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Selected AbstractsMajor sites for the differentiation of V,14+ NKT cells inferred from the V-J junctional sequences of the invariant T-cell receptor ,,chainFEBS JOURNAL, Issue 1 2001Michio Shimamura CD1d-restricted mouse NK1.1+ TCR,,+ natural killer T (NKT) cells predominantly use an invariant TCR ,,chain encoded by V,14 and J,281 gene segments with a one-nucleotide N region. We found that NKT cells generated in the culture of fetal liver precursors possessed V,14-J,281 junctions that could be produced without the action of terminal deoxyribonucleotidyl transferase (TdT), indicating that NKT cells derived from fetal liver precursors are distinguishable from those from adult precursors with TdT expression. In fact, the frequency of the fetal-form sequences decreased with ageing. Surprisingly, the fetal-type sequences were predominantly observed in the lymphoid organs of athymic mice with the exception of bone marrow, where a sequence peculiar to the organ, with TdT-involved conversion from the invariant junction, was frequently present. These findings suggest that there are two independent sites of V,14+ NKT cell development, the hematopoietic organs throughout life (the developing liver and adult bone marrow) and, principally, the mature thymus. [source] Ageing, oxidative stress, and mitochondrial uncouplingACTA PHYSIOLOGICA, Issue 4 2004M.-E. Harper Abstract Mitochondria are a cell's single greatest source of reactive oxygen species. Reactive oxygen species are important for many life sustaining processes of cells and tissues, but they can also induce cell damage and death. If their production and levels within cells is not effectively controlled, then the detrimental effects of oxidative stress can accumulate. Oxidative stress is widely thought to underpin many ageing processes, and the oxidative stress theory of ageing is one of the most widely acknowledged theories of ageing. As well as being the major source of reactive oxygen species, mitochondria are also a major site of oxidative damage. The purpose of this review is a concise and current review of the effects of oxidative stress and ageing on mitochondrial function. Emphasis is placed upon the roles of mitochondrial proton leak, the uncoupling proteins, and the anti-ageing effects of caloric restriction. [source] Src-dependent phosphorylation of Scar1 promotes its association with the Arp2/3 complexCYTOSKELETON, Issue 1 2006Hazel Ardern Abstract The WAVE/Scar proteins regulate actin polymerisation at the leading edge of motile cells via activation of the Arp2/3 complex in response to extracellular cues. Within cells they form part of a pentameric complex that is thought to regulate their ability to interact and activate the Arp2/3 complex. However, the exact mechanism for this is not known. We set out to assess whether phosphorylation of Scar1 by the non-receptor tyrosine kinase Src may influence the function of Scar1 and its ability to regulate Arp2/3-mediated actin polymerisation. We show that Scar1 is phosphorylated by Src in vitro and in vivo and identify tyrosine 125 as the major site in Scar1 to be phosphorylated in cells. Src-dependent phosphorylation of Scar1 on tyrosine 125 enhances its ability to bind to the Arp2/3 complex and regulates its ability to control actin polymerisation in cells. Thus, Src may act as an intermediary to regulate the activity of the Arp2/3 complex in response to external stimuli, via modulation of its interaction with WAVE/Scar proteins. Cell Motil. Cytoskeleton, 2006. © 2005 Wiley-Liss, Inc. [source] Morphine modulation of temporomandibular joint-responsive units in superficial laminae at the spinomedullary junction in female rats depends on estrogen statusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2008A. Tashiro Abstract The influence of analgesic agents on neurons activated by stimulation of the temporomandibular joint (TMJ) region is not well defined. The spinomedullary junction [trigeminal subnucleus caudalis (Vc)/C1,2] is a major site of termination for TMJ sensory afferents. To determine whether estrogen status influences opioid-induced modulation of TMJ units, the classical opioid analgesic, morphine, was given to ovariectomized (OvX) rats and OvX rats treated for 2 days with low-dose (LE2) or high-dose (HE2) 17,-estradiol-3-benzoate. Under thiopental anesthesia, TMJ units in superficial and deep laminae at the Vc/C1,2 junction were activated by injection of ATP (1 mm) directly into the joint space. In superficial laminae, morphine inhibited evoked activity in units from OvX and LE2 rats in a dose-related and naloxone-reversible manner, whereas units from HE2 rats were not inhibited. By contrast, in deep laminae, morphine reduced TMJ-evoked unit activity similarly in all groups. Morphine reduced the background activity of units in superficial and deep laminae and resting arterial pressure similarly in all groups. Morphine applied to the dorsal surface of the Vc/C1,2 junction inhibited all units independently of E2 treatment. Quantitative polymerase chain reaction and immunoblots revealed a similar level of expression for ,-opioid receptors at the Vc/C1,2 junction in LE2 and HE2 rats. These results indicated that estrogen status differentially affected morphine modulation of TMJ unit activity in superficial, but not deep, laminae at the Vc/C1,2 junction in female rats. The site(s) for estrogen influence on morphine-induced modulation of TMJ unit activity was probably outside the medullary dorsal horn. [source] Evidence for vesicular glutamate transporter synapses onto gonadotropin-releasing hormone and other neurons in the rat medial preoptic areaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2003J. Kiss Abstract The medial preoptic area is a key structure in the control of reproduction. Several data suggest that excitatory amino acids are involved in the regulation of this function and the major site of this action is the medial preoptic region. Data concerning the neuromorphology of the glutamatergic innervation of the medial preoptic area are fragmentary. The present investigations were focused on: (i) the morphology of the vesicular glutamate transporter 1 (VGluT1)- and vesicular glutamate transporter 2 (VGluT2)-immunoreactive nerve terminals, which are considered to be specific to presumed glutamatergic neuronal elements, in the medial preoptic area of rat; and (ii) the relationship between these glutamate transporter-positive endings and the gonadotropin-releasing hormone (GnRH) neurons in the region. Single- and double-label immunocytochemistry was used at the light and electron microscopic level. There was a weak to moderate density of VGluT1- and a moderate to intense density of VGluT2-immunoreactive elements in the medial preoptic area. Electron microscopy revealed that both VGluT1- and VGluT2-immunoreactive boutons made asymmetric type synaptic contacts with unlabelled neurons. VGluT2-labelled, but not VGluT1-labelled, axon terminals established asymmetric synaptic contacts on GnRH-immunostained neurons, mainly on their dendrites. The present findings are the first electron microscopic examinations on the glutamatergic innervation of the rat medial preoptic area. They provide direct neuromorphological evidence for the existence of direct glutamatergic innervation of GnRH and other neurons in the rat medial preoptic area. [source] A New Class of Enehydroxylamino Ketones , (R)-2-(1-Hydroxy-4,4,5,5-tetraalkylimidazolidin-2-ylidene)ethanones: Synthesis and ReactionsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 4 2004Vladimir A. Reznikov Abstract Three approaches to the synthesis of (R)-2-(1-hydroxy-4,4,5,5-tetraalkylimidazolidin-2-ylidene)ethanones 1 are described: (a) condensation of 1,2-bishydroxylamines with ,-ketoaldehyde synthons, (b) treatment of metallated 1-hydroxy-2-methyl-4,5-dihydroimidazoles with esters, and (c) 1,3-dipolar cycloaddition between 1-hydroxy-4,5-dihydroimidazole-3-oxide and DMAD. The reactivity of 1 with electrophiles has been studied. The exocyclic methylene (enamine) carbon atom is shown to be the major site of electrophilic attack. Synthesized chloro-substituted 1-hydroxy-2-acetylideneimidazolidines react with sodium cyanide to form the corresponding nitriles. Oxidation of these nitriles occurs with formation of persistent vinyl nitroxides, which are of interest as potential paramagnetic ligands. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004) [source] Lipopolyamine treatment increases the efficacy of intoxication with saporin and an anticancer saporin conjugateFEBS JOURNAL, Issue 18 2007Sandra E. Geden Saporin is a type I ribosome-inactivating protein that is often appended with a cell-binding domain to specifically target and kill cancer cells. Urokinase plasminogen activator (uPA)-saporin, for example, is an anticancer toxin that consists of a chemical conjugate between the human uPA and native saporin. Both saporin and uPA-saporin enter the target cell by endocytosis and must then escape the endomembrane system to reach the cytosolic ribosomes. The latter process may represent a rate-limiting step for intoxication and would therefore directly affect toxin potency. In the present study, we document two treatments (shock with dimethylsulfoxide and lipopolyamine coadministration) that generate substantial cellular sensitization to saporin/uPA-saporin. With the use of lysosome-endosome X (LEX)1 and LEX2 mutant cell lines, an endosomal trafficking step preceding cargo delivery to the late endosomes was identified as a major site for the dimethylsulfoxide-facilitated entry of saporin into the cytosol. Dimethylsulfoxide and lipopolyamines are known to disrupt the integrity of endosome membranes, so these reagents could facilitate the rapid movement of toxin from permeabilized endosomes to the cytosol. However, the same pattern of toxin sensitization was not observed for dimethylsulfoxide- or lipopolyamine-treated cells exposed to diphtheria toxin, ricin, or the catalytic A chain of ricin. The sensitization effects were thus specific for saporin, suggesting a novel mechanism of saporin translocation by endosome disruption. Lipopolyamines have been developed as in vivo gene therapy vectors; thus, lipopolyamine coadministration with uPA-saporin or other saporin conjugates could represent a new approach for anticancer toxin treatments. [source] Molecular cloning of the Matrix Gla Protein gene from Xenopus laevisFEBS JOURNAL, Issue 7 2002Functional analysis of the promoter identifies a calcium sensitive region required for basal activity To analyze the regulation of Matrix Gla Protein (MGP) gene expression in Xenopus laevis, we cloned the xMGP gene and its 5, region, determined their molecular organization, and characterized the transcriptional properties of the core promoter. The Xenopus MGP (xMGP) gene is organized into five exons, one more as its mammalian counterparts. The first two exons in the Xenopus gene encode the DNA sequence that corresponds to the first exon in mammals whereas the last three exons show homologous organization in the Xenopus MGP gene and in the mammalian orthologs. We characterized the transcriptional regulation of the xMGP gene in transient transfections using Xenopus A6 cells. In our assay system the identified promoter was shown to be transcriptionally active, resulting in a 12-fold induction of reporter gene expression. Deletional analysis of the 5, end of the xMGP promoter revealed a minimal activating element in the sequence from ,70 to ,36 bp. Synthetic reporter constructs containing three copies of the defined regulatory element delivered 400-fold superactivation, demonstrating its potential for the recruitment of transcriptional activators. In gel mobility shift assays we demonstrate binding of X. laevis nuclear factors to an extended regulatory element from ,180 to ,36, the specificity of the interaction was proven in competition experiments using different fragments of the xMGP promoter. By this approach the major site of factor binding was demonstrated to be included in the minimal activating promoter fragment from ,70 to ,36 bp. In addition, in transient transfection experiments we could show that this element mediates calcium dependent transcription and increasing concentrations of extracellular calcium lead to a significant dose dependent activation of reporter gene expression. [source] Non-receptor tyrosine kinase CSK-1 controls pharyngeal muscle organization in Caenorhabditis elegansGENES TO CELLS, Issue 3 2009Nozomu Takata C-terminal Src kinase (Csk) is a non-receptor type of tyrosine kinase, and serves as an essential negative regulator of Src family tyrosine kinases (SFKs) in vertebrates. However, analyses of Csk and SFKs from primitive animals suggest that the Csk-mediated mechanisms regulating SFK activity might diverge between evolutional branches, different tissues or SFK family members. We examined in vivo roles of CSK-1, a Caenorhabditis elegans orthologue of Csk, by generating animals lacking csk-1 function. Although some csk-1 mutants died during embryogenesis, the majority of mutants died during the first stage of larval development. In csk-1 mutants, the function of pharyngeal muscles, the major site of CSK-1 expression, was severely damaged. The pumping of pharyngeal grinder cells became arrhythmic, causing disabled feeding. Electron microscopy showed that pharyngeal muscle filaments were disorientated in the csk-1 mutants. These indicate that CSK-1 is crucial for proper organization of pharyngeal muscles. However, the growth arrest phenotype in csk-1 mutants could not be suppressed by src-1 and/or src-2 mutation, and SRC-1 was not significantly activated in the csk-1 mutants. These results suggest that CSK-1 has an essential function in organization of pharyngeal muscle filaments that does not require C. elegans SFKs. [source] Natural killer cells in infection and inflammation of the lungIMMUNOLOGY, Issue 2 2009Fiona J. Culley Summary The lungs are a major site of entry of pathogens into the body and thus require rapid and effective innate responses to prevent pathogens establishing infection and to limit their spread. Additionally, the immune response in the lung must be tightly regulated such that pathogens are cleared, but immunopathology and chronic inflammation are prevented. In this review, I consider the role of natural killer (NK) cells in pulmonary infection and inflammation, specifically their contributions to influenza, tuberculosis, asthma and chronic obstructive pulmonary disease (COPD), which are major causes of morbidity and mortality world-wide. Despite evidence of the importance of NK cells in these diseases, there are still major gaps in our understanding of how their function is regulated in this unique tissue environment. Understanding how different beneficial and detrimental effector functions of NK cells are triggered will be crucial if NK cells are to be exploited therapeutically in respiratory disease. [source] Skeletal muscle glucose uptake during exercise: A focus on reactive oxygen species and nitric oxide signalingIUBMB LIFE, Issue 5 2009Troy L. Merry Abstract Like insulin, muscle contraction (in vitro or in situ) and exercise increase glucose uptake into skeletal muscle. However, the contraction/exercise pathway of glucose uptake in skeletal muscle is an independent pathway to that of insulin. Indeed, skeletal muscle glucose uptake is normal during exercise in those who suffer from insulin resistance and diabetes. Thus, the pathway of contraction-mediated glucose uptake into skeletal muscle provides an attractive potential target for pharmaceutical treatment and prevention of such conditions, especially as skeletal muscle is the major site of impaired glucose disposal in insulin resistance. The mechanisms regulating skeletal muscle glucose uptake during contraction have not been fully elucidated. Potential regulators include Ca2+ (via CaMK's and/or CaMKK), AMPK, ROS, and NO signaling, with some redundancy likely to be evident within the system. In this review, we attempt to briefly synthesize current evidence regarding the potential mechanisms involved in regulating skeletal muscle glucose uptake during contraction, focusing on ROS and NO signaling. While reading this review, it will become clear that this is an evolving field of research and that much more work is required to elucidate the mechanism(s) regulating skeletal muscle glucose uptake during contraction. © 2009 IUBMB IUBMB Life 61(5): 479,484, 2009 [source] Nitric Oxide: The "Second Messenger" of InsulinIUBMB LIFE, Issue 5 2000Nighat N. Kahn Abstract Incubation of various tissues, including heart, liver, kidney, muscle, and intestine from mice and erythrocytes or their membrane fractions from humans, with physiologic concentration of insulin resulted in the activation of a membrane-bound nitric oxide synthase (NOS). Activation of NOS and synthesis of NO were stimulated by the binding of insulin to specific receptors on the cell surface. A Lineweaver-Burk plot of the enzymatic activity demonstrated that the stimulation of NOS by insulin was related to the decrease in the Km for L-arginine, the substrate for NOS, with a simultaneous increase of Vmax. Addition of NG-nitro-L-arginine methyl ester (LNAME), a competitive inhibitor of NOS, to the reaction mixture completely inhibited the hormone-stimulated NO synthesis in all tissues. Furthermore, NO had an insulin-like effect in stimulating glucose transport and glucose oxidation in muscle, a major site for insulin action. Addition of NAME to the reaction mixture completely blocked the stimulatory effect of insulin by inhibiting both NO production and glucose metabolism, without affecting the hormone-stimulated tyrosine or phosphatidylinositol 3-kinases of the membrane preparation. Injection of NO in alloxan-induced diabetic mice mimicked the effect of insulin in the control of hyperglycemia (i.e., lowered the glucose content in plasma). However, injection of NAME before the administration of insulin to diabetic-induced and nondiabetic mice inhibited not only the insulin-stimulated increase of NO in plasma but also the glucose-lowering effect of insulin. [source] The development and evolution of the pharyngeal archesJOURNAL OF ANATOMY, Issue 1-2 2001ANTHONY GRAHAM abstract A muscularised pharynx, with skeletal support, serving the dual functions of feeding and respiration, is a fundamental vertebrate characteristic. Embryologically, the pharyngeal apparatus has its origin in a series of bulges that form on the lateral surface of the embryonic head, the pharyngeal arches, whose development is complex. These structures are composed of a number of disparate embryonic cell types: ectoderm, endoderm, neural crest and mesoderm, whose development must be coordinated to generate the functional adult apparatus. In the past, most studies have emphasised the role played by the neural crest, which generates the skeletal elements of the arches, in directing pharyngeal arch development, but it has also become apparent that the endoderm plays a prominent role in directing arch development. Neural crest cells are not required for arch formation, their regionalisation nor to some extent their sense of identity. Furthermore, the endoderm is the major site of expression of a number of important signalling molecules, and this tissue has been shown to be responsible for promoting the formation of particular components of the arches. Thus vertebrate pharyngeal morphogenesis can now be seen to be a more complex process than was previously believed, and must result from an integration of both neural crest and endodermal patterning mechanisms. Interestingly, this also mirrors the fact that the evolutionary origin of pharyngeal segmentation predates that of the neural crest, which is an exclusively vertebrate characteristic. As such, the evolution of the vertebrate pharynx is also likely to have resulted from an integration between these 2 patterning systems. Alterations in the interplay between neural crest and endodermal patterning are also likely to be responsible for the evolutionary that occurred to the pharyngeal region during subsequent vertebrate evolution. [source] GABAA receptor associated proteins: a key factor regulating GABAA receptor functionJOURNAL OF NEUROCHEMISTRY, Issue 2 2007Zi-Wei Chen Abstract ,-Aminobutyric acid (GABA), an important inhibitory neurotransmitter in both vertebrates and invertebrates, acts on GABA receptors that are ubiquitously expressed in the CNS. GABAA receptors also represent a major site of action of clinically relevant drugs, such as benzodiazepines, barbiturates, ethanol, and general anesthetics. It has been shown that the intracellular M3-M4 loop of GABAA receptors plays an important role in regulating GABAA receptor function. Therefore, studies of the function of receptor intracellular loop associated proteins become important for understanding mechanisms of regulating receptor activity. Recently, several labs have used the yeast two-hybrid assay to identify proteins interacting with GABAA receptors, for example, the interaction of GABAA receptor associated protein (GABARAP) and Golgi-specific DHHC zinc finger protein (GODZ) with , subunits, PRIP, phospholipase C-related, catalytically inactive proteins (PRIP-1) and (PRIP-2) with GABARAP and receptor ,2 and , subunits, Plic-1 with some , and , subunits, radixin with the ,5 subunit, HAP1 with the ,1 subunit, GABAA receptor interacting factor-1 (GRIF-1) with the ,2 subunit, and brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) with the ,3 subunit. These proteins have been shown to play important roles in modulating the activities of GABAA receptors ranging from enhancing trafficking, to stabilizing surface and internalized receptors, to regulating modification of GABAA receptors. This article reviews the current studies of GABAA receptor intracellular loop-associated proteins. [source] Stereospecific reduction of the original anticancer drug oracin in rat extrahepatic tissuesJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 7 2003Barbora Szotáková ABSTRACT The liver is the major site of drug metabolism in the body. However, many drugs undergo metabolism in extrahepatic sites and in the gut wall and lumen. In this study, the distribution and activity of reductases in rat that reduced potential cytostatic oracin to its principal metabolite 11-dihydrooracin (DHO) were investigated. The extension and stereospecificity of oracin reduction to DHO were tested in microsomal and cytosolic fractions from the liver, kidney, heart, lung and wall of small intestine, caecum and large intestine. Intestinal bacterial reduction of oracin was studied as well. The amount of DHO enantiomers was measured by HPLC with Chiralcel OD-R as chiral column. Reductive biotransformation of oracin was mostly stereospecific for (+)-DHO, but the enantiomeric ratio differed significantly among individual tissues and subcellular fractions (from 56% (+)-DHO in heart microsomes to 92% (+)-DHO in liver cytosol). Stereospecificity for (-)-DHO (60%) was observed in bacterial oracin reduction in the lumen of small intestine, caecum and large intestine. Shift of the (+)-DHO/(-)-DHO enantiomeric ratio from 90:10 (in liver subcellular fractions) to 60:40 (in-vivo) clearly demonstrated the importance of the contribution of extrahepatic metabolism to the total biotransformation of oracin to DHO. [source] Nicotine Decreases Blood Alcohol Concentrations in Adult Rats: A Phenomenon Potentially Related to Gastric FunctionALCOHOLISM, Issue 8 2006Scott E. Parnell Background: In spite of the fact that drinking and smoking often occur together, little is known about the pharmacokinetic interaction between alcohol and nicotine. Previous research in neonatal rats demonstrated that nicotine reduces blood alcohol concentrations (BACs) if alcohol and nicotine are administered simultaneously. However, it is unclear whether such a phenomenon can be observed in adult subjects, given the fact that there is an ontogenetic difference in alcohol metabolism. Methods: A range of nicotine doses (0, 0.25, 0.5, 1, 2, 4, and 6 mg/kg) were administered individually with an alcohol dose (4 g/kg) via intragastric (IG) intubation to adult female rats, and the resultant BACs were measured at various time points following drug administration. Furthermore, the hypothesis that nicotine's role in reducing BACs is mediated through factors related to gastric function was examined by comparing the resultant BACs after an IG intubation or intraperitoneal (IP) injection of alcohol. Results: The results from this study showed significant nicotine dose,related decreases in BACs with 0.5, 1, 2, 4, and 6 mg/kg doses of nicotine at the various time points assessed. This effect, however, occurred only when alcohol was administered via IG intubation, but not after an IP injection of alcohol. Conclusions: These results suggest that the nicotine-induced decrease in BAC may be related to gastric function. One possible explanation was related to nicotine's action in delaying gastric emptying. The longer the alcohol was retained in the stomach, the more likely that the alcohol would be metabolized by gastric alcohol dehydrogenase before its absorption into the bloodstream by the small intestine (the major site of alcohol absorption). [source] EGF-induced EGF-receptor and MAP kinase phosphorylation in goat cumulus cells during in vitro maturationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2005Laurence Gall Abstract EGF has been shown to influence meiotic maturation and development competence of oocyte in various mammalian species. We previously reported, in goat, that the EGF receptor (EGF-R) was present both on cumulus cells and oocytes. Here, EGF-induced signaling was investigated during the in vitro maturation process in goat cumulus,oocyte complexes (COCs). Cumulus cells and oocytes were subjected to Western immunoblotting analysis using anti-MAP kinase, anti-phosphotyrosine, anti-phospho MAP kinase, and anti-phospho EGF-R antibodies. We demonstrated that treatment with EGF during the in vitro maturation process induced rapid tyrosine phosphorylation of EGF-R in a time and concentration dependent manner in cumulus cells. A similar pattern of activation by phosphorylation was observed for MAP kinase upon EGF stimulation. AG 1478, an inhibitor of the EGF kinase, suppressed EGF-stimulated phosphorylation of EGF-R and also affected the MAP kinase activation. Treatment with the MEK inhibitor PD 98059 abolished EGF-induced MAP kinase activation. We did not observe oocyte EGF-R phosphorylation in our experiments during the in vitro maturation process. Our data indicate, in goat cumulus cells, that activation of EGF-R by EGF triggers signaling through the MAP kinase pathway during in vitro maturation. This supports the hypothesis that the major site of action for EGF, that regulates oocyte maturation, is the cumulus cell. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] Lentivirus vector-mediated gene transfer to the developing bronchiolar airway epithelium in the fetal lambTHE JOURNAL OF GENE MEDICINE, Issue 6 2007Ze-Yan Yu Abstract Background Development of effective and durable gene therapy for treatment of the respiratory manifestations of cystic fibrosis remains a formidable challenge. Obstacles include difficulty in achieving efficient gene transfer to mature airway epithelium and the need to stably transduce self-renewing epithelial progenitor cells in order to avoid loss of transgene expression through epithelial turnover. Targeting the developing airway epithelium during fetal life offers the prospect of circumventing these challenges. Methods In the current study we investigated vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped HIV-1-derived lentivirus vector-mediated gene transfer to the airway epithelium of mid-gestation fetal lambs, both in vitro and in vivo. In the in vitro studies epithelial sheet explants and lung organ culture were used to examine transduction of the proximal and more distal airway epithelium, respectively. For the in vivo studies, vector was delivered directly into the proximal airway. Results We found that even during the early pseudoglandular and canalicular phases of lung development, occurring through mid-gestation, the proximal bronchial airway epithelium was relatively mature and highly resistant to lentivirus-mediated transduction. In contrast, the more distal bronchiolar airway epithelium was relatively permissive for transduction although the absolute levels achieved remained low. Conclusion This result is promising as the bronchiolar airway epithelium is a major site of pathology in the cystic fibrosis airway, and much higher levels of transduction are likely to be achieved by developing strategies that increase the amount of vector reaching the more distal airway after intratracheal delivery. Copyright © 2007 John Wiley & Sons, Ltd. [source] Glutamine metabolism: Role in acid-base balance,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 5 2004Lynn Taylor Abstract The intent of this review is to provide a broad overview of the interorgan metabolism of glutamine and to discuss in more detail its role in acid-base balance. Muscle, adipose tissue, and the lungs are the primary sites of glutamine synthesis and release. During normal acid-base balance, the small intestine and the liver are the major sites of glutamine utilization. The periportal hepatocytes catabolize glutamine and convert ammonium and bicarbonate ions to urea. In contrast, the perivenous hepatocytes are capable of synthesizing glutamine. During metabolic acidosis, the kidney becomes the major site of glutamine extraction and catabolism. This process generates ammonium ions that are excreted in the urine to facilitate the excretion of acids and bicarbonate ions that are transported to the blood to partially compensate the acidosis. The increased renal extraction of glutamine is balanced by an increased release from muscle and liver and by a decreased utilization in the intestine. During chronic acidosis, this adaptation is sustained, in part, by increased renal expression of genes that encode various transport proteins and key enzymes of glutamine metabolism. The increased levels of phosphoenolpyruvate carboxykinase result from increased transcription, while the increase in glutaminase and glutamate dehydrogenase activities result from stabilization of their respective mRNAs. Where feasible, this review draws upon data obtained from studies in humans. Studies conducted in model animals are discussed where available data from humans is either lacking or not firmly established. Because there are quantitative differences in tissue utilization and synthesis of glutamine in different mammals, the review will focus more on common principles than on quantification. [source] Positron emission tomography of [18F]-big endothelin-1 reveals renal excretion but tissue-specific conversion to [18F]-endothelin-1 in lung and liverBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2010Peter Johnström Background and purpose:, Big endothelin-1 (ET-1) circulates in plasma but does not bind to ET receptors until converted to ET-1 by smooth muscle converting enzymes. We hypothesized that tissue-specific conversion of [18F]-big ET-1 to [18F]-ET-1 could be imaged dynamically in vivo within target organs as binding to ET receptors. Methods:, [18F]-big ET-1 conversion imaged in vivo following infusion into rats using positron emission tomography (PET). Key results:, [18F]-big ET-1 was rapidly cleared from the circulation (t1/2= 2.9 ± 0.1 min). Whole body microPET images showed highest uptake of radioactivity in three major organs. In lungs and liver, time activity curves peaked within 2.5 min, then plateaued reaching equilibrium after 10 min, with no further decrease after 120 min. Phosphoramidon did not alter half life of [18F]-big ET-1 but uptake was reduced in lung (42%) and liver (45%) after 120 min, consistent with inhibition of enzyme conversion and reduction of ET-1 receptor binding. The ETA antagonist, FR139317 did not alter half-life of [18F]-big ET-1 (t1/2= 2.5 min) but radioactivity was reduced in all tissues except for kidney consistent with reduction in binding to ETA receptors. In kidney, however, the peak in radioactivity was higher but time to maximum accumulation was slower (,30 min), which was increased by phosphoramidon, reflecting renal excretion with low conversion and binding to ET receptors. Conclusions and implications:, A major site for conversion was within the vasculature of the lung and liver, whereas uptake in kidney was more complex, reflecting excretion of [18F]-big ET-1 without conversion to ET-1. This article is part of a themed section on Imaging in Pharmacology. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00685.x [source] The role of mitochondria in ageing and carcinogenesisCLINICAL & EXPERIMENTAL DERMATOLOGY, Issue 4 2006M. A. Birch-Machin Summary Mitochondria can perform multiple cellular functions including energy production, cell proliferation and apoptosis. These organelles contain their own genetic material, mitochondrial DNA (mtDNA), which is maternally inherited. Although much smaller than the nuclear genome, mtDNA is equally important, as it has been hypothesized to play a crucial role in ageing and carcinogenesis. This is partly due to the fact that mitochondria represent the major site for the generation of cellular oxidative stress and play a key role in mediating programmed cell death (apoptosis). Damage to mtDNA is therefore an important contributor to human ageing, cancer and neurodegenerative diseases. The most relevant footprints of mtDNA damage are point mutations of single bases, or deletions of the 16.5-kb mitochondrial genome. This review will focus on the key roles of mitochondrial function and mtDNA in oxidative stress production and as a mediator of apoptosis, and on the use of mtDNA as a biomarker of sun exposure. This will be related to the contribution of mitochondria and mtDNA in the ageing process and cancer, with a specific focus on human skin. In conclusion, it is likely that the interplay between nuclear and mitochondrial genes may hold the final understanding of the mitochondrial role in these disease processes. [source] The FRAX tool in French women: How well does it describe the real incidence of fracture in the OFELY cohortJOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2010Elisabeth Sornay-Rendu Abstract The FRAX tool estimates an individual's fracture probability over 10 years from clinical risk factors with or without bone mineral density (BMD) measurement. The aim of our study was to compare the predicted fracture probabilities and the observed incidence of fracture in French women during a 10-year follow-up. The probabilities of fracture at four major sites (hip, clinical spine, shoulder, or wrist) and at the hip were calculated with the FRAX tool in 867 women aged 40 years and over from the Os des Femmes de Lyon (OFELY) cohort. The incidence of fracture was observed over 10 years. Thus 82 women sustained 95 incident major osteoporotic (OP) fractures including 17 fractures at the hip. In women aged at least 65 years (n,=,229), the 10-year predicted probabilities of fracture with BMD were 13% for major OP fractures and 5% for hip fractures, contrasting with 3.6% and 0.5% in women younger than 65 years (p,<,.0001). The predicted probabilities of both major OP and hip fractures were significantly higher in women with osteoporosis (n,=,77, 18% and 10%) and osteopenia (n= 390, 6% and 2%) compared with women with normal BMD (n,=,208, 3% and <1%; p,<,.0001. The predicted probabilities of fracture were two and five times higher in women who sustained an incident major OP fracture and a hip fracture compared with women who did not (p,<,.0001). Nevertheless, among women aged at least 65 years with low BMD values (T -score , ,1; n,=,199), the 10-year predicted probability of major OP fracture with BMD was 48% lower than the observed incidence of fractures (p,<,.01). A 10-year probability of major OP fracture higher than 12% identified more women with incident fractures than did BMD in the osteoporotic range (p,<,.05). In French women from the OFELY cohort, the observed incidence of fragility fractures over 10 years increased with age following a pattern similar to the predicted probabilities given by the FRAX tool. However, in women aged at least 65 years with low BMD, the observed incidence of fractures was substantially higher than the predicted probability. © 2010 American Society for Bone and Mineral Research. [source] Physicochemical, Nutritional, and Functional Characterization of Fruits Xoconostle (Opuntia matudae) Pears from Central-México RegionJOURNAL OF FOOD SCIENCE, Issue 6 2010Salvador H. Guzmán-Maldonado Abstract:, Xoconostle cv. Cuaresmeño (Opuntia matudae) has attracted domestic and international industry attention; however, variations of composition from xoconostle structures have not been evaluated. Industries discard the pulp (endocarp) and peel (pericarp) as wastes and utilize the skin (mesocarp), which is the edible portion. The physicochemical, nutritional, and functional characterization of structures from xoconostle pear from 3 major sites of production in Mexico were assessed. Skin yield ranged from 58% to 64% and was higher to that of peel (22% to 24%) and pulp (12% to 18%) yields. pH, °Brix, and acidity were similar among xoconostle structures. Total fiber showed by peel (18.23% to 20.37%) was 2-fold higher than that of skin. Protein and ether extract were higher in xoconostle pulp compared to that showed by peel and skin. Iron content of xoconostle peel (6 to 9.6 mg/100 g, DWB) was higher to that of skin and pulp and prickly pear pulp. Soluble phenols of peel (840 to 863 mg GAE/100 g, DWB) were almost similar to that of skin (919 to 986 mg GAE/100 g, dry weigh basis); meanwhile, ascorbic acid concentration of skin was 2-fold higher compared to that of peel. The phenolic fraction of xoconostle structures consisted of gallic, vanillic, and 4-hydroxybenzoic acids; catechin, epicatechin, and vanillin were also identified by high-performance liquid chromatography,didoe array detection (HPLC-DAD). Xoconostle peel showed higher antioxidant activity (TEAC) compared to that of skin (2-fold) and pulp (6-fold) of commonly consumed fruits and vegetables. The potential of xoconostle peel and pulp for the production of feed or food is promissory. Practical Application:, Outstanding nutritional and functional properties of xoconostle cv. Cuaresmeño fruits are demonstrated. Increased consumption could contribute positively to improve the diet of rural and urban consumers. The high fiber, mineral, and antioxidant components of xoconostle peel and pulp suggest that these fruit structures, which are currently discarded as waste, have promissory use as feed or food by industry. [source] Contribution of ROCK in contraction of trabecular meshwork: Proposed mechanism for regulating aqueous outflow in monkey and human eyesJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 4 2005Emi Nakajima Abstract Aqueous outflow in the conventional outflow pathway is regulated by the contraction and relaxation of the ciliary muscle (CM) and the trabecular meshwork (TM). Rho-associated coiled coil-forming protein kinase (ROCK) is thought to regulate actomyosin-based contractility in many types of cells by phosphorylation of ROCK substrates. In animal models, ROCK inhibitor Y-39983 relaxed CM and TM and decreased intraocular pressure (IOP). Thus, ROCK is implicated in the regulation of aqueous outflow and IOP. However, the site of action of ROCK in monkey and man is unknown. In the present communication, RT-PCR analysis of monkey tissues showed higher levels of mRNAs for ROCK and ROCK substrates in TM compared to CM. Human TM also showed higher levels of mRNAs for ROCK and ROCK substrates compared to CM. Differences between TM and CM in human were not as high as in monkey. ROCK inhibitor Y-39983 led to a dose-dependent relaxation of carbachol-induced, contracted TM from monkey. In contrast, Y-39983 was only slightly effective in relaxing CM. Our results suggested that TM was one of the major sites for regulating IOP by ROCK. ROCK inhibitor Y-39983 might be a candidate drug for lowering IOP by increasing conventional outflow and producing fewer side effects on accommodation and miosis. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:701,708, 2005 [source] Structure, function, and regulation of renal organic anion transportersMEDICINAL RESEARCH REVIEWS, Issue 6 2002Guofeng You Abstract Renal elimination of anionic drugs, xenobiotics, and toxins is necessary for the survival of mammalian species. This process is mediated by vectorial transport from blood to urine through the cooperative functions of specific transporters in the basolateral and apical membranes of the proximal tubule epithelium. The first step of this process is the extraction of organic anions from the peritubular blood plasma into proximal tubule cells largely through the organic anion transporter (OAT) pathway. Therefore, the OAT pathway is one of the major sites for body drug clearance/detoxification. As a result, it is also the site for drug,drug interaction and drug-induced nephrotoxicity. To maximize therapeutic efficacy and minimize toxicity, the structure-function relationships of OATs and their regulation must be defined. The recent cloning and identification of OATs have paved the way for such investigations. This review summarizes the available data on the general properties of OATs, focusing in particular on the recent progress made from the author's laboratory as well as from other's, on the molecular characterization of the structure-function relationships of OATs and their regulatory mechanisms. © 2002 Wiley Periodicals, Inc. Med Res Rev, 22, No. 6, 602,616, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/med.10019 [source] High-throughput analysis in drug discovery: application of liquid chromatography/ion-trap mass spectrometry for simultaneous cassette analysis of ,-1a antagonists and their metabolites in mouse plasmaRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 8 2001Zongwei Cai The application of liquid chromatography/ion-trap mass spectrometry for simultaneous quantification of multiple drugs and detection of their metabolites for in vitro experiments was reported recently. In the current study, the use of these techniques was extended to in vivo pharmacokinetic (PK) studies of ,-1a antagonists. In combination with limited time-point PK, greatly increased throughput was demonstrated for the in vivo screening and investigation of in vivo,in vitro correlation. In addition to quantitative analyses, the technique allowed simultaneous detection of major in vivo metabolites without having to reanalyze the plasma samples. The drugs were individually dosed in mice intravenously via tail vein injection and the blood samples were collected 5,min and 2,h after dosing. After the plasma samples for the different drugs had been prepared separately, they were pooled for cassette analysis. The concentrations of five test compounds in the plasma samples at 2,h ranged from 36,1062,ng/mL, whereas their 5-min plasma levels were similar. From the same cassette analysis, major metabolites in the samples were also detected simultaneously through the interpretation of full-scan mass spectra. The metabolite identification confirmed the results from a previous report that the major sites of metabolism are hydroxylation of the phenyl ring not bearing the alkylsulfonamide substitutent, piperidine N-dealkylation, and N-demethylation of the alkylsulfonamide group. Copyright © 2001 John Wiley & Sons, Ltd. [source] Determination of zinc incorporation in the Zn-substituted gallophosphate ZnULM-5 by multiple wavelength anomalous dispersion techniquesACTA CRYSTALLOGRAPHICA SECTION B, Issue 3 2010M. Helliwell The location of isomorphously substituted zinc over eight crystallographically different gallium sites has been determined in a single-crystal study of the gallophosphate ZnULM-5, GaZn(PO4)14(HPO4)2(OH)2F7, [H3N{CH2}6NH3]4, 6H2O, in an 11 wavelength experiment, using data from Station 9.8, SRS Daresbury. The measurement of datasets around the K edges of both Ga and Zn, as well as two reference datasets away from each absorption edge, was utilized to selectively exploit dispersive differences of each metal atom type in turn, which allowed the major sites of Zn incorporation to be identified as the metal 1 and 3 sites, M1 and M3. The preferential substitution of Zn at these sites probably arises because they are located in double four-ring (D4R) building units which can relax to accommodate the incorporation of hetero atoms. As the crystal is non-centrosymmetric, with space group P21212, it was also possible to use anomalous differences to corroborate the results obtained from the dispersive differences. These results were obtained firstly from difference Fourier maps, calculated using a phase set from the refined structure from data measured at the Zr K edge. Also, refined dispersive and anomalous occupancies, on an absolute scale, could be obtained using the program MLPHARE, allowing estimates for the Zn incorporation of approximately 22 and 18 at. % at the M1 and M3 sites to be obtained. In addition, f, and f,, values for Ga and Zn at each wavelength could be estimated both from MLPHARE results, and by refinement in JANA2006. The fully quantitative determinations of the dispersive and anomalous coefficients for Ga and Zn at each wavelength, as well as metal atom occupancies over the eight metal atom sites made use of the CCP4's MLPHARE program as well as SHELXL and JANA2006. The results by these methods agree closely, and JANA2006 allowed the ready determination of standard uncertainties on the occupancy parameters, which were for M1 and M3, 20.6,(3) and 17.2,(3),at %, respectively. [source] Immunohistolocalization and Gene Expression of the Secretory Carbonic Anhydrase Isozymes (CA-VI) in Canine Oral Mucosa, Salivary Glands and OesophagusANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 1 2007T. Kasuya Summary The immunohistolocalization of secretory carbonic anhydrase isoenzymes (CA-VI) in canine salivary glands, parotid, submandibular, sublingual and zygomatic glands, oral and oesophageal mucosa was studied using a specific antiserum against a canine CA-VI. In addition, the gene expression of CA-VI from the same tissue was studied using a real-time reverse-transcriptase polymerase chain reaction. In all salivary glands and oesophageal gland, immunostaining intensely localized CA-VI antiserum throughout the cytoplasm of serous acinar cells, including serous demilune and ductal epithelial cells. In contrast, no immunoreaction localized CA-VI in the mucous acinar cells of the gland. CA-VI gene transcripts were also detected in the same areas. The physiological significance of secretory CA-VI in the oral and oesophageal cavity is thought to play a highly specialized role in the maintenance of bicarbonate level in saliva and to protect mucosa from acid injury. It is shown that the major sites of the CA-VI secretion in dogs were in serous (demilune) secretory cells in all four major salivary glands and oesophageal glands in particular. [source] Glutamine metabolism: Role in acid-base balance,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 5 2004Lynn Taylor Abstract The intent of this review is to provide a broad overview of the interorgan metabolism of glutamine and to discuss in more detail its role in acid-base balance. Muscle, adipose tissue, and the lungs are the primary sites of glutamine synthesis and release. During normal acid-base balance, the small intestine and the liver are the major sites of glutamine utilization. The periportal hepatocytes catabolize glutamine and convert ammonium and bicarbonate ions to urea. In contrast, the perivenous hepatocytes are capable of synthesizing glutamine. During metabolic acidosis, the kidney becomes the major site of glutamine extraction and catabolism. This process generates ammonium ions that are excreted in the urine to facilitate the excretion of acids and bicarbonate ions that are transported to the blood to partially compensate the acidosis. The increased renal extraction of glutamine is balanced by an increased release from muscle and liver and by a decreased utilization in the intestine. During chronic acidosis, this adaptation is sustained, in part, by increased renal expression of genes that encode various transport proteins and key enzymes of glutamine metabolism. The increased levels of phosphoenolpyruvate carboxykinase result from increased transcription, while the increase in glutaminase and glutamate dehydrogenase activities result from stabilization of their respective mRNAs. Where feasible, this review draws upon data obtained from studies in humans. Studies conducted in model animals are discussed where available data from humans is either lacking or not firmly established. Because there are quantitative differences in tissue utilization and synthesis of glutamine in different mammals, the review will focus more on common principles than on quantification. [source] Dynamics of growth and dissemination of Salmonella in vivoCELLULAR MICROBIOLOGY, Issue 10 2010Kathryn G. Watson Summary The last decade has witnessed increasing research on dissemination of bacterial pathogens in their hosts and on the processes that underlie bacterial spread and growth during organ colonization. Here, we discuss work on the mouse model of human typhoid fever caused by Salmonella enterica serovar Typhimurium. This has revealed the use of several routes of systemic dissemination that result in colonization and growth within the spleen and liver, the major sites of bacterial proliferation. We also highlight techniques that enable in vivo analysis of the infecting population at the spatiotemporal and single cell levels. These approaches have provided more detailed insights into the events underlying the dynamics of Salmonella replication, spread and clearance within host organs and tissues. [source] | |||||||||||||||||||||||||||||||