Home About us Contact
|
Home About us Contact | ||
|
|
||
Intact Tissue (intact + tissue)
Selected AbstractsImproved workup for glycosaminoglycan disaccharide analysis using CE with LIF detectionELECTROPHORESIS, Issue 22 2008Alicia M. Hitchcock Abstract This work describes improved workup and instrumental conditions to enable robust, sensitive glycosaminoglycan (GAG) disaccharide analysis from complex biological samples. In the process of applying CE with LIF to GAG disaccharide analysis in biological samples, we have made improvements to existing methods. These include (i) optimization of reductive amination conditions, (ii) improvement in sensitivity through the use of a cellulose cleanup procedure for the derivatization, and (iii) optimization of separation conditions for robustness and reproducibility. The improved method enables analysis of disaccharide quantities as low as 1,pmol prior to derivatization. Biological GAG samples were exhaustively digested using lyase enzymes, the disaccharide products and standards were derivatized with the fluorophore 2-aminoacridone and subjected to reversed polarity CE-LIF detection. These conditions resolved all known chondroitin sulfate (CS) disaccharides or 11 of 12 standard heparin/heparan sulfate disaccharides, using 50,mM phosphate buffer, pH 3.5, and reversed polarity at 30,kV with 0.3,psi pressure. Relative standard deviation in migration times of CS ranged from 0.1 to 2.0% over 60 days, and the relative standard deviations of peak areas were less than 3.2%, suggesting that the method is reproducible and precise. The CS disaccharide compositions are similar to those obtained by our group using tandem MS. The reversed polarity CE-LIF disaccharide analysis protocol yields baseline resolution and quantification of heparin/heparan sulfate and CS/dermatan sulfate disaccharides from both standard preparations and biologically relevant proteoglycan samples. The improved CE-LIF method enables disaccharide quantification of biologically relevant proteoglycans from small samples of intact tissue. [source] Direct Measurement of Hormone-Induced Acidification in Intact BoneJOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2000Glenn S. Belinsky Abstract Previous findings have shown that osteoblasts respond to parathyroid hormone (PTH) with an increase in extracellular acidification rate (ECAR) in addition to the known effect of PTH to increase local acidification by osteoclasts. We, therefore, investigated use of the Cytosensor to measure the ECAR response of whole intact bone to PTH employing microphysiometry. The Cytosensor measures a generic metabolic increase of cells to various agents. Using neonatal mouse calvaria, we found that the area surrounding the sagittal suture was particularly responsive to PTH. In this bone, the increase in ECAR was slower to develop (6 minutes) and more persistent than in cultured human osteoblast-like SaOS-2 cells and was preceded by a brief decrease in ECAR Salmon calcitonin also produced an increase in ECAR in this tissue but with a different pattern than that elicited by PTH. Because PTH stimulates osteoclastic bone resorption in mouse calvaria via a cyclic adenosine monophosphate (cAMP)-mediated mechanism, we showed that the adenylyl cyclase activator forskolin also stimulated ECAR in this tissue. When the protein kinase A (PKA) pathway was activated by maintaining a high intracellular concentration of cAMP using N6 -2,-0-dibutyryladenosine-cAMP (db-cAMP), there was a reduction of PTH-induced acidification, while isobutylmethylxanthine pretreatment potentiated the PTH-induced acidification, consistent with a PKA-mediated pathway. Thapsigargin and the protein kinase C (PKC) activator phorbol myristate acetate had no effect on the PTH-induced increase in ECAR in calvaria, indicating that PKC does not play a major role in the ECAR response in intact bone. These results indicate the utility of using microphysiometry to study ECAR responses in intact tissue and should enable elucidation of the relative importance of extracellular acidification by osteoblasts and osteoclasts to the anabolic and catabolic activities of PTH, respectively. [source] Neonatal Alcohol-Induced Region-Dependent Changes in Rat Brain Neurochemistry Measured by High-Resolution Magnetic Resonance SpectroscopyALCOHOLISM, Issue 10 2008Shonagh K. O'Leary-Moore Background:, Maternal drinking during pregnancy can lead to a range of deleterious outcomes in the developing offspring that have been collectively termed fetal alcohol spectrum disorders (FASDs). There is interest and recognized value in using non-invasive neuroimaging techniques such as magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) to characterize, respectively, structural and biochemical alterations in individuals with FASDs. To date, however, results with MRS have been inconsistent regarding the degree and/or nature of abnormalities. Methods:, High-resolution magic angle spinning (HR-MAS) proton (1H) MRS is an ex vivo neuroimaging technique that can acquire spectra in small punches of intact tissue, providing clinically relevant neurochemical information about discrete brain regions. In this study, HR-MAS 1H MRS was used to examine regional neurochemistry in frontal cortex, striatum, hippocampus, and cerebellum of young rats previously exposed to ethanol as neonates. Key neurochemicals of interest included N-acetyl-aspartate (NAA), glutamate, GABA, glutamine, creatine, choline and myo -inositol. Results:, Daily neonatal alcohol exposure from postnatal day 4 (PN4) through PN9 significantly reduced levels of NAA and taurine in the cerebellum and striatum, and induced sex-dependent reductions in cerebellar glutamate when measured on PN16. In addition, myo -inositol was significantly increased in cerebellum. The frontal cortex and hippocampus were virtually unaffected by this neonatal alcohol exposure. Conclusion:, Results of this research may have implications for understanding the underlying neurobiology associated with FASDs and aid in testing treatments in the future. Ongoing studies are assessing the developmental persistence of and/or maturational recovery from these changes. [source] Investigation of metabolite changes in the transition from pre-invasive to invasive cervical cancer measured using 1H and 31P magic angle spinning MRS of intact tissueNMR IN BIOMEDICINE, Issue 2 2009Sonali S. De Silva Abstract The aim of this study was to determine the metabolic changes in the transition from pre-invasive to invasive cervical cancer using high-resolution magic angle spinning (HR-MAS) MRS. Biopsy specimens were obtained from women with histologically normal cervix (n,=,5), cervical intraepithelial neoplasia (CIN; mild, n,=,5; moderate/severe, n,=,40), and invasive cancer (n,=,23). 1H HR-MAS MRS data were acquired using a Bruker Avance 11.74,T spectrometer (Carr,Purcell,Meiboom,Gill sequence; TR,=,4.8,s; TE,=,135,ms; 512 scans; 41,min acquisition). 31P HR-MAS spectra were obtained from the normal subjects and cancer patients only (as acetic acid applied before tissue sampling in patients with CIN impaired spectral quality) using a 1H-decoupled pulse-acquire sequence (TR,=,2.82,s; 2048 scans; 96,min acquisition). Peak assignments were based on values reported in the literature. Peak areas were measured using the AMARES algorithm. Estimated metabolite concentrations were compared between patient diagnostic categories and tissue histology using independent samples t tests. Comparisons based on patient category at diagnosis showed significantly higher estimated concentrations of choline (P,=,0.0001) and phosphocholine (P,=,0.002) in tissue from patients with cancer than from patients with high-grade dyskaryosis, but no differences between non-cancer groups. Division by histology of the sample also showed increases in choline (P,=,0.002) and phosphocholine (P,=,0.002) in cancer compared with high-grade CIN tissue. Phosphoethanolamine was increased in cancer compared with normal tissue (P,=,0.0001). Estimated concentrations of alanine (P,=,0.01) and creatine (P,=,0.008) were significantly reduced in normal tissue from cancer patients compared with normal tissue from non-cancer patients. The estimated concentration of choline was significantly increased in CIN tissue from cancer patients compared with CIN tissue from non-cancer patients (P,=,0.0001). Estimated concentrations of choline-containing metabolites increased from pre-invasive to invasive cervical cancer. Concurrent metabolite depletion occurs in normal tissue adjacent to cancer tissue. Copyright © 2008 John Wiley & Sons, Ltd. [source] Increased blood,brain barrier permeability of morphine in a patient with severe brain lesions as determined by microdialysisACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 3 2001R. Bouw Intracerebral microdialysis was utilised to obtain information regarding how morphine is transported across the blood,brain barrier (BBB). In a patient with a severe brain injury, we measured simultaneously unbound extracellular fluid (ECF) concentrations of morphine in human brain and in subcutaneous fat tissue, which were compared to morphine levels in arterial blood. This report shows an increase in morphine levels near the trauma site in the brain compared to uninjured brain tissue. The half-life of morphine in uninjured and injured brain tissue of 178 min and 169 min, respectively, were comparable but were longer than in blood (64 min) and adipose tissue (63 min). This indicates that morphine is retained in brain tissue for a longer time than what could be expected from the blood concentration,time profile. These results show the potential of the microdialysis technique in providing new information regarding the pharmacokinetics of drug in the human brain close to the trauma site and in macroscopically intact tissue. [source] Proteomic analysis of core breakdown disorder in Conference pears (Pyrus communis L.)PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 12 2007Romina Pedreschi Abstract 2-DE was applied to study core breakdown disorder in controlled atmosphere stored 'Conference' pears. This physiological disorder is characterized by internal browning of the fruit tissue and the development of cavities. Suitable protein phenol extraction/ammonium acetate-methanol precipitation and 2-DE protocols for a wide pH range were established for pear tissue. The protein expression profiles of healthy, sound (intact tissue of pears with core breakdown) and brown tissue were analyzed with the univariate non-parametric Kolmogorov-Smirnov test and multivariate statistical techniques such as principal component analysis and partial least square discriminant analysis. Both statistical approaches revealed interesting differentially expressed proteins between healthy and disordered pears. LC-ESI-MS/MS identification of differentially expressed proteins between healthy and sound tissue revealed their participation in the energy metabolism, the antioxidant system and ethylene biosynthesis. Up-regulated characteristic proteins in brown tissue were mainly involved in energy metabolism and defense mechanisms. Proteomics coupled to univariate and multivariate statistical techniques seems to be an efficient approach to get a better insight into the different mechanisms and pathways leading to the core breakdown disorder. [source] Development and validation of a gas chromatography/mass spectrometry method for the metabolic profiling of human colon tissueRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 4 2009Mainak Mal In this study, a gas chromatography/mass spectrometry (GC/MS) method was developed and validated for the metabolic profiling of human colon tissue. Each colon tissue sample (20,mg) was ultra-sonicated with 1,mL of a mixture of chloroform/methanol/water in the ratio of 20:50:20 (v/v/v), followed by centrifugation, collection of supernatant, drying, removal of moisture using anhydrous toluene and finally derivatization using N -methyl- N -trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS). A volume of 1,µL of the derivatized mixture was injected into the GC/MS system. A total of 53 endogenous metabolites were separated and identified in the GC/MS chromatogram, all of which were selected to evaluate the sample stability and precision of the method. Of the identified endogenous metabolites 19 belonging to diverse chemical classes and covering a wide range of the GC retention times (Rt) were selected to investigate the quantitative linearity of the method. The developed GC/MS method demonstrated good reproducibility with intra- and inter-day precision within relative standard deviation (RSD) of ±15%. The metabolic profiles of the intact tissue were determined to be stable (100,±,15%) for up to 90 days at ,80°C. Satisfactory results were also obtained in the case of other stability-indicating studies such as freeze/thaw cycle stability, bench-top stability and autosampler stability. The developed method showed a good linear response for each of the 19 analytes tested (r2,>,0.99). Our GC/MS metabolic profiling method was successfully applied to discriminate biopsied colorectal cancer (CRC) tissue from their matched normal tissue obtained from six CRC patients using orthogonal partial least-squares discriminant analysis [two latent variables, R2Y,=,0.977 and Q2 (cumulative),=,0.877]. Copyright © 2009 John Wiley & Sons, Ltd. [source] Pharmacological characterization of a Bombyx mori ,-adrenergic-like octopamine receptor stably expressed in a mammalian cell lineARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2010Jia Huang Abstract Series of agonists and antagonists were examined for their actions on a Bombyx mori,-adrenergic-like octopamine receptor (OAR) stably expressed in HEK-293 cells. The rank order of potency of the agonists was clonidine>naphazoline>tolazoline in Ca2+ mobilization assays, and that of the antagonists was chlorpromazine>yohimbine. These findings suggest that the B. mori OAR is more closely related to the class-1 OAR in the intact tissue than to the other classes. N,-(4-Chloro- o -tolyl)- N -methylformamidine (DMCDM) and 2-(2,6-diethylphenylimino)imidazolidine (NC-5) elevated the intracellular calcium concentration ([Ca2+]i) with EC50s of 92.8,µM and 15.2,nM, respectively. DMCDM and NC-5 led to increases in intracellular cAMP concentration ([cAMP]i) with EC50s of 234,nM and 125,nM, respectively. The difference in DMCDM potencies between the cAMP and Ca2+ assays might be due to "functional selectivity." The Ca2+ and cAMP assay results for DMCDM suggest that the elevation of [cAMP]i, but not that of [Ca2+]i, might account for the insecticidal effect of formamidine insecticides. © 2009 Wiley Periodicals, Inc. [source] In vivo Distribution of Bismuth in the Mouse Brain: Influence of Long-Term Survival and Intracranial Placement on the Uptake and Transport of Bismuth in Neuronal TissueBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 3 2005Agnete Larsen In medicine, bismuth-compounds have long been used to remedy gastrointestinal disorders; lately in combination with antibiotics to treat Helicobacter pylori associated peptic ulcers. An epidemic episode of bismuth-induced encephalopathy in France in the 1970s revealed the neurotoxic potential of bismuth. This incidence, involving almost 1000 patients, remains unexplained and the contribution of other factors besides bismuth has been postulated. Recently an autometallographic technique made it possible to detect bismuth in morphologically intact tissue. In the present study, autometallographicly detectable bismuth was seen throughout the brain following intraperitoneal and intracranial exposure. The neuronal staining pattern seems highly organized with some areas heavily stained and others with low or no staining. Long-term (8 months) intraperitoneal exposure led to higher bismuth uptake than short-term (2 weeks) exposure. Following both intraperitoneal and intracranial exposure, high amounts of bismuth were found in the reticular and hypothalamic nuclei, in the oculomotor and hypoglossal nuclei and in Purkinje cells. Within the central nervous system (CNS) retrograde axonal transport was seen after intracranial bismuth exposure. Axonal transport seems to influence the distribution of bismuth as the highest uptake of bismuth after intraperitoneal exposure was seen in the facial and the trigeminal motor nuclei, i.e. neurones with processes outside the blood-brain barrier, whereas these nuclei contained no bismuth following ic exposure. Ultrastructurally, accumulation of bismuth was seen in lysosomes. [source] Inhibitory effect of 1,8-cineole on guinea-pig airway challenged with ovalbumin involves a preferential action on electromechanical couplingCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 11 2009Vasco PD Bastos Summary 11,8-Cineole is a terpenoid constituent of essential oils with anti-inflammatory properties. It reduces the neural excitability, functions as an antinociceptive agent and has myorelaxant actions in guinea-pig airways. The aim of the present study was to investigate the mechanism underlying the myorelaxant effects of 1,8-cineole in guinea-pig isolated trachea from either naïve guinea-pigs or ovalbumin (OVA)-sensitized animals subjected to antigenic challenge. 2Isometric recordings were made of the tone of isolated tracheal rings. Rings with an intact epithelium relaxed beyond basal tone in the presence of 1,8-cineole (6.5 × 10,6 to 2 × 10,2 mol/L) in a concentration-dependent manner (P < 0.001, anova) with a pD2 value of 2.23 (95% confidence interval 2.10,2.37). Removal of the epithelium or pretreatment of intact tissue for 15 min with 50 µmol/L NG -nitro- l -arginine methyl ester, 5 mmol/L tetraethylammonium, 0.5 µmol/L tetrodotoxin or 5 µmol/L propranolol did not alter the potency (pD2) or the maximal myorelaxant effect (Emax) of 1,8-cineole. 31,8-Cineole also significantly decreased the Schultz-Dale contraction induced by OVA, mainly in preparations from OVA-sensitized animals submitted to antigen challenge. 1,8-Cineole decreased tracheal hyperresponsiveness to KCl and carbachol caused by antigen challenge and almost abolished the concentration,response curves to KCl, whereas it had little effect on the concentration,response curves to carbachol. Under Ca2+ -free conditions and in the presence of 10,4 mol/L acetylcholine, neither 1,8-cineole (6.5 × 10,3 mol/L) nor verapamil (1 × 10,5 mol/L) affected Ca2+ -induced contractions, but they almost abolished Ba2+ -induced contractions. 4In conclusion, the findings of the present study show that 1,8-cineole is a tracheal myorelaxant that acts preferentially on contractile responses elicited electromechanically. [source] Expression of stem cell pluripotency factors during regeneration in newtsDEVELOPMENTAL DYNAMICS, Issue 6 2009Nobuyasu Maki Abstract In this study, we present data indicating that mammalian stem cell pluripotency-inducing factors are expressed during lens and limb regeneration in newts. The apparent expression even in intact tissues and the ensued regulation during regeneration raises the possibility that these factors might regulate tissue-specific reprogramming and regeneration. Furthermore, these factors should enable us to understand the similarities and differences between animal regeneration in the newt and stem cell strategies in mammals. Developmental Dynamics 238:1613,1616, 2009. © 2009 Wiley-Liss, Inc. [source] Virtual biopsy of the joint tissues using near-infrared, reflectance confocal microscopy.MICROSCOPY RESEARCH AND TECHNIQUE, Issue 10 2006A pilot study Abstract Standard noninvasive imaging techniques applied to joints provide gross morphological features, insufficient for assessing histological detail. On the other hand, biopsying is invasive, time consuming, and may involve unwanted processing artifacts. Near-infrared reflectance confocal microscopy is a technique that allows serial, high-resolution optical sectioning through intact tissues without employing exogenous fluorescent stains. The aim of this work was to evaluate the potential utility of near-infrared reflectance confocal microscopy for providing immediate histological information on meniscus, articular cartilage, epiphyseal plate, bone, muscle, and tendon. Images from near-infrared reflectance confocal microscopy were compared with mirror routine histology sections. Characteristic architectural features were readily visualized in the three dimensions of space. Additionally, the use of experimental contrast agents highlighted the localization of nuclei. Limitations include penetration depth and minor optical artifacts. In conclusion, near-infrared reflectance confocal microscopy is a useful technique for immediate, nondestructive, serial "virtual" sectioning through intact tissues, being thus a potential adjunct to current imaging techniques in orthopedics. Microsc. Res. Tech., 2006. © 2006 Wiley-Liss, Inc. [source] Cannabinoid signalling in the enteric nervous systemNEUROGASTROENTEROLOGY & MOTILITY, Issue 9 2009J. J. Galligan Abstract, Cannabinoid signalling is an important mechanism of synaptic modulation in the nervous system. Endogenous cannabinoids (anandamide and 2-arachidonyl-glycerol) are synthesized and released via calcium-activated biosynthetic pathways. Exogenous cannabinoids and endocannabinoids act on CB1 and CB2 receptors. CB1 receptors are neuronal receptors which couple via G-proteins to inhibition of adenylate cyclase or to activation or inhibition of ion channels. CB2 receptors are expressed by immune cells and cannabinoids can suppress immune function. In the central nervous system, the endocannabinoids may function as retrograde signals released by the postsynaptic neuron to inhibit neurotransmitter release from presynaptic nerve terminals. Enteric neurons also express CB receptors. Exogenously applied CB receptor agonists inhibit enteric neuronal activity but it is not clear if endocannabinoids released by enteric neurons can produce similar responses in the enteric nervous system (ENS). In this issue of Neurogastroenterology and Motility, Boesmans et al. show that CB1 receptor activation on myenteric neurons maintained in primary culture can suppress neuronal activity, inhibit synaptic transmission and mitochondrial transport along axons. They also provide initial evidence that myenteric neurons (or other cell types present in the cultures) release endocannabinoids and which activate CB1 receptors constitutively. These data provide new information about targets for cannabinoid signalling in the ENS and highlight the potential importance of CB receptors as drug targets. It is necessary that future work extends these interesting findings to intact tissues and ideally to the in vivo setting. [source] | ||||||||||||||||