Dependence on the lipophilicity of maleimide derivatives in their inhibitory action upon chemotaxis in neutrophils

Summary Modification of polymorphonuclear neutrophils by a series of maleimide derivatives with various degrees of lipophilicity and hydrophilicity indicated that hydrophilic reagents had little effect on chemotaxis, whereas the degree of the inhibitory effect of lipophilic reagents on the chemotaxis was parallel to the degree of their lipophilicity.This work was supported in part by a grant-in-aid for Scientific Research (No.267043) from the Ministry of Education, Science and Culture, Japan, awarded to T.Y.     

A possible preferential inhibition of chemotaxis of polymorphonuclear neutrophils by a chemical modification.

The modification of neutrophils with amino group blocking reagents of different chemical specificities showed that dansyl chloride caused inhibition of chemotaxis without suppression of random movement. Dansylated neutrophils, like control cells, ingested bacteria. Neither the stimulated cyanide-insensitive respiration, nor lactate production during phagocytosis, was affected significantly by dansylation as compared with the inhibition of directed movement.     

A possible preferential inhibition of chemotaxis of polymorphonuclear neutrophils by a chemical modification

Summary The modification of neutrophils with amino group blocking reagents of different chemical specificities showed that dansyl chloride caused inhibition of chemotaxis without suppression of random movement. Dansylated neutrophils, like control cells, ingested bacteria. Neither the stimulated cyanide-insensitive respiration, nor lactate production during phagocytosis, was affected significantly by dansylation as compared with the inhibition of directed movement.     

Affinity of 1,2-substituted oxytocin analogues to the uterus receptor: Free-Wilson and Hansch analysis.

The analysis of pA2 values for 1,2-substituted oxytocin analogues suggests a significant resonance effect of p-substituted groups in 2-tyrosine when the hormone binds to its uterus receptor, whereas the N-terminal amino group exerts less clearly characterized effects (participation of its lipophilicity and molecular volume can be assumed.     

Transduction of the chemotactic cAMP signal across the plasma membrane ofDictyostelium cells

AggregatingDictyostelium cells secrete cAMP during cell aggregation. cAMP induces two fast responses, the production of more cAMP (relay) and directed cell locomotion (chemotaxis). Extracellular cAMP binds to G-protein-coupled receptors leading to the activation of second messenger pathways, including the activation of adenylyl cyclase, guanylyl cyclase, phospholipase C and the opening of plasma membrane Ca²⁺ channels. Many genes encoding these sensory transduction proteins have been cloned and null mutants of nearly all components have been characterized in detail. Undoubtedly, activation of adenylyl cyclase is the most complex, involving G-proteins, a soluble protein called CRAC and components of the MAP kinase pathway. Null mutants in this pathway do not aggregate, but can exhibit chemotaxis and develop normally when supplied with exogenous cAMP. The pathways leading to the activation of phospholipase C were identified, but unexpectedly, deletion of the phospholipase C gene has no effect on chemotaxis and development, nor on intracellular Ins(1,4,5)P₃ levels; the metabolism of this second messenger will be discussed in some detail. Activation of guanylyl cyclase is G-protein-dependent and essential for chemotaxis. Analysis of a collection of chemotactic mutants reveals that most mutants are defective in either the production or intracellular detection of cGMP, thereby placing this second messenger at the center of chemotactic signal transduction. Analysis of the cAMP-mediated opening of plasma membrane calcium channels in signal transduction mutants suggests that it has two components, one that depends on G-proteins and intracellular cGMP and one that is G-protein-independent.     

Affinity of 1,2-substituted oxytocin analogues to the uterus receptor: Free-Wilson and Hansch analysis

Summary The analysis of pA₂ values for 1,2-substituted oxytocin analogues suggests a significant resonance effect of psubstituted groups in 2-tyrosine when the hormone binds to its uterus receptor, whereas the N-terminal amino group exerts less clearly characterized effects (participation of its lipophilicity and molecular volume can be assumed).Supported by the Swiss National Science Foundation, grant No. 3.040.76.     

Bacterial chemoreceptors and chemoeffectors

Bacteria use chemotaxis signaling pathways to sense environmental changes. Escherichia coli chemotaxis system represents an ideal model that illustrates fundamental principles of biological signaling processes. Chemoreceptors are crucial signaling proteins that mediate taxis toward a wide range of chemoeffectors. Recently, in deep study of the biochemical and structural features of chemoreceptors, the organization of higher-order clusters in native cells, and the signal transduction mechanisms related to the on–off signal output provides us with general insights to understand how chemotaxis performs high sensitivity, precise adaptation, signal amplification, and wide dynamic range. Along with the increasing knowledge, bacterial chemoreceptors can be engineered to sense novel chemoeffectors, which has extensive applications in therapeutics and industry. Here we mainly review recent advances in the E. coli chemotaxis system involving structure and organization of chemoreceptors, discovery, design, and characterization of chemoeffectors, and signal recognition and transduction mechanisms. Possible strategies for changing the specificity of bacterial chemoreceptors to sense novel chemoeffectors are also discussed.     

Isoelectric focusing study of radiation damage to dry conalbumin.

The effect of gamma-rays on iron-free conalbumin was studied by isoelectric focusing technique. The results obtained can be interpreted on the basis of radiation-induced conformational changes of the macromolecules through a cleavage of disulphide bridges. Treatments with reagents acting on disulphide bridges lead to isoelectric focusing patterns that confirm this hypothesis.     

Lack of effect of colchicine on human neutrophil chemotaxis under agarose

Summary Human polymorphonuclear neutrophil chemotaxis under agarose was assessed following in vitro treatment of cells with colchicine. No inhibition of random migration or chemotaxis was detected. The discrepancy between these results and reports utilizing micropore filter assays could be due to the need for cell deformability in the latter.Supported by a grant from the Veterans Administration and Research grant AM 19349 from the NIAMDD, NIH.     

Purinergic regulation of neutrophil chemotaxis

Chemotaxis allows polymorphonuclear neutrophils (PMN) to rapidly reach infected and inflamed sites. However, excessive influx of PMN damages host tissues. Better knowledge of the mechanisms that control PMN chemotaxis may lead to improved treatments of inflammatory diseases. Recent findings suggest that ATP and adenosine are involved in PMN chemotaxis. Therefore, these purinergic signaling processes may be suitable targets for novel therapeutic approaches to ameliorate host tissue damage.     

The effect of sulfhydryl reagents upon the activity of 40S ribosomal subunits

Summary p-Chloromercuribenzoate inhibited the poly (U)-dependent binding of Phe-tRNA to the 40S ribosomal subunit but displayed no inhibitory effect on the binding of poly (U) to the ribosome. Other sulfhydryl reagents tested, likeN-ethylmaleimide and iodoacetamide, did not affect the binding of Phe-tRNA to the small ribosomal subunit.     

Physicochemical profiling in drug research: a brief survey of the state-of-the-art of experimental techniques

This review begins with a general presentation of the new paradigm of drug discovery, with its emphasis on the rapid identification and elimination of compounds with unsuitable physicochemical and pharmacokinetic properties. The focus of the paper is on the various experimental methods used to determine such key physicochemical properties as ionization, lipophilicity and distribution in isotropic and anisotropic systems, solubility, and permeability across artificial membranes. Both traditional and high-throughput methods are presented and their limits highlighted. The text concludes with the trade-off between quantity/speed in high-throughput screening techniques versus greater data quality in the more labor-intensive methods. Received 23 April 2002; received after revision 25 June 2002; accepted 11 July 2002 RID="*" ID="*"Corresponding author.     

Relation between stimulus intensity and neutrophil chemotactic response

Summary The effect of chemotactic peptides which lack chemokinetic activity has been investigated. The neutrophil response is proportional to the logarithm of the stimulus intensity, or alternatively a power function with an exponent of 0.3. Equal responses are obtained for equal ratios between the peptide concentration in the lower compartment and the threshold concentration. The significance of Weber-Fechner's law in leucocyte chemotaxis is discussed.We thank Miss B. Zanolari, Mrs M. Marti, Mrs Hauck and Mr Dietz for excellent technical assistance. This work was supported by the Swiss National Foundation for Scientific Research.     

Role of the conserved glutamine 291 in the rat γ-aminobutyric acid transporter rGAT-1

We investigated the role of the Q291 glutamine residue in the functioning of the rat γ-aminobutyric acid (GABA) transporter GAT-1. Q291 mutants cannot transport GABA or give rise to transient, leak and transport-coupled currents even though they are targeted to the plasma membrane. Coexpression experiments of wild-type and Q291 mutants suggest that GAT-1 is a functional monomer though it requires oligomeric assembly for membrane insertion. We determined the accessibility of Q291 by investigating the impact of impermeant sulfhydryl reagents on cysteine residues engineered in close proximity to Q291. The effect of these reagents indicates that Q291 faces the external aqueous milieu. The introduction of a steric hindrance close to Q291 by means of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide modification of C74A/T290C altered the affinity of the mutant for cations. Taken together, these results suggest that this irreplaceable residue is involved in the interaction with sodium or in maintaining the cation accessibility to the transporter. Received 24 October 2005; accepted 11 November 2005     

Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene6: in vitro and in vivo studies

The present study was undertaken to develop an efficient non-viral gene delivery system for cardiovascular gene therapy. We investigated transfection efficiency and toxic properties of the new transfection reagent, FuGene6, and compared it with two other transfection reagents, Tfx-50 and LipoTaxi. For in vivo experiments, the plasmid was delivered intramuscularly via transplantation of fibroblasts transfected with plasmid and FuGene6. Conditions for efficient gene delivery were initially studied in vitro. Human and rabbit fibroblasts were isolated from skin, cultured and transfected with phVEGF165 or pCMVbeta gal plasmids, coding for vascular endothelial growth factor (VEGF) or beta-galactosidase, respectively. The effect of the DNA amount and the DNA:transfection reagent ratio on plasmid uptake were studied. Of the transfection reagents tested, only FuGene6 provided high-efficiency and dose-dependent plasmid transfer both for cell-localised (beta-galactosidase) and secreted (VEGF) gene products. When analysed with an MTT assay, FuGene6 showed no toxicity at low doses. Optimised conditions were applied for in vivo reporter gene delivery. Rabbits were injected intramuscularly with ex vivo-transfected fibroblasts. As in in vitro studies, ex vivo-transfected fibroblasts showed highly efficient gene expression in vivo. Tissue sections were analysed with macrophage-specific immunostaining. No signs of inflammation were seen in the region of fibroblast injection. This study demonstrates that FuGene6 is a highly efficient transfection reagent that may be useful for in vitro non-viral transfection of primary human and rabbit fibroblasts and for in vivo therapeutic non-viral gene delivery.     

Effects of prolactin (PRL) on gonadotropin release in mice with congenital PRL deficiency

Summary In prolactin (PRL)-deficient male dwarf mice, treatment with PRL stimulates the release of FSH without affecting plasma LH levels. We now report that this effect of PRL is not mediated by the testes and that PRL does not modify FSH or LH release in female dwarf mice.This work was supported by the National Institute of Child Health and Human Development through a grand HD12642 and RIA Core of grant HD10202. We thank NIAMDD and Drs G.D. Niswender and L.E. Reichert, Jr, for reagents used in radioimmunoassays.     

Modification of rat polymorphonuclear chemotaxis during an acute inflammatory reaction]

The appearance of a serum factor which inhibited the chemotaxis of polymorphonuclears was demonstrated during various acute inflammatory reactions. The activity of this factor was studied in vivo and in vitro.     

二茂铁基三级醇的合成

将RMgCl（3倍量）和ZnCl2（1倍量）混合原位生成高效亲核性烷基化试剂有机锌酸复合物，来对二茂铁基酮进行烷基化反应，制备二茂铁基三级醇。获得了对二茂铁基酮的高效烷基化结果，如i-PrMgCl与二茂铁基乙酮的烷基化产率从20％提高到91％，还原副产物产率相应从36％降低至0％。较炕基锂和格氏试剂，前者能够有效地进行二茂铁基酮的烷基化，该方法将成为非常好的通用烷基化方法，并为工业化大量生产二茂铁基三级醇探索了道路。     

EP4 receptor stimulation down-regulates human eosinophil function

Accumulation of eosinophils in tissue is a hallmark of allergic inflammation. Here we observed that a selective agonist of the PGE₂ receptor EP4, ONO AE1-329, potently attenuated the chemotaxis of human peripheral blood eosinophils, upregulation of the adhesion molecule CD11b and the production of reactive oxygen species. These effects were accompanied by the inhibition of cytoskeletal rearrangement and Ca²⁺ mobilization. The involvement of the EP4 receptor was substantiated by a selective EP4 antagonist, which reversed the inhibitory effects of PGE₂ and the EP4 agonist. Selective kinase inhibitors revealed that the inhibitory effect of EP4 stimulation on eosinophil migration depended upon activation of PI 3-kinase and PKC, but not cAMP. Finally, we found that EP4 receptors are expressed by human eosinophils, and are also present on infiltrating leukocytes in inflamed human nasal mucosa. These data indicate that EP4 agonists might be a novel therapeutic option in eosinophilic diseases.     

Molecular basis of localized responses during chemotaxis in amoebae and leukocytes

Free-living amoebae as well as mammalian leukocytes sense chemoattractants with seven helix receptors linked to G-proteins. The cells respond by extending pseudopods and moving in the direction of the highest concentration. Recent studies using GFP-tagged proteins in Dictyostelium have shown that the directional response becomes sharply localized downstream of the receptors and G-proteins but upstream of the actin cytoskeleton. These studies together with the isolation novel genes by insertional mutagenesis in Dictyostelium are leading to a new understanding of chemotaxis in eucaryotic cells.