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Vivo Analysis (vivo + analysis)
Kinds of Vivo Analysis Selected AbstractsParathyroid hormone (PTH),induced bone gain is blunted in SOST overexpressing and deficient miceJOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2010Ina Kramer Abstract Intermittent parathyroid hormone (PTH) treatment is a potent bone anabolic principle that suppresses expression of the bone formation inhibitor Sost. We addressed the relevance of Sost suppression for PTH-induced bone anabolism in vivo using mice with altered Sost gene dosage. Six-month-old Sost overexpressing and 2-month-old Sost deficient male mice and their wild-type littermates were subjected to daily injections of 100,µg/kg PTH(1,34) or vehicle for a 2-month period. A follow-up study was performed in Sost deficient mice using 40 and 80,µg/kg PTH(1,34). Animals were sacrificed 4 hours after the final PTH administration and Sost expression in long bone diaphyses was determined by qPCR. Bone changes were analyzed in vivo in the distal femur metaphysis by pQCT and ex vivo in the tibia and lumbar spine by DXA. Detailed ex vivo analyses of the femur were performed by pQCT, µCT, and histomorphometry. Overexpression of Sost resulted in osteopenia and Sost deletion in high bone mass. As shown before, PTH suppressed Sost in wild-type mice. PTH treatment induced substantial increases in bone mineral density, content, and cortical thickness and in aging wild-type mice also led to cancellous bone gain owing to amplified bone formation rates. PTH-induced bone gain was blunted at all doses and skeletal sites in Sost overexpressing and deficient mice owing to attenuated bone formation rates, whereas bone resorption was not different from that in PTH-treated wild-type controls. These data suggest that suppression of the bone formation inhibitor Sost by intermittent PTH treatment contributes to PTH bone anabolism. © 2010 American Society for Bone and Mineral Research [source] Glucose-dependent insulinotropic polypeptide (GIP) and its receptor (GIPR): Cellular localization, lesion-affected expression, and impaired regenerative axonal growthJOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2009Bettina A. Buhren Abstract Glucose-dependent insulinotropic polypeptide (GIP) was initially described to be rapidly regulated by endocrine cells in response to nutrient ingestion, with stimulatory effects on insulin synthesis and release. Previously, we demonstrated a significant up-regulation of GIP mRNA in the rat subiculum after fornix injury. To gain more insight into the lesion-induced expression of GIP and its receptor (GIPR), expression profiles of the mRNAs were studied after rat sciatic nerve crush injury in 1) affected lumbar dorsal root ganglia (DRG), 2) spinal cord segments, and 3) proximal and distal nerve fragments by means of quantitative RT-PCR. Our results clearly identified lesion-induced as well as tissue type-specific mRNA regulation of GIP and its receptor. Furthermore, comprehensive immunohistochemical stainings not only confirmed and exceeded the previous observation of neuronal GIP expression but also revealed corresponding GIPR expression, implying putative modulatory functions of GIP/GIPR signaling in adult neurons. In complement, we also observed expression of GIP and its receptor in myelinating Schwann cells and oligodendrocytes. Polarized localization of GIPR in the abaxonal Schwann cell membranes, plasma membrane-associated GIPR expression of satellite cells, and ependymal GIPR expression strongly suggests complex cell type-specific functions of GIP and GIPR in the adult nervous system that are presumably mediated by autocrine and paracrine interactions, respectively. Notably, in vivo analyses with GIPR-deficient mice suggest a critical role of GIP/GIPR signal transduction in promoting spontaneous recovery after nerve crush, insofar as traumatic injury of GIPR-deficient mouse sciatic nerve revealed impaired axonal regeneration compared with wild-type mice. © 2009 Wiley-Liss, Inc. [source] The tumor suppressor parafibromin is required for posttranscriptional processing of histone mRNAMOLECULAR CARCINOGENESIS, Issue 3 2010Leslie J. Farber Abstract Parafibromin, encoded by the gene HRPT2, is a tumor suppressor protein associated with the RNA polymerase II-associated complex, Paf1 complex. HRPT2 mutations were first identified in patients with the multiple endocrine neoplasia syndrome, hyperparathyroidism-jaw tumor (HPT-JT) syndrome, and have also been found in sporadic parathyroid and renal tumors. However, the mechanisms by which parafibromin suppresses tumor formation remain unknown. In this study, we identify a novel role of parafibromin in the regulation of replication-dependent histones. Both in vitro and in vivo analyses reveal a posttranscriptional role of parafibromin in histone mRNA processing. Downregulation of parafibromin through RNA interference or in vivo mutations lead to uncleaved histone mRNA with polyadenylated tails. These results indicate that parafibromin regulates the 3, processing of histone RNA, an essential component of the cell cycle. © 2009 Wiley-Liss, Inc. [source] Temporal separation of distinct differentiation pathways by a dual specificity Rap-Phr system in Bacillus subtilisMOLECULAR MICROBIOLOGY, Issue 1 2007Wiep Klaas Smits Summary In bacterial differentiation, mechanisms have evolved to limit cells to a single developmental pathway. The establishment of genetic competence in Bacillus subtilis is controlled by a complex regulatory circuit that is highly interconnected with the developmental pathway for spore formation, and the two pathways appear to be mutually exclusive. Here we show by in vitro and in vivo analyses that a member of the Rap family of proteins, RapH, is activated directly by the late competence transcription factor ComK, and is capable of inhibiting both competence and sporulation. Importantly, RapH is the first member of the Rap family that demonstrates dual specificity, by dephosphorylating the Spo0F,P response regulator and inhibiting the DNA-binding activity of ComA. The protein thus acts at the stage where competence is well initiated, and prevents initiation of sporulation in competent cells as well as contributing to the escape from the competent state. A deletion of rapH induces both differentiation pathways and interferes with their temporal separation. Together, these results indicate that RapH is an integral part of a multifactorial regulatory circuit affecting the cell's decision between distinct developmental pathways. [source] Delineation of pilin domains required for bacterial association into microcolonies and intestinal colonization by Vibrio choleraeMOLECULAR MICROBIOLOGY, Issue 4 2000Thomas J. Kirn The toxin-co-regulated pilus (TCP), a type 4 pilus that is expressed by epidemic strains of Vibrio cholerae O1 and O139, is required for colonization of the human intestine. The TCP structure is assembled as a polymer of repeating subunits of TcpA pilin that form long fibres, which laterally associate into bundles. Previous passive immunization studies have suggested that the C-terminal region of TcpA is exposed on the surface of the pilus fibre and has a critical role in mediating the colonization functions of TCP. In the present study, we have used site-directed mutagenesis to delineate two domains within the C-terminal region that contribute to TCP structure and function. Alterations in the first domain, termed the structural domain, result in altered pilus stability or morphology. Alterations in the second domain, termed the interaction domain, affect colonization and/or infection by CTX-bacteriophage without affecting pilus morphology. In vitro and in vivo analyses of the tcpA mutants revealed that a major function of TCP is to mediate bacterial interaction through direct pilus,pilus contact required for microcolony formation and productive intestinal colonization. The importance of this function is supported by the finding that intragenic suppressor mutations that restore colonization ability to colonization-deficient mutants simultaneously restore pilus-mediated bacterial interactions. The alterations resulting from the suppressor mutations also provide insight into the molecular interactions between pilin subunits within and between pilus fibres. [source] REVIEW ARTICLE: Uterine NK Cells, Spiral Artery Modification and the Regulation of Blood Pressure During Mouse PregnancyAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 6 2010Suzanne D. Burke Citation Burke SD, Barrette VF, Gravel J, Carter ALI, Hatta K, Zhang J, Chen Z, Leno-Durán E, Bianco J, Leonard S, Murrant C, Adams MA, Anne Croy B. Uterine NK cells, spiral artery modification and the regulation of blood pressure during mouse pregnancy. Am J Reprod Immunol 2010 Reproductive success in mammals involves coordinated changes in the immune and cardiovascular as well as in the neuroendocrine and reproductive systems. This review addresses studies that identify potential links for NK cells and T cells with the local and systemic cardiovascular adaptations of pregnancy. The studies reviewed have utilized immunohistochemisty and in vivo analyses of vascular parameters by ultrasound, chronic monitoring of hemodynamics via radiotelemetric recording and intravital microscopy. At the uterine level, functional subsets of uterine natural killer cells were identified. These included subsets expressing molecules important for vasoregulation, in addition to those previously identified for angiogenesis. Spiral arteries showed conducted responses that could account for conceptus control of vasoactivity and mouse gestational blood pressure 5-phase pattern. Vascular immunology is an emerging transdisciplinary field, critical for both reproductive immunology and cardiovascular disease. [source] Expression and function of CXCL16 in a novel model of goutARTHRITIS & RHEUMATISM, Issue 8 2010Jeffrey H. Ruth Objective To better define the activity of soluble CXCL16 in the recruitment of polymorphonuclear neutrophils (PMNs) in vivo, utilizing a novel animal model of gout involving engraftment of SCID mice with normal human synovial tissue (ST) injected intragraft with gouty human synovial fluid (SF). Methods For in vitro studies, a modified Boyden chemotaxis system was used to identify CXCL16 as an active recruitment factor for PMNs in gouty SF. Migration of PMNs could be reduced by neutralization of CXCL16 activity in gouty SF. For in vivo analyses, fluorescent dye,tagged PMNs were injected intravenously into SCID mice while, simultaneously, diluted gouty SF containing CXCL16, or depleted of CXCL16 by antibody blocking, was administered intragraft. In addition, the receptor for CXCL16, CXCR6, was inhibited by incubating PMNs with a neutralizing anti-CXCR6 antibody prior to injection into the mouse chimeras. Recruitment of PMNs to the gouty SF,injected normal human ST was then examined in this SCID mouse chimera system. Results CXCL16 concentrations were highly elevated in gouty SF, and PMNs were observed to migrate in response to CXCL16 in vitro. Normal human ST,SCID mouse chimeras injected intragraft with gouty SF that had been depleted of CXCL16 during PMN transfer showed a significant reduction of 50% in PMN recruitment to engrafted tissue as compared with that after administration of sham-depleted gouty SF. Similar findings were achieved when PMNs were incubated with a neutralizing anti-CXCR6 antibody before injection into chimeras. Conclusion Overall, the results of this study outline the effectiveness of the human,SCID mouse chimera system as a viable animal model of gout, serving to identify the primary function of CXCL16 as a significant mediator of in vivo recruitment of PMNs to gouty SF. [source] Characterization of peach thaumatin-like proteins and their identification as major peach allergensCLINICAL & EXPERIMENTAL ALLERGY, Issue 9 2010A. Palacín Summary Background Peach is the most important fruit related to food allergy in the Mediterranean area. Pru p 3, its lipid transfer protein, has been described as the principal allergen responsible for cross-reactivities with other foods and pollen and the severity of clinical symptoms. However, the involvement of other allergenic families cannot be ruled out. Thaumatin-like proteins (TLPs) have been described as food allergen in several fruits, such as apple, cherry, kiwi and banana, and pollen. Objective To identify members of the TLP family in peach fruit and to characterize putative allergens. Methods Through two-dimensional (2D) electrophoresis of peach extract and immunodetections with a pool of peach-allergic patients, IgE-binding spots were identified and the corresponding proteins purified and characterized as allergens by in vitro and in vivo assays. Three isoforms, belonging to the TLP family, were purified by different chromatographic systems and characterized by N -terminal amino acid sequences, molecular weight determination (MALDI) and enzymatic activity analysis (,-1,3-gluconase test and inhibition growth of fungi). In the same way, their IgE-binding capacity and allergenic activity were tested by ELISA assays, basophil activation tests and skin prick tests (SPT). Results Two peach-TLPs, Pru p 2.0101 and Pru p 2.0201, were identified as IgE-binding spots by 2D electrophoresis. Another peach-TLP, Pru p 2.0301, was cloned and produced as recombinant protein in a yeast system. The three isoforms were purified and characterized as TLPs by immunoblotting with anti-chestnut TLP antibodies and anti-plant N -asparagine complex glycan (anti-cross-reactive carbohydrate determinant). All of them showed ,-1,3-glucanase activity and inhibition of fungal growth. The three TLPs were recognized by around 50% of the sera from 31 patients analysed in ELISA experiments. All three gave a positive response to an SPT and/or in basophil activation experiments. Conclusion Three isoforms, belonging to the TLP family, were identified in peach as principal allergens. Their prevalence, observed in in vitro, ex vivo and in vivo analyses, suggests that they are important allergens and should therefore be included in the routine diagnosis of peach allergy, at least in the Mediterranean area. Cite this as: A. Palacín, L. Tordesillas, P. Gamboa, R. Sanchez-Monge, J. Cuesta-Herranz, M. L. Sanz, D. Barber, G. Salcedo and A. Díaz-Perales, Clinical & Experimental Allergy, 2010 (40) 1422,1430. [source] In vivo analysis of MT-based vesicle transport by confocal reflection microscopyCYTOSKELETON, Issue 2 2009Imre Gáspár Abstract The use of confocal reflection microscopy (CRM) for the in vivo analysis of microtubule (MT) mediated transport of lipid droplets in the developing Drosophila egg primordia is described here. The developing Drosophila oocytes are ideal objects to study MT-mediated transport in vivo: transport of e.g. the lipid droplets can be conveniently, selectively and sensitively monitored through CRM and the egg primordia are readily available for physical, chemical and/or genetic manipulations. CRM is a non-destructive way to follow vesicle movement and allows high frame rate image recording. When combined with fluorescence imaging, CRM offers simultaneous visualization of the cargo and the protein(s) of interest, i.e. a motor or a cargo adapter, thus allowing a better understanding of MT-mediated transport and spatiotemporal coordination of the transport machinery. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] In vivo analysis reveals different apoptotic pathways in pre- and postmigratory cerebellar granule cells of rabbitDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2004Laura Lossi Abstract Naturally occurring neuronal death (NOND) has been described in the postnatal cerebellum of several species, mainly affecting the cerebellar granule cells (CGCs) by an apoptotic mechanism. However, little is known about the cellular pathway(s) of CGC apoptosis in vivo. By immunocytochemistry, in situ detection of fragmented DNA, electron microscopy, and Western blotting, we demonstrate here the existence of two different molecular mechanisms of apoptosis in the rabbit postnatal cerebellum. These two mechanisms affect CGCs at different stages of their maturation and migration. In the external granular layer, premigratory CGCs undergo apoptosis upon phosphorylation of checkpoint kinase 1 (Chk1), and hyperphosphorylation of retinoblastoma protein. In postmigratory CGCs within the internal granular layer, caspase 3 and to a lesser extent 7 and 9 are activated, eventually leading to poly-ADP-ribose polymerase-1 (PARP-1) cleavage and programmed cell death. We conclude that NOND of premigratory CGCs is linked to activation of DNA checkpoint and alteration of normal cell cycle, whereas in postmigratory CGCs apoptosis is, more classically, dependent upon caspase 3 activation. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 437,452, 2004 [source] Targeted replacement of rodent CCR2 with the human orthologue CCR2B: A mouse model for in vivo analysis of human target-selective small molecule MCP-1 receptor antagonistsDRUG DEVELOPMENT RESEARCH, Issue 4 2002Haydn M. Prosser Abstract Rodent models for testing the efficacy of lead compounds are often invalidated by species selectivity of the compounds. The advent of mouse embryonic stem cell technology has allowed the development of genetically engineered mouse strains that incorporate a specific human gene in place of the orthologous mouse gene, a so-called knock-in mouse. This study describes the generation and validation of a mutant mouse line that expresses human CCR2B as a functional substitute for murine CCR2. The human CCR2B knock-in mice are viable and appear normal. In vitro assays indicate that the CCR2B knock-in is functionally expressed, giving a macrophage chemotactic profile in response to JE or MCP-1 that is similar to human peripheral blood monocytes rather than that of a murine macrophage cell line. In addition, the human selective CCR2B antagonist, SB-399721, was a more potent inhibitor of CCR2B knock-in macrophages in response to hMCP-1 than JE. The ability of the human CCR2B gene to functionally substitute for the mouse orthologue in vivo is demonstrated by a normal inflammatory response to intraperitoneal thioglycollate injection. Drug Dev. Res. 55:197,209, 2002. © 2002 Wiley-Liss, Inc. [source] Differential role of IL-2R signaling for CD8+ T cell responses in acute and chronic viral infectionsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2007Martin Abstract IL-2 is a cytokine with multiple and even divergent functions; it has been described as a key cytokine for in vitro T cell proliferation but is also essential for down-regulating T cell responses by inducing activation-induced cell death as well as regulatory T cells. The in vivo analysis of IL-2 function in regulating specific T cell responses has been hampered by the fact that mice deficient in IL-2 or its receptors develop lymphoproliferative diseases and/or autoimmunity. Here we generated chimeric mice harboring both IL-2R-competent and IL-2R-deficient T cells and assessed CD8+ T cell induction, function and maintenance after acute or persistent viral infections. Induction and maintenance of CD8+ T cells were relatively independent of IL-2R signaling during acute/resolved viral infection. In marked contrast, IL-2 was crucial for secondary expansion of memory CD8+ T cells and for the maintenance of virus-specific CD8+ T cells during persistent viral infections. Thus, depending on the chronicity of antigen exposure, IL-2R signaling is either essential or largely dispensable for induction and maintenance of virus-specific CD8+ T cell responses. [source] Analysis of the essential oil of the aerial parts of Viola etrusca from Monte Labbro (South Tuscany, Italy) and in vivo analysis of flower volatiles using SPMEFLAVOUR AND FRAGRANCE JOURNAL, Issue 2 2002Guido Flamini Abstract The composition of the essential oil of the endemic Viola etrusca Erben from Monte Labbro (Italy) has been studied for the first time. It was made up almost exclusively of methyl salicylate (96%), together with many other mono- and sesquiterpenes and non-terpenic alcohols and aldehydes. In addition, the SPME technique has been used to evaluate the volatiles emitted in vivo by different-coloured flowers. Copyright © 2002 John Wiley & Sons, Ltd. [source] Novel pathogenic mechanism suggested by ex vivo analysis of MCT8 (SLC16A2) mutations,HUMAN MUTATION, Issue 1 2009W. Edward Visser Abstract Monocarboxylate transporter 8 (MCT8; approved symbol SLC16A2) facilitates cellular uptake and efflux of 3,3,,5-triiodothyronine (T3). Mutations in MCT8 are associated with severe psychomotor retardation, high serum T3 and low 3,3,,5,-triiodothyronine (rT3) levels. Here we report three novel MCT8 mutations. Two subjects with the F501del mutation have mild psychomotor retardation with slightly elevated T3 and normal rT3 levels. T3 uptake was mildly affected in F501del fibroblasts and strongly decreased in fibroblasts from other MCT8 patients, while T3 efflux was always strongly reduced. Moreover, type 3 deiodinase activity was highly elevated in F501del fibroblasts, whereas it was reduced in fibroblasts from other MCT8 patients, probably reflecting parallel variation in cellular T3 content. Additionally, T3-responsive genes were markedly upregulated by T3 treatment in F501del fibroblasts but not in fibroblasts with other MCT8 mutations. In conclusion, mutations in MCT8 result in a decreased T3 uptake in skin fibroblasts. The much milder clinical phenotype of patients with the F501del mutation may be correlated with the relatively small decrease in T3 uptake combined with an even greater decrease in T3 efflux. If fibroblasts are representative of central neurons, abnormal brain development associated with MCT8 mutations may be the consequence of either decreased or increased intracellular T3 concentrations. Hum Mutat 0,1-10, 2008. © 2008 Wiley-Liss, Inc. [source] In vivo analysis of gut function and disease changes in a zebrafish larvae model of inflammatory bowel disease: A feasibility studyINFLAMMATORY BOWEL DISEASES, Issue 7 2010Angeleen Fleming PhD Abstract Background: The aim of this study was to develop a model of inflammatory bowel disease (IBD) in zebrafish larvae, together with a method for the rapid assessment of gut morphology and function in vivo thereby enabling medium-throughput compound screening. Methods: Assays were performed using larval zebrafish from 3,8 days postfertilization (d.p.f.) in 96-well plates. Gut morphology and peristalsis were observed in vivo using fluorescent imaging following ingestion of fluorescent dyes. IBD was induced by addition of 2,4,6-trinitrobenzenesulfonic acid (TNBS) to the medium within the well. Pathology was assessed in vivo using fluorescent imaging and postmortem by histology, immunohistochemistry, and electron microscopy. Therapeutic compounds were evaluated by coadministration with TNBS. Results: A novel method of investigating gut architecture and peristalsis was devised using fluorescent imaging of live zebrafish larvae. Archetypal changes in gut architecture consistent with colitis were observed throughout the gut. Significant changes in goblet cell number and tumor necrosis factor alpha (TNF-,) antibody staining were used to quantify disease severity and rescue. Prednisolone and 5-amino salicylic acid treatment ameliorated the disease changes. Candidate therapeutic compounds (NOS inhibitors, thalidomide, and parthenolide) were assessed and a dissociation was observed between efficacy assessed using a single biochemical measure (TNF-, staining) versus an assessment of the entire disease state. Conclusions: Gut physiology and pathology relevant to human disease state can be rapidly modeled in zebrafish larvae. The model is suitable for medium-throughput chemical screens and is amenable to genetic manipulation, hence offers a powerful novel premammalian adjunct to the study of gastrointestinal disease. (Inflamm Bowel Dis 2010) [source] Wear particle analysis of highly crosslinked polyethylene isolated from a failed total hip arthroplastyJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 2 2008Yukihide Minoda Abstract Polyethylene wear particles are one of the most important factors affecting the results of total hip arthroplasty (THA). To reduce wear generation and to achieve better long-term results of THA, highly crosslinked polyethylene (HXPE) has recently been introduced and come into wide use. Thus far, however, there have been no reports on in vivo analysis of HXPE wear particles. We isolated HXPE wear particles from periprosthetic tissue of a failed THA and analyzed using scanning electron microscope. The number of particles was 5.33 × 107 g,1. Particle size (equivalent circle diameter) was 0.66 ± 0.40 ,m (mean ± standard error). Aspect ratio and roundness were 1.37 ± 0.26 and 1.44 ± 0.67, respectively. All the particles were round shaped, and "fibrils" or "shreds" were not detected. Thus far, this was the first report on in vivo wear particle analysis of HXPE. HXPE generated less, smaller, and rounder particles, compared with the corresponding reported values for particles generated from conventional polyethylene. These characteristics might affect macrophage response, osteolysis, and long-term results of THA with HXPE. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2008 [source] Guidelines for assessment of bone microstructure in rodents using micro,computed tomographyJOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2010Mary L Bouxsein Abstract Use of high-resolution micro,computed tomography (µCT) imaging to assess trabecular and cortical bone morphology has grown immensely. There are several commercially available µCT systems, each with different approaches to image acquisition, evaluation, and reporting of outcomes. This lack of consistency makes it difficult to interpret reported results and to compare findings across different studies. This article addresses this critical need for standardized terminology and consistent reporting of parameters related to image acquisition and analysis, and key outcome assessments, particularly with respect to ex vivo analysis of rodent specimens. Thus the guidelines herein provide recommendations regarding (1) standardized terminology and units, (2) information to be included in describing the methods for a given experiment, and (3) a minimal set of outcome variables that should be reported. Whereas the specific research objective will determine the experimental design, these guidelines are intended to ensure accurate and consistent reporting of µCT-derived bone morphometry and density measurements. In particular, the methods section for papers that present µCT-based outcomes must include details of the following scan aspects: (1) image acquisition, including the scanning medium, X-ray tube potential, and voxel size, as well as clear descriptions of the size and location of the volume of interest and the method used to delineate trabecular and cortical bone regions, and (2) image processing, including the algorithms used for image filtration and the approach used for image segmentation. Morphometric analyses should be based on 3D algorithms that do not rely on assumptions about the underlying structure whenever possible. When reporting µCT results, the minimal set of variables that should be used to describe trabecular bone morphometry includes bone volume fraction and trabecular number, thickness, and separation. The minimal set of variables that should be used to describe cortical bone morphometry includes total cross-sectional area, cortical bone area, cortical bone area fraction, and cortical thickness. Other variables also may be appropriate depending on the research question and technical quality of the scan. Standard nomenclature, outlined in this article, should be followed for reporting of results. © 2010 American Society for Bone and Mineral Research [source] Functional and molecular MR imaging of angiogenesis: Seeing the target, seeing it workJOURNAL OF CELLULAR BIOCHEMISTRY, Issue S39 2002Michal NeemanArticle first published online: 16 JAN 200 Abstract Intensive research over the last years led to the discovery of multiple molecular pathways and intricate regulatory network controlling the growth and regression of blood vessels in general and angiogenesis in particular. The difficulties in elucidation of the regulation of angiogenesis, stems from the inherent complexity due to participation of many cell types, under a dominant impact of physiological and environmental effects of flow, perfusion, and oxygenation. Major advances were achieved with the use of sophisticated transgenic mice models engineered so as to provide spatially and temporally controlled expression of specific factors alone or in combination. In vivo analysis of these models frequently requires the use of non-invasive imaging modalities for measurement of functional parameters of the vasculature along with dynamic molecular information. Optical methods are extensively applied for the study of angiogenesis [Brown et al., 2001] but provide very limited tissue penetration. MRI offers the advantage of being non-invasive with uniform and relatively high spatial resolution for deep tissues. Multiple MRI approaches for monitoring angiogenesis were developed over the last years, each looking at a particular step in the process. The aim of this paper is to analyze the clinical, pharmaceutical, and biological needs for imaging of angiogenesis, and to critically evaluate the strengths and weaknesses of functional and molecular imaging for monitoring angiogenesis. The inherent problem of validation of different measures of angiogenesis, and the advantages and limitations associated with application of MRI based methods, as surrogates for other measurements of angiogenesis will be discussed. The terms molecular imaging and functional imaging are frequently loosely defined with a significant overlap between the two. For the sake of this paper we will apply a narrower definition of both terms, where molecular imaging will apply to methods directed towards detection of specific biological molecules that participate directly in (regulation of) a physiological process; while functional imaging will be used to describe those methods that aim to detect the physiological response to a defined (molecular) stimulus. J. Cell. Biochem. Suppl. 39: 11,17, 2002. © 2002 Wiley-Liss, Inc. [source] LIGHT REGULATION OF PHYCOBILISOME BIOSYNTHESIS AND CONTROL BY A PHYTOCHROME-LIKE PHOTORECEPTORJOURNAL OF PHYCOLOGY, Issue 2000K. Terauchi Ambient light quality changes dramatically affect the composition of light harvesting structures, the phycobilisomes, in many cyanobacterial species. In the cyanobacterium Fremyella diplosiphon, shifts in the ratio of red to green light lead to transcriptional changes and altered synthesis of several phycobilisome components. This process is called complementary chromatic adaptation (CCA). These two colors have opposite effects: red light activates an operon encoding the biliprotein phycocyanin (PC) and inactivates the operon encoding phycoerythrin (PE), whereas green light activates PE synthesis and shuts down PC synthesis. The effects of red and green light on CCA are photoreversible. Thus, CCA is similar to transcriptional processes that are controlled by phytochromes, a family of eukaryotic red/far red photoreversible photoreceptors. We are using molecular genetics to determine the mechanisms by which F. diplosiphon senses changes in the color of light of its environment. Initial mutant generation and complementation lead to the discovery of three CCA regulatory components that are part of a complex two component system. The most interesting of these is RcaE (regulator of chromatic adaptation), a histidine kinase-class protein containing a region in its amino-terminal half with similarity to the chromophore binding domains of phytochromes. Within this region, RcaE contains a cysteine residue in a similar location as that used for covalent attachment of the open-chain tetrapyrrole chromophore in phytochromes. We will present recent data characterizing RcaE, including in vivo analysis of the chromophore that is attached to RcaE, as well as results from our recent isolation of a new CCA regulatory component. [source] Spo0A directly controls the switch from acid to solvent production in solvent-forming clostridiaMOLECULAR MICROBIOLOGY, Issue 5 2000Adriana Ravagnani The spo0A genes of Clostridium beijerinckii NCIMB 8052 and Clostridium cellulolyticum ATCC 35319 were isolated and characterized. The C-terminal DNA-binding domains of the predicted products of spo0A from these two organisms, as well as 16 other taxonomically diverse species of Bacillus and Clostridium, show extensive amino acid sequence conservation (56% identity, 65% similarity over 104 residues). A 12-amino-acid motif (SRVERAIRHAIE) that forms the putative DNA recognition helix is particularly highly conserved, suggesting a common DNA target. Insertional inactivation of spo0A in C. beijerinckii blocked the formation of solvents (as well as spores and granulose). Sequences resembling Spo0A-binding motifs (TGNCGAA) are found in the promoter regions of several of the genes whose expression is modulated at the onset of solventogenesis in Clostridium acetobutylicum and C. beijerinckii. These include the upregulated adc gene, encoding acetoacetate decarboxylase (EC 4.1.1.4), and the downregulated ptb gene, encoding phosphotransbutyrylase (EC 2.3.1.c). In vitro gel retardation experiments using C. acetobutylicum adc and C. beijerinckii ptb promoter fragments and recombinant Bacillus subtilis and C. beijerinckii Spo0A suggested that adc and ptb are directly controlled by Spo0A. The binding affinity was reduced when the 0A boxes were destroyed, and enhanced when they were modified to conform precisely to the consensus sequence. In vivo analysis of wild-type and mutagenized promoters transcriptionally fused to the gusA reporter gene in C. beijerinckii validated this hypothesis. Post-exponential phase expression from the mutagenized adc promoter was substantially reduced, whereas expression from the mutagenized ptb promoter was not shut down at the end of exponential growth. [source] In vivo analysis of the post-natal development of normal mouse brain by DTINMR IN BIOMEDICINE, Issue 4 2007Pierre Larvaron Abstract The water diffusion characteristics of wild-type mouse brains have been studied in vivo by DTI to follow developmental changes. Here, axial (,//) and radial (,,) diffusivities and fractional anisotropy were measured from the fifth day of life (P5) and at three other post-natal ages (P12, P19 and P54). Magnetic resonance images were collected from a single sagittal slice in the middle of the two hemispheres; ROI were chosen in nine different structures of both grey and white matter. Fractional anisotropy (FA) from P5 onwards distinguished structures of both white and grey matter, even though myelination had yet to occur. Between P5 and P54, a significant increase in FA was observed in the genu of the corpus callosum due to a significant decrease in ,, whereas ,// remained stable. Many other significant variations of ,// and ,, were measured in different structures. They were substantially correlated with axon and myelin maturation which are responsible for the main evolutions of the brain during its post-natal development. These quantitative data show that in vivo characterization of the anatomy and microstructure of the normal mouse brain during development is possible. The normative data will greatly improve the characterization of abnormal development in the transgenic mouse brain. Copyright © 2006 John Wiley & Sons, Ltd. [source] Prostaglandin E2 is activated by airway injury and regulates fibroblast cytoskeletal dynamics,THE LARYNGOSCOPE, Issue 7 2009Vlad C. Sandulache MD Abstract Objectives/Hypothesis: To characterize the activation of cyclooxygenase (COX)-2/prostaglandin (PG) E2 signaling during airway mucosal repair and its subsequent role during the wound healing process. Study Design: Prospective animal study. Methods: The subglottis was approached via cricothyroidotomy. Sham airways were closed, and wounded airways were subjected to laser injury and closed. Subglottic tissue was harvested at 12 hours, 24 hours, 48 hours, and 72 hours postinjury. Secretions were collected preoperatively and at time of sacrifice. Inflammatory gene expression was analyzed using quantitative reverse transcriptase polymerase chain reaction. Subglottic/tracheal explants were exposed to exogenous IL-1, in the presence or absence of COX inhibitors. Explant-produced PGE2 levels were assayed using enzyme linked immunoassays. Human airway fibroblast migration and collagen contraction were assayed in the presence or absence of prostaglandin E2. Results: Laser injury triggers a rapid, dose-dependent increase in mucosal IL-1, and COX-2 gene expression, with an anatomical distribution proportional to the distance from the site of injury. Gene upregulation correlates with dose-dependent increases in PGE2 mucosal secretion levels. Ex vivo analysis indicates IL-1, is responsible for the activation of the COX-2 / PGE2 pathway. Prostaglandin E2 differentially inhibits airway fibroblast migration and contraction in a specific, dose-dependent manner. Conclusions: PGE2 is activated during mucosal inflammation and acts to decrease fibroplastic activity in the mucosal wound bed. During subglottic stenosis (SGS) development, the levels of PGE2 generated in response to injury may be insufficient to blunt the intrinsically fibroplastic phenotype of SGS fibroblasts, resulting in excessive scarring. Laryngoscope, 2009 [source] In vivo analysis of the lumenal binding protein (BiP) reveals multiple functions of its ATPase domainTHE PLANT JOURNAL, Issue 6 2007Christopher James Snowden Summary The endoplasmic reticulum (ER) chaperone binding protein (BiP) binds exposed hydrophobic regions of misfolded proteins. Cycles of ATP hydrolysis and nucleotide exchange on the ATPase domain were shown to regulate the function of the ligand-binding domain in vitro. Here we show that ATPase mutants of BiP with defective ATP-hydrolysis (T46G) or ATP-binding (G235D) caused permanent association with a model ligand, but also interfered with the production of secretory, but not cytosolic, proteins in vivo. Furthermore, the negative effect of BiP(T46G) on secretory protein synthesis was rescued by increased levels of wild-type BiP, whereas the G235D mutation was dominant. Unexpectedly, expression of a mutant BiP with impaired ligand binding also interfered with secretory protein production. Although mutant BiP lacking its ATPase domain had no detrimental effect on ER function, expression of an isolated ATPase domain interfered with secretory protein synthesis. Interestingly, the inhibitory effect of the isolated ATPase was alleviated by the T46G mutation and aggravated by the G235D mutation. We propose that in addition to its role in ligand release, the ATPase domain can interact with other components of the protein translocation and folding machinery to influence secretory protein synthesis. [source] Mature antigen-experienced T helper cells synthesize and secrete the B cell chemoattractant CXCL13 in the inflammatory environment of the rheumatoid jointARTHRITIS & RHEUMATISM, Issue 11 2008Antonio Manzo Objective Synovial B cells play a critical role in rheumatoid arthritis (RA), being involved in autoantibody synthesis, T cell activation, and cytokine production. CXCL13 is a B cell chemoattractant that is instrumental in synovial B cell organization; the regulatory determinants of CXCL13 in inflammation are poorly characterized. This study was undertaken to investigate the functional involvement of synovial T cells in the ectopic expression of CXCL13 in RA. Methods CXCL13 production and regulation were addressed using immunohistochemistry, in situ hybridization, quantitative polymerase chain reaction, multicolor flow cytometry, and enzyme-linked immunosorbent assay, by in situ,ex vivo analysis and in vitro functional assays with rheumatoid synovial tissue and primary cells. Results CXCL13 messenger RNA and protein expression and spontaneous CXCL13 secretion were detected in RA synovial fluid T cells but were not detected (or were detected only occasionally) in peripheral blood T cells. Analysis of tissue expression confirmed cytoplasm localization of CXCL13 in T lymphocytes infiltrating B cell follicles and small perivascular aggregates. Multicolor characterizations in synovial fluid demonstrated CXCL13 expression in antigen-experienced T helper cells, frequently characterized by terminal differentiation and the lack of the follicular helper T cell markers CXCR5 and BCL6 protein. In vitro functional assays revealed the enhancing effect of T cell receptor,CD28 engagement on CXCL13 production and secretion in primary cells. Conclusion Our findings define a new functional property of synovial T cells, demonstrating their active involvement in the local production of B cell chemoattractants, and support a direct contribution of the adaptive immune system and antigen-dependent signals in the mechanisms of B cell localization in RA. [source] CD56-expressing T cells that have features of senescence are expanded in rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 1 2007Joshua J. Michel Objective T cells deficient in CD28 expression have been implicated in the pathogenesis of rheumatoid arthritis (RA). Given that CD28-null T cells are functionally heterogeneous, we undertook this study to screen for novel receptors on these cells. Methods Seventy-two patients with RA (ages 35,84 years) and 53 healthy persons (32 young controls ages 19,34 years, 21 older controls ages 39,86 years) were recruited. Phenotypes and proliferative capacity of T cells from fresh leukocytes and of long-term cultures were monitored by flow cytometry. Lung biopsy specimens from patients with RA-associated interstitial pneumonitis (IP) were examined by immunohistochemistry. Receptor functionality was assessed by crosslinking bioassays. Results Chronic stimulation of CD28+ T cells in vitro yielded progenies that lacked CD28 but that gained CD56. Ex vivo analysis of leukocytes from patients with extraarticular RA showed a higher frequency of CD56+,CD28-null T cells than in patients with disease confined to the joints or in healthy controls. CD56+,CD28-null T cells had nil capacity for proliferation, consistent with cellular senescence. CD56+ T cells had skewed T cell receptor (TCR) ,/,-chain usage and restricted TCR third complementarity-determining region spectra. Histologic studies showed that CD56+ T cells were components of cellular infiltrates in RA-associated IP. CD56 crosslinking on T cells sufficiently induced cytokine production, although CD56/TCR coligation induced higher production levels. Conclusion Chronic activation of T cells induces counterregulation of CD28 and CD56 expression. The loss of CD28 is accompanied by the gain of CD56 that confers TCR-independent and TCR-dependent activation pathways. We propose that accumulation of CD56+ T cells in RA contributes to maladaptive immune responses and that CD56+ T cells are potential targets for therapy. [source] Human ovarian surface epithelial cells immortalized with hTERT maintain functional pRb and p53 expressionCELL PROLIFERATION, Issue 5 2007N. F. Li Normal human ovarian surface epithelial (OSE) cells, which are thought to be the origin of most of human ovarian carcinomas, have a very limited lifespan in culture. Establishment of immortalized OSE cell lines has, in the past, required inactivation of pRb and p53 functions. However, this often leads to increased chromosome instability during prolonged culture. Materials and Methods:,In this study, we have used a retroviral infection method to overexpress human telomerase reverse transcriptase (hTERT) gene, in primary normal OSE cells, under optimized culture conditions. Results:,In vitro and in vivo analysis of hTERT-immortalized cell lines confirmed their normal epithelial characteristics. Gene expression profiles and functional analysis of p16INK4A, p15INK4B, pRb and p53 confirmed the presence of their intact functions. Our study suggests that inactivation of pRb and p53 is not necessary for OSE immortalization. Furthermore, down-regulation of p15INK4B in the immortalized cells may indicate a functional role for this protein in them. Conclusion:,These immortal OSE cell lines are likely to be an important tool for studying human OSE biology and carcinogenesis. [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] | ||||||||||||||||