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.     

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.     

Efficient cell proliferation and predominant accumulation of ɛ-globin mRNA in human leukemic K562 cells which produce mostly Hb Gower 1

Summary Long-term cultures of K562(S) cells in 50–75 M hemin allow the selection of hemin-resistant K562 cells together with cells which proliferate efficiently while fully induced to express the human embryonic globin genes, as the hemoglobin Gower 1 (₂₂) is the predominant hemoglobin produced. Our experiments demonstrate that these K562 cells accumulate mostly -globin mRNA (-globin mRNA/-globin mRNA=2.9) suggesting that the control of hemoglobin expression is at a pretranslational level.We thank Dr Irene Bozzoni (Centro degli Acidi Nucleici, Università di Roma) for the pXCR7 probe. Address for reprint request: R.G. Centro Studi Biochimici sul Morbo di Cooley, Via Borsari 46, I-44100 Ferrara.     

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.     

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.     

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.     

Human and rodent type 1 11β-hydroxysteroid dehydrogenases are 7β-hydroxycholesterol dehydrogenases involved in oxysterol metabolism

Interconversion between cortisone and the glucocorticoid receptor ligand cortisol is carried out by 11-hydroxysteroid dehydrogenase (11-HSD)isozymes and constitutes a medically important example of pre-receptor control of steroid hormones. The enzyme 11-HSD type 1 (11-HSD1) catalyzes the conversion of cortisone to its active receptor-binding derivative cortisol, whereas 11-HSD type 2 performs the reverse reaction. Specific inhibitors against the type 1 enzyme lower intracellular levels of glucocorticoid hormone, with an important clinical application in insulin resistance and other metabolic disorders. We report here on the in vitro oxysterol-metabolizing properties of human and rodent 11-HSD1. The enzyme, either as full-length, membrane-attached, or as a transmembrane domain-deleted, soluble form, mediates exclusively conversion between 7-ketocholesterol and 7-hydroxycholesterol with similar k_cat values as observed with glucocorticoid hormones. Thus, human, rat, and mouse 11-HSD1 have dual enzyme activities like the recently described 7-hydroxysteroid dehydrogenase/11-hydroxysteroid dehydrogenase from hamster liver, but differ fundamentally from the latter in that 7-OH rather than 7-OH dehydrogenase constitutes the second activity. These results demonstrate an enzymatic origin of species differences in 7-oxysterol metabolism, establish the origin of endogenous 7-OH cholesterol in humans, and point to a possible involvement of 11-HSD1 in atherosclerosis.Received 30 December 2003; received after revision 16 February 2004; accepted 16 February 2004     

Neurosteroid enhances glutamate release in rat prelimbic cortex via activation of α1-adrenergic and σ1 receptors

The present paper studied the effect and mechanism of neurosteroid pregnenolone sulfate (PREGS) on spontaneous glutamate release using electrophysiological and biochemical methods combined with a pharmacological approach. The results suggested that PREGS had a selective enhancing effect on spontaneous glutamate release in the prelimbic cortex and the hippocampus but not in the striatum. The effect of PREGS in the prelimbic cortex appeared to be via modulation of ₁-adrenergic and ₁ receptors, but in the hippocampus it might be dependent on ₁ receptors only. The activation of ₁-adrenergic receptors synergized ₁ receptor activation in the prelimbic cortex. Intracellular calcium released from the endoplasmic reticulum, protein kinase C, adenylyl cyclase and protein kinase A played a key role in the effect of PREGS. Intracellular calcium, protein kinase C and adenylyl cyclase might be upstream events in the activation of protein kinase A after PREGS.Received 7 January 2005; received after revision 19 February 2005; accepted 22 February 2005 Available online 29 March 2005     

Complex formation betweenγ-immunoglobulin and calmodulin in calcium-free conditions

We show that -immunoglobulin (IgG) binds calmodulin (CaM) in a Ca²⁺-independent manner, with Kd value of (1.7±0.5)×10^–7M. A single IgG molecule maximally bound 10 CaM molecules. The binding is to the heavy chain or Fab portion, but not the Fc portion, of the IgG molecules. Ca²⁺ greatly diminished the interaction between IgG and CaM, with IC₅₀=8–9M. These data give a novel insight into protein-protein interactions.     

Naloxone prevents the analgesic action of α-MSH in mice

Summary -MSH (0.1, 1, 10 g) was administered intracerebroventricularly and its action on pain sensitivity was investigated by the hot-plate method in mice. -MSH produced dose-dependent analgesia and this analgesic effect was prevented by naloxone (1 mg/kg, s.c.). It is possible that -MSH may play a role in the mechanism of pain through endogeneous opioid systems.     

Reduction of high affinity glutamate uptake in rat hippocampus by two polyamine-like toxins isolated from the venom of the predatory waspphilanthus triangulum F

Summary Two components of the venom of the predatory waspPhilanthus triangulum F. significantly reduce — to a greater or less extent — the high affinity uptake of glutamate in rat hippocampus. A concentration of 10 M -PTX caused a reduction of 74%, while the other component, -PTX, at the same concentration, caused a reduction of 18%. Hence the effect of -PTX on high affinity glutamate uptake in the hippocampus is comparable with its effect on high affinity glutamate uptake in insect neuromuscular junctions. Contrary to our previous findings that -PTX has no effect on high affinity glutamate uptake in insect glutamatergic terminal axons, however, -PTX significantly reduces high affinity glutamate uptake in the hippocampus, albeit less effectively than -PTX.     

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.     

α1-Adrenergic stimulation of ketogenesis and fatty acid oxidation is associated with inhibition of lipogenesis in rat hepatocytes

Summary The effect of norepinephrine on fatty acid synthesis (³H₂O incorporation into fatty acids), on fatty acid oxidation to CO₂ and on ketogenesis was studied in isolated hepatocytes of fed rats. After incubation with norepinephrine (50 M), lipogenesis was lower (5.7±1.1 nmoles³H₂O incorporated into fatty acids/mg dry weight/30 min) than in controls (7.5±1.7; n=6, p<0.02). In contrast, (1-¹⁴C) palmitate conversion into total ketone bodies was increased to 10.9±1.8 nmoles/mg/30 min with norepinephrine, vs 8.5±1.6 in controls (p<0.05), and more (1-¹⁴C) palmitate was converted to¹⁴CO₂ with norepinephrine than in controls (1.48±0.10 nmoles/mg/30 min vs 1.06±0.11, p<0.05). The inhibitory effect of norepinephrine on lipogenesis was abolished by addition of the ₁-receptor blocker prazosin, but not by ₂ or -blockers. The results demonstrate that the ketogenic effect of norepinephrine is coupled with an inhibitory effect on lipogenesis which may be explained by diminished activity of acetyl-CoA carboxylase, diminished formation of malonyl-CoA and decreased activity of carnitine palmitoyl transferase I.     

The tumor necrosis factor α of the bony fish seabream exhibits the in vivo proinflammatory and proliferative activities of its mammalian counterparts,yet it functions in a species-specific manner

Information on the bioactivities of non-mammalian cytokines is scant due to the lack of the recombinant molecules and specific antibodies. We produced the mature predicted peptide of tumor necrosis factor (TNF) from the bony fish gilthead seabream (Sparus aurata L.) (sbTNF), and its biological role was determined in vitro and in vivo. We first demonstrated by analytical size-exclusion chromatography that sbTNF is an oligomeric protein but the dimer appears to predominate over the trimeric form, in contrast to mammalian TNF. Intraperitoneal injection of native sbTNF resulted in (i) priming of the respiratory burst of the peritoneal exudate and head-kidney (HK) leukocytes, the latter being the bone marrow equivalent in fish; (ii) rapid recruitment of phagocytic granulocytes to the injection site, and (iii) induction of granulopoiesis in the HK. Interestingly, sbTNF was able to induce a strong proliferation of HK cells in vitro, whereas human TNF did not. Conversely, sbTNF was not cytotoxic for murine L929 fibroblasts.Received 12 February 2004; received after revision 15 March 2004; accepted 29 March 2004     

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.     

Lack of tolerance development to tumor necrosis factor α inside the central nervous system

Tumor necrosis factor (TNF) was repeatedly microinfused into the lateral ventricle of guinea pig brains at a dose of 200 ng, 4 times within 150 min, at intervals of 3 days. In comparison to guinea pigs infused with solvent according to the same time schedule, the animals responded to TNF with pronounced fevers. The quantity of the fever response was the same after each of the 4 microinfusions of TNF. Three days after the last infusion of TNF or solvent all animals received an intramuscular injection of bacterial lipopolysaccharide (LPS). The fever in response to LPS was the same in both groups. Thus, the reported development of tolerance to repeated systemic administration of TNF^1–3 does not develop inside the blood-brain barrier. Also, the febrile response to LPS is not influenced by repeated central pre-treatment with TNF, whereas repeated peripheral treatment does have an effect.     

A2 adenosine receptors in human glomerular mesangial cells

A₂ adenosine receptors were characterized in human glomerular mesangial cells using [³H] 5-N-ethylcarboxamidoadenosine (NECA) as a tracer. There was a single group of receptor sites with a K_D of 184 nM, and a number of sites of 317 fmol/mg of cell protein. Adenosine agonists increased 5-nucleotidase activity via A₂ receptor stimulation. The specific A₂ agonist-NECA, at 0.1 and 1 m, was a potent inhibitor of DNA synthesis.     

Effect of acute administration of Δ1-tetrahydrocannabinol on β-endorphin levels in plasma and brain tissue of the rat

Summary Acute treatment with ¹-tetrahydrocannabinol (¹-THC) elevated the concentration of -endorphin-like immunoreactivity (-ELIR) in plasma and in the hypothalamus, but not in the hippocampus of rats habituated to the injection procedure. These effects were not obtained with the psychotropically inert analog of (¹-THC), cannabidiol. In animals that had not been habituated to the injection procedure, placebo treatment induced a decrease in hippocampal -ELIR.The authors acknowledge the skillful technical assistance of Mrs Willeke Logtenberg.     

Chromosomal location of α-amylase structural genes in rye (Secale cereale L.)

Summary Rye (Secale cereale L.) -amylase isozymes are controlled by at least four loci located on the 5R (three) and 7R (one) chromosomes. In the case of Imperial and King II cultivars, two of the three 5R chromosome loci could be specifically located on the 5Rl chromosome arm; the other one was located on the 5RS. The locus of 7R chromosome was located on the 7RL chromosome arm of Imperial rye.     

Alpha-crystallin: an ATP-independent complete molecular chaperone toward sorbitol dehydrogenase

-Crystallin, the major component of the vertebrate lens, is known to interact with proteins undergoing denaturation and to protect them from aggregation phenomena. Bovine lens sorbitol dehydrogenase (SDH) was previously shown to be completely protected by -crystallin from thermally induced aggregation and inactivation. Here we report that -crystallin, in the presence of the SDH pyridine cofactor NAD(H), can exert a remarkable chaperone action by favoring the recovery of the enzyme activity from chemically denaturated SDH up to 77%. Indeed, even in the absence of the cofactor, -crystallin present at a ratio with SDH of 20:1 (w:w) allows a recovery of 35% of the enzyme activity. The effect of ATP in enhancing -crystallin-promoted SDH renaturation appears to be both nonspecific and to not involve hydrolysis phenomena, thus confirming that the chaperone action of -crystallin is not dependent on ATP as energy donor.Received 28 October 2004; received after revision 22 December 2004; accepted 10 January 2005