Chronophin is a glial tumor modifier involved in the regulation of glioblastoma growth and invasiveness.

Glioblastoma is the most aggressive primary brain tumor in adults. Although the rapid recurrence of glioblastomas after treatment is a major clinical challenge, the relationships between tumor growth and intracerebral spread remain poorly understood. We have identified the cofilin phosphatase chronophin (gene name: pyridoxal phosphatase, PDXP) as a glial tumor modifier. Monoallelic PDXP loss was frequent in four independent human astrocytic tumor cohorts and increased with tumor grade. We found that aberrant PDXP promoter methylation can be a mechanism leading to further chronophin downregulation in glioblastomas, which correlated with shorter glioblastoma patient survival. Moreover, we observed an inverse association between chronophin protein expression and cofilin phosphorylation levels in glioma tissue samples. Chronophin-deficient glioblastoma cells showed elevated cofilin phosphorylation, an increase in polymerized actin, a higher directionality of cell migration, and elevated in vitro invasiveness. Tumor growth of chronophin-depleted glioblastoma cells xenografted into the immunodeficient mouse brain was strongly impaired. Our study suggests a mechanism whereby the genetic and epigenetic alterations of PDXP resulting in altered chronophin expression may regulate the interplay between glioma cell proliferation and invasion.

The chaperone protein 14-3-3eta interacts with the nicotinic acetylcholine receptor alpha 4 subunit. Evidence for a dynamic role in subunit stabilization.

By using the large cytoplasmic domain of the nicotinic acetylcholine receptor (AChR) alpha4 subunit as a bait in the yeast two-hybrid system, we isolated the first cytosolic protein, 14-3-3eta, known to interact directly with neuronal AChRs. 14-3-3eta is a member of a family of proteins that function as regulatory or chaperone/ scaffolding/adaptor proteins. 14-3-3eta interacted with the recombinant alpha4 subunit alone in tsA 201 cells following activation of cAMP-dependent protein kinase by forskolin. The interaction of 14-3-3eta with recombinant alpha4 subunits was abolished when serine 441 of the alpha4 subunit was mutated to alanine (alpha4(S441A)). The surface levels of recombinant wild-type alpha4beta2 AChRs were approximately 2-fold higher than those of mutant alpha4(S441A)beta2 AChRs. The interaction significantly increased the steady state levels of the alpha4 subunit and alpha4beta2 AChRs but not that of the mutant alpha4(S441A) subunit or mutant alpha4(S441A)beta2 AChRs. The EC50 values for activation by acetylcholine were not significantly different for alpha4beta2 AChRs and alpha4(S441A)beta2 AChRs coexpressed with 14-3-3eta in oocytes following treatment with forskolin. 14-3-3 coimmunopurified with native alpha4 AChRs from brain. These results support a role for 14-3-3 in dynamically regulating the expression levels of alpha4beta2 AChRs through its interaction with the alpha4 subunit.

Assembly and ligand binding properties of the water-soluble extracellular domains of the glutamate receptor 1 subunit.

High resolution structural studies of models of glutamate receptors (GluRs) have been limited to monomeric models of the ligand-binding site. To obtain oligomeric models of glutamate receptors that can reveal more complete structural information, we examined the assembly and ligand binding properties of two truncated versions of the GluR1 subunit. The first version, GluR1-WS, consisted of only the N-terminal extracellular segment (Ala(1)-Glu(520)) bridged by a synthetic linker to the second extracellular domain (Asn(615)-Gly(790)). The second version, GluR1-M1, consisted of the first N-terminal extracellular domain (Ala(1)-Glu(520)) bridged by a synthetic linker to a second segment containing the second extracellular domain, the third transmembrane domain, and the intracellular C-terminal domain (Asn(615)-Leu(889)). When expressed in Xenopus oocytes, GluR-WS was secreted and water-soluble; GluR1-M1 was displayed on the surface of oocytes. GluR1-WS exhibited a velocity sedimentation profile that was consistent with assembly of homooligomers and bound the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid with high affinity. These findings show that the extracellular domains of GluR1 that are sufficient for ligand binding apparently are sufficient for subunit assembly and might be a suitable target for structural studies of a water-soluble GluR1 oligomer.

Telomere length inversely correlates with pulse pressure and is highly familial.

There is evidence that telomeres, the ends of chromosomes, serve as clocks that pace cellular aging in vitro and in vivo. In industrialized nations, pulse pressure rises with age, and it might serve as a phenotype of biological aging of the vasculature. We therefore conducted a twin study to investigate the relation between telomere length in white blood cells and pulse pressure while simultaneously assessing the role of genetic factors in determining telomere length. We measured by Southern blot analysis the mean length of the terminal restriction fragments (TRF) in white blood cells of 49 twin pairs from the Danish Twin Register and assessed the relations of blood pressure parameters with TRF. TRF length showed an inverse relation with pulse pressure. Both TRF length and pulse pressure were highly familial. We conclude that telomere length, which is under genetic control, might play a role in mechanisms that regulate pulse pressure, including vascular aging.

Cellular proliferation and telomerase activity in CHRF-288-11 cells.

Telomerase activity is detected in many immortalized cell lines. Recent studies suggest that terminal differentiation of some of these cell lines is associated with a reduction in telomerase activity. However, the question remains whether the reduction in telomerase activity results from terminal differentiation or from cessation of cellular proliferation. This was explored in the megakaryocytic cell line CHRF-288-11. Cells were treated with phorbol 12-myristate 13-acetate (PMA), which induces terminal differentiation of CHRF-288-11 cells, EGTA, serum depletion, and okadaic acid. All treatments resulted in cessation of proliferation. Except for okadaic acid, these treatments also induced inhibition of telomerase within 7 days. Restoring the original growth conditions of cells treated with PMA, EGTA and serum depletion resulted in the reversal of telomerase inhibition and an acceleration of proliferation. Apparent inhibition of telomerase was observed to follow the cessation of proliferation, whereas enhanced telomerase activity was noted to precede acceleration in proliferation. Thus, telomerase activity usually reflects the proliferative status rather than the differentiated status of CHRF-288-11 cells.

Physiological and molecular characterization of the Na+/Ca2+ exchanger in human platelets.

In this report, we have demonstrated that Na+/Ca2+ exchanger activity in a human megakaryocytic cell line (CHRF-288 cells) is K+ dependent, similar to the properties previously described for Na+/Ca2+ exchange activity in human platelets. With the use of RT-PCR techniques and mRNA, the exchanger expressed in CHRF-288 cells was found to be identical to that expressed in human retinal rods. Northern blot analysis of the mRNA for the human retinal rod exchanger in CHRF-288 cells revealed a major transcript at 5.8 kb with two minor bands at 4.9 and 6.8 kb. mRNA for the retinal rod exchanger was also identified in human platelets. Using Ba2+ influx as a measure of Na+/Ca2+ exchange activity in human platelets, we have demonstrated that exchange activity is driven by the transmembrane gradient for K+ as well as that for Na+. We propose that the K+ dependence of the platelet Na+/Ca2+ exchanger could make platelets especially sensitive to daily fluctuations in salt intake.

Synchrony in telomere length of the human fetus.

Telomere length, measured by terminal restriction fragments, was examined in tissues from human fetuses of gestational ages estimated as 15-19 weeks. The length of telomeres was similar in most fetal tissues. However, there were significant variations in telomere length among fetuses, with no apparent relationship between gestational age and telomere length. We conclude that synchrony in telomere length exists among tissues of the human fetus. This synchrony is apparently lost during extrauterine life.

Shortened telomere length in white blood cells of patients with IDDM.

IDDM is a polygenic and autoimmune disorder in which subsets of white blood cells (WBCs) are engaged in the destruction of beta-cells of the pancreas. The mechanisms that account for the abnormal behavior of these cells in IDDM are not fully understood. By measuring the mean length of telomeres of WBCs from patients with IDDM, we tested the concept that telomeres might play a role in IDDM. We examined the lengths of the terminal restriction fragments (TRFs) of DNA of WBCs from 234 white men comprising 54 patients with IDDM, 74 patients with NIDDM, and 106 control subjects. When adjusted for age, the TRF length from WBCs of patients with IDDM was significantly shorter than that of nondiabetic control subjects (mean +/- SE: 8.6 +/- 0.1 vs. 9.2 +/- 0.1, P = 0.002). No significant difference was observed between the TRF length from WBCs of patients with NIDDM versus nondiabetic subjects. Neither the duration nor the complications of IDDM (i.e., nephropathy and hypertension) had an effect on the TRF length of WBCs from patients with IDDM. The shortened TRF length of WBCs of patients with IDDM likely reflects a marked reduction in the TRF length of subsets of WBCs that play a role in the pathogenesis of IDDM.

Linkage analysis using platelet-activating factor Ca2+ response in transformed lymphoblasts.

Epstein-Barr virus-transformed lymphoblasts from patients with essential hypertension demonstrate enhanced G protein-mediated cytosolic free calcium ([Ca2+]i) response to platelet-activating factor (PAF). To map genes responsible for variation in G protein-coupled signaling, we used this cellular phenotype for a linkage study of transformed cell lines from the Centre d'Etude du Polymorphisme Humain (CEPH) reference pedigrees. The PAF-evoked change in [Ca2+]i ranged from 20 to 392 mmol/L and was highly reproducible within each cell line. PAF-elicited [Ca2+]i responses were obtained in lymphoblastic cell lines from five densely mapped pedigrees of the CEPH collection. Using PAF-evoked [Ca2+]i responses as a quantitative trait, two-point sibpair linkage analyses were conducted using 5150 markers from the Collaborative Human Linkage Center (CHLC) database. Nine loci, located on chromosomes 1, 4, 10, 11, 13, 16, and 17, were suggestive of linkage, with values of P < 7.4 x 10(-4). Multipoint linkage analysis produced a significant linkage finding (P = 2.1 x 10(-5) in one family at D16S151, with suggestive linkage results for seven additional markers spanning a 40-cM interval of chromosome 16. Multipoint analysis produced suggestive findings of linkage to eight loci from two distinct regions of chromosome 11 in another family. These results indicate that loci involved in the control of G protein-mediated mechanisms, suggested to be involved in the pathophysiology of essential hypertension, can be identified using cell lines from general pedigrees selected without any knowledge of the blood pressure status of the donors. This strategy represents an approach to rapidly and inexpensively mapping loci related to common, complex disorders, using phenotypes that are stable in immortalized lymphoblasts together with existing reference pedigree cell lines and genotype databases.

Action of azelastine on intracellular Ca2+ in cultured airway smooth muscle.

Azelastine, a novel antiasthmatic/antiallergic agent, was tested for Ca2+ antagonistic properties in cultured rabbit airway smooth muscle, vascular smooth muscle and cardiocytes. In airway smooth muscle cells, the basal cytosolic free calcium content was 195 +/- 72 nM (mean +/- S.D., n = 18). These basal values were decreased by azelastine with an IC50 value of 1.1 +/- 0.3 x 10(-4) M. Endothelin-1 (10(-7) M) induced a rapid increase in free cytosolic calcium up to 806 +/- 314 nM, which returned to normal levels in 3-5 min. This was fully blocked by azelastine in a concentration-dependent manner, with an IC50 value of 6.7 +/- 2.9 x 10(-5) M. Moreover, azelastine fully blocked histamine-induced calcium mobilization (IC50 = 7 x 10(-5) M). In cultured vascular smooth muscle cells and cardiocytes, azelastine was unable to decrease the basal cytosolic free calcium content or inhibit agonist-induced calcium mobilization. Therefore, at therapeutic levels, a specific, mild inhibition of calcium mobilization in airway smooth muscle may be one component of the antiasthmatic action of azelastine.

Stimulatory action of endothelin-1 on membrane Na+ transport in vascular smooth muscle cells in culture.

Endothelin-1 was able to induce an immediate and transient increase in cytosolic free Ca2+ concentrations in the A10 cell line of vascular smooth muscle. This was associated with a strong stimulation of the Na+:H+ exchange, the Na+, K+ pump and the [Na+,K+,Cl-]-cotransport system. Pump stimulation appeared to be secondary to sodium entry through Na+:H+ exchange because it was absent in Na+ loaded cells and in the presence of ethyl-isopropyl-amiloride. Cotransport stimulation was blocked by indomethacin, suggesting the involvement of a cyclooxygenase product. In conclusion, the monovalent ionic perturbations associated to the vasoconstrictor and mitogenic actions of endothelin-1 are counterbalanced by activation of the Na+,K+ pump and the [Na+,K+,Cl-]-cotransport system.

Stimulation of Na(+)-H+ exchange, the Na(+)-K+ pump and Na+,K+,Cl- cotransport by endothelin-1 in cultured vascular smooth muscle cells.

Endothelin-1 concentrations higher than 10(-10) mol/l were able to strongly stimulate Na(+)-H+ exchange, the Na(+)-K+ pump and the Na+,K+Cl- cotransport system in A10 vascular smooth muscle cells. The stimulation of Na(+)-H+ exchange was more marked in fresh than in H(+)-loaded cells, suggesting an increase in the apparent affinity for internal H+. Pump stimulation appeared to be secondary to sodium entry through Na(+)-H+ exchange because it was absent in (1) Na(+)-loaded cells and (2) in the presence of ethyl-isopropylamiloride (EIPA). Stimulation of cotransport was blocked by indomethacin, suggesting the involvement of a cyclo-oxygenase product. In conclusion, the action of endothelin-1 in vascular smooth muscle induces the onset of negative feedback mechanisms which enhance the ability of the vascular cells to regulate disturbances in Na+,K+ and Cl- contents.

Inhibitory action of norepinephrine on sodium transport in vascular smooth muscle cells in culture.

Cultured vascular smooth muscle cells from porcine aortas incubated in Na+ -free medium rapidly release their intracellular Na+ contents (Nai) (23 +/- 4% of baseline after 60 min incubation, mean +/- SEM of 18 experiments). Total Nai release was inhibited by 35-40% after addition of ouabain and by 60-70% after addition of ouabain + bumetanide. Norepinephrine inhibited ouabain and bumetanide-sensitives Na+ efflux with an IC50 of about 10(-9)-10(-8) M. Addition of the alpha-adrenergic agonist phenylephrine (10 microM) to the cells mimicked the inhibitory action of norepinephrine on Nai release. Conversely, the beta-adrenergic agonist isoproterenol was without effect on Nai release. Simultaneous addition of 10 microM norepinephrine and the alpha-adrenergic antagonist phentolamine prevented any effect of norepinephrine on the rate of Nai decline. In A-10 cultured vascular smooth muscle cells, the alpha-adrenergic agonist phenylephrine (10 microM) inhibited 40.0 +/- 8.1% of ouabain-sensitive Rb+ influx and 70.7 +/- 6.9% of bumetanide-sensitive Rb+ influx (mean +/- SEM of three experiments). 50% inhibition of bumetanide-sensitive Rb+ influx was obtained with about 5 x 10(-7) M of phenylephrine. Our results show that in vascular smooth muscle cells a [Na+, K+, Cl-]-cotransport system is able to catalyze outward Na+ movements (in Na+ -free media) of a similar order of magnitude to those of the Na+, K+ pump and that alpha-adrenergic stimulation markedly inhibits Na+ efflux (and Rb+ influx) through these two transport systems.