Detection of pyrimidine 5′-nucleotidase deficiency using1H-or31P-nuclear magnetic resonance

Summary We describe here a further Japanese family with pyrimidine 5-nucleotidase (P5N) deficiency diagnosed using a nuclear magnetic resonance (NMR) spectrum, in Kumamoto prefecture where two families having the disease have been reported before. The specific spectra in¹H-NMR of P5N deficient erythrocytes were due to three methyl protons of CDP-choline at 3.22 ppm and to H-2, H-8 and ribose-1 of pyrimidine nucleotide phosphate(s) in the lower fields (at 5.82 and 8.00 ppm). The other specificities in³¹P-NMR spectra were due to CDP-choline, CDP-ethanolamine and UDP-glucose. Those spectra were not detected in other types of hemolytic anemia.     

Antiproliferative effects of 4′, 5′-unsaturated adenine nucleosides on leukemia L 1210 cells in vitro

Summary Several 4, 5-unsaturated adenine nucleosides were shown to have antiproliferative activity against L 1210 leukemia cells in vitro. The active nucleosides were cytotoxic to the L 1210 cells as demonstrated by Trypan Blue uptake. The cytotoxicity was not induced by alterations in the ribonucleoside and deoxyribonucleoside triphosphate levels of the L 1210 cells.     

Changes of the level of proteinase inhibitors in rat plasma during turpentine-induced inflammation

Summary The levels of rat plasma -macroglobulins, -cysteine proteinase inhibitor, haptoglobin and antipapain activity were studied during the acute-phase reaction after an injection of -pinen. An increase in concentration of all the compounds examined was observed.     

Preferential hydrophobic interactions are responsible for a preference of D-amino acids in the aminoacylation of 5′-AMP with hydrophobic amino acids

We have studied the chemistry of aminoacyl AMP to model reactions at the 3 terminus of aminoacyl tRNA for the purpose of understanding the origin of protein synthesis. The present studies relate to the D, L preference in the esterification of 5-AMP. All N-acetyl amino acids we studied showed faster reaction of the D-isomer, with a generally decreasing preference for D-isomer as the hydrophobicity of the amino acid decreased. The -branched amino acids, Ile and Val, showed an extreme preference for D-isomer. Ac-Leu, the -branched amino acid, showed a slightly low D/L ratio relative to its hydrophobicity. The molecular basis for these preferences for D-isomer is understandable in the light of our previous studies and seems to be due to preferential hydrophobic interaction of the D-isomer with ademine. The preference for hydrophobic D-amino acids can be decreased by addition of an organic solvent to the reaction medium. Conversely, peptidylation with Ac-PhePhe shows a preference for the LL isomer over the DD isomer.     

Axillary 5α-androst-16-en-3-one in men and women: Relationships with olfactory acuity to odorous 16-androstenes

Summary Axillary 5-androst-16-en-3-one (5-androstenone) levels were found to be significantly higher in men than in women but do not vary between left and right axillae, are not related to age, handedness or degree of hirsutism (in women) nor to anosmia to this steroid. In men (but not in women), levels are related linearly to axillary cholesterol concentrations but not to squalene. Olfactory thresholds for 5-androstenone varied widely, the lowest recorded being 0.2 ppb, but there was no difference in thresholds between men and women. Women (70%) found the smell repellant but anosmia did not differ greatly between men and women (9–20%). Anosmia to the smell of 5-androst-16-en-3-ol was most marked in women (90%) rather than in men (45%). Axillary 5-androstenone values were generally consistent with the musky or strong smells of male axillary extracts, compared with the sweet smell of those from female subjects.Supported by the Herbert Dunhill Trust.     

Structure of the porcine thyrotropin receptor: a 200 kilodalton glycoprotein heterocomplex

Summary We have determined that the porcine thyroidal TSH receptor is a glycoprotein heterotetramer composed of two M_r 35,000 () covalently linked subunits which interact noncovalently with two copies of (M_r 66,000) chains.Acknowledgments. This work was supported by grants from the Medical Research Council of Canada.     

Effect of dipyridamole on glomerular mesangial cell ecto-5′-nucleotidase expression

Although dipyridamole has been extensively studied as an anti-aggregating agent, its mechanism of action has not been elucidated. Cultured mesangial cells were treated with dipyridamole 1–100 M from 6–72 h. Ecto-5-nucleotidase activity approximately doubled (from 115±11 to 226±14 nmol/min/mg) after treatment with 100 M dipyridamole for 72 h. This effect was concentration- and time-dependent. Cycloheximide, an inhibitor of protein synthesis, did not alter basal 5-nucleotidase activity. However, it prevented stimulation by 5 M dipyridamole. Adenosine availability at the receptor sites was increased by dipyridamole and S-(p-nitrobenzyl)-6-thioinosine (NBTI), which inhibit adenosine uptake into the cell. Addition of dipyridamole or NBTI to the adenosine-treated mesagial cells produced an additive increase in ecto-5-nucleotidase activity. Dipyridamole, through its effect on extracellular adenosine and ecto-5-nucleotidase, may have an influence upon regulation of the glomerular microcirculation.     

Effect of glucose and phloretin-2′-β-D-glucose (phloridzin) on in vitro fertilization of mouse ova

Summary The fertilization ratio of mouse ova in vitro decreased when glucose concentration in the medium was lowered. However, the addition of phloretin-2--D-glucose (phloridzin), known as a glucose uptake inhibitor, restored the fertilization ratio back to the control level. The glucose moiety of the phloridzin seemed to be responsible for this effect.     

Thyroid function parameters during a selenium repletion/depletion study in phenylketonuric subjects

Phenylketonuric (PKU) subjects have a limited supply of selenium (Se) in their phenylalanine-restricted diet. A Se repletion (1 g Se/kg/day)/depletion study was conducted in PKU children to determine the effect of Se on thyroid function parameters.The initial plasma Se concentration (mean±SD: 0.26±0.12 mol/L, p<0.00003, n=10) and glutathione peroxidase (GSH-Px) activity (140±58 U/L, p<0.00003, n=10) were significantly lower compared to agematched controls. After 14 weeks of supplementation, the plasma Se concentration (mean ±SD: 0.74±0.20 mol/L) normalized (normal range: 0.57–1.15 mol/L, mean ±SD: 0.76±0.13 mol/L, n=32) and remained stable thereafter during repletion. Plasma GSH-Px activity reached normal values after 18 weeks of supplementation (312±57 U/L; normal range: 238–492 U/L, mean ±SD: 345±54 U/L, n=32) and increased significantly for up to eight weeks thereafter (332±52 U/L). Individual and mean thyroid parameters were initially normal in all cases. The mean concentrations of plasma thyroxine (T₄: p<0.025), free T₄ (FT₄: p<0.01) and reverse triiodothyronine (rT₃: p<0.005) decreased to 75% of their initial value within three weeks of Se supplementation and remained stable thereafter, within a normal physiological range during selenium supplementation. They increased back to their initial values three weeks (T₄: p<0.05, FT₄: p<0.05) and six weeks (rT₃: p<0.025) respectively, after the end of the supplementation. In conclusion, Se supplementation modifies thyroid function parameters in Se-deficient PKU subjects most likely by an increase in activity of type I 5-deiodinase (5-DIase I).Preliminary results of this study were presented as posters on the 4th International Congress on Trace Elements in Medicine and Biology, Chamonix, France (1993) and the 4th Joint Meeting of the Lawson Wilkens Pediatric Endocrine Society and the European Society for Paediatric Endocrinology, San Francisco, California, Ped Res (1993) 33: S93 (abstract 537). Support in part by Research Grants of Nutricia BV, (The Netherlands).     

Age distribution of circulatingα-interferon

Summary A sensitive radioimmunoassay showed that circulating -interferon in the plasma of healthy individuals was low in children and reached the highest level in the young adult, then declined gradually with age. Circulating -interferon was 0.201±0.059 ng/ml in males (n=19) and 0.184±0.076 ng/ml in females (n=14) at ages 30–39 years old. It was noted that circulating -interferon was maintained up to a certain level even in elderly individuals.     

Age- and sex-related differences in the content of prothymosinα in rat tissues

Differences in the tissue content of prothymosin during the early postnatal development of male and female rats are reported. Thymus and spleen have been found to contain significantly higher amounts of prothymosin in the newborn and prepubertal animals, as compared to adults, whereas liver has been found to contain low levels of prothymosin throughout development. These findings indicate a functional association of prothymosin with the proliferating lymphoid tissues of the young rat.     

Antidiuretic hormone involvement in the release of α-melanocyte-stimulating hormone by hyperosmotic stimuli

Summary In the normal Wistar rat, the plasma -MSH level was raised by hypertonic saline injection (as compared with control rats injected with isotonic saline). No such rise in -MSH followed hypertonic saline administration in the Brattleboro (hereditary diabetes insipidus) animal (compared to isotonic saline injected controls). It is suggested that, in the rat, endogenous antidiuretic hormone is involved in the secretory response of the pars intermedia to osmotic stimuli.     

Enzymatic formation of potential anticancer and antiviral inosine analogues

Theoretically, inosine analogues should act as effective inhibitors of tumor cell proliferation and viral replication. To acquire a broad spectrum of new candidate inosine analogues, a rapid, facile, quantitative and stereoselective method for deaminating potential antitumor and antiviral adenine analogues previously synthesized in our laboratory was developed. A novel 5-adenylic acid deaminase, with relaxed substrate requirements, fromAspergillus species was utilized to deaminate four hexofuranosyladenine nucleosides and five adenine nucleoside dialdehydes to their corresponding inosine analogues. The fastest rates of deamination for the hexofuranosyl nucleosides were for the compounds where the vicinal hydroxyl groups on the sugars are oriented in the erythro configuration. For rapid deamination of the adenine nucleoside dialdehydes, theR configuration at the proximal carbon atom is preferred, while the nature of the group on the distal carbon atom has no significant effect on the rate or extent of deamination.     

Ethanol and opioid receptor signalling

Summary Ethanol may modulate endogenous opioid systems by disrupting opioid receptor signalling. Low concentrations of ethanol slightly potentiate -opioid receptor binding by increasing receptor B_max, and, in some cases, chronic ethanol exposure decreases the density or affinity of the -opioid receptors. By contrast, high concentrations of ethanol acutely decrease -opioid receptor binding by decreasing receptor affinity, whereas chronic exposure of animals and neuronal cell lines to lower concentrations of ethanol leads to possibly adaptive increases in the density or affinity of the -opioid receptors. In the neuronal cell line NG108-15, ethanol does not up-regulate the -opioid receptor by blocking receptor degradation or endocytosis, but protein synthesis is required for this response. Up-regulation of the -opioid receptor renders ethanol-treated NG108-15 cells 3.5-fold more sensitive to opioid inhibition of adenylyl cyclase. Long-term treatment with ethanol also increases maximal opioid inhibition in NG108-15 cells, possibly by decreasing levels of G_s and its mRNA. Ethanol differentially modulates signal transduction proteins in three additional neuronal cell lines, N18TG2, N4TG1, and N1E-115. Ethanol-treated N18TG2 cells show the least up-regulation of the -opioid receptor, little heterologous desensitization of adenylyl cyclase, and no changes in G_s or G_i. By contrast, ethanol-treated N1E-115 cells show the largest up-regulation of the -opioid receptor, the most heterologous desensitization of adenylyl cyclase, and concentration-dependent decreases in G_s and increases in G_i. Further analysis of these related neuronal cell lines may help to identify the molecular elements that endow some, but not all, neuronal cells with the capacity to adapt to ethanol.     

Evidence for the extranuclear localization of thymosins in thymus

A new radioimmunoassay has been developed for thymosin ₄ by generating rabbit polyclonal antibodies against the synthetic N-terminal peptide fragment 1–15 coupled to KLH. The synthetic analogue [Tyr¹²]-thymosin ₄ (1–15) was used as tracer. This radioimmunoassay, with a useful range of 10–1000 pmoles, showed cross-reactivity with the second homologous -thymosin of man and rat (thymosin ₁₀) but not of calf (thymosin ₉). This radioimmunoassay, together with an improved radioimmunoassay for the N-terminus of parathymosin , was employed for the measurement of the levels of thymosin ₄ and parathymosin in nuclear and extranuclear extracts of calf thymus. The bulk of these polypeptides was found in the extranuclear material whereas only traces were observed in the nuclear environment, which indicates the extranuclear localisation of - and -thymosins.     

The expression levels of three raft-associated molecules in cultivated vascular cells are dependent on culture conditions

Relaying a signal across the plasma membrane requires functional connections between the partner molecules. Membrane microdomains or lipid rafts provide an environment in which such specific interactions can take place. The integrity of these sites is often taken for granted when signalling pathways are investigated in cell culture. However, it is well known that smooth muscle and endothelial cells undergo cytoskeletal rearrangements during monolayer culturing. Likewise affected – and with potentially important consequences for signalling events – is the organization of the plasma membrane. The expression levels of three raft markers were massively upregulated, and raft-associated 5-nucleotidase activity increased in conventional monolayer cultures as compared with a spheroidal coculture model, shown to promote the differentiation of endothelial cells. Our data point to a shift of raft components in monolayer cultures and demonstrate potential advantages of the spheroid coculture system for investigation of raft-mediated signalling events in endothelial cells.Received 4 August 2003; received after revision 18 September 2003; accepted 25 September 2003     

Tissue-specific induction of intestinal glutathione S-transferases by αβ-unsaturated carbonyl compounds

Summary Glutathione S-transferase activity in rat intestinal mucosa was increased by the injection of -unsaturated carbonyl compounds such as phorone and diethylmaleate, but that in the liver and kidney was not. Since the administration of cycloheximide completely blocked the increase of the enzyme activity by phorone, the increase of the activity may be due to de novo synthesis rather than enzyme activation.