RESUMEN
PURPOSE: The goal of this study was to assess neuropeptide levels in patients with dry eye disease (DED) and investigate their correlations with clinical characteristics. METHODS: This study included 38 eyes of 38 patients diagnosed with DED (DED group) and 38 eyes of 38 healthy volunteers without DED (control group). Ocular surface evaluation was performed. The severity of dry eye symptoms and signs in the DED group was graded. Neuropeptides [substance P (SP), alpha-melanocyte-stimulating hormone (α-MSH), ß-endorphin, neurotensin, and oxytocin] and inflammatory cytokines levels were measured in basal tears. The link between neuropeptides and clinical parameters was investigated using Spearman rank correlation. RESULTS: Overall, 76.3% of patients in the DED group showed dry eye symptoms and signs that were inconsistent in severity. Compared with the control group, the DED group showed higher levels of SP, α-MSH, and oxytocin in tears (P = 0.012, P = 0.030, and P = 0.006, respectively), but similar levels of ß-endorphin and neurotensin (P = 0.269 and P = 0.052). The levels of SP, α-MSH, and oxytocin were elevated in DED patients with higher grading of symptoms than clinical signs (all P < 0.05). SP, α-MSH, and oxytocin levels in tears were positively correlated with Ocular Surface Disease Index scores, frequency of sensitivity to light, and frequency of blurred vision (all P < 0.05). CONCLUSIONS: The increased tear levels of SP, α-MSH, and oxytocin may be linked to ocular discomfort in DED. Neuropeptides may play a key role in the development of DED, especially in DED patients with more severe symptoms than clinical signs.
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Síndromes de Ojo Seco , Neurotensina , Humanos , Neurotensina/análisis , alfa-MSH/análisis , Oxitocina/análisis , betaendorfina/análisis , Síndromes de Ojo Seco/diagnóstico , Lágrimas/químicaRESUMEN
The endogenous tridecapeptide neurotensin (NT) has emerged as an important inhibitory modulator of pain transmission, exerting its analgesic action through the activation of the G protein-coupled receptors, NTS1 and NTS2. Whereas both NT receptors mediate the analgesic effects of NT, NTS1 activation also produces hypotension and hypothermia, which may represent obstacles for the development of new pain medications. In the present study, we implemented various chemical strategies to improve the metabolic stability of the biologically active fragment NT(8-13) and assessed their NTS1/NTS2 relative binding affinities. We then determined their ability to reduce the nociceptive behaviors in acute, tonic, and chronic pain models and to modulate blood pressure and body temperature. To this end, we synthesized a series of NT(8-13) analogs carrying a reduced amide bond at Lys8-Lys9 and harboring site-selective modifications with unnatural amino acids, such as silaproline (Sip) and trimethylsilylalanine (TMSAla). Incorporation of Sip and TMSAla respectively in positions 10 and 13 of NT(8-13) combined with the Lys8-Lys9 reduced amine bond (JMV5296) greatly prolonged the plasma half-life time over 20 h. These modifications also led to a 25-fold peptide selectivity toward NTS2. More importantly, central delivery of JMV5296 was able to induce a strong antinociceptive effect in acute (tail-flick), tonic (formalin), and chronic inflammatory (CFA) pain models without inducing hypothermia. Altogether, these results demonstrate that the chemically-modified NT(8-13) analog JMV5296 exhibits a better therapeutic profile and may thus represent a promising avenue to guide the development of new stable NT agonists and improve pain management.
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Dolor Agudo/tratamiento farmacológico , Analgesia , Analgésicos/farmacología , Conducta Animal/efectos de los fármacos , Dolor Crónico/tratamiento farmacológico , Neurotensina/farmacología , Dolor Nociceptivo/tratamiento farmacológico , Analgésicos/química , Animales , Modelos Animales de Enfermedad , Masculino , Neurotensina/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
Electrochemiluminescence immunoassays are usually carried out through "on-electrode" strategy, i.e., sandwich-type immunoassay format, the sensitivity of which is restricted by two key bottlenecks: (1) the number of signal labels is limited and (2) only a part of signal labels could participate in the electrode reaction. In this Perspective, we discuss the development of an "in-electrode" Faraday-cage-type concept-based immunocomplex immobilization strategy. The biggest difference from the traditional sandwich-type one is that the designed "in-electrode" Faraday-cage-type immunoassay uses a conductive two-dimensional (2-D) nanomaterial simultaneously coated with signal labels and a recognition component as the detection unit, which could directly overlap on the electrode surface. In such a case, electrons could flow freely from the electrode to the detection unit, the outer Helmholtz plane (OHP) of the electrode is extended, and thousands of signal labels coated on the 2-D nanomaterial are all electrochemically "effective." Thus, then, the above-mentioned bottlenecks obstructing the improvement of the sensitivity in sandwich-type immunoassay are eliminated, and as a result a much higher sensitivity of the Faraday-cage-type immunoassay can be obtained. And, the applications of the proposed versatile "in-electrode" Faraday-cage-type immunoassay have been explored in the detection of target polypeptide, protein, pathogen, and microRNA, with the detection sensitivity improved tens to hundreds of times. Finally, the outlook and challenges in the field are summarized. The rise of Faraday-cage-type electrochemiluminescence immunoassay (FCT-ECLIA)-based biosensing strategies opens new horizons for a wide range of early clinical identification and diagnostic applications.
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Anticuerpos/química , Técnicas Biosensibles , Técnicas Electroquímicas , Inmunoensayo , Nanoestructuras/química , Electrodos , Óxido Ferrosoférrico/química , Oro/química , Grafito/química , Humanos , Límite de Detección , Luminiscencia , MicroARNs/análisis , Neurotensina/análisis , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/aislamiento & purificación , Vibrio vulnificus/química , Vibrio vulnificus/aislamiento & purificación , Factores de Transcripción p300-CBP/análisisRESUMEN
Neurotensin (Nts) is a neuropeptide implicated in the regulation of many facets of physiology, including cardiovascular tone, pain processing, ingestive behaviors, locomotor drive, sleep, addiction and social behaviors. Yet, there is incomplete understanding about how the various populations of Nts neurons distributed throughout the brain mediate such physiology. This knowledge gap largely stemmed from the inability to simultaneously identify Nts cell bodies and manipulate them in vivo. One means of overcoming this obstacle is to study NtsCre mice crossed onto a Cre-inducible green fluorescent reporter line (NtsCre;GFP mice), as these mice permit both visualization and in vivo modulation of specific populations of Nts neurons (using Cre-inducible viral and genetic tools) to reveal their function. Here we provide a comprehensive characterization of the distribution and relative densities of the Nts-GFP populations observed throughout the male NtsCre;GFP mouse brain, which will pave the way for future work to define their physiologic roles. We also compared the distribution of Nts-GFP neurons with Nts-In situ Hybridization (Nts-ISH) data from the adult mouse brain. By comparing these data sets we can distinguish Nts-GFP populations that may only transiently express Nts during development but not in the mature brain, and hence which populations may not be amenable to Cre-mediated manipulation in adult NtsCre;GFP mice. This atlas of Nts-GFP neurons will facilitate future studies using the NtsCre;GFP line to describe the physiological functions of individual Nts populations and how modulating them may be useful to treat disease.
Asunto(s)
Encéfalo/metabolismo , Neuronas/metabolismo , Neurotensina/análisis , Animales , Atlas como Asunto , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Neurotensina/genéticaRESUMEN
Neurotensin (NTS) is a neuropeptide distributed in central nervous and digestive systems. In this study, the significant association between ectopic NTS expression and tumor invasion was confirmed in hepatocellular carcinoma (HCC). In primary HCC tissues, the NTS and neurotensin receptor 1 (NTR1) co-expression (NTS+NTR1+) is a poor prognostic factor correlated with aggressive biological behaviors and poor clinical prognosis. Enhanced epithelial-to-mesenchymal transition (EMT) features, including decreased E-cadherin, increased ß-catenin translocation and N-cadherin expression, were identified in NTS+NTR1+ HCC tissues. Varied NTS-responsible HCC cell lines were established using NTR1 genetically modified Hep3B and HepG2 cells which were used to elucidate the molecular mechanisms regulating NTS-induced EMT and tumor invasion in vitro. Results revealed that inducing exogenous NTS stimulation and enhancing NTR1 expression promoted tumor invasion rather than proliferation by accelerating EMT in HCC cells. The NTS-induced EMT was correlated with the remarkable increase in Wnt1, Wnt3, Wnt5, Axin, and p-GSK3ß expression and was significantly reversed by blocking the NTS signaling via the NTR1 antagonist SR48692 or by inhibiting the activation of the Wnt/ß-catenin pathway via specific inhibitors, such as TSW119 and DKK-1. SR48692 also inhibited the metastases of NTR1-overexpressing HCC xenografts in the lungs in vivo. This finding implied that NTS may be an important stimulus to promote HCC invasion and metastasis both in vitro and in vivo, and NTS signaling enhanced the tumor EMT and invasion potentials by activating the canonical Wnt/ß-catenin signaling pathway. Therefore, NTS may be a valuable therapeutic target to prevent tumor progression in HCC.
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Carcinoma Hepatocelular/patología , Transición Epitelial-Mesenquimal , Neoplasias Hepáticas/patología , Neurotensina/fisiología , Receptores de Neurotensina/fisiología , Vía de Señalización Wnt/fisiología , Adulto , Anciano , Línea Celular Tumoral , Femenino , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Neurotensina/análisis , Receptores de Neurotensina/análisisRESUMEN
Metabolomics data provide unprecedented opportunities to decipher metabolic mechanisms by analyzing hundreds to thousands of metabolites. Data quality concerns and complex batch effects in metabolomics must be appropriately addressed through statistical analysis. This study developed an integrated analysis tool for metabolomics studies to streamline the complete analysis flow from initial data preprocessing to downstream association analysis. We developed Statistical Metabolomics Analysis-An R Tool (SMART), which can analyze input files with different formats, visually represent various types of data features, implement peak alignment and annotation, conduct quality control for samples and peaks, explore batch effects, and perform association analysis. A pharmacometabolomics study of antihypertensive medication was conducted and data were analyzed using SMART. Neuromedin N was identified as a metabolite significantly associated with angiotensin-converting-enzyme inhibitors in our metabolome-wide association analysis (p = 1.56 × 10(-4) in an analysis of covariance (ANCOVA) with an adjustment for unknown latent groups and p = 1.02 × 10(-4) in an ANCOVA with an adjustment for hidden substructures). This endogenous neuropeptide is highly related to neurotensin and neuromedin U, which are involved in blood pressure regulation and smooth muscle contraction. The SMART software, a user guide, and example data can be downloaded from http://www.stat.sinica.edu.tw/hsinchou/metabolomics/SMART.htm .
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Metabolómica , Interfaz Usuario-Computador , Análisis de Varianza , Cromatografía de Gases y Espectrometría de Masas , Internet , Neurotensina/análisis , Fragmentos de Péptidos/análisis , Renina/antagonistas & inhibidores , Renina/metabolismoRESUMEN
A new-concept of an "in-electrode" Faraday cage-type electrochemiluminescence immunoassay (ECLIA) method for the ultrasensitive detection of neurotensin (NT) was reported with capture antibody (Ab1)-nanoFe3O4@graphene (GO) and detector antibody (Ab2)&N-(4-aminobutyl)-N-ethylisoluminol (ABEI)@GO, which led to about 1000-fold improvement in sensitivity by extending the Helmholtz plane (OHP) of the proposed electrode assembly effectively.
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Electroquímica/métodos , Electrodos , Inmunoensayo/métodos , Anticuerpos/metabolismo , Grafito/química , Límite de Detección , Mediciones Luminiscentes , Luminol/análogos & derivados , Luminol/química , Neurotensina/análisisRESUMEN
Obtaining maximal sensitivity of nano UHPLC-MS/MS methods is primordial to quantify picomolar concentrations of neuropeptides in microdialysis samples. Since aspecific adsorption of peptides to Eppendorf tubes, pipette tips and UHPLC vials is detrimental for method sensitivity, a strategy is presented to reduce adsorption of these peptides during standard preparation. Within this respect, all procedural steps from dissolution of the lyophilized powder until the injection of the sample onto the system are investigated. Two peptides of the neuromedin family, i.e. neuromedin B and neuromedin N, and a neuromedin N-related neuropeptide, neurotensin, are evaluated. The first part of this study outlines a number of parameters which are known to affect peptide solubility. The main focus of the second part involves the optimization of the sample composition in the UHPLC vial by using design of experiments. Contradictory findings are observed concerning the influence of acetonitrile, salts and matrix components. They are found important for injection of the peptides into the system, but crucially need to be excluded from the dilution solvent. Furthermore, the type of surface material, temperature and the pipetting protocol considerably affect the adsorption phenomenon. Statistical analysis on the results of the central composite design reveals that the highest peptide responses are obtained with the injection solvent consisting of 13.1% V/V ACN and 4.4% V/V FA. This aspect of the optimization strategy can be identified as the main contributor to the gain in method sensitivity. Since the reduction of peptide adsorption and the optimization of the injection solvent resulted in a clear and quantifiable signal of the three peptides, optimization of both issues should be considered in the early stage of method development, in particular when the analysis of low-concentration peptide solutions is envisaged.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Neurotensina/análisis , Fragmentos de Péptidos/análisis , Espectrometría de Masas en Tándem/métodos , Adsorción , Fenómenos Químicos , Solventes/química , Propiedades de SuperficieRESUMEN
In order to improve the sensitivity of CE-ESI-MS for the analysis of brain-gut peptides, a solid-phase extraction combined with field-amplified sample injection was used for the pre-concentration of the brain-gut peptides. Compared with the conventional pressure injection method, the sensitivity in the detection of brain-gut peptides was improved more than 100-fold. The possible factors affecting sample stacking, such as the sample matrix, the composition and the length of the water column, the types and the volumes of eluent, have been investigated in detail. Under the optimum conditions, the detectable concentration of brain-gut peptides was found to be as low as 0.02 µM. A linear response concentration for the detection was developed in the range of 0.08-25 µmol/L. A real sample of human cerebrospinal fluids, which was spiked with brain-gut peptides, was also examined in order to evaluate the reliability of the proposed approach. The recovery of the method was in a range from 69.2 to 85.4%. The method was found to be reliable, accurate and potentially applicable for clinical drug analysis.
Asunto(s)
Electroforesis Capilar/métodos , Neurotensina/análisis , Extracción en Fase Sólida/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Tetragastrina/análisis , Humanos , Límite de Detección , Neurotensina/líquido cefalorraquídeo , Fragmentos de Péptidos , Análisis de Regresión , Reproducibilidad de los Resultados , Tetragastrina/líquido cefalorraquídeoRESUMEN
Deregulated steroids are involved in different hormone-dependent tumors, including benign and malignant uterine neoplasms. Leiomyomas (LM) are estrogen and progesterone-dependent benign tumors, whereas "bizarre or atypical LMs" (AL) are considered a subgroup of LM and clinically benign, although their malignant potential is suspect. Uterine leiomyosarcomas (LMS) are malignant smooth muscle tumors, and ovarian steroids may control their growth. Estrogen effects are mediated by 2 receptors, estrogen receptors (ER) α and ß, and the ratio of both receptors seems to be a critical parameter in the estrogen-mediated carcinogenic process. Estradiol induces the expression of neurotensin (NTS), and the coupling of this peptide with its high-affinity receptor, NTS1, has been involved in the regulation of tumoral cell growth. Given the importance of these markers in tumor development, we aim to determine the status of ERα and ERß in the myometrium and LM, AL, and LMS, concomitantly with the expression of NTS/NTS receptor 1 in these tumors. For that purpose, we use immunohistochemistry for all markers analyzed and in-situ hybridization to detect NTS mRNA. These data suggest that LMS are estrogen-dependent tumors, which may use NTS as an autocrine growth factor. In addition, the phenotype of AL with regard to ERα and ERß status and NTS expression is closer to LMS than LM; thus, a potential malignization of this tumor is feasible.
Asunto(s)
Receptor alfa de Estrógeno/análisis , Receptor beta de Estrógeno/análisis , Leiomioma/química , Leiomiosarcoma/química , Neurotensina/análisis , Neoplasias Uterinas/química , Núcleo Celular/química , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Músculo Liso/química , Músculo Liso/ultraestructura , Miometrio/química , Receptores de Neurotensina/análisisRESUMEN
A new method for the determination of the peptide hormones and their fragments by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection and transient pseudo-isotachophoresis (pseudo-tITP) preconcentration was established in this study. The LIF detector used an argon ion laser with excitation wavelength at 488 nm and emission wavelength at 535 nm. Fluorescein isothiocyanate (FITC) was used as precolumn derivatization reagent to label cholecystokinin tetrapeptide (CCK-4), neurotensin (NT), neurotensin hexapeptide (NT(8-13)), and neurokinin B (NKB). Borate (10 mmol/L, pH 9.0) was selected as derivatization medium to get the high efficiency. When the addition of 70% (v/v) methanol and 1% (m/v) sodium chloride (NaCl) to the sample matrix, and with borate buffer (110 mM, pH 9.5) and 20% (v/v) methanol as running buffer, a preconcentration based on the pseudo-tITP afforded 100-fold improvement in peak heights compared with the traditional hydrodynamic injection (2.3% capillary volume). The detection limits (signal/noise=3) based on peak height were found to be 0.04, 0.1, 0.2, and 0.08 nmol/L for NT(8-13), NT, NKB, and CCK-4, respectively. The method was validated and applied to qualitative analysis of NT and NT(8-13) in human cerebrospinal fluid sample.
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Electroforesis Capilar/métodos , Hormonas Peptídicas/aislamiento & purificación , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/líquido cefalorraquídeo , Moléculas de Adhesión Celular/aislamiento & purificación , Fluoresceína-5-Isotiocianato/química , Fluorescencia , Humanos , Láseres de Gas , Neuroquinina B/análisis , Neuroquinina B/líquido cefalorraquídeo , Neuroquinina B/aislamiento & purificación , Neurotensina/análisis , Neurotensina/líquido cefalorraquídeo , Neurotensina/aislamiento & purificación , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/líquido cefalorraquídeo , Fragmentos de Péptidos/aislamiento & purificación , Hormonas Peptídicas/líquido cefalorraquídeo , Hormonas Peptídicas/química , Proteínas Tirosina Quinasas Receptoras/análisis , Proteínas Tirosina Quinasas Receptoras/líquido cefalorraquídeo , Proteínas Tirosina Quinasas Receptoras/aislamiento & purificación , Hemorragia Subaracnoidea/líquido cefalorraquídeoRESUMEN
We have studied here the cellular distribution of several regulatory peptides in hormone-producing cells of the human pituitary during the fetal period. Immunohistochemistry was used to show the expression of several regulatory peptides, namely Angiotensin-II, Neurotensin and Galanin, at successive gestational stages and their co-localization with hormones in the human fetal adenohypophysis. Somatotrophs, gonadotrophs and thyrotrophs were differentiated earliest. At gestational week 9, Angiotensin-II immunoreactivity was co-localized only with growth hormone immunoreactivity in somatotrophs, one of the first hormone-producing cells to differentiate. This co-localization remained until week 37. Neurotensin immunoreactivity was present in gonadotrophs and thyrotrophs in week 23, after FSH and TSH hormone differentiation. Galanin immunoreactivity was present in all hormone-producing cell types except corticotrophs. The different pro-opiomelanocortin-derived peptides were detected at different stages of gestation and adrenocorticotrophic hormone immunoreaction was the last to be detected. Our results show an interesting relationship between regulatory peptides and hormones during human fetal development, which could imply that these peptides play a regulatory role in the development of pituitary function.
Asunto(s)
Angiotensina II/análisis , Galanina/análisis , Neurotensina/análisis , Adenohipófisis/embriología , Hormona Adrenocorticotrópica/análisis , Diferenciación Celular , Corticotrofos/química , Edad Gestacional , Gonadotrofos/química , Humanos , Inmunohistoquímica , Adenohipófisis/química , Somatotrofos/química , Tirotrofos/químicaRESUMEN
The suprachiasmatic nuclei (SCN) are necessary and sufficient for the maintenance of circadian rhythms in primate and other mammalian species. The human dorsomedial SCN contains populations of non-species-specific vasopressin and species-specific neurotensin neurons. We made time-series recordings of core body temperature and locomotor activity in 19 elderly, male, end-stage dementia patients and 8 normal elderly controls. Following the death of the dementia patients, neuropathological diagnostic information and tissue samples from the hypothalamus were obtained. Hypothalamic tissue was also obtained from eight normal control cases that had not had activity or core temperature recordings previously. Core temperature was analysed for parametric, circadian features, and activity was analysed for non-parametric and parametric circadian features. These indices were then correlated with the degree of degeneration seen in the SCN (glia/neuron ratio) and neuronal counts from the dorsomedial SCN (vasopressin, neurotensin). Specific loss of SCN neurotensin neurons was associated with loss of activity and temperature amplitude without increase in activity fragmentation. Loss of SCN vasopressin neurons was associated with increased activity fragmentation but not loss of amplitude. Evidence for a circadian rhythm of vasopressinergic activity was seen in the dementia cases but no evidence was seen for a circadian rhythm in neurotensinergic activity. These results provide evidence that the SCN is necessary for the maintenance of the circadian rhythm in humans, information on the role of neuronal subpopulations in subserving this function and the utility of dementia in elaborating brain-behaviour relationships in the human.
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Enfermedad de Alzheimer/patología , Núcleo Talámico Mediodorsal/patología , Neuronas/patología , Anciano , Enfermedad de Alzheimer/metabolismo , Análisis de Varianza , Regulación de la Temperatura Corporal , Estudios de Casos y Controles , Recuento de Células , Ritmo Circadiano , Humanos , Inmunohistoquímica , Masculino , Núcleo Talámico Mediodorsal/metabolismo , Actividad Motora , Neuroglía/patología , Neuronas/metabolismo , Neurotensina/análisis , Sueño , Vasopresinas/análisisRESUMEN
We report an efficient and streamlined way to improve the analysis and identification of peptides and proteins in complex mixtures of soluble proteins, cell lysates, etc. By using the shotgun proteomics methodology combined with bioaffinity purification we can remove or minimize the interference contamination of a complex tryptic digest and so avoid the time-consuming separation steps before the final MS analysis. We have proved that by means of enzymatic fragmentation (endoproteinases with Arg-C or/and Lys-C specificity) connected with the isolation of specific peptides we can obtain a simplified peptide mixture for easier identification of the entire protein. A new bioaffinity sorbent was developed for this purpose. Anhydrotrypsin (AHT), an inactive form of trypsin with an affinity for peptides with arginine (Arg) or lysine (Lys) at the C-terminus, was immobilized onto micro/nanoparticles with superparamagnetic properties (silica magnetite particles (SiMAG)-Carboxyl, Chemicell, Germany). This AHT carrier with a determined binding capacity (26.8 nmol/mg of carrier) was tested with a model peptide, human neurotensin, and the resulting MS spectra confirmed the validity of this approach.
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Reactores Biológicos , Cromatografía de Afinidad/métodos , Magnetismo , Neurotensina/análisis , Péptidos/análisis , Péptidos/química , Tripsina/química , Cromatografía de Afinidad/instrumentación , Enzimas Inmovilizadas/química , Humanos , Ligandos , Metaloendopeptidasas/química , Nanopartículas/química , Proteómica , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Serina Endopeptidasas/química , Dióxido de Silicio/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Factores de Tiempo , Tripsina/aislamiento & purificaciónRESUMEN
The main purpose of this study was to evaluate the effects of early dexamethasone treatment on pain-related peptides at an early stage in the development of neuropathic pain induced by implantation of a sciatic nerve cuff in Sprague Dawley rats (body weight 250 to 350 g). The rats were tested for touch sensitivity with the use of von Frey filaments before and 3 d after cuff implantation (n = 12) or sham surgery (n = 6). Half of the cuff-implanted rats received dexamethasone, 1 mg/kg intraperitoneally, 1 h after surgery. Spinal cords were collected on the 3rd day after surgery, and the lumbar enlargement was processed for the detection of selected peptides (neurotensin, substance P, cholecystokinin [CCK], vasoactive intestinal peptide, and calcitonin gene-related peptide) by means of liquid chromatography and tandem mass spectrometry. The right sciatic nerve of each rat was collected, fixed, and stained for histopathological evaluation. Except for neurotensin, all the peptides showed an increased concentration with neuropathic pain; however, the differences were significant (P < 0.05) only for substance P and CCK. In the animals treated with dexamethasone, mechanical allodynia was less pronounced (P < 0.01), and only the concentration of substance P was decreased significantly (P < 0.05). Sciatic nerve sections showed a decrease in C (P < 0.01) and Adelta (P < 0.03) fibres with neuropathic pain and a nearly normal percentage of C fibres after dexamethasone treatment. The dexamethasone-treated animals also had less inflammation detectable microscopically at the nerve constriction site compared with cuff-implanted animals that were not treated with dexamethasone. Our results suggest that in the early stages of neuropathic pain induced by an inflammatory process, dexamethasone may be a useful treatment and that substance P plays an important role in pain perception.
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Antiinflamatorios/uso terapéutico , Colecistoquinina/metabolismo , Dexametasona/uso terapéutico , Neuropatía Ciática/veterinaria , Médula Espinal/metabolismo , Sustancia P/metabolismo , Animales , Calcitonina/análisis , Calcitonina/metabolismo , Colecistoquinina/análisis , Cromatografía Liquida , Constricción , Modelos Animales de Enfermedad , Masculino , Espectrometría de Masas , Fibras Nerviosas/metabolismo , Neurotensina/análisis , Neurotensina/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Nervio Ciático/lesiones , Neuropatía Ciática/tratamiento farmacológico , Médula Espinal/patología , Sustancia P/análisis , Factores de Tiempo , Resultado del TratamientoRESUMEN
This study assesses the action of hypercortisolism on the hormone and peptide periadenoma region of removed ACTH-producing microadenoma. Our findings show that cortisol excess affects both ACTH and GH production, with no immunoreaction for these hormones. The remaining pituitary hormones (TSH, FSH and PRL) and POMC-derived peptides (betaEnd, alphaMSH and betaMSH) were not modified. Likewise, we observed pituitary immunoreactive cells for Neurotensin (NT), Intestinal vasoactive peptide (VIP), Substance P (SP) and Angiotensin-II (Ang-II). The colocalization demonstrated that NT was expressed in thyrotrope and gonadotrope cells, VIP in gonadotrope cells and SP in corticotrope cells. The results about Ang-II were inconclusive. On the other hand, immunoreaction for the NPY and Gal peptides were not present. In the adenomatous cells, the peptide NT is present in ACTH cells as well as SP. These results suggest a peptide regulation of pituitary cells in the pathological state that can differ between normal and tumoural cells of the same pituitary.
Asunto(s)
Adenoma Hipofisario Secretor de ACTH/química , Adenoma/química , Síndrome de Cushing/etiología , Neuropéptidos/análisis , Hormonas Hipofisarias/análisis , Adenoma Hipofisario Secretor de ACTH/complicaciones , Adenoma Hipofisario Secretor de ACTH/patología , Adenoma/complicaciones , Adenoma/patología , Hormona Adrenocorticotrópica/análisis , Adulto , Angiotensina II/análisis , Corticotrofos/química , Síndrome de Cushing/metabolismo , Síndrome de Cushing/patología , Femenino , Gonadotrofos/química , Hormona de Crecimiento Humana/análisis , Humanos , Inmunohistoquímica , Neurotensina/análisis , Sustancia P/análisis , Tirotrofos/química , Péptido Intestinal Vasoactivo/análisisRESUMEN
A matrix assisted laser desorption/ionization time-of-flight mass spectrometer has been built with an ion source that can be operated in either constant-energy or constant-momentum acceleration modes. A decreasing electric field distribution in the ion-accelerating region makes it possible to direct ions onto a space-focal plane in either modes of operation. Ions produced in the constant-momentum mode have velocities and, thus, flight times that are linearly dependent on mass and kinetic energies that are inversely dependent on mass. The linear mass dispersion doubles mass resolving power of ions accelerated with space-focusing conditions in constant-momentum mode. The mass-dependent kinetic energy is exploited to disperse ions according to mass in a simple kinetic energy filter constructed from two closely spaced, oblique ion reflectors. Focusing velocity of ions of the same mass can substantially improve ion selection for subsequent post source decay or tandem time-of-flight analyses.
Asunto(s)
Péptidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Angiotensina I/análisis , Hormona Liberadora de Gonadotropina/análisis , Cinética , Neurotensina/análisis , Sustancia P/análisisRESUMEN
High concentrations of urea and guanidine hydrochloride are commonly used for the denaturation of protein, which was digested by enzymatic proteolysis for the identification by MS analysis. The presence of these contaminants seriously suppresses the ion signal of analytes in MALDI-TOF MS analysis. Herein, a novel MALDI matrix, 3, 4-diaminobenzophenone (DABP), has been found with high tolerance for these contaminants in MALDI MS analysis. The ion signal of analyte insulin can be detected in the presence of 2 M guanidine hydrochloride and 1.5 M urea using DABP as matrix. The tryptic digest of BSA (400 fmol) in 1 M guanidine hydrochloride or 1 M urea was successfully analyzed without any pretreatment prior to MS analysis. Furthermore, it has been found that this matrix can also effectively suppress the cation ion adduction of the peptides in the presence of high concentrations of metal ions in sample solution.
Asunto(s)
Benzofenonas/química , Péptidos/análisis , Fenilendiaminas/química , Proteínas/análisis , Soluciones/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Cationes , Guanidina/química , Insulina/análisis , Metales/química , Neurotensina/análisis , Sensibilidad y Especificidad , Albúmina Sérica Bovina/análisis , Tripsina/metabolismo , Urea/químicaRESUMEN
The distribution of neurotensin-containing cell bodies and fibers has been observed in the central and peripheral nervous system, including sensory ganglia, but no description has been found in the peripheral auditory system. Here, we investigated the presence of neurotensin immunoreactivity in the cochlea of the adult Wistar rat. Strong neurotensin immunoreactivity was detected in the cytoplasm of the inner hair cells (IHC) and Deiters' cells of the organ of Corti. Outer hair cells (OHC) show weak immunoreaction. Neurotensin immunoreactivity was also found in the neurons and fibers of the spiral ganglia. Quantitative microdensitometric image analysis of the neurotensin immunoreactivity showed a strong immunoreaction in the hair cells of organ of Corti and a moderate to strong labeling in the spiral ganglion neurons. A series of double immunolabeling experiments demonstrated a strong neurotensin immunoreactivity in the parvalbumin immunoreactive IHC and also in the calbindin immunoreactive Deiters' cells. Weak neurotensin immunoreactivity was seen in the calbindin positive OHC. Neurofilament and parvalbumin immunoreactive neurons and fibers in the spiral ganglia showed neurotensin immunoreactivity. Calbindin immunoreactivity was not detected in the spiral ganglion neurons, which are labeled by neurotensin immunoreactivity. The presence of neurotensin in the cochlea may be related to its modulation of neurotransmission in the peripheral auditory pathway.
Asunto(s)
Neuronas/química , Neurotensina/análisis , Órgano Espiral/química , Animales , Células Ciliadas Auditivas/química , Células Ciliadas Auditivas/inmunología , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Masculino , Microscopía Confocal , Microscopía Fluorescente , Neuronas/inmunología , Neurotensina/biosíntesis , Neurotensina/inmunología , Órgano Espiral/inmunología , Ratas , Ratas Wistar , Organismos Libres de Patógenos Específicos , Ganglio Espiral de la Cóclea/química , Ganglio Espiral de la Cóclea/inmunologíaRESUMEN
Bacillary dysentery arises when Shigella invades the colonic and rectal mucosae of the human gut and elicits a strong inflammatory response, which may lead to life-threatening complications. Hence, downregulation of the host inflammatory response is an appealing therapeutical alternative. The gastrointestinal tract is densely innervated, and nerve endings are often found in the vicinity of leukocytes. We have assessed the impact of experimental Shigella infection on levels of neuropeptides in the intestinal mucosa of rabbits. Ligated small intestinal loops were created in rabbits, and either live, pathogenic Shigella flexneri, a nonpathogenic mutant of Shigella, or NaCl was injected into the loops. Infection was allowed to proceed for 8 or 16 h, after which the rabbits were sacrificed and intestinal biopsies collected. Tissue destruction, fluid secretion and degree of bacterial invasion were monitored. Intestinal biopsies were homogenized, and levels of the neuropeptides calcitonin gene-related peptide, substance P, peptide YY (PYY), vasoactive intestinal peptide, somatostatin, galanin, motilin and neurotensin were measured by radioimmunoassay. Loops exposed to invasive Shigella had 5.7 times lower levels of PYY (P = 0.0095) than loops exposed to NaCl, after 16 h of infection. The levels of the other neuropeptides tested were unchanged. Inhibition of nicotinic cholinergic neurotransmission partly protected the intestinal mucosa from destruction elicited by invasive Shigella. These findings indicate that a tissue-invasive bacterium such as Shigella, which is strictly localized to the intestinal mucosa, activates intramural nerve reflexes that presumably involve a nicotinic synapse as well as the neuropeptide PYY.