Home About us Contact
|
Home About us Contact | ||
|
|
||
Several Tissues (several + tissue)
Selected AbstractsConnective tissue growth factor and cardiac fibrosisACTA PHYSIOLOGICA, Issue 3 2009A. Daniels Abstract Cardiac fibrosis is a major pathogenic factor in a variety of cardiovascular diseases and refers to an excessive deposition of extracellular matrix components in the heart, which leads to cardiac dysfunction and eventually overt heart failure. Evidence is accumulating for a crucial role of connective tissue growth factor (CTGF) in fibrotic processes in several tissues including the heart. CTGF orchestrates the actions of important local factors evoking cardiac fibrosis. The central role of CTGF as a matricellular protein modulating the fibrotic process in cardiac remodelling makes it a possible biomarker for cardiac fibrosis and a potential candidate for therapeutic intervention to mitigate fibrosis in the heart. [source] Regionalized expression of ADAM13 during chicken embryonic developmentDEVELOPMENTAL DYNAMICS, Issue 3 2007Juntang Lin Abstract ADAMs are a family of membrane proteins possessing a disintegrin domain and a metalloprotease domain, which have functions in cell,cell adhesion, cell,matrix adhesion, and protein shedding, respectively. ADAMs are involved in morphogenesis and tissue formation during embryonic development. In the present study, chicken ADAM13 was cloned and identified, and its expression was investigated by semiquantitative reverse transcriptase-polymerase chain reaction and in situ hybridization during chicken embryonic development. Our results show that ADAM13 expression is temporally and spatially regulated in chicken embryos. At early developmental stages, ADAM13 is expressed in the head mesenchyme, which later develops into the craniofacial skeleton, in the branchial arches, and in the meninges surrounding the brain. Furthermore, ADAM13 mRNA was also detected in several tissues and organs, such as the somites and their derived muscles, the meninges surrounding the spinal cord, the dorsal aorta, the developing kidney, and several digestive organs. Developmental Dynamics 236:862,870, 2007. © 2007 Wiley-Liss, Inc. [source] Zac1 is expressed in progenitor/stem cells of the neuroectoderm and mesoderm during embryogenesis: Differential phenotype of the Zac1-expressing cells during developmentDEVELOPMENTAL DYNAMICS, Issue 2 2005Tony Valente Abstract Zac1, a new zinc-finger protein that regulates both apoptosis and cell cycle arrest, is abundantly expressed in many neuroepithelia during early brain development. In the present work, we study the expression of Zac1 during early embryogenesis and we determine the cellular phenotype of the Zac1-expressing cells throughout development. Our results show that Zac1 is expressed in the progenitor/stem cells of several tissues (nervous system, skeleton, and skeletal muscle), because they colocalize with several progenitor/stem markers (Nestin, glial fibrillary acidic protein, FORSE-1, proliferating cell nuclear antigen, and bromodeoxyuridine). In postnatal development, Zac1 is expressed in all phases of the life cycle of the chondrocytes (from proliferation to apoptosis), in some limbic ,-aminobutyric acid-ergic neuronal subpopulations, and during developmental myofibers. Therefore, the intense expression of Zac1 in the progenitor/stem cells of different cellular lineages during the proliferative cycle, before differentiation into postmitotic cells, suggests that Zac1 plays an important role in the control of cell fate during neurogenesis, chondrogenesis, and myogenesis. Developmental Dynamics 233:667,679, 2005. © 2005 Wiley-Liss, Inc. [source] Retinoic acid, a regeneration-inducing moleculeDEVELOPMENTAL DYNAMICS, Issue 2 2003Malcolm Maden Abstract Retinoic acid (RA) is the biologically active metabolite of vitamin A. It is a low molecular weight, lipophilic molecule that acts on the nucleus to induce gene transcription. In amphibians and mammals, it induces the regeneration of several tissues and organs and these examples are reviewed here. RA induces the "super-regeneration" of organs that can already regenerate such as the urodele amphibian limb by respecifying positional information in the limb. In organs that cannot normally regenerate such as the adult mammalian lung, RA induces the complete regeneration of alveoli that have been destroyed by various noxious treatments. In the mammalian central nervous system (CNS), which is another tissue that cannot regenerate, RA does not induce neurite outgrowth as it does in the embryonic CNS, because one of the retinoic acid receptors, RAR,2, is not up-regulated. When RAR,2 is transfected into the adult spinal cord in vitro, then neurite outgrowth is stimulated. In all these cases, RA is required for the development of the organ, in the first place suggesting that the same gene pathways are likely to be used for both development and regeneration. This suggestion, therefore, might serve as a strategy for identifying potential tissue or organ targets that have the capacity to be stimulated to regenerate. Developmental Dynamics 226:237,244, 2003.© 2003 Wiley-Liss, Inc. [source] Sorting nexin-14, a gene expressed in motoneurons trapped by an in vitro preselection methodDEVELOPMENTAL DYNAMICS, Issue 4 2001Patrick Carroll Abstract A gene-trap strategy was set up in embryonic stem (ES) cells with the aim of trapping genes expressed in restricted neuronal lineages. The vector used trap genes irrespective of their activity in undifferentiated totipotent ES cells. Clones were subjected individually to differentiation in a system in which ES cells differentiated into neurons. Two ES clones in which the trapped gene was expressed in ES-derived neurons were studied in detail. The corresponding cDNAs were cloned, sequenced, and analysed by in situ hybridisation on wild-type embryo sections. Both genes are expressed in the nervous system. One gene, YR-23, encodes a large intracellular protein of unknown function. The second clone, YR-14, represents a sorting nexin (SNX14) gene whose expression in vivo coincides with that of LIM-homeodomain Islet-1 in several tissues. Sorting nexins are proteins associated with the endoplasmic reticulum (ER) and may play a role in receptor trafficking. Gene trapping followed by screening based on in vitro preselection of differentiated ES recombinant clones, therefore, has the potential to identify integration events in subsets of genes before generation of mouse mutants. © 2001 Wiley-Liss, Inc. [source] Cardiovascular effects of the thiazolidinedionesDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 2 2006Rehan Qayyum Abstract Thiazolidinediones, used for the treatment of diabetes mellitus type 2, modulate gene expression by binding to nuclear transcription factor, peroxisome proliferator-activated receptor-gamma. Peroxisome proliferator,activated receptor-gamma is expressed in several tissues, therefore, thiazolidinediones have biological effects on multiple organ systems. Here, we describe evidence that thiazolidinediones have beneficial effects on the cardiovascular system independent of their antidiabetic effect. Studies in animals have clearly shown that thiazolidinediones decrease blood pressure, left ventricular hypertrophy, development of atherosclerotic lesions, and protect myocardium from ischemia/reperfusion injury. Although relatively few studies in humans have been reported, the preponderance of available evidence suggests a beneficial effect of thiazolidinediones. Thus, by modulating gene expression, thiazolidinediones may provide a novel method for the prevention and treatment of cardiovascular diseases. Copyright © 2005 John Wiley & Sons, Ltd. [source] Elevated cerebrospinal fluid adiponectin and adipsin levels in patients with multiple sclerosis: a Finnish co-twin studyEUROPEAN JOURNAL OF NEUROLOGY, Issue 2 2010A. Hietaharju Background and purpose:, The aim of this study was to investigate the levels of three adipocytokines: leptin, adiponectin and adipsin, in serum and cerebrospinal fluid (CSF) of twins discordant for multiple sclerosis (MS). Adipose tissue is an important component connecting immune system and several tissues and organs including CNS. Fat cells produce adipocytokines, which seem to have a role in various autoimmune disorders including MS. Methods:, Plasma samples were collected from twelve twins and CSF samples from four twins discordant for MS. The concentrations of interleukine (IL)-6, adiponectin, adipsin and leptin in plasma and CSF samples were determined by enzyme immuno assay. Results:, A significant difference was seen in the adipocytokine levels in CSF samples. Twins with MS had higher concentrations of adiponectin (P = 0.039) and adipsin (P = 0.039), than their asymptomatic co-twins. Conclusion:, As adiponectin and adipsin levels in CSF did not correlate with their levels in plasma, it seems that there could be a secondary intrathecal synthesis of these adipocytokines in MS. [source] Retention of the duplicated cellular retinoic acid-binding protein 1 genes (crabp1a and crabp1b) in the zebrafish genome by subfunctionalization of tissue-specific expressionFEBS JOURNAL, Issue 14 2005Rong-Zong Liu The cellular retinoic acid-binding protein type I (CRABPI) is encoded by a single gene in mammals. We have characterized two crabp1 genes in zebrafish, designated crabp1a and crabp1b. These two crabp1 genes share the same gene structure as the mammalian CRABP1 genes and encode proteins that show the highest amino acid sequence identity to mammalian CRABPIs. The zebrafish crabp1a and crabp1b were assigned to linkage groups 25 and 7, respectively. Both linkage groups show conserved syntenies to a segment of the human chromosome 15 harboring the CRABP1 locus. Phylogenetic analysis suggests that the zebrafish crabp1a and crabp1b are orthologs of the mammalian CRABP1 genes that likely arose from a teleost fish lineage-specific genome duplication. Embryonic whole mount in situ hybridization detected zebrafish crabp1b transcripts in the posterior hindbrain and spinal cord from early stages of embryogenesis. crabp1a mRNA was detected in the forebrain and midbrain at later developmental stages. In adult zebrafish, crabp1a mRNA was localized to the optic tectum, whereas crabp1b mRNA was detected in several tissues by RT-PCR but not by tissue section in situ hybridization. The differential and complementary expression patterns of the zebrafish crabp1a and crabp1b genes imply that subfunctionalization may be the mechanism for the retention of both crabp1 duplicated genes in the zebrafish genome. [source] Group IID heparin-binding secretory phospholipase A2 is expressed in human colon carcinoma cells and human mast cells and up-regulated in mouse inflammatory tissuesFEBS JOURNAL, Issue 11 2002Makoto Murakami Group IID secretory phospholipase A2 (sPLA2 -IID), a heparin-binding sPLA2 that is closely related to sPLA2 -IIA, augments stimulus-induced cellular arachidonate release in a manner similar to sPLA2 -IIA. Here we identified the residues of sPLA2 -IID that are responsible for heparanoid binding, are and therefore essential for cellular function. Mutating four cationic residues in the C-terminal portion of sPLA2 -IID resulted in abolition of its ability to associate with cell surface heparan sulfate and to enhance stimulus-induced delayed arachidonate release, cyclooxygenase-2 induction, and prostaglandin generation in 293 cell transfectants. As compared with several other group II subfamily sPLA2s, which were equally active on A23187- and IL-1-primed cellular membranes, sPLA2 -IID showed apparent preference for A23187-primed membranes. Several human colon carcinoma cell lines expressed sPLA2 -IID and sPLA2 -X constitutively, the former of which was negatively regulated by IL-1. sPLA2 -IID, but not other sPLA2 isozymes, was expressed in human cord blood-derived mast cells. The expression of sPLA2 -IID was significantly altered in several tissues of mice with experimental inflammation. These results indicate that sPLA2 -IID may be involved in inflammation in cell- and tissue-specific manners under particular conditions. [source] Characterization of a novel silkworm (Bombyx mori) phenol UDP-glucosyltransferaseFEBS JOURNAL, Issue 3 2002Teresa Luque Sugar conjugation is a major pathway for the inactivation and excretion of both endogenous and exogenous compounds. We report here the molecular cloning and functional characterization of a phenol UDP-glucosyltransferase (UGT) from the silkworm, Bombyx mori, which was named BmUGT1. The complete cDNA clone is 1.6 kb, and the gene is expressed in several tissues of fifth-instar larvae, including fat body, midgut, integument, testis, silk gland and haemocytes. The predicted protein comprises 520 amino acids and has ,,30% overall amino-acid identity with other members of the UGT family. The most conserved region of the protein is the C-terminal half, which has been implicated in binding the UDP-sugar. BmUGT1 was expressed in insect cells using the baculovirus expression system, and a range of compounds belonging to diverse chemical groups were assessed as potential substrates for the enzyme. The expressed enzyme had a wide substrate specificity, showing activity with flavonoids, coumarins, terpenoids and simple phenols. These results support a role for the enzyme in detoxication processes, such as minimizing the harmful effects of ingested plant allelochemicals. This work represents the first instance where an insect ugt gene has been associated with a specific enzyme activity. [source] Generation of mice harboring a Sox6 conditional null allele,GENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 5 2006Bogdan Dumitriu Abstract Sox6 belongs to the family of Sry-related HMG box transcription factors, which determine cell fate and differentiation in various lineages. Sox6 is expressed in several tissues, including cartilage, testis, neuronal, and erythropoietic tissues. Mice lacking Sox6 have revealed critical roles for Sox6 in several of these tissues, but their multiple defects and early lethality has limited studies in specific cell types and in postnatal mice. We show here that we have generated mice harboring a Sox6 conditional null allele (Sox6fl+) by flanking the second coding exon with loxP sites. This allele encodes wildtype Sox6 protein, is expressed normally, and is efficiently converted into a null allele (Sox6fl,) by Cre-mediated recombination in somatic and germ cells. Sox6fl+/fl+ mice are indistinguishable from wildtype mice, and Sox6fl,/fl, mice from Sox6,/, mice. These Sox6 conditional null mice will thus be valuable for further uncovering the roles of Sox6 in various processes in vivo. genesis 44:219,224, 2006. Published 2006 Wiley-Liss, Inc. [source] Protection against Fas-induced liver apoptosis in transgenic mice expressing cyclooxygenase 2 in hepatocytes,HEPATOLOGY, Issue 3 2007Marta Casado Cyclooxygenase-2 (COX-2) is upregulated in many cancers, and the prostanoids synthesized increase proliferation, improve angiogenesis, and inhibit apoptosis in several tissues. To explore the function of COX-2 in liver, transgenic (Tg) mice were generated containing a fusion gene (LIVhCOX-2) consisting of human COX-2 cDNA under the control of the human ApoE promoter. Six lines were developed; all of them expressed the LIVhCOX-2 transgene selectively in hepatocytes. The Tg mice exhibited a normal phenotype, and the increased levels of PGE2 found were due to the constitutively expressed COX-2. Histological analysis of different tissues and macroscopic examination of the liver showed no differences between wild-type (Wt) and Tg animals. However, Tg animals were resistant to Fas-mediated liver injury, as demonstrated by low levels of plasmatic aminotransferases, a lesser caspase-3 activation, and Bax levels and an increase in Bcl-2, Mcl-1, and xIAP proteins, when compared with the Wt animals. Moreover, the resistance to Fas-mediated apoptosis is suppressed in the presence of COX-2,selective inhibitors, which prevented prostaglandin accumulation in the liver of Tg mice. Conclusion: These results demonstrate that expression of COX-2,dependent prostaglandins exerted a protection against liver apoptosis. (HEPATOLOGY 2007;45:631,638.) [source] Expression of an Aedes aegypti cation-chloride cotransporter and its Drosophila homologuesINSECT MOLECULAR BIOLOGY, Issue 4 2003V. Filippov Abstract Insects maintain haemolymph homeostasis under different environmental conditions by modulating the concentrations of Na+, K+ and Cl, ions. One group of proteins involved in ion transport across cell membranes consists of cation-chloride cotransporters that form a family of structurally similar proteins. Although much is known about these proteins in mammalian systems, our understanding of them in insects is lacking. The recent sequencing of two insect genomes, Drosophila and Anopheles, enabled us to identify globally members of the family of cation chloride cotransporters in these insects. Using RT-PCR we monitored the transcription of members of this family in development and in several tissues. Our analyses showed that transcription of these genes differ considerably from the ubiquitously and highly expressed CG5594 gene to the almost silent gene CG31547. Comparison of Drosophila CG12773 and its Aedes homologue AaeCG12773 showed that they have similar transcript expression profiles. Immunohistochemical analysis of AaeCG1277 gene expression revealed that it is highly expressed in the gut of larvae and female adults but not in Malpighian tubules. A more detailed analysis showed that this protein is localized predominantly in the basolateral membrane of these tissues. This expression pattern confirmed the results of RT-PCR analysis. We also created a mutant for one of the genes, CG10413, in Drosophila using P-element excision. Analysis of this mutant showed this protein does not appear to be essential for development. [source] PPAR, and PP2A are involved in the proapoptotic effect of conjugated linoleic acid on human hepatoma cell line SK-HEP-1INTERNATIONAL JOURNAL OF CANCER, Issue 11 2007Giuliana Muzio Abstract Conjugated linoleic acid (CLA), found in dairy products, in beef and lamb has been demonstrated to possess anticancer properties protecting several tissues from developing cancer. Moreover, it has been shown to modulate apoptosis in several cancer cell lines. The aim of this study was to investigate which signaling transduction pathways were modulated in CLA-induced apoptosis in human hepatoma SK-HEP-1 cells. The cells exposed to CLA were evaluated for PPAR,, PP2A, pro-apoptotic proteins Bak, Bad and caspases, and anti-apoptotic proteins Bcl-2 and Bcl-XL. Cells were also treated with okadaic acid, a PP2A inhibitor, or with Wy-14643, a specific PPAR, agonist. The CLA-induced apoptosis was concomitant to the increase of percentage of cells in the S phase, PPAR,, PP2A and pro-apoptotic proteins; simultaneously, antiapoptotic proteins decreased. Inhibition of PP2A prevented apoptosis, and PPAR, agonist showed similar effect as CLA. The increased PP2A could be responsible for the dephosphorylation of Bcl-2 and Bad, permitting apoptotic activity of Bax and Bad. The increase of caspase 8 and 9 suggested that both the intrinsic and extrinsic apoptotic pathways were induced. PP2A was probably increased by PPAR,, since putative PPRE sequences were found in genes encoding its subunits. In conclusion, CLA induces apoptosis in human hepatoma SK-HEP-1 cells, by increasing PPAR,, PP2A and pro-apoptotic proteins. © 2007 Wiley-Liss, Inc. [source] Chronic constriction injury induces aquaporin-2 expression in the dorsal root ganglia of ratsJOURNAL OF ANATOMY, Issue 5 2009Barbara Buffoli Abstract Aquaporins are a family of water channel proteins involved in water homeostasis in several tissues. Current knowledge of aquaporin expression in the nervous system is very limited. Therefore the first aim of this study was to assess, by immunohistochemistry and immunoblotting analysis, the presence and localization of aquaporin-2 in the spinal cord and dorsal root ganglia of naïve adult rats. In addition, we evaluated aquaporin-2 expression in response to chronic constriction injury of the sciatic nerve, a model of neuropathic pain. Our results showed that aquaporin-2 expression was not detectable either in the spinal cord or the dorsal root ganglia of naïve rats. However, we showed for the first time an increase of aquaporin-2 expression in response to chronic constriction injury treatment in small-diameter dorsal root ganglia neurons but no expression in the lumbar spinal cord. These data support the hypothesis that aquaporin-2 expression is involved in inflammatory neuropathic nerve injuries, although its precise role remains to be determined. [source] Nitrous Oxide Determination in Postmortem Biological Samples: A Case of Serial Fatal Poisoning in a Public Hospital,JOURNAL OF FORENSIC SCIENCES, Issue 1 2010Diana Poli Ph.D. Abstract:, In a public hospital, eight cases of fatal poisoning by nitrous oxide (N2O) occurred under oxygen administration, due to an erroneous swapping of the lines in the gas system. The aim of the study was to clarify the factors involved in asphyxia by characterizing gases from different lines and measuring N2O concentrations in postmortem biological samples from bodies exhumed. Analyses carried out on the gas system confirmed the erroneous substitution of O2 line with N2O and air line with O2. Consequently, high N2O amounts were revealed in several tissues and gaseous biological samples. All specimens were analyzed by headspace gas chromatography technique. A rigorous quantitative analysis was possible only in blood (11.29,2152.04 mg/L) and urine (95.11 mg/L) and in air samples from stomach and trachea (from 5.28 to 83.63 g/m3). This study demonstrates that N2O can be detected in biological samples even 1 month after death. [source] New light on the biology and developmental potential of haematopoietic stem cells and progenitor cellsJOURNAL OF INTERNAL MEDICINE, Issue 4 2009M. Sigvardsson Abstract. Even though stem cells have been identified in several tissues, one of the best understood somatic stem cells is the bone marrow residing haematopoietic stem cell (HSC). These cells are able to generate all types of blood cells found in the periphery over the lifetime of an animal, making them one of the most profound examples of tissue-restricted stem cells. HSC therapy also represents one of the absolutely most successful cell-based therapies applied both in the treatment of haematological disorders and cancer. However, to fully explore the clinical potential of HSCs we need to understand the molecular regulation of cell maturation and lineage commitment. The extensive research effort invested in this area has resulted in a rapid development of the understanding of the relationship between different blood cell lineages and increased understanding for how a balanced composition of blood cells can be generated. In this review, several of the basic features of HSCs, as well as their multipotent and lineage-restricted offspring, are addressed, providing a current view of the haematopoietic development tree. Some of the basic mechanisms believed to be involved in lineage restriction events including activities of permissive and instructive external signals are also discussed, besides transcription factor networks and epigenetic alterations to provide an up-to-date view of early haematopoiesis. [source] Gadolinium-based contrast agents and their potential role in the pathogenesis of nephrogenic systemic fibrosis: The role of excess ligandJOURNAL OF MAGNETIC RESONANCE IMAGING, Issue 5 2008Martin A. Sieber PhD Abstract Purpose To investigate the role of excess ligand present in gadolinium (Gd) -based contrast agents in the development of nephrogenic systemic fibrosis (NSF). Using a dosing regimen to simulate the exposure seen in patients with severe renal impairment, we investigated the effect of excess ligand on Gd-deposition and the depletion of endogenous ions. Materials and Methods Gadodiamide and gadoversetamide were formulated with 0%, 5%, and 10% excess ligand. Forty-two, healthy, male Hannover Wistar rats received daily intravenous injections of each formulation over a period of 20 days. At the end of the study, histopathological analysis of the skin was performed and the concentrations of Gd, Zn, and Cu were measured in several tissues. The levels of Zn in the urine were also measured. Results The most severe skin lesions were observed after injection of formulations containing 0% free ligand and in those animals with the highest Gd concentrations in the skin. There were no significant reductions in the levels of Zn or Cu observed in the skin; however, the levels of Zn in the urine were elevated following administration of formulations with the highest amount of excess ligand. Conclusion Our findings suggest that there is an inverse correlation between the amount of excess ligand present in Gd-containing contrast agents and the amount of Gd in the tissue, and further underline the importance of the inherent stability of these agents in the development of NSF. J. Magn. Reson. Imaging 2008;27:955,962. © 2008 Wiley-Liss, Inc. [source] Non-homologous DNA end joining in the mature rat brainJOURNAL OF NEUROCHEMISTRY, Issue 6 2002Keqin Ren Abstract Recent evidence suggests that DNA double strand breaks (DSBs) are introduced in neurons during the course of normal development, and that repair of such DSBs is essential for neuronal survival. Here we describe a non-homologous DNA end joining (NHEJ) system in the adult rat brain that may be used to repair DNA DSBs. In the brain NHEJ system, blunt DNA ends are joined with lower efficiency than cohesive or non-matching protruding ends. Moreover, brain NHEJ is blocked by DNA ligase inhibitors or by dATP and can occur in the presence or absence of exogenously added ATP. Comparison of NHEJ activities in several tissues showed that brain and testis share similar mechanisms for DNA end joining, whereas the activity in thymus seems to utilize different mechanisms than in the nervous system. The developmental profile of brain NHEJ showed increasing levels of activity after birth, peaking at postnatal day 12 and then gradually decreasing along with age. Brain distribution analysis in adult animals showed that NHEJ activity is differentially distributed among different regions. We suggest that the DNA NHEJ system may be utilized in the postnatal brain for the repair of DNA double strand breaks introduced within the genome in the postnatal brain. [source] Therapeutic benefits of intrathecal protein therapy in a mouse model of amyotrophic lateral sclerosisJOURNAL OF NEUROSCIENCE RESEARCH, Issue 13 2008Yasuyuki Ohta Abstract When fused with the protein transduction domain (PTD) derived from the human immunodeficiency virus TAT protein, proteins can cross the blood,brain barrier and cell membrane and transfer into several tissues, including the brain, making protein therapy feasible for various neurological disorders. We have constructed a powerful antiapoptotic modified Bcl-XL protein (originally constructed from Bcl-XL) fused with PTD derived from TAT (TAT-modified Bcl-XL), and, to examine its clinical effectiveness in a mouse model of familial amyotrophic lateral sclerosis (ALS), transgenic mice expressing human Cu/Zn superoxide dismutase (SOD1) bearing a G93A mutation were treated by intrathecal infusion of TAT-modified Bcl-XL. We demonstrate that intrathecally infused TAT-fused protein was effectively transferred into spinal cord neurons, including motor neurons, and that intrathecal infusion of TAT-modified Bcl-XL delayed disease onset, prolonged survival, and improved motor performance. Histological studies show an attenuation of motor neuron loss and a decrease in the number of cleaved caspase 9-, cleaved caspase 3-, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in the lumbar cords of TAT-modified Bcl-XL -treated G93A mice. Our results indicate that intrathecal protein therapy using a TAT-fused protein is an effective clinical tool for the treatment of ALS. © 2008 Wiley-Liss, Inc. [source] Immunocytochemical analysis of the circadian clock protein in mouse hepatocytesMICROSCOPY RESEARCH AND TECHNIQUE, Issue 5 2003Manuela Malatesta Abstract Many biochemical, physiological, and behavioral processes in organisms ranging from prokaryotes to humans exhibit circadian rhythms, defined as cyclic oscillations of about 24 hours. The mechanism of the cellular circadian clock relies on interlocking positive and negative transcriptional/translational feedback loops based on the regulated expression of several genes. Clock is one of these genes and its transcript, CLOCK protein, is a transcription factor belonging to the bHLH-PAS family. In mammals the clock gene is expressed in several tissues, including the liver. In the present study, we analyzed by means of quali-quantitative immunoelectron microscopy the fine intracellular distribution of the CLOCK protein in mouse hepatocytes during the daily cycle. We demonstrated that CLOCK protein is mostly located in the cell nucleus, where it accumulates on perichromatin fibrils, representing the in situ form of nascent pre-mRNA, while condensed chromatin and nucleoli contain lower amounts of protein. Moreover, we found that CLOCK protein shows circadian oscillations in these nuclear compartments, peaking in late afternoon. At this time the hepatic transcriptional rate reaches the maximal level, thus suggesting an important role of CLOCK protein in the regulation of liver gene expression. Microsc. Res. Tech. 61:414,418, 2003. © 2003 Wiley-Liss, Inc. [source] pFARs, Plasmids free of antibiotic resistance markers, display high-level transgene expression in muscle, skin and tumour cellsTHE JOURNAL OF GENE MEDICINE, Issue 4 2010Corinne Marie Abstract Background Nonviral gene therapy requires a high yield and a low cost production of eukaryotic expression vectors that meet defined criteria such as biosafety and quality of pharmaceutical grade. To fulfil these objectives, we designed a novel antibiotic-free selection system. Methods The proposed strategy relies on the suppression of a chromosomal amber mutation by a plasmid-borne function. We first introduced a nonsense mutation into the essential Escherichia coli thyA gene, resulting in thymidine auxotrophy. The bacterial strain was optimized for the production of small and novel plasmids free of antibiotic resistance markers (pFARs) and encoding an amber suppressor t-RNA. Finally, the potentiality of pFARs as eukaryotic expression vectors was assessed by monitoring luciferase activities after electrotransfer of LUC-encoding plasmids into various tissues. Results The introduction of pFARs into the optimized bacterial strain restored normal growth to the auxotrophic mutant and allowed an efficient production of monomeric supercoiled plasmids. The electrotransfer of LUC-encoding pFAR into muscle led to high luciferase activities, demonstrating an efficient gene delivery. In transplanted tumours, transgene expression levels were superior after electrotransfer of the pFAR derivative compared to a plasmid carrying a kanamycin resistance gene. Finally, in skin, whereas luciferase activities decreased within 3 weeks after intradermal electrotransfer of a conventional expression vector, sustained luciferase expression was observed with the pFAR plasmid. Conclusions Thus, we have designed a novel strategy for the efficient production of biosafe plasmids and demonstrated their potentiality for nonviral gene delivery and high-level transgene expression in several tissues. Copyright © 2010 John Wiley & Sons, Ltd. [source] Downstream utrophin enhancer is required for expression of utrophin in skeletal muscleTHE JOURNAL OF GENE MEDICINE, Issue 6 2008Jun Tanihata Abstract Background Duchenne muscular dystrophy is caused by the absence of the muscle cytoskeletal protein dystrophin. Utrophin is an autosomal homologue of dystrophin, and overexpression of utrophin is expected to compensate for the dystrophin deficit. We previously reported that the 5.4-kb 5,-flanking region of the utrophin gene containing the A-utrophin core promoter did not drive transgene expression in heart and skeletal muscle. To clarify the regulatory mechanism of utrophin expression, we generated a nuclear localization signal-tagged LacZ transgenic (Tg) mouse, in which the LacZ gene was driven by the 129-bp downstream utrophin enhancer (DUE) and the 5.4-kb 5,-flanking region of the utrophin promoter. Methods Two Tg lines were established. The levels of transgene mRNA expression in several tissues were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative RT-PCR. Cryosections of several tissues were stained with haematoxylin and eosin and X-gal. Results The transgene expression patterns were consistent with endogenous utrophin in several tissues including heart and skeletal muscle. Transgene expression was also up-regulated more in regenerating muscle than in nonregenerating muscle. Moreover, utrophin expression was augmented in the skeletal muscle of DUE Tg/dystrophin-deficient mdx mice through cross-breeding experiments. We finally established cultures of primary myogenic cells from this Tg mouse and found that utrophin up-regulation during muscle differentiation depends on the DUE motif. Conclusions Our results showed that DUE is indispensable for utrophin expression in skeletal muscle and heart, and primary myogenic cells from this Tg mice provide a high through-put screening system for drugs that up-regulate utrophin expression. Copyright © 2008 John Wiley & Sons, Ltd. [source] The utrophin promoter A drives high expression of the transgenic LacZ gene in liver, testis, colon, submandibular gland, and small intestineTHE JOURNAL OF GENE MEDICINE, Issue 2 2005Joji Takahashi Abstract Background Duchenne muscular dystrophy (DMD) is caused by the absence of the muscle cytoskeletal protein dystrophin. Utrophin is an autosomal homologue of dystrophin, and overexpression of the protein is expected to compensate for the defect of dystrophin. The utrophin gene has two promoters, A and B, and promoter A of the utrophin gene is a possible target of pharmacological interventions for DMD because A-utrophin is up-regulated in dystrophin-deficient mdx skeletal and cardiac muscles. To investigate the utrophin promoter A activity in vivo, we generated nuclear localization signal-tagged LacZ transgenic mice, where the LacZ gene was driven by the 5-kb flanking region of the A- utrophin gene. Methods Four transgenic lines were established by mating four independent founders with C57BL/6J mice. The levels of mRNA for ,-galactosidase in several tissues were examined by RT-PCR. Cryosections from several tissues were stained with hematoxylin and eosin (H&E) and with 5-bromo-4-chloro-3-indolyl-,- D -galactopyranoside (X-Gal). Results The 5-kb upstream region of the A- utrophin gene showed high transcriptional activity in liver, testis, colon, submandibular gland, and small intestine, consistent with the endogenous expression of utrophin protein. Surprisingly, the levels of both ,-gal protein and mRNA for the transgene in cardiac and skeletal muscles were extremely low, even in nuclei near the neuromuscular junctions. These results indicate that the regulation of the utrophin gene in striated muscle is different from that in non-muscle tissues. Conclusions Our results clearly showed that the utrophin A promoter is not sufficient to drive expression in muscle, but other regulatory elements are required. Copyright © 2004 John Wiley & Sons, Ltd. [source] Secretion and cell volume regulation by salivary acinar cells from mice lacking expression of the Clcn3 Cl, channel geneTHE JOURNAL OF PHYSIOLOGY, Issue 1 2002Jorge Arreola Salivary gland acinar cells shrink when Cl, currents are activated following cell swelling induced by exposure to a hypotonic solution or in response to calcium-mobilizing agonists. The molecular identity of the Cl, channel(s) in salivary cells involved in these processes is unknown, although ClC-3 has been implicated in several tissues as a cell-volume-sensitive Cl, channel. We found that cells isolated from mice with targeted disruption of the Clcn3 gene undergo regulatory volume decrease in a fashion similar to cells from wild-type littermates. Consistent with a normal regulatory volume decrease response, the magnitude and the kinetics of the swell-activated Cl, currents in cells from ClC-3-deficient mice were equivalent to those from wild-type mice. It has also been suggested that ClC-3 is activated by Ca2+ -calmodulin-dependent protein kinase II; however, the magnitude of the Ca2+ -dependent Cl, current was unchanged in the Clcn3,/- animals. In addition, we observed that ClC-3 appeared to be highly expressed in the smooth muscle cells of glandular blood vessels, suggesting a potential role for this channel in saliva production by regulating blood flow, yet the volume and ionic compositions of in vivo stimulated saliva from wild-type and null mutant animals were comparable. Finally, in some cells ClC-3 is an intracellular channel that is thought to be involved in vesicular acidification and secretion. Nevertheless, the protein content of saliva was unchanged in Clcn3,/- mice. Our results demonstrate that the ClC-3 Cl, channel is not a major regulator of acinar cell volume, nor is it essential for determining the secretion rate and composition of saliva. [source] Identification and characterization of microRNAs from porcine skeletal muscleANIMAL GENETICS, Issue 2 2010S. S. Xie Summary MicroRNAs (miRNAs) are a class of non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. There is increasing evidence to suggest that miRNAs participate in muscle development in mice and humans; however, few studies have focused on miRNAs in porcine muscle tissue. Here, we experimentally detected and identified conserved and unique miRNAs from porcine skeletal muscle. Fifty-seven distinct miRNAs were identified, of which 39 have not been reported earlier in the pig. Of these, two miRNAs appear to be novel and pig-specific. Surprisingly, these two differ only by a single nucleotide. A part of their primary transcript was cloned and confirmed by sequencing analysis. Alignment of the two sequences using ClustalW showed that the precursor sequences were almost identical, but the flanking sequences were different, indicating that these two novel miRNAs may represent rapidly evolving miRNAs in the pig genome. The expression patterns of eight miRNAs were characterized by real-time polymerase chain reaction of eight pig tissue samples. The ssc-let-7e and ssc-miR-181b miRNAs were expressed in all tissues analysed. The ssc-let-7c, ssc-miR-125b, ssc-miR-new1 and ssc-miR-new2 miRNAs were expressed in several tissues, while ssc-miR-122 and ssc-miR-206 were specifically expressed in the liver and muscle respectively. Our results add to existing data on porcine miRNAs and are useful for investigating the biological functions of miRNAs in porcine skeletal muscle development. [source] Purification, crystallization and preliminary X-ray crystallographic analysis of a cysteine-rich secretory protein (CRISP) from Naja atra venomACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2004Yu-Ling Wang Cysteine-rich secretory proteins (CRISPs) play an important role in the innate immune system and are transcriptionally regulated by androgens in several tissues. The proteins are mostly found in the epididymis and granules of mammals, whilst a number of snake venoms also contain CRISP-family proteins. The natrin protein from the venom of Naja atra (Taiwan cobra), which belongs to a family of CRISPs and has a cysteine-rich C-terminal amino-acid sequence, has been purified using a three-stage chromatography procedure and crystals suitable for X-ray analysis have been obtained using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.58,Å resolution using synchrotron radiation; the crystals belong to space group C2221, with unit-cell parameters a = 59.172, b = 65.038, c = 243.156,Å. There are two protein molecules in the asymmetric unit and the Matthews coefficient is estimated to be 2.35,Å3,Da,1, corresponding to a solvent content of 47.60%. [source] Tissue distribution of the novel DPP-4 inhibitor BI 1356 is dominated by saturable binding to its target in ratsBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 5 2009Holger Fuchs Abstract BI 1356 (INN: linagliptin) is an inhibitor of dipeptidyl peptidase-4 (DPP-4). This study investigated whether saturable binding of BI 1356 to its target DPP-4 occurs in tissues and whether drug accumulation occurs at these sites in vivo. In order to test these hypotheses, the tissue distribution of BI 1356 was determined in wild-type and DPP-4 deficient rats at different dose levels by means of whole body autoradiography and measurement of tissue radioactivity concentrations after single i.v. dosing of [14C]-radio labeled BI 1356. The accumulation behavior of drug-related radioactivity in tissues was further explored in an oral repeat dose study. Tissue levels of [14C]BI 1356 related radioactivity were markedly lower in all investigated tissues of the DPP-4 deficient rats and the difference of the dose-dependent increase of radioactivity tissue levels between both rat strains indicates that tissue distribution at low doses of BI 1356 is dominated by binding of BI 1356 to DPP-4 in tissues. As the binding to DPP-4 is strong but reversible, the tissue binding results in a long terminal half-life in several tissues including plasma. The binding capacity to DPP-4 is, however, limited. In the rat, saturation of DPP-4 binding is suggested at an intravenous dose above 0.01,0.1,mg/kg [14C]BI 1356. As the DPP-4 binding capacity is saturated already at low doses, accumulation of BI 1356 in tissues is unlikely, despite the long persistence of low amounts in the body. Copyright © 2009 John Wiley & Sons, Ltd. [source] Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytesBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2010K Varani Background and purpose:, Adenosine is an endogenous modulator, interacting with four G-protein coupled receptors (A1, A2A, A2B and A3) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes. Experimental approach:, mRNA, Western blotting, saturation and competition binding experiments, cyclic AMP, p38 mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-,B activation, tumour necrosis factor , (TNF-,) and interleukin-8 (IL-8) release were assessed in human synoviocytes isolated from patients with osteoarthritis. Key results:, mRNA and protein for A1, A2A, A2B and A3 adenosine receptors are expressed in human synoviocytes. Standard adenosine agonists and antagonists showed affinity values in the nanomolar range and were coupled to stimulation or inhibition of adenylyl cyclase. Activation of A2A and A3 adenosine receptors inhibited p38 MAPK and NF-,B pathways, an effect abolished by selective adenosine antagonists. A2A and A3 receptor agonists decreased TNF-, and IL-8 production. The phosphoinositide 3-kinase or Gs pathways were involved in the functional responses of A3 or A2A adenosine receptors. Synoviocyte A1 and A2B adenosine receptors were not implicated in the inflammatory process whereas stimulation of A2A and A3 adenosine receptors was closely associated with a down-regulation of the inflammatory status. Conclusions and implications:, These results indicate that A2A and A3 adenosine receptors may represent a potential target in therapeutic modulation of joint inflammation. [source] Heat shock proteins as emerging therapeutic targetsBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2005Csaba Sõti Chaperones (stress proteins) are essential proteins to help the formation and maintenance of the proper conformation of other proteins and to promote cell survival after a large variety of environmental stresses. Therefore, normal chaperone function is a key factor for endogenous stress adaptation of several tissues. However, altered chaperone function has been associated with the development of several diseases; therefore, modulators of chaperone activities became a new and emerging field of drug development. Inhibition of the 90 kDa heat shock protein (Hsp)90 recently emerged as a very promising tool to combat various forms of cancer. On the other hand, the induction of the 70 kDa Hsp70 has been proved to be an efficient help in the recovery from a large number of diseases, such as, for example, ischemic heart disease, diabetes and neurodegeneration. Development of membrane-interacting drugs to modify specific membrane domains, thereby modulating heat shock response, may be of considerable therapeutic benefit as well. In this review, we give an overview of the therapeutic approaches and list some of the key questions of drug development in this novel and promising therapeutic approach. British Journal of Pharmacology (2005) 146, 769,780. doi:10.1038/sj.bjp.0706396 [source] | |||||||||||||||||||||||||