Splice variant-dependent regulation of beta-cell sodium-calcium exchange by acyl-coenzyme As

The sodium-calcium exchanger isoform 1 (NCX1) is intimately involved in the regulation of calcium (Ca(2+)) homeostasis in many tissues including excitation-secretion coupling in pancreatic beta-cells. Our group has previously found that intracellular long-chain acyl-coenzyme As (acyl CoAs) are potent regulators of the cardiac NCX1.1 splice variant. Despite this, little is known about the biophysical properties of beta-cell NCX1 splice variants and the effects of intracellular modulators on their important physiological function in health and disease. Here, we show that the forward-mode activity of beta-cell NCX1 splice variants is differentially modulated by acyl-CoAs and is dependent both upon the intrinsic biophysical properties of the particular NCX1 splice variant as well as the side chain length and degree of saturation of the acyl-CoA moiety. Notably, saturated long-chain acyl-CoAs increased both peak and total NCX1 activity, whereas polyunsaturated long-chain acyl-CoAs did not show this effect. Furthermore, we have identified the exon within the alternative splicing region that bestows sensitivity to acyl-CoAs. We conclude that the physiologically relevant forward-mode activity of NCX1 splice variants expressed in the pancreatic beta-cell are sensitive to acyl-CoAs of different saturation and alterations in intracellular acyl-CoA levels may ultimately lead to defects in Ca(2+)-mediated exocytosis and insulin secretion.     

A new method for surface staining large slices of fixed brain using a copper phthalocyanine dye

Here we describe a method for gross staining of gray matter in slices of formaldehyde-fixed human brain. After protection of white matter with 4% phenol at 60°C for 5 min followed by a cold water wash, the gray matter was stained for 10-15 min at 20-25°C with 1% aqueous copper(II) phthalocyanine tetrasulfonic acid tetrasodium salt (CPTS). The staining resisted all attempts to be washed from the gray matter. Stained slices can be stored indefinitely in slightly acidified water, or plastinated as permanent dry specimens.     

Endostatin binds tropomyosin. A potential modulator of the antitumor activity of endostatin

The mechanism of action of Endostatin, an endogenous inhibitor of angiogenesis and tumor growth, remains unknown. We utilized phage-display technology to identify polypeptides that mimic the binding domains of proteins with which Endostatin interacts. A conformed peptide (E37) was identified that shares an epitope with human tropomyosin implicating tropomyosin as an Endostatin-binding protein. We show that recombinant human Endostatin binds tropomyosin in vitro and to tropomyosin-associated microfilaments in a variety of endothelial cell types. The most compelling evidence that tropomyosin modulates the activity of Endostatin was demonstrated when E37 blocked greater than 84% of the tumor-growth inhibitory activity of Endostatin in the B16-BL6 metastatic melanoma model. We conclude that the E37 peptide mimics the Endostatin-binding epitope of tropomyosin and blocks the antitumor activity of Endostatin by competing for Endostatin binding. We postulate that the Endostatin interaction with tropomyosin results in disruption of microfilament integrity leading to inhibition of cell motility, induction of apoptosis, and ultimately inhibition of tumor growth.     

A novel SCN1A mutation associated with generalized epilepsy with febrile seizures plus--and prevalence of variants in patients with epilepsy

We recently described mutations of the neuronal sodium-channel alpha-subunit gene, SCN1A, on chromosome 2q24 in two families with generalized epilepsy with febrile seizures plus (GEFS+) type 2. To assess the contribution that SCN1A makes to other types of epilepsy, 226 patients with either juvenile myoclonic epilepsy, absence epilepsy, or febrile convulsions were screened by conformation-sensitive gel electrophoresis and manual sequencing of variants; the sample included 165 probands from multiplex families and 61 sporadic cases. The novel mutation W1204R was identified in a family with GEFS+. Seven other coding changes were observed; three of these are potential disease-causing mutations. Two common haplotypes, with frequencies of .67 and .33, were defined by five single-nucleotide polymorphisms (SNPs) spanning a 14-kb region of linkage disequilibrium. An SNP located 18 bp upstream of the splice-acceptor site for exon 3 was observed in 7 of the 226 patients but was not present in 185 controls, suggesting possible association with a disease mutation. This work has confirmed the role of SCN1A in GEFS+, by identification of a novel mutation in a previously undescribed family. Although a few candidate disease alleles were identified, the patient survey suggests that SCN1A is not a major contributor to idiopathic generalized epilepsy. The SCN1A haplotypes and SNPs identified here will be useful in future association and linkage studies.     

Partial restoration of lutropin activity by an intersubunit disulfide bond: implications for structure/function studies

Gonadal function is controlled by lutropins and follitropins, heterodimeric cystine knot proteins that have nearly identical alpha-subunits. These heterodimeric proteins are stabilized by a portion of the hormone-specific beta-subunit termed the "seatbelt" that is wrapped around alpha-subunit loop 2 (alpha 2). Here we show that replacing human chorionic gonadotropin (hCG) alpha 2 residue Lys51 with cysteine or alanine nearly abolished its lutropin activity, an observation that implies that alpha Lys51 has a key role in hormone activity. The activity of the heterodimer containing alpha K51C, but not that containing alpha K51A, was increased substantially when beta-subunit seatbelt residue beta Asp99 was converted to cysteine. As had been reported by others, heterodimers containing alpha K51C and beta D99C were crosslinked by a disulfide. The finding that an intersubunit disulfide restored some of the activity lost by replacing alpha Lys51 suggests that this residue is not crucial for receptor binding or signaling and also that hCG and related hormones may be particularly sensitive to mutations that alter interactions between their subunits. We propose the unique structures of hCG and related family members may permit some subunit movement in the heterodimer, making it difficult to deduce key residues involved in receptor contacts simply by correlating the activities of hormone analogs with their amino acid sequences.     

Hypomorphic expression of Dkk1 in the doubleridge mouse: dose dependence and compensatory interactions with Lrp6

doubleridge is a transgene-induced mouse mutation displaying forelimb postaxial polysyndactyly. We have cloned the doubleridge transgene insertion site and demonstrate that doubleridge acts in cis from a distance of 150 kb to reduce the expression of dickkopf 1 (Dkk1), the secreted Wnt antagonist. Expression of Dkk1 from the doubleridge allele ranges from 35% of wild-type level in E7.0 head to <1% of wild type in E13.5 tail. doubleridge homozygotes and doubleridge/null compound heterozygotes are viable. An allelic series combining the wild-type, doubleridge and null alleles of Dkk1 demonstrates the effect of varying Dkk1 concentration on development of limb, head and vertebrae. Decreasing expression of Dkk1 results in hemivertebral fusions in progressively more anterior positions, with severity increasing from tail kinks to spinal curvature. We demonstrated interaction between Dkk1 and the Wnt coreceptors Lrp5 and Lrp6 by analysis of several types of double mutants. The polydactyly of Dkk1(d/d) mice was corrected by reduced expression of Lrp5 or Lrp6. The posterior digit loss and axial truncation characteristic of Lrp6 null mice was partially corrected by reduction of Dkk1. Similarly, the anterior head truncation characteristic of Dkk1 null mice was rescued by reduction of Lrp6. These compensatory interactions between Dkk1 and Lrp6 demonstrate the importance of correctly balancing positive and negative regulation of Wnt signaling during mammalian development.     

Enzymes in pancreatic islets that use NADP(H) as a cofactor including evidence for plasma membrane aldehyde reductase

Recent evidence of a pyruvate malate shuttle capable of transporting a large amount of NADPH equivalents out of mitochondria in pancreatic islets suggests that cytosolic NADP(H) plays a role in beta cell metabolism. To obtain clues about these processes the activities of several NADPHutilizing enzymes were estimated in pancreatic islets. Low levels of pyrroquinolone quinone (PQQ) and low levels of enzyme activity that reduce PQQ were found in islets. Low activities of palmitoylCoA and stearoylCoA desaturases were also detected. Significant activities of glutathione reductase, aldose reductase (EC.1.1.1.21) and aldehyde reductase (EC.1.1.1.2) were present in islets. Potent inhibitors of aldehyde and aldose reductases inhibited neither glucoseinduced insulin release nor glucose metabolism in islets indicating that these reductases are not directly involved in glucoseinduced insulin reaction. Over 90% of aldose reductase plus aldehyde reductase enzyme activity was present in the cytosol. Kinetic and chromatographic studies indicated that 60–70% of this activity in cytosol was due to aldehyde reductase and the remainder due to aldose reductase. Aldehyde reductaselike enzyme activity, as well as aldose reductase immunoreactivity, was detected in rat islet plasma membrane fractions purified by a polyethylene glycolDextran gradient or by a sucrose gradient. This is interesting in view of the fact that voltagegated potassium channel beta subunits that contain aldehyde and aldose reductaselike NADPH-binding motifs have been detected in plasma membrane fractions of islets [Receptors and Channels 7: 237–243, 2000] and suggests that NADPH might have a yet unknown function in regulating activity of these potassium channels. Reductases may be present in cytosol to protect the insulin cell from molecules that cause oxidative injury.     

Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons

Long-term potentiation (LTP) of excitatory transmission in the hippocampus likely contributes to learning and memory. The mechanisms underlying LTP at these synapses are not well understood, although phosphorylation and redistribution of AMPA receptors may be responsible for this form of synaptic plasticity. We show here that miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons reliably demonstrate LTP when postsynaptic NMDA receptors are briefly stimulated with glycine. LTP of these synapses is accompanied by a rapid insertion of native AMPA receptors and by increased clustering of AMPA receptors at the surface of dendritic membranes. Both LTP and glycine-facilitated AMPA receptor insertion are blocked by intracellular tetanus toxin (TeTx), providing evidence that AMPA receptors are inserted into excitatory synapses via a SNARE-dependent exocytosis during LTP.