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.     

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.     

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     

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.     

Determination of erythrocyte pyrimidine 5′-nucleotidase activity by31P nuclear magnetic resonance: Comparison of normal controls and multiple sclerosis patients

Summary A technique to assay erythrocyte pyrimidine 5-nucleotidase activity in situ using³¹P nuclear magnetic resonance spectroscopy is presented. The assay is chemically specific, simple and applicable to untreated lysates. A comparison of enzyme levels in normal controls and in multiple sclerosis patients employing the assay yielded no significant differences between both groups. Difficulties encountered in the quantitative analysis of the assay using¹H-NMR spectroscopy are briefly discussed.     

Flow-induced release of adenosine 5′-triphosphate from endothelial cells of the rat mesenteric arterial bed

Adenosine 5-triphosphate (ATP) was released into the perfusate of rat isolated mesenteric arterial beds during each of two consecutive increases in flow. There was no significant difference between the amounts of ATP released on each occasion. Substance P was also released into the perfusate by increased flow, although its release was more variable. Removal of the endothelium of the mesenteric vessels with sodium deoxycholate led to a significant reduction (74%) in the amount of ATP released compared with the release before the endothelium had been removed. This suggests that the ATP released into the mesenteric arterial perfusate during increased flow arises from endothelial cells.     

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.     

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 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.     

Hb F in sickle cell anemia

We have reviewed the methodology for an accurate quantitation of Hb F in the blood of patients with sickle cell anemia, values observed in hundreds of patients of different (racial or ethnic) backgrounds and with differences in severity of the disease, and the various factors that affect the level of Hb F. The latter include sex, age, genetic background or chromosomal haplotypes, variations in the sequences of the locus control region(s) 5 to the -globin gene, and the presence of an chain deficiency or -thalassemia. Finally, a few remarks about agents effective in increasing the in vivo Hb F synthesis are also included.     

Steroid hormones enhanced sister-chromatid exchange in cultured CHO cells

Summary The influence of steroid hormones on the induction of sister-chromatid exchange (SCE) in cultured CHO cells was studied. It was observed that estradiol-17 , estriol, estrone and ethynyl estradiol treatments enhanced SCE rates compared to the controls. Overall, these compounds produced a dose response effect. The importance of a detailed study on the long-term genetic effects of steroids on mammalian cells is emphasized.This work was supported by NIH-Minority Biomedical Research Support Program (MBRS), Grant No. RR 08124-11. The author is thankful for the technical assistance provided by NIH-MBRS student, Hoang Duong.     

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.     

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.     

Adenosine uptake in pyrimidine 5′-nucleotidase deficient human erythrocytes via a high affinity transport system

Summary Using a pulse-labeling technique,¹⁴C-adenosine uptake into pyrimidine 5-nucleotidase (P5N) deficient erythrocytes (RBC) was found to be impaired. The Lineweaver-Burk plot showed K_m values of 2.0×10^–3 mM and 0.2 ×10^–3 mM for normal RBC and P5N deficient RBC, respectively. These results indicate that P5N is one of regulators of the adenosine transport system and/or is associated with adenosine carrier protein.     

Hypoxia and estrogen receptor profile influence the responsiveness of human breast cancer cells to estradiol and antiestrogens

Angiogenesis activation mediated by vascular endothelial growth factor (VEGF) is one of the factors that can cause antiestrogen treatment failure in estrogen receptor (ER)?positive breast cancer patients. Since VEGF synthesis is modulated not only by hypoxia but also by steroid hormones, we investigated the relationship between hypoxic and estrogenic/antiestrogenic stimuli in two human breast cancer cell lines expressing both ER6α and ERβ (MCF7) or only ERβ (MDA-MB231). In both cell lines, the VEGF level was significantly influenced by hypoxic conditions and in antiestrogen-responsive MCF7 cells, this effect was not counteracted by tamoxifen or ICI 182,780, thus providing an experimental explanation for the resistance to endocrine treatment observed in patients with ER-positive tumors. In MDA-MB231 cells, estradiol significantly reduced the VEGF level, suggesting that through the ERβ isoform it may function as a negative modulator of VEGF synthesis under hypoxia, and providing evidence for a complex interplay of the estrogen-dependent and hypoxia-dependent pathways.