The effect of force applied to the left paratracheal oesophagus on air entry into the gastric antrum during positive-pressure ventilation using a facemask.

Cricoid force is widely applied to decrease the risk of pulmonary aspiration and gastric antral insufflation of air during positive-pressure ventilation, yet its efficacy remains controversial. We compared manual oesophageal compression at the low left paratracheal and cricoid levels for the prevention of gastric antral air insufflation during positive-pressure ventilation by facemask in patients scheduled for elective surgery under general anaesthesia. After gaining written consent, participants were randomly allocated by sealed envelope to one of three groups: oesophageal compression by 30 N paratracheal force (paratracheal group); oesophageal compression by 30 N cricoid force (cricoid group); or no oesophageal compression (control group). Gastric insufflation of air was assessed before and after positive-pressure ventilation by ultrasound measurement of the antral cross-sectional area and/or presence of air artefacts in the antrum. The primary outcome measure was the proportion of participants with ultrasound evidence of gastric insufflation. We recruited 30 patients into each group. Before facemask ventilation, no air artefacts were visible in the antrum in any of the participants. After facemask ventilation of the participant's lungs, no air artefacts were seen in the paratracheal group, compared with six subjects in the cricoid group and eight subjects in the control group (p = 0.012). Our results suggest that oesophageal compression can be achieved by the application of manual force at the low left paratracheal level and that this is more effective than cricoid force in preventing air entry into the gastric antrum during positive-pressure ventilation by facemask.

Defect in skeletal muscle phosphatidylinositol-3-kinase in obese insulin-resistant mice.

Activation of phosphatidylinositol-3-kinase (PI3K) is one of the earliest postreceptor events in the insulin signaling pathway. Incubation of soleus muscles from lean mice with 50 nM insulin caused a 3-10-fold increase in antiphosphotyrosine-immunoprecipitable PI3K (antiPTyr-PI3K) activity within 2 min in muscle homogenates as well as both the cytosolic and membrane fractions. Insulin did not affect total PI3K activity. Both the antiPTyr-PI3K stimulation and activation of insulin receptor tyrosine kinase were dependent on hormone concentration. In muscles from obese, insulin-resistant mice, there was a 40-60% decrease in antiPTyr-PI3K activity after 2 min of insulin that was present equally in the cytosolic and membrane fractions. A significant reduction in insulin sensitivity was also observed. The defect appears to result from alterations in both insulin receptor and postreceptor signaling. Starvation of obese mice for 48 h, which is known to reverse insulin resistance, normalized the insulin response of both PI3K and the receptor tyrosine kinase. The results demonstrate that: (a) antiPTyr-PI3K activity is responsive to insulin in mouse skeletal muscle, (b) both the insulin responsiveness and sensitivity of this activity are blunted in insulin-resistant muscles from obese mice, (c) these alterations result from a combination of insulin receptor and postreceptor defects, and (d) starvation restores normal insulin responses.

Diverse effects of Glut 4 ablation on glucose uptake and glycogen synthesis in red and white skeletal muscle.

The ability of muscles from Glut 4-null mice to take up and metabolize glucose has been studied in the isolated white EDL and red soleus muscles. In EDL muscles from male or female Glut 4-null mice, basal deoxyglucose uptake was lower than in control muscles and was not stimulated by insulin. In parallel, glycogen synthesis and content were decreased. Soleus muscles from male Glut 4-null mice took up twice more deoxyglucose in the absence of insulin than control muscles, but did not respond to insulin. In females, soleus deoxyglucose uptake measured in the absence of hormone was similar in Glut 4-null mice and in control mice. This uptake was stimulated twofold in Glut 4-null mice and threefold in control mice. Basal glycogen synthesis was increased by 4- and 2.2-fold in male and female null mice, respectively, compared to controls, and insulin had no or small (20% stimulation over basal) effect. These results indicate that while EDL muscles behaved as expected, soleus muscles were able to take up a large amount of glucose in the absence (males) or the presence of insulin (females). Whether this is due to a change in Glut 1 intrinsic activity or targeting and/or to the appearance of another glucose transporter remains to be determined.

Potential role of Rab4 in the regulation of subcellular localization of Glut4 in adipocytes.

A role for Rab4 in the translocation of the glucose transporter Glut4 induced by insulin has been recently proposed. To study more directly the role of this small GTPase, freshly isolated adipocytes were transiently transfected with the cDNAs of both an epitope-tagged Glut4-myc and Rab4, a system which allows direct measurement of the concentration of Glut4 molecules at the cell surface. When cells were cotransfected with Glut4-myc and Rab4, the concentration of Glut4-myc at the cell surface decreased in parallel with the increased expression of Rab4, suggesting that Rab4 participates in the intracellular retention of Glut4. In parallel, the amount of Rab4 associated with the Glut4-containing vesicles increased. When Rab4 was moderately overexpressed, the number of Glut4-myc molecules recruited to the cell surface in response to insulin was similar to that observed in mock-transfected cells, and thus the insulin efficiency was increased. When Rab4 was expressed at a higher level, the amount of Glut4-myc present at the cell surface in response to insulin decreased. Since the overexpressed protein was predominantly cytosolic, this suggests that the cytosolic Rab4 might complex some factor(s) necessary for insulin action. This hypothesis was strengthened by the fact that Rab4 deltaCT, a Rab4 mutant lacking the geranylgeranylation sites, inhibited insulin-induced recruitement of Glut4-myc to the cell surface, even when moderately overexpressed. Rab3D was without effect on Glut4-myc subcellular distribution in basal or insulin-stimulated conditions. While two mutated proteins unable to bind GTP did not decrease the number of Glut4-myc molecules in basal or insulin-stimulated conditions at the plasma membrane, the behavior of a mutated Rab4 protein without GTPase activity was similar to that of the wild-type Rab4 protein, indicating that GTP binding but not its hydrolysis was required for the observed effects. Altogether, our results suggest that Rab4, but not Rab3D, participates in the molecular mechanism involved in the subcellular distribution of the Glut4 molecules both in basal and in insulin-stimulated conditions in adipocytes.

Spontaneous hypoglycaemia in the presence of both anti-insulin antibody and anti-insulin receptor antibody.

Beside insulinoma, alternative causes of hyperinsulinaemic hypoglycaemia include the rare autoimmune syndrome related to spontaneous autoantibodies either to insulin or to insulin receptor. We describe a case of hypoglycaemia with high insulinemia in which insulinoma could not be evidenced. Surprisingly, we found in the patient's serum both insulin autoantibodies and insulin receptor autoantibodies. Available data eventually supported the predominant role of insulin autoantibodies rather than insulin receptor autoantibodies in the mechanism of hypoglycaemia of this patient. Insulin antibodies were present in high titre. Most of the insulin in serum was bound to the insulin antibodies and free insulin was slightly increased. HLA typing displayed DR4 haplotype, known to be strongly linked to the insulin autoimmune syndrome. The patient's serum was able to inhibit insulin binding to its receptor in a cultured cell line overexpressing insulin receptors both in experiments with native serum and with serum depleted from insulin antibodies. However, we could not demonstrate that the insulin receptor antibodies had insulin mimicking effect. We have no obvious explanation for the presence of these two antibodies in the same patient. Possible hypotheses might involve an idiotype-anti-idiotype mechanism or a poly-autoimmune disease.

Effect of IGF-I on phosphatidylinositol 3-kinase in soleus muscle of lean and insulin-resistant obese mice.

Insulin and IGF-I induced a similar stimulation of glucose transport in isolated soleus muscle. These actions require phosphatidylinositol (PI) 3-kinase activation since the PI 3-kinase inhibitor, wortmannin, blocked the stimulation by both peptides. We compared IGF-I with insulin in the ability to activate PI 3-kinase in the isolated soleus muscle from lean and gold thioglucose-induced obese insulin-resistant mice. In muscles from lean mice, IGF-I and insulin were able to activate PI 3-kinase with a similar time course, the effects being maximal within 3-5 min of stimulation. However, the IGF-I concentrations required to obtain similar effects on PI 3-kinase were about 10 times higher than the corresponding insulin doses. To determine through which receptor IGF-I was activating PI 3-kinase, the ability of IGF-I to activate both its own receptor and insulin receptor was simultaneously measured. Whatever the dose used (100 or 500 nmol/l), IGF-I activated to a nearly similar extent both the tyrosine kinase activity of its own receptor and that of the insulin receptor, suggesting that IGF-I was not only activating its receptor but was also able to stimulate the insulin receptor kinase. In muscles of obese insulin-resistant mice, although the defect of PI 3-kinase activation in response to IGF-I was relatively less pronounced (45%) than in response to insulin (70%) when compared with lean mice, PI 3-kinase stimulation was still markedly altered in response to IGF-I.

Wortmannin inhibits the action of insulin but not that of okadaic acid in skeletal muscle: comparison with fat cells.

To look for the possible involvement of phosphatidylinositol-3-kinase (PI3-kinase) in insulin action in muscle, we have used wortmannin, described as a specific inhibitor of the enzyme, and compared its effect in muscle and in adipose cells. Both in intact mouse soleus muscle and in isolated rat adipocytes, wortmannin blocked insulin effect on glucose uptake, without markedly altering basal glucose uptake. In adipocyte, this effect results from a blockade of the translocation process because wortmannin inhibited the stimulatory action of insulin on both the Glut 4 movement from the internal compartment to the plasma membranes and the Rab4 departure from the microsomes. In a similar fashion, two other insulin effects, the activation of glycogen synthase and the stimulation of amino acid uptake, were blocked by wortmannin in skeletal muscle. Lipogenesis from acetate was also inhibited by wortmannin in adipocytes. By contrast, wortmannin did not affect muscle deoxglucose uptake when it was stimulated either by okadaic acid or by the protein kinase C activator tumor promoting agent. These results suggest that, in muscle and adipocyte, PI3-kinase inhibition causes a blockade of all insulin effects studied. By contrast, wortmannin did not affect the same responses elicited in muscle by okadaic acid or tumor promoting agent.

Potential involvement of the carboxy-terminus of the Glut 1 transporter in glucose transport.

The role of the carboxy-terminal domain of the Glut 1 glucose transporter was investigated using an antipeptide antibody to the C-terminal part of the molecule. The study was performed in fibroblasts transfected with the cDNA coding for the human insulin receptor. These cells acutely respond to insulin for glucose transport. Using antipeptide antibodies to Glut 1 and Glut 4, we first established that these cells expressed only Glut 1. Then, to define the role of the C-terminal part of Glut 1 in glucose transport, the antibodies were loaded into the cells by electroporation. When anti-Glut 1 immunoglobulins were introduced into the cells, a 60% increase in basal deoxyglucose and 3-O-methylglucose transport was observed compared to that in cells electroporated with nonimmune immunoglobulins. The stimulatory action of the antipeptide was not due to an increase in the total amount of transporters. It was found only at low glucose concentrations, suggesting that the affinity of the transporter, rather than its maximal capacity, was changed. Finally, the effect of antibody was additive to that of insulin. The interaction between the anti-Glut 1 antibody and the carboxy-tail of the transporter seems to lead to an increase in the intrinsic activity of the transporter, suggesting that this part of the molecule could be implicated in the regulation of glucose uptake.

Focal adhesion kinase and Src mediate integrin regulation of insulin receptor phosphorylation.

We show here that phosphorylation of the insulin receptor and insulin receptor substrate-1 is increased when suspended cells are replated on fibronectin. This is not due to decreased numbers of cell surface receptors, alteration of insulin binding, or stimulation of a phosphatase activity in non-adherent cells. Expression of Src together with focal adhesion kinase (FAK) in suspended cells restores insulin-induced receptor autophosphorylation to levels observed in fibronectin-attached cells. Conversely, expression of dominant-negative mutants of either Src or FAK abolishes potentiation of insulin receptor phosphorylation by cell adhesion. The results suggest that both Src and FAK participate in integrin-mediated regulation of insulin receptor signal.

Effects of insulin, insulin-like growth factor-I (IGF-I) and progesterone on glucose and amino acid uptake in Xenopus laevis oocytes.

In Xenopus oocytes, insulin or IGF-I activated glucose transport and maturation, but not amino acid transport, as measured by the uptake of alanine. Glucose transporter identical or closely related to the mammalian erythroid/brain transporter (Glut-1/EGT) were found in oocyte membranes. The EC50 for stimulation of glucose uptake and of maturation were similar (1-5 x 10(-8) M for insulin and 2-8 x 10(-10) M for IGF-I), confirming that these effects were mediated through IGF-I receptors. Other agents, such as phorbol 12-myristate 13-acetate (TPA) (0.5 microM) and vanadate (2 mM) evoked only part of the insulin effect on glucose uptake (50% and 65%, respectively), without being additive to insulin. In contrast, progesterone at 1 microM, a potent inducer of maturation, inhibited at least partially the insulin-induced glucose uptake. Uptake of alanine and glucose was decreased after prolonged incubations (3-6 h) with agents that trigger maturation, and was dramatically reduced in oocytes that have undergone maturation (unfertilized eggs). Maturation is thus accompanied by a reduction in glucose and amino acid transports. These result further document the validity of Xenopus oocytes as a model to study insulin and IGF-I signalling.

Effected of insulin and insulin-like growth factor-I on glucose transport and its transporters in soleus muscle of lean and obese mice.

The mechanisms underlying insulin and insulin-like growth factor-I (IGF-I) action on glucose transport share similar processes leading to Glut 4 translocation after respective receptor activation. Among these steps are phosphorylation of insulin receptor substrate-1 (IRS-1) and activation of phosphatidylinositol-3-kinase (P13-kinase). This enzyme could be involved in stimulated glucose transport in muscle, since its inhibitor, wortmannin, blocks the hormonal effect in muscle. P13-kinase is activated by insulin and IGF-I in a rapid and transient manner in incubated soleus muscles. When P13-kinase activation was studied in muscle of obese insulin-resistant mice, there was a marked alteration in the response to insulin both in vivo and in vitro. P13-kinase activation by IGF-I was also altered in obese mice, although to a lesser degree.

Structure of mycoside F, a family of trehalose-containing glycolipids of Mycobacterium fortuitum.

Nuclear magnetic resonance spectroscopy, fast-atom bombardment mass spectrometry, gas chromatography-mass spectrometry, as well as chemical degradations were used to elucidate the structure of the major glycolipids of Mycobacterium fortuitum. Three main glycoconjugates were detected and their structures established as 2,3-diacyl, 2,3,4- and 2,3,6-triacyl trehalose. The characteristic infrared spectrum which led to their original designation as mycoside F, a family of glycolipids limited in distribution to M. fortuitum, was due to the nature of the fatty acyl substiuents identified primarily as 2-methyl-octadecen-2-oyl. The antigenic glycolipids typified the biovar. fortuitum, thus allowing its easy recognition from the C-mycoside glycopeptidolipid-containing biovar. peregrinum.

A reverse transcription-polymerase chain reaction method to analyze porcine cytokine gene expression.

A reverse transcription-polymerase chain reaction (RT-PCR) method was developed in order to provide a highly sensitive, rapid, and simple means of simultaneously measuring the expression of porcine cytokines in immune cell populations. Oligonucleotide primers were designed to amplify porcine cytokine cDNA from genes encoding IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, TNF-beta and the housekeeping genes beta-actin and cyclophilin by PCR. Primers were chosen from different exons to detect for possible genomic DNA contamination of samples. To validate RT-PCR, unstimulated and concanavalin A (ConA) stimulated porcine peripheral blood mononuclear cells (PBMCs) were cultured from 2 h to 72 h, RNA was extracted and reverse transcribed, and cDNA was amplified using the different primer sets. Band intensities of PCR products were quantified by densitometric scanning and values were normalized against cyclophilin. For each of the cytokines, the kinetics of gene expression were similar among PBMCs isolated from different animals and could be grouped into two main patterns. Lymphocyte derived cytokines (IL-2, IL-4, IFN-gamma, and TNF-beta) exhibited low level expression in unstimulated cells and increased expression in ConA-stimulated PBMCs. IFN-gamma and IL-2 mRNA levels peaked at 24 h and returned to baseline by 72 h, whereas IL-4 and TNF-beta mRNA levels did not return to baseline by 72 h. In contrast, substantial mRNA levels for inflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-12, and TNF-alpha) and IL-10 were detected from both unstimulated and ConA-stimulated PBMCs. Results indicate that RT-PCR is a sensitive and convenient method to monitor cytokine mRNA expression in porcine samples.

[Pleomorphic lobular intraepithelial neoplasia: clinical, histological and prognostic study of nine cases]. / Néoplasie lobulaire intra-épithéliale de type pléomorphe : analyse clinique, histologique et pronostique d'une série de neuf cas.

INTRODUCTION: Retrospective clinical, histological and prognostic study of nine cases of lobular intraepithelial neoplasia in its pleomorphic subtype (LIN-P). PATIENTS AND METHODS: Analysis of our center database with selection of nine cases of LIN-P from 140 files of patients who underwent surgical excision with a final diagnosis of LIN (2000 to 2011). The medical files were reviewed with a re-analysis of the mammograms and the histological slides. The outcome of the patients was also analyzed according to their clinical and operative data. RESULTS AND DISCUSSION: The average age at diagnosis was 63 years (later than common LIN [LIN-C]). All patients had mammograms classified ACR 4 and 5, mainly due to the presence of microcalcifications (seven cases) with a case of opacity associated with microcalcifications, and two other cases with only isolated opacities. The preoperative diagnosis of these lesions was difficult: five cases on nine core needle biopsies were reviewed and reclassified LIN-P after finding the presence of LIN-P on the surgical specimen. Associated invasive lesions were found in 55% of core needle biopsy and in 33% of cases of surgical resection specimen. The treatment included a wide surgical excision (five lumpectomies and four mastectomies with a patient who had two lumpectomies) with margins of more than 2mm: we noted a recurrence of LIN-P only in one case where margins were very close (1mm). The analysis of our cases confirms that LIN-P if they probably share the same origin as the LIN-C represent a particular form constituting a true precancerous condition warranting at least a wide surgical excision.

Gestational nicotine exposure modifies myelin gene expression in the brains of adolescent rats with sex differences.

Myelination defects in the central nervous system (CNS) are associated with various psychiatric disorders, including drug addiction. As these disorders are often observed in individuals prenatally exposed to cigarette smoking, we tested the hypothesis that such exposure impairs central myelination in adolescence, an important period of brain development and the peak age of onset of psychiatric disorders. Pregnant Sprague Dawley rats were treated with nicotine (3 mg kg(-1) per day; gestational nicotine (GN)) or gestational saline via osmotic mini pumps from gestational days 4-18. Both male and female offsprings were killed on postnatal day 35 or 36, and three limbic brain regions, the prefrontal cortex (PFC), caudate putamen and nucleus accumbens, were removed for measurement of gene expression and determination of morphological changes using quantitative real-time PCR (qRT-PCR) array, western blotting and immunohistochemical staining. GN altered myelin gene expression at both the mRNA and protein levels, with striking sex differences. Aberrant expression of myelin-related transcription and trophic factors was seen in GN animals, which correlated highly with the alterations in the myelin gene expression. These correlations suggest that these factors contribute to GN-induced alterations in myelin gene expression and also indicate abnormal function of oligodendrocytes (OLGs), the myelin-producing cells in the CNS. It is unlikely that these changes are attributable solely to an alteration in the number of OLGs, as the cell number was changed only in the PFC of GN males. Together, our findings suggest that abnormal brain myelination underlies various psychiatric disorders and drug abuse associated with prenatal exposure to cigarette smoke.

ZnO particles of wurtzite structure as a component in ZnO/carbon nanotube composite.

Composites based on carbon nanotubes and ZnO particles with needle shapes were prepared for applications in energy storage. Depending on the temperature (85 or 25 °C) at which the reaction between NaOH and ZnCl(2) was carried out, particles with two different morphologies: needle-shaped (NS) and double-pyramid-shaped (DPS), respectively, are obtained. Scanning electron microscopy, photoluminescence, UV-Vis absorption spectroscopy, x-ray diffraction and Raman light scattering studies reveal that the NS and DPS particles belong to ZnO with wurtzite (WZ) structure and ε-Zn (OH)(2) as precursors of ZnO, respectively. Using the ZnO/carbon nanotube composite as a negative electrode and an electrolytic solution containing LiPF(6), the charge-discharge characteristics of rechargeable lithium ions cells were determined. Additional information concerning the electrochemical reactions at the interface of the two electrodes was obtained by cyclic voltammetry.

Amino acids require glucose to enhance, through phosphoinositide-dependent protein kinase 1, the insulin-activated protein kinase B cascade in insulin-resistant rat adipocytes.

AIMS/HYPOTHESIS: Amino acids are well known to activate the mammalian target of the rapamycin (mTOR) pathway in synergy with insulin to regulate cell functions. Despite recent important advances, the mTOR signalling pathway is poorly understood. Our previous results revealed a new pathway in which amino acids permit insulin-induced activation of the protein kinase B (PKB)/mTOR pathway in freshly isolated adipocytes when phosphatidylinositol 3-kinase (PI3K) is inhibited. The aim of this study was to further investigate this pathway at the molecular level. METHODS: We studied the effect of amino acids on PKB phosphorylation in different cellular models or in freshly isolated adipocytes incubated in different buffers, after a time course of insulin and amino acids and in the presence of pharmacological inhibitors. To investigate the potential role of amino acids in insulin action, the effect on glucose transport in obese rat adipocytes following a high-fat diet was assessed. RESULTS: Insulin-induced PKB phosphorylation is restored by amino acids in the presence of wortmannin in adipose tissue explants and freshly isolated adipocytes, but not in cultured adipocytes or hepatocytes. Moreover, amino acids require the presence of glucose to phosphorylate PKB and to partially rescue glucose transport in a PI3K-independent manner. The results also suggest that the amino acids act through the phosphoinositide-dependent protein kinase 1. In addition, amino acids were seen to improve insulin-stimulated glucose transport in adipocytes from high-fat-fed rats. CONCLUSIONS/INTERPRETATION: This study suggests that amino acids could enhance adipocyte insulin signalling in pathophysiological situations such as insulin resistance associated with obesity.

Object-centered representation for species systematics and identification in living systems in nature.

An artificial intelligence application to identification in biological taxonomy is presented. An object-centered representation is described using SHIRKA, a knowledge-based system developed in France. The capacities of this system, relating to a classification algorithm, are described. An application to the identification of the trees and lianas of the evergreen forests of the Western Ghats (India) is presented to illustrate the problems encountered in the development of the knowledge base. A model of a systematist's reasoning, using evolutionary principles, is the basis of this work. Finally the efficiency of the system is discussed.

The insulin receptor activation process involves localized conformational changes.

The molecular process by which insulin binding to the receptor alpha-subunit induces activation of the receptor beta-subunit with ensuing substrate phosphorylation remains unclear. In this study, we aimed at approaching this molecular mechanism of signal transduction and at delineating the cytoplasmic domains implied in this process. To do this, we used antipeptide antibodies to the following sequences of the receptor beta-subunit: (i) positions 962-972 in the juxtamembrane domain, (ii) positions 1247-1261 at the end of the kinase domain, and (iii) positions 1294-1317 and (iv) positions 1309-1326, both in the receptor C terminus. We have previously shown that insulin binding to its receptor induces a conformational change in the beta-subunit C terminus. Here, we demonstrate that receptor autophosphorylation induces an additional conformational change. This process appears to be distinct from the one produced by ligand binding and can be detected in at least three different beta-subunit regions: the juxtamembrane domain, the kinase domain, and the C terminus. Hence, the cytoplasmic part of the receptor beta-subunit appears to undergo an extended conformational change upon autophosphorylation. By contrast, the insulin-induced change does not affect the juxtamembrane domain 962-972 nor the kinase domain 1247-1261 and may be limited to the receptor C terminus. Further, we show that the hormone-dependent conformational change is maintained in a kinase-deficient receptor due to a mutation at lysine 1018. Therefore, during receptor activation, the ligand-induced change could precede ATP binding and receptor autophosphorylation. We propose that insulin binding leads to a transient receptor form that may allow ATP binding and, subsequently, autophosphorylation. The second conformational change could unmask substrate-binding sites and stabilize the receptor in an active conformation.