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The aim of this prospective study is to elucidate the relationship between the Voice Handicap Index (VHI) and several voice laboratory measurements in the network of the multidimensional voice assessment. Fifty-eight patients were included. Each patient replies to the questionnaire and performs a voice assessment during the same time. The following parameters were measured: minimum frequency, maximum frequency, range, minimum intensity, subglottic pressure, mean flow, maximum phonation time, jitter, and dysphonia severity index. Regarding the relationship with the scores of the VHI, poor correlations with the minimal frequency for all the scores except the emotional one (total and subscales) and with the range for only the physical one are found. Seventeen questions correlate with the voice laboratory measurements we performed, with a decreased distribution between physical, functional, and emotional subscales. We observe that acoustic parameter is correlated with the emotional subscale, the parameters of the profile range are more often involved in the emotional subscale, as is the minimal frequency, but never with the physical subscale, and all the subscales are interesting despite the smaller number of differences with the emotional one. The VHI and the laboratory measurements give independent informations in practice.

Recent studies indicate that renal gluconeogenesis is substantially stimulated in patients with type 2 diabetes, but the mechanism that is responsible for such stimulation remains unknown. Therefore, this study tested the hypothesis that renal gluconeogenesis is intrinsically elevated in the Zucker diabetic fatty rat, which is considered to be an excellent model of type 2 diabetes. For this, isolated renal proximal tubules from diabetic rats and from their lean nondiabetic littermates were incubated in the presence of physiologic gluconeogenic precursors. Although there was no increase in substrate removal and despite a reduced cellular ATP level, a marked stimulation of gluconeogenesis was observed in diabetic relative to nondiabetic rats, with near-physiologic concentrations of lactate (38%), glutamine (51%) and glycerol (66%). This stimulation was caused by a change in the fate of the substrate carbon skeletons resulting from an increase in the activities and mRNA levels of the key gluconeogenic enzymes that are common to lactate, glutamine, and glycerol metabolism, i.e., mainly of phosphoenolpyruvate carboxykinase and, to a lesser extent, of glucose-6-phosphatase and fructose-1,6-bisphosphatase. Experimental evidence suggests that glucocorticoids and cAMP were two factors that were responsible for the long-term stimulation of renal gluconeogenesis observed in the diabetic rats. These data provide the first demonstration in an animal model that renal gluconeogenesis is upregulated by a long-term mechanism during type 2 diabetes. Together with the increased renal mass (38%) observed, they lend support to the view so far based only on in vivo studies performed in humans that renal gluconeogenesis may be stimulated by and crucially contribute to the hyperglycemia of type 2 diabetes.

Exposure of neutrophils to chemoattractant induces cell polarization and migration. These behaviors require the asymmetric activation of distinct signaling pathways and cytoskeletal elements in the protruding pseudopod at the front of cells and the retracting uropod at the rear. An important outstanding question is, how does the organization of the plasma membrane participate in establishing asymmetry during polarization and migration? To answer this question, we investigated the function of cholesterol, a lipid known to influence membrane organization. Using controlled cholesterol depletion, we found that a cholesterol-dependent membrane organization enabled cell polarization and migration by promoting uropod function and suppressing ectopic pseudopod formation. At a mechanistic level, we showed that cholesterol was directly required for suppressing inappropriate activation of the pseudopod-promoting Gi/PI3-kinase signaling pathway. Furthermore, cholesterol was required for dampening Gi-dependent negative feedback on the RhoA signaling pathway, thus enabling RhoA activation and uropod function. Our findings suggest a model in which a cholesterol-dependent membrane organization plays an essential role in the establishment of cellular asymmetry by balancing the activation and segregating the localization of competing pseudopod- and uropod-inducing signaling pathways during neutrophil polarization and migration.

Dynamic connections between actin filaments and the plasma membrane are crucial for the regulation of blood platelet functions. Protein complexes associated with alphaIIbbeta3 integrin-based cytoskeleton structures are known to play a role in these processes. However, mechanisms involving lateral organizations of the plasma membrane remain to be investigated. Here, we demonstrate that a large fraction of platelet lipid rafts specifically associates with the actin cytoskeleton upon activation. This association was inhibited by antagonists of fibrinogen-alphaIIbbeta3 binding and did not occur in type I Glanzman's thrombasthenic platelets. The raft-cytoskeleton interaction is a reversible process correlating with the intensity and stability of platelet aggregation. Although only a minor fraction of alphaIIbbeta3 was recovered in rafts upon activation, this integrin specifically upregulated the level of PtdIns(4,5)P2 in membrane microdomains and induced the recruitment of several actin-modulating proteins known to directly or indirectly interact with this lipid. Controlled disruption of rafts did not affect alphaIIbbeta3-mediated platelet aggregation in response to high concentrations of thrombin but significantly inhibited fibrin clot retraction. We propose that rafts participate in the organization of membrane-cytoskeleton interactions where alphaIIbbeta3-mediated tension forces apply during the late phase of platelet activation.

•  The cellular tolerance to nickel (Ni), zinc (Zn) and cadmium (Cd) of two poly-hyperaccumulators, Arabidopsis halleri and Thlaspi caerulescens, was investigated in order to compare their cellular phenotypes toward various metal ion exposures. •  Protoplasts were kept for 24 h on solutions containing increasing concentrations of the metal ions, and a viability test was performed. Zinc loading of the protoplasts was investigated with Arabidopsis lyrata and A. halleri protoplasts using the Zn fluorescent indicator Newport green diacetate. •  Only T. caerulescens protoplasts showed a clear tolerance to Ni. On the other hand, protoplasts from both hyperaccumulators displayed a very high and constitutive Zn tolerance and an inducible Cd tolerance. The vacuolar storage of Zn was confirmed, but no Zn accumulation at all was observed in A. halleri protoplasts after Zn exposure. •  Specific metal tolerances were found at the cellular level in the hyperaccumulating plants, highlighting that specific adaptations to metal ions exist in the cells as well as in the whole plants.

The aim of this prospective investigation was to validate a French version of the Voice Handicap Index (VHI). A population of 52 normal subjects and 63 patients with voice disorders replied to the questionnaire at the day of their first consultation and ten to thirty days after the consultation (before any treatment). Reproducing the methodology of the American authors, the test-retest reliability and the internal consistency reliability were measured. The validity and the sensibility related to a non dysphonic population were also analysed. Regarding the normal subjects, the maximum scores for total score and subscale scores were 20 (total), 12 (physical), 7 (functional), 6 (Emotional). These scores are statistically different when compared with pathological subjects (p < 0.00001). Test-Retest stability of the pathological subjects was found to be satisfactory for both total score and subscale scores (r > 0.87). From this data set, the critical difference scores were derived for the VHI total score (15 points), for the physical subscale (9 points) and for the functional and emotional.subscales (6 points each). As for as the internal consistency reliability, Cronbach's alpha is correct (r > 7) for the pathological subjects. Yet, the analysis of the validity reply by reply, reveals some abnormalities. In conclusion, the validity of the French translation of the VHI is confirmed but the results prompt us to improve the quality of the translation.

Src homology domain 2-containing inositol 5-phosphatases 1 and 2 (SHIP1 and SHIP2) are capable of dephosphorylating the second messenger PtdIns(3,4,5) P3 (phosphatidylinositol 3,4,5-trisphosphate) and interacting with several signalling proteins. SHIP1 is essentially expressed in haematopoietic cells, whereas SHIP2, a closely related enzyme, is ubiquitous. In the present study, we show that SHIP1 and SHIP2 are expressed as functional PtdIns(3,4,5) P3 5-phosphatases in human blood platelets and are capable of interacting when these two lipid phosphatases are co-expressed, either naturally (platelets and A20 B lymphoma cells) or artificially (COS-7 cells). Using COS-7 cells transfected with deletion mutants of SHIP2, we demonstrate that the Src homology domain 2 of SHIP2 is the minimal and sufficient protein motif responsible for the interaction between the two phosphatases. These results prompted us to investigate the relative importance of SHIP1 and SHIP2 in the control of PtdIns(3,4,5) P3 levels in platelets using homozygous or heterozygous SHIP1- or SHIP2-deficient mice. Our results strongly suggest that SHIP1, rather than SHIP2, plays a major role in controlling PtdIns(3,4,5) P3 levels in response to thrombin or collagen activation of mouse blood platelets.

Human platelets express the receptor for immunoglobulin G, FcgammaRIIa, that triggers cell aggregation upon interaction with immune complexes. Here, we report that the rapid tyrosine phosphorylation of the Linker for Activation of T-cell (LAT) in human platelets stimulated by FcgammaRIIa cross-linking was followed by its complete dephosphorylation in an alphaIIb/beta3 integrin-dependent manner. Concomitant to LAT dephosphorylation, the protein tyrosine phosphatase 1B (PTP1B) was activated through a mechanism involving its proteolysis by calpains downstream of integrins. Both PTP1B and LAT were associated with the actin cytoskeleton complex formed during platelet aggregation. Moreover, phospho-LAT appeared as a good substrate of activated PTP1B in vitro and these two proteins interacted upon platelet activation by FcgammaRIIa cross-linking. The permeant substrate-trapping PTP1B (TAT-PTP1B D181A) partly inhibited LAT dephosphorylation in human platelets, strongly suggesting that this tyrosine phosphatase was involved in this regulatory pathway. Using a pharmacological inhibitor, we provide evidence that PTP1B activation and LAT dephosphorylation processes were required for irreversible platelet aggregation. Altogether, our results demonstrate that PTP1B plays an important role in the integrin-mediated dephosphorylation of LAT in human platelets and is involved in the control of irreversible aggregation upon FcgammaRIIa stimulation.

Among the various hematopoi;etic cells, platelets are critical for maintaining the integrity of the vascular system. They must be rapidly activated by sequential and coordinated mechanisms in order to efficiently prevent haemorrhage upon vascular injury. Several signal transduction pathways lead to platelet activation in vitro and in vivo, among them, several are initiated via receptors or co-receptors containing immuno-receptor tyrosine-based activation motifs (ITAM) which trigger downstream signalling like the immune receptors in lymphocytes. However, in contrast to immune cells for which the role of lipid rafts in signalling has largely been described, the involvement of laterally segregated membrane microdomains in platelet activation has been investigated only recently. The results obtained until now strongly suggest that early steps of platelet activation via the collagen receptor GpVI or via FcgammaRIIa occur preferentially in these microdomains where specific proteins efficiently organize key downstream signalling pathways. In addition, lipid rafts also contribute to platelet activation via heterotrimeric G-protein-coupled receptors. They are sites where the phosphoinositide (PI) metabolism is highly active, leading to a local generation of lipid second messengers such as phosphatidylinositol 3,4,5-trisphosphate. Here, evidence is accumulating that cholesterol-enriched membrane microdomains are part of a general process that contributes to the efficiency and the coordination of platelet activation mechanisms. Here we will discuss the biochemical and functional characterizations of human platelet rafts and their potential impact in platelet physiopathology.

The involvement of platelet FcgammaRIIa in heparin-associated thrombocytopenia (HIT) is now well established. However, the precise sequence of molecular events initiated by FcgammaRIIa cross-linking in platelets remains partly characterized. We investigated here the role of lipid rafts in the spatio-temporal organization of the FcgammaRIIa-dependent signaling events. Upon cross-linking, FcgammaRIIa relocated in rafts where the kinase Lyn and the adapter LAT were among the major phosphotyrosyl proteins. Upon stimulation by HIT sera, the second messenger phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) accumulated in rafts in a P(2)Y(12) adenosine diphosphate (ADP) receptor-dependent manner. PtdIns(3,4,5)P(3) was then essential to specifically recruit phospholipase Cgamma2 (PLCgamma2) to these membrane microdomains. Controlled disruption of rafts by methyl beta-cyclodextrin reversibly abolished PtdIns(3,4,5)P(3) production, PLC activation and platelet responses induced by FcgammaRIIa cross-linking without affecting the tyrosine phosphorylation events. This work demonstrates that platelet rafts are essential for the integration of a key signaling complex leading to the rapid production of PtdIns(3,4,5)P(3) and in turn PLCgamma2 activation during HIT.

Glycosphingolipid- and cholesterol-enriched membrane microdomains, called rafts, can be isolated from several mammalian cells, including platelets. These microdomains appear to play a critical role in signal transduction in several hematopoietic cells, but their function in blood platelets remains unknown. Herein, we first characterized the lipid composition, including the fatty acid composition of phospholipids, of human platelet rafts. Then their role in platelet activation process was investigated. Interestingly, thrombin stimulation led to morphological changes of rafts correlating with the production of lipid second messengers in these microdomains. Indeed, we could demonstrate for the first time that a large part of the stimulation-dependent production of phosphatidic acid and phosphoinositide 3-kinase products was concentrated in rafts. Moreover, cholesterol depletion with methyl-beta-cyclodextrin disrupted platelet rafts, dramatically decreased the agonist-dependent production of these lipid signaling molecules, and impaired platelet secretion and aggregation. Cholesterol repletion restored the physiological platelet responses. Altogether our data indicate that rafts are highly dynamic platelet membrane structures involved in critical signaling mechanisms linked to the production of lipid second messengers. The demonstration of phosphatidylinositol 3,4,5-trisphosphate production in rafts may have general implications for the understanding of the role of this key second messenger found ubiquitously in higher eucaryotic cells.

Platelets are critical for the maintenance of the integrity of the vascular system and are the first line of defence against haemorrhage. When they encounter a subendothelial matrix exposed by injury to a vessel, platelets adhere, are activated, and become adhesive for other platelets so that they aggregate. alpha IIb/beta 3, a platelet-specific integrin, is largely prominent amongst the adhesion receptors and is essential for platelet aggregation. The ligands for alpha IIb/beta 3 are the multivalent adhesive proteins fibrinogen and von Willebrand factor. In resting platelets, alpha IIb/beta 3 is normally in a low activation state, unable to interact with soluble fibrinogen. Stimulation of platelets with various agonists will induce a conformational change in alpha IIb/beta 3 (inside-out signalling), which is then able to bind soluble fibrinogen resulting in the onset of platelet aggregation. However, fibrinogen binding to its membrane receptor is not simply a passive event allowing the formation of intercellular bridges between platelets. Indeed, a complex signalling pathway triggered by integrin ligation and clustering (outside-in signalling) will regulate the extent of irreversible platelet aggregation and clot retraction. Amongst the signalling enzymes activated downstream of alpha IIb/beta 3 engagement, phosphoinositide 3-kinase plays an important role in the control of the irreversible phase of aggregation.

SH2-containing inositol-5-phosphatase 1 (SHIP1) was originally identified as a 145 kDa protein that became tyrosine-phosphorylated in response to multiple cytokines. It is now well established that SHIP1 is specifically expressed in haemopoietic cells and is important as a negative regulator of signalling. We found recently that SHIP1 was present in human blood platelets as an Ins(1,3,4, 5)P(4)-phosphatase and a PtdIns(3,4,5)P(3)-5-phosphatase that became tyrosine-phosphorylated and was relocated to the cytoskeleton in an integrin-dependent manner. Here we report biochemical and pharmacological evidence that the tyrosine kinase pp60(c-src) is constitutively associated with SHIP1 and is involved in its tyrosine phosphorylation downstream of integrin engagement in thrombin-activated human platelets. The use of cytochalasin D allowed us to demonstrate that the actin cytoskeleton reorganization induced on thrombin stimulation was not required for its integrin-mediated phosphorylation. Moreover, the integrin-dependent relocation of SHIP1 to the cytoskeleton did not require its tyrosine phosphorylation. These results suggest that SHIP1 is first recruited to the integrin-linked signalling complexes and then becomes tyrosine-phosphorylated through a Src-kinase-dependent mechanism but independently of the actin cytoskeleton reorganization.

A large urban university medical center used a unique set of strategies in preparation for its triennial survey conducted by the Joint Commission on Accreditation of Healthcare Organizations. Although the medical center had experienced many Joint Commission surveys, this particular survey was the first to include associated physician offices in multiple diverse settings. To address these challenges, the hospital developed a structure and process that engaged all levels of staff and ensured a successful survey outcome.