Éducation précoce pour les enfants Sourds : les effets du dépistage à Saint-Étienne.

BACKGROUND: Launched in 2014, the national screening program for permanent neonatal deafness has reconfigured the care of Deaf children. By bringing forward the age of the start of care, it promotes language development and allows these children to be referred to dedicated services more quickly. OBJECTIVE: In this context of epidemiological reorganization and technological change (cochlear implantation in particular), the Family Support and Early Education Services of the Loire (Safep 42) propose a reflection of the type “evaluation of professional practices” around the mediations proposed in its service and the “educational garden” framework. METHOD: This mixed-method work combines composite data: activity reports, observations, interviews, experience capture, evaluation of professional practices. RESULTS: A decrease in the age of entry (linked to early detection) and an increase in the age of exit (due to the prevalence of children with associated disorders or complexity factors) led Safep 42 to adapt by creating new mediation, while maintaining the support provided for older children, particularly the educational garden. Safep is currently reaching its maximum capacity: in addition to this tension, there is a shortage of outpatient speech therapists and an increase in the number of deafness situations associated with multiple disabilities. The service has had to give up some of its tasks in order to be able to continue to provide proper care for the children in its care. DISCUSSION: The rehabilitation and family support program is accompanied by a longer period of care for the children and this data must be recorded by the funding authorities.

Dynamic dentin: A quantitative microscopic assessment of age and spatial changes to matrix architecture, peritubular dentin, and collagens types I and III.

To investigate age and site-related changes to human dentin collagen, sound human teeth collected from donors aged 13-29 (young) and 50-74 (aged) years (n = 9/group) were cut to shallow and deep sites. Dentin collagen orientation and fibril bundling was investigated using the Picrosirius Red (PSR) stain observed under cross-polarized light microscopy (Pol), and collagen distribution was investigated using Confocal Laser Scanning Microscopy (CLSM). Collagen types III to I distribution in peritubular dentin (PTD) was revealed using Herovici stain and brightfield microscopy. Image analysis software and linear mixed modelling quantified outcomes. In situ dentin collagen was observed using Xenon Plasma Focussed Ion Beam Scanning Electron Microscopy (Xe PFIB-SEM). The PSR-Pol analysis revealed less coherently aligned and more bundled collagen fibrils in aged dentin (P = 0.005). Deep inner dentin collagen in both groups were less coherently aligned with reduced bundling. Regardless of age, CLSM showed collagen distribution remained stable; and more collagen type III was detectable in PTD located in inner dentin (Young: P = 0.006; Aged: P = 0.008). Observations following Xe PFIB-SEM cross-sectioning showed apatite-like deposits surrounding large intratubular collagen fibers, and evidence of smaller intertubular dentin collagen fibrils in situ. In conclusion, aging changes collagen network architecture, but not distribution or content.

Early prosthetic joint infection due to Ureaplasma urealyticum: Benefit of 16S rRNA gene sequence analysis for diagnosis.

Detection of the most frequently bacteria involved in prosthetic joint infection (PJI) is usually performed by conventional cultures. We report a case of early PJI due to Ureaplasma urealyticum, diagnosed by 16S rRNA gene sequence analysis, which highlights the interest of molecular methods if fastidious bacteria are involved in PJI.

N-acetyl-cysteine increases cellular dysfunction in progressive chronic kidney damage after acute kidney injury by dampening endogenous antioxidant responses.

Oxidative stress and mitochondrial dysfunction exacerbate acute kidney injury (AKI), but their role in any associated progress to chronic kidney disease (CKD) remains unclear. Antioxidant therapies often benefit AKI, but their benefits in CKD are controversial since clinical and preclinical investigations often conflict. Here we examined the influence of the antioxidant N-acetyl-cysteine (NAC) on oxidative stress and mitochondrial function during AKI (20-min bilateral renal ischemia plus reperfusion/IR) and progression to chronic kidney pathologies in mice. NAC (5% in diet) was given to mice 7 days prior and up to 21 days post-IR (21d-IR). NAC treatment resulted in the following: prevented proximal tubular epithelial cell apoptosis at early IR (40-min postischemia), yet enhanced interstitial cell proliferation at 21d-IR; increased transforming growth factor-ß1 expression independent of IR time; and significantly dampened nuclear factor-like 2-initiated cytoprotective signaling at early IR. In the long term, NAC enhanced cellular metabolic impairment demonstrated by increased peroxisome proliferator activator-γ serine-112 phosphorylation at 21d-IR. Intravital multiphoton microscopy revealed increased endogenous fluorescence of nicotinamide adenine dinucleotide (NADH) in cortical tubular epithelial cells during ischemia, and at 21d-IR that was not attenuated with NAC. Fluorescence lifetime imaging microscopy demonstrated persistent metabolic impairment by increased free/bound NADH in the cortex at 21d-IR that was enhanced by NAC. Increased mitochondrial dysfunction in remnant tubular cells was demonstrated at 21d-IR by tetramethylrhodamine methyl ester fluorimetry. In summary, NAC enhanced progression to CKD following AKI not only by dampening endogenous cellular antioxidant responses at time of injury but also by enhancing persistent kidney mitochondrial and metabolic dysfunction.

Comparison of lateral and skyline fluoroscopic views for detection of prominent screws in distal radius fractures plating: results of an ultrasonographic study.

INTRODUCTION: Extensor tendon rupture is a recognized complication of volar plate fixation of distal radius fractures due to screws protruding past the dorsal cortex. The aim of this study was to compare the Skyline view with traditional lateral fluoroscopic views using ultrasonography as a reference standard in the postoperative assessment. MATERIALS AND METHODS: A monocentric prospective study was conducted to identify screws penetrating the dorsal cortex after volar plating of distal radius fractures. PATIENTS AND INTERVENTION: Intraoperative anteroposterior (AP) and lateral views were used for group A (28 patients). AP, lateral and skyline fluoroscopic views were used for Group B (40 patients). Prominent screws were changed. MAIN OUTCOME MEASUREMENTS: Ultrasound was done 6 months postoperatively to evaluate the number and length of prominent dorsal screws and any signs of extensor tenosynovitis. RESULTS: The number of prominent dorsal screws exceeding 1 mm was 14 in group A (14.9%), and 16 screws (11.8%) in group B (p = 0.487). Average length of prominent dorsal screw was 1.9 mm (range 1-2.1 mm) for group A and 2.4 mm (range 1.1-4.8 mm) for group B (p = 0.534). The number of patients with extensor tenosynovitis was 11 for group A and 12 for group B (p = 0.66). CONCLUSIONS: The Skyline view does not provide sensitive and reliable detection of the dorsal screw penetration. Intraoperative ultrasound might be a better tool to detect screw prominence. LEVEL OF EVIDENCE: III, case-control study.

Medication at discharge in an orthopaedic surgical ward: quality of information transmission and implementation of a medication reconciliation form.

Background Medication reconciliation (MedRec) at discharge is a cumbersome but necessary process to reduce the risk of medication errors at transitions of care. The main barriers to implementing such a process are the large number of professionals involved and a lack of collaboration among caregivers. Objective This study was designed to assess the need for a medication reconciliation form at discharge in an orthopaedic surgical ward. Setting The study was conducted in the orthopaedic surgery ward among inpatients at a 407-bed French teaching hospital. Method We first performed a retrospective audit to evaluate the quality of discharge medication information in the medical record, after which a 5-week prospective study was conducted in 2013. All patients admitted to the orthopaedic surgery unit who had at least two chronic diseases and three medications underwent MedRec at discharge. We designed a Best Possible Medication at Discharge List (BPMDL) to be completed by hospital staff and transmitted to community caregivers. Mean outcome measures We assessed the completeness of medication information in the medical records, discrepancies between medications noted on the BPMDL and those prescribed on the discharge order, and the value of the BPMDL for stakeholders. Results Thirty patients were included in the study. Only 4 % of medical records contained a discharge summary with complete medication information. In 67 % of cases, treatment discontinuations at admission were justified, and medications were reintroduced before discharge, while 107 treatments (45 %) were added but not prescribed on discharge orders. Discontinuations prior to discharge were justified in 60 % of cases (treatments were ended or supportive treatment was required during hospitalization). An average of 2.1 treatments were prescribed on discharge orders (vs. 9.4 prescribed on the BPMDL). Patients, general practitioners (GP), and physicians in long-term care settings (PLTCS) rated the format, content, and readability of the BPMDL as satisfactory, and it was found to be of value for patients and PLTCS to support medication information. Because of the very low response rate among GP (10 %), we were unable to determine their satisfaction with the MedRec discharge process. Conclusion The transmission of patient medication information at discharge is often lacking. As such, the BPMDL appears to have value to both patients and community health providers. Because this study was conducted within a single surgical unit, further study in other surgical wards is needed to assess generalizability.

Reducing medication errors at admission: 3 cycles to implement, improve and sustain medication reconciliation.

BACKGROUND: In France, medication errors are the third leading cause of serious adverse events. Many studies have shown the positive impact of medication reconciliation (MR) on reducing medication errors at admission but this practice is still rarely implemented in French hospitals. OBJECTIVE: Implement and sustain a MR process at admission in two surgery units. The quality improvement approach used to meet this objective is described. SETTING: The gastrointestinal surgery and orthopedic surgery departments of a 407 inpatient bed French teaching hospital. METHODS: A step by step collaborative approach based on plan-do-study-act (PDSA) cycles. Three cycles were successively performed with regular feedback during multidisciplinary meetings. MAIN OUTCOME MEASURE: mean unintended medication discrepancies (UMDs) per patients at admission. RESULTS: The three PDSA cycles and the monitoring phase allowed to implement, optimize and sustain a MR process in the two surgery units. Cycle 1, by showing a rate of 0.65 UMDs at admission (95 % CI 0.39-0.91), underlined the need for a MR process; cycle 2 showed how the close-collaboration between pharmacy and surgery units could help to reduce mean UMDs per patients at admission (0.18; 95 % CI 0.09-0.27) (p < 0.001); finally, cycle 3 allowed the optimization of the MR process by reducing the delays of the best possible medication history availability. CONCLUSIONS: This work highlights how a collaborative quality-improvement approach based on PDSA cycles can meet the challenge of implementing MR to improve medication management at admission.

Multiphoton fluorescence microscopy of the live kidney in health and disease.

The structural and functional heterogeneity of the kidney ensures a diversity of response in health and disease. Multiphoton microscopy has improved our understanding of kidney physiology and pathophysiology by enabling the visualization of the living kidney in comparison with the static view of previous technologies. The use of multiphoton microscopy with rodent models in conjunction with endogenous fluorescence and exogenous infused dyes permits the measurement of renal processes, such as glomerular permeability, juxtaglomerular apparatus function, tubulointerstitial function, tubulovascular interactions, vascular flow rate, and the intrarenal renin-angiotensin-aldosterone system. Subcellular processes, including mitochondrial dynamics, reactive oxygen species production, cytosolic ion concentrations, and death processes apoptosis and necrosis, can also be measured by multiphoton microscopy. This has allowed valuable insight into the pathophysiology of diabetic nephropathy, renal ischemia-reperfusion injury, hypertensive nephropathy, as well as inflammatory responses of the kidney. The current review presents an overview of multiphoton microscopy with a focus on techniques for imaging the kidney and gives examples of instances where multiphoton microscopy has been utilized to study renal pathophysiology in the living kidney. With continued advancements in the field of biological optics and increased adoption in experimental nephrology, multiphoton microscopy will undoubtedly continue to create new paradigms in kidney disease.

Portal, but not lobular, macrophages express matrix metalloproteinase-9: association with the ductular reaction and fibrosis in chronic hepatitis C.

BACKGROUND: Liver macrophages are a heterogeneous cell population that produces factors involved in fibrogenesis and matrix turnover, including matrix metalloproteinase (MMP) -9. During liver injury, their close proximity to hepatic progenitor cells and the ductular reaction may enable them to regulate liver repair and fibrosis. AIMS: To enumerate and characterise liver macrophages in patients with chronic hepatitis C, to determine whether a distinct population of macrophages is associated with the ductular reaction and portal fibrosis. METHODS: Immunostaining for macrophage markers (CD68, CD163, CCR2), the ductular reaction (keratin-7) and MMP-9 was performed in liver biopsy sections from patients with chronic hepatitis C virus (HCV) (n = 85). RESULTS: Portal tracts were more densely populated with macrophages (10.5 ± 0.36 macrophages/HPF) than lobules (7.2 ± 0.16 macrophages/HPF, P < 0.001) and macrophages were found in close proximity to the ductular reaction. ≥30% of portal and periductal macrophages expressed MMP-9 and these were significantly associated with increasing stage of fibrosis (rs  = 0.58, 0.68, respectively, both P < 0.001). In contrast, MMP-9(+) macrophages were largely absent in lobular regions and non-diseased liver. Hepatic MMP-9 mRNA levels and gelatinolytic activity were significantly associated with stage of fibrosis (rs  = 0.47, rs  = 0.89, respectively, both P < 0.001). Furthermore, a second distinct CCR2(+) macrophage population was localised to the centrilobular regions and was predominantly absent from portal and periductal areas. CONCLUSIONS: These findings demonstrate significant regional differences in macrophage phenotypes, suggesting that there are at least two populations of liver macrophages. We propose that these populations have distinct contributions to the pathogenesis of chronic HCV-related liver disease.

Oxidative stress and cell senescence combine to cause maximal renal tubular epithelial cell dysfunction and loss in an in vitro model of kidney disease.

BACKGROUND: The incidence and cost of chronic kidney disease (CKD) are increasing. Renal tubular epithelial cell dysfunction and attrition, involving increased apoptosis and cell senescence, are central to the pathogenesis of CKD. The aim here was to use an in vitro model to investigate the separate and cumulative effects of oxidative stress, mitochondrial dysfunction and cell senescence in promoting loss of renal mass. METHODS: Human kidney tubular epithelial cells (HK2) were treated with moderate hydrogen peroxide (H2O2) for oxidative stress, with or without cell cycle inhibition (apigenin, API) for cell senescence. Adenosine triphosphate (ATP) and oxidative stress were measured by ATP assay, lipid peroxidation, total antioxidant capacity, mitochondrial function with confocal microscopy, MitoTracker Red CMXRos and live cell imaging with JC-1. In parallel, cell death and injury (i.e. apoptosis and Bax/Bcl-XL expression, lactate dehydrogenase), cell senescence (SA-ß-galactosidase) and renal regenerative ability (cell proliferation), and their modulation with the anti-oxidant N-acetyl-cysteine (NAC) were investigated. RESULTS: H2O2 and API, separately, increased oxidative stress and mitochondrial dysfunction, apoptosis and cell senescence. Although API caused cell senescence, it also induced oxidative stress at levels similar to H2O2 treatment alone, indicating that senescence and oxidative stress may be intrinsically linked. When H2O2 and API were delivered concurrently, their detrimental effects on renal cell loss were compounded. The antioxidant NAC attenuated apoptosis and senescence, and restored regenerative potential to the kidney. CONCLUSION: Oxidative stress and cell senescence both cause mitochondrial destabilization and cell loss and contribute to the development of the cellular characteristics of CKD.

Syntaxin 11 binds Vti1b and regulates late endosome to lysosome fusion in macrophages.

Syntaxin 11 (Stx11) is a SNARE protein enriched in cells of the immune system. Loss or mutation of Stx11 results in familial hemophagocytic lymphohistiocytosis type-4 (FHL-4), an autosomal recessive disorder of immune dysregulation characterized by high levels of inflammatory cytokines along with defects in T-cell and natural killer cell function. We show here Stx11 is located on endosomal membranes including late endosomes and lysosomes in macrophages. While Stx11 did not form a typical trans-SNARE complex, it did bind to the Q-SNARE Vti1b and was able to regulate the availability of Vti1b to form the Q-SNARE complexes Stx6/Stx7/Vtib and Stx7/Stx8/Vti1b. The mutant form of Stx11 sequestered Vti1b from forming the Q-SNARE complex that mediates late endosome to lysosome fusion. Depletion of Stx11 in activated macrophages leads to an accumulation of enlarged late endocytic compartments, increased trafficking to the cell surface and inhibition of late endosome to lysosome fusion. These phenotypes are rescued by the expression of an siRNA-resistant Stx11 construct in Stx11-depleted cells. Our results suggest that by regulating the availability of Vti1b, Stx11 regulates trafficking steps between late endosomes, lysosomes and the cell surface in macrophages.

Cytokine secretion is distinct from secretion of cytotoxic granules in NK cells.

NK cells are renowned for their ability to kill virally infected or transformed host cells by release of cytotoxic granules containing granzymes and perforin. NK cells also have important regulatory capabilities chiefly mediated by secretion of cytokines, such as IFN-gamma and TNF. The secretory pathway for the release of cytokines in NK cells is unknown. In this study, we show localization and trafficking of IFN-gamma and TNF in human NK cells in compartments and vesicles that do not overlap with perforin or other late endosome granule markers. Cytokines in post-Golgi compartments colocalized with markers of the recycling endosome (RE). REs are functionally required for cytokine release because inactivation of REs or mutation of RE-associated proteins Rab11 and vesicle-associated membrane protein-3 blocked cytokine surface delivery and release. In contrast, REs are not needed for release of perforin from preformed granules but may be involved at earlier stages of granule maturation. These findings suggest a new role for REs in orchestrating secretion in NK cells. We show that the cytokines IFN-gamma and TNF are trafficked and secreted via a different pathway than perforin. Although perforin granules are released in a polarized fashion at lytic synapses, distinct carriers transport both IFN-gamma and TNF to points all over the cell surface, including within the synapse, for nonpolarized release.

[Laboratory diagnosis of Fabry disease: historical perspectives and recent breakthroughs]. / Le diagnostic biologique de la maladie de Fabry: aspects historiques et évolutions récentes.

Due to its high sensitivity and specificity, determination of the c-galactosidase A activity in leukocytes is the gold standard to confirm the diagnosis of Fabry disease in hemizygous moles. In contrast, heterozygous females cannot be dependably diagnosed by this method and genotyping should always be carried out. Two recent breakthroughs have contributed to the rise of screening programs for Fabry disease: first, the use of filter papers to collect blood or urine spots from individuals in at-risk populations for Fabry disease, second, the development of moss spectrometry Substrate accumulation and enzyme activity can be measured on urine and blood samples, respectively Moss spectrometry allows the qualitative and quantitative determination of the accumulation of the undegraded substrate (globotriaosylceromide) in urine, but also the measurement of enzyme activities in blood samples for the simultaneous diagnosis of several inborn errors of metabolism, among which lysosomal storage diseases and in particular Fabry disease.

[Non-guideline use of innovative and expensive drugs in pediatrics: assessing clinical practice]. / Utilisation hors recommandations des médicaments innovants et coûteux en pédiatrie.

OBJECTIVE: The objective of this study was to analyze in a pediatric hospital the use of expensive drugs as part of the new activity-based system (T2A) of funding for French public hospitals. We identified and analyzed the therapeutic use of these drugs in indications not included in the expert recommendations issued to accompany this change, with the goal of proposing specific pediatric recommendations. METHOD: Analysis of prescriptions from May through September 2005 showed that 259 patients received expensive drugs subject to special reimbursement. The computerized prescription system enabled us to monitor and validate prescriptions daily. Indications for these expensive drugs were ranked by relevance. RESULTS: The prescriptions analyzed covered 26 expensive drugs. Among the 344 "patient-drugs", 80% were expensive drugs for an accepted therapeutic use, 5% for a pertinent therapeutic use (under evaluation), and 15% for "off-label" uses (2% "not approved" and 13% for indications not considered by the recommendations). CONCLUSION: This study showed that some therapeutic uses not approved by the official recommendations are nevertheless justified. Gathering data from other pediatric hospitals is essential to determine the need for pediatric clinical trials.

Atmospheric pressure photoionization coupled to porous graphitic carbon liquid chromatography for the analysis of globotriaosylceramides. Application to Fabry disease.

Globotriaosylceramides (Gb(3)) are biological compounds implicated in Fabry disease, a lysosomal storage disease due to the deficient activity of alpha-D-galactosidase A, which results in an accumulation of Gb(3) in many organs. The naturally occurring samples are composed of mixtures of several molecular species differing by the structure of the alkyl chains and the nature of the sphingoid base. Atmospheric pressure photoionization mass spectrometry (APPI-MS) proved to be an efficient method for the analysis of globotriaosylceramide molecular species, both in direct injection and by coupling with liquid chromatography (LC). In the positive ion mode, in-source fragmentations yield very precious information that can be used to determine the structure of the alkyl chains. In the negative ion mode, the chloroform solvent participates to the analyte ionization by forming an adduct with chloride ions generated in situ. Combination of LC on a Porous Graphitic Carbon stationary phase and APPI-MS allowed the detection of a great number of species from biological samples isolated from Fabry patients. This method could be an interesting analytical tool for the biochemical investigation of (sphingo) lipid metabolism.

Individual palmitoyl residues serve distinct roles in H-ras trafficking, microlocalization, and signaling.

H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopalmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cholesterol-dependent and cholesterol-independent microdomains. In contrast, monopalmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-palmitate is weaker. Thus, membrane affinity of a palmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopalmitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.

Fast fingerprinting by MALDI-TOF mass spectrometry of urinary sediment glycosphingolipids in Fabry disease.

Fabry disease (FD) is an X-linked inborn error of glycosphingolipid (GSL) metabolism, caused by a deficiency of the lysosomal alpha-galactosidase A, which results in high levels in lysosomes and biological fluids of globotriaosylceramide (Gb3) and digalactosylceramide (Ga2), also known as galabiosylceramide. We report here a detailed study of the molecular species of GSLs in urinary samples obtained from hemizygous and heterozygous patients by use of matrix-assisted laser desorption ionisation and tandem mass spectrometry (MALDI-MS-MS). Twenty-two and fifteen molecular species were identified in the globotriaosylceramide and digalabiosylceramide series, respectively. The major sphingoid base was sphingosine (d18:1), and dihydrosphingosine (C18:0) and sphingadienine (d18:2) were also present. The molecular profiles obtained by MALDI-TOF-MS enabled us to show significant differences between GSLs composition for young, adult or atypic hemizygote and heterozygote patients. Thus, MALDI-TOF-MS and MS-MS proved a powerful tool for screening a population of patients with clinical signs suggestive of FD by direct and rapid GSL fingerprinting and identification, and for study of the biological processes occurring in glycosphingolipid accumulation.

H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis.

Endocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wild-type Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and endocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.

Inhibition of lipid raft-dependent signaling by a dystrophy-associated mutant of caveolin-3.

Specific point mutations in caveolin-3, a predominantly muscle-specific member of the caveolin family, have been implicated in limb-girdle muscular dystrophy and in rippling muscle disease. We examined the effect of these mutations on caveolin-3 localization and function. Using two independent assay systems, Raf activation in fibroblasts and neurite extension in PC12 cells, we show that one of the caveolin-3 point mutants, caveolin-3-C71W, specifically inhibits signaling by activated H-Ras but not by K-Ras. To gain insights into the effect of the mutant protein on H-Ras signaling, we examined the localization of the mutant proteins in fibroblastic cells and in differentiating myotubes. Unlike the previously characterized caveolin-3-DGV mutant, the inhibitory caveolin-3-C71W mutant reached the plasma membrane and colocalized with wild type caveolins. In BHK cells, caveolin-3-C71W associated with caveolae and in differentiating muscle cells with the developing T-tubule system. In contrast, the caveolin-3-P104L mutant accumulated in the Golgi complex and had no effect on H-Ras-mediated Raf activation. Inhibition by caveolin-3-C71W was rescued by cholesterol addition, suggesting that the mutant protein perturbs cholesterol-rich raft domains. Thus, we have demonstrated that a naturally occurring caveolin-3 mutation can inhibit signaling involving cholesterol-sensitive raft domains.