Nucleoside exchange catalysed by the cytoplasmic 5''-nucleotidase.

The inhibition of the cytoplasmic 5'-nucleotidase (EC 3.1.3.5) by its product, inosine, was studied with a partially purified preparation of the enzyme from rat liver. Inhibition of Pi production was found to be due to exchange of the inosine moiety between inosine and IMP. Exchange was not catalysed by reversal of the hydrolytic reaction, suggesting, instead, the mediation of an enzyme-phosphate intermediate. Two models for the catalytic mechanism are proposed and rate equations for the dependence of Pi production on inosine concentration are derived. The experimentally determined dependence was consistent with a mechanism in which hydrolysis of the enzyme-phosphate intermediate occurred only when it was unoccupied by inosine. This conclusion suggests that inosine analogues that cannot participate in exchange should inhibit the enzyme. Such inhibitors might be useful in defining the enzyme's physiological role or as pharmacological agents to decrease breakdown of purine nucleotides. The possibility that nucleoside exchange provides an alternative route for the phosphorylation of mutagenic or cytotoxic nucleoside analogues should also be considered.     

Petrosal bones of metatherian mammals from the Late Palaeocene of Itaboraí (Brazil), and a cladistic analysis of petrosal features in metatherians

Metatherian petrosal bones were recovered from the early Late Palaeocene Itaboraí, Brazil, and are formally described. A cladistic analysis of the distribution of 56 petrosal and basicranial characters among extant and fossil metatherians was conducted, resulting in seven parsimonious trees. Relationships among metatherian ingroup taxa are congruent with current understanding of metatherian phylogeny. Metatheria is diagnosed by eight petrosal synapomorphies: stapedial artery absent in adults; reduced, intramural prootic canal; extrabullar internal carotid artery; inferior petrosal sinus between petrosal, basisphenoid, and basioccipital; cava supracochleare and epiptericum completely separated; reduction of the lateral flange; reduction of the anterior lamina; separation of the jugular foramen from the opening for the inferior petrosal sinus. The Palaeocene taxa Mayulestes , Pucadelphy s, and Andinodelphys from Tiupampa, and Petrosal Type II from Itaboraí are the sister groups of all other South American and Australian metatherians. This analysis confirms previous results showing the South American 'monito del monte' Dromiciops nested within the Australasian radiation. Within this australidelphian clade, Dromiciops is closely related to the dasyurids. The South American Caenolestes appears more closely related to the Australidelphia than to the South American didelphids. The Petrosal Types I, III, IV and V from Itaboraí are the stem taxa of the clade Australidelphia plus Caenolestes . The significant synapomorphies supporting this relationship are: enlargement of the fossa subarcuata that produces a bulbous ventral aspect of the mastoid and loss of post-temporal canal.  Journal compilation © 2007 The Linnean Society of London, Zoological Journal of the Linnean Society , 2007, 150 , 85–115. No claim to original French government works.     

Dosage response evaluation of luprostiol administered to pregnant sows

Two trials were completed to investigate the effects of luprostiol in swine. The first trial was to evaluate parturition induced by various dosages of luprostiol compared with those of lutalyse or vehicle. Sows were assigned by random allotment to one of the following treatments on Day 112 of gestation: Group A, control (0 mg luprostiol); Group B (1.88 mg luprostiol); Group C (3.75 mg luprostiol); Group D (7.5 mg luprostiol); Group E (15 mg luprostiol); Group F (10 mg lutalyse). All prostaglandin-treated groups farrowed earlier than the controls (P<0.05), with Groups D (26.3 h), E (31.0 h) and F (25.8 h) having the shortest treatment-to-first-pig intervals, and Groups A (76.0 h), B (54.4 h) and C (40.0 h) having the longest intervals. Luprostiol-treated sows had the shortest farrowing time (P<0.05; range = 3.2 to 3.9 h). Significant differences were found for the time (min) between births: Group A (32.1), Group B (28.4), Group F (35.5) took longer than Group C (20.2), Group D (21.0) and Group E (21.6). In a second trial, 20 crossbred pregnant sows received either vehicle or luprostiol (7.5 mg) on Day 112 of gestation. Progesterone concentrations declined rapidly (P<0.05) in luprotiol treated females but were unchanged in control females during the 24-h collection period. The results of these trials show 7.5 mg luprostiol to be the most effective dose for inducing farrowing.     

Nitroprusside differentially inhibits ADP-stimulated calcium influx and mobilization in human platelets.

1. The effect of nitroprusside on cGMP concn., cAMP concn., shape change, aggregation, intracellular free Ca2+ concn. (by quin-2 fluorescence) and Mn2+ entry (by quenching of quin-2) was investigated in human platelets incubated with 1 mM-Ca2+ or 1 mM-EGTA. 2. Nitroprusside (10 nM-10 microM) caused similar concentration-dependent increases in platelet cGMP concn. and was without effect on cAMP concn. in the presence of extracellular Ca2+ or EGTA. 3. In ADP (3-6 microM)-stimulated platelets, nitroprusside caused 50% inhibition of shape change at 0.4 microM (+Ca2+) or 1.3 microM (+EGTA), aggregation at 0.09 microM (+Ca2+) and of increased intracellular Ca2+ at 0.02 microM (+Ca2+) or 2.1 microM (+EGTA). Entry of 1 mM-Mn2+ (-Ca2+) was inhibited by 80% by 5 microM-nitroprusside. 4. In ionomycin (20-500 nM)-stimulated platelets, nitroprusside (10 nM-100 microM) did not inhibit shape change or intracellular-Ca2+-increase responses, and only partially inhibited aggregation. 5. In phorbol myristate acetate (10 nM)-stimulated platelets, neither shape change nor aggregation was inhibited by 5 microM-nitroprusside. 6. The data demonstrate that nitroprusside inhibits ADP-mediated Ca2+ influx more potently than Ca2+ mobilization. Nitroprusside appears not to influence Ca2+ efflux or sequestration and not to affect the sensitivity of the activation mechanism to intracellular Ca2+ concn. or activation of protein kinase C.     

Stimulation of prostaglandin-dependent macrophage suppressor cells by the subcutaneous injection of polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs), in the form of pure linoleic, linolenic, or arachidonic acid, were injected subcutaneously into male C57Bl/6 mice daily for 10 days. Injection of 3.6 mg/day of PUFA resulted in up to a two- to threefold increase in spleen weight. Spleen cell response to mitogens was reduced by about 70%; mixed lymphocyte responses were reduced by about 90% when compared to normal values. In admixture experiments, spleen cells from PUFA treated mice suppressed the mitogen induced blastogenic response of control spleen cells by up to 90%. Fractionation of spleen cells from PUFA treated mice by G-10 adherence resulted in an enrichment of suppressive activity in the adherent cells. The suppressive effect of G-10 adherent cells was abolished by the addition of indomethacin as well as by depletion of macrophages by treatments with agents such as carbonyl iron and leucine methyl ester. These studies indicate that the administration of PUFA has marked immunosuppressive effects in mice. These effects may be related to increased prostaglandin production and appear to be mediated by a macrophage type cell.     

Adenosine formation and release from neonatal-rat heart cells in culture.

The incorporation of [3H]adenosine (10 microM) into neonatal-rat heart cell nucleotides was inhibited in a concentration-dependent manner, such that 50% inhibition was obtained with 0.75 microM-dipyridamole, 0.26 microM-hexobendine or 0.22 microM-dilazep. Adenosine formation was accelerated 2.5-fold to 2.1 +/- 0.3 nmol/10(7) cells in 10 min when cells were incubated with a combination of 30 mM-2-deoxyglucose and 2 micrograms of oligomycin/ml. Of the newly formed adenosine, 6 +/- 2% was in the cells. Dipyridamole, hexobendine or dilazep (10 microM) increased the amount of adenosine in the cells and decreased that in the medium such that 45-50% of the newly formed adenosine was in the cells. Antibodies which inhibited ecto-5'-nucleotidase by 98.7 +/- 0.3% did not alter the rate of adenosine formation or its distribution between cells and medium. We conclude that adenosine was formed in the cytoplasm during catabolism of cellular ATP and was released via the dipyridamole-sensitive symmetric nucleoside transporter.     

Regulation of fibronectin biosynthesis by glucocorticoids in human fibrosarcoma cells and normal fibroblasts

When treated with the synthetic glucocorticoid dexamethasone, HT1080 human fibrosarcoma cells show changes in morphology, adhesion, and the extracellular matrix. Dexamethasone treatment results in a tenfold increase in the rate of fibronectin biosynthesis in HT1080 cells and a twofold increase in untransformed, normal human fibroblasts. Maximal induction levels are attained within one cell generation, while decay of the response requires several cell cycles. Pulse-chase studies showed that most of the newly synthesized fibronectin is secreted into the medium. The glucocorticoid antagonist, RU-486, blocks the dexamethasone-induced changes but does not alter the basal rate of fibronectin production. Therefore, fibronectin biosynthesis appears to be controlled by two distinct mechanisms--one, regulating basal rates of fibronectin production, which is transformation-sensitive and glucocorticoid-independent; and another, which is mediated by the glucocorticoid receptor, resulting in elevated rates of fibronectin biosynthesis upon dexamethasone treatment both in normal fibroblasts and in HT1080 cells.     

The mechanism of adenosine production in rat polymorphonuclear leucocytes.

Adenosine production in intact rat polymorphonuclear leucocytes was studied during 2-deoxyglucose-induced ATP catabolism. A cell-free system containing the cytosolic 5'-nucleotidase (EC 3.1.3.5) as the only phosphohydrolase was also studied. The rate of adenosine formation in both intact cells and the cell-free system showed a similar dependence on energy charge (([ATP] + 1/2 [ADP]/([ATP] + [ADP] + [AMP])), being maximal only at values close to 0.8. Sufficient cytosolic 5'-nucleotidase was present in intact cells to explain the observed rate of adenosine formation. We conclude that the cytosolic 5'-nucleotidase is responsible for adenosine production in rat polymorphonuclear leucocytes. This mechanism provides a direct biochemical link between the energy status of a cell and the rate of adenosine formation.     

Synchronization of estrus after treatment with Luprostiol in beef cows and in beef and dairy heifers

Four trials were conducted to study synchronous estrous response in beef cows and in beef and dairy heifers to Luprostiol (13, thia-PG-F(2)alpha analog) in comparison with other prostaglandin products. In Trial 1, 60 virgin beef heifers were observed for estrus for 5 d and artificially inseminated. Heifers not observed in estrus within 5 d were randomly assigned to receive 15 mg Luprostiol or 25 mg Lutalyse. In Trial 2, 75 multiparous, lactating beef cows were randomly assigned to receive either 15 mg Luprostiol, 25 mg Lutalyse or 500 mcg Estrumate. All cows received a second injection of the respective treatment 11 d later. In Trial 3, 96 multiparous, lactating beef cows were randomly assigned to receive 15 mg Luprostiol or 25 mg Lutalyse. All cows received a second injection of the respective treatment 11 d later. In Trial 4, virgin dairy heifers were palpated per rectum. Seventy-seven heifers with a palpable corpus luteum (CL) were randomly assigned to receive 15 mg Luprostiol or 500 mcg Estrumate. In all trials animals were artificially inseminated 12 h following observed estrus. Estrous response during the 5-d synchronized period was 44% for Luprostiol and 42% for Lutalyse treated heifers in Trial 1. It was 52, 56 and 60%, respectively, for Luprostiol, Lutalyse and Estrumate treated cows in Trial 2; 23% for Luprostiol and 19% for Lutalyse treated cows in Trial 3; and 68% for Luprostiol and 70% for Estrumate treated heifers in Trial 4. Treatment with Luprostiol results in a similar synchronous estrous response as with the other prostaglandin products used in these studies.