Plasma lipid profiles discriminate bacterial from viral infection in febrile children.

Fever is the most common reason that children present to Emergency Departments. Clinical signs and symptoms suggestive of bacterial infection are often non-specific, and there is no definitive test for the accurate diagnosis of infection. The 'omics' approaches to identifying biomarkers from the host-response to bacterial infection are promising. In this study, lipidomic analysis was carried out with plasma samples obtained from febrile children with confirmed bacterial infection (n = 20) and confirmed viral infection (n = 20). We show for the first time that bacterial and viral infection produces distinct profile in the host lipidome. Some species of glycerophosphoinositol, sphingomyelin, lysophosphatidylcholine and cholesterol sulfate were higher in the confirmed virus infected group, while some species of fatty acids, glycerophosphocholine, glycerophosphoserine, lactosylceramide and bilirubin were lower in the confirmed virus infected group when compared with confirmed bacterial infected group. A combination of three lipids achieved an area under the receiver operating characteristic (ROC) curve of 0.911 (95% CI 0.81 to 0.98). This pilot study demonstrates the potential of metabolic biomarkers to assist clinicians in distinguishing bacterial from viral infection in febrile children, to facilitate effective clinical management and to the limit inappropriate use of antibiotics.

Exploring strengths and limits of urinary D-chiro inositol phosphoglycans (IPG-P) as a screening test for preeclampsia: A systematic review and meta-analysis.

Preeclampsia is a severe complication of human pregnancy as it leads to significant maternal and perinatal mortality and morbidity worldwide. A prompt recognition of women that develop this syndrome can improve clinical management, increase surveillance and, finally, improve outcomes. Different methods (based on history, ultrasound, serum and urinary biomarkers) were proposed a screening tests for this disease but their performance showed limited results. Urinary inositol phosphoglycans P-type (IPG-P) were shown to identify in advance most of the women who will develop preeclampsia in case-control and longitudinal studies, so we undertook a systematic review and meta-analysis of published studies. Seven studies met the entry criteria so were evaluated. All case-control studies showed excellent statistical performances in a quality statistical assessment. The meta-analysis considered three longitudinal, prospective studies that showed high sensitivity and specificity with ranges of 0.82- 0.99 and 0.90-1.00, respectively. Univariate measures of accuracy revealed a positive and negative likelihood ratio respectively of 3.61 (95% CI 1.56-5.67) and -2.35 (95% CI -3.79 to -0.91). By univariate approach, we found a pooled logarithm of diagnostic odds ratio of 6.15 (95% CI 2.64-9.67). A limitation of this analysis is that, although conducted in different settings (UK, Italy, France, South Africa, and Mauritius) and different clinical groups, they were based on a single academic group. According to our findings, IPG-P test showed very encouraging results as a rapid noninvasive screening test for preeclampsia. Further studies are needed to verify and to validate the reported findings.

Pioglitazone Therapy Increases Insulin-Stimulated Release of d-Chiro-Inositol-Containing Inositolphosphoglycan Mediator in Women with Polycystic Ovary Syndrome.

BACKGROUND: Insulin resistance in women with polycystic ovary syndrome (PCOS) may be mediated, in part, by a deficiency in the insulin-stimulated release of a d-chiro-inositol-inositolphosphoglycan (DCI-IPG) mediator of insulin action. Supporting this idea, several studies have reported improved insulin sensitivity in both lean and obese women with PCOS after oral administration of DCI. Pioglitazone improves insulin sensitivity in women with PCOS, but it is unknown whether this may be contributed by enhanced insulin-stimulated release of the DCI-IPG second messenger. The study aimed to determine if pioglitazone increases release of biologically active DCI-IPG per unit insulin released during an oral glucose tolerance test (OGTT). METHODS: A randomized, double-blind placebo-controlled trial was conducted in 32 women with PCOS at a tertiary referral center in Venezuela. The intervention comprised administration of pioglitazone 45 mg daily or matched placebo for 6 months. Outcome measures included area under curves (AUC) of DCI-IPG (AUCDCI-IPG), insulin (AUCinsulin), and the ratio of AUCDCI-IPG to AUCinsulin during a 2-hr OGTT. RESULTS: After treatment with pioglitazone, AUCinsulin during the OGTT decreased and whole body insulin sensitivity, as determined by the Matsuda index, increased significantly only in the pioglitazone group. The ratio of AUCDCI-IPG/AUCinsulin increased in the pioglitazone group by 1.85-fold (P < 0.0001) with no significant change in the placebo group. Change in Matsuda index correlated with change in DCI-IPG released per unit of insulin during OGTT (r = 0.47, P < 0.01). CONCLUSION: In women with PCOS, pioglitazone increased insulin-stimulated release of the DCI-IPG second messenger of insulin action, which may contribute to its insulin-sensitizing effect in these women.

The maternal ITPK1 gene polymorphism is associated with neural tube defects in a high-risk Chinese population.

BACKGROUND: Epidemiological surveys and animal studies have revealed that inositol metabolism is associated with NTDs, but the mechanisms are not clear. Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a pivotal regulatory enzyme in inositol metabolic pathway. The objective was to assess the potential impact of the maternal ITPK1 genotypes on the inositol parameter and on the NTD risk in a NTD high-risk area in China. METHODOLOGY/RESULTS: A case-control study of pregnant women affected with NTDs (n = 200) and controls (n = 320) was carried out. 13 tag SNPs of ITPK1 were selected and genotyped by the Sequenom MassArray system. We found that 4 tag SNPs were statistically significant in spina bifida group (P<0.05). MACH was used to impute the un-genotyped SNPs in ITPK1 locus and showed that 3 meaningful SNPs in the non-coding regions were significant. We also predicted the binding capacity of transcription factors in the positive SNPs using the bioinformatics method and found that only rs3783903 was located in the conserved sequence of activator protein-1 (AP-1). To further study the association between biochemical values and genotypes, maternal plasma inositol hexakisphosphate (IP6) levels were also assessed using LC-MS. The maternal plasma IP6 concentrations in the spina bifida subgroup were 7.1% lower than control (136.67 vs. 147.05 ng mL(-1), P<0.05), and significantly lower in rs3783903 GG genotype than others (P<0.05). EMSA showed a different allelic binding capacity of AP-1 in rs3783903, which was affected by an A→G exchange. The RT-PCR suggested the ITPK1 expression was decreased significantly in mutant-type of rs3783903 compared with wild-type in the 60 healthy pregnancies (P<0.05). CONCLUSIONS/SIGNIFICANCE: These results suggested that the maternal rs3783903 of ITPK1 might be associated with spina bifida, and the allele G of rs3783903 might affect the binding of AP-1 and the decrease of maternal plasma IP6 concentration in this Chinese population.

Detection of myo-inositol tris pyrophosphate (ITPP) in equine following an administration of ITPP.

Myo-Inositol tris pyrophosphate (ITPP) is a powerful allosteric modulator of haemoglobin that increases oxygen-releasing capacity of red blood cells. It is capable of crossing the red blood cell membrane unlike its open polyphosphate analog myo-inositol hexakisphosphate (IHP). Systemic administration of ITPP enhanced the exercise capacity in mice. There have been rumours of its abuse in the horse racing industry to enhance the performance of racing horses. In this paper, the detection of ITPP in equine plasma and urine after an administration of ITPP is reported. A Standardbred mare was administered 200 mg of ITPP intravenously. Urine and plasma samples were collected up to 120 h post administration and analyzed for ITPP by liquid chromatography-tandem mass spectrometry. ITPP was detected in post administration plasma samples up to 6 hours. The peak concentration was detected at 5 min post administration. In urine, ITPP was detected up to 24 h post administration. The peak concentration was detected at 1.5 h post administration.

Detection of myo-inositol trispyrophosphate in equine urine and plasma by hydrophillic interaction chromatography-tandem mass spectrometry.

Myo-inositol trispyrophosphate (ITPP) is a new drug capable of increasing the amount of oxygen in hypoxic tissues. Studies have shown that administration of ITPP increases the maximal exercise capacity in normal mice as well as mice with severe heart failure. The properties of ITPP make it an ideal candidate as a doping agent to enhance performance in racehorses. While there have been speculations in the horseracing industry that the covert use of ITPP is already widespread, no reported method exists for the detection of ITPP in equine biological samples. ITPP is a difficult-to-detect drug due to its hydrophilic nature; the complexity of equine biological matrices also adds to the problem. This paper describes for the first time a method for the detection and confirmation of ITPP in equine urine and plasma. ITPP was isolated from the sample matrices by solid-phase extraction and the extract was analyzed by hydrophilic interaction chromatography-tandem mass spectrometry. ITPP could be detected at low ppb levels in both fortified equine plasma and urine with good precision, fast instrumental turnaround time, and negligible matrix interferences. To our knowledge, this is the first report of a validated method for the detection and unequivocal confirmation of low levels of ITPP in any biological fluid.

Insulin-stimulated release of D-chiro-inositol-containing inositolphosphoglycan mediator correlates with insulin sensitivity in women with polycystic ovary syndrome.

Some actions of insulin are mediated by inositolphosphoglycan (IPG) mediators. Deficient release of a putative D-chiro-inositol-containing (DCI) IPG mediator may contribute to insulin resistance in women with polycystic ovary syndrome (PCOS). Previously, we demonstrated that oral DCI supplementation improved ovulation and metabolic parameters in women with PCOS. However, whether oral DCI mediates an increase in the release of the DCI-IPG mediator and an improvement in insulin sensitivity in women with PCOS is unknown. We conducted a randomized controlled trial of DCI supplementation vs placebo in 11 women with PCOS who were assessed at 2 time points 6 weeks apart. Plasma DCI, DCI-IPG release during oral glucose tolerance test (AUC(DCI-IPG)), and insulin sensitivity (S(i)) by frequently sampled intravenous glucose tolerance test were assessed at baseline and end of study. The study was terminated early because of a sudden unavailability of the study drug. However, in all subjects without regard to treatment assignment, there was a positive correlation between the change in AUC(DCI-IPG)/AUC(insulin) ratio and the change in S(i) during the 6-week period (r = 0.69, P = .02), which remained significant after adjustment for body mass index (P = .022) and after further adjustment for body mass index and treatment allocation (P = .0261). This suggests that, in women with PCOS, increased glucose-stimulated DCI-IPG release is significantly correlated with improved insulin sensitivity. The significant relationship between DCI-IPG release and insulin sensitivity suggests that the DCI-IPG mediator may be a target for therapeutic interventions in PCOS.

Inositol phosphoglycan P-type in healthy and preeclamptic pregnancies.

An association between inositol phosphoglycan P-type (P-IPG) and preeclampsia has been demonstrated over recent years. This molecule can mediate many of the metabolic and growth promoting effects of insulin. Dysregulation of the mediator family is associated with insulin resistance. An increased concentration of P-IPG has been reported in preeclamptic placenta, although its precursor (GPI) was undetectable in those placental samples. Insulin administration, that induces P-IPG release in normal human placenta, was shown not to cause production/release of the mediator from preeclamptic placental tissue as a consequence of a disturbed insulin signalling. Amniotic fluid is enriched of this mediator, with further increase during preeclampsia. We have found that the fetus released increasing amounts of P-IPG in the urine between 13 and 18 weeks of gestation, reaching a plateau beyond 20 weeks. Cord blood of infants of preeclamptic mothers showed an increased content of soluble P-IPG compared to controls and to the mother.

P-type inositol phosphoglycans in serum and amniotic fluid in active pre-eclampsia.

OBJECTIVES: Abnormal secretion of P-type inositol phosphoglycans (IPG-P) has been described in maternal urine of pre-eclamptic women. The aim of this study was to determine the origin of production of IPG-P. We examined the IPG-P content of maternal and fetal serum, maternal urine and amniotic fluid in both normal pregnancy and pre-eclampsia. DESIGN: Established extraction and bioactivity assay techniques were used to compare total IPG-P levels in serum samples, and a polyclonal-antibody-based ELISA to assay the amniotic fluid and urine samples in matched pairs of women. SUBJECTS: Eleven women with pre-eclampsia requiring caesarean section (subjects), 11 pregnant women requiring elective caesarean section for reasons other than pre-eclampsia (controls). RESULTS: Our data confirm the abnormal level of IPG-P in maternal urine during pre-eclampsia. Moreover, IPG-P levels were higher in umbilical sera than in maternal sera samples. Amniotic fluid as well as urine ELISA results were significantly higher in the pre-eclamptic group compared with normal controls. Total IPG-P bioactivity in serum did not vary between serum compartments in normal pregnancy. Uterine vein IPG-P levels were lower in pre-eclampsia when compared with normal pregnancy. A possible correlation was observed between urine and amniotic fluid levels in normal women. No correlation was observed between measured blood levels and those in urine and amniotic fluid. CONCLUSIONS: It is hypothesized that steady state equilibrium of IPG-P in serum in normal pregnancy is disrupted in pre-eclampsia. Additionally, an abnormal IPG-P sub-fraction, detectable in urine and amniotic fluid, may be present and involved in the pathophysiology of the syndrome, although sites of production of this abnormal form remain unclear.

Inositol tripyrophosphate: a new membrane permeant allosteric effector of haemoglobin.

Nine inositol tripyrophosphate (ITPP) salts have been synthesized. Their ability to act as allosteric effectors of haemoglobin (Hb) has been measured in vitro with free Hb and whole blood. All the synthesized compounds bound to free Hb and were also able to cross, to a certain extent, the plasma membrane of the red blood cells (RBCs) in whole blood samples, lowering the affinity of Hb for oxygen. The oxy-haemoglobin dissociation curves were significantly shifted towards higher values of oxygen partial pressures, both for free Hb and for intracellular Hb in whole blood.

Metformin therapy increases insulin-stimulated release of D-chiro-inositol-containing inositolphosphoglycan mediator in women with polycystic ovary syndrome.

Some actions of insulin are mediated by putative inositolphosphoglycan mediators, and a deficiency in D-chiro-inositol-containing inositolphosphoglycan (DCI-IPG) may contribute to insulin resistance in women with polycystic ovary syndrome (PCOS). Furthermore, similar effects of DCI and metformin, an insulin-sensitizing drug, have been demonstrated in PCOS women. To determine whether metformin improves insulin actions by increasing biologically active DCI-IPG in women with PCOS, we analyzed DCI-IPG during an oral glucose tolerance test in 19 obese women with PCOS before and after 4-8 wk of metformin or placebo. After treatment, the mean (+/-SE) area under the curve (AUC) during the oral glucose tolerance test of insulin (AUC(insulin)) decreased significantly more in the metformin group, compared with the placebo group [-3574 +/- 962 vs. +1367 +/- 1021 micro IU/min.ml (-26 +/- 7 vs. +10 +/- 7 nmol/min.liter), P = 0.003], but the AUC of DCI-IPG (AUC(DCI-IPG)) decreased similarly in both groups (-1452 +/- 968 vs. -2207 +/- 1021%/min, P = 0.60). However, the ratio of AUC(DCI-IPG)/AUC(insulin) increased by 160% after metformin and decreased by 29% after placebo (P = 0.002 between groups). Moreover, metformin seemed to improve the positive correlation between AUC(DCI-IPG) and AUC(insulin) but not placebo (r = 0.32, P = 0.68 at baseline; r = 0.52, P = 0.12 after metformin; and r = -0.39, P = 0.30 after placebo). We conclude that in obese women with PCOS, metformin may improve the action of insulin in part by improving insulin-mediated release of DCI-IPG mediators, as evidenced by increased bioactive DCI-IPG released per unit of insulin.

A possible role of HSP70 in mediating cardioprotection in patients undergoing CABG.

Heat shock protein 70 (HSP70) has been reported to be involved in myocardial self-preservation system. This study shows direct evidence of the effect of HSP70 on lymphocytes during ischemia and reperfusion in CABG (coronary artery bypass graft) surgery. Lymphocytes were separated from the blood obtained from 10 patients undergoing CABG at different time intervals. (i) Baseline samples (drawn before onset of bypass), (ii) ischemic samples (30 min after cross-clamp) and (iii) reperfusion samples (10 min after the cross clamp removal) were incubated with recombinant HSP70 and the cells were harvested after 36 h. The effect of HSP70 was monitored by measuring second messengers such as intracellular calcium, protein kinase C (PKC) and inositol triphosphate (IP3). In addition CD69 expression was also measured. The results showed a significant decrease in intracellular calcium and CD69 expression in ischemia and further in reperfusion samples as compared to their respective untriggered controls. PKC and IP3 levels however remained unaffected. The protective effect of HSP70 during ischemia and reperfusion could thus be attributed to decreasing intracellular calcium and CD69 expression. This study could therefore provide a mechanism of cardioprotection afforded by HSP70.

Analysis of highly phosphorylated inositols in avian and crocodilian erythrocytes.

Both morphological and paleontological characteristics support the hypothesis of a monophyletic origin of crocodilian and avian groups. However, while the erythrocytes of all birds studied to date are reported to contain high levels of inositol pentakisphosphate (InsP(5)), which acts as an allosteric effector of hemoglobin, this molecule has not been reported in crocodilian erythrocytes. In this study we compare the highly phosphorylated inositols in crocodilian and avian erythrocytes using a particularly sensitive analytical procedure. Our aim was to obtain new data which might provide further evidence for the monophyletic origin, or otherwise, of crocodiles and birds. We studied three avian and three crocodilian species. The erythrocytes of the three bird species contained low levels of inositol-3,4,5,6-tetrakisphosphate and inositol-1,3,4,6-tetrakisphosphate, thought to be precursors of Ins(1,3,4,5,6)P(5). The crocodilian erythrocytes studied contained Ins(1,3,4,5,6)P(5) and InsP(6) in higher concentrations than those found in mammal erythrocytes and in other more active cells such as macrophages. Our data provide further evidence of the similarity between crocodilian and avian groups and agree with the hypothesis that both groups evolved from a common ancestor. The process by which the function of inositol phosphates changed from that of intracellular signaling to hemoglobin allosteric effector is discussed.

A straightforward method for analysis of highly phosphorylated inositols in blood cells by high-performance liquid chromatography.

Soluble inositol polyphosphates are found in many cells. The trisphosphate isomers, mainly inositol-1,4,5-trisphosphate, have been extensively studied because of their involvement in signal transduction. However, higher phosphorylated inositols are less frequently studied and their physiological role is poorly understood. Among these, only the myo-inositol-1,3,4,5,6-pentakisphosphate (Ins1,3,4,5,6P5), an important component of bird erythrocytes, has been intensively studied in comparative studies because it is a potent allosteric effector of hemoglobin and decreases its affinity to oxygen. We have developed a procedure for the analysis of inositol polyphosphates and other phosphate compounds in vertebrate blood cells based on a quick and accurate HPLC separation coupled to metal-dye detection. The procedure includes acid extraction of cellular phosphates, acid elimination and concentration of the extract, HPLC separation of phosphate compounds, and quantification by coupled highly sensitive metal-dye detection. The method is especially useful for analyses of highly phosphorylated inositols and for red cell comparative studies. Using the described method we have quantified Ins1,3,4,5,6P5 and the low quantities of InsP6 found in bird erythrocytes. We also identified traces of Ins3,4,5,6P4 and Ins1,3,4,6P4. Moreover, by applying the method in cultured murine macrophages, we have found changes of highly phosphorylated inositols when these cells are activated by lipopolysaccharide.

Hyperactive phosphoinositide signaling pathway in platelets of depressed patients: effect of desipramine treatment.

There is some evidence to suggest that certain neurotransmitter receptors, such as adrenergic and serotonergic receptors and receptor-linked signaling systems, may be altered in depression. Serotonin(2A) and alpha(2)-adrenergic receptors are linked to the phosphoinositide (PI) signaling system in platelets and brain. To examine if the PI signaling system is altered in depression, we studied thrombin- and sodium fluoride-stimulated inositol phosphate(1) (IP(1)) formation before and during desipramine (DMI) treatment in platelets of depressed patients and normal control subjects. We determined thrombin- and sodium fluoride-stimulated IP(1) formation in platelets obtained from hospitalized depressed patients during a drug-free baseline period and after 6 weeks of DMI treatment, and drug-free non-hospitalized normal control subjects. Depressed subjects were diagnosed according to DSM-IV criteria, and severity of illness was assessed with the Hamilton Depression Rating Scale. We observed that thrombin-stimulated IP(1) formation in platelets of depressed patients was significantly higher compared with that of normal control subjects. There were no significant differences in sodium fluoride-stimulated IP(1) formation between depressed patients and normal control subjects. We also did not find any significant effect of treatment with DMI on either thrombin- or sodium fluoride-stimulated IP(1) formation in platelets of depressed patients, which continued to be significantly higher after 6 weeks of treatment with DMI, compared with normal control values. Our studies found a hyperactive PI signaling system in platelets of depressed patients. This hyperactive system may be related either to an increased number of thrombin receptors or to a generalized overstimulation of this pathway; however, since we did not observe any differences in sodium fluoride-stimulated IP(1) formation, it appears that, although the sites distal to the receptors may be altered, this abnormality is probably not related to the abnormalities in G proteins.

The flavonoids quercetin and catechin synergistically inhibit platelet function by antagonizing the intracellular production of hydrogen peroxide.

BACKGROUND: Epidemiologic studies have shown an inverse relation between moderate consumption of red wine and cardiovascular disease. Studies have shown that red wine and its component flavonoids inhibit in vivo platelet activation, but the underlying mechanism has not yet been identified. OBJECTIVE: Because we showed previously that collagen-induced platelet aggregation is associated with a burst of hydrogen peroxide, which in turn contributes to stimulating the phospholipase C pathway, the aim of this study was to investigate whether flavonoids synergize in inhibiting platelet function and interfere with platelet function by virtue of their antioxidant effect. DESIGN: We tested the effect of 2 flavonoids, quercetin and catechin, on collagen-induced platelet aggregation and hydrogen peroxide and on platelet adhesion to collagen. RESULTS: Catechin (50-100 micromol/L) and quercetin (10-20 micromol/L) inhibited collagen-induced platelet aggregation and platelet adhesion to collagen. The combination of 25 micromol catechin/L and 5 micromol quercetin/L, neither of which had any effect on platelet function when used alone, significantly inhibited collagen-induced platelet aggregation and platelet adhesion to collagen. Such a combination strongly inhibited collagen-induced hydrogen peroxide production, calcium mobilization, and 1,3,4-inositol triphosphate formation. CONCLUSIONS: These data indicate that flavonoids inhibit platelet function by blunting hydrogen peroxide production and, in turn, phospholipase C activation and suggest that the synergism among flavonoids could contribute to an understanding of the relation between the moderate consumption of red wine and the decreased risk of cardiovascular disease.

Distinct localization and function of (1,4,5)IP(3) receptor subtypes and the (1,3,4,5)IP(4) receptor GAP1(IP4BP) in highly purified human platelet membranes.

Platelet activation is associated with an increase of cytosolic Ca(++) levels. The (1,4,5)IP(3) receptors [(1,4,5)IP(3)R] are known to mediate Ca(++) release from intracellular stores of many cell types. Currently there are at least 3 distinct subtypes of (1,4, 5)IP(3)R-type I, type II, and type III-with suggestions of distinct roles in Ca(++) elevation. Specific receptors for (1,3,4,5)IP(4) belonging to the GAP1 family have also been described though their involvement with Ca(++) regulation is controversial. In this study we report that platelets contain all 3 subtypes of (1,4,5)IP(3)R but in different amounts. Type I and type II receptors are predominant. In studies using highly purified platelet plasma (PM) and intracellular membranes (IM) we report a distinct localization of these receptors. The PM fractions were found to contain the type III (1,4,5)IP(3)R and GAP1(IP4BP) in contrast to IM, which contained type I (1,4,5)IP(3)R. The type II receptor exhibited a dual distribution. In studies examining the labeling of surface proteins with biotin in intact platelets only the type III (1,4,5)IP(3)R was significantly labeled. Immunogold studies of ultracryosections of human platelets showed significantly more labeling of the PM with the type III receptor antibodies than with type I receptor antibodies. Ca(++) flux studies were carried out with the PM to demonstrate in vitro function of inositol phosphate receptors. Ca(++) release activities were present with both (1,4,5)IP(3) and (1, 3,4,5)IP(4) (EC(50) = 1.3 and 0.8 micromol/L, respectively). Discrimination of the Ca(++)-releasing activities was demonstrated with cyclic adenosine monophosphate (cAMP)-dependent protein kinase (cAMP-PK) specifically inhibiting (1,4,5)IP(3) but not (1,3,4, 5)IP(4)-induced Ca(++) flux. In experiments with both PM and intact platelets, the (1,4,5)IP(3)Rs but not GAP1(IP4BP) were found to be substrates of cAMP-PK and cGMP-PK. Thus the Ca(++) flux property of (1,3,4,5)IP(4) is insensitive to cAMP-PK. These studies suggest distinct roles for the (1,4,5)IP(3)R subtypes in Ca(++) movements, with the type III receptor and GAP1(IP4BP) associated with cation entry in human platelets and the type I receptor involved with Ca(++) release from intracellular stores.

Thrombopoietin potentiates collagen receptor signaling in platelets through a phosphatidylinositol 3-kinase-dependent pathway.

Collagen activates platelets through a tyrosine kinase-dependent pathway, involving phospholipase Cgamma2. Functional responses such as aggregation and secretion induced by collagen are potentiated by preincubation with thrombopoietin (TPO). In this study, we show that collagen and thrombopoietin activate the phosphatidylinositol 3-kinase (PI 3-kinase) pathway and that this contributes to their respective actions. The structurally distinct inhibitors of PI 3-kinase, wortmannin, and LY294002, completely inhibit formation of phosphatidylinositol 3,4,5-trisphosphate by collagen. This leads to a substantial reduction in the formation of inositol phosphates and phosphatidic acid, 2 indices of PLC activity, and the consequent inhibition of intracellular Ca(++) [Ca(++)](i), aggregation and secretion. Potentiation of the collagen response by TPO is prevented in the presence of wortmannin and LY294002. However, when the 2 PI 3-kinase inhibitors are given after the addition of TPO but before the collagen, recovery of potentiation is observed. This suggests that potentiation is mediated through activation of PI 3-kinase. TPO stimulates aggregation of platelets from a low percentage of donors and this is also blocked by wortmannin. These results suggest that the PI 3-kinase pathway plays an important role in signaling by collagen and in the priming action of TPO.

Lipid analysis by matrix-assisted laser desorption and ionization mass spectrometry: A methodological approach.

Whereas matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has gained high importance in the field of protein analysis, surprisingly few studies were published about the use of MALDI for lipid analysis. Lipids, however, are well-suited for MALDI since all experiments can be performed in a sole organic phase and, thus, extremely homogeneous matrix/analyte mixtures are formed. We report here for the first time the application of MALDI-TOF-MS for the analysis of diacylglycerols, phosphatidylcholines, and (poly)phosphoinositides. It is shown that in a matrix of 2,5-dihydroxybenzoic acid the molecular ions (M + 1) of phosphatidylcholines as well as the corresponding adducts of different phosphoinositides are easily detected even in complex mixtures, and thus, detailed data on the fatty acid composition are provided. In contrast, diacylglycerols are mainly detected as the corresponding sodium or potassium adducts, but not as the protonated forms. Fragmentation reactions of fatty acids on the double bonds and on the polar lipid head group are observed to a minor extent in the spectra of all investigated lipids. Generally, choline derivatives are most sensitive toward further fragmentation reactions. Due to its very high sensitivity (up to picomolar concentrations) MALDI-TOF-MS can be used for the direct investigation of biologically relevant lipid mixtures occurring, e.g. , in cell membranes. The analysis of the lipid composition of neutrophilic granulocytes is given as a representative example for future applications.

Involvement of cyclic AMP generation in the inhibition of respiratory burst by 2-phenyl-4-quinolone (YT-1) in rat neutrophils.

The inhibitory effect of 2-phenyl-4-quinolone (YT-1) on respiratory burst in rat neutrophils was investigated, and the underlying mechanism of action was assessed. YT-1 caused a concentration-dependent inhibition of the rate of O2.- release from rat neutrophils in response to formylmethionyl-leucyl-phenylalanine (fMLP), but not to phorbol 12-myristate 13-acetate (PMA), with an IC50 value of 60.7+/-8.2 microM. A comparable effect was also demonstrated in the inhibition of O2 consumption. Unlike superoxide dismutase, YT-1 had no effect on O2.- generation in the xanthine-xanthine oxidase system and during dihydroxyfumaric acid autoxidation. The fMLP-induced inositol trisphosphate (IP3) formation was unaffected by YT-1. In addition, YT-1 did not affect the initial spike of [Ca2+]i, but it accelerated the rate of [Ca2+]i decline in cells in response to fMLP. YT-1 was found to have little effect on the activity of neutrophil cytosolic protein kinase C (PKC). YT-1 increased the cellular cyclic AMP level, while having no effect on the cyclic GMP level. In addition, YT-1 increased neutrophil cytosolic protein kinase A (PKA) activity, but had no direct effect on the enzyme activity of pure porcine heart PKA. When neutrophils were treated with (8R,9S,11S)-(-)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetra hydro-8,11-epoxy- 1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinde n-1-one, (KT 5720), a PKA inhibitor, the inhibition of O2.- generation by YT-1, as well as by prostaglandin E1 (PGE1) and dibutyryl cyclic AMP, was attenuated effectively. YT-1 did not activate the adenylate cyclase associated with neutrophil particulate fraction but inhibited the cytosolic phosphodiesterase (PDE) activity in a concentration-dependent manner. Neutrophils treated with YT-1 had a more pronounced increase in cellular cyclic AMP level by PGE1. Moreover, the ability of PGE1 to inhibit the respiratory burst in neutrophils was greatly enhanced by YT-1. These results suggest that the increase in cellular cyclic AMP levels by YT-1 through the inhibition of PDE (probably PDE4 isoenzyme) activity is involved in its inhibition of fMLP-induced respiratory burst in rat neutrophils.