Biomedical subjects

Alex J Laude

Publications and source records attributed to Alex J Laude.

5 recordsLinked to original sources

PubMed2005

Rapid functional assays of recombinant IP3 receptors.

Functional assays of inositol 1,4,5-trisphosphate receptors (IP3R) currently use 45Ca2+ release methods, fluorescent Ca2+ indicators within either the ER or cytosol, or electrophysiological analyses of IP3R in the nuclear envelope or artificial bilayers. None of the methods is presently amenable to the rapid, high-throughput quantitative analyses of IP3R function needed to address the structural determinants of IP3R behavior. We use a low-affinity Ca2+ indicator (Mag-fluo-4) to measure free [Ca2+] within the ER of permeabilized DT40 cells expressing only rat type 1 IP(3)R, and establish that the indicator is capable of reliably reporting the Ca(2+) release evoked by IP3. A 96-well fluorescence plate reader equipped for automated fluid additions (FlexStation, Molecular Devices) is used to monitor IP3-evoked Ca2+ release. The method allows quick and economical functional assays of recombinant IP3R in small volumes (< or = 100 microl).

Animals↗

PubMed2003

Synthesis and Ca2+-mobilizing activity of purine-modified mimics of adenophostin A: a model for the adenophostin-Ins(1,4,5)P3 receptor interaction.

The synthesis of a series of adenophostin A analogues modified at C-6 and C-2 of adenine is described. The target compounds were synthesized by a convergent route involving a modified Vorbrüggen condensation of either 6-chloropurine or 2,6-dichloropurine with a protected disaccharide, yielding two versatile intermediates capable of undergoing substitution with a range of nucleophiles. The new analogues showed a range of abilities to mobilize Ca(2+) from the intracellular stores of permeabilized hepatocytes and are among the first totally synthetic compounds to approach the activity of adenophostin A. In agreement with the biological results, docking studies of adenophostin A using the recently reported X-ray crystal structure of the type 1 Ins(1,4,5)P(3) receptor binding core suggested that, in likely binding modes of adenophostin A, the area around N(6) may be relatively open, identifying this region of the adenophostin A molecule as a promising target for further elaboration. The docking results also point to specific interactions involving residues within the binding domain of the Ins(1,4,5)P(3) receptor that may be involved in the molecular recognition of the adenophostins.

Adenosine↗

PubMed2002

Modulation of IP(3)-sensitive Ca(2+) release by 2,3-butanedione monoxime.

We describe the actions of 2,3-butanedione monoxime (BDM) on calcium responses in secretory cells. Our studies were prompted by the widespread use of BDM as a myosin-ATPase inhibitor. Application of 10 mM BDM almost completely inhibited agonist-evoked amylase secretion from mouse pancreatic acinar cells. This action might be interpreted as indicating a role for myosin in secretion. However, BDM alone elicited a calcium response in single cells and this calcium signal was sufficient to activate calcium-dependent chloride currents. Furthermore, in some cases, BDM potentiated agonist-evoked calcium signals but almost always blocked agonist-evoked calcium oscillations. These effects of BDM were not due to an action on calcium influx pathways but rather to direct effects on IP(3)-sensitive stores. We conclude that BDM cannot be used for unequivocal identification of the involvement of myosin motors in a cellular response. Further, our evidence suggests that BDM can act directly to modify the opening of IP(3) receptors.

Acetylcholine↗

PubMedDate not supplied

Dimers of D-myo-inositol 1,4,5-trisphosphate: design, synthesis, and interaction with Ins(1,4,5)P3 receptors.

The design and synthesis of dimeric versions of the intracellular signaling molecule d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] are reported. Ins(1,4,5)P(3) dimers in a range of sizes were constructed by conjugation of a partially protected 2-O-(2-aminoethyl)-Ins(1,4,5)P(3) intermediate with activated oligo- and poly(ethylene glycol) (PEG) tethers, to give benzyl-protected dimers with amide or carbamate linkages. After deprotection, the resulting water-soluble Ins(1,4,5)P(3) dimers were purified by ion-exchange chromatography. The interaction of the Ins(1,4,5)P(3) dimers with tetrameric Ins(1,4,5)P(3) receptors was explored, using equilibrium [(3)H]Ins(1,4,5)P(3)-binding to membranes from cerebellum, and (45)Ca(2+)-release from permeabilized hepatocytes. The results showed that dimers, even when they incorporate large PEG tethers, interact potently with Ins(1,4,5)P(3) receptors, and that the shorter dimers are more potent than Ins(1,4,5)P(3) itself. A very small dimer, consisting of two Ins(1,4,5)P(3) motifs joined by a short N,N'-diethylurea spacer, was synthesized. Preliminary studies of (45)Ca(2+) release from the intracellular stores of permeabilized hepatocytes showed this shortest dimer to be almost as potent as adenophostin A, the most potent Ins(1,4,5)P(3) receptor ligand known. Possible interpretations of this result are considered in relation to the recently disclosed X-ray crystal structure of the type 1 Ins(1,4,5)P(3) receptor core binding domain.

Animals↗

PubMedDate not supplied

Plasma membrane microdomains: organization, function and trafficking.

The plasma membrane consists of a mosaic of functional microdomains facilitating a variety of physiological processes associated with the cell surface. In most cells, the majority of the cell surface is morphologically featureless, leading to difficulties in characterizing its organization and microdomain composition. The reliance on indirect and perturbing techniques has led to vigorous debate concerning the nature and even existence of some microdomains. Recently, increasing technical sophistication has been applied to study cell surface compartmentalization providing evidence for small, short-lived clusters that may be much less than 50 nm in diameter. Lipid rafts and caveolae are cholesterol-dependent, highly ordered microdomains that have received most attention in recent years, yet their precise roles in regulating functions such as cell signalling remain to be determined. Endocytosis of lipid rafts/caveolae follows a clathrin-independent route to both early endosomes and non-classical caveosomes. The observation that a variety of cellular pathogens localize to and internalize with these microdomains provides an additional incentive to characterize the organization, dynamics and functions of these domains.

Biological Transport↗