary creatinine concentration. RESULTS: Male and female newborns, as well as children in whom the samples were obtained in the first or second day of life, did not differ significantly in terms of their sKIM-1 and uKIM-1 levels. Gestational age correlated inversely with sKIM-1 and positively with uKIM-1, but not with uKIM-1/cr. No correlation was found with birth weight and gender. CONCLUSION: This is the first report of sKIM-1 and uKIM-1 levels in healthy full-term newborns during the first postnatal days. The data from healthy newborns may serve as the reference values for future studies in the youngest children.

Purine nucleoside phosphorylases: properties, functions, and clinical aspects.

The ubiquitous purine nucleoside phosphorylases (PNPs) play a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effects on B-cell function. This review updates the properties of the enzymes from eukaryotes and a wide range of prokaryotes, including a tentative classification of the enzymes from various sources, based on three-dimensional structures in the solid state, subunit composition, amino acid sequences, and substrate specificities. Attention is drawn to the compelling need of quantitative experimental data on subunit composition in solution, binding constants, and stoichiometry of binding; order of ligand binding and release; and its possible relevance to the complex kinetics exhibited with some substrates. Mutations responsible for PNP deficiency are described, as well as clinical methods, including gene therapy, for corrections of this usually fatal disease. Substrate discrimination between enzymes from different sources is also being profited from for development of tumour-directed gene therapy. Detailed accounts are presented of design of potent inhibitors, largely nucleosides and acyclonucleosides, their phosphates and phosphonates, particularly of the human erythrocyte enzyme, some with Ki values in nanomolar and picomolar range, intended for induction of the immunodeficient state for clinical applications, such as prevention of host-versus-graft response in organ transplantations. Methods of assay of PNP activity are reviewed. Also described are applications of PNP from various sources as tools for the enzymatic synthesis of otherwise inaccessible therapeutic nucleoside analogues, as coupling enzymes for assays of orthophosphate in biological systems in the micromolar and submicromolar ranges, and for coupled assays of other enzyme systems.

Interactions of calf spleen purine nucleoside phosphorylase with antiviral acyclic nucleoside phosphonate inhibitors: kinetics and emission studies.

Association between calf spleen purine nucleoside phosphorylase and a series of phosphonylalkoxyalkyl derivatives of purine bases was studied by inhibition kinetics and fluorimetric titrations. Dissociation constants, determined by fluorimetric titration in phosphate-free conditions, were lower than inhibition constants in 1 mM phosphate, and inhibition was still weaker in 50 mM phosphate, in accord with the postulated bisubstrate analogue character of this class of inhibitors.

[The value of the head-up tilt table test for with unexplained syncope in children and young adolescents]. / Znaczenie próby pionizacyjnej w diagnostyce nawracajacych stanów napadowych pod postacia zaburzen swiadomosci wystepujacych u dzieci i mlodziezy.

UNLABELLED: Syncope occurs in about 15% of children and young adolescents. The diagnosis of syncope of unknown origin is frequently difficult. In 1986, Kenny et al. introduced the Head-up Tilt Table Test (HUT), which enables to reproduce syncope. The aim of the study was to evaluate HUT in diagnosis of syncope in children and young adolescents. Ninety five children and young adolescents (57 females, 38 males, age range 7-18 years) with recurrent syncope of unexplained etiology were referred for HUT. The study group was divided into two subgroups: A--with history consistent with vasovagal syncope (VVS) and B--with non-characteristic symptoms for VVS. HUT was performed according to the Westminster protocol. The patient was tilted at 60 degree for 45 min. or until syncope occurred. Positive response to HUT was 36%. Negative outcome occurred in 59%. Non-diagnostic HUT was observed in 5%. The vasodepressive type of VVS was recognised in 35%, cardioinhibitory in 12% and mixed in 53%. In group A positive response of HUT occurred in 65% of pts., negative in 31%. In group B positive HUT was observed in 4% of pts. and negative in 89%. CONCLUSIONS: 1. In children and young adolescents head-up tilt test is a very useful diagnostic method. 2. In patients referred for the head-up tilt test the history of syncope should be taken into consideration.

[Acid-base balance in hypotrophic neonates in the first hours of life]. / Równowaga kwasowo-zasadowa u noworodków hipotroficznych w pierwszych godzinach zycia.

Our purpose was to present parameters of acid-base status at 30, 90 and 150 minutes after delivery in cases of hypotrophic infants. Significantly lowered values of pH, HCO3 and BE were found as compared to results of normal term infants. Parameters are consistent with metabolic acidosis. Such conditions require appropriate treatment.

Nicotinamide riboside, an unusual, non-typical, substrate of purified purine-nucleoside phosphorylases.

Nicotinamide 1-beta-D-riboside (Nir), the cationic, reducible moiety of the coenzyme NAD+, has been confirmed as an unusual substrate for purified purine-nucleoside phosphorylase (PNP) from a mammalian source (calf spleen). It is also a substrate of the enzyme from Escherichia coli. The Km values at pH 7, 1.48 mM and 0.62 mM, respectively, were 1-2 orders of magnitude higher than for the natural substrate inosine, but the Vmax values were comparable, 96% and 35% that for Ino. The pseudo first-order rate constants, Vmax/Km, were 1.1% and 2.5% for the calf spleen and E. coli enzymes. The aglycon, nicotinamide, was neither a substrate nor an inhibitor of PNP. Nir was a weak inhibitor of inosine phosphorolysis catalyzed by both enzymes, with Ki values close to the Km for its phosphorolysis, consistent with simple competitive inhibition; this was further confirmed by Dixon plots. Phosphorolysis of the fluorescent positively charged substrate 7-methylguanosine was also inhibited in a competitive manner by both Ino and Nir. Phosphorolysis of Nir by both enzymes was inhibited competitively by several specific inhibitors of calf spleen and E. coli PNP, with Ki values similar to those for inhibition of other natural substrates. The pH dependence of the kinetic constants for the phosphorolysis of Nir and of a variety of other substrates, was extensively investigated, particularly in the alkaline pH range, where Nir exhibited abnormally high substrate activity relative to the reduced reaction rates of both enzymes towards other anionic or neutral substrates. The overall results are discussed in relation to present concepts regarding binding and phosphorolysis of substrates by PNP based on crystallographic data of enzyme-inhibitor complexes, and current studies on enzymatic and nonenzymatic mechanisms of the cleavage of the Nir glycosidic bond.

Kinetics of phosphorolysis of 3-(beta-D-ribofuranosyl)adenine and 3-(beta-D-ribofuranosyl)hypoxanthine, non-conventional substrates of purine-nucleoside phosphorylase.

The properties of two non-conventional substrates of the calf-spleen and Escherichia coli purine nucleoside phosphorylases (PNP), 3-(beta-D-ribofuranosyl)adenine (RibfAde) and 3-(beta-D-ribofuranosyl)hypoxanthine (RibfHyp), are described. In contrast to Ado, RibfAde is a substrate for the mammalian enzyme. With the calf enzyme, the pseudo-first-order rate constants (Vmax/K(m)) for phosphorolysis of RibfAde and RibfHyp are 3% and 13%, respectively, that for phosphorolysis of Ino, while for E. coli PNP the corresponding values are 22% and 30%, respectively. The Michaelis constants (K(m)) for RibfAde were 800 microM (calf PNP) and 150 microM (E. coli PNP). For RibfHyp, the corresponding K(m) values were 220 microM and 260 microM. Two well-characterized inhibitors of calf spleen PNP [9-(2-fluoro-3,4-dihydroxybutyl)guanine] and E. coli PNP (formycin A) were found to inhibit phosphorolysis of RibfAde and RibfHyp with the same inhibition constants as for Ino. Moreover, the inhibition was competitive, which indicates that phosphorolysis of 3-beta-nucleosides occurs at the same active site as for the natural substrate Ino. In particular, the substrate properties of both 3-beta-nucleosides are consistent with their binding to the enzyme in the conformation anti to the imidazole ring about the glycosidic bond, which is superimposable on the structure of natural 9-beta-nucleosides in the conformation anti to the pyrimidine ring. The results are examined in relation to present concepts regarding the binding of substrates and inhibitors at the active site(s) of these enzymes.

[Determination of benzoic acid in nonalcoholic carbonated soft drinks using the HPLC method]. / Oznaczanie kwasu benzoesowego w napojach bezalkoholowych gazowanych metoda chromatografii cieczowej.

A method for benzoic acid determination in soft drinks by liquid chromatography using UV detector is presented. The method makes the possibility of benzoic acid determination with recovery about 95% and with variability index about 2.6%.

Purine nucleoside phosphorylase: inhibition by purine N(7)- and N(9)-acyclonucleosides; and substrate properties of 7-beta-D-ribofuranosylguanine and 7-beta-D-ribofuranosylhypoxanthine.

A series of 10 N(7)- and N(9)-acyclonucleosides of guanine and 8-substituted guanines (8-Br, 8-SH and 8-NH2), and two N(7)-acyclonucleosides of hypoxanthine, were tested for their ability to inhibit purine nucleoside phosphorylase (PNP) (E.C. 2.4.2.1) from human erythrocytes and rabbit kidney. The acyclic chains contained a nitrogen in place of a carbon at the 3', 4' or 5' position and, in one case, an ether oxygen at the 2' position. Most striking was the finding that one of the N(7)-acyclonucleoside analogues, 7-[(1,3-dihydroxypropyl-2)amino]ethylguanine, proved to be a 3-fold more effective inhibitor than its corresponding N(9) counterpart, with Ki = 5 vs 14 microM for the human enzyme and 0.7 vs 2.3 microM for the rabbit enzyme. Both analogues, as well as the others examined, inhibited phosphorolysis competitively with respect to nucleoside substrates (inosine with the human enzyme and guanosine with the rabbit enzyme). The foregoing logically led to the finding that the 7-beta-D-ribosides of guanine (N7Guo) and hypoxanthine (N7Ino) were weak substrates of PNP from human erythrocytes, calf spleen and E. coli. With the human enzyme the pseudo-first-order rate constants (Vmax/Km) for phosphorolysis of N7Guo and N7Ino were 0.08 and 0.02% that for Ino. The Michaelis constants (Km) for N7Guo were 27 (calf PNP), 108 (human PNP) and 450 microM (E. coli PNP). For N7Ino the corresponding Km values were 1.52, 1.26 and 0.64 mM. Four previously well-characterized N(9)-acyclonucleoside inhibitors of calf spleen PNP were found to inhibit phosphorolysis of N7Ino by the same enzyme 2-10-fold more effectively than the parent Ino. The overall results, along with the known excellent substrate properties of N(7)-alkyl- Guo and Ino (Bzowska et al. J Biol Chem 263, 9212-9217, 1988), were examined in relation to present concepts regarding binding of substrates and inhibitors at the active site(s) of these enzymes.

Ultrastructure of the hypothalamo-neurohypophysial system in rats exposed to lead. I. Magnocellular nuclei.

The effect of lead on ultrastructure of supraoptic and paraventricular neurons was studied in rats allowed to drink lead acetate solution instead of water during 6 weeks. Generally, the neurosecretory neurons appeared to be sensitive to lead dependently on its concentrations, but the ultrastructural patterns of the individual neurons varied from cell to cell and suggested the co-existence of the neurons in different functional states such as the increased secretory activity, adaptive responses or cellular degeneration.

Ultrastructure of the hypothalamo-neurohypophysial system in rats exposed to lead. II. Neurohypophysis.

Ultrastructure of neurohypophysis was examined in rats allowed to drink for 6 weeks only solution of lead acetate. It was found the increased number of neurosecretory granules in axonal terminals, the signs of granulolysis in Herring bodies and the presence of axonal terminals with atypical, heterogeneous contents. The possible mechanisms of alterations observed in the whole hypothalamoneurohypophysial system affected by lead were discussed.

Linear free energy relationships for N(7)-substituted guanosines as substrates of calf spleen purine nucleoside phosphorylase. Possible role of N(7)-protonation as an intermediary in phosphorolysis.

Quantitative structure-activity relationships (QSAR) for a series of N(7)-substituted guanosines as substrates for calf spleen purine nucleoside phosphorylase (PNP) were developed, and compared with those for acid hydrolysis of these analogues. There is no correlation between the rates for enzymatic phosphorolysis and acid hydrolysis, indicating that for the enzymatic reaction labilization of the glycosidic bond is not the only, nor the predominant, effect of N(7)-substitution. Multiple regression analysis of the enzymatic process revealed that optimal substrate properties (minimal Michaelis constant) are associated with the Taft electronic constant equal zero and a substituent size, parametrized by the Taft steric constant, smaller than that for a methyl group. These results support the hypothesis of protonation of the N(7)-position of the base by the enzyme as a catalytic mechanism for calf spleen PNP. Attention is drawn to the postulated similar mechanism of action of other purine N-glycosidases, including plant antiviral proteins which function as RNA N-glycosidases, and possibly some DNA N-glycosidases which function as repair enzymes.

Formycins A and B and some analogues: selective inhibitors of bacterial (Escherichia coli) purine nucleoside phosphorylase.

Formycin B (FB), a moderate inhibitor (Ki approximately 100 microM) of mammalian purine nucleoside phosphorylase (PNP), and formycin A (FA), which is totally inactive vs. the mammalian enzyme, are both effective inhibitors of the bacterial (Escherichia coli) enzyme (Ki approximately 5 microM). Examination of a series of N-methyl analogues of FA and FB led to the finding that N(6)-methyl-FA, virtually inactive vs. the mammalian enzyme, is the most potent inhibitor of E. coli purine nucleoside phosphorylase (Ki approximately 0.3 uM) at neutral pH. Inhibition is competitive not only with respect to Ino, but also relative to 7-methyl-Guo and 7-methyl-Ado, as substrates. Both oxoformycins A and B are relatively poor inhibitors. For the most potent inhibitor, N(6)-methyl-FA, it was shown that the enzyme preferentially binds the neutral, and not the cationic, form. In accordance with this the neutral, but not the cationic form, of the structurally related N(1)-methyl-Ado was found to be an excellent substrate. Reported data on tautomerism of formycins were profited from, and extended, to infer which tautomeric species and ionic forms are the active inhibitors. A commercially available (Sigma) bacterial PNP, of unknown origin, was shown to differ from the E. coli enzyme by its inability to phosphorylase Ado; this enzyme was also resistant to FA and FB. These findings have been extended to provide a detailed comparison of the substrate/inhibitor properties of PNP from various microorganisms.

Acyclonucleoside analogue inhibitors of mammalian purine nucleoside phosphorylase.

A series of about 60 purine acyclonucleosides, most with guanine as the aglycone and a 4-carbon chain as the acyclic moiety, was examined for ability to inhibit purine nucleoside phosphorylase from human erythrocytes and calf spleen. Compounds with shorter and longer acyclic chains were less effective inhibitors. Synthetic procedures are described. About 25 of the analogues were competitive inhibitors (relative to inosine or 7-methylguanosine as substrates) with Ki values in the range of 2 to 100 microM. The more potent ones (Ki 2-5 microM) included guanine as the aglycone, with various substituents at C(2') of the acyclic chain and hydroxyls at C(3') and C(4'). In one instance, 9-(2-fluoro-3,4-dihydroxybutyl)guanine, the (+)erythro enantiomer was 10-fold more effective than its (-) counterpart (2.5 microM vs 27 microM). Replacement of guanine by 8-bromo- or 8-aminoguanine enhanced affinity for the enzyme by an order of magnitude or more; 7-deazaacyclovir was also 10-fold more effective than acyclovir. With some of the inhibitors, Ki (human)/Ki (calf) varied over the range 0.4 to 4, reflecting differences between the two enzymes; nonetheless, the much more stable, and commercially available, calf spleen enzyme is recommended for preliminary screening of potential inhibitors of the human or other unstable enzymes. The overall results provide useful indications for the synthesis of potentially more potent inhibitors of the enzyme, by simultaneous modifications of the aglycone and the acyclic chains.

Properties of purine nucleoside phosphorylase (PNP) of mammalian and bacterial origin.

Purine nucleoside phosphorylase (PNP), from calf spleen, human erythrocytes and E. coli have been examined with regard to structural requirements of substrates and inhibitors. Kinetic parameters (Km, Vmax/Km) for a variety of N(1) and/or N(7)-methylated analogues of guanosine, inosine and adenosine have been evaluated for all three enzymes. The substrate and/or inhibitor properties of purine riboside, 1,6-dihydropurine riboside, some deazapurine nucleosides: 3-deaza- and 7-deazainosine, 1,3-dideazapurine riboside (ribobenzimidazole), and a variety of acyclonucleosides, have been determined with mammalian and bacterial enzymes. Overall results indicate distinct similarities of kinetic properties and structural requirements of the two mammalian enzymes, although there are some differences as well. The N(1) and O6 of the purine ring are necessary for substrate-inhibitor activity and constitute a binding site for the mammalian (but not the bacterial) enzymes. Moreover, nucleosides lacking the N(3) undergo phosphorolysis and those lacking N(7) are inhibitors (but not substrates). Methylation of the ring N(7) leads to two overlapping effects: labilization of the glycosidic bond, and impediment to protonation at this site by the enzyme, a postulated prerequisite for enzymatic phosphorolysis. It is proposed that a histidine interacts with N(1) as a donor and O6 as an acceptor. Alternatively N(1)-H and C(2)-NH2 may serve as donors for hydrogen bonds with a glutamate residue. The less specific E. coli enzyme phosphorolyses all purine ring modified nucleosides but 7-deazainosine which is only an inhibitor. On the other hand, the bacterial enzyme exhibits decreased activity towards N(7)-methylated nucleosides and lack of affinity for a majority of the tested acyclonucleoside inhibitors of the mammalian enzymes. The foregoing results underline the fundamental differences between mammalian and bacterial enzymes, including variations in the binding sites for the purine ring.

[Trichomonas vaginitis at different life stages of women]. / Rzesistkowica pochwowa w róznych okresach zycia organizmu zenskiego.

Using of fresh slides and culture T. vaginalis was found in 1094 persons (3.20%). It has been seen the morphological differentiation of T. vaginalis connected with the age, physiological state of macroorganisms and the clinic picture of trichomoniasis. In vivo, the spheroidal as well as ameboid forms of T. vaginalis were observed. The latter ones, characteristic in acute trichomoniasis, were often seen in pregnant women. On the other hand, nonmobile round-shaped forms of T. vaginalis occurred in vaginal contents of girls, women in child-bed and old (5 years after menopause) women. It seems, that morphologically + variability of T. vaginalis depends on changes of specific environment of human vagina, which is very sensitive to hormonal+ responses. It may be considered as adaptation of parasite to different biological conditions of vaginal environment.

The effect of zinc on the toxic action of lead after administration of ethanol to rats.

The aim of this paper was to investigate if zinc may counteract toxic symptoms of poisoning with lead and ethanol in rats, reflected by some biochemical changes in the blood. Wistar rats received lead in drinking water (500 micrograms/ml) for 6 weeks, followed by zinc (240 micrograms/ml) for 2 weeks. During the last 108 h of the experiments the rats received every 12 h an intragastric dose of 5 g/kg of ethanol. In rats receiving zinc together with ethanol the blood levels of zinc, iron and transferrin saturation index were depressed and the latent iron binding capacity and delta-aminolevulinic acid dehydratase activity were elevated. Zinc protected rats against the action of lead on the following hematologic parameters: hemoglobin concentration, hematocrit, iron concentration, latent iron binding capacity and delta-aminolevulinic acid dehydratase activity in the blood. Ethanol administration counteracts the protective effect of zinc on iron concentration and total iron binding capacity, while the favorable action of zinc is maintained in respect of hematocrit, hemoglobin concentration and transferrin saturation index.