The hydrolysis of lysophospholipids and nucleotides by autotaxin (NPP2) involves a single catalytic site

Autotaxin (NPP2) is a tumor cell motility-stimulating factor that displays both a nucleotide pyrophosphatase/phosphodiesterase activity and a recently described lysophospholipase D activity. The hydrolysis of nucleotides is a metal-assisted reaction that occurs via a nucleotidylated threonine in the catalytic site. We show here that the catalytic site threonine and the metal-coordinating residues are also essential for the hydrolysis of lysophospholipids. In comparing the substrate specificity of NPP2 and the closely related NPP1 and NPP3, we found that only NPP2 displayed a lysophospholipase D activity, whereas NPP1 and NPP3 had a much higher nucleotide pyrophosphatase activity.     

Comparison of progesterone concentration determination by 12 non-isotopic immunoassays and gas chromatography/mass spectrometry in 99 human serum samples

A single serum progesterone determination may be highly predictive for early pregnancy and in vitro fertilisation and embryo-transfer outcomes. We therefore compared 12 direct non-isotopic progesterone immunoassays with gas-chromatography/mass spectrometry (GC/MS). For each assay, data from the analysis of 99 individual sera were compared with data obtained by GC/MS, using regression and bias plot analyses and the ratio method. We observed a larger difference in concentration between high and low values and a broader distribution of results for immunoassays than for GC/MS. All immunoassays displayed bias in the calibration process and a lack of specificity and/or sensitivity, to various degrees. We tried to identify the parameters of the assay procedure that might contribute to these discrepancies. None of the criteria investigated (antibodies, control and preparation of calibrators, blocking agents and choice of tracer) had a significant effect when studied alone.     

Angiotensin II is bound to both receptors AT1 and AT2, parallel to the transmembrane domains and in an extended form

We have applied photoaffinity labelling methods combined with site-directed mutagenesis towards the two principal angiotensin II (AnglI) receptors AT1 and AT2 in order to determine contact points between AngII and the two receptors. We have first identified the receptor contact points between an N- and a C-terminal residue of the AngII molecule and the AT1 receptor and constructed with this stereochemical restriction a molecular model of AT1. A similar approach with a modified procedure of photoaffinity labelling has allowed us now to determine contact points also in the AT2 receptor. Molecular modelling of AT2 on the rhodopsin scaffold and energy minimisation of AngII binding into this AT2 model produced a model strikingly similar to the AT11 structure. Superposition of the experimentally obtained contact points of AngII with AT2 upon this model revealed excellent congruence between the experimental and modelling results. Conclusions: (i) athough AT1 and AT2 have quite low sequence homology, they both bind AngII with similar affinity and in an almost identical fashion, as if the ligand dictates the way it has to be bound, and (ii) in its bound form, AngII adopts an extended conformation in both AT1 and AT2, contrary to all previous predictions.     

Phosphorylation-dependent interaction between the splicing factors SAP155 and NIPP1

NIPP1 is a ubiquitously expressed nuclear protein that functions both as a regulator of protein Ser/Thr phosphatase-1 and as a splicing factor. The N-terminal part of NIPP1 consists of a phosphothreonine-interacting Forkhead-associated (FHA) domain. We show here that the FHA domain of NIPP1 interacts in vitro and in vivo with a TP dipeptide-rich fragment of the splicing factor SAP155/SF3b(155), a component of the U2 small nuclear ribonucleoprotein particle. The NIPP1-SAP155 interaction was entirely dependent on the phosphorylation of specific TP motifs in SAP155. Mutagenesis and competition studies revealed that various phosphorylated TP motifs competed for binding to the same site in the FHA domain. The SAP155 kinases in cell lysates were blocked by the Ca(2+) chelator EGTA and by the cyclin-dependent protein kinase inhibitor roscovitine. The phosphorylation level of SAP155 was dramatically increased during mitosis, and accordingly the activity of SAP155 kinases was augmented in mitotic lysates. We discuss how the interaction between NIPP1 and SAP155 could contribute to spliceosome (dis)assembly and the catalytic steps of splicing.     

The protein phosphatase-1 regulator NIPP1 is also a splicing factor involved in a late step of spliceosome assembly

NIPP1 is a ubiquitous regulator of protein phosphatase-1 (PP1) and is targeted to the splicing factor storage sites (speckles) in the nucleus by its forkhead-associated domain. We show here that NIPP1 is also a component of the spliceosomes in HeLa cell-splicing extracts and that the interaction with the spliceosomes requires a functional forkhead-associated domain. The in vitro splicing of beta-globin pre-mRNA was not affected by exogenous wild type NIPP1 but was blocked by mutants that lacked residues 225-329. The inhibition by these dominant negative mutants resulted from a block in a late phase of spliceosome assembly, i.e. at the transition between the B-complex and the C-complex. Site-directed mutagenesis furthermore showed that this spliceosomal function of NIPP1 was unrelated to its ability to bind PP1 or RNA. Our data suggest that NIPP1 can function independently as a splicing factor and a phosphatase regulator.     

Decreased hemoglobin concentrations,hyperparasitemia, and severe malaria are associated with increased Plasmodium falciparum gametocyte carriage

To determine factors influencing gametocyte carriage, a cross-sectional study was conducted among 512 patients admitted for Plasmodium falciparum malaria. After adjustments for potential confounders, hemoglobin concentrations were lower in gametocyte carriers 10.5 (+/-2.5) than in patients without gametocytes 12.5 (+/-2.3) (P < 0.0001). Hemoglobin concentrations were negatively correlated with peak gametocyte counts (Spearman's p = -0.37, P < 0.0001) and gametocyte carriage durations (Spearman's p = -(0.30, P < 0.0001). Adjustments for the duration of the malaria episode and other potential confounders did not alter the association (P < 0.0001). After adjustment for potential confounders, the median asexual parasitemia was higher in patients with gametocytes than in patients without gametocytes (P = 0.003). Severe malaria cases were more likely to have gametocytes (65%) than malaria with hyperparasitemia (38%) or mild malaria (31%) (P = 0.0001). These findings suggest that events surrounding anemia and tissue hypoxia stimulate Plasmodium falciparum gametocytogenesis.     

A simplified method for the detection of Y chromosome microdeletions in infertile men using a multiplex sequence-tagged site-based amplification

Sixteen sequence-tagged sites (STSs) were combined in five amplification reactions, to screen for deletions of DNA fragments located within the AZFa, AZFb, and AZFc regions of the Y chromosome. This multiplex strategy is fast and reliable, and most of the azoospermia-associated deletions reported so far are detected with this simplified method. Internal control STSs are included that allow discrimination between deletion and failure of amplification.     

Chronic carbon monoxide enhanced IbTx-sensitive currents in rat resistance pulmonary artery smooth muscle cells

Exogenous carbon monoxide (CO) can induce pulmonary vasodilation by acting directly on pulmonary artery (PA) smooth muscle cells. We investigated the contribution of K+ channels to the regulation of resistance PA resting membrane potential on control (PAC) rats and rats exposed to CO for 3 wk at 530 parts/million, labeled as PACO rats. Whole cell patch-clamp experiments revealed that the resting membrane potential of PACO cells was more negative than that of PAC cells. This was associated with a decrease of membrane resistance in PACO cells. Additional analysis showed that outward current density in PACO cells was higher (50% at +60 mV) than in PAC cells. This was linked to an increase of iberiotoxin (IbTx)-sensitive current. Chronic CO hyperpolarized membrane of pressurized PA from -46.9 +/- 1.2 to -56.4 +/- 2.6 mV. Additionally, IbTx significantly depolarized membrane of smooth muscle cells from PACO arteries but not from PAC arteries. The present study provides initial evidence of an increase of Ca2+-activated K+ current in smooth muscle cells from PA of rats exposed to chronic CO.     

Muscarinic M2 receptors in acetylcholine-isoproterenol functional antagonism in human isolated bronchus

The muscarinic functional antagonism of isoproterenol relaxation and the contribution of muscarinic M2 receptors were examined in human isolated bronchus. In intact tissues, acetylcholine (ACh) precontraction decreased isoproterenol potency and maximal relaxation (-log EC50 shift = -1.49 +/- 0.16 and E(max) inhibition for 100 microM ACh = 30%) more than the same levels of histamine contraction. The M2 receptor-selective antagonist methoctramine (1 microM) reduced this antagonism in ACh- but not histamine-contracted tissues. Similar results were obtained for forskolin-induced relaxation. After selective inactivation of M3 receptors with 4-diphenylacetoxy-N-(2-chloroethyl)piperadine hydrochloric acid (30 nM), demonstrated by abolition of contractile and inositol phosphate responses to ACh, muscarinic recontractile responses were obtained in U-46619-precontracted tissues fully relaxed with isoproterenol. Methoctramine antagonized recontraction, with pK(B) (6.9) higher than in intact tissues (5.4), suggesting participation of M2 receptors. In M3-inactivated tissues, methoctramine augmented the isoproterenol relaxant potency in U-46619-contracted bronchus and reversed the ACh-induced inhibition of isoproterenol cAMP accumulation. These results indicate that M2 receptors cause indirect contraction of human bronchus by reversing sympathetically mediated relaxation and contribute to cholinergic functional antagonism.