Chemotactic peptide, calcium and guanine nucleotide regulation of phospholipase C activity in membranes from DMSO-differentiated HL60 cells

Membranes prepared from DMSO-differentiated HL60 cells labeled with [3H]inositol hydrolyze polyphosphoinositides in a Ca2+-dependent manner, generating inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). Incubation of membranes with GTP or GTP gamma S reduces the concentration of Ca2+ required for activation. This nucleotide effect is potentiated by formyl-Met-Leu-Phe (FMLP). Pertussis toxin inhibits FMLP-induced augmentation, but not the induction of IP2/IP3 formation by GTP or GTP gamma S. These results suggest that differentiated HL60 cells contain a membrane-associated phospholipase C that degrades polyphosphoinositides and that activation of this enzyme is mediated by at least two guanine nucleotide binding proteins, one of which is linked to FMLP receptors and is pertussis toxin sensitive.     

Identification of phospholipid platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in human amniotic fluid and urine

Human amniotic fluid and fetal urine were examined for the presence of phospholipid platelet-activating factor (PAF). PAF was detected in lipid extracts of some samples of amniotic fluid obtained from women in labor but it was undetectable in samples of amniotic fluid obtained before the onset of labor. PAF was identified by chromatographic mobility, platelet aggregation and chemical modifications. LysoPAF was also present in amniotic fluid at higher concentrations than those of PAF. Both PAF and lysoPAF were identified also in newborn and adult urine.     

Phosphatidylinositol-specific phospholipase-C of platelets: association with 1,2-diacyglycerol-kinase and inhibition by cyclic-AMP.

Horse platelets prelabeled with [¹⁴C]arachidonate (AA) rapidly degrade [¹⁴C]phosphatidylinositol (PI) to [¹⁴C]1,2-diacylglycerol (DG) upon treatment with deoxycholate (DOC). This phospholipase-C (PLC) activity is specific for PI since other phospholipids or neutral lipids are not affected. Although exogenous Ca²⁺ is not required for activity, EGTA or EDTA abolishes PI degradation. Addition of Mg²⁺ (1 mM) and ATP (1 mM) results in phosphorylation of the DG and production of phosphatidic acid (PA). Higher concentrations of DOC inhibit DG-kinase. These observations, together with the fact that different platelet agonists induce a rapid degradation of PI and production of PA, indicate that PLC and DG-kinase activities are intimately linked. Incubation of platelets with dibutyryl cyclic-AMP, cyclic AMP-phosphodiesterase inhibitors and pyridoxal-5′-phosphate, which prevent platelet aggregation, inhibits the DOC-dependent conversion of PI to DG. The activity of PLC may play a central role in mediating platelet function and aggregation.     

Phosphatidylinositol metabolism in rat hepatocytes stimulated by glycogenolytic hormones. Effects of angiotensin, vasopressin, adrenaline, ionophore A23187 and calcium-ion deprivation

1. The effects on phosphatidylinositol metabolism of three Ca(2+)-mobilizing glycogenolytic hormones, namely angiotensin, vasopressin and adrenaline, have been investigated by using rat hepatocytes. 2. All three hormones stimulate both phosphatidylinositol breakdown and the labelling of this lipid with (32)P. 3. The response to angiotensin occurs quickly, requires a high concentration of the hormone and is prevented by [1-sarcosine, 8-isoleucine]angiotensin, a specific angiotensin antagonist that does not prevent the responses to vasopressin and to adrenaline. This response therefore seems to be mediated by angiotensin-specific receptors. 4. [1-Deaminocysteine,2-phenylalanine,7-(3,4-didehydroproline),8-arginine] vasopressin, a vasopressin analogue with enhanced antidiuretic potency, is relatively ineffective at stimulating phosphatidylinositol metabolism. This suggests that the hepatic vasopressin receptors that stimulate phosphatidylinositol breakdown are different in their ligand selectivity from the antidiuretic vasopressin receptors that activate renal adenylate cyclase. 5. Incubation of hepatocytes with ionophore A23187, a bivalent-cation ionophore, neither mimicked nor appreciably changed the effects of vasopressin on phosphatidylinositol metabolism, suggesting that phosphatidylinositol breakdown is not controlled by changes in the cytosol Ca(2+) concentration. This conclusion was supported by the observation that hormonal stimulation of phosphatidylinositol breakdown and resynthesis persists in cells incubated for a substantial period in EGTA, although this treatment somewhat decreased the phosphatidylinositol response of the hepatocyte. The phosphatidylinositol response of the hepatocyte therefore appears not to be controlled by changes in cytosol [Ca(2+)], despite the fact that this ion is thought to be the second messenger by which the same hormones control glycogenolysis. 6. These results may be an indication that phosphatidylinositol breakdown is an integral reaction in the stimulus-response coupling sequence(s) that link(s) activation of alpha-adrenergic, vasopressin and angiotensin receptors to mobilization of Ca(2+) in the rat hepatocyte.     

Artificial insemination in dromedary camels

Artificial insemination (AI) is an important technique in all domestic species to ensure rapid genetic progress. The use of AI has been reported in camelids although insemination trials are rare. This could be because of the difficulties involved in collecting as well as handling the semen due to the gelatinous nature of the seminal plasma. In addition, as all camelids are induced ovulators, the females need to be induced to ovulate before being inseminated.     

Human cord blood-derived mast cells synthesize and release I-309 in response to IgE

Mast cells are the central mediating cells of allergic reactions. Binding of allergen specific IgE to high affinity IgE receptor (Fcepsilon RI) and subsequent binding of allergen by the IgE causes receptor cross-linking and activation. In a study examining the differential gene expression in human cord blood-derived mast cells (CBMCs) mediated by activation of Fcepsilon RI both with IgE and IgE followed by cross-linking with alpha-IgE, the chemokine I-309 was found to be upregulated. I-309 is the ligand for the CCR8 receptor and is responsible for chemoattraction of TH2 type T-cells. Interestingly, I-309 RNA and protein levels were elevated not only in response to IgE/alpha-IgE activation but also by IgE alone. In addition, the I-309 levels were augmented by growth of the CBMCs in the presence of the proinflammatory cytokine IL-4. GM-CSF and MIP-1alpha secretion was also induced by IgE. These results suggest that IgE, through the production and release of cytokines such as I-309, GM-CSF and MIP-1alpha could promote an inflammatory reaction in the absence of antigen stimulation of mast cells.     

Human ADAM33: protein maturation and localization

ADAM33 (a disintegrin and metalloprotease) was recently found to be a novel asthma susceptibility gene. Domain-specific antibodies were used to study its expression and processing. When the pro-domain and catalytic domain were expressed by a stable-transfected cell line, the pro-domain was removed by cleavage within a putative furin cleavage site. The catalytic domain was active in an alpha(2)-macroglobulin complex formation assay and mutation of the catalytic site glutamic acid (E346A) eliminated activity. In transient transfections using the full-length protein, a pro-form and mature form were detectable and alternate glycosylation was demonstrated at sites within the catalytic domain. ADAM33 was detected on the cell surface, with the majority of protein detected intracellularly. The E346A mutation had no significant effect on protein processing. Endogenous ADAM33 was detected in bronchus tissue, bronchial smooth muscle cells, and MRC-5 fibroblasts, consistent with a role in the pathophysiology of asthma.     

Microarray profile of differentially expressed genes in a monkey model of allergic asthma

Background

Inhalation of Ascaris suum antigen by allergic monkeys causes an immediate bronchoconstriction and delayed allergic reaction, including a pulmonary inflammatory infiltrate. To identify genes involved in this process, the gene-expression pattern of allergic monkey lungs was profiled by microarrays. Monkeys were challenged by inhalation of A. suum antigen or given interleukin-4 (IL-4) treatment; lung tissue was collected at 4, 18 or 24 h after antigen challenge or 24 h after IL-4. Each challenged monkey lung was compared to a pool of normal, unchallenged monkey lungs.

Results

Of the approximately 40,000 cDNAs represented on the microarray, expression levels of 169 changed by more than 2.5-fold in at least one of the pairwise probe comparisons; these cDNAs encoded 149 genes, of which two thirds are known genes. The largest number of regulated genes was observed 4 h after challenge. Confirmation of differential expression in the original tissue was obtained for 95% of a set of these genes using real-time PCR. Cluster analysis revealed at least five groups of genes with unique expression patterns. One cluster contained genes for several chemokine mediators including eotaxin, PARC, MCP-1 and MCP-3. Genes involved in tissue remodeling and antioxidant responses were also identified as regulated by antigen and IL-4 or by antigen only.

Conclusion

This study provides a large-scale profile of gene expression in the primate lung following allergen or IL-4 challenge. It shows that microarrays, with real-time PCR, are a powerful tool for identifying and validating differentially expressed genes in a disease model.     

Nanostructured transducer surfaces for electrochemical biosensor construction—Interfacing the sensing component with the electrode

For fabrication of effective electrochemical biosensors, interfacing the biomolecular receptor with the underlying transducer represents a critical step. The actual approach taken depends on the tethering layer covering the transducer, which is typically either a conducting polymeric matrix, or a thin film, such as an alkanethiol monolayer. Non-specific immobilisation methods can be either covalent, or non-covalent affinity attachment, with multipoint electrostatic attachment of the sensing biomolecule to either a polyanionic or polycationic layer representing the most common approach. Many specific affinity immobilisation strategies exist, but the majority make use of one of two binding systems. The first relies on the specific and strong affinity between biotin and proteins of the avidin family, with both bioreceptor and transducer bearing pendant biotins and avidin used as the crosslinker. The second approach employs a metal chelating group on the transducer to which can be bound a polyhistidine tag present on the N- or C-terminus of the receptor protein and which can be introduced genetically, when the expression sequence for a recombinant proteins is designed.