A short in-frame deletion in NTRK1 tyrosine kinase domain caused by a novel splice site mutation in a patient with congenital insensitivity to pain with anhidrosis

Background: Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive genetic disease characterized by the lack of reaction to noxious stimuli and anhidrosis. It is caused by mutations in the NTRK1 gene, which encodes the high affinity tyrosine kinase receptor I for Neurotrophic Growth Factor (NGF). -- Case Presentation: We present the case of a female patient diagnosed with CIPA at the age of 8 months. The patient is currently 6 years old and her psychomotor development conforms to her age (RMN, SPECT and psychological study are in the range of normality). PCR amplification of DNA, followed by direct sequencing, was used to investigate the presence of NTRK1 gene mutations. Reverse transcriptase (RT)-PCR amplification of RNA, followed by cloning and sequencing of isolated RT-PCR products was used to characterize the effect of the mutations on NTRK1 mRNA splicing. The clinical diagnosis of CIPA was confirmed by the detection of two splice-site mutations in NTRK1, revealing that the patient was a compound heterozygote at this gene. One of these alterations, c.574+1G > A, is located at the splice donor site of intron 5. We also found a second mutation, c.2206-2 A > G, not previously reported in the literature, which is located at the splice acceptor site of intron 16. Each parent was confirmed to be a carrier for one of the mutations by DNA sequencing analysis. It has been proposed that the c.574+1G > A mutation would cause exon 5 skipping during NTRK1 mRNA splicing. We could confirm this prediction and, more importantly, we provide evidence that the novel c.2206-2A > G mutation also disrupts normal NTRK1 splicing, leading to the use of an alternative splice acceptor site within exon 17. As a consequence, this mutation would result in the production of a mutant NTRK1 protein with a seven aminoacid in-frame deletion in its tyrosine kinase domain. --Conclusions: We present the first description of a CIPA-associated NTRK1 mutation causing a short interstitial deletion in the tyrosine kinase domain of the receptor. The possible phenotypical implications of this mutation are discussed.

Increased expression of cystine/glutamate antiporter in multiple sclerosis

Background: Glutamate excitotoxicity contributes to oligodendrocyte and tissue damage in multiple sclerosis (MS). Intriguingly, glutamate level in plasma and cerebrospinal fluid of MS patients is elevated, a feature which may be related to the pathophysiology of this disease. In addition to glutamate transporters, levels of extracellular glutamate are controlled by cystine/glutamate antiporter x(c)(-), an exchanger that provides intracellular cystine for production of glutathione, the major cellular antioxidant. The objective of this study was to analyze the role of the system x(c)(-) in glutamate homeostasis alterations in MS pathology. -- Methods: Primary cultures of human monocytes and the cell line U-937 were used to investigate the mechanism of glutamate release. Expression of cystine glutamate exchanger (xCT) was quantified by quantitative PCR, Western blot, flow cytometry and immunohistochemistry in monocytes in vitro, in animals with experimental autoimmune encephalomyelitis (EAE), the animal model of MS, and in samples of MS patients. -- Results and discussion: We show here that human activated monocytes release glutamate through cystine/glutamate antiporter x(c)(-) and that the expression of the catalytic subunit xCT is upregulated as a consequence of monocyte activation. In addition, xCT expression is also increased in EAE and in the disease proper. In the later, high expression of xCT occurs both in the central nervous system (CNS) and in peripheral blood cells. In particular, cells from monocyte-macrophage-microglia lineage have higher xCT expression in MS and in EAE, indicating that immune activation upregulates xCT levels, which may result in higher glutamate release and contribution to excitotoxic damage to oligodendrocytes. -- Conclusions: Together, these results reveal that increased expression of the cystine/glutamate antiporter system x(c)(-) in MS provides a link between inflammation and excitotoxicity in demyelinating diseases.

Mechanisms Responsible for the Trophic Effect of Beta-Adrenoceptors on the I-to Current Density in Type 1 Diabetic Rat Cardiomyocytes

Background/Aims: In diabetic ventricular myocytes, transient outward potassium current (I-to) amplitude is severely reduced because of the impaired catecholamine release that characterizes diabetic autonomic neuropathy. Sympathetic nervous system exhibits a trophic effect on I-to since incubation of myocytes with noradrenaline restores current amplitude via beta-adrenoceptor (beta AR) stimulation. Here, we investigate the intracellular signalling pathway though which incubation of diabetic cardiomyocytes with the beta AR agonist isoproterenol recovers I-to amplitude to normal values. Methods: Experiments were performed in ventricular myocytes isolated from streptozotocin-diabetic rats. I-to current was recorded by using the patch-clamp technique. Kv4 channel expression was determined by immunofluorescence. Protein-protein interaction was determined by coimmunoprecipitation. Results: Stimulation of beta AR activates first a G alpha s protein, adenylyl cyclase and Protein Kinase A. PKA-phosphorylated receptor then switches to the G alpha i protein. This leads to the activation of the beta AR-Kinase-1 and further receptor phosphorylation and arrestin dependent internalization. The internalized receptor-arrestin complex recruits and activates cSrc and the MAPK cascade, where Ras, c-Raf1 and finally ERK1/2 mediate the increase in Kv4.2 and Kv4.3 protein abundance in the plasma membrane. Conclusion: beta(2)AR stimulation activates a G alpha s and G alpha i protein dependent pathway where the ERK1/2 modulates the Ito current amplitude and the density of the Kv4.2 and Kv4.2 channels in the plasma membrane upon sympathetic stimulation in diabetic heart.

A Temperature Sensor Based on a Polymer Optical Fiber Macro-Bend

The design and development of a plastic optical fiber (POF) macrobend temperature sensor is presented. The sensor has a linear response versus temperature at a fixed bend radius, with a sensitivity of 1.92.10(-3) (degrees C)(-1). The sensor system used a dummy fiber-optic sensor for reference purposes having a resolution below 0.3 degrees C. A comprehensive experimental analysis was carried out to provide insight into the effect of different surrounding media on practical macro-bend POF sensor implementation. Experimental results are successfully compared with bend loss calculations.

A Self-Referencing Intensity Based Polymer Optical Fiber Sensor for Liquid Detection

A novel self-referencing fiber optic intensity sensor based on bending losses of a partially polished polymer optical fiber (POF) coupler is presented. The coupling ratio (K) depends on the external liquid in which the sensor is immersed. It is possible to distinguish between different liquids and to detect their presence. Experimental results for the most usual liquids found in industry, like water and oil, are given. K value increases up to 10% from the nominal value depending on the liquid. Sensor temperature dependence has also been studied for a range from 25 degrees C (environmental condition) to 50 degrees C. Any sector requiring liquid level measurements in flammable atmospheres can benefit from this intrinsically safe technology.

The prevalence of diabetes-related complications and multimorbidity in the population with type 2 diabetes mellitus in the Basque Country

Background: Type 2 diabetes mellitus is associated with a diverse range of pathologies. The aim of the study was to determine the incidence of diabetes-related complications, the prevalence of coexistent chronic conditions and to report multimorbidity in people with type 2 diabetes living in the Basque Country. Methods: Administrative databases, in four cross sections (annually from 2007 to 2011) were consulted to analyse 149,015 individual records from patients aged >= 35 years with type 2 diabetes mellitus. The data observed were: age, sex, diabetes-related complications (annual rates of acute myocardial infarction, major amputations and avoidable hospitalisations), diabetes-related pathologies (prevalence of ischaemic heart disease, renal failure, stroke, heart failure, peripheral neuropathy, foot ulcers and diabetic retinopathy) and other unrelated pathologies (44 diseases). Results: The annual incidence for each condition progressively decreased during the four-year period: acute myocardial infarction (0.47 to 0.40%), major amputations (0.10 to 0.08%), and avoidable hospitalisations (5.85 to 5.5%). The prevalence for diabetes-related chronic pathologies was: ischaemic heart disease (11.5%), renal failure (8.4%), stroke (7.0%), heart failure (4.3%), peripheral neuropathy (1.3%), foot ulcers (2.0%) and diabetic retinopathy (7.2%). The prevalence of multimorbidity was 90.4%. The highest prevalence for other chronic conditions was 73.7% for hypertension, 13.8% for dyspepsia and 12.7% for anxiety. Conclusions: In the type 2 diabetes mellitus population living in the Basque Country, incidence rates of diabetes complications are not as high as in other places. However, they present a high prevalence of diabetes related and unrelated diseases. Multimorbidity is very common in this group, and is a factor to be taken into account to ensure correct clinical management.

Specific Interaction with Cardiolipin Triggers Functional Activation of Dynamin-Related Protein 1

Dynamin-Related Protein 1 (Drp1), a large GTPase of the dynamin superfamily, is required for mitochondrial fission in healthy and apoptotic cells. Drp1 activation is a complex process that involves translocation from the cytosol to the mitochondrial outer membrane (MOM) and assembly into rings/spirals at the MOM, leading to membrane constriction/division. Similar to dynamins, Drp1 contains GTPase (G), bundle signaling element (BSE) and stalk domains. However, instead of the lipid-interacting Pleckstrin Homology (PH) domain present in the dynamins, Drp1 contains the so-called B insert or variable domain that has been suggested to play an important role in Drp1 regulation. Different proteins have been implicated in Drp1 recruitment to the MOM, although how MOM-localized Drp1 acquires its fully functional status remains poorly understood. We found that Drp1 can interact with pure lipid bilayers enriched in the mitochondrion-specific phospholipid cardiolipin (CL). Building on our previous study, we now explore the specificity and functional consequences of this interaction. We show that a four lysine module located within the B insert of Drp1 interacts preferentially with CL over other anionic lipids. This interaction dramatically enhances Drp1 oligomerization and assembly-stimulated GTP hydrolysis. Our results add significantly to a growing body of evidence indicating that CL is an important regulator of many essential mitochondrial functions.

Influence of Extracellular Matrix Components on the Expression of Integrins and Regeneration of Adult Retinal Ganglion Cells

Purpose Retinal ganglion cells (RGCs) are exposed to injury in a variety of optic nerve diseases including glaucoma. However, not all cells respond in the same way to damage and the capacity of individual RGCs to survive or regenerate is variable. In order to elucidate factors that may be important for RGC survival and regeneration we have focussed on the extracellular matrix (ECM) and RGC integrin expression. Our specific questions were: (1) Do adult RGCs express particular sets of integrins in vitro and in vivo? (2) Can the nature of the ECM influence the expression of different integrins? (3) Can the nature of the ECM affect the survival of the cells and the length or branching complexity of their neurites? Methods Primary RGC cultures from adult rat retina were placed on glass coverslips treated with different substrates: Poly-L-Lysine (PL), or PL plus laminin (L), collagen I (CI), collagen IV (CIV) or fibronectin (F). After 10 days in culture, we performed double immunostaining with an antibody against beta III-Tubulin to identify the RGCs, and antibodies against the integrin subunits: alpha V, alpha 1, alpha 3, alpha 5, beta 1 or beta 3. The number of adhering and surviving cells, the number and length of the neurites and the expression of the integrin subunits on the different substrates were analysed. Results PL and L were associated with the greatest survival of RGCs while CI provided the least favourable conditions. The type of substrate affected the number and length of neurites. L stimulated the longest growth. We found at least three different types of RGCs in terms of their capacity to regenerate and extend neurites. The different combinations of integrins expressed by the cells growing on different substrata suggest that RGCs expressed predominantly alpha 1 beta 1 or alpha 3 beta 1 on L, alpha 1 beta 1 on CI and CIV, and alpha 5 beta 3 on F. The activity of the integrins was demonstrated by the phosphorylation of focal adhesion kinase (FAK). Conclusions Adult rat RGCs can survive and grow in the presence of different ECM tested. Further studies should be done to elucidate the different molecular characteristics of the RGCs subtypes in order to understand the possible different sensitivity of different RGCs to damage in diseases like glaucoma in which not all RGCs die at the same time.