Mechanism of desensitization of neutrophil response to N-formylmethionylleucylphenylalanine by slow rate of receptor occupancy. Studies on changes in Ca2+ concentration and phosphatidylinositol turnover

Previous studies on the regulation of responses of neutrophils to fMet-Leu-Phe have demonstrated the relevance of the role of the rate of occupation of the receptors by the stimulant. When this rate is decreased by presenting the peptide to neutrophils over a period of time by means of an infusion pump, the activation of the respiratory burst and of the secretion is greatly depressed or is absent. This paper deals with further investigations on the mechanisms of this desensitization, which previous results have shown to consist of an uncoupling between the ligand-receptor complexes and the target for cell responses, caused by the deceleration of the initial rate of occupation of the receptors. The data presented here demonstrate that this desensitization is not linked to the formation of a negative intermediate such as cAMP, but is associated with: (i) a depression of the rate and magnitude of the phosphatidylinositol response (activation of phospahtidylinositol turnover measured as modification of incorporation of [³²P]P_i and [³H]glycerol into phosphatidylinositol and phosphatidic acid); (ii) a deceleration of the rate of the release of bound Ca²⁺, without a decrease in the total quantity of Ca²⁺ liberated (measured as fluorescence changes of chlorotetracycline treated neutrophils); (iii) a slower rise of cytosolic free Ca²⁺ concentration [Ca²⁺]_i, without a decrease in the magnitude of the final increase of [Ca²⁺]_i (monitored with Quin 2). These findings, which are discussed in relation to the recent hypotheses on the transduction reactions of receptor-mediated stimuli for neutrophil responses, are consistent with a mechanism of desensitization involving decreased production of diacylglycerol by the hydrolysis of phosphatidylinositol and deficient activation of Ca²⁺-phospholipid-dependent protein kinase C.     

The effects of monensin on blood-borne arrest and glycosylation of BL/VL3 lymphoma cells.

We have previously shown that inhibitors of N-glycan processing alter both the cell surface carbohydrates and the homing properties in lymphoid cells. We have now studied the effects of the ionophore monensin (MON) on these parameters. Arrest in the spleen of [111In]-labelled BL/VL3 murine T lymphoma cells, injected intravenously was clearly reduced if the cells had been cultured for 24 h in the presence of monensin (0.1-1.0 microgram ml-1). We have characterized glycopeptides from BL/VL3 murine T lymphoma cells. Following labelling with tritiated precursors (fucose, mannose, galactose, glucosamine), surface glycopeptides from BL/VL3 murine T lymphoma cells, were released by trypsin and separated by gel filtration on Bio-Gel P6 and by affinity chromatography on immobilized lectins. After treatment with MON, a class of high molecular mass glycopeptides was no longer found. There were less complex and more high mannose glycans, as a consequence of a reduction of terminal glycosylation (sialylation, fucosylation or incorporation of N-acetyl-glucosamine). Similar findings were obtained with immunoprecipitated Thy-1 antigen. However, as estimated by flow cytometry analysis, the cell surface expression of Thy-1 was not reduced in MON-treated cells. Taken together our results show that cell surface oligosaccharides are modified dramatically, but that at least, certain cell surface antigens are present in normal amounts. It is tempting to speculate that changes in glycosylation account for the abnormal homing properties of MON-treated cells.     

Population structure of the melon fly, Bactrocera cucurbitae, in Reunion Island

The melon fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) is an agricultural pest of major significance worldwide that primarily attacks cucurbit crops. In Reunion Island, it represents the main tephritid pest on cucurbits. In this paper, we provide a genetic characterization of populations of B. cucurbitae from Reunion Island and investigate their geographical origin using ten microsatellite loci at two mitochondrial gene fragments. Microsatellites reveal the occurrence of three different genetic clusters of B. cucurbitae in Reunion Island, all clearly distinguishable from their African and Asian relatives. These three clusters are sympatric and show no signs of recent bottlenecks. Levels of gene flow among clusters are relatively high, yet gene flow also occurs with populations from the African continent and, to a lesser extent, from Asia. The B. cucurbitae clusters show distinct distributions across eastern and western locations in Reunion Island (but not at different altitudes or between wild and cultivated host plants or between sampling periods), and their abundance is also correlated with the average amount of rainfall. Microsatellite and sequence analyses suggest Africa as the most probable source area for populations of B. cucurbitae in Reunion Island.     

TORC1 Determines Fab1 Lipid Kinase Function at Signaling Endosomes and Vacuoles

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Spatial and temporal variations of assemblages in a Mediterranean coralligenous reef and relationships with surface orientation

The structure, distribution, and temporal changes of epibenthic assemblages of a Mediterranean coralligenous reef were investigated using a multifactorial sampling design. The distribution of taxa on vertical walls and down-facing surfaces of overhangs and crevices was analysed at ten sites along 2 km of rocky reefs, south of Livorno (Ligurian Sea, Italy). The temporal variations were analysed between two periods (1995–1996 and 1997–1998) and among four sampling times within each period. Most of the space was dominated by prostrate seaweeds (including Peyssonnelia rubra, P. rosa-marina, and Mesophyllum lichenoides), turf-forming seaweeds, and the red coral Corallium rubrum. The cover of a variety of other invertebrates, mainly sponges and bryozoans, was less than 2%. All taxa were found on both vertical and down-facing surfaces. However, seaweeds dominated the vertical surfaces (mean cover >97%), while C. rubrum and other invertebrates dominated down-facing surfaces (mean density of C. rubrum >16 colonies dm⁻²). Although there was some fluctuation in the abundance of taxa, no obvious patterns were observed. These results support the model of limited temporal variability in Mediterranean coralligenous reefs, possibly related to the slow growth rates of the most abundant taxa and the reduced seasonality of physical conditions.     

The riboflavin/FAD cycle in rat liver mitochondria.

Here we provide evidence that mitochondria isolated from rat liver can synthesize FAD from riboflavin that has been taken up and from endogenous ATP. Riboflavin uptake takes place via a carrier-mediated process, as shown by the inverse relationship between fold accumulation and riboflavin concentration, the saturation kinetics [riboflavin Km and Vmax values were 4.4+/-1.3 microM and 35+/-5 pmol x min(-1) (mg protein)(-1), respectively] and the inhibition shown by the thiol reagent mersalyl, which cannot enter the mitochondria. FAD synthesis is due to the existence of FAD synthetase (EC 2.7.7.2), localized in the matrix, which has as a substrate pair mitochondrial ATP and FMN synthesized from taken up riboflavin via the putative mitochondrial riboflavin kinase. In the light of certain features, including the protein thermal stability and molecular mass, mitochondrial FAD synthetase differs from the cytosolic isoenzyme. Apparent Km and apparent Vmax values for FMN were 5.4+/-0.9 microM and 22.9+/-1.4 pmol x min(-1) x (mg matrix protein)(-1), respectively. Newly synthesized FAD inside the mitochondria can be exported from the mitochondria in a manner sensitive to atractyloside but insensitive to mersalyl. The occurrence of the riboflavin/FAD cycle is proposed to account for riboflavin uptake in mitochondria biogenesis and riboflavin recovery in mitochondrial flavoprotein degradation; both are prerequisites for the synthesis of mitochondrial flavin cofactors.     

Dual mechanism of activation of plant plasma membrane Ca2+-ATPase by acidic phospholipids: Evidence for a phospholipid binding site which overlaps the calmodulin-binding site

The effect of phospholipids on the activity of isoform ACA8 of Arabidopsis thaliana plasma membrane (PM) Ca²⁺-ATPase was evaluated in membranes isolated from Saccharomyces cerevisiae strain K616 expressing wild type or mutated ACA8 cDNA. Acidic phospholipids stimulated the basal Ca²⁺-ATPase activity in the following order of efficiency: phosphatidylinositol 4-monophosphate>phosphatidylserine>phosphatidylcholine?phosphatidylethanolamine?0. Acidic phospholipids increased V_max-Ca²⁺ and lowered the value of K_0.5-Ca²⁺ below the value measured in the presence of calmodulin (CaM). In the presence of CaM acidic phospholipids activated ACA8 by further decreasing its K_0.5-Ca²⁺ value. Phosphatidylinositol 4-monophosphate and, with lower efficiency, phosphatidylserine bound peptides reproducing ACA8 N-terminus (aa 1–116). Single point mutation of three residues (A56, R59 and Y62) within the sequence A56-T63 lowered the apparent affinity of ACA8 for phosphatidylinositol 4-monophosphate by two to three fold, indicating that this region contains a binding site for acidic phospholipids. However, the N-deleted mutant Δ74-ACA8 was also activated by acidic phospholipids, indicating that acidic phospholipids activate ACA8 through a complex mechanism, involving interaction with different sites. The striking similarity between the response to acidic phospholipids of ACA8 and animal plasma membrane Ca²⁺-ATPase provides new evidence that type 2B Ca²⁺-ATPases share common regulatory properties independently of structural differences such as the localization of the terminal regulatory region at the N- or C-terminal end of the protein.     

Levels of 15-keto-1344-dihydro-PFG2α, Progesterone and Oestradiol-17β after Induced Ovulations in Llamas and Alpacas

Six llamas and 6 alpacas were mated to vasectomized males; ovulation and corpus luteum formation followed. Progesterone in blood was elevated from day 5 and reached maximum concentrations of 10–20 nmol/1 on day 7–8. A rapid decline in progesterone levels occurred on day 9–10 in connection with repeated surge releases of prostaglandin F2α. Oestradiol-17β levels were > 100–200 pmol/1 during oestrus when the animals were mated. These high levels might have been caused by coital stimulation. A temporary increase was detected in connection with the rise in progesterone levels in the early luteal phase. With this exception levels of oestradiol stayed low, 20–40 pmol/1 during the luteal phase but rose in most animals after luteolysis to 40–60 pmol/1.     

Effects of the infusion with ACTH or CRH on the secretory activity of rat adrenal cortex

Six-hour infusion with ACTH or CRH induced a dose-dependent rise in the plasma concentrations of ACTH, corticosterone (B) and aldosterone (A). Positive linear correlations between the plasma levels of ACTH and B or A were found in both ACTH-or CRH-infused animals. Regression curves for B were similar in both groups of animals, while the regression line for A was significantly (P less than 0.05) steeper in CRH-than in ACTH-treated rats. These findings suggest that, in the rat, the mechanism underlying the CRH-induced stimulation of A secretion does not exclusively involve the enhancement of ACTH release.