Integrin signalling regulates the nuclear localization and function of the lysophosphatidic acid receptor-1 (LPA1) in mammalian cells

We show that LPA1 (lysophosphatidic acid receptor-1) is constitutively localized in the nucleus of mammalian cells. LPA1 also traffics from cell membranes to the nucleus in response to LPA (lysophosphatidic acid). Several lines of evidence suggest an important role for cell-matrix interaction in regulating the constitutive nuclear localization of LPA1. First, the RGDS peptide, which blocks cell matrix-induced integrin clustering and cytoskeletal rearrangement, reduced the number of cells containing LPA1 in the nucleus. Secondly, a higher proportion of cells contained nuclear LPA1 when adhesion on fibronectin-coated glass was compared with adherence to polylysine-coated glass. Thirdly, pre-treatment of cells with the Rho kinase inhibitor (Y27632) or the myosin light chain kinase inhibitor (ML9) reduced the number of cells containing nuclear LPA1. The addition of LPA and/or Ki16425 (which binds to LPA1) to isolated nuclei containing LPA1 induced the phosphorylation of several proteins with molecular masses of 34, 32, 14 and 11 kDa. These findings demonstrate that trafficking of LPA1 to the nucleus is influenced by cell-matrix interactions and that nuclear LPA1 may be involved in regulating intranuclear protein phosphorylation and signalling.     

Nitric oxide modulation of ET(B) receptor-induced vasopressin release by rat and mouse hypothalamo-neurohypophyseal explants

Endothelin (ET) peptides stimulate vasopressin (AVP) secretion via ET(B) receptors at hypothalamic loci. Nitric oxide modulates the actions of ET in the cardiovascular system and also influences neurotransmission and specifically suppresses firing of magnocellular neurons. The purpose of these studies was to ascertain whether nitric oxide, generated in response to ET(B) receptor stimulation, buffers the stimulatory effect of ET and suppresses AVP release. Studies were performed using a pharmacological approach in hypothalamo-neurohypophyseal explants from rats, and an alternative strategy using explants from mice with an inactivating mutation of neuronal NOS (nNOS-/-) and their wild-type parent strain. Whole explants in standard culture or only the hypothalamus of compartmentalized explants was exposed to the ET(B) selective agonist, IRL 1620 (10(-13) to 10(-8) M). Rat and wild-type mouse explants displayed similar responses, although absolute basal release rates were higher from murine explants. Maximal AVP release at 0.1 nM IRL 1620 was 311 +/- 63 (rat) and 422 +/- 112% basal x explant(-1) x h(-1) (mouse). Sodium nitroprusside (SNP; 0.1 mM) suppressed maximal AVP release to basal values. N(omega)-nitro-L-arginine methyl ester (L-NAME, 0.1 microM), which did not itself stimulate AVP secretion, more than doubled the response to 1 pM IRL 1620, from 136 +/- 28 to 295 +/- 49% basal x explant(-1) x h(-1) (P < 0.05) by rat explants. Explants from wild-type mice responded similarly. Explants from nNOS-/- mice had higher basal AVP secretory rate in response to 1 pM IRL 1620: 271 +/- 48 compared with 150 +/- 24% basal x explant(-1) x h(-1) (P < 0.05) from wild-type murine explants. In the nNOS-/-, SNP suppressed stimulated release, and L-NAME exerted no additional stimulatory effect: 243 +/- 38% basal x explant(-1) x h(-1). Thus nitric oxide inhibits the AVP secretory response induced by ET(B) receptor activation within the hypothalamo-neurohypophyseal system and is generated primarily by the nNOS isoform. The modulation of AVP secretion by ET and also nitric oxide can take place independently from their effects on cerebral blood flow, systemic hemodynamics, or the arterial baroreflex.     

Design and expression of a dimeric form of human immunodeficiency virus type 1 antibody 2G12 with increased neutralization potency

The antigen-binding fragment of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) antibody 2G12 has an unusual three-dimensional (3D) domain-swapped structure with two aligned combining sites that facilitates recognition of its carbohydrate epitope on gp120. When expressed as an intact immunoglobulin G (IgG), 2G12 formed typical IgG monomers containing two combining sites and a small fraction of a higher-molecular-weight species, which showed a significant increase in neutralization potency (50- to 80-fold compared to 2G12 monomer) across a range of clade A and B strains of HIV-1. Here we show that the higher-molecular-weight species corresponds to a 2G12 dimer containing four combining sites and present a model for how intermolecular 3D domain swapping could create a 2G12 dimer. Based on the structural model for a 3D domain-swapped 2G12 dimer, we designed and tested a series of 2G12 mutants predicted to increase the ratio of 2G12 dimer to monomer. We report a mutation that effectively increases the 2G12 dimer/monomer ratio without decreasing the expression yield. Increasing the proportion of 2G12 dimer compared to monomer could lead to a more potent reagent for gene therapy or passive immunization.     

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples

Background  

Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals.     

Augmenting the activity of antifungal agents against aspergilli using structural analogues of benzoic acid as chemosensitizing agents

A number of benzoic acid analogues showed antifungal activity against strains of Aspergillus flavus, Aspergillus fumigatus and Aspergillus terreus, causative agents of human aspergillosis, in in vitro bioassays. Structure-activity analysis revealed that antifungal activities of benzoic and gallic acids were increased by addition of a methyl, methoxyl or chloro group at position 4 of the aromatic ring, or by esterification of the carboxylic acid with an alkyl group, respectively. Thymol, a natural phenolic compound, was a potent chemosensitizing agent when co-applied with the antifungal azole drugs fluconazole and ketoconazole. The thymol-azole drug combination demonstrated complete inhibition of fungal growth at dosages far lower than the drugs alone. Co-application of thymol with amphotericin B had an additive effect on all strains of aspergilli tested with the exception of two of three strains of A. terreus, where there was an antagonistic effect. Use of two mitogen-activated protein kinase (MAPK) mutants of A. fumigatus, sakAΔ and mpkCΔ, having gene deletions in the oxidative stress response pathway, indicated antifungal and/or chemosensitization activity of the benzo analogues was by disruption of the oxidative stress response system. Results showed that both these genes play overlapping roles in the MAPK system in this fungus. The potential of safe, natural compounds or analogues to serve as chemosensitizing agents to enhance efficacy of commercial antifungal agents is discussed.     

Reactive oxygen species-mediated regulation of the Na+-H+ exchanger 1 gene expression connects intracellular redox status with cells' sensitivity to death triggers

We have previously demonstrated that a slight increase in intracellular superoxide (O2*-) anion confers resistance to death stimuli. Using pharmacological and molecular approaches to manipulate intracellular O2*-, here we report that an increase in intracellular O2*- anion induces Na+/H+ exchanger 1 (NHE-1) gene promoter activity resulting in increased NHE-1 protein expression, which strongly correlates with the resistance of cells to death stimuli. In contrast, exposure to exogenous hydrogen peroxide suppressed NHE-1 promoter activity and gene expression, and increased cell sensitivity to death triggers. Furthermore, the increase in cell sensitivity to death upon downregulation of NHE-1 gene expression correlates with reduced capacity of cells to recover from an acid load, while survival upon overexpression of NHE-1 appears independent of its pump activity. These findings indicate that NHE-1 is a redox-regulated gene, and provide a novel intracellular target for the redox control of cell death sensitivity.     

Effectiveness of potassium sulfate in mitigating salt-induced adverse effects on different physio-biochemical attributes in sunflower (Helianthus annuus L.)

To assess whether foliar application of K+S as potassium sulfate (K₂SO₄) could alleviate the adverse effects of salt on sunflower (Helianthus annuus L. cv. SF-187) plants, a greenhouse experiment was conducted. There were two NaCl levels (0 and 150 mM) applied to the growth medium and six levels of K+S as K₂SO₄ (NS (no spray), WS (spray of water+0.1% Tween 20 solution), 0.5% K+0.21% S, 1.0% K+0.41% S, 1.5% K+0.62% S, and 2.0% K+0.82% S in 0.1% Tween-20 solution) applied two times foliarly to non-stressed and salt-stressed sunflower plants. Salt stress markedly repressed the growth, yield, photosynthetic pigments, water relations and photosynthetic attributes, quantum yield (F_v/F_m), leaf and root K⁺, Mg²⁺, P, Ca²⁺, N as well as K⁺/Na⁺ ratios, while it enhanced the cell membrane permeability, and leaf and root Na⁺ and Cl⁻ concentrations. Foliar application of potassium sulfate significantly improved growth, achene yield, photosynthetic and transpiration rates, stomatal conductance, water use efficiency, leaf turgor and enhanced shoot and leaf K⁺ of the salt-stressed sunflower plants, but it did not improve leaf and root Na⁺, Cl⁻, Mg²⁺, P, Ca²⁺, N as well as K⁺/Na⁺ ratios. The most effective dose of K+S for improving growth and achene yield was found to be 1.5% K+0.62% S and 1% K+0.41% S, respectively. Improvement in growth of sunflower plants due to exogenously applied K₂SO₄ was found to be linked to enhanced photosynthetic capacity, water use efficiency, leaf turgor and relative water content.     

Combined affinity and rate constant distributions of ligand populations from experimental surface binding kinetics and equilibria

The present article considers the influence of heterogeneity in a mobile analyte or in an immobilized ligand population on the surface binding kinetics and equilibrium isotherms. We describe strategies for solving the inverse problem of calculating two-dimensional distributions of rate and affinity constants from experimental data on surface binding kinetics, such as obtained from optical biosensors. Although the characterization of a heterogeneous population of analytes binding to uniform surface sites may be possible under suitable experimental conditions, computational difficulties currently limit this approach. In contrast, the case of uniform analytes binding to heterogeneous populations of surface sites is computationally feasible, and can be combined with Tikhonov-Phillips and maximum entropy regularization techniques that provide the simplest distribution that is consistent with the data. The properties of this ligand distribution analysis are explored with several experimental and simulated data sets. The resulting two-dimensional rate and affinity constant distributions can describe well experimental kinetic traces measured with optical biosensors. The use of kinetic surface binding data can give significantly higher resolution than affinity distributions from the binding isotherms alone. The shape and the level of detail of the calculated distributions depend on the experimental conditions, such as contact times and the concentration range of the analyte. Despite the flexibility introduced by considering surface site distributions, the impostor application of this model to surface binding data from transport limited binding processes or from analyte distributions can be identified by large residuals, if a sufficient range of analyte concentrations and contact times are used. The distribution analysis can provide a rational interpretation of complex experimental surface binding kinetics, and provides an analytical tool for probing the homogeneity of the populations of immobilized protein.