Abstract-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. This process also occurs in vivo because an inverse relation exists between telomere length in replicating somatic cells and the age of human beings who have donated these cells (References 5 through 9; reviewed in References 1 through 4). Thus, the replicative history of somatic cells is a major determinant of telomere length. Another determinant of telomere length is heredity, since the high variability in this parameter among human beings is to a large extent genetically determined. 5 Recent experimental data support the concept that telomeres might serve as "biological clocks," pacing not only life span at the cellular level but also aging at the systemic level. These data show that (1) the prevention of telomere attrition by the forced expression in cultured somatic cells of the catalytic component of telomerase, the reverse transcriptase that adds telomere repeats onto the ends of chromosomes, postpones replicative senescence 10,11 and (2) the "knockout" of telomerase in the mouse amplifies some characteristics associated with systemic aging in later generations of mice that exhibit substantially shortened telomere length. 12 At least 2 fundamental questions therefore arise with respect to the clinical implications of telomere biology. First, can the length of telomeres serve as an in vivo indicator of biological aging of replicating somatic cells in different organ systems of humans? A related question is: Is the aging of tissues from persons who are genetically endowed with long telomeres likely to occur later in life or at a slower pace than of tissues from persons who inherit short telomeres? Second, which biological parameters can serve as indicators of aging in human beings, since for obvious reasons chronological age (which is determined by calendar time) is a poor criterion for biological aging?In light of these considerations, this work had 2 goals. The first goal was driven by the following concept. Since in industrialized nations pulse pressure increases with age, 13 pulse pressure might serve as a phenotype of cardiovascular agin...

By using the large cytoplasmic domain of the nicotinic acetylcholine receptor (AChR) ␣4 subunit as a bait in the yeast two-hybrid system, we isolated the first cytosolic protein, 14-3-3, known to interact directly with neuronal AChRs. 14-3-3 is a member of a family of proteins that function as regulatory or chaperone/ scaffolding/adaptor proteins. 14-3-3 interacted with the recombinant ␣4 subunit alone in tsA 201 cells following activation of cAMP-dependent protein kinase by forskolin. The interaction of 14-3-3 with recombinant ␣4 subunits was abolished when serine 441 of the ␣4 subunit was mutated to alanine (␣4 S441A ). The surface levels of recombinant wild-type ␣4␤2 AChRs were ϳ2-fold higher than those of mutant ␣4 S441A ␤2 AChRs. The interaction significantly increased the steady state levels of the ␣4 subunit and ␣4␤2 AChRs but not that of the mutant ␣4 S441A subunit or mutant ␣4 S441A ␤2 AChRs. The EC 50 values for activation by acetylcholine were not significantly different for ␣4␤2 AChRs and ␣4 S441A ␤2 AChRs coexpressed with 14-3-3 in oocytes following treatment with forskolin. 14-3-3 coimmunopurified with native ␣4 AChRs from brain. These results support a role for 14-3-3 in dynamically regulating the expression levels of ␣4␤2 AChRs through its interaction with the ␣4 subunit. Neuronal nicotinic acetylcholine receptors (AChR)1 are a family of ligand-gated, cation-selective, homo-or heteropentameric ion channels expressed in the peripheral and central nervous system (1, 2). A multitude of neuronal AChR subtypes assembled from different combinations of ␣2-␣9 and ␤2-␤4 subunits have been identified (3,4 (7), and show attenuated self-administration of nicotine (8) suggesting that ␣4␤2 AChRs have a role in mediating addiction to nicotine. The normal and pathophysiological functions mediated by ␣4␤2 AChRs are of significant importance to human health. Some inherited forms of epilepsy, such as the autosomal dominant nocturnal frontal lobe epilepsies, are caused by ␣4␤2 AChRs harboring at least two separate mutations within their ␣4 subunit (9 -12). Most recently, ␣4␤2 AChRs, among other ␤2 subunit-containing AChRs, have been implicated in neuronal survival during aging, as surmised from the neurodegeneration observed in ␤2-subunit knock-out mice (13).The ␣4 subunit, like the other AChR subunits, consists of an extracellular N-terminal domain, followed by three transmembrane domains (M1-M3), a large cytoplasmic domain, a fourth transmembrane domain (M4), and a short extracellular C terminus. The large cytoplasmic domain is highly divergent among the various subunits, and this sequence divergence presumably provides the diversity necessary for different AChR subtypes to interact directly with cytosolic proteins of different function. To identify such proteins associated with ␣4␤2 AChRs, we used the large cytoplasmic domain of the ␣4 subunit as a bait to screen a mouse brain cDNA yeast two-hybrid library. Here we describe the isolation of a known protein termed 14-3-3. The 14-3-3 proteins family consists of sev...

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.

Class IB phosphoinositide 3-kinase ␥ (PI3K␥) elicits various immunologic and cardiovascular responses; however, the molecular basis for this signal heterogeneity is unclear. PI3K␥ consists of a catalytic p110␥ and a regulatory p87 PIKAP (p87, also p84) or p101 subunit. Hitherto p87 and p101 are generally assumed to exhibit redundant functions in receptor-induced and G protein ␤␥ (G␤␥)-mediated PI3K␥ regulation. Here we investigated the molecular mechanism for receptor-dependent p87/p110␥ activation. By analyzing GFP-tagged proteins expressed in HEK293 cells, PI3K␥-complemented bone marrow-derived mast cells (BMMCs) from p110␥ -/-mice, and purified recombinant proteins reconstituted to lipid vesicles, we elucidated a novel pathway of p87-dependent, G protein-coupled receptor (GPCR)-induced PI3K␥ activation. Although p101 strongly interacted with G␤␥, thereby mediating PI3K␥ membrane recruitment and stimulation, p87 exhibited only a weak interaction, resulting in modest kinase activation and lack of membrane recruitment. Surprisingly, Ras-GTP substituted the missing G␤␥-dependent membrane recruitment of p87/p110␥ by direct interaction with p110␥, suggesting the indispensability of Ras for activation of p87/p110␥. Consequently, interference with Ras signaling indeed selectively blocked p87/p110␥, but not p101/ p110␥, kinase activity in HEK293 and BMMC cells, revealing an important crosstalk between monomeric and trimeric G proteins for p87/p110␥ activation. Our data display distinct signaling requirements of p87 and p101, conferring signaling specificity to PI3K␥ that could open up new possibilities for therapeutic intervention.confocal life cell imaging ͉ G protein ͉ receptor signaling ͉ mast cells

The calcium sensor protein visinin-like protein-1 (VILIP-1) was isolated from a brain cDNA yeast twohybrid library using the large cytoplasmic domain of the ␣4 subunit as a bait. VILIP-1 is a myristoylated calcium sensor protein that contains three functional calcium binding EF-hand motifs. The ␣4 subunit residues 302-339 were found to be essential for the interaction with VILIP-1. VILIP-1 coimmunopurified with detergent-solubilized recombinant ␣4␤2 acetylcholine receptors (AChRs) expressed in tsA201 cells and with native ␣4 AChRs isolated from brain. Coexpression of VILIP-1 with recombinant ␣4␤2 AChRs up-regulated their surface expression levels ϳ2-fold and increased their agonist sensitivity to acetylcholine ϳ3-fold. The modulation of the recombinant ␣4␤2 AChRs by VILIP-1 was attenuated in VILIP-1 mutants that lacked the ability to be myristoylated or to bind calcium. Collectively, these results suggest that VILIP-1 represents a novel modulator of ␣4␤2 AChRs that increases their surface expression levels and agonist sensitivity in response to changes in the intracellular levels of calcium.Neuronal nicotinic acetylcholine receptors (AChRs) 1 are members of a gene superfamily of ligand-gated ion channels. In vertebrates, neuronal AChRs are composed of subunits ␣2-␣10 and ␤2-␤4 (for review, see Ref. 1). AChR subunits have a large cytoplasmic domain between their third and fourth transmembrane domain whose amino acid sequence is highly divergent among the various subunits (2). The full functional unit of AChRs, like those of the N-methyl-D-aspartate receptors (3), is likely to include proteins that associate with this large cytoplasmic domain and modulate AChR functions.To identify proteins associated with ␣4 AChRs, we used bait consisting of the large cytoplasmic domain of the ␣4 subunit to screen a mouse brain cDNA yeast two-hybrid library. In this paper, we describe the isolation of VILIP-1, a member of the visinin-like protein family of calcium sensor proteins, by such a screen. VILIP-1 is a member of a superfamily of neuronal calcium sensor proteins. This superfamily has been classified into the five subfamilies termed group I-V. The recoverins belong to group I, the frequenins and neuronal calcium sensor (NCS-1) to group II, the VILIPs, hippocalcin, and neurocalcins to group III, NCS-2 to group IV, and guanylyl cyclase-activating proteins and GC-inhibiting proteins to group V (for review, see Ref. 4).The VILIP family is comprised of three members, VILIP-1, VILIP-2, and VILIP-3 (5-10). The members of this family contain 4 EF-hand motifs, of which only EF-hand 2, 3, and 4 are thought to be functional because EF-hand 1 lacks two oxygencontaining side chain residues crucial for binding calcium. A glycine residue at the second position on the polypeptide chain is myristoylated. Interestingly, within most, but not all members of this calcium sensor protein family, the myristoyl moiety is sequestered and exposed through a rapid conformational change that unmasks it in response to alterations in cellular levels of c...

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.

The cellular and molecular mechanisms orchestrating the complex process by which bone marrow megakaryocytes form and release platelets remain poorly understood. Mature megakaryocytes generate long cytoplasmic extensions, proplatelets, which have the capacity to generate platelets. Although microtubules are the main structural component of proplatelets and microtubule sliding is known to drive proplatelet elongation, the role of actin dynamics in the process of platelet formation has remained elusive. Here, we tailored a mouse model lacking all ADF/ n-cofilin-mediated actin dynamics in megakaryocytes to specifically elucidate the role of actin filament turnover in platelet formation. We demonstrate, for the first time, that in vivo actin filament turnover plays a critical role in the late stages of platelet formation from megakaryocytes and the proper sizing of platelets in the periphery. Our results provide the genetic proof that platelet production from megakaryocytes strictly requires dynamic changes in the actin cytoskeleton. (Blood. 2010;116(10):1767-1775) IntroductionThe formation of anucleated blood platelets from their bone marrow resident precursors, the megakaryocytes (MKs), is a unique process in mammalian physiology. 1 Terminally differentiated MKs, the largest cells evolving from hematopoietic stem cells, are polyploid and contain a highly organized cytoplasm with a membranous network, the demarcation membrane system (DMS), which converts into cytoplasmic protrusions during proplatelet formation. 1,2 According to the current model, the tips of these proplatelet protrusions extend into the lumen of a vessel in bone marrow sinusoids and are shed by shear forces generated by the blood flow. Recently, the first direct visualization of this process in situ in the bone marrow of mice using 2-photon microscopy revealed that the released fragments are larger in size than normal platelets. In addition, beaded proplatelets and barbell-shaped platelet pairs were observed in the blood, leading to the hypothesis that final shaping and sizing of platelets may occur in the circulation. 3 In line with this, very recent observations indicate that platelet progeny formation occurs in whole blood, suggesting that thrombopoiesis continues in the bloodstream. 4 Proplatelet formation relies on coordinated cytoskeletal rearrangements with an essential involvement of tubulin-dependent processes. 5 The extension of cytoplasmic protrusions from MKs is mechanically driven by tubulin sliding and assembly. In contrast, the role of actin, the other major cytoskeletal component in platelets, has remained elusive in platelet biogenesis from MKs. In mature platelets, the importance of actin filament dynamics, mainly the uncapping and elongation of actin filaments, for platelet activation and shape changes, has been well documented. 6 A mouse model for the filamentous actin (F-actin) severing protein gelsolin provided in vivo evidence for a physiologic role of actin dynamics in platelet activation. 7 However, surprisingly, gelsolin-d...

1. Selective protein-protein interactions between neurotransmitter transporters and their synaptic targets play important roles in regulating chemical neurotransmission. We screened a yeast two-hybrid library with bait containing the C-terminal amino acids of VGLUT1 and obtained clones that encode endophilin 1 and endophilin 3, proteins considered to play an integral role in glutamatergic vesicle formation. 2. Using a modified yeast plasmid vector to enable more cost-effective screens, we analyzed the selectivity and specificity of this interaction. Endophilins 1 and 3 selectively recognize only VGLUT1 as the C-terminus of VGLUT2 and VGLUT3 do not interact with either endophilin isoform. We mutagenized four conserved stretches of primary sequence in VGLUT1 that includes two polyproline motifs (Pro1, PPAPPP, and Pro2, PPRPPPP), found only in VGLUT1, and two conserved stretches (SEEK, SYGAT), found also in VGLUT2 and VGLUT3. The absence of the VGLUT conserved regions does not affect VGLUT1-endophilin association. Of the two polyproline stretches, only one (Pro2) is required for binding specificity to both endophilin 1 and endophilin 3. 3. We also show that endophilin 1 and endophilin 3 co-localize with VGLUT1 in synaptic terminals of differentiated rat neocortical neurons in primary culture. These results indicate that VGLUT1 and both endophilins are enriched in a class of excitatory synaptic terminals in cortical neurons and there, may interact to play an important role affecting the vesicular sequestration and synaptic release of glutamate.