Telomere dysfunction induces metabolic and mitochondrial compromise.

Telomere dysfunction activates p53-mediated cellular growth arrest, senescence and apoptosis to drive progressive atrophy and functional decline in high-turnover tissues. The broader adverse impact of telomere dysfunction across many tissues including more quiescent systems prompted transcriptomic network analyses to identify common mechanisms operative in haematopoietic stem cells, heart and liver. These unbiased studies revealed profound repression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC-1α and PGC-1ß, also known as Ppargc1a and Ppargc1b, respectively) and the downstream network in mice null for either telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. Consistent with PGCs as master regulators of mitochondrial physiology and metabolism, telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species. In the setting of telomere dysfunction, enforced Tert or PGC-1α expression or germline deletion of p53 (also known as Trp53) substantially restores PGC network expression, mitochondrial respiration, cardiac function and gluconeogenesis. We demonstrate that telomere dysfunction activates p53 which in turn binds and represses PGC-1α and PGC-1ß promoters, thereby forging a direct link between telomere and mitochondrial biology. We propose that this telomere-p53-PGC axis contributes to organ and metabolic failure and to diminishing organismal fitness in the setting of telomere dysfunction.

Familial influences on the full range of variability in attention and activity levels during adolescence: A longitudinal twin study.

To investigate familial influences on the full range of variability in attention and activity across adolescence, we collected maternal ratings of 339 twin pairs at ages 12, 14, and 16, and estimated the transmitted and new familial influences on attention and activity as measured by the Strengths and Weaknesses of Attention-Deficit/Hyperactivity Disorder Symptoms and Normal Behavior Scale. Familial influences were substantial for both traits across adolescence: genetic influences accounted for 54%-73% (attention) and 31%-73% (activity) of the total variance, and shared environmental influences accounted for 0%-22% of the attention variance and 13%-57% of the activity variance. The longitudinal stability of individual differences in attention and activity was largely accounted for by familial influences transmitted from previous ages. Innovations over adolescence were also partially attributable to familial influences. Studying the full range of variability in attention and activity may facilitate our understanding of attention-deficit/hyperactivity disorder's etiology and intervention.

Effects of nicotine deprivation and replacement on BOLD-fMRI response to smoking cues as a function of DRD4 VNTR genotype.

INTRODUCTION: Reactivity to smoking cues is an important factor in the motivation to smoke and has been associated with the dopamine receptor 4 variable number tandem repeat (DRD4 exon III VNTR) polymorphism. However, little is known about the associated neural mechanisms. METHODS: Non-treatment-seeking Caucasian smokers completed overnight abstinence and viewed smoking and neutral cues during 2 separate functional magnetic resonance imaging scans while wearing either a nicotine or placebo patch (order randomized) and were genotyped for the DRD4 VNTR. We conducted mixed-effects repeated-measures analyses of variance (within-subject factor: nicotine or placebo patch; between-subject factor: DRD4 long [L: ≥ 1 copy of ≥ 7 repeats] or short [S: 2 copies ≤ 6 repeats] genotype) of 6 a priori regions of interest. RESULTS: Relative to neutral cues, smoking cues elicited greater activity in bilateral ventral striatum and left amygdala during nicotine replacement and deactivation in these regions during nicotine deprivation. A patch × DRD4 interaction was observed in the left amygdala, an area associated with appetitive reinforcement and relapse risk, such that S allele carriers demonstrated greater activation on active patch than on placebo patch. CONCLUSIONS: Brain systems associated with reward salience may become primed and overreactive at nicotine replacement doses intended for the first step of smoking cessation and may become inhibited during nicotine withdrawal in DRD4 S but not in DRD4 L carriers. These findings are consistent with the role of these regions in drug reinforcement and suggest a differential influence of nicotine replacement on amygdala activation in the association of incentive salience with smoking stimuli across DRD4 genotypes.

Exogenous control of mammalian gene expression via modulation of translational termination.

Here, we describe a system for the exogenous control of gene expression in mammalian cells that relies on the control of translational termination. To achieve gene regulation, we modified protein-coding sequences by introduction of a translational termination codon just downstream from the initiator AUG codon. Translation of the resulting mRNA leads to potent reduction in expression of the desired gene product. Expression of the gene product can be controlled by treating cells that express the mRNA with either aminoglycoside antibiotics or several nonantibiotic compounds. We show that the extent of regulation of gene expression can be substantial (60-fold) and that regulation can be achieved in the case of a variety of different genes, in different cultured cell lines and in primary cells in vivo. This gene regulation strategy offers significant advantages over existing methods for controlling gene expression and should have both immediate experimental application and possible clinical application.

VEGF modulates erythropoiesis through regulation of adult hepatic erythropoietin synthesis.

Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60-75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1alpha-independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte-endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.

A phase I trial of adeno-associated virus serotype 1-γ-sarcoglycan gene therapy for limb girdle muscular dystrophy type 2C.

γ-Sarcoglycanopathy or limb girdle muscular dystrophy type 2C is an untreatable disease caused by autosomal recessively inherited mutations of the γ-sarcoglycan gene. Nine non-ambulatory patients (two males, seven females, mean age 27 years; range 16-38 years) with del525T homozygous mutation of the γ-sarcoglycan gene and no γ-sarcoglycan immunostaining on muscle biopsy were divided into three equal groups to receive three escalating doses of an adeno-associated virus serotype 1 vector expressing the human γ-sarcoglycan gene under the control of the desmin promoter, by local injection into the extensor carpi radialis muscle. The first group received a single injection of 3 × 10(9) viral genomes in 100 µl, the second group received a single injection of 1.5 × 10(10) viral genomes in 100 µl, and the third group received three simultaneous 100-µl injections at the same site, delivering a total dose of 4.5 × 10(10) viral genomes. No serious adverse effects occurred during 6 months of follow-up. All nine patients became adeno-associated virus serotype 1 seropositive and one developed a cytotoxic response to the adeno-associated virus serotype 1 capsid. Thirty days later, immunohistochemical analysis of injected-muscle biopsy specimens showed γ-sarcoglycan expression in all three patients who received the highest dose (4.7-10.5% positively stained fibres), while real-time polymerase chain reaction detected γ-sarcoglycan messenger RNA. In one patient, γ-sarcoglycan protein was detected by western blot. For two other patients who received the low and intermediate doses, discrete levels of γ-sarcoglycan expression (<1% positively stained fibres) were also detectable. Expression of γ-sarcoglycan protein can be induced in patients with limb girdle muscular dystrophy type 2C by adeno-associated virus serotype 1 gene transfer, with no serious adverse effects.

Effects of HIV and early life stress on amygdala morphometry and neurocognitive function.

Both HIV infection and high levels of early life stress (ELS) have been related to abnormalities in frontal-subcortical structures, yet the combined effects of HIV and ELS on brain structure and function have not been previously investigated. In this study we assessed 49 non-demented HIV-seropositive (HIV+) and 47 age-matched HIV-seronegative healthy control (HC) adults. Levels of ELS exposure were quantified and used to define four HIV-ELS groups: HC Low-ELS (N = 20); HC High-ELS (N = 27); HIV+ Low-ELS (N = 24); HIV+ High-ELS (N = 25). An automated segmentation tool measured volumes of brain structures known to show HIV-related or ELS-related effects; a brief neurocognitive battery was administered. A significant HIV-ELS interaction was observed for amygdala volumes, which was driven by enlargements in HIV+ High-ELS participants. The HIV+ High-ELS group also demonstrated significant reductions in psychomotor/processing speed compared with HC Low-ELS. Regression analyses in the HIV+ group revealed that amygdala enlargements were associated with higher ELS, lower nadir CD4 counts, and reduced psychomotor/processing speed. Our results suggest that HIV infection and high ELS interact to increase amygdala volume, which is associated with neurocognitive dysfunction in HIV+ patients. These findings highlight the lasting neuropathological influence of ELS and suggest that high ELS may be a significant risk factor for neurocognitive impairment in HIV-infected individuals.

Mitotic spindle destabilization and genomic instability in Shwachman-Diamond syndrome.

Deficiencies in the SBDS gene result in Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome associated with leukemia predisposition. SBDS encodes a highly conserved protein previously implicated in ribosome biogenesis. Using human primary bone marrow stromal cells (BMSCs), lymphoblasts, and skin fibroblasts, we show that SBDS stabilized the mitotic spindle to prevent genomic instability. SBDS colocalized with the mitotic spindle in control primary BMSCs, lymphoblasts, and skin fibroblasts and bound to purified microtubules. Recombinant SBDS protein stabilized microtubules in vitro. We observed that primary BMSCs and lymphoblasts from SDS patients exhibited an increased incidence of abnormal mitoses. Similarly, depletion of SBDS by siRNA in human skin fibroblasts resulted in increased mitotic abnormalities and aneuploidy that accumulated over time. Treatment of primary BMSCs and lymphoblasts from SDS patients with nocodazole, a microtubule destabilizing agent, led to increased mitotic arrest and apoptosis, consistent with spindle destabilization. Conversely, SDS patient cells were resistant to taxol, a microtubule stabilizing agent. These findings suggest that spindle instability in SDS contributes to bone marrow failure and leukemogenesis.

Neural correlates of inhibitory control in adult attention deficit/hyperactivity disorder: evidence from the Milwaukee longitudinal sample.

Only a few studies have investigated the neural substrate of response inhibition in adult attention deficit hyperactivity disorder (ADHD) using Stop-Signal and Go/No-Go tasks. Inconsistencies and methodological limitations in the existing literature have resulted in limited conclusions regarding underlying pathophysiology. We examined the neural basis of response inhibition in a group of adults diagnosed with ADHD in childhood and who continue to meet criteria for ADHD. Adults with ADHD (n=12) and controls (n=12) were recruited from an ongoing longitudinal study and were matched for age, IQ, and education. Individuals with comorbid conditions were excluded. Functional magnetic resonance imaging (fMRI) was used to identify and compare the brain activation patterns during correct trials of a response-inhibition task (Go/No-Go). Our results showed that the control group recruited a more extensive network of brain regions than the ADHD group during correct inhibition trials. Adults with ADHD showed reduced brain activation in the right frontal eye field, pre-supplementary motor area, left precentral gyrus, and the inferior parietal lobe bilaterally. During successful inhibition of an inappropriate response, adults with ADHD display reduced activation in fronto-parietal networks previously implicated in working memory, goal-oriented attention, and response selection. This profile of brain activation may be specifically associated with ADHD in adulthood.

Soluble receptor-mediated selective inhibition of VEGFR and PDGFRbeta signaling during physiologic and tumor angiogenesis.

The simultaneous targeting of both endothelial cells and pericytes via inhibition of VEGF receptor (VEGFR) and PDGFbeta receptor (PDGFRbeta) signaling, respectively, has been proposed to enhance the efficacy of antiangiogenic tumor therapy. Clinical and preclinical modeling of combined VEGFR and PDGFRbeta signaling inhibition, however, has used small molecule kinase inhibitors with inherently broad substrate specificities, precluding detailed examination of this hypothesis. Here, adenoviral expression of a soluble VEGFR2/Flk1 ectodomain (Ad Flk1-Fc) in combination with a soluble ectodomain of PDGFRbeta (Ad sPDGFRbeta) allowed highly selective inhibition of these pathways. The activity of Ad sPDGFRbeta was validated in vitro against PDGF-BB and in vivo with near-complete blockade of pericyte recruitment in the angiogenic corpus luteum, resulting in prominent hemorrhage, thus demonstrating an essential function for PDGF signaling during ovarian angiogenesis. Combination therapy with Ad PDGFRbeta and submaximal doses of Ad Flk1-Fc produced modest additive antitumor effects; however, no additivity was observed with maximal VEGF inhibition in numerous s.c. models. Notably, VEGF inhibition via Ad Flk1-Fc was sufficient to strongly suppress tumor endothelial and pericyte content as well as intratumoral PDGF-B mRNA, obscuring additive Ad sPDGFRbeta effects on pericytes or tumor volume. These studies using highly specific soluble receptors suggest that additivity between VEGFR and PDGFRbeta inhibition depends on the strength of VEGF blockade and appears minimal under conditions of maximal VEGF antagonism.

The ARRIVE guidelines, a welcome improvement to standards for reporting animal research.

Here we introduce the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines, produced by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), which are published in this issue of the journal with our endorsement, and will be incorporated into our Instructions to Authors.

Effects of nicotine withdrawal on verbal working memory and associated brain response.

Previous literature has reported effects of nicotine withdrawal on brain function during cognitive tasks such as verbal working memory (VWM). Mechanisms of these withdrawal effects have not been clearly identified. Functional neuroimaging offers an objective method to examine brain mechanisms associated with observable behavior and subjective reports. To investigate these mechanisms, 12 smokers were administered a 2-Back VWM challenge during two functional magnetic resonance imaging sessions. Participants abstained from smoking prior to both sessions; however, they applied a nicotine patch before one session and a placebo patch prior to the other. Among regions that exhibited a significant response to the 2-Back during either session, withdrawal was associated with significantly greater deactivation in left and right temporal poles and left medial frontal gyrus. The magnitude of task-related activation showed a significant inverse relationship to craving in the majority of regions during placebo administration. Also, individual brain responses varied more during placebo, suggesting inefficient neural processing. Results suggest that differences in brain response to a VWM challenge during abstinence may be attributed to increased craving. Further deactivation of regions associated with the default network (medial frontal and anterior temporal clusters) during the placebo condition suggests further suspension of default activity, possibly to compensate for inefficient neural processing.

Exogenous control of mammalian gene expression through modulation of RNA self-cleavage.

Recent studies on the control of specific metabolic pathways in bacteria have documented the existence of entirely RNA-based mechanisms for controlling gene expression. These mechanisms involve the modulation of translation, transcription termination or RNA self-cleavage through the direct interaction of specific intracellular metabolites and RNA sequences. Here we show that an analogous RNA-based gene regulation system can effectively be designed for mammalian cells via the incorporation of sequences encoding self-cleaving RNA motifs into the transcriptional unit of a gene or vector. When correctly positioned, the sequences lead to potent inhibition of gene or vector expression, owing to the spontaneous cleavage of the RNA transcript. Administration of either oligonucleotides complementary to regions of the self-cleaving motif or a specific small molecule results in the efficient induction of gene expression, owing to inhibition of self-cleavage of the messenger RNA. Efficient regulation of transgene expression is shown in a variety of mammalian cell lines and live animals. In conjunction with other emerging technologies, this methodology may be particularly applicable to the development of gene regulation systems tailored to any small inducer molecule, and provide a novel means of biological sensing in vivo that may have an important application in the regulated delivery of protein therapeutics.

Genetically engineered T cells to target EGFRvIII expressing glioblastoma.

Glioblastoma remains a significant therapeutic challenge, warranting further investigation of novel therapies. We describe an immunotherapeutic strategy to treat glioblastoma based on adoptive transfer of genetically modified T-lymphocytes (T cells) redirected to kill EGFRvIII expressing gliomas. We constructed a chimeric immune receptor (CIR) specific to EGFRvIII, (MR1-zeta). After in vitro selection and expansion, MR1-zeta genetically modified primary human T-cells specifically recognized EGFRvIII-positive tumor cells as demonstrated by IFN-gamma secretion and efficient tumor lysis compared to control CIRs defective in EGFRvIII binding (MRB-zeta) or signaling (MR1-delzeta). MR1-zeta expressing T cells also inhibited EGFRvIII-positive tumor growth in vivo in a xenografted mouse model. Successful targeting of EGFRvIII-positive tumors via adoptive transfer of genetically modified T cells may represent a new immunotherapy strategy with great potential for clinical applications.

A mammalian cell-based assay for screening inhibitors of RNA cleavage.

RNA cleavage is a catalytic reaction which defines many types of RNA processing events, including those of metabolite-sensing riboswitch, self-splicing introns, mRNA splicing, tRNA processing, polyA-cleavage, and various small ribozymes such as hairpin and hammerhead ribozyme. In this chapter, we describe a general methodology for developing a mammalian cell-based high-throughput screening assay useful for identifying small molecules capable of inhibiting RNA cleavage in mammalian cells. In the specific assay described, a plasmid DNA vector in which the expression of a luciferase reporter gene is controlled by hammerhead ribozyme cleavage was stably introduced into the human 293 cell line. Such a cell line enabled the rapid screening of chemical compound libraries and the identification of cell membrane-permeable inhibitory molecules capable of blocking ribozyme cleavage. The general strategy described later could in principle be adapted to identify small molecule inhibitors of many types of RNA cleavage reactions.

Imaging phonological similarity effects on verbal working memory.

Studies of verbal working memory (VWM) report that performance declines as the phonemic similarity of stimuli increases. To determine how phonological similarity affects brain function during VWM, "standard" and "similarity" versions of the 2-Back task were presented to 34 healthy participants during functional magnetic resonance imaging (FMRI). Letter consonants presented during similarity blocks rhymed, while consonants did not rhyme during standard blocks. Empirical ROIs were identified from significant 2-Back-related activity observed during either condition. A priori ROIs were selected from functional neuroimaging literature on phonological processing. Although VWM-related activity was not modulated by similarity in any of four regions recruited (dorsolateral prefrontal, posterior parietal, anterior insular, and supplementary motor cortices), four of five regions of deactivation exhibited significantly greater deactivation during the similarity compared to the standard condition (posterior cingulate, paracentral lobule, posterior insula, and parahippocampal gyrus). In a priori phonological processing-related ROIs, similarity did not affect observed increases in activity (supplementary motor area, Broca's area, and cerebellum), while two of the three regions exhibiting decreased activity (near Wernicke's area and Heschel's Gyrus) also exhibited more deactivation during similarity. Accuracy was lower during the similarity 2-Back, positively related to activity within recruited VWM-related ROIs, and inversely related to activity in regions of VWM-related deactivation. Based on known functions of these ROIs, we conclude that language, audition, and self-reflection processes may disengage during phonological interference, while activity levels are maintained in regions recruited during VWM processing. Similarity effects likely include suspension of attention to unrelated and distracting processes to improve concentration.

Successful inhibition of intracranial human glioblastoma multiforme xenograft growth via systemic adenoviral delivery of soluble endostatin and soluble vascular endothelial growth factor receptor-2: laboratory investigation.

OBJECT: Glioblastoma multiforme (GBM) is characterized by neovascularization, raising the question of whether angiogenic blockade may be a useful therapeutic strategy for this disease. It has been suggested, however, that, to be useful, angiogenic blockade must be persistent and at levels sufficient to overcome proangiogenic signals from tumor cells. In this report, the authors tested the hypothesis that sustained high concentrations of 2 different antiangiogenic proteins, delivered using a systemic gene therapy strategy, could inhibit the growth of established intracranial U87 human GBM xenografts in nude mice. METHODS: Mice harboring established U87 intracranial tumors received intravenous injections of adenoviral vectors encoding either the extracellular domain of vascular endothelial growth factor receptor-2-Fc fusion protein (Ad-VEGFR2-Fc) alone, soluble endostatin (Ad-ES) alone, a combination of Ad-VEGFR2-Fc and Ad-ES, or immunoglobulin 1-Fc (Ad-Fc) as a control. RESULTS: Three weeks after treatment, magnetic resonance imaging-based determination of tumor volume showed that treatment with Ad-VEGFR2-Fc, Ad-ES, or Ad-VEGFR2-Fc in combination with Ad-ES, produced 69, 59, and 74% growth inhibition, respectively. Bioluminescent monitoring of tumor growth revealed growth inhibition in the same treatment groups to be 62, 74, and 72%, respectively. Staining with proliferating cell nuclear antigen and with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling showed reduced tumor cell proliferation and increased apoptosis in all antiangiogenic treatment groups. CONCLUSIONS: These results suggest that systemic delivery and sustained production of endostatin and soluble VEGFR2 can slow intracranial glial tumor growth by both reducing cell proliferation and increasing tumor apoptosis. This work adds further support to the concept of using antiangiogenesis therapy for intracranial GBM.

Long-lived antitumor CD8+ lymphocytes for adoptive therapy generated using an artificial antigen-presenting cell.

PURPOSE: Antitumor lymphocytes can be generated ex vivo unencumbered by immunoregulation found in vivo. Adoptive transfer of these cells is a promising therapeutic modality that could establish long-term antitumor immunity. However, the widespread use of adoptive therapy has been hampered by the difficulty of consistently generating potent antitumor lymphocytes in a timely manner for every patient. To overcome this, we sought to establish a clinical grade culture system that can reproducibly generate antigen-specific cytotoxic T lymphocytes (CTL). EXPERIMENTAL DESIGN: We created an off-the-shelf, standardized, and renewable artificial antigen-presenting cell (aAPC) line that coexpresses HLA class I, CD54, CD58, CD80, and the dendritic cell maturation marker CD83. We tested the ability of aAPC to generate tumor antigen-specific CTL under optimal culture conditions. The number, phenotype, effector function, and in vitro longevity of generated CTL were determined. RESULTS: Stimulation of CD8(+) T cells with peptide-pulsed aAPC generated large numbers of functional CTL that recognized a variety of tumor antigens. These CTLs, which possess a phenotype consistent with in vivo persistence, survived ex vivo for prolonged periods of time. Clinical grade aAPC(33), produced under current Good Manufacturing Practices guidelines, generated sufficient numbers of CTL within a short period of time. These CTL specifically lysed a variety of melanoma tumor lines naturally expressing a target melanoma antigen. Furthermore, antitumor CTL were easily generated in all melanoma patients examined. CONCLUSIONS: With clinical grade aAPC(33) in hand, we are now poised for clinical translation of ex vivo generated antitumor CTL for adoptive cell transfer.

Engineering vascularized skeletal muscle tissue.

One of the major obstacles in engineering thick, complex tissues such as muscle is the need to vascularize the tissue in vitro. Vascularization in vitro could maintain cell viability during tissue growth, induce structural organization and promote vascularization upon implantation. Here we describe the induction of endothelial vessel networks in engineered skeletal muscle tissue constructs using a three-dimensional multiculture system consisting of myoblasts, embryonic fibroblasts and endothelial cells coseeded on highly porous, biodegradable polymer scaffolds. Analysis of the conditions for induction and stabilization of the vessels in vitro showed that addition of embryonic fibroblasts increased the levels of vascular endothelial growth factor expression in the construct and promoted formation and stabilization of the endothelial vessels. We studied the survival and vascularization of the engineered muscle implants in vivo in three different models. Prevascularization improved the vascularization, blood perfusion and survival of the muscle tissue constructs after transplantation.

Structural requirements of SLP-76 in signaling via the high-affinity immunoglobulin E receptor (Fc epsilon RI) in mast cells.

The adapter SLP-76 plays an essential role in Fc epsilon RI signaling, since SLP-76(-/-) bone marrow-derived mast cells (BMMC) fail to degranulate and release interleukin-6 (IL-6) following Fc epsilon RI ligation. To define the role of SLP-76 domains and motifs in Fc epsilon RI signaling, SLP-76(-/-) BMMC were retrovirally transduced with SLP-76 and SLP-76 mutants. The SLP-76 N-terminal and Gads binding domains, but not the SH2 domain, were critical for Fc epsilon RI-mediated degranulation and IL-6 secretion, whereas all three domains are essential for T-cell proliferation following T-cell receptor (TCR) ligation. Unexpectedly, the three tyrosine residues in SLP-76 critical for TCR signaling, Y112, Y128, and Y145, were not essential for IL-6 secretion, but were required for degranulation and mitogen-activated protein kinase activation. Furthermore, a Y112/128F SLP-76 mutant, but not a Y145F mutant, strongly reconstituted mast cell degranulation, suggesting a critical role for Y145 in Fc epsilon RI-mediated exocytosis. These results point to important differences in the function of SLP-76 between T cells and mast cells.