Influence of mobbing (workplace bullying) on depressive symptoms: a longitudinal study among employees working with people with intellectual disabilities.

BACKGROUND: The problem of mobbing has attracted a great deal of attention over the past few years. This concern has increased the study of the phenomena, which has resulted in many scientific publications. Mobbing has been characterised as an emerging risk at work. The aim of this longitudinal study was to analyse the influence of mobbing on depressive symptoms in a sample of employees working with people with intellectual disabilities (ID). METHOD: The sample consisted of 372 Spanish employees working with people with ID at 61 job centres in the Valencian Community (Spain). Seventy-nine (21.2%) participants were men, and 293 were (78.8%) women. Mobbing was evaluated by the Mobbing-UNIPSICO scale, and depressive symptoms were measured using the Zung Self Rating Depression Scale. Using analyses of variance (anova), we tested the differences in depressive symptoms according to the mobbing criteria indicated by Leymann, that is, frequency and duration at Time 1 and Time 2. RESULTS: Employees who met the mobbing criteria: frequency (at least once a week) and duration (at least 6 months) at the two study times presented significantly higher levels of depressive symptoms than employees who met mobbing criteria at Time 1, but did not meet any criteria for mobbing at Time 2, and employees who did not meet any criteria for mobbing at Time 1 or Time 2. CONCLUSIONS: We conclude that permanence of mobbing from Time 1 to Time 2 increases depressive symptoms.

The mitochondrial RNA granule modulates manganese-dependent cell toxicity.

Prolonged manganese exposure causes manganism, a neurodegenerative movement disorder. The identity of adaptive and nonadaptive cellular processes targeted by manganese remains mostly unexplored. Here we study mechanisms engaged by manganese in genetic cellular models known to increase susceptibility to manganese exposure, the plasma membrane manganese efflux transporter SLC30A10 and the mitochondrial Parkinson's gene PARK2. We found that SLC30A10 and PARK2 mutations as well as manganese exposure compromised the mitochondrial RNA granule composition and function, resulting in disruption of mitochondrial transcript processing. These RNA granule defects led to impaired assembly and function of the mitochondrial respiratory chain. Notably, cells that survived a cytotoxic manganese challenge had impaired RNA granule function, thus suggesting that this granule phenotype was adaptive. CRISPR gene editing of subunits of the mitochondrial RNA granule, FASTKD2 or DHX30, as well as pharmacological inhibition of mitochondrial transcription-translation, were protective rather than deleterious for survival of cells acutely exposed to manganese. Similarly, adult Drosophila mutants with defects in the mitochondrial RNA granule component scully were safeguarded from manganese-induced mortality. We conclude that impairment of the mitochondrial RNA granule function is a protective mechanism for acute manganese toxicity.

ADP ribosylation factor 1 is required for synaptic vesicle budding in PC12 cells.

Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein-T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O'Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041-1049). A peptide spanning the effector domain of human ARF1 (2-17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

Neuroendocrine synaptic vesicles are formed in vitro by both clathrin-dependent and clathrin-independent pathways.

In the neuroendocrine cell line, PC12, synaptic vesicles can be generated from endosomes by a sorting and vesiculation process that requires the heterotetrameric adaptor protein AP3 and a small molecular weight GTPase of the ADP ribosylation factor (ARF) family. We have now discovered a second pathway that sorts the synaptic vesicle-associated membrane protein (VAMP) into similarly sized vesicles. For this pathway the plasma membrane is the precursor rather than endosomes. Both pathways require cytosol and ATP and are inhibited by GTPgammaS. The second pathway, however, uses AP2 instead of AP3 and is brefeldin A insensitive. The AP2-dependent pathway is inhibited by depletion of clathrin or by inhibitors of clathrin binding, whereas the AP3 pathway is not. The VAMP-containing, plasma membrane-derived vesicles can be readily separated on sucrose gradients from transferrin (Tf)-containing vesicles generated by incubating Tf-labeled plasma membrane preparations at 37 degreesC. Dynamin- interacting proteins are required for the AP2-mediated vesiculation from the plasma membrane, but not from endosomes. Thus, VAMP is sorted into small vesicles by AP3 and ARF1 at endosomes and by AP2 and clathrin at the plasma membrane.

BLOC-1 complex deficiency alters the targeting of adaptor protein complex-3 cargoes.

Mutational analyses have revealed many genes that are required for proper biogenesis of lysosomes and lysosome-related organelles. The proteins encoded by these genes assemble into five distinct complexes (AP-3, BLOC-1-3, and HOPS) that either sort membrane proteins or interact with SNAREs. Several of these seemingly distinct complexes cause similar phenotypic defects when they are rendered defective by mutation, but the underlying cellular mechanism is not understood. Here, we show that the BLOC-1 complex resides on microvesicles that also contain AP-3 subunits and membrane proteins that are known AP-3 cargoes. Mouse mutants that cause BLOC-1 or AP-3 deficiencies affected the targeting of LAMP1, phosphatidylinositol-4-kinase type II alpha, and VAMP7-TI. VAMP7-TI is an R-SNARE involved in vesicle fusion with late endosomes/lysosomes, and its cellular levels were selectively decreased in cells that were either AP-3- or BLOC-1-deficient. Furthermore, BLOC-1 deficiency selectively altered the subcellular distribution of VAMP7-TI cognate SNAREs. These results indicate that the BLOC-1 and AP-3 protein complexes affect the targeting of SNARE and non-SNARE AP-3 cargoes and suggest a function of the BLOC-1 complex in membrane protein sorting.

Genetic analysis of the neuronal and ubiquitous AP-3 adaptor complexes reveals divergent functions in brain.

Neurons express adaptor (AP)-3 complexes assembled with either ubiquitous (beta3A) or neuronal-specific (beta3B) beta3 isoforms. However, it is unknown whether these complexes indeed perform distinct functions in neuronal tissue. Here, we explore this hypothesis by using genetically engineered mouse models lacking either beta3A- or beta3B-containing AP-3 complexes. Somatic and neurological phenotypes were specifically associated with the ubiquitous and neuronal adaptor deficiencies, respectively. At the cellular level, AP-3 isoforms were localized to distinct neuronal domains. beta3B-containing AP-3 complexes were preferentially targeted to neuronal processes. Consistently, beta3B deficiency compromised synaptic zinc stores assessed by Timm's staining and the synaptic vesicle targeting of membrane proteins involved in zinc uptake (ZnT3 and ClC-3). Surprisingly, despite the lack of neurological symptoms, beta3A-deficient mouse brain possessed significantly increased synaptic zinc stores and synaptic vesicle content of ZnT3 and ClC-3. These observations indicate that the functions of beta3A- and beta3B-containing complexes are distinct and divergent. Our results suggest that concerted nonredundant functions of neuronal and ubiquitous AP-3 provide a mechanism to control the levels of selected membrane proteins in synaptic vesicles.

A v-SNARE participates in synaptic vesicle formation mediated by the AP3 adaptor complex.

Reconstitution of synaptic vesicle formation in vitro has revealed a pathway of synaptic vesicle biogenesis from endosomes that requires the heterotetrameric adaptor complex AP3. Because synaptic vesicles have a distinct protein composition, the AP3 complex should selectively recognize some or all of the synaptic vesicle proteins. Here we show that one element of this recognition process is the v-SNARE, VAMP-2, because tetanus toxin, which cleaves VAMP-2, inhibited the formation of synaptic vesicles and their coating with AP3 in vitro. Mutant tetanus toxin and botulinum toxins, which cleave t-SNAREs, did not inhibit synaptic vesicle production. AP3-containing complexes isolated from coated vesicles could be immunoprecipitated by a VAMP-2 antibody. These data imply that AP3 recognizes a component of the fusion machinery, which may prevent the production of inert synaptic vesicles.

The AP-3 complex required for endosomal synaptic vesicle biogenesis is associated with a casein kinase Ialpha-like isoform.

The formation of small vesicles is mediated by cytoplasmic coats the assembly of which is regulated by the activity of GTPases, kinases, and phosphatases. A heterotetrameric AP-3 adaptor complex has been implicated in the formation of synaptic vesicles from PC12 endosomes (). When the small GTPase ARF1 is prevented from hydrolyzing GTP, we can reconstitute AP-3 recruitment to synaptic vesicle membranes in an assembly reaction that requires temperatures above 15 degrees C and the presence of ATP suggesting that an enzymatic step is involved in the coat assembly. We have now found an enzymatic reaction, the phosphorylation of the AP-3 adaptor complex, that is linked with synaptic vesicle coating. Phosphorylation occurs in the beta3 subunit of the complex by a kinase similar to casein kinase 1alpha. The kinase copurifies with neuronal-specific AP-3. In vitro, purified casein kinase I selectively phosphorylates the beta3A and beta3B subunit at its hinge domain. Inhibiting the kinase hinders the recruitment of AP-3 to synaptic vesicles. The same inhibitors that prevent coat assembly in vitro also inhibit the formation of synaptic vesicles in PC12 cells. The data suggest, therefore, that the mechanism of AP-3-mediated vesiculation from neuroendocrine endosomes requires the phosphorylation of the adaptor complex at a step during or after AP-3 recruitment to membranes.

The neuronal form of adaptor protein-3 is required for synaptic vesicle formation from endosomes.

Heterotetrameric adaptor complexes vesiculate donor membranes. One of the adaptor protein complexes, AP-3, is present in two forms; one form is expressed in all tissues of the body, whereas the other is restricted to brain. Mice lacking both the ubiquitous and neuronal forms of AP-3 exhibit neurological disorders that are not observed in mice that are mutant only in the ubiquitous form. To begin to understand the role of neuronal AP-3 in neurological disease, we investigated its function in in vitro assays as well as its localization in neural tissue. In the presence of GTPgammaS both ubiquitous and neuronal forms of AP-3 can bind to purified synaptic vesicles. However, only the neuronal form of AP-3 can produce synaptic vesicles from endosomes in vitro. We also identified that the expression of neuronal AP-3 is limited to varicosities of neuronal-like processes and is expressed in most axons of the brain. Although the AP-2/clathrin pathway is the major route of vesicle production and the relatively minor neuronal AP-3 pathway is not necessary for viability, the absence of the latter could lead to the neurological abnormalities seen in mice lacking the expression of AP-3 in brain. In this study we have identified the first brain-specific function for a neuronal adaptor complex.

Microtubules and calibers in developing axons.

The caliber and microtubular content of growing axons were assessed with the electron microscope in the sural nerve of the rat from birth until the age of 63 days (young adult). The caliber of both nonmedullated and myelinated fibers increased throughout the period of observation. At birth, nonmedullated fibers smaller than 0.2 micron2 represented 69% of the population; at day 63 less than 7% were that small. Irrespective of the age of the rat, the number of microtubule profiles of nonmedullated fibers increased with the cross-sectional area of the axon although their packing decreased. In myelinated fibers of a given caliber, the packing of microtubules increased with time, and by day 63 the density had reached its final value. In nonmedullated fibers of a given caliber, a similar trend was observed after day 31; i.e., fibers showed a small but consistent increase in density. However, before that, and in contrast to myelinated fibers, nonmedullated fibers of defined calibers exhibited a transient increase in the microtubular density. Notwithstanding, during the history of an individual fiber the packing of its microtubules may decrease continuously until stabilizing, because the developing fiber is increasing its caliber and hence decreasing its microtubular density. In the 5-day-old rats, the caliber spectra of myelinated and nonmedullated axons overlapped in the 0.49-0.83 micron2 range and their microtubular densities were similar. We conclude that the microtubular content of developing axons correlates with caliber, an architectural feature, and is largely independent of growth and of its myelinated condition. We propose that the cytoskeletal rather than the transport function commands the organization of axonal microtubules.

Calibers and microtubules of nerve fibers: differential effect of undernutrition in developing and adult rats.

Sural nerves of 9-week-old rats undernourished since birth, and of adult rats food-restricted for 27 and 48 days, were studied to explore the effect of severe undernutrition on the caliber and microtubules of axons in growing and non-growing animals. In 9-week-old undernourished rats, the number and caliber of myelinated fibers were normal while the cross-sectional area of non-medullated fibers was 29% smaller than controls. By contrast, in adult undernourished rats the cross-sectional area of myelinated fibers was affected sooner and to a greater extent (-28%) than that of non-medullated fibers (-23%). Regardless of age, in both controls and in undernourished rats non-medullated fibers of equal caliber had similar microtubular content. The same was found in 3-microns myelinated axons. These findings indicate that food restriction affects proportionately caliber and microtubules of axons. It is proposed that the anatomy of the axon is in a dynamic equilibrium and that microtubules participate in the specification of the axonal caliber.

The WASH complex, an endosomal Arp2/3 activator, interacts with the Hermansky-Pudlak syndrome complex BLOC-1 and its cargo phosphatidylinositol-4-kinase type IIα.

Vesicle biogenesis machinery components such as coat proteins can interact with the actin cytoskeleton for cargo sorting into multiple pathways. It is unknown, however, whether these interactions are a general requirement for the diverse endosome traffic routes. In this study, we identify actin cytoskeleton regulators as previously unrecognized interactors of complexes associated with the Hermansky-Pudlak syndrome. Two complexes mutated in the Hermansky-Pudlak syndrome, adaptor protein complex-3 and biogenesis of lysosome-related organelles complex-1 (BLOC-1), interact with and are regulated by the lipid kinase phosphatidylinositol-4-kinase type IIα (PI4KIIα). We therefore hypothesized that PI4KIIα interacts with novel regulators of these complexes. To test this hypothesis, we immunoaffinity purified PI4KIIα from isotope-labeled cell lysates to quantitatively identify interactors. Strikingly, PI4KIIα isolation preferentially coenriched proteins that regulate the actin cytoskeleton, including guanine exchange factors for Rho family GTPases such as RhoGEF1 and several subunits of the WASH complex. We biochemically confirmed several of these PI4KIIα interactions. Of importance, BLOC-1 complex, WASH complex, RhoGEF1, or PI4KIIα depletions altered the content and/or subcellular distribution of the BLOC-1-sensitive cargoes PI4KIIα, ATP7A, and VAMP7. We conclude that the Hermansky-Pudlak syndrome complex BLOC-1 and its cargo PI4KIIα interact with regulators of the actin cytoskeleton.

Neurodevelopmental disorders: mechanisms and boundary definitions from genomes, interactomes and proteomes.

Neurodevelopmental disorders such as intellectual disability, autism spectrum disorder and schizophrenia lack precise boundaries in their clinical definitions, epidemiology, genetics and protein-protein interactomes. This calls into question the appropriateness of current categorical disease concepts. Recently, there has been a rising tide to reformulate neurodevelopmental nosological entities from biology upward. To facilitate this developing trend, we propose that identification of unique proteomic signatures that can be strongly associated with patient's risk alleles and proteome-interactome-guided exploration of patient genomes could define biological mechanisms necessary to reformulate disorder definitions.

[Body composition assessment in patients with chronic renal failure]. / Evaluación de la composición corporal en pacientes con insuficiencia renal crónica.

INTRODUCTION: Assessment of body composition is paramount in early assessment of nutritional status impairments due to excess or deficit. There are, however, few field reliable methods for this objective for patients with chronic renal failure (CRF.). OBJECTIVE: To assess the reliability of the estimations of body composition by different methods as compared to dual energy X-ray absorptiometry (DEXA) as the gold standard method in patients with CRF and on regular chronic haemodialysis. PATIENTS AND METHODS: We assessed body composition in 30 haemodialysis patients (46.9 +/- 15.1 years (18-76); BMI 25.9 +/- 5.7 kg/m(2) (18.1-41.5)), observing agreement in the percentage of fat mass (%FM) between the sum of the 4 folds (SP; calibrator Lange) and bioimpedantiometry by using different equations (BIA; Biodynamics 450) versus DEXA (Lunar DPX-L). RESULTS: (X +/- SD) By BMI, 3 subjects had low weight (10%), 14 normal weight (46.7%), 7 overweight (23.3%), and 6 obesity (20%). The %FM with SP (30.7 +/- 7.1%) significantly differed from DEXA (27.3 +/- 10.3%; p < 0.001). With BIA there was a significant difference in %FM with the Deurenberg and Formica equations. The %FM obtained with the manufacturer's equations (Segal, Lukaski and Kyle) did not show a significant difference from DEXA. With Kyle's equation we observed a better agreement (difference with DEXA: -0.58 +/- 4.2%). CONCLUSIONS: We found a low percentage of patients with low weight as compared to previous studies. The skin folds show low reliability to estimate the fat mass. The bioimpedantiometry, using Kile's equation may be a good filed method to assess haemodialysis patients.

Evaluación de la composición corporal en pacientes con insuficiencia renal crónica / Body composition assessment in patients with chronic renal failure

Introducción: La evaluación de la composición corporal es de gran importancia en la pesquisa temprana de alteraciones en el estado nutricional por déficit o por exceso, sin embargo existen pocos métodos de campo confiables para este objetivo en pacientes con insuficiencia renal crónica (IRC). Objetivo: Evaluar la confiabilidad de estimaciones de composición corporal con distintos métodos en comparación con absorciometría de rayos X de doble energía (DEXA) como método de referencia, en pacientes portadores de IRC sometidos a hemodiálisis crónica periódica. Pacientes y métodos: Se evaluó la composición corporal en 30 pacientes en hemodiálisis (46,9 ± 15,1 años (18-76); IMC 25,9 ± 5,7 kg/m2 (18,1-41,5)), observando la concordancia en el porcentaje de masa grasa (%MG) entre sumatoria de 4 pliegues (SP; calibrador Lange®) y bioimpedanciometría usando distintas ecuaciones (BIA; Biodynamics® 450) contra DEXA (Lunar DPX-L). Resultados: (X ± DE) Según IMC, 3 individuos tenían bajo peso (10%), 14 normopeso (46,7%), 7 sobrepeso (23,3%) y 6 obesidad (20%). El %MG con SP (30,7 ± 7,1%) difirió significativamente de DEXA (27,3 ± 10,3%; p < 0,001). Para BIA hubo diferencia significativa en %MG con ecuaciones de Deurenberg y Formica. El %MG obtenido con ecuaciones del equipo, de Segal, Lukaski y Kyle, no mostró diferencia significativa con DEXA. Con Kyle se observó la mejor concordancia (diferencia con DEXA: -0,58 ± 4,2%). Conclusiones: Se encontró un bajo porcentaje de pacientes con bajo peso con respecto a estudios previos. Los pliegues cutáneos muestran una baja confiabilidad para estimar la masa grasa. La bioimpedanciometría, utilizando la ecuación de Kyle, podría ser un buen método de campo para la evaluación de pacientes en hemodiálisis (AU)

Introduction: Assessment of body composition is paramount in early assessment of nutritional status impairments due to excess or deficit. There are, however, few field reliable methods for this objective for patients with chronic renal failure (CRF.). Objective: To assess the reliability of the estimations of body composition by different methods as compared to dual energy X-ray absorptiometry (DEXA) as the gold standard method in patients with CRF and on regular chronic haemodialysis. Patients and methods: We assessed body composition in 30 haemodialysis patients (46.9 ± 15.1 years (18-76); BMI 25.9 ± 5.7 kg/m2 (18.1-41.5)), observing agreement in the percentage of fat mass (%FM) between the sum of the 4 folds (SP; calibrator Lange®) and bioimpedantiometry by using different equations (BIA; Biodynamics® 450) versus DEXA (Lunar DPX-L). Results: (X ± SD) By BMI, 3 subjects had low weight (10%), 14 normal weight (46.7%), 7 overweight (23.3%), and 6 obesity (20%). The %FM with SP (30.7 ± 7.1%) significantly differed from DEXA (27.3 ± 10.3%; p < 0.001). With BIA there was a significant difference in %FM with the Deurenberg and Formica equations. The %FM obtained with the manufacturer's equations (Segal, Lukaski and Kyle) did not show a significant difference from DEXA. With Kyle's equation we observed a better agreement (difference with DEXA: -0.58 ± 4.2%). Conclusions: We found a low percentage of patients with low weight as compared to previous studies. The skin folds show low reliability to estimate the fat mass. The bioimpedantiometry, using Kile's equation may be a good filed method to assess haemodialysis patient (AU)

A function for the AP3 coat complex in synaptic vesicle formation from endosomes.

Synaptic vesicles can be coated in vitro in a reaction that is ARF-, ATP-, and temperature-dependent and requires synaptic vesicle membrane proteins. The coat is largely made up of the heterotetrameric complex, adaptor protein 3, recently implicated in Golgi-to-vacuole traffic in yeast. Depletion of AP3 from brain cytosol inhibits small vesicle formation from PC12 endosomes in vitro. Budding from washed membranes can be reconstituted with purified AP3 and recombinant ARF1. We conclude that AP3 coating is involved in at least one pathway of small vesicle formation from endosomes.

Beta 2-adrenergic receptor endocytic pathway is controlled by a saturable mechanism distinct from that of transferrin receptor.

Agonist-dependent internalization is an important phase of beta 2-adrenergic receptor (beta 2AR) regulation. Recent reports have indicated that early steps of beta 2AR endocytosis may involve mechanisms different from those which regulate the internalization of constitutively recycling receptors, such as transferrin receptor (TfR). In the present study, we addressed this issue by comparing, in the same cells, the endocytic pathway of beta 2AR with that of the TfR. Upon incubation at 15 degrees C, activated beta 2ARs accumulated in peripheral endosomes of HEK-293 cells while they were targeted to perinuclear organelles at 37 degrees C. The temperature block was not specific to beta 2ARs, since both peripheral and perinuclear beta 2AR-containing endosomes comigrated on sucrose gradients with those containing transferrin receptors and were loaded with horseradish peroxidase-coupled transferrin. Endocytosis of beta 2ARs was saturable in HEK-293 cells and did not increase upon overexpression of beta-arrestin 1. TfR endocytosis was unaffected by the simultaneous internalization of overexpressed beta 2AR, indicating that the limiting components which regulate endocytosis of these two receptors are different. In conclusion, ligand activated beta 2AR and constitutively recycling receptors, such as TfR, enter the endocytic pathway via distinct saturable mechanisms but converge in the same endosomal compartments. Our results also indicate that a still unidentified component(s) controls beta 2AR endocytosis.

Epidermal growth factor receptor in synaptic fractions of the rat central nervous system.

Functional relationships between epidermal growth factor (EGF) and neural tissues have of late attracted increasing interest. However, in spite of reported EGF effects on neurons, the expression of the EGF receptor (EGF-R) has not yet been unambiguously demonstrated in these cells. This 170-kDa protein bears an intracellular tyrosine kinase domain in which activity is ligand-dependent. We give definitive evidence here for its presence in neonatal and adult rat neurons showing also, for the first time, its binding and functional tyrosine kinase activities in the synaptic region. Immunohistochemistry using a polyclonal antibody prepared against the receptor purified from rat liver showed positive staining localized exclusively to neurons without regionalization to any particular brain zone. Binding studies made in Percoll-obtained synaptosomes revealed specific high affinity 125I-EGF binding sites (Kd, 1.42 x 10(-10) +/- 0.58 M) accounting for 17% of total binding and a great majority of low affinity (Kd, 2.55 x 10(-9) +/- 0.35 M) binding sites. Higher binding capacity was found in synaptosomal fractions obtained from newborn rats. The identity of the synaptosomal EGF binding activity with the 170-kDA EGF-R protein was demonstrated by cross-linking experiments. Furthermore, EGF-Affi-Prep affinity chromatography adsorbs a 170-kDa protein with EGF-R immunoreactivity from whole homogenates of adult rat brain. Phosphorylation assays made in freeze-thawed or intact synaptosomes showed EGF-induced tyrosine phosphorylation in the range of 170-, 126-150-, 124-, 113-, 98-, and 70-kDa proteins including the EGF-R. Thus, the EGF-R/EGF regulatory system could have a role in synaptic function that remains to be explored.

Ciprofibrate, a carcinogenic peroxisome proliferator, increases the phosphorylation of epidermal-growth-factor receptor in isolated rat hepatocytes.

Ciprofibrate, a hypolipidaemic drug with carcinogenic and peroxisome-proliferation effects in rat liver, was found to increase the phosphorylation of epidermal-growth-factor receptor in 32P-labeled isolated rat hepatocytes. This effect was suppressed by protein-kinase-C inhibitors, and was accompanied by an almost complete inhibition of the receptor autophosphorylation normally induced by its ligand. However, in vitro experiments showed that protein-kinase-C phosphorylation of purified epidermal-growth-factor receptor was activated by ciprofibroyl-CoA, the acyl-CoA derivative of the drug, but not by the unmodified drug. Neither compound affected the ligand induction of epidermal-growth-factor-receptor autophosphorylation in isolated liver membranes. These results suggest that metabolically produced ciprofibroyl-CoA in liver cells would activate protein-kinase-C and produce changes in epidermal-growth-factor-receptor function.

Vesiculation and sorting from PC12-derived endosomes in vitro.

Formation of small vesicles resembling synaptic vesicles can be reconstituted in vitro by incubating labeled homogenates of PC12 cells with ATP and two cytoplasmic proteins, AP3 and ARF1 [Faúndez, V., Horng, J.-T. & Kelly, R. B. (1998) Cell 93, 423-432]. To determine whether AP3 was mediating budding from plasma membranes or endosomes the organelle that generated the synaptic vesicles was characterized. The budding activity was enriched in organelles that labeled at 15 degrees C, but not at 4 degrees C, that excluded a marker of plasma membranes and that contained internalized transferrin, indicating that the precursor was an endosome. Vesicles formed from the endosomal precursor in vitro excluded transferrin. We conclude that ARF-mediated vesiculation into synaptic vesicle-sized organelles uses an endosomal precursor and occurs simultaneously in vitro with sorting of synaptic vesicle proteins from other membrane protein constituents of the endosome.