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Sample records for adult wild-type mouse

  1. Porphyrin Interactions with Wild Type and Mutant Mouse Ferrochelatase

    SciTech Connect

    Ferreira, Gloria C.; Franco, Ricardo; Lu, Yi; Ma, Jian-Guo; Shelnutt, John A.

    1999-05-19

    Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes Fe2+ chelation into protoporphyrin IX. Resonance Raman and W-visible absorbance spectroscopes of wild type and engineered variants of murine ferrochelatase were used to examine the proposed structural mechanism for iron insertion into protoporphyrin by ferrochelatase. The recombinant variants (i.e., H207N and E287Q) are enzymes in which the conserved amino acids histidine-207 and glutamate-287 of murine ferrochelatase were substituted with asparagine and glutamine, respectively. Both of these residues are at the active site of the enzyme as deduced from the Bacillus subtilis ferrochelatase three-dimensional structure. Addition of free base or metalated porphyrins to wild type ferrochelatase and H207N variant yields a quasi 1:1 complex, possibly a monomeric protein-bound species. In contrast, the addition of porphyrin (either free base or metalated) to E287Q is sub-stoichiometric, as this variant retains bound porphyrin in the active site during isolation and purification. The specificity of porphyrin binding is confirmed by the narrowing of the structure-sensitive resonance Raman lines and the vinyl vibrational mode. Resonance Raman spectra of free base and metalated porphyrins bound to the wild type ferrochelatase indicate a nonplanar distortion of the porphyrin macrocycle, although the magnitude of the distortion cannot be determined without first defining the specific type of deformation. Significantly, the extent of the nonplanar distortion varies in the case of H207N- and E287Q-bound porphyrins. In fact, resonance Raman spectral decomposition indicates a homogeneous ruffled distortion for the nickel protoporphyrin bound to the wild type ferrochelatase, whereas both a planar and ruffled conformations are present for the H207N-bound porphyrin. Perhaps more revealing is the unusual resonance , 3 Raman spectrum of the endogenous E287Q-bound porphyrin, which has

  2. Retinal ganglion cell responses to voltage and current stimulation in wild-type and rd1 mouse retinas

    NASA Astrophysics Data System (ADS)

    Goo, Yong Sook; Ye, Jang Hee; Lee, Seokyoung; Nam, Yoonkey; Ryu, Sang Baek; Kim, Kyung Hwan

    2011-06-01

    Retinal prostheses are being developed to restore vision for those with retinal diseases such as retinitis pigmentosa or age-related macular degeneration. Since neural prostheses depend upon electrical stimulation to control neural activity, optimal stimulation parameters for successful encoding of visual information are one of the most important requirements to enable visual perception. In this paper, we focused on retinal ganglion cell (RGC) responses to different stimulation parameters and compared threshold charge densities in wild-type and rd1 mice. For this purpose, we used in vitro retinal preparations of wild-type and rd1 mice. When the neural network was stimulated with voltage- and current-controlled pulses, RGCs from both wild-type and rd1 mice responded; however the temporal pattern of RGC response is very different. In wild-type RGCs, a single peak within 100 ms appears, while multiple peaks (approximately four peaks) with ~10 Hz rhythm within 400 ms appear in RGCs in the degenerated retina of rd1 mice. We find that an anodic phase-first biphasic voltage-controlled pulse is more efficient for stimulation than a biphasic current-controlled pulse based on lower threshold charge density. The threshold charge densities for activation of RGCs both with voltage- and current-controlled pulses are overall more elevated for the rd1 mouse than the wild-type mouse. Here, we propose the stimulus range for wild-type and rd1 retinas when the optimal modulation of a RGC response is possible.

  3. Wild type microglia do not arrest pathology in mouse models of Rett syndrome

    PubMed Central

    Wang, Jieqi; Wegener, Jan Eike; Huang, Teng-Wei; Sripathy, Smitha; De Jesus-Cortes, Hector; Xu, Pin; Tran, Stephanie; Knobbe, Whitney; Leko, Vid; Britt, Jeremiah; Starwalt, Ruth; McDaniel, Latisha; Ward, Chris; Parra, Diana; Newcomb, Benjamin; Lao, Uyen; Flowers, David A.; Cullen, Sean; Jorstad, Nikolas L; Yang, Yue; Glaskova, Lena; Vigneau, Sebastian; Kozlitina, Julia; Reichardt, Sybille D.; Reichardt, Holger M.; Gärtner, Jutta; Bartolomei, Marisa S.; Fang, Min; Loeb, Keith; Keene, C. Dirk; Bernstein, Irwin; Goodell, Margaret; Brat, Daniel J.

    2015-01-01

    Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by mutations in the X chromosomal gene Methyl-CpG-binding Protein 2 (MECP2) (1). RTT treatment so far is symptomatic. Mecp2 disruption in mice phenocopies major features of the syndrome (2) that can be reversed upon re-expression of Mecp2 (3. It has recently been reported that transplantation of wild type (WT) bone marrow (BMT) into lethally irradiated Mecp2tm1.1Jae/y mice prevented neurologic decline and early death by restoring microglial phagocytic activity against apoptotic targets (4). Based on this report, clinical trials of BMT for patients with RTT have been initiated (5). We aimed to replicate and extend the BMT experiments in three different RTT mouse models but found that despite robust microglial engraftment, BMT from WT donors did not rescue early death or ameliorate neurologic deficits. Furthermore, early and specific genetic expression of Mecp2 in microglia did not rescue Mecp2-deficient mice. In conclusion our experiments do not support BMT as therapy for RTT. PMID:25993969

  4. Wild-Type Mouse Models to Screen Antisense Oligonucleotides for Exon-Skipping Efficacy in Duchenne Muscular Dystrophy

    PubMed Central

    Cao, Limin; Han, Gang; Gu, Ben; Yin, HaiFang

    2014-01-01

    A readily available animal model is essential for rapidly identifying effective treatments for Duchenne muscular dystrophy (DMD), a devastating neuromuscular disorder caused by the lack of dystrophin protein, which results from frame-disrupting mutations in the DMD gene. Currently, the mdx mouse is the most commonly used model for antisense oligonucleotide (AO)-mediated exon skipping pre-clinical studies, with a mild phenotype. However, the accessibility of mdx mouse colonies particularly in developing countries can constrain research. Therefore in this study we explore the feasibility of using wild-type mice as models to establish exon-skipping efficiency of various DMD AO chemistries and their conjugates. Four different strains of wild-type mice and six different AO chemistries were investigated intramuscularly and the results indicated that the same exon-skipping efficiency was achieved for all tested AOs as that from mdx mice. Notably, levels of exon-skipping obtained in C57BL6 and C3H and mdx mice were most closely matched, followed by ICR and BALB/C mice. Systemic validation revealed that wild-type mice are less responsive to AO-mediated exon skipping than mdx mice. Our study provides evidence for the first time that wild-type mice can be appropriate models for assessing DMD AO exon-skipping efficiency with similar sensitivity to that of mdx mice and this finding can further accelerate the development of effective DMD AOs. PMID:25365558

  5. Beta Cell Formation in vivo Through Cellular Networking, Integration and Processing (CNIP) in Wild Type Adult Mice.

    PubMed

    Doiron, Bruno; Hu, Wenchao; DeFronzo, Ralph A

    2016-01-01

    Insulin replacement therapy is essential in type 1 diabetic individuals and is required in ~40- 50% of type 2 diabetics during their lifetime. Prior attempts at beta cell regeneration have relied upon pancreatic injury to induce beta cell proliferation, dedifferentiation and activation of the embryonic pathway, or stem cell replacement. We report an alternative method to transform adult non-stem (somatic) cells into pancreatic beta cells. The Cellular Networking, Integration and Processing (CNIP) approach targets cellular mechanisms involved in pancreatic function in the organ's adult state and utilizes a synergistic mechanism that integrates three important levels of cellular regulation to induce beta cell formation: (i) glucose metabolism, (ii) membrane receptor function, and (iii) gene transcription. The aim of the present study was to induce pancreatic beta cell formation in vivo in adult animals without stem cells and without dedifferentiating cells to recapitulate the embryonic pathway as previously published (1-3). Our results employing CNIP demonstrate that: (i) insulin secreting cells can be generated in adult pancreatic tissue in vivo and circumvent the problem of generating endocrine (glucagon and somatostatin) cells that exert deleterious effects on glucose homeostasis, and (ii) longterm normalization of glucose tolerance and insulin secretion can be achieved in a wild type diabetic mouse model. The CNIP cocktail has the potential to be used as a preventative or therapeutic treatment or cure for both type 1 and type 2 diabetes. PMID:26696016

  6. A PCR-based assay for the wild-type dystrophin gene transferred into the mdx mouse.

    PubMed

    Shrager, J B; Naji, A; Kelly, A M; Stedman, H H

    1992-10-01

    Myoblast transfer has emerged as a promising treatment for inherited myopathies such as Duchenne muscular dystrophy (DMD). Further development of the technique's therapeutic potential requires an experimental system in which issues of graft rejection can be clearly discriminated from those related to myoblast biology. Here we report the development and initial application of a quantitative assay for myogenic cells bearing a wild-type dystrophin gene following transfer into the mdx mouse. The technique relies upon the ability of a mutagenizing polymerase chain reaction (PCR) primer to create a new restriction site in the amplification production of the wild-type, but not the mdx dystrophin gene. The ratio of host to donor cells can be determined from muscle biopsies as small as 1 mg, regardless of donor H-2 background. This simple technique should allow a number of basic questions related to myoblast and direct gene transfer to be addressed using the mdx mouse model. PMID:1357549

  7. Activation of ganglion cells in wild-type and rd1 mouse retinas with monophasic and biphasic current pulses

    NASA Astrophysics Data System (ADS)

    Jensen, Ralph J.; Rizzo, Joseph F. III

    2009-06-01

    We and other research groups are designing an electronic retinal prosthesis to provide vision for patients who are blind due to photoreceptor degeneration. In this study, we examined the effect of stimulus waveform on the amount of current needed to activate retinal ganglion cells (RGCs) when the retinal neural network is stimulated. Isolated retinas of wild-type and rd1 mice were stimulated with cathodal and anodal monophasic current pulses of 1 ms duration and symmetric biphasic current pulses (1 ms per phase) delivered through an electrode that was located subretinally. For both wild-type and rd1 mouse retinas, cathodal current pulses were least effective in activating most RGCs. The median threshold current for a cathodal current pulse was 2.0-4.4 fold higher than the median threshold current for either an anodal or a biphasic current pulse. In wild-type mouse retinas, the median threshold current for activating RGCs with anodal current pulses was 23% lower than that with biphasic current pulses. In rd1 mouse retinas, the median threshold currents for anodal and biphasic current pulses were about the same. However, the variance in thresholds of rd1 RGCs for biphasic pulse stimulation was much smaller than for anodal pulse stimulation. Thus, a symmetric biphasic current pulse may be the best stimulus for activating the greatest number of RGCs in retinas devoid of photoreceptors.

  8. Experimental Infection of Adults With Recombinant Wild-Type Human Metapneumovirus

    PubMed Central

    Talaat, Kawsar R.; Karron, Ruth A.; Thumar, Bhagvanji; McMahon, Bridget A.; Schmidt, Alexander C.; Collins, Peter L.; Buchholz, Ursula J.

    2013-01-01

    Background. Human metapneumovirus (HMPV) causes lower respiratory tract infections in young children. rHMPV-SHs is a recombinant HMPV (rHMPV) based on a biologically derived wild-type HMPV strain. We characterized its infectivity and immunogenicity in healthy adults to determine whether it would be suitable for use as the parent virus for the development of live attenuated rHMPV vaccines. Methods. Twenty-one healthy adults were inoculated intranasally with 106 plaque-forming units of rHMPV-SHs. Respiratory symptoms and shedding of challenge virus were assessed. Neutralizing antibody responses, serum immunoglobulin G and A, and nasal wash specimen immunoglobulin A antibody responses to the HMPV F protein were also measured. Induction of nasal cytokines was assessed with electrochemiluminescence assays. Results. Nine subjects (43%) were infected with challenge virus as determined by virus detection and/or ≥4-fold rise in serum antibody titers. Peak viral shedding occurred on days 7–9 after infection. Four weeks after inoculation, 35% of subjects had any antibody response. Six of 9 infected subjects had respiratory symptoms, and 3 had headache after inoculation. Cytokine patterns differed considerably between subjects with similar illness severity and viral shedding. Conclusions. The rHMPV-SHs virus is infectious and is a suitable parent virus for development of live-attenuated HMPV vaccine candidates. Clinical Trials Registration. NCT01109329. PMID:23908489

  9. Continuum Diffusion Reaction Rate Calculations of Wild-Type and Mutant Mouse Acetylcholinesterase: Adaptive Finite Element Analysis

    PubMed Central

    Song, Yuhua; Zhang, Yongjie; Bajaj, Chandrajit L.; Baker, Nathan A.

    2004-01-01

    As described previously, continuum models, such as the Smoluchowski equation, offer a scalable framework for studying diffusion in biomolecular systems. This work presents new developments in the efficient solution of the continuum diffusion equation. Specifically, we present methods for adaptively refining finite element solutions of the Smoluchowski equation based on a posteriori error estimates. We also describe new, molecular-surface-based models, for diffusional reaction boundary criteria and compare results obtained from these models with the traditional spherical criteria. The new methods are validated by comparison of the calculated reaction rates with experimental values for wild-type and mutant forms of mouse acetylcholinesterase. The results show good agreement with experiment and help to define optimal reactive boundary conditions. PMID:15345536

  10. Protective efficacy of low-dose amantadine in adults challenged with wild-type influenza A virus.

    PubMed Central

    Sears, S D; Clements, M L

    1987-01-01

    The prophylactic efficacy of a low dose (100 mg) of amantadine hydrochloride against experimental challenge with influenza A/Texas/1/85 (H1N1) wild-type virus was determined in healthy adult volunteers in a placebo-controlled, double-blind, randomized trial. No side effects of the 100-mg dose were observed in the amantadine-treated volunteers. Compared with placebo, 100 mg of amantadine significantly reduced the frequency of illness (9 of 22 versus 2 of 22 volunteers, P less than 0.04) and provided 78% protection against influenza illness. The two ill volunteers in the amantadine group had rhinitis only, whereas most of the ill placebo controls developed both systemic and upper-respiratory-tract illness. Wild-type virus was recovered from 50% of the amantadine-treated volunteers, compared with 82% of the placebo controls. Of note, the infected amantadine recipients shed 100 times less virus and shed virus for half as many days as did the infected placebo recipients. Although amantadine restricted viral replication, it did not interfere with the development of an antibody response to influenza virus. These results indicate that in adults experimentally challenged with influenza wild-type virus, 100 mg of amantadine is effective both in the prevention of influenza illness and in the restriction of virus replication. PMID:3435099

  11. Prognosis and management of adult wild type gastrointestinal stromal tumours (GISTs): A pooled analysis and review of literature.

    PubMed

    Bhatt, N R; Collins, D; Crotty, P; Ridgway, P F

    2016-09-01

    A pooled review was performed to determine survival in adult WT GIST (Wild Type GastroIntestinal Stromal Tumours) and compare the same with pediatric WT GISTs. Electronic databases were searched using the terms "Wild type" AND "GIST". Eighty-two adult patients from 14 studies were included in the pooled analysis. Cumulative survival was greater than 50% in both age groups, hence medial survival could not be computed. Mean survival in adults was 15.7 years ± 0.78 and in children was 18.8 years ± 1.3 (p = 0.241). Median disease free survival in adults was 10 years while 5-year overall survival was 88%. There was no statistically significant difference in the survival between the two groups (p = 0.241). Overall survival in adults with WT GISTs is favourable compared to other adult GIST subtypes likely reflects a common molecular pathway similar to pediatric GIST. PMID:27566016

  12. Viscoelasticity in wild-type and vinculin-deficient (5.51) mouse F9 embryonic carcinoma cells examined by atomic force microscopy and rheology.

    PubMed

    Goldmann, W H; Ezzell, R M

    1996-07-10

    We have been studying mouse F9 embryonic carcinoma cells which contain no detectable vinculin protein (5.51 cells), and compared them with F9 wild-type cells. Employing atomic force microscopy, we probed the elastic properties of individual F9 wild-type and 5.51 cells by measuring the dynamic response of controlled loads of the cantilever tip. An elastic modulus (Young) of approximately 3.8 and approximately 2.5 kPa was calculated for wild-type and 5.51 cells, respectively. Using disc rheometry, we detected a marked change in shear of a 1000g pellet of approximately 55 x 10(6) cells between wild-type and 5.51 mutants. These differences are attributed to the loss of vinculin and altered cytoskeletal organization in these cells. PMID:8660960

  13. Inducible differentiation and morphogenesis of bipotential liver cell lines from wild-type mouse embryos.

    PubMed

    Strick-Marchand, Hélène; Weiss, Mary C

    2002-10-01

    This work shows that hepatic cell lines reproducibly can be derived from E14 embryos of many mouse inbred strains. These bipotential mouse embryonic liver (BMEL) cell lines present a mixed morphology, containing both epithelial and palmate-like cells, and an uncoupled phenotype, expressing hepatocyte transcription factors (HNF1alpha, HNF4alpha, GATA4) but not functions (apolipoproteins, albumin). BMEL cells are bipotential: under inducing conditions they express hepatocyte and bile duct functions. In addition, they can undergo morphogenesis in Matrigel culture to form bile duct units. When returned to basal culture conditions, the differentiated cells revert, within a few days, to an undifferentiated state. The ensemble of markers expressed by BMEL cells implies that they originate from hepatoblasts, the endodermal precursors of the liver. In conclusion, the establishment of a simple and reproducible method to isolate from any mouse embryo bipotential hepatic cell lines that exhibit the properties of transit stem cells provides a novel paradigm for investigation of hepatic cell lineage relationships. PMID:12297826

  14. Transcriptional regulatory program in wild-type and retinoblastoma gene-deficient mouse embryonic fibroblasts during adipocyte differentiation

    PubMed Central

    2011-01-01

    Background Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb) was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs), and retinoblastoma gene-deficient MEFs (Rb-/- MEFs). Findings Comparative analysis of the expression profiles of 3T3-L1 cells and wild-type MEFs revealed genes involved specifically in early regulation of the adipocyte differentiation as well as secreted factors and signaling molecules regulating the later phase of differentiation. In an attempt to identify transcription factors regulating adipogenesis, bioinformatics analysis of the promoters of coordinately and highly expressed genes was performed. We were able to identify a number of high-confidence target genes for follow-up experimental studies. Additionally, combination of experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1. To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs) for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis of 64 deregulated genes showed that the Rb-/- MEF model exhibits a brown(-like) adipocyte phenotype. Additionally, the analysis results indicate a different or additional role for pRb family member involvement in the lineage commitment. Conclusion In this study a number of commonly modulated genes during adipogenesis in 3T3-L1 cells and MEFs, potential transcriptional regulation mechanisms, and differentially regulated targets during adipocyte differentiation of Rb

  15. Comparative microarray analysis of basal gene expression in mouse Hepa-1c1c7 wild-type and mutant cell lines.

    PubMed

    Fong, C J; Burgoon, L D; Zacharewski, T R

    2005-08-01

    Hepa-1c1c7 wild-type and benzo[a]pyrene-resistant derived mutant cell lines have been used to elucidate pathways and mechanisms involving the aryl hydrocarbon receptor (AhR). However, there has been little focus on other biological processes which may differ between the isolated lines. In this study, mouse cDNA microarrays representing 4858 genes were used to examine differences in basal gene expression between mouse Hepa-1c1c7 wild-type and c1 (truncated Cyp1a1 protein), c4 (AhR nuclear translocator, ARNT, deficient), and c12 (low AhR levels) mutant cell lines. Surprisingly, c1 mutants exhibited the greatest number of gene expression changes compared to wild-type cells, followed by c4 and c12 lines, respectively. Differences in basal gene expression were consistent with cell line specific variations in morphology, mitochondrial activity, and proliferation rate. MTT and direct cell count assays indicate both c4 and c12 mutants exhibit increased proliferative activity when compared to wild-type cells, while the c1 mutants exhibited decreased activity. This study further characterizes Hepa-1c1c7 wild-type and mutant cells and identifies significant differences in biological processes that should be considered when conducting comparative mechanistic studies with these lines. PMID:15888666

  16. Wild-type SOD1 overexpression accelerates disease onset of a G85R SOD1 mouse

    PubMed Central

    Wang, Lijun; Deng, Han-Xiang; Grisotti, Gabriella; Zhai, Hong; Siddique, Teepu; Roos, Raymond P.

    2009-01-01

    Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (FALS), and ∼25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase type 1 (SOD1). Mutant (MT) SOD1 is thought to be pathogenic because it misfolds and aggregates. A number of transgenic mice have been generated that express different MTSOD1s as transgenes and exhibit an ALS-like disease. Although one study found that overexpression of human wild-type (WT) SOD1 did not affect disease in G85R transgenic mice, more recent reports claim that overexpression of WTSOD1 in other MTSOD1 transgenic mice hastened disease, raising a possibility that the effect of WTSOD1 overexpression in this FALS mouse model is mutant-specific. In order to clarify this issue, we studied the effect of WTSOD1 overexpression in a G85R transgenic mouse that we recently generated. We found that G85R/WTSOD1 double transgenic mice had an acceleration of disease onset and shortened survival compared with G85R single transgenic mice; in addition, there was an earlier appearance of pathological and immunohistochemical abnormalities. The spinal cord insoluble fraction from G85R/WTSOD1 mice had evidence of G85R–WTSOD1 heterodimers and WTSOD1 homodimers (in addition to G85R homodimers) with intermolecular disulfide bond cross-linking. These studies suggest that WTSOD1 can be recruited into disease-associated aggregates by redox processes, providing an explanation for the accelerated disease seen in G85R mice following WTSOD1 overexpression, and suggesting the importance of incorrect disulfide-linked protein as key to MTSOD1 toxicity. PMID:19233858

  17. Susceptibility of Different Mouse Wild Type Strains to Develop Diet-Induced NAFLD/AFLD-Associated Liver Disease

    PubMed Central

    Fengler, Vera H. I.; Macheiner, Tanja; Kessler, Sonja M.; Czepukojc, Beate; Gemperlein, Katja; Müller, Rolf; Kiemer, Alexandra K.; Magnes, Christoph; Haybaeck, Johannes; Lackner, Carolin; Sargsyan, Karine

    2016-01-01

    Although non-alcoholic and alcoholic fatty liver disease have been intensively studied, concerning pathophysiological mechanisms are still incompletely understood. This may be due to the use of different animal models and resulting model-associated variation. Therefore, this study aimed to compare three frequently used wild type mouse strains in their susceptibility to develop diet-induced features of non-alcoholic/alcoholic fatty liver disease. Fatty liver disease associated clinical, biochemical, and histological features in C57BL/6, CD-1, and 129Sv WT mice were induced by (i) high-fat diet feeding, (ii) ethanol feeding only, and (iii) the combination of high-fat diet and ethanol feeding. Hepatic and subcutaneous adipose lipid profiles were compared in CD-1 and 129Sv mice. Additionally hepatic fatty acid composition was determined in 129Sv mice. In C57BL/6 mice dietary regimens resulted in heterogeneous hepatic responses, ranging from pronounced steatosis and inflammation to a lack of any features of fatty liver disease. Liver-related serum biochemistry showed high deviations within the regimen groups. CD-1 mice did not exhibit significant changes in metabolic and liver markers and developed no significant steatosis or inflammation as a response to dietary regimens. Although 129Sv mice showed no weight gain, this strain achieved most consistent features of fatty liver disease, apparent from concentration alterations of liver-related serum biochemistry as well as moderate steatosis and inflammation as a result of all dietary regimens. Furthermore, the hepatic lipid profile as well as the fatty acid composition of 129Sv mice were considerably altered, upon feeding the different dietary regimens. Accordingly, diet-induced non-alcoholic/alcoholic fatty liver disease is most consistently promoted in 129Sv mice compared to C57BL/6 and CD-1 mice. As a conclusion, this study demonstrates the importance of genetic background of used mouse strains for modeling diet

  18. CYP1A1 and CYP1A2 expression: comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines.

    PubMed

    Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W

    2009-05-15

    Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how "human-like" can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1_CYP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+)_severe-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs. PMID:19285097

  19. CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

    SciTech Connect

    Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

    2009-05-15

    Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1{sub C}YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+){sub s}evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.

  20. Patterns of differential gene expression in adult rotation-resistant and wild-type western corn rootworm digestive tracts

    PubMed Central

    Chu, Chia-Ching; Zavala, Jorge A; Spencer, Joseph L; Curzi, Matías J; Fields, Christopher J; Drnevich, Jenny; Siegfried, Blair D; Seufferheld, Manfredo J

    2015-01-01

    The western corn rootworm (WCR,Diabrotica virgifera virgifera LeConte) is an important pest of corn. Annual crop rotation between corn and soybean disrupts the corn-dependent WCR life cycle and is widely adopted to manage this pest. This strategy selected for rotation-resistant (RR) WCR with reduced ovipositional fidelity to corn. Previous studies revealed that RR-WCR adults exhibit greater tolerance of soybean diets, different gut physiology, and host–microbe interactions compared to rotation-susceptible wild types (WT). To identify the genetic mechanisms underlying these phenotypic changes, a de novo assembly of the WCR adult gut transcriptome was constructed and used for RNA-sequencing analyses of RNA libraries from different WCR phenotypes fed with corn or soybean diets. Global gene expression profiles of WT- and RR-WCR were similar when feeding on corn diets, but different when feeding on soybean. Using network-based methods, we identified gene modules transcriptionally correlated with the RR phenotype. Gene ontology enrichment analyses indicated that the functions of these modules were related to metabolic processes, immune responses, biological adhesion, and other functions/processes that appear to correlate to documented traits in RR populations. These results suggest that gut transcriptomic divergence correlated with brief soybean feeding and other physiological traits may exist between RR- and WT-WCR adults. PMID:26240606

  1. Patterns of differential gene expression in adult rotation-resistant and wild-type western corn rootworm digestive tracts.

    PubMed

    Chu, Chia-Ching; Zavala, Jorge A; Spencer, Joseph L; Curzi, Matías J; Fields, Christopher J; Drnevich, Jenny; Siegfried, Blair D; Seufferheld, Manfredo J

    2015-08-01

    The western corn rootworm (WCR,Diabrotica virgifera virgifera LeConte) is an important pest of corn. Annual crop rotation between corn and soybean disrupts the corn-dependent WCR life cycle and is widely adopted to manage this pest. This strategy selected for rotation-resistant (RR) WCR with reduced ovipositional fidelity to corn. Previous studies revealed that RR-WCR adults exhibit greater tolerance of soybean diets, different gut physiology, and host-microbe interactions compared to rotation-susceptible wild types (WT). To identify the genetic mechanisms underlying these phenotypic changes, a de novo assembly of the WCR adult gut transcriptome was constructed and used for RNA-sequencing analyses of RNA libraries from different WCR phenotypes fed with corn or soybean diets. Global gene expression profiles of WT- and RR-WCR were similar when feeding on corn diets, but different when feeding on soybean. Using network-based methods, we identified gene modules transcriptionally correlated with the RR phenotype. Gene ontology enrichment analyses indicated that the functions of these modules were related to metabolic processes, immune responses, biological adhesion, and other functions/processes that appear to correlate to documented traits in RR populations. These results suggest that gut transcriptomic divergence correlated with brief soybean feeding and other physiological traits may exist between RR- and WT-WCR adults. PMID:26240606

  2. Proteomic Analysis of Wild-type and Mutant Huntingtin-associated Proteins in Mouse Brains Identifies Unique Interactions and Involvement in Protein Synthesis*

    PubMed Central

    Culver, Brady P.; Savas, Jeffrey N.; Park, Sung K.; Choi, Jeong H.; Zheng, Shuqiu; Zeitlin, Scott O.; Yates, John R.; Tanese, Naoko

    2012-01-01

    Huntington disease is a neurodegenerative disorder caused by a CAG repeat amplification in the gene huntingtin (HTT) that is reflected by a polyglutamine expansion in the Htt protein. Nearly 20 years of research have uncovered roles for Htt in a wide range of cellular processes, and many of these discoveries stemmed from the identification of Htt-interacting proteins. However, no study has employed an impartial and comprehensive strategy to identify proteins that differentially associate with full-length wild-type and mutant Htt in brain tissue, the most relevant sample source to the disease condition. We analyzed Htt affinity-purified complexes from wild-type and HTT mutant juvenile mouse brain from two different biochemical fractions by tandem mass spectrometry. We compared variations in protein spectral counts relative to Htt to identify those proteins that are the most significantly contrasted between wild-type and mutant Htt purifications. Previously unreported Htt interactions with Myo5a, Prkra (PACT), Gnb2l1 (RACK1), Rps6, and Syt2 were confirmed by Western blot analysis. Gene Ontology analysis of these and other Htt-associated proteins revealed a statistically significant enrichment for proteins involved in translation among other categories. Furthermore, Htt co-sedimentation with polysomes in cytoplasmic mouse brain extracts is dependent upon the presence of intact ribosomes. Finally, wild-type or mutant Htt overexpression inhibits cap-dependent translation of a reporter mRNA in an in vitro system. Cumulatively, these data support a new role for Htt in translation and provide impetus for further study into the link between protein synthesis and Huntington disease pathogenesis. PMID:22556411

  3. Functional interaction between mouse erbB3 and wild-type rat c-neu in transgenic mouse mammary tumor cells

    PubMed Central

    Kim, Aeree; Liu, Bolin; Ordonez-Ercan, Dalia; Alvarez, Kathy M; Jones, Lynn D; McKimmey, Christine; Edgerton, Susan M; Yang, XiaoHe; Thor, Ann D

    2005-01-01

    Introduction Co-expression of several receptor tyrosine kinases (RTKs), including erbB2 and erbB3, is frequently identified in breast cancers. A member of the RTK family, the kinase-deficient erbB3 can activate downstream signaling via heterodimer formation with erbB2. We studied the expression of RTK receptors in mammary tumors from the wild-type (wt) rat c-neu transgenic model. We hypothesized that physical and functional interactions between the wt rat neu/ErbB2 transgene and mouse ErbB3-encoded proteins could occur, activating downstream signaling and promoting mammary oncogenesis. Methods Immunohistochemical and Western blot analyses were performed to study the expression of rat c-neu/ErbB2 and mouse erbB3 in mammary tumors and tumor-derived cell lines from the wt rat c-neu transgenic mice. Co-immunoprecipitation methods were employed to quantitate heterodimerization between the transgene-encoded protein erbB2 and the endogenous mouse erbB3. Tumor cell growth in response to growth factors, such as Heregulin (HRG), epidermal growth factor (EGF), or insulin-like growth factor-1 (IGF-1), was also studied. Post-HRG stimulation, activation of the RTK downstream signaling was determined by Western blot analyses using antibodies against phosphorylated Akt and mitogen-activated protein kinase (MAPK), respectively. Specific inhibitors were then used with cell proliferation assays to study the phosphoinositide-3 kinase (PI-3K)/Akt and MAPK kinase (MEK)/MAPK pathways as possible mechanisms of HRG-induced tumor cell proliferation. Results Mammary tumors and tumor-derived cell lines frequently exhibited elevated co-expression of erbB2 and erbB3. The transgene-encoded protein erbB2 formed a stable heterodimer complex with endogenous mouse erbB3. HRG stimulation promoted physical and functional erbB2/erbB3 interactions and tumor cell growth, whereas no response to EGF or IGF-1 was observed. HRG treatment activated both the Akt and MAPK pathways in a dose- and time

  4. Mouse model of the OPRM1 (A118G) polymorphism: differential heroin self-administration behavior compared with wild-type mice.

    PubMed

    Zhang, Yong; Picetti, Roberto; Butelman, Eduardo R; Ho, Ann; Blendy, Julie A; Kreek, Mary Jeanne

    2015-04-01

    Mu-opioid receptors (MOPRs) are the target of heroin and other prescription opioids, which are currently responsible for massive addiction morbidity in the US. The gene coding for the human MOPR (OPRM1) has an important functional single nucleotide polymorphism (SNP), A118G. The OPRM1 A118G genotype results in substantially increased risk of heroin addiction in humans; however, the neurobiological mechanism for this increased risk is not fully understood. This study examined heroin self-administration (SA) behavior in A112G (G/G) mice, harboring a functionally equivalent SNP in Oprm1 with a similar amino acid substitution, in extended (4 h) SA sessions. Adult male and female G/G mice and 'wild-type' litter mates (A/A) were allowed to self-administer heroin (0.25 mg/kg/unit dose, FR1 with a nose poke response) for 4 h/day, for 10 consecutive days. Half of the mice then continued in a heroin dose-response study, while extinction from heroin SA was studied in the other half. In vivo microdialysis was used to measure acute heroin-induced increases of striatal dopamine in the GG vs AA genotypes. Male and female G/G mice responded for heroin significantly more (and thus had greater intake) than A/A mice, in the initial 10 days of heroin SA, and in the subsequent dose-response study. There were no significant differences in extinction of SA between the A/A and G/G mice. Heroin-induced increases in striatal dopamine levels are higher in the GG mice than in the AA mice. Both male and female G/G mice self-administered more heroin than did A/A mice over a 10-day period, possibly because of the greater increases of heroin-induced striatal dopamine in the GG mice. Furthermore, G/G male mice escalated the amount of heroin self-administration across 10 extended-access sessions more than A/A male mice did. These are the first studies to examine the acquisition of heroin SA in this mouse model. These studies may lead to a better understanding of the neurobiological and behavioral

  5. Mouse Model of the OPRM1 (A118G) Polymorphism: Differential Heroin Self-Administration Behavior Compared with Wild-Type Mice

    PubMed Central

    Zhang, Yong; Picetti, Roberto; Butelman, Eduardo R; Ho, Ann; Blendy, Julie A; Kreek, Mary Jeanne

    2015-01-01

    Mu-opioid receptors (MOPRs) are the target of heroin and other prescription opioids, which are currently responsible for massive addiction morbidity in the US. The gene coding for the human MOPR (OPRM1) has an important functional single nucleotide polymorphism (SNP), A118G. The OPRM1 A118G genotype results in substantially increased risk of heroin addiction in humans; however, the neurobiological mechanism for this increased risk is not fully understood. This study examined heroin self-administration (SA) behavior in A112G (G/G) mice, harboring a functionally equivalent SNP in Oprm1 with a similar amino acid substitution, in extended (4 h) SA sessions. Adult male and female G/G mice and ‘wild-type' litter mates (A/A) were allowed to self-administer heroin (0.25 mg/kg/unit dose, FR1 with a nose poke response) for 4 h/day, for 10 consecutive days. Half of the mice then continued in a heroin dose–response study, while extinction from heroin SA was studied in the other half. In vivo microdialysis was used to measure acute heroin-induced increases of striatal dopamine in the GG vs AA genotypes. Male and female G/G mice responded for heroin significantly more (and thus had greater intake) than A/A mice, in the initial 10 days of heroin SA, and in the subsequent dose–response study. There were no significant differences in extinction of SA between the A/A and G/G mice. Heroin-induced increases in striatal dopamine levels are higher in the GG mice than in the AA mice. Both male and female G/G mice self-administered more heroin than did A/A mice over a 10-day period, possibly because of the greater increases of heroin-induced striatal dopamine in the GG mice. Furthermore, G/G male mice escalated the amount of heroin self-administration across 10 extended-access sessions more than A/A male mice did. These are the first studies to examine the acquisition of heroin SA in this mouse model. These studies may lead to a better understanding of the neurobiological and

  6. Prednisolone-induced differential gene expression in mouse liver carrying wild type or a dimerization-defective glucocorticoid receptor

    PubMed Central

    2010-01-01

    Background Glucocorticoids (GCs) control expression of a large number of genes via binding to the GC receptor (GR). Transcription may be regulated either by binding of the GR dimer to DNA regulatory elements or by protein-protein interactions of GR monomers with other transcription factors. Although the type of regulation for a number of individual target genes is known, the relative contribution of both mechanisms to the regulation of the entire transcriptional program remains elusive. To study the importance of GR dimerization in the regulation of gene expression, we performed gene expression profiling of livers of prednisolone-treated wild type (WT) and mice that have lost the ability to form GR dimers (GRdim). Results The GR target genes identified in WT mice were predominantly related to glucose metabolism, the cell cycle, apoptosis and inflammation. In GRdim mice, the level of prednisolone-induced gene expression was significantly reduced compared to WT, but not completely absent. Interestingly, for a set of genes, involved in cell cycle and apoptosis processes and strongly related to Foxo3a and p53, induction by prednisolone was completely abolished in GRdim mice. In contrast, glucose metabolism-related genes were still modestly upregulated in GRdim mice upon prednisolone treatment. Finally, we identified several novel GC-inducible genes from which Fam107a, a putative histone acetyltransferase complex interacting protein, was most strongly dependent on GR dimerization. Conclusions This study on prednisolone-induced effects in livers of WT and GRdim mice identified a number of interesting candidate genes and pathways regulated by GR dimers and sheds new light onto the complex transcriptional regulation of liver function by GCs. PMID:20525385

  7. Comparative Analyses of Transcriptional Profiles in Mouse Organs Using a Pneumonic Plague Model after Infection with Wild-Type Yersinia pestis CO92 and Its Braun Lipoprotein Mutant

    PubMed Central

    Galindo, Cristi L.; Moen, Scott T.; Kozlova, Elena V.; Sha, Jian; Garner, Harold R.; Agar, Stacy L.; Chopra, Ashok K.

    2009-01-01

    We employed Murine GeneChips to delineate the global transcriptional profiles of the livers, lungs, and spleens in a mouse pneumonic plague infection model with wild-type (WT) Y. pestis CO92 and its Braun lipoprotein (Δlpp) mutant with reduced virulence. These organs showed differential transcriptional responses to infection with WT Y. pestis, but the overall host functional processes affected were similar across all three tissues. Gene expression alterations were found in inflammation, cytokine signaling, and apoptotic cell death-associated genes. Comparison of WT and Δlpp mutant-infected mice indicated significant overlap in lipopolysaccharide- (LPS-) associated gene expression, but the absence of Lpp perturbed host cell signaling at critical regulatory junctions resulting in altered immune response and possibly host cell apoptosis. We generated a putative signaling pathway including major inflammatory components that could account for the synergistic action of LPS and Lpp and provided the mechanistic basis of attenuation caused by deletion of the lpp gene from Y. pestis in a mouse model of pneumonic plague. PMID:20145715

  8. Differential requirement for wild-type Flt3 in leukemia-initiation among mouse models of human leukemia

    PubMed Central

    Kamezaki, Kenjiro; Luchsinger, Larry; Snoeck, Hans-Willem

    2014-01-01

    FLT3 is one of the most frequently mutated genes in acute leukemias. However, the role in leukemogenesis of wt Flt3, which is highly expressed in many hematological malignancies, is unclear. We show here that in mouse models established by retroviral transduction of leukemic fusion proteins deletion of Flt3 strongly inhibits MLL-ENL and to lesser extent p210BCR-ABL-induced leukemogenesis, but has no effect in MLL-AF9 or AML1-ETO9a models. Flt3 acts at the level of leukemic stem cells (LSCs), as a fraction of LSCs in MLL-ENL, but not in MLL-AF9-induced leukemia, expressed Flt3 in vivo, and Flt3 expression on LSCs was associated with leukemia development in this model. Furthermore, efficiency of MLL-ENL, but not of MLL-AF9-induced leukemia induction was significantly enhanced after transduction of Flt3+ compared to Flt3− wt myeloid progenitors. However, Flt3 is not required for immortalization of bone marrow cells in vitro by MLL-ENL and does not affect colony-formation by MLL-ENL LSCs in vitro, suggesting that in vitro models do not reflect the in vivo biology of MLL-ENL leukemia with respect to Flt3 requirement. We conclude that wt Flt3 plays a role in leukemia initiation in vivo, which is, however, not universal. PMID:24269847

  9. Comparison of dendritic calcium transients in juvenile wild type and SOD1(G93A) mouse lumbar motoneurons.

    PubMed

    Quinlan, Katharina A; Lamano, Jonathan B; Samuels, Julienne; Heckman, C J

    2015-01-01

    Previous studies of spinal motoneurons in the SOD1 mouse model of amyotrophic lateral sclerosis have shown alterations long before disease onset, including increased dendritic branching, increased persistent Na(+) and Ca(2+) currents, and impaired axonal transport. In this study dendritic Ca(2+) entry was investigated using two photon excitation fluorescence microscopy and whole-cell patch-clamp of juvenile (P4-11) motoneurons. Neurons were filled with both Ca(2+) Green-1 and Texas Red dextrans, and line scans performed throughout. Steps were taken to account for different sources of variability, including (1) dye filling and laser penetration, (2) dendritic anatomy, and (3) the time elapsed from the start of recording. First, Ca(2+) Green-1 fluorescence was normalized by Texas Red; next, neurons were reconstructed so anatomy could be evaluated; finally, time was recorded. Customized software detected the largest Ca(2+) transients (area under the curve) from each line scan and matched it with parameters above. Overall, larger dendritic diameter and shorter path distance from the soma were significant predictors of larger transients, while time was not significant up to 2 h (data thereafter was dropped). However, Ca(2+) transients showed additional variability. Controlling for previous factors, significant variation was found between Ca(2+) signals from different processes of the same neuron in 3/7 neurons. This could reflect differential expression of Ca(2+) channels, local neuromodulation or other variations. Finally, Ca(2+) transients in SOD1(G93A) motoneurons were significantly smaller than in non-transgenic motoneurons. In conclusion, motoneuron processes show highly variable Ca(2+) transients, but these transients are smaller overall in SOD1(G93A) motoneurons. PMID:25914627

  10. Expression pattern of matrix metalloproteinase and TIMP genes in fibroblasts derived from Ets-1 knock-out mice compared to wild-type mouse fibroblasts.

    PubMed

    Hahne, Jens Claus; Fuchs, Tanja; El Mustapha, Haddouti; Okuducu, Ali Fuat; Bories, Jean Christophe; Wernert, Nicolas

    2006-07-01

    Matrix-degrading proteases play a key role in normal development, wound healing, many diseases such as rheumatoid arthritis and, in particular, tumour invasion. In invasive tumours, these enzymes are expressed by fibroblasts of the tumour stroma. Their expression and activity are tightly regulated at several levels, an important one being transcription. Previous in vitro and in vivo findings pointed to a major role of the Ets-1 transcription factor for this level of regulation. In the present study, we tried to prove this role in fibroblasts. We stimulated wild-type mouse fibroblasts with physiological doses of basic fibroblast growth factor (bFGF, known to induce different proteases and expressed by tumour cells) and compared the results to those obtained in Ets-1 -/- fibroblasts derived from Ets-1 knock-out mice. We found that basal Ets-1 levels are necessary not only for a fast induction of MMPs 2, 3 and 13 by bFGF but also for maintenance of the bFGF-induced expression of tissue inhibitors of metalloproteinases (TIMPs) 1, 2 and 3, which are known not only to inhibit but also participate as activators of certain pro-MMPs. PMID:16786167

  11. Mitochondria are more numerous and smaller in pink-eyed dilution melanoblasts and melanocytes than in wild-type melanocytes in the neonatal mouse epidermis.

    PubMed

    Hirobe, Tomohisa; Ishizuka, Kenji; Ogawa, Shigeru; Abe, Hiroyuki

    2008-11-01

    Abstract The mouse pink-eyed dilution (p) locus is known to control the melanin content in melanocytes. However, it was not known whether the p gene is involved in regulating the proliferation and differentation of melanocytes during development, especially the biogenesis of melanosomes and other organelles. Epidermal cell suspensions of neonatal dorsal skin derived from mice wild type for the p locus (black, C57BL/10JHir-P/P) and their congenic mutant phenotype (pink-eyed dilution, C57BL/10JHir-p/p) were cultured in serum-free melanocyte-proliferation medium (MDMD). The supplement of additional L-tyrosine (Tyr) into the MDMD stimulated the differentiation of p/p melanoblasts into melanocytes. Electron microscopy revealed that in p/p melanoblasts and melanocytes treated with L-Tyr, the number of stage II and III melanosomes dramatically increased. Moreover, p/p melanoblasts possessed smaller but more numerous mitochondria than P/P melanocytes. The treatment of p/p melanoblasts and melanocytes with L-Tyr decreased the number of mitochondria. The supplement of 2, 4-dinitrophenol (DNP), an inhibitor of mitochondrial function, into the MDMD stimulated both the proliferation and differentiation of p/p melanoblasts. Simultaneous treatment of DNP and L-Tyr dramatically stimulated the differetiation of p/p melanocytes. These results suggest that L-Tyr and some unknown factors related to mitochondrial function may influence the differentiation of melanoblasts in the epidermis of p/p mice. PMID:19267617

  12. Mouse model of human RPE65 P25L hypomorph resembles wild type under normal light rearing but is fully resistant to acute light damage.

    PubMed

    Li, Yan; Yu, Shirley; Duncan, Todd; Li, Yichao; Liu, Pinghu; Gene, Erelda; Cortes-Pena, Yoel; Qian, Haohua; Dong, Lijin; Redmond, T Michael

    2015-08-01

    Human RPE65 mutations cause a spectrum of blinding retinal dystrophies from severe early-onset disease to milder manifestations. The RPE65 P25L missense mutation, though having <10% of wild-type (WT) activity, causes relatively mild retinal degeneration. To better understand these mild forms of RPE65-related retinal degeneration, and their effect on cone photoreceptor survival, we generated an Rpe65/P25L knock-in (KI/KI) mouse model. We found that, when subject to the low-light regime (∼100 lux) of regular mouse housing, homozygous Rpe65/P25L KI/KI mice are morphologically and functionally very similar to WT siblings. While mutant protein expression is decreased by over 80%, KI/KI mice retinae retain comparable 11-cis-retinal levels with WT. Consistently, the scotopic and photopic electroretinographic (ERG) responses to single-flash stimuli also show no difference between KI/KI and WT mice. However, the recovery of a-wave response following moderate visual pigment bleach is delayed in KI/KI mice. Importantly, KI/KI mice show significantly increased resistance to high-intensity (20 000 lux for 30 min) light-induced retinal damage (LIRD) as compared with WT, indicating impaired rhodopsin regeneration in KI/KI. Taken together, the Rpe65/P25L mutant produces sufficient chromophore under normal conditions to keep opsins replete and thus manifests a minimal phenotype. Only when exposed to intensive light is this hypomorphic mutation manifested physiologically, as its reduced expression and catalytic activity protects against the successive cycles of opsin regeneration underlying LIRD. These data also help define minimal requirements of chromophore for photoreceptor survival in vivo and may be useful in assessing a beneficial therapeutic dose for RPE65 gene therapy in humans. PMID:25972377

  13. Aerobic Glycolysis in the Frontal Cortex Correlates with Memory Performance in Wild-Type Mice But Not the APP/PS1 Mouse Model of Cerebral Amyloidosis

    PubMed Central

    Harris, Richard A.; Tindale, Lauren; Lone, Asad; Singh, Olivia; Macauley, Shannon L.; Stanley, Molly; Holtzman, David M.; Bartha, Robert

    2016-01-01

    regions of the brain. Here we detected an age-dependent decline in the expression of aerobic glycolysis enzymes and a concomitant decrease in lactate levels within the frontal cortex of wild-type mice. Improved memory performance in wild-type mice correlated with elevated expression of aerobic glycolysis enzymes. Surprisingly, lactate levels remained elevated with age and increased aerobic glycolysis enzyme expression correlated with poorer memory performance in APP/PS1 mice. These findings suggest that while lactate production is beneficial for memory in the healthy aging brain, it might be detrimental in an Alzheimer's disease context. PMID:26865611

  14. Larval Population Density Alters Adult Sleep in Wild-Type Drosophila melanogaster but Not in Amnesiac Mutant Flies

    PubMed Central

    Chi, Michael W.; Griffith, Leslie C.; Vecsey, Christopher G.

    2014-01-01

    Sleep has many important biological functions, but how sleep is regulated remains poorly understood. In humans, social isolation and other stressors early in life can disrupt adult sleep. In fruit flies housed at different population densities during early adulthood, social enrichment was shown to increase subsequent sleep, but it is unknown if population density during early development can also influence adult sleep. To answer this question, we maintained Drosophila larvae at a range of population densities throughout larval development, kept them isolated during early adulthood, and then tested their sleep patterns. Our findings reveal that flies that had been isolated as larvae had more fragmented sleep than those that had been raised at higher population densities. This effect was more prominent in females than in males. Larval population density did not affect sleep in female flies that were mutant for amnesiac, which has been shown to be required for normal memory consolidation, adult sleep regulation, and brain development. In contrast, larval population density effects on sleep persisted in female flies lacking the olfactory receptor or83b, suggesting that olfactory signals are not required for the effects of larval population density on adult sleep. These findings show that population density during early development can alter sleep behavior in adulthood, suggesting that genetic and/or structural changes are induced by this developmental manipulation that persist through metamorphosis. PMID:25116571

  15. Piper betle induces phase I & II genes through Nrf2/ARE signaling pathway in mouse embryonic fibroblasts derived from wild type and Nrf2 knockout cells

    PubMed Central

    2014-01-01

    Background Nuclear factor-erythroid 2 p45 related factor 2 (Nrf2) is a primary transcription factor, protecting cells from oxidative stress by regulating a number of antioxidants and phase II detoxifying enzymes. Dietary components such as sulforaphane in broccoli and quercetin in onions have been shown to be inducers of Nrf2. Piper betle (PB) grows well in tropical climate and the leaves are used in a number of traditional remedies for the treatment of stomach ailments and infections among Asians. The aim of this study was to elucidate the effect of Piper betle (PB) leaves extract in Nrf2 signaling pathway by using 2 types of cells; mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) and Nrf2 knockout (N0) mice. Methods WT and N0 cells were treated with 5 and 10 μg/ml of PB for 10 and 12-h for the determination of nuclear translocation of Nrf2 protein. Luciferase reporter gene activity was performed to evaluate the antioxidant response element (ARE)-induction by PB. Real-time PCR and Western blot were conducted on both WT and N0 cells after PB treatment for the determination of antioxidant enzymes [superoxide dismutase (SOD1) and heme-oxygenase (HO-1)], phase I oxidoreductase enzymes [NAD(P)H: quinone oxidoreductase (NQO1)] and phase II detoxifying enzyme [glutathione S-transferase (GST)]. Results Nuclear translocation of Nrf2 by PB in WT cells was better after 10 h incubation compared to 12 h. Real time PCR and Western blot analysis showed increased expressions of Nrf2, NQO1 and GSTA1 genes with corresponding increases in glutathione, NQO1 and HO-1 proteins in WT cells. Reporter gene ARE was stimulated by PB as shown by ARE/luciferase assay. Interestingly, PB induced SOD1 gene and protein expressions in N0 cells but not in WT cells. Conclusion The results of this study confirmed that PB activated Nrf2-ARE signaling pathway which subsequently induced some phase I oxidoreductase, phase II detoxifying and antioxidant genes expression via ARE reporter

  16. A human papillomavirus type 18 E6/E7 transgene sensitizes mouse lens cells to human wild-type p53-mediated apoptosis.

    PubMed

    Nakamura, T; Williams-Simons, L; Westphal, H

    1997-06-26

    We have studied the concerted action of factors that influence the balance between cell proliferation and cell death in the developing lens of transgenic mice. We show that a human papillomavirus type 18 (HPV18) E6/E7 transgene that predominantly expresses the viral E7 gene product triggers apoptosis in a dose dependent manner, and causes retardation of lens growth or microphakia. E7 is known to inactivate pRB, the product of the retinoblastoma gene, and to enhance the action of p53. Our earlier work had demonstrated that over-expression of p53 itself can cause apoptosis of lens cells, and that a mutant p53 allele can interfere with this process. In the present study, we examined lenses that simultaneously express different constellations of the HPV18 E6/E7, wild-type and mutant human p53, and wild-type human pRB transgenes. We observed that lens cells expressing the HPV18 transgene are more sensitive to wild-type human p53 action than normal lens cells. As a result, there is severe microphakia in lenses that express both the HPV18 and the wild-type p53 transgenes. By contrast, apoptosis was reduced in lenses that co-expressed the HPV18 and either the pRB or the mutant p53 transgene. We conclude that levels of wild-type p53 are critical, and that any excess of p53 or suppression of pRB can cause cell death. Our results encourage attempts to counteract the deleterious action of human papillomaviruses in cervical cancer by a combination of measures that decrease cell proliferation and enhance apoptosis. PMID:9223662

  17. Early Life Inorganic Lead Exposure Induces Testicular Teratoma and Renal and Urinary Bladder Preneoplasia in Adult Metallothionein-Knockout Mice but Not in Wild Type Mice

    PubMed Central

    Tokar, Erik J.; Diwan, Bhalchandra A.; Waalkes, Michael P.

    2010-01-01

    Inorganic lead compounds are carcinogenic in animals and have carcinogenic potential in humans. In mice, lead (Pb) is a transplacental carcinogen in the kidney. Metallothionein (MT) is a metal-binding protein that can reduce the toxicity of various metals, including Pb, either by direct sequestration or as an antioxidant for metals that generate reactive oxygen species. Although MT appears to reduce Pb carcinogenicity in adult mice it is unknown how MT deficiency may affect Pb carcinogenicity from early life exposure. Thus, groups (n = 10) of pregnant MT-I/II double knockout (MT-null) or 129/SVJ MT wild type (WT) mice were exposed to Pb acetate in the drinking water (0, 2000, 4000 ppm Pb) from gestation day 8 through birth and during lactation. Maternal drinking water Pb exposure continued to weaning at 4 weeks of age and the male offspring were then directly exposed to Pb until 8 weeks of age and observed until 2 years old. High dose (4000 ppm) but not low dose (2000 ppm) Pb reduced survival in the latter part of the study in both MT-null and WT mice. In MT-null mice, but not WT, early life Pb exposure caused a dose-related increase in testicular teratomas, to a maximum incidence of 28% compared to control (4%). Pb-induced renal cystic hyperplasia, considered preneoplastic, were a prominent occurrence in MT-null mice but nearly absent in WT mice. Pb dose-related increases in renal cystic hyperplasia occurred in adult MT-null with early life exposure with maximal incidence of 52%. Pb-treated MT-null mice also showed dose-related increases in urinary bladder hyperplasia with occasional papilloma that were absent in WT mice. Thus, MT deficiency made mice more sensitive to early life Pb exposure with regard to testes tumors, and renal and urinary bladder preneoplastic lesions. PMID:20600549

  18. Action Potentials are required for nitric oxide dependent LTP in CA1 neurons of adult GluR1 knockout and Wild-type mice

    PubMed Central

    Phillips, Keith G.; Hardingham, Neil R.; Fox, Kevin

    2009-01-01

    Neocortical LTP consists of both pre- and postsynaptic components that rely on nitric oxide (NO) and GluR1 respectively. In this study, we found that hippocampal LTP, induced by theta-burst stimulation in mature (> 8 week old) GluR1 knockout mice was almost entirely NO-dependent and involved both the α splice variant of NO synthase-1 (αNOS-1) and the NO synthase-3 (NOS-3) isoforms of NO synthase. Theta-burst induced LTP was also partly NO-dependent in wild-type mice, and made up approximately 50% of the potentiation 2 hours post-tetanus. Theta-burst stimulation reliably produced postsynaptic spikes including a high probability of complex spikes. Inhibition of postsynaptic somatic spikes with intracellular QX314 or local TTX application prevented LTP in the GluR1 knockout mice and also blocked the NO-component of LTP in wild-types. We conclude that theta-burst stimulation is particularly well suited to producing the somatic postsynaptic spikes required for NO-dependent LTP. PMID:19109486

  19. An Escherichia coli Nissle 1917 missense mutant colonizes the streptomycin-treated mouse intestine better than the wild type but is not a better probiotic.

    PubMed

    Adediran, Jimmy; Leatham-Jensen, Mary P; Mokszycki, Matthew E; Frimodt-Møller, Jakob; Krogfelt, Karen A; Kazmierczak, Krystyna; Kenney, Linda J; Conway, Tyrrell; Cohen, Paul S

    2014-02-01

    Previously we reported that the streptomycin-treated mouse intestine selected for two different Escherichia coli MG1655 mutants with improved colonizing ability: nonmotile E. coli MG1655 flhDC deletion mutants that grew 15% faster in vitro in mouse cecal mucus and motile E. coli MG1655 envZ missense mutants that grew slower in vitro in mouse cecal mucus yet were able to cocolonize with the faster-growing flhDC mutants. The E. coli MG1655 envZ gene encodes a histidine kinase that is a member of the envZ-ompR two-component signal transduction system, which regulates outer membrane protein profiles. In the present investigation, the envZP41L gene was transferred from the intestinally selected E. coli MG1655 mutant to E. coli Nissle 1917, a human probiotic strain used to treat gastrointestinal infections. Both the E. coli MG1655 and E. coli Nissle 1917 strains containing envZP41L produced more phosphorylated OmpR than their parents. The E. coli Nissle 1917 strain containing envZP41L also became more resistant to bile salts and colicin V and grew 50% slower in vitro in mucus and 15% to 30% slower on several sugars present in mucus, yet it was a 10-fold better colonizer than E. coli Nissle 1917. However, E. coli Nissle 1917 envZP41L was not better at preventing colonization by enterohemorrhagic E. coli EDL933. The data can be explained according to our "restaurant" hypothesis for commensal E. coli strains, i.e., that they colonize the intestine as sessile members of mixed biofilms, obtaining the sugars they need for growth locally, but compete for sugars with invading E. coli pathogens planktonically. PMID:24478082

  20. Dietary DHA supplementation causes selective changes in phospholipids from different brain regions in both wild type mice and the Tg2576 mouse model of Alzheimer's disease.

    PubMed

    Bascoul-Colombo, Cécile; Guschina, Irina A; Maskrey, Benjamin H; Good, Mark; O'Donnell, Valerie B; Harwood, John L

    2016-06-01

    Alzheimer's disease (AD) is of major concern in ageing populations and we have used the Tg2576 mouse model to understand connections between brain lipids and amyloid pathology. Because dietary docosahexaenoic acid (DHA) has been identified as beneficial, we compared mice fed with a DHA-supplemented diet to those on a nutritionally-sufficient diet. Major phospholipids from cortex, hippocampus and cerebellum were separated and analysed. Each phosphoglyceride had a characteristic fatty acid composition which was similar in cortex and hippocampus but different in the cerebellum. The biggest changes on DHA-supplementation were within ethanolamine phospholipids which, together with phosphatidylserine, had the highest proportions of DHA. Reciprocal alterations in DHA and arachidonate were found. The main diet-induced alterations were found in ethanolamine phospholipids, (and included their ether derivatives), as were the changes observed due to genotype. Tg mice appeared more sensitive to diet with generally lower DHA percentages when on the standard diet and higher relative proportions of DHA when the diet was supplemented. All four major phosphoglycerides analysed showed age-dependent decreases in polyunsaturated fatty acid contents. These data provide, for the first time, a detailed evaluation of phospholipids in different brain areas previously shown to be relevant to behaviour in the Tg2576 mouse model for AD. The lipid changes observed with genotype are consistent with the subtle alterations found in AD patients, especially for the ethanolamine phospholipid molecular species. They also emphasise the contrasting changes in fatty acid content induced by DHA supplementation within individual phospholipid classes. PMID:26968097

  1. Dietary DHA supplementation causes selective changes in phospholipids from different brain regions in both wild type mice and the Tg2576 mouse model of Alzheimer's disease

    PubMed Central

    Bascoul-Colombo, Cécile; Guschina, Irina A.; Maskrey, Benjamin H.; Good, Mark; O'Donnell, Valerie B.; Harwood, John L.

    2016-01-01

    Alzheimer's disease (AD) is of major concern in ageing populations and we have used the Tg2576 mouse model to understand connections between brain lipids and amyloid pathology. Because dietary docosahexaenoic acid (DHA) has been identified as beneficial, we compared mice fed with a DHA-supplemented diet to those on a nutritionally-sufficient diet. Major phospholipids from cortex, hippocampus and cerebellum were separated and analysed. Each phosphoglyceride had a characteristic fatty acid composition which was similar in cortex and hippocampus but different in the cerebellum. The biggest changes on DHA-supplementation were within ethanolamine phospholipids which, together with phosphatidylserine, had the highest proportions of DHA. Reciprocal alterations in DHA and arachidonate were found. The main diet-induced alterations were found in ethanolamine phospholipids, (and included their ether derivatives), as were the changes observed due to genotype. Tg mice appeared more sensitive to diet with generally lower DHA percentages when on the standard diet and higher relative proportions of DHA when the diet was supplemented. All four major phosphoglycerides analysed showed age-dependent decreases in polyunsaturated fatty acid contents. These data provide, for the first time, a detailed evaluation of phospholipids in different brain areas previously shown to be relevant to behaviour in the Tg2576 mouse model for AD. The lipid changes observed with genotype are consistent with the subtle alterations found in AD patients, especially for the ethanolamine phospholipid molecular species. They also emphasise the contrasting changes in fatty acid content induced by DHA supplementation within individual phospholipid classes. PMID:26968097

  2. Complete Genome Sequence and Comparative Analysis of the Wild-type Commensal Escherichia coli Strain SE11 Isolated from a Healthy Adult

    PubMed Central

    Oshima, Kenshiro; Toh, Hidehiro; Ogura, Yoshitoshi; Sasamoto, Hiroyuki; Morita, Hidetoshi; Park, Sang-Hee; Ooka, Tadasuke; Iyoda, Sunao; Taylor, Todd D.; Hayashi, Tetsuya; Itoh, Kikuji; Hattori, Masahira

    2008-01-01

    We sequenced and analyzed the genome of a commensal Escherichia coli (E. coli) strain SE11 (O152:H28) recently isolated from feces of a healthy adult and classified into E. coli phylogenetic group B1. SE11 harbored a 4.8 Mb chromosome encoding 4679 protein-coding genes and six plasmids encoding 323 protein-coding genes. None of the SE11 genes had sequence similarity to known genes encoding phage- and plasmid-borne virulence factors found in pathogenic E. coli strains. The comparative genome analysis with the laboratory strain K-12 MG1655 identified 62 poorly conserved genes between these two non-pathogenic strains and 1186 genes absent in MG1655. These genes in SE11 were mostly encoded in large insertion regions on the chromosome or in the plasmids, and were notably abundant in genes of fimbriae and autotransporters, which are cell surface appendages that largely contribute to the adherence ability of bacteria to host cells and bacterial conjugation. These data suggest that SE11 may have evolved to acquire and accumulate the functions advantageous for stable colonization of intestinal cells, and that the adhesion-associated functions are important for the commensality of E. coli in human gut habitat. PMID:18931093

  3. The effect of mild traumatic brain injury on peripheral nervous system pathology in wild-type mice and the G93A mutant mouse model of motor neuron disease.

    PubMed

    Evans, T M; Jaramillo, C A; Sataranatarajan, K; Watts, L; Sabia, M; Qi, W; Van Remmen, H

    2015-07-01

    Traumatic brain injury (TBI) is associated with a risk of neurodegenerative disease. Some suggest a link between TBI and motor neuron disease (MND), including amyotrophic lateral sclerosis (ALS). To investigate the potential mechanisms linking TBI to MND, we measured motor function and neuropathology following mild-TBI in wild-type and a transgenic model of ALS, G93A mutant mice. Mild-TBI did not alter the lifespan of G93A mice or age of onset; however, rotarod performance was impaired in G93A verses wild-type mice. Grip strength was reduced only in G93A mice after mild-TBI. Increased electromyography (EMG) abnormalities and markers of denervation (AchR, Runx1) indicate that mild-TBI may result in peripheral effects that are exaggerated in G93A mice. Markers of inflammation (cell edema, astrogliosis and microgliosis) were detected at 24 and 72h in the brain and spinal cord in wild-type and G93A mice. Levels of F2-isoprostanes, a marker of oxidative stress, were increased in the spinal cord 24h post mild-TBI in wild-type mice but were not affected by TBI in G93A mice. In summary, our data demonstrate that mild-TBI induces inflammation and oxidative stress and negatively impacts muscle denervation and motor performance, suggesting mild-TBI can potentiate motor neuron pathology and influence the development of MND in mice. PMID:25921732

  4. Cathepsin B-dependent motor neuron death after nerve injury in the adult mouse

    SciTech Connect

    Sun, Li; Wu, Zhou; Baba, Masashi; Peters, Christoph; Uchiyama, Yasuo; Nakanishi, Hiroshi

    2010-08-27

    Research highlights: {yields} Cathepsin B (CB), a lysosomal cysteine protease, is expressed in neuron and glia. {yields} CB increased in hypogrossal nucleus neurons after nerve injury in adult mice. {yields} CB-deficiency significantly increased the mean survival ratio of injured neurons. {yields} Thus, CB plays a critical role in axotomy-induced neuronal death in adult mice. -- Abstract: There are significant differences in the rate of neuronal death after peripheral nerve injury between species. The rate of neuronal death of motor neurons after nerve injury in the adult rats is very low, whereas that in adult mice is relatively high. However, the understanding of the mechanism underlying axotomy-induced motor neuron death in adult mice is limited. Cathepsin B (CB), a typical cysteine lysosomal protease, has been implicated in three major morphologically distinct pathways of cell death; apoptosis, necrosis and autophagic cell death. The possible involvement of CB in the neuronal death of hypogrossal nucleus (HGN) neurons after nerve injury in adult mice was thus examined. Quantitative analyses showed the mean survival ratio of HGN neurons in CB-deficient (CB-/-) adult mice after nerve injury was significantly greater than that in the wild-type mice. At the same time, proliferation of microglia in the injured side of the HGN of CB-/- adult mice was markedly reduced compared with that in the wild-type mice. On the injured side of the HGN in the wild-type adult mice, both pro- and mature forms of CB markedly increased in accordance with the increase in the membrane-bound form of LC3 (LC3-II), a marker protein of autophagy. Furthermore, the increase in CB preceded an increase in the expression of Noxa, a major executor for axotomy-induced motor neuron death in the adult mouse. Conversely, expression of neither Noxa or LC3-II was observed in the HGN of adult CB-/- mice after nerve injury. These observations strongly suggest that CB plays a critical role in axotomy

  5. Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras.

    PubMed

    Keighren, Margaret A; Flockhart, Jean H; West, John D

    2016-01-01

    The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1(-/-) null mouse embryos die but a previous study showed that some homozygous Gpi1(-/-) null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1(-/-)↔Gpi1(c/c) chimaera with functional Gpi1(-/-) null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1(-/-) null cells in adult Gpi1(-/-)↔Gpi1(c/c) chimaeras and determine if Gpi1(-/-) null germ cells are functional. Analysis of adult Gpi1(-/-)↔Gpi1(c/c) chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1(-/-) null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1(-/-) null oocytes in one female Gpi1(-/-)↔Gpi1(c/c) chimaera were functional and provided preliminary evidence that one male putative Gpi1(-/-)↔Gpi1(c/c) chimaera produced functional spermatozoa from homozygous Gpi1(-/-) null germ cells. Although the male chimaera was almost certainly Gpi1(-/-)↔Gpi1(c/c), this part of the study is considered preliminary because only blood was typed for GPI. Gpi1(-/-) null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1(-/-) null germ cells, it successfully identified functional Gpi1(-/-) null oocytes and revealed that some Gpi1(-/-) null cells could survive in many adult tissues. PMID:27103217

  6. Hair cell replacement in adult mouse utricles after targeted ablation of hair cells with diphtheria toxin.

    PubMed

    Golub, Justin S; Tong, Ling; Ngyuen, Tot B; Hume, Cliff R; Palmiter, Richard D; Rubel, Edwin W; Stone, Jennifer S

    2012-10-24

    We developed a transgenic mouse to permit conditional and selective ablation of hair cells in the adult mouse utricle by inserting the human diphtheria toxin receptor (DTR) gene into the Pou4f3 gene, which encodes a hair cell-specific transcription factor. In adult wild-type mice, administration of diphtheria toxin (DT) caused no significant hair cell loss. In adult Pou4f3(+/DTR) mice, DT treatment reduced hair cell numbers to 6% of normal by 14 days post-DT. Remaining hair cells were located primarily in the lateral extrastriola. Over time, hair cell numbers increased in these regions, reaching 17% of untreated Pou4f3(+/DTR) mice by 60 days post-DT. Replacement hair cells were morphologically distinct, with multiple cytoplasmic processes, and displayed evidence for active mechanotransduction channels and synapses characteristic of type II hair cells. Three lines of evidence suggest replacement hair cells were derived via direct (nonmitotic) transdifferentiation of supporting cells: new hair cells did not incorporate BrdU, supporting cells upregulated the pro-hair cell gene Atoh1, and supporting cell numbers decreased over time. This study introduces a new method for efficient conditional hair cell ablation in adult mouse utricles and demonstrates that hair cells are spontaneously regenerated in vivo in regions where there may be ongoing hair cell turnover. PMID:23100430

  7. Podocyte-Specific Overexpression of Wild Type or Mutant Trpc6 in Mice Is Sufficient to Cause Glomerular Disease

    PubMed Central

    Kairath, Pamela; Carmona-Mora, Paulina; Molina, Jessica; Carpio, J. Daniel; Ruiz, Phillip; Mezzano, Sergio A.; Li, Jing; Wei, Changli; Reiser, Jochen; Young, Juan I.; Walz, Katherina

    2010-01-01

    Mutations in the TRPC6 calcium channel (Transient receptor potential channel 6) gene have been associated with familiar forms of Focal and Segmental Glomerulosclerosis (FSGS) affecting children and adults. In addition, acquired glomerular diseases are associated with increased expression levels of TRPC6. However, the exact role of TRPC6 in the pathogenesis of FSGS remains to be elucidated. In this work we describe the generation and phenotypic characterization of three different transgenic mouse lines with podocyte-specific overexpression of the wild type or any of two mutant forms of Trpc6 (P111Q and E896K) previously related to FSGS. Consistent with the human phenotype a non-nephrotic range of albuminuria was detectable in almost all transgenic lines. The histological analysis demonstrated that the transgenic mice developed a kidney disease similar to human FSGS. Differences of 2–3 folds in the presence of glomerular lesions were found between the non transgenic and transgenic mice expressing Trpc6 in its wild type or mutant forms specifically in podocytes. Electron microscopy of glomerulus from transgenic mice showed extensive podocyte foot process effacement. We conclude that overexpression of Trpc6 (wild type or mutated) in podocytes is sufficient to cause a kidney disease consistent with FSGS. Our results contribute to reinforce the central role of podocytes in the etiology of FSGS. These mice constitute an important new model in which to study future therapies and outcomes of this complex disease. PMID:20877463

  8. Developmental Divergence of Sleep-Wake Patterns in Orexin Knockout and Wild-Type Mice

    PubMed Central

    Coleman, Cassandra M.; Johnson, Eric D.; Shaw, Cynthia

    2008-01-01

    Narcolepsy, a disorder characterized by fragmented bouts of sleep and wakefulness during the day and night as well as cataplexy, has been linked in humans and non-human animals to the functional integrity of the orexinergic system. Adult orexin knockout mice and dogs with a mutation of the orexin receptor exhibit symptoms that mirror those seen in narcoleptic humans. As with narcolepsy, infant sleep-wake cycles in humans and rats are highly fragmented, with consolidated bouts of sleep and wakefulness developing gradually. Based on these common features of narcoleptics and infants, we hypothesized that the development of sleep-wake fragmentation in orexin knockout mice would be expressed as a developmental divergence between knockouts and wild-types, with the knockouts lagging behind the wild-types. We tested this hypothesis by recording the sleep-wake patterns of infant orexin knockout and wild-type mice across the first three postnatal weeks. Both knockouts and wild-types exhibited age-dependent, and therefore orexin-independent, quantitative and qualitative changes in sleep-wake patterning. At 3 weeks of age, however, by which time the sleep and wake bouts of the wild-types had consolidated further, the knockouts lagged behind the wild-types and exhibited significantly more bout fragmentation. These findings suggest the possibility that the fragmentation of behavioral states that characterizes narcolepsy in adults reflects reversion back toward the more fragmented sleep-wake patterns that characterize infancy. PMID:17284193

  9. Wild type p53 reactivation: from lab bench to clinic.

    PubMed

    Selivanova, Galina

    2014-08-19

    The p53 tumor suppressor is the most frequently inactivated gene in cancer. Several mouse models have demonstrated that the reconstitution of the p53 function suppresses the growth of established tumors. These facts, taken together, promote the idea of p53 reactivation as a strategy to combat cancer. This review will focus on recent advances in the development of small molecules which restore the function of wild type p53 by blocking its inhibitors Mdm2 and MdmX or their upstream regulators and discuss the impact of different p53 functions for tumor prevention and tumor eradication. Finally, the recent progress in p53 research will be analyzed concerning the role of p53 cofactors and cellular environment in the biological response upon p53 reactivation and how this can be applied in clinic. PMID:24726725

  10. Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras

    PubMed Central

    Keighren, Margaret A.; Flockhart, Jean H.

    2016-01-01

    ABSTRACT The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1−/− null mouse embryos die but a previous study showed that some homozygous Gpi1−/− null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1−/−↔Gpi1c/c chimaera with functional Gpi1−/− null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1−/− null cells in adult Gpi1−/−↔Gpi1c/c chimaeras and determine if Gpi1−/− null germ cells are functional. Analysis of adult Gpi1−/−↔Gpi1c/c chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1−/− null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1−/− null oocytes in one female Gpi1−/−↔Gpi1c/c chimaera were functional and provided preliminary evidence that one male putative Gpi1−/−↔Gpi1c/c chimaera produced functional spermatozoa from homozygous Gpi1−/− null germ cells. Although the male chimaera was almost certainly Gpi1−/−↔Gpi1c/c, this part of the study is considered preliminary because only blood was typed for GPI. Gpi1−/− null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1−/− null germ cells, it successfully identified functional Gpi1−/− null oocytes and revealed that some Gpi1−/− null cells could survive in many adult tissues. PMID:27103217

  11. The heparan sulphate deficient Hspg2 exon 3 null mouse displays reduced deposition of TGF-β1 in skin compared to C57BL/6 wild type mice.

    PubMed

    Shu, Cindy; Smith, Susan M; Melrose, James

    2016-06-01

    This was an observational study where we examined the role of perlecan HS on the deposition of TGF-β1 in C57BL/6 and Hspg2(∆3-/∆3-) perlecan exon 3 null mouse skin. Despite its obvious importance in skin repair and tissue homeostasis no definitive studies have immunolocalised TGF-β1 in skin in WT or Hspg2(∆3-/∆3-) perlecan exon 3 null mice. Vertical parasagittal murine dorsal skin from 3, 6 and 12 week old C57BL/6 and Hspg2(∆3-/∆3-) mice were fixed in neutral buffered formalin, paraffin embedded and 4 μm sections stained with Mayers haematoxylin and eosin (H & E). TGF-β1 was immunolocalised using a rabbit polyclonal antibody, heat retrieval and the Envision NovaRED detection system. Immunolocalisation of TGF-β1 differed markedly in C57BL/6 and Hspg2(∆3-/∆3-) mouse skin, ablation of exon 3 of Hspg2 resulted in a very severe reduction in the deposition of TGF-β1 in skin 3-12 weeks postnatally. The reduced deposition of TGF-β1 observed in the present study would be expected to impact detrimentally on the remodelling and healing capacity of skin in mutant mice compounding on the poor wound-healing properties already reported for perlecan exon 3 null mice due to an inability to signal with FGF-2 and promote angiogenic repair processes. TGF-β1 also has cell mediated effects in tissue homeostasis and matrix stabilisation a reduction in TGF-β1 deposition would therefore be expected to detrimentally impact on skin homeostasis in the perlecan mutant mice. PMID:27098652

  12. Abca7 deletion does not affect adult neurogenesis in the mouse

    PubMed Central

    Li, Hongyun; Karl, Tim; Garner, Brett

    2016-01-01

    ATP-binding cassette transporter A7 (ABCA7) is highly expressed in the brain. Recent genome-wide association studies (GWAS) have identified ABCA7 single nucleotide polymorphisms (SNPs) that increase Alzheimer's disease (AD) risk, however, the mechanisms by which ABCA7 may control AD risk remain to be fully elucidated. Based on previous research suggesting that certain ABC transporters may play a role in the regulation of neurogenesis, we conducted a study of cell proliferation and neurogenic potential using cellular bromodeoxyuridine (BrdU) incorporation and doublecortin (DCX) immunostaining in adult Abca7 deficient mice and wild-type-like (WT) littermates. In the present study counting of BrdU-positive and DCX-positive cells in an established adult neurogenesis site in the dentate gyrus (DG) indicated there were no significant differences when WT and Abca7 deficient mice were compared. We also measured the area occupied by immunohistochemical staining for BrdU and DCX in the DG and the subventricular zone (SVZ) of the same mice and this confirmed that ABCA7 does not play a significant role in the regulation of cell proliferation or neurogenesis in the adult mouse. PMID:26792809

  13. Abca7 deletion does not affect adult neurogenesis in the mouse.

    PubMed

    Li, Hongyun; Karl, Tim; Garner, Brett

    2016-01-01

    ATP-binding cassette transporter A7 (ABCA7) is highly expressed in the brain. Recent genome-wide association studies (GWAS) have identified ABCA7 single nucleotide polymorphisms (SNPs) that increase Alzheimer's disease (AD) risk, however, the mechanisms by which ABCA7 may control AD risk remain to be fully elucidated. Based on previous research suggesting that certain ABC transporters may play a role in the regulation of neurogenesis, we conducted a study of cell proliferation and neurogenic potential using cellular bromodeoxyuridine (BrdU) incorporation and doublecortin (DCX) immunostaining in adult Abca7 deficient mice and wild-type-like (WT) littermates. In the present study counting of BrdU-positive and DCX-positive cells in an established adult neurogenesis site in the dentate gyrus (DG) indicated there were no significant differences when WT and Abca7 deficient mice were compared. We also measured the area occupied by immunohistochemical staining for BrdU and DCX in the DG and the subventricular zone (SVZ) of the same mice and this confirmed that ABCA7 does not play a significant role in the regulation of cell proliferation or neurogenesis in the adult mouse. PMID:26792809

  14. Overexpression of Wild-Type Murine Tau Results in Progressive Tauopathy and Neurodegeneration

    PubMed Central

    Adams, Stephanie J.; Crook, Richard J.P.; DeTure, Michael; Randle, Suzanne J.; Innes, Amy E.; Yu, Xin Z.; Lin, Wen-Lang; Dugger, Brittany N.; McBride, Melinda; Hutton, Mike; Dickson, Dennis W.; McGowan, Eileen

    2009-01-01

    Here, we describe the generation and characterization of a novel tau transgenic mouse model (mTau) that overexpresses wild-type murine tau protein by twofold compared with endogenous levels. Transgenic tau expression was driven by a BAC transgene containing the entire wild-type mouse tau locus, including the endogenous promoter and the regulatory elements associated with the tau gene. The mTau model therefore differs from other tau models in that regulation of the genomic mouse transgene mimics that of the endogenous gene, including normal exon splicing regulation. Biochemical data from the mTau mice demonstrated that modest elevation of mouse tau leads to tau hyperphosphorylation at multiple pathologically relevant epitopes and accumulation of sarkosyl-insoluble tau. The mTau mice show a progressive increase in hyperphosphorylated tau pathology with age up to 15 to 18 months, which is accompanied by gliosis and vacuolization. In contrast, older mice show a decrease in tau pathology levels, which may represent hippocampal neuronal loss occurring in this wild-type model. Collectively, these results describe a novel model of tauopathy that develops pathological changes reminiscent of early stage Alzheimer’s disease and other related neurodegenerative diseases, achieved without overexpression of a mutant human tau transgene. This model will provide an important tool for understanding the early events leading to the development of tau pathology and a model for analysis of potential therapeutic targets for sporadic tauopathies. PMID:19717642

  15. Prion-Specific Antibodies Produced in Wild-Type Mice.

    PubMed

    Heegaard, Peter M H; Bergström, Ann-Louise; Andersen, Heidi Gertz; Cordes, Henriette

    2015-01-01

    Peptide-specific antibodies produced against synthetic peptides are of high value in probing protein structure and function, especially when working with challenging proteins, including not readily available, non-immunogenic, toxic, and/or pathogenic proteins. Here, we present a straightforward method for production of mouse monoclonal antibodies (MAbs) against peptides representing two sites of interest in the bovine prion protein (boPrP), the causative agent of bovine spongiform encephalopathy ("mad cow disease") and new variant Creutzfeldt-Jakob's disease (CJD) in humans, as well as a thorough characterization of their reactivity with a range of normal and pathogenic (misfolded) prion proteins. It is demonstrated that immunization of wild-type mice with ovalbumin-conjugated peptides formulated with Freund's adjuvant induces a good immune response, including high levels of specific anti-peptide antibodies, even against peptides very homologous to murine protein sequences. In general, using the strategies described here for selecting, synthesizing, and conjugating peptides and immunizing 4-5 mice with 2-3 different peptides, high-titered antibodies reacting with the target protein are routinely obtained with at least one of the peptides after three to four immunizations with incomplete Freund's adjuvant. PMID:26424281

  16. Elucidation of the atherosclerotic disease process in apo E and wild type mice by vibrational spectroscopy

    NASA Astrophysics Data System (ADS)

    Adar, Fran; Jelicks, Linda; Naudin, Coralie; Rousseau, Denis; Yeh, Syun-ru

    2004-07-01

    Raman and FTIR microprobe spectroscopy have been used to characterize the atherosclerotic process in Apo E and wild type mice. The Apo E null mouse is being studied in parallel with a healthy strain as a model of the human atherosclerotic disease. Preliminary Raman microprobe spectra have been recorded from the lumen of the aorta vessels from a normal black mouse (C57BL/6J) and the apo E null mouse fed on a normal chow diet. Spectra were also recorded from another normal mouse fed breeder chow containing a much higher content of fats. In the Raman spectra the fat cells exhibited spectra typical of esterified triglycerides while the wall tissue had spectra dominated by Amide I and III modes and the phenylalanine stretch at 1003 cm-1 of protein. The FTIR spectra showed the typical Amide I and II bands of protein and the strong >C=O stretch of the triglycerides. In addition, there were morphologically distinct regions of the specimens indicating a surprising form of calcification in one very old mouse (wild type), and free fatty acid inclusions in the knock out mouse. The observation of these chemistries provide new information for elucidation of the molecular mechanisms of the development of atherosclerosis.

  17. Peroxisomes in wild-type and rosy mutant Drosophila melanogaster.

    PubMed Central

    Beard, M E; Holtzman, E

    1987-01-01

    This study shows that peroxisomes are abundant in the Malpighian tubule and gut of wild-type Oregon R Drosophila melanogaster and that the peroxisomal population of the rosy-506 eye-color mutant differs from that of the wild type. Catalase activity in wild-type flies is demonstrable in bodies of appearance and centrifugal behavior comparable to the peroxisomes of vertebrate tissues. Xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.1.3.22) activity of the Malpighian tubule of wild-type flies is demonstrable cytochemically in bodies like those containing catalase. The rosy-506 mutant flies, with a deletion in the structural gene for xanthine dehydrogenase (xanthine:NAD+ oxidoreductase, EC 1.1.1.204), lack cytochemically demonstrable peroxisomal xanthine oxidase activity. In addition, peroxisomes in the rosy-506 mutants show less intense cytochemical staining for catalase than those in wild-type flies, and biochemical assays indicate that catalase in the rosy mutant is much more accessible to substrate in the absence of detergent than in the wild type. Thus, the rosy-506 mutation appears to affect peroxisomes and may mimic aspects of the defects of peroxisomes in some human metabolic disorders. Images PMID:3118368

  18. Rhythmic Ganglion Cell Activity in Bleached and Blind Adult Mouse Retinas

    PubMed Central

    Menzler, Jacob; Channappa, Lakshmi; Zeck, Guenther

    2014-01-01

    In retinitis pigmentosa – a degenerative disease which often leads to incurable blindness- the loss of photoreceptors deprives the retina from a continuous excitatory input, the so-called dark current. In rodent models of this disease this deprivation leads to oscillatory electrical activity in the remaining circuitry, which is reflected in the rhythmic spiking of retinal ganglion cells (RGCs). It remained unclear, however, if the rhythmic RGC activity is attributed to circuit alterations occurring during photoreceptor degeneration or if rhythmic activity is an intrinsic property of healthy retinal circuitry which is masked by the photoreceptor’s dark current. Here we tested these hypotheses by inducing and analysing oscillatory activity in adult healthy (C57/Bl6) and blind mouse retinas (rd10 and rd1). Rhythmic RGC activity in healthy retinas was detected upon partial photoreceptor bleaching using an extracellular high-density multi-transistor-array. The mean fundamental spiking frequency in bleached retinas was 4.3 Hz; close to the RGC rhythm detected in blind rd10 mouse retinas (6.5 Hz). Crosscorrelation analysis of neighbouring wild-type and rd10 RGCs (separation distance <200 µm) reveals synchrony among homologous RGC types and a constant phase shift (∼70 msec) among heterologous cell types (ON versus OFF). The rhythmic RGC spiking in these retinas is driven by a network of presynaptic neurons. The inhibition of glutamatergic ganglion cell input or the inhibition of gap junctional coupling abolished the rhythmic pattern. In rd10 and rd1 retinas the presynaptic network leads to local field potentials, whereas in bleached retinas additional pharmacological disinhibition is required to achieve detectable field potentials. Our results demonstrate that photoreceptor bleaching unmasks oscillatory activity in healthy retinas which shares many features with the functional phenotype detected in rd10 retinas. The quantitative physiological differences advance the

  19. Effects of chronic variable stress on cognition and Bace1 expression among wild-type mice.

    PubMed

    Cordner, Z A; Tamashiro, K L K

    2016-01-01

    Stressful life events, activation of the hypothalamic-pituitary-adrenal (HPA) axis and glucocorticoids are now thought to have a role in the development of several neurodegenerative and psychiatric disorders including Alzheimer's disease (AD) through mechanisms that may include exacerbation of cognitive impairment, neuronal loss, and beta-amyloid (Aβ) and tau neuropathology. In the current study, we use a wild-type mouse model to demonstrate that chronic variable stress impairs cognitive function and that aged mice are particularly susceptible. We also find that stress exposure is associated with a 1.5- to 2-fold increase in the expression of Bace1 in the hippocampus of young adult mice and the hippocampus, prefrontal cortex and amygdala of aged mice. Further, the increased expression of Bace1 was associated with decreased methylation of several CpGs in the Bace1 promoter region. In a second series of experiments, exposure to environmental enrichment (EE) prevented the stress-related changes in cognition, gene expression and DNA methylation. Together, these findings re-affirm the adverse effects of stress on cognition and further suggest that aged individuals are especially susceptible. In addition, demonstrating that chronic stress results in decreased DNA methylation and increased expression of Bace1 in the brain may provide a novel link between stress, Aβ pathology and AD. Finally, understanding the mechanisms by which EE prevented the effects of stress on cognition and Bace1 expression will be an important area of future study that may provide insights into novel approaches to the treatment of AD. PMID:27404286

  20. Histomorphological Phenotyping of the Adult Mouse Brain.

    PubMed

    Mikhaleva, Anna; Kannan, Meghna; Wagner, Christel; Yalcin, Binnaz

    2016-01-01

    This article describes a series of standard operating procedures for morphological phenotyping of the mouse brain using basic histology. Many histological studies of the mouse brain use qualitative approaches based on what the human eye can detect. Consequently, some phenotypic information may be missed. Here we describe a quantitative approach for the assessment of brain morphology that is simple and robust. A total of 78 measurements are made throughout the brain at specific and well-defined regions, including the cortex, the hippocampus, and the cerebellum. Experimental design and timeline considerations, including strain background effects, the importance of sectioning quality, measurement variability, and efforts to correct human errors are discussed. © 2016 by John Wiley & Sons, Inc. PMID:27584555

  1. Wild-type KRAS inhibits oncogenic KRAS-induced T-ALL in mice.

    PubMed

    Staffas, A; Karlsson, C; Persson, M; Palmqvist, L; Bergo, M O

    2015-05-01

    The role of hyperactive RAS signaling is well established in myeloid malignancies but less clear in T-cell malignancies. The Kras2(LSL)Mx1-Cre (KM) mouse model expresses endogenous KRAS(G12D) in hematopoietic cells and is widely used to study mechanisms and treatment of myeloproliferative neoplasms (MPN). The model displays an intriguing shift from MPN to acute T-cell leukemia (T-ALL) after transplantation to wild-type mice, but the mechanisms underlying this lineage shift is unknown. Here, we show that KRAS(G12D) increases proliferation of both myeloid and T-cell progenitors, but whereas myeloid cells differentiate, T-cell differentiation is inhibited at early stages. Secondary mutations in the expanded pool of T-cell progenitors accompany T-ALL development, and our results indicate that the shift from myeloid to T-lymphoid malignancy after transplantation is explained by the increased likelihood for secondary mutations when the tumor lifespan is increased. We demonstrate that tumor lifespan increases after transplantation because primary KM mice die rapidly, not from MPN, but from KRAS(G12D) expression in nonhematopoietic cells, which causes intestinal bleeding and severe anemia. We also identify loss of the wild-type KRAS allele as a secondary mutation in all T-ALL cells and provide evidence that wild-type KRAS acts as a tumor suppressor in the T-cell lineage in mice. PMID:25371176

  2. ATM localization and gene expression in the adult mouse eye

    PubMed Central

    Leemput, Julia; Masson, Christel; Bigot, Karine; Errachid, Abdelmounaim; Dansault, Anouk; Provost, Alexandra; Gadin, Stéphanie; Aoufouchi, Said; Menasche, Maurice

    2009-01-01

    Purpose High levels of metabolism and oxygen consumption in most adult murine ocular compartments, combined with exposure to light and ultraviolet (UV) radiation, are major sources of oxidative stress, causing DNA damage in ocular cells. Of all mammalian body cells, photoreceptor cells consume the largest amount of oxygen and generate the highest levels of oxidative damage. The accumulation of such damage throughout life is a major factor of aging tissues. Several multiprotein complexes have recently been identified as the major sensors and mediators involved in the maintenance of DNA integrity. The activity of these complexes initially seemed to be restricted to dividing cells, given their ultimate role in major cell cycle checkpoints. However, it was later established that they are also active in post-mitotic cells. Recent findings demonstrate that the DNA damage response (DDR) is essential for the development, maintenance, and normal functioning of the adult central nervous system. One major molecular factor in the DDR is the protein, ataxia telangiectasia mutated (ATM). It is required for the rapid induction of cellular responses to DNA double-strand breaks. These cytotoxic DNA lesions may be caused by oxidative damage. To understand how ATM prevents oxidative stress and participates in the maintenance of genomic integrity and cell viability of the adult retina, we determined the ATM expression patterns and studied its localization in the adult mouse eye. Methods Atm gene expression was analyzed by RT–PCR experiments and its localization by in situ hybridization on adult mouse ocular and cerebellar tissue sections. ATM protein expression was determined by western blot analysis of proteins homogenates extracted from several mouse tissues and its localization by immunohistochemistry experiments performed on adult mouse ocular and cerebellar tissue sections. In addition, subcellular localization was realized by confocal microscopy imaging of ocular tissue

  3. A Comprehensive Atlas of the Adult Mouse Penis

    PubMed Central

    Phillips, Tiffany R.; Wright, David K.; Gradie, Paul E.; Johnston, Leigh A.; Pask, Andrew J.

    2016-01-01

    Mice are routinely used to study the development of the external genitalia and, in particular, the process of male urethral closure. This is because misplacement of the male penile urethra, or hypospadias, is amongst the most common birth defects reported in humans. While mice present a tractable model to study penile development, several structures differ between mice and humans, and there is a lack of consensus in the literature on their annotation and developmental origins. Defining the ontology of the mouse prepuce is especially important for the relevance and interpretation of mouse models of hypospadias to human conditions. We have developed a detailed annotation of the adult mouse penis that addresses these differences and enables an accurate comparison of murine and human hypospadias phenotypes. Through MRI data, gross morphology and section histology, we define the origin of the mouse external and internal prepuces, their relationship to the single human foreskin as well as provide a comprehensive view of the various structures of the mouse penis and their associated muscle attachments within the body. These data are combined to annotate structures in a novel 3D adult penis atlas that can be downloaded, viewed at any angle, and manipulated to examine the relationship of various structures. PMID:26112156

  4. Taurine in drinking water recovers learning and memory in the adult APP/PS1 mouse model of Alzheimer's disease

    PubMed Central

    Kim, Hye Yun; Kim, Hyunjin V.; Yoon, Jin H.; Kang, Bo Ram; Cho, Soo Min; Lee, Sejin; Kim, Ji Yoon; Kim, Joo Won; Cho, Yakdol; Woo, Jiwan; Kim, YoungSoo

    2014-01-01

    Alzheimer's disease (AD) is a lethal progressive neurological disorder affecting the memory. Recently, US Food and Drug Administration mitigated the standard for drug approval, allowing symptomatic drugs that only improve cognitive deficits to be allowed to accelerate on to clinical trials. Our study focuses on taurine, an endogenous amino acid found in high concentrations in humans. It has demonstrated neuroprotective properties against many forms of dementia. In this study, we assessed cognitively enhancing property of taurine in transgenic mouse model of AD. We orally administered taurine via drinking water to adult APP/PS1 transgenic mouse model for 6 weeks. Taurine treatment rescued cognitive deficits in APP/PS1 mice up to the age-matching wild-type mice in Y-maze and passive avoidance tests without modifying the behaviours of cognitively normal mice. In the cortex of APP/PS1 mice, taurine slightly decreased insoluble fraction of Aβ. While the exact mechanism of taurine in AD has not yet been ascertained, our results suggest that taurine can aid cognitive impairment and may inhibit Aβ-related damages. PMID:25502280

  5. Hes3 expression in the adult mouse brain is regulated during demyelination and remyelination.

    PubMed

    Toutouna, Louiza; Nikolakopoulou, Polyxeni; Poser, Steven W; Masjkur, Jimmy; Arps-Forker, Carina; Troullinaki, Maria; Grossklaus, Sylvia; Bosak, Viktoria; Friedrich, Ulrike; Ziemssen, Tjalf; Bornstein, Stefan R; Chavakis, Triantafyllos; Androutsellis-Theotokis, Andreas

    2016-07-01

    Hes3 is a component of the STAT3-Ser/Hes3 Signaling Axis controlling the growth and survival of neural stem cells and other plastic cells. Pharmacological activation of this pathway promotes neuronal rescue and behavioral recovery in models of ischemic stroke and Parkinson's disease. Here we provide initial observations implicating Hes3 in the cuprizone model of demyelination and remyelination. We focus on the subpial motor cortex of mice because we detected high Hes3 expression. This area is of interest as it is impacted both in human demyelinating diseases and in the cuprizone model. We report that Hes3 expression is reduced at peak demyelination and is partially restored within 1 week after cuprizone withdrawal. This raises the possibility of Hes3 involvement in demyelination/remyelination that may warrant additional research. Supporting a possible role of Hes3 in the maintenance of oligodendrocyte markers, a Hes3 null mouse strain shows lower levels of myelin basic protein in undamaged adult mice, compared to wild-type controls. We also present a novel method for culturing the established oligodendrocyte progenitor cell line oli-neu in a manner that maintains Hes3 expression as well as its self-renewal and differentiation potential, offering an experimental tool to study Hes3. Based upon this approach, we identify a Janus kinase inhibitor and dbcAMP as powerful inducers of Hes3 gene expression. We provide a new biomarker and cell culture method that may be of interest in demyelination/remyelination research. PMID:27018293

  6. Differential proteomic responses of selectively bred and wild-type Sydney rock oyster populations exposed to elevated CO2.

    PubMed

    Thompson, E L; O'Connor, W; Parker, L; Ross, P; Raftos, D A

    2015-03-01

    Previous work suggests that larvae from Sydney rock oysters that have been selectively bred for fast growth and disease resistance are more resilient to the impacts of ocean acidification than nonselected, wild-type oysters. In this study, we used proteomics to investigate the molecular differences between oyster populations in adult Sydney rock oysters and to identify whether these form the basis for observations seen in larvae. Adult oysters from a selective breeding line (B2) and nonselected wild types (WT) were exposed for 4 weeks to elevated pCO2 (856 μatm) before their proteomes were compared to those of oysters held under ambient conditions (375 μatm pCO2 ). Exposure to elevated pCO2 resulted in substantial changes in the proteomes of oysters from both the selectively bred and wild-type populations. When biological functions were assigned, these differential proteins fell into five broad, potentially interrelated categories of subcellular functions, in both oyster populations. These functional categories were energy production, cellular stress responses, the cytoskeleton, protein synthesis and cell signalling. In the wild-type population, proteins were predominantly upregulated. However, unexpectedly, these cellular systems were downregulated in the selectively bred oyster population, indicating cellular dysfunction. We argue that this reflects a trade-off, whereby an adaptive capacity for enhanced mitochondrial energy production in the selectively bred population may help to protect larvae from the effects of elevated CO2 , whilst being deleterious to adult oysters. PMID:25689603

  7. Gene expression of the IGF pathway family distinguishes subsets of gastrointestinal stromal tumors wild type for KIT and PDGFRA.

    PubMed

    Beadling, Carol; Patterson, Janice; Justusson, Emily; Nelson, Dylan; Pantaleo, Maria A; Hornick, Jason L; Chacón, Matias; Corless, Christopher L; Heinrich, Michael C

    2013-02-01

    Gastrointestinal stromal tumors (GISTs) arise from the interstitial cells of Cajal (ICCs) and are the most common mesenchymal neoplasm of the gastrointestinal tract. While the majority of GISTs harbor activating mutations in either the v-kit Hardy-Zuckerman feline sarcoma viral oncogene homolog (KIT) or platelet-derived growth factor receptor alpha (PDGFRA) tyrosine kinases, approximately 10-15% of adult GISTs and 85% of pediatric GISTs lack such mutations. These "wild-type" GISTs have been reported to express high levels of the insulin-like growth factor 1 receptor (IGF1R), and IGF1R-targeted therapy of wild-type GISTs is being evaluated in clinical trials. However, it is not clear that all wild-type GISTs express IGF1R, because studies to date have predominantly focused on a particular subtype of gastric wild-type GIST that is deficient in the mitochondrial succinate dehydrogenase (SDH) complex. This study of a series of 136 GISTs, including 72 wild-type specimens, was therefore undertaken to further characterize wild-type GIST subtypes based on the relative expression of transcripts encoding IGF1R. Additional transcripts relevant to GIST biology were also evaluated, including members of the IGF-signaling pathway (IGF1, IGF2, and insulin receptor [INSR]), neural markers (CDH2[CDH: Cadherin], neurofilament, light polypeptide, LHX2 [LHX: LIM homeobox], and KIRREL3 [KIRREL: kin of IRRE like]), KIT, PDGFRA, CD34, and HIF1A. Succinate dehydrogenase complex, subunit B protein expression was also assessed as a measure of SDH complex integrity. In addition to the previously described SDH-deficient, IGF1R(high) wild-type GISTs, other SDH-intact wild-type subpopulations were defined by high relative expression of IGF1R, neural markers, IGF1 and INSR, or low IGF1R coupled with high IGF2. These results underscore the complexity and heterogeneity of wild-type GISTs that will need to be factored into molecularly-targeted therapeutic strategies. PMID:24133624

  8. Modest increased sensitivity to radiation oncogenesis in ATM heterozygous versus wild-type mammalian cells

    NASA Technical Reports Server (NTRS)

    Smilenov, L. B.; Brenner, D. J.; Hall, E. J.

    2001-01-01

    Subpopulations that are genetically predisposed to radiation-induced cancer could have significant public health consequences. Individuals homozygous for null mutations at the ataxia telangiectasia gene are indeed highly radiosensitive, but their numbers are very small. Ataxia Telangiectasia heterozygotes (1-2% of the population) have been associated with somewhat increased radiosensitivity for some end points, but none directly related to carcinogenesis. Here, intralitter comparisons between wild-type mouse embryo fibroblasts and mouse embryo fibroblasts carrying ataxia telangiectasia mutated (ATM) null mutation indicate that the heterozygous cells are more sensitive to radiation oncogenesis than their normal, litter-matched, counterparts. From these data we suggest that Ataxia Telangiectasia heterozygotes could indeed represent a societally-significant radiosensitive human subpopulation.

  9. Time-lapse imaging of neuroblast migration in acute slices of the adult mouse forebrain.

    PubMed

    Khlghatyan, Jivan; Saghatelyan, Armen

    2012-01-01

    There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB). In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes using blood vessels as a structural support and a source of molecular factors required for migration. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network. In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of

  10. Prolactin Stimulates Precursor Cells in the Adult Mouse Hippocampus

    PubMed Central

    Walker, Tara L.; Vukovic, Jana; Koudijs, Margaretha M.; Blackmore, Daniel G.; Mackay, Eirinn W.; Sykes, Alex M.; Overall, Rupert W.; Hamlin, Adam S.; Bartlett, Perry F.

    2012-01-01

    In the search for ways to combat degenerative neurological disorders, neurogenesis-stimulating factors are proving to be a promising area of research. In this study, we show that the hormonal factor prolactin (PRL) can activate a pool of latent precursor cells in the adult mouse hippocampus. Using an in vitro neurosphere assay, we found that the addition of exogenous PRL to primary adult hippocampal cells resulted in an approximate 50% increase in neurosphere number. In addition, direct infusion of PRL into the adult dentate gyrus also resulted in a significant increase in neurosphere number. Together these data indicate that exogenous PRL can increase hippocampal precursor numbers both in vitro and in vivo. Conversely, PRL null mice showed a significant reduction (approximately 80%) in the number of hippocampal-derived neurospheres. Interestingly, no deficit in precursor proliferation was observed in vivo, indicating that in this situation other niche factors can compensate for a loss in PRL. The PRL loss resulted in learning and memory deficits in the PRL null mice, as indicated by significant deficits in the standard behavioral tests requiring input from the hippocampus. This behavioral deficit was rescued by direct infusion of recombinant PRL into the hippocampus, indicating that a lack of PRL in the adult mouse hippocampus can be correlated with impaired learning and memory. PMID:22973440

  11. Adult mouse brain gene expression patterns bear an embryologic imprint

    PubMed Central

    Zapala, Matthew A.; Hovatta, Iiris; Ellison, Julie A.; Wodicka, Lisa; Del Rio, Jo A.; Tennant, Richard; Tynan, Wendy; Broide, Ron S.; Helton, Rob; Stoveken, Barbara S.; Winrow, Christopher; Lockhart, Daniel J.; Reilly, John F.; Young, Warren G.; Bloom, Floyd E.; Lockhart, David J.; Barlow, Carrolee

    2005-01-01

    The current model to explain the organization of the mammalian nervous system is based on studies of anatomy, embryology, and evolution. To further investigate the molecular organization of the adult mammalian brain, we have built a gene expression-based brain map. We measured gene expression patterns for 24 neural tissues covering the mouse central nervous system and found, surprisingly, that the adult brain bears a transcriptional “imprint” consistent with both embryological origins and classic evolutionary relationships. Embryonic cellular position along the anterior–posterior axis of the neural tube was shown to be closely associated with, and possibly a determinant of, the gene expression patterns in adult structures. We also observed a significant number of embryonic patterning and homeobox genes with region-specific expression in the adult nervous system. The relationships between global expression patterns for different anatomical regions and the nature of the observed region-specific genes suggest that the adult brain retains a degree of overall gene expression established during embryogenesis that is important for regional specificity and the functional relationships between regions in the adult. The complete collection of extensively annotated gene expression data along with data mining and visualization tools have been made available on a publicly accessible web site (www.barlow-lockhart-brainmapnimhgrant.org). PMID:16002470

  12. Gene expression of the IGF pathway family distinguishes subsets of gastrointestinal stromal tumors wild type for KIT and PDGFRA

    PubMed Central

    Beadling, Carol; Patterson, Janice; Justusson, Emily; Nelson, Dylan; Pantaleo, Maria A.; Hornick, Jason L.; Chacón, Matias; Corless, Christopher L.; Heinrich, Michael C.

    2013-01-01

    Gastrointestinal stromal tumors (GISTs) arise from the interstitial cells of Cajal (ICCs) and are the most common mesenchymal neoplasm of the gastrointestinal tract. While the majority of GISTs harbor activating mutations in either the v-kit Hardy-Zuckerman feline sarcoma viral oncogene homolog (KIT) or platelet-derived growth factor receptor alpha (PDGFRA) tyrosine kinases, approximately 10–15% of adult GISTs and 85% of pediatric GISTs lack such mutations. These “wild-type” GISTs have been reported to express high levels of the insulin-like growth factor 1 receptor (IGF1R), and IGF1R-targeted therapy of wild-type GISTs is being evaluated in clinical trials. However, it is not clear that all wild-type GISTs express IGF1R, because studies to date have predominantly focused on a particular subtype of gastric wild-type GIST that is deficient in the mitochondrial succinate dehydrogenase (SDH) complex. This study of a series of 136 GISTs, including 72 wild-type specimens, was therefore undertaken to further characterize wild-type GIST subtypes based on the relative expression of transcripts encoding IGF1R. Additional transcripts relevant to GIST biology were also evaluated, including members of the IGF-signaling pathway (IGF1, IGF2, and insulin receptor [INSR]), neural markers (CDH2[CDH: Cadherin], neurofilament, light polypeptide, LHX2 [LHX: LIM homeobox], and KIRREL3 [KIRREL: kin of IRRE like]), KIT, PDGFRA, CD34, and HIF1A. Succinate dehydrogenase complex, subunit B protein expression was also assessed as a measure of SDH complex integrity. In addition to the previously described SDH-deficient, IGF1Rhigh wild-type GISTs, other SDH-intact wild-type subpopulations were defined by high relative expression of IGF1R, neural markers, IGF1 and INSR, or low IGF1R coupled with high IGF2. These results underscore the complexity and heterogeneity of wild-type GISTs that will need to be factored into molecularly-targeted therapeutic strategies. PMID:24133624

  13. Combined effect of temperature and zinc on Caenorhabditis elegans wild type and daf-21 mutant strains.

    PubMed

    Wang, Yunbiao; Ezemaduka, Anastasia N

    2014-04-01

    Heavy metal pollution in aquatic ecosystems is a far reaching environmental problem. The possible influences of heavy metal exposure and the potential harm to organisms when combined with other environmental stressors such as temperature have been largely unexplored. An aquatic toxicity test of Caenorhabditis elegans was performed to estimate the 24h median lethal concentration (LC50) of different zinc concentrations at different temperatures (15°C, 20°C, 25°C, and 30°C). We also examined the time course thermotolerance on wild type (N2) and daf-21 null (JT6130) adults exposed to 6.1mM zinc at 37°C. Hsp90 protein expression level in response to the combined effect of temperature and zinc toxicity was also investigated by both Western blots and ELISA. Our results show that C. elegans wild type nematodes exhibit severe lethal toxicity after a 24h exposure to zinc at higher temperatures. In addition, the expression level of Hsp90 was highly inhibited in adult worms subjected to zinc stress. This toxicity assay at different temperatures provides insight into organism response to combined effects of temperature and zinc toxicity. PMID:24679967

  14. Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

    PubMed Central

    Graham, Evan Lee; Balla, Cristina; Franchino, Hannabeth; Melman, Yonathan

    2013-01-01

    The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease. PMID:24084584

  15. "Wild type" GIST: Clinicopathological features and clinical practice.

    PubMed

    Wada, Ryuichi; Arai, Hiroki; Kure, Shoko; Peng, Wei-Xia; Naito, Zenya

    2016-08-01

    Gastrointestinal stromal tumor (GIST) is a mesenchymal tumor of the gastrointestinal tract. Mutation of KIT and PDGFRA genes is implicated in the tumorigenesis. Approximately 10% of GISTs do not harbor mutation of these genes, and they are designated as "wild type" GIST. They are classified into succinate dehydrogenase (SDH)-deficient and non-SDH-deficient groups. SDH-deficient group includes Carney triad and Carney Stratakis syndrome. The patients are young women. Tumors occur in the antrum of the stomach, and tumor cells are epithelioid. Lymph node metastasis is frequent. The non-SDH-deficient group includes neurofibromatosis (NF) type 1 and GISTs with mutations of BRAF, KRAS, and PIK3CA and with the ETV6-NTRK3 fusion gene. GIST in NF occurs in the small intestine, and tumor cells are spindle shaped. GIST with BRAF mutation arises in the small intestine. Attention to the age, gender, family history and other neoplasms may raise the prediction of syndromic disease. Location of the tumor, morphology, and pleomorphism of the tumor cells are further informative. Lymphovascular invasion should be carefully evaluated. The determination of KIT expression is essential for the diagnosis. When wild type GIST is suspected, intensive genetic analysis is required. Further, a careful and long-time observation is recommended. PMID:27427238

  16. Mechanical Testing of Mouse Carotid Arteries: from Newborn to Adult

    PubMed Central

    Amin, Mazyar; Le, Victoria P.; Wagenseil, Jessica E.

    2012-01-01

    The large conducting arteries in vertebrates are composed of a specialized extracellular matrix designed to provide pulse dampening and reduce the work performed by the heart. The mix of matrix proteins determines the passive mechanical properties of the arterial wall1. When the matrix proteins are altered in development, aging, disease or injury, the arterial wall remodels, changing the mechanical properties and leading to subsequent cardiac adaptation2. In normal development, the remodeling leads to a functional cardiac and cardiovascular system optimized for the needs of the adult organism. In disease, the remodeling often leads to a negative feedback cycle that can cause cardiac failure and death. By quantifying passive arterial mechanical properties in development and disease, we can begin to understand the normal remodeling process to recreate it in tissue engineering and the pathological remodeling process to test disease treatments. Mice are useful models for studying passive arterial mechanics in development and disease. They have a relatively short lifespan (mature adults by 3 months and aged adults by 2 years), so developmental3 and aging studies4 can be carried out over a limited time course. The advances in mouse genetics provide numerous genotypes and phenotypes to study changes in arterial mechanics with disease progression5 and disease treatment6. Mice can also be manipulated experimentally to study the effects of changes in hemodynamic parameters on the arterial remodeling process7. One drawback of the mouse model, especially for examining young ages, is the size of the arteries. We describe a method for passive mechanical testing of carotid arteries from mice aged 3 days to adult (approximately 90 days). We adapt a commercial myograph system to mount the arteries and perform multiple pressure or axial stretch protocols on each specimen. We discuss suitable protocols for each age, the necessary measurements and provide example data. We also include

  17. Exploration and visualization of connectivity in the adult mouse brain.

    PubMed

    Feng, David; Lau, Chris; Ng, Lydia; Li, Yang; Kuan, Leonard; Sunkin, Susan M; Dang, Chinh; Hawrylycz, Michael

    2015-02-01

    The Allen Mouse Brain Connectivity Atlas is a mesoscale whole brain axonal projection atlas of the C57Bl/6J mouse brain. All data were aligned to a common template in 3D space to generate a comprehensive and quantitative database of inter-areal and cell-type-specific projections. A suite of computational tools were developed to search and visualize the projection labeling experiments, available at http://connectivity.brain-map.org. We present three use cases illustrating how these publicly-available tools can be used to perform analyses of long range brain region connectivity. The use cases make extensive use of advanced visualization tools integrated with the atlas including projection density histograms, 3D computed anterograde and retrograde projection paths, and multi-specimen projection composites. These tools offer convenient access to detailed axonal projection information in the adult mouse brain and the ability to perform data analysis and visualization of projection fields and neuroanatomy in an integrated manner. PMID:25637033

  18. Differential Apoptosis Radiosensitivity of Neural Progenitors in Adult Mouse Hippocampus.

    PubMed

    Li, Yu-Qing; Cheng, Zoey; Wong, Shun

    2016-01-01

    Mammalian tissue-specific stem cells and progenitors demonstrate differential DNA damage response. Neural progenitors in dentate gyrus of the hippocampus are known to undergo apoptosis after irradiation. Using a mouse model of hippocampal neuronal development, we characterized the apoptosis sensitivity of the different neural progenitor subpopulations in adult mouse dentate gyrus after irradiation. Two different bromodeoxyuridine incorporation paradigms were used for cell fate mapping. We identified two apoptosis sensitive neural progenitor subpopulations after irradiation. The first represented non-proliferative and non-newborn neuroblasts and immature neurons that expressed doublecortin, calretinin or both. The second consisted of proliferative intermediate neural progenitors. The putative radial glia-like neural stem cells or type-1 cells, regardless of proliferation status, were apoptosis resistant after irradiation. There was no evidence of radiation-induced apoptosis in the absence of the Trp53 (p53) gene but absence of Cdkn1a (p21) did not alter the apoptotic response. Upregulation of nuclear p53 was observed in neuroblasts after irradiation. We conclude that adult hippocampal neural progenitors may demonstrate differential p53-dependent apoptosis sensitivity after irradiation. PMID:27331809

  19. Differential Apoptosis Radiosensitivity of Neural Progenitors in Adult Mouse Hippocampus

    PubMed Central

    Li, Yu-Qing; Cheng, Zoey; Wong, Shun

    2016-01-01

    Mammalian tissue-specific stem cells and progenitors demonstrate differential DNA damage response. Neural progenitors in dentate gyrus of the hippocampus are known to undergo apoptosis after irradiation. Using a mouse model of hippocampal neuronal development, we characterized the apoptosis sensitivity of the different neural progenitor subpopulations in adult mouse dentate gyrus after irradiation. Two different bromodeoxyuridine incorporation paradigms were used for cell fate mapping. We identified two apoptosis sensitive neural progenitor subpopulations after irradiation. The first represented non-proliferative and non-newborn neuroblasts and immature neurons that expressed doublecortin, calretinin or both. The second consisted of proliferative intermediate neural progenitors. The putative radial glia-like neural stem cells or type-1 cells, regardless of proliferation status, were apoptosis resistant after irradiation. There was no evidence of radiation-induced apoptosis in the absence of the Trp53 (p53) gene but absence of Cdkn1a (p21) did not alter the apoptotic response. Upregulation of nuclear p53 was observed in neuroblasts after irradiation. We conclude that adult hippocampal neural progenitors may demonstrate differential p53-dependent apoptosis sensitivity after irradiation. PMID:27331809

  20. A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

    PubMed Central

    Webb, Carol F.; Wirsig-Wiechmann, Celeste R.; Lakiza, Olga; Obara, Tomoko

    2015-01-01

    Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. PMID:26111446

  1. High Pathogenicity of Wild-Type Measles Virus Infection in CD150 (SLAM) Transgenic Mice

    PubMed Central

    Sellin, Caroline I.; Davoust, Nathalie; Guillaume, Vanessa; Baas, Dominique; Belin, Marie-Françoise; Buckland, Robin; Wild, T. Fabian; Horvat, Branka

    2006-01-01

    Measles virus (MV) infection causes an acute childhood disease, associated in certain cases with infection of the central nervous system and development of a severe neurological disease. We have generated transgenic mice ubiquitously expressing the human protein SLAM (signaling lymphocytic activation molecule), or CD150, recently identified as an MV receptor. In contrast to all other MV receptor transgenic models described so far, in these mice infection with wild-type MV strains is highly pathogenic. Intranasal infection of SLAM transgenic suckling mice leads to MV spread to different organs and the development of an acute neurological syndrome, characterized by lethargy, seizures, ataxia, weight loss, and death within 3 weeks. In addition, in this model, vaccine and wild-type MV strains can be distinguished by virulence. Furthermore, intracranial MV infection of adult transgenic mice generates a subclinical infection associated with a high titer of MV-specific antibodies in the serum. Finally, to analyze new antimeasles therapeutic approaches, we created a recombinant soluble form of SLAM and demonstrated its important antiviral activity both in vitro and in vivo. Taken together, our results show the high susceptibility of SLAM transgenic mice to MV-induced neurological disease and open new perspectives for the analysis of the implication of SLAM in the neuropathogenicity of other morbilliviruses, which also use this molecule as a receptor. Moreover, this transgenic model, in allowing a simple readout of the efficacy of an antiviral treatment, provides unique experimental means to test novel anti-MV preventive and therapeutic strategies. PMID:16775330

  2. MicroRNA-128-3p impaired water maze learning by suppressing Doublecortin expression in both wild type and Aβ-42 infused mice.

    PubMed

    Chen, Jinlong; Li, Wen; Li, Yuan; He, Songwei; Li, Lingyu; Liang, Lining; Song, Yancheng; Qin, Dajiang; Zheng, Hui

    2016-07-28

    MicroRNA-128-3p (miR-128) is a brain-enriched microRNA reported to target Doublecortin (Dcx), a key transcriptional factor during adult neurogenesis. However, the downstream physiological effects of this miR-128-DCX axis remain unclear. Here we demonstrated that miR-128 could suppress Dcx expression by complementally binding to the -849 to -856 region of the 3'UTR of mouse Dcx. During differentiation of neural stem cells, over-expressing miR-128 with a lentivirus system inhibited the up-regulation of Dcx on Day 5, subsequently decreasing the percentage of TuJ+ cells on Day 16. Administration of the lentivirus encoding miR-128 into mouse hippocampi significantly impaired water maze learning after 14days, which could be attenuated when the Dcx-encoding virus was delivered simultaneously. In addition, similar changes including miR-128 up-regulation, Dcx down-regulation and learning defects were observed after a 14-day infusion of Aβ-42, which were also partially reversed by over-expressing Dcx. Collectively, the regulation axis from miR-128 to Dcx is critical for hippocampus-related contextual learning not only in wild type, but also in mice infused with Aβ-42. PMID:27222923

  3. A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

    SciTech Connect

    Webb, Carol F.; Ratliff, Michelle L.; Powell, Rebecca; Wirsig-Wiechmann, Celeste R.; Lakiza, Olga; Obara, Tomoko

    2015-08-07

    Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. - Highlights: • An ARID3a-deficient mouse kidney cell line expresses multiple progenitor markers. • This cell line spontaneously forms multiple nephron-like structures in vitro. • This cell line formed mouse kidney structures in immunocompromised medaka fish kidneys. • Our data identify a novel model system for studying kidney development.

  4. RUNX1B Expression Is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis

    PubMed Central

    Draper, Julia E.; Sroczynska, Patrycja; Tsoulaki, Olga; Leong, Hui Sun; Fadlullah, Muhammad Z. H.; Miller, Crispin; Kouskoff, Valerie; Lacaud, Georges

    2016-01-01

    The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny. RUNX1 also plays vital roles in adult mice, in regulating the correct specification of numerous blood lineages. Akin to the other mammalian Runx genes, Runx1 has two promoters P1 (distal) and P2 (proximal) which generate distinct protein isoforms. The activities and specific relevance of these two promoters in adult hematopoiesis remain to be fully elucidated. Utilizing a dual reporter mouse model we demonstrate that the distal P1 promoter is broadly active in adult hematopoietic stem and progenitor cell (HSPC) populations. By contrast the activity of the proximal P2 promoter is more restricted and its upregulation, in both the immature Lineage- Sca1high cKithigh (LSK) and bipotential Pre-Megakaryocytic/Erythroid Progenitor (PreMegE) populations, coincides with a loss of erythroid (Ery) specification. Accordingly the PreMegE population can be prospectively separated into “pro-erythroid” and “pro-megakaryocyte” populations based on Runx1 P2 activity. Comparative gene expression analyses between Runx1 P2+ and P2- populations indicated that levels of CD34 expression could substitute for P2 activity to distinguish these two cell populations in wild type (WT) bone marrow (BM). Prospective isolation of these two populations will enable the further investigation of molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. Having characterized the extensive activity of P1, we utilized a P1-GFP homozygous mouse model to analyze the impact of the complete absence of Runx1 P1 expression in adult mice and observed strong defects in the T cell lineage. Finally, we investigated how the leukemic fusion protein AML1-ETO9a might influence Runx1 promoter usage. Short-term AML1-ETO9a induction in BM resulted in preferential P2 upregulation, suggesting its expression may be important to

  5. Cytidine is a novel substrate for wild-type concentrative nucleoside transporter 2.

    PubMed

    Nagai, Katsuhito; Nagasawa, Kazuki; Koma, Mineto; Hotta, Ayumi; Fujimoto, Sadaki

    2006-08-25

    Nucleoside transporter (NT) plays key roles in the physiology of nucleosides and the pharmacology of its analogues in mammals. We previously cloned Na+/nucleoside cotransporter CNT2 from mouse M5076 ovarian sarcoma cells, the peptide encoded by it differing from that by the previously reported mouse CNT2 in five substitutions, and observed that the transporter can take up cytidine, like CNT1 and CNT3. In the present study, we examined which of the two aforementioned CNT2 is the normal one, and whether or not cytidine is transported via the previously reported CNT2. The peptide encoded by CNT2 derived from mouse intestine, liver, spleen, and ovary was identical to that previously reported. The uptake of [3H]cytidine, but not [3H]thymidine, by Cos-7 cells transfected with CNT2 cDNA obtained from mouse intestine was much greater than that by mock cells, as in the case of [3H]uridine, a typical substrate of NT. [3H]Cytidine and [3H]uridine were taken up via CNT2, in temperature-, extracellular Na+-, and substrate concentration-dependent manners. The uptake of [3H]cytidine and [3H]uridine mediated by CNT2 was significantly inhibited by the variety of nucleosides used in this study, except for thymidine, and inhibition of the [3H]uridine uptake by cytidine was competitive. The [3H]uridine uptake via CNT2 was significantly decreased by the addition of cytarabin or gemcitabine, antimetabolites of cytidine analogue. These results indicated that the previously reported mouse CNT2 is the wild-type one, and cytidine is transported mediated by the same recognition site on the CNT2 with uridine, and furthermore, cytidine analogues may be substrates for the transporter. PMID:16828706

  6. An anatomic gene expression atlas of the adult mouse brain.

    PubMed

    Ng, Lydia; Bernard, Amy; Lau, Chris; Overly, Caroline C; Dong, Hong-Wei; Kuan, Chihchau; Pathak, Sayan; Sunkin, Susan M; Dang, Chinh; Bohland, Jason W; Bokil, Hemant; Mitra, Partha P; Puelles, Luis; Hohmann, John; Anderson, David J; Lein, Ed S; Jones, Allan R; Hawrylycz, Michael

    2009-03-01

    Studying gene expression provides a powerful means of understanding structure-function relationships in the nervous system. The availability of genome-scale in situ hybridization datasets enables new possibilities for understanding brain organization based on gene expression patterns. The Anatomic Gene Expression Atlas (AGEA) is a new relational atlas revealing the genetic architecture of the adult C57Bl/6J mouse brain based on spatial correlations across expression data for thousands of genes in the Allen Brain Atlas (ABA). The AGEA includes three discovery tools for examining neuroanatomical relationships and boundaries: (1) three-dimensional expression-based correlation maps, (2) a hierarchical transcriptome-based parcellation of the brain and (3) a facility to retrieve from the ABA specific genes showing enriched expression in local correlated domains. The utility of this atlas is illustrated by analysis of genetic organization in the thalamus, striatum and cerebral cortex. The AGEA is a publicly accessible online computational tool integrated with the ABA (http://mouse.brain-map.org/agea). PMID:19219037

  7. Structure and age-dependent development of the turkey liver: a comparative study of a highly selected meat-type and a wild-type turkey line.

    PubMed

    Hünigen, Hana; Mainzer, Kathleen; Hirschberg, Ruth M; Custodis, Pia; Gemeinhardt, Ole; Al Masri, Salah; Richardson, Kenneth C; Hafez, Hafez Mohamed; Plendl, Johanna

    2016-04-01

    In this study the macroscopic and microscopic structure of the liver of a fast growing, meat-type turkey line (British United turkeys BUT Big 6, n=25) and a wild-type turkey line (Wild Canadian turkey, n=48) were compared at the age of 4, 8, 12, 16, and 20 wk. Because the growth plates of long bones were still detectable in the 20-week-old wild-type turkeys, indicating immaturity, a group of 8 wild-type turkeys at the age of 24 wk was included in the original scope of the study. Over the term of the study, the body and liver weights of birds from the meat-type turkey line increased at a faster rate than those of the wild-type turkey line. However, the relative liver weight of the meat-type turkeys declined (from 2.7 to 0.9%) to a greater extent than that of the wild-type turkeys (from 2.8 to 1.9%), suggesting a mismatch in development between muscle weights and liver weights of the meat-type turkeys. Signs of high levels of fat storage in the liver were detected in both lines but were greater in the wild-type turkey line, suggesting a better feed conversion by the extreme-genotype birds i.e., meat-type birds. For the first time, this study presents morphologic data on the structure and arrangement of the lymphatic tissue within the healthy turkey liver, describing two different types of lymphatic aggregations within the liver parenchyma, i.e., aggregations with and without fibrous capsules. Despite differences during development, both adult meat-type and adult wild-type turkeys had similar numbers of lymphatic aggregations. PMID:26908884

  8. Function of GATA Factors in the Adult Mouse Liver

    PubMed Central

    Zheng, Rena; Rebolledo-Jaramillo, Boris; Zong, Yiwei; Wang, Liqing; Russo, Pierre; Hancock, Wayne; Stanger, Ben Z.; Hardison, Ross C.; Blobel, Gerd A.

    2013-01-01

    GATA transcription factors and their Friend of Gata (FOG) cofactors control the development of diverse tissues. GATA4 and GATA6 are essential for the expansion of the embryonic liver bud, but their expression patterns and functions in the adult liver are unclear. We characterized the expression of GATA and FOG factors in whole mouse liver and purified hepatocytes. GATA4, GATA6, and FOG1 are the most prominently expressed family members in whole liver and hepatocytes. GATA4 chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) identified 4409 occupied sites, associated with genes enriched in ontologies related to liver function, including lipid and glucose metabolism. However, hepatocyte-specific excision of Gata4 had little impact on gross liver architecture and function, even under conditions of regenerative stress, and, despite the large number of GATA4 occupied genes, resulted in relatively few changes in gene expression. To address possible redundancy between GATA4 and GATA6, both factors were conditionally excised. Surprisingly, combined Gata4,6 loss did not exacerbate the phenotype resulting from Gata4 loss alone. This points to the presence of an unusually robust transcriptional network in adult hepatocytes that ensures the maintenance of liver function. PMID:24367609

  9. Crystal structure of wild-type human procathepsin K.

    PubMed

    Sivaraman, J; Lalumière, M; Ménard, R; Cygler, M

    1999-02-01

    Cathepsin K is a lysosomal cysteine protease belonging to the papain superfamily. It has been implicated as a major mediator of osteoclastic bone resorption. Wild-type human procathepsin K has been crystallized in a glycosylated and a deglycosylated form. The latter crystals diffract better, to 3.2 A resolution, and contain four molecules in the asymmetric unit. The structure was solved by molecular replacement and refined to an R-factor of 0.194. The N-terminal fragment of the proregion forms a globular domain while the C-terminal segment is extended and shows substantial flexibility. The proregion interacts with the enzyme along the substrate binding groove and along the proregion binding loop (residues Ser138-Asn156). It binds to the active site in the opposite direction to that of natural substrates. The overall binding mode of the proregion to cathepsin K is similar to that observed in cathepsin L, caricain, and cathepsin B, but there are local differences that likely contribute to the specificity of these proregions for their cognate enzymes. The main observed difference is in the position of the short helix alpha3p (67p-75p), which occupies the S' subsites. As in the other proenzymes, the proregion utilizes the S2 subsite for anchoring by placing a leucine side chain there, according to the specificity of cathepsin K toward its substrate. PMID:10048321

  10. Crystal structure of wild-type human procathepsin K.

    PubMed Central

    Sivaraman, J.; Lalumière, M.; Ménard, R.; Cygler, M.

    1999-01-01

    Cathepsin K is a lysosomal cysteine protease belonging to the papain superfamily. It has been implicated as a major mediator of osteoclastic bone resorption. Wild-type human procathepsin K has been crystallized in a glycosylated and a deglycosylated form. The latter crystals diffract better, to 3.2 A resolution, and contain four molecules in the asymmetric unit. The structure was solved by molecular replacement and refined to an R-factor of 0.194. The N-terminal fragment of the proregion forms a globular domain while the C-terminal segment is extended and shows substantial flexibility. The proregion interacts with the enzyme along the substrate binding groove and along the proregion binding loop (residues Ser138-Asn156). It binds to the active site in the opposite direction to that of natural substrates. The overall binding mode of the proregion to cathepsin K is similar to that observed in cathepsin L, caricain, and cathepsin B, but there are local differences that likely contribute to the specificity of these proregions for their cognate enzymes. The main observed difference is in the position of the short helix alpha3p (67p-75p), which occupies the S' subsites. As in the other proenzymes, the proregion utilizes the S2 subsite for anchoring by placing a leucine side chain there, according to the specificity of cathepsin K toward its substrate. PMID:10048321

  11. An improved isolation procedure for adult mouse cardiomyocytes.

    PubMed

    Pinz, Ilka; Zhu, Ming; Mende, Ulrike; Ingwall, Joanne S

    2011-09-01

    Isolated adult mouse cardiomyocytes are an important tool in cardiovascular research, but are challenging to prepare. Because the energy supply determines cell function and viability, we compared total creatine ([Cr]) and [ATP] in isolated cardiomyocytes with the intact mouse heart. Isolated myocytes suffered severe losses of Cr (-70%) and ATP (-53%). Myocytes were not able to replete [Cr] during a 5 h incubation period in medium supplemented with 1 mM Cr. In contrast, adding 20 mM Cr to the digestion buffers was sufficient to maintain normal [Cr]. Supplementing buffers with 5 mM of inosine (Ino) and adenosine (Ado) to prevent loss of cellular nucleosides partially protected against loss of ATP. To test whether maintaining [ATP] and [Cr] improves contractile function, myocytes were challenged by varying pacing rate from 0.5 to 10 Hz and by adding isoproterenol (Iso) at 5 and 10 Hz. All groups performed well up to 5 Hz, showing a positive cell shortening-frequency relationship; however, only 16% of myocytes isolated under standard conditions were able to sustain pacing with Iso challenge at 10 Hz. In contrast, 30-50% of the myocytes with normal Cr levels were able to contract and maintain low diastolic [Ca(2+)]. Cell yield also improved in Cr and the Cr/Ino/Ado-treated groups (85-90% vs. 70-75% rod shaped in untreated myocytes). These data suggest that viability and performance of isolated myocytes are improved when they are protected from the severe loss of Cr and ATP during the isolation, making them an even better research tool. PMID:21327944

  12. Biosafety of recombinant and wild type nucleopolyhedroviruses as bioinsecticides.

    PubMed

    Ashour, Mohamed-Bassem; Ragheb, Didair A; El-Sheikh, El-Sayed A; Gomaa, El-Adarosy A; Kamita, Shizuo G; Hammock, Bruce D

    2007-06-01

    The entomopathogenic Autographa californica (Speyer) nucleopolyhedrovirus (AcMNPV) has been genetically modified to increase its speed of kill. The potential adverse effects of a recombinant AcMNPV (AcAaIT) as well as wild type AcMNPV and wild type Spodoptera littoralis NPV (SlNPV) were studied. Cotton plants were treated with these viruses at concentrations that were adjusted to resemble the recommended field application rate (4 x 10(12) PIBs/feddan, feddan = 4,200 m2) and 3rd instar larvae of S. littoralis were allowed to feed on the contaminated plants. SDS-PAGE, ELISA, and DNA analyses were used to confirm that larvae that fed on these plants were virus-infected. Polyhedra that were purified from the infected larvae were subjected to structural protein analysis. A 32 KDa protein was found in polyhedra that were isolated from all of the viruses. Subtle differences were found in the size and abundance of ODV proteins. Antisera against polyhedral proteins isolated from AcAaIT polyhedra were raised in rabbits. The terminal bleeds from rabbits were screened against four coating antigens (i.e., polyhedral proteins from AcAaIT, AcAaIT from field-infected larvae (AcAaIT-field), AcMNPV, and SlNPV) using a two-dimensional titration method with the coated antigen format. Competitive inhibition experiments were conducted in parallel to optimize antibody and coating antigen concentrations for ELISA. The IC50 values for each combination ranged from 1.42 to 163 microg/ml. AcAaIT-derived polyhedrin gave the lowest IC50 value, followed by those of SlNPV, AcAaIT-field, and AcMNPV. The optimized ELISA system showed low cross reactivity for AcMNPV (0.87%), AcAaIT-field (1.2%), and SlNPV (4.0%). Genomic DNAs isolated from AcAaIT that were passaged in larvae of S. littoralis that were reared in the laboratory or field did not show any detectable differences. Albino rats (male and female) that were treated with AcAaIT, AcMNPV or SlNPV (either orally or by intraperitoneal injection at

  13. Impaired olfactory bulb neurogenesis depends on the presence of human wild-type alpha-synuclein.

    PubMed

    May, V E L; Nuber, S; Marxreiter, F; Riess, O; Winner, B; Winkler, J

    2012-10-11

    Synucleinopathies including Parkinson's disease (PD) are characterized by the accumulation of alpha-synuclein (α-syn) within neural cell bodies and their processes. Transgenic mice overexpressing human wild-type or mutant forms of α-syn under the control of different promoters were developed to analyse the underlying neuropathology of PD. One of the earliest clinical symptoms associated with PD is olfactory impairment. The generation of new neurons persists up to adulthood in mammals, in particular the olfactory bulb (OB). In order to assess this process in relation to α-syn accumulation, we used mice overexpressing human wild-type α-syn under the regulatable control (tet-off) of the calcium/calmodulin-dependent protein kinase IIα-promoter (CaMKII). We observed a decrease in OB neurogenesis in transgenic animals compared to controls using 5-bromo-2'-deoxyuridine (BrdU) to label newly generated cells (neuron-specific nuclear protein; NeuN). After cessation of transgene expression we detected an increase in newly generated cells both in granular (GCL) and glomerular (GLOM) layers of the OB. This led to a rescue of newly generated neurons (BrdU(+)/NeuN(+)) within the GLOM with a distinct specificity for the dopaminergic subpopulation. In contrast, we did not detect a cell-specific rescue of neuronal cells in the GCL suggesting diverse effects of alpha-synucleinopathy in both interneuronal layers of the OB. Colabelling of BrdU with glial markers showed that a differentiation into neither astroglia nor microglia attributed to the observed phenotype in the GCL. In particular, BrdU(+) particles located within microglial cells were predominantly associated close to the membrane therefore the resembling phagocytosed nuclear fragments of BrdU(+) cells. Thus, our study further contributes insights into α-syn accumulation as a causative player in the impairment of adult neurogenesis and emphasizes its diverse role in cell renewal of distinct OB cell layers. PMID:22814000

  14. Posttranslational Modifications in Type I Collagen from Different Tissues Extracted from Wild Type and Prolyl 3-Hydroxylase 1 Null Mice*

    PubMed Central

    Pokidysheva, Elena; Zientek, Keith D.; Ishikawa, Yoshihiro; Mizuno, Kazunori; Vranka, Janice A.; Montgomery, Nathan T.; Keene, Douglas R.; Kawaguchi, Tatsuya; Okuyama, Kenji; Bächinger, Hans Peter

    2013-01-01

    Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils. PMID:23861401

  15. Visualization of Melanosome Dynamics within Wild-Type and Dilute Melanocytes Suggests a Paradigm for Myosin V Function In Vivo

    PubMed Central

    Wu, Xufeng; Bowers, Blair; Rao, Kang; Wei, Qin; Hammer, John A.

    1998-01-01

    Unlike wild-type mouse melanocytes, where melanosomes are concentrated in dendrites and dendritic tips, melanosomes in dilute (myosin Va−) melanocytes are concentrated in the cell center. Here we sought to define the role that myosin Va plays in melanosome transport and distribution. Actin filaments that comprise a cortical shell running the length of the dendrite were found to exhibit a random orientation, suggesting that myosin Va could drive the outward spreading of melanosomes by catalyzing random walks. In contrast to this mechanism, time lapse video microscopy revealed that melanosomes undergo rapid (∼1.5 μm/s) microtubule-dependent movements to the periphery and back again. This bidirectional traffic occurs in both wild-type and dilute melanocytes, but it is more obvious in dilute melanocytes because the only melanosomes in their periphery are those undergoing this movement. While providing an efficient means to transport melanosomes to the periphery, this component does not by itself result in their net accumulation there. These observations, together with previous studies showing extensive colocalization of myosin Va and melanosomes in the actin-rich periphery, suggest a mechanism in which a myosin Va–dependent interaction of melanosomes with F-actin in the periphery prevents these organelles from returning on microtubules to the cell center, causing their distal accumulation. This “capture” model is supported by the demonstration that (a) expression of the myosin Va tail domain within wild-type cells creates a dilute-like phenotype via a process involving initial colocalization of tail domains with melanosomes in the periphery, followed by an ∼120-min, microtubule-based redistribution of melanosomes to the cell center; (b) microtubule-dependent melanosome movement appears to be damped by myosin Va; (c) intermittent, microtubule-independent, ∼0.14 μm/s melanosome movements are seen only in wild-type melanocytes; and (d) these movements do

  16. Mitochondrially targeted wild-type p53 induces apoptosis in a solid human tumor xenograft model

    PubMed Central

    Palacios, Gustavo; Crawford, Howard C.; Vaseva, Angelina; Moll, Ute M.

    2013-01-01

    Classic but also novel roles of p53 are becoming increasingly well characterized. We previously showed that ex vivo retroviral transfer of mitochondrially targeted wild type p53 (mitop53) in the Eμ-myc mouse lymphoma model efficiently induces tumor cell killing in vivo. In an effort to further explore the therapeutic potential of mitop53 for its pro-apoptotic effect in solid tumors, we generated replication-deficient recombinant human Adenovirus type 5 vectors. We show here that adenoviral delivery of mitop53 by intratumoral injection into HCT116 human colon carcinoma xenograft tumors in nude mice is surprisingly effective, resulting in tumor cell death of comparable potency to conventional p53. These apoptotic effects in vivo were confirmed by Ad5-mitop53 mediated cell death of HCT116 cells in culture. Together, these data provide encouragement to further explore the potential for novel mitop53 proteins in cancer therapy to execute the shortest known circuitry of p53 death signaling. PMID:18719383

  17. Magnetic Resonance Spectroscopy discriminates the response to microglial stimulation of wild type and Alzheimer's disease models.

    PubMed

    Pardon, Marie-Christine; Yanez Lopez, Maria; Yuchun, Ding; Marjańska, Małgorzata; Prior, Malcolm; Brignell, Christopher; Parhizkar, Samira; Agostini, Alessandra; Bai, Li; Auer, Dorothee P; Faas, Henryk M

    2016-01-01

    Microglia activation has emerged as a potential key factor in the pathogenesis of Alzheimer's disease. Metabolite levels assessed by magnetic resonance spectroscopy (MRS) are used as markers of neuroinflammation in neurodegenerative diseases, but how they relate to microglial activation in health and chronic disease is incompletely understood. Using MRS, we monitored the brain metabolic response to lipopolysaccharides (LPS)-induced microglia activation in vivo in a transgenic mouse model of Alzheimer's disease (APP/PS1) and healthy controls (wild-type (WT) littermates) over 4 hours. We assessed reactive gliosis by immunohistochemistry and correlated metabolic and histological measures. In WT mice, LPS induced a microglial phenotype consistent with activation, associated with a sustained increase in macromolecule and lipid levels (ML9). This effect was not seen in APP/PS1 mice, where LPS did not lead to a microglial response measured by histology, but induced a late increase in the putative inflammation marker myoinositol (mI) and metabolic changes in total creatine and taurine previously reported to be associated with amyloid load. We argue that ML9 and mI distinguish the response of WT and APP/PS1 mice to immune mediators. Lipid and macromolecule levels may represent a biomarker of activation of healthy microglia, while mI may not be a glial marker. PMID:26813748

  18. Female Adult Mouse Cardiomyocytes Are Protected Against Oxidative Stress

    PubMed Central

    Wang, Fangfei; He, Quan; Sun, Ying; Dai, Xiangguo; Yang, Xiao-Ping

    2010-01-01

    Premenopausal women have less cardiovascular disease and lower cardiovascular morbidity and mortality than men the same age. Our previous studies showed that female mice have lower mortality and better preserved cardiac function after myocardial infarction. However, the precise cellular and molecular mechanisms responsible for such a sex difference are not well established. Using cultured adult mouse cardiomyocytes (ACMs), we tested the hypothesis that the survival advantage of females stems from activated estrogen receptors (ER) and Akt survival signaling pathways. ACMs were isolated from male and female C57BL/6J mice and treated with hydrogen peroxide (H2O2, 100 μM) for 30 min. Cell survival was indicated by rod ratio (rod shaped cells/total cells) and cell death by lactate dehydrogenase (LDH) release and positive staining of Annexin-V (AV+, a marker for apoptosis) and propidium iodide (PI+, a marker for necrosis). In response to H2O2, female ACMs exhibited a higher rod ratio, lower LDH release and fewer AV+ and PI+ cells compared to males. Phospho-Akt was greater in females both at baseline and after H2O2 stimulation. The downstream molecule of Akt, phosphor-GSK-3β (inactivation), was also higher while caspase-3 activity was lower in females in response to H2O2. Bcl-2 did not differ between genders. ERα was the dominant isoform in females, whereas ERβ was low but similar in both genders. Our findings demonstrate that female ACMs have a greater survival advantage when challenged with oxidative stress-induced cell death. This may be attributable to activation of Akt and inhibition of GSK-3β and caspase-3 through an ERα-mediated mechanism. PMID:20212261

  19. Spaceflight influences both mucosal and peripheral cytokine production in PTN-Tg and wild type mice.

    PubMed

    McCarville, Justin L; Clarke, Sandra T; Shastri, Padmaja; Liu, Yi; Kalmokoff, Martin; Brooks, Stephen P J; Green-Johnson, Julia M

    2013-01-01

    Spaceflight is associated with several health issues including diminished immune efficiency. Effects of long-term spaceflight on selected immune parameters of wild type (Wt) and transgenic mice over-expressing pleiotrophin under the human bone-specific osteocalcin promoter (PTN-Tg) were examined using the novel Mouse Drawer System (MDS) aboard the International Space Station (ISS) over a 91 day period. Effects of this long duration flight on PTN-Tg and Wt mice were determined in comparison to ground controls and vivarium-housed PTN-Tg and Wt mice. Levels of interleukin-2 (IL-2) and transforming growth factor-beta1 (TGF-β1) were measured in mucosal and systemic tissues of Wt and PTN-Tg mice. Colonic contents were also analyzed to assess potential effects on the gut microbiota, although no firm conclusions could be made due to constraints imposed by the MDS payload and the time of sampling. Spaceflight-associated differences were observed in colonic tissue and systemic lymph node levels of IL-2 and TGF-β1 relative to ground controls. Total colonic TGF-β1 levels were lower in Wt and PTN-Tg flight mice in comparison to ground controls. The Wt flight mouse had lower levels of IL-2 and TGF-β1 compared to the Wt ground control in both the inguinal and brachial lymph nodes, however this pattern was not consistently observed in PTN-Tg mice. Vivarium-housed Wt controls had higher levels of active TGF-β1 and IL-2 in inguinal lymph nodes relative to PTN-Tg mice. The results of this study suggest compartmentalized effects of spaceflight and on immune parameters in mice. PMID:23874826

  20. Overexpression of ALS-associated p.M337V human TDP-43 in mice worsens disease features compared to wild-type human TDP-43 mice.

    PubMed

    Janssens, Jonathan; Wils, Hans; Kleinberger, Gernot; Joris, Geert; Cuijt, Ivy; Ceuterick-de Groote, Chantal; Van Broeckhoven, Christine; Kumar-Singh, Samir

    2013-08-01

    Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis (ALS), while wild-type TDP-43 is a pathological hallmark of patients with sporadic ALS and frontotemporal lobar degeneration (FTLD). Various in vitro and in vivo studies have also demonstrated toxicity of both mutant and wild-type TDP-43 to neuronal cells. To study the potential additional toxicity incurred by mutant TDP-43 in vivo, we generated mutant human TDP-43 (p.M337V) transgenic mouse lines driven by the Thy-1.2 promoter (Mt-TAR) and compared them in the same experimental setting to the disease phenotype observed in wild-type TDP-43 transgenic lines (Wt-TAR) expressing comparable TDP-43 levels. Overexpression of mutant TDP-43 leads to a worsened dose-dependent disease phenotype in terms of motor dysfunction, neurodegeneration, gliosis, and development of ubiquitin and phosphorylated TDP-43 pathology. Furthermore, we show that cellular aggregate formation or accumulation of TDP-43 C-terminal fragments (CTFs) are not primarily responsible for development of the observed disease phenotype in both mutant and wild-type TDP-43 mice. PMID:23475610

  1. Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells

    SciTech Connect

    Amacher, S.L.; Goodman, L.J.; Bravo, D.A.; Wong, K.Y.; Goldfine, I.D.; Hawley, D.M.; Firestone, G.L. )

    1989-10-01

    Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by (125I) insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways.

  2. Wild-Type and Non-Wild-Type Mycobacterium tuberculosis MIC Distributions for the Novel Fluoroquinolone Antofloxacin Compared with Those for Ofloxacin, Levofloxacin, and Moxifloxacin

    PubMed Central

    Yu, Xia; Wang, Guirong; Chen, Suting; Wei, Guomei; Shang, Yuanyuan; Dong, Lingling; Schön, Thomas; Moradigaravand, Danesh; Peacock, Sharon J.

    2016-01-01

    Antofloxacin (AFX) is a novel fluoroquinolone that has been approved in China for the treatment of infections caused by a variety of bacterial species. We investigated whether it could be repurposed for the treatment of tuberculosis by studying its in vitro activity. We determined the wild-type and non-wild-type MIC ranges for AFX as well as ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), using the microplate alamarBlue assay, of 126 clinical Mycobacterium tuberculosis strains from Beijing, China, of which 48 were OFX resistant on the basis of drug susceptibility testing on Löwenstein-Jensen medium. The MIC distributions were correlated with mutations in the quinolone resistance-determining regions of gyrA (Rv0006) and gyrB (Rv0005). Pharmacokinetic/pharmacodynamic (PK/PD) data for AFX were retrieved from the literature. AFX showed lower MIC levels than OFX but higher MIC levels than LFX and MFX on the basis of the tentative epidemiological cutoff values (ECOFFs) determined in this study. All strains with non-wild-type MICs for AFX harbored known resistance mutations that also resulted in non-wild-type MICs for LFX and MFX. Moreover, our data suggested that the current critical concentration of OFX for Löwenstein-Jensen medium that was recently revised by the World Health Organization might be too high, resulting in the misclassification of phenotypically non-wild-type strains with known resistance mutations as wild type. On the basis of our exploratory PK/PD calculations, the current dose of AFX is unlikely to be optimal for the treatment of tuberculosis, but higher doses could be effective. PMID:27324769

  3. Wild-Type and Non-Wild-Type Mycobacterium tuberculosis MIC Distributions for the Novel Fluoroquinolone Antofloxacin Compared with Those for Ofloxacin, Levofloxacin, and Moxifloxacin.

    PubMed

    Yu, Xia; Wang, Guirong; Chen, Suting; Wei, Guomei; Shang, Yuanyuan; Dong, Lingling; Schön, Thomas; Moradigaravand, Danesh; Parkhill, Julian; Peacock, Sharon J; Köser, Claudio U; Huang, Hairong

    2016-09-01

    Antofloxacin (AFX) is a novel fluoroquinolone that has been approved in China for the treatment of infections caused by a variety of bacterial species. We investigated whether it could be repurposed for the treatment of tuberculosis by studying its in vitro activity. We determined the wild-type and non-wild-type MIC ranges for AFX as well as ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), using the microplate alamarBlue assay, of 126 clinical Mycobacterium tuberculosis strains from Beijing, China, of which 48 were OFX resistant on the basis of drug susceptibility testing on Löwenstein-Jensen medium. The MIC distributions were correlated with mutations in the quinolone resistance-determining regions of gyrA (Rv0006) and gyrB (Rv0005). Pharmacokinetic/pharmacodynamic (PK/PD) data for AFX were retrieved from the literature. AFX showed lower MIC levels than OFX but higher MIC levels than LFX and MFX on the basis of the tentative epidemiological cutoff values (ECOFFs) determined in this study. All strains with non-wild-type MICs for AFX harbored known resistance mutations that also resulted in non-wild-type MICs for LFX and MFX. Moreover, our data suggested that the current critical concentration of OFX for Löwenstein-Jensen medium that was recently revised by the World Health Organization might be too high, resulting in the misclassification of phenotypically non-wild-type strains with known resistance mutations as wild type. On the basis of our exploratory PK/PD calculations, the current dose of AFX is unlikely to be optimal for the treatment of tuberculosis, but higher doses could be effective. PMID:27324769

  4. Loss of Sigma Factor RpoN Increases Intestinal Colonization of Vibrio parahaemolyticus in an Adult Mouse Model

    PubMed Central

    Whitaker, W. Brian; Richards, Gary P.

    2014-01-01

    Vibrio parahaemolyticus is the leading cause of bacterial seafood-borne gastroenteritis worldwide, yet little is known about how this pathogen colonizes the human intestine. The alternative sigma factor RpoN/sigma-54 is a global regulator that controls flagellar synthesis, as well as a wide range of nonflagellar genes. We constructed an in-frame deletion mutation in rpoN (VP2670) in V. parahaemolyticus RIMD2210633, a clinical serogroup O3:K6 isolate, and examined the effects in vivo using a streptomycin-treated mouse model of colonization. We confirmed that deletion of rpoN rendered V. parahaemolyticus nonmotile, and it caused reduced biofilm formation and an apparent defect in glutamine synthetase production. In in vivo competition assays between the rpoN mutant and a wild-type RIMD2210633 strain marked with the β-galactosidase gene lacZ (WBWlacZ), the mutant colonized significantly more proficiently. Intestinal persistence competition assays also demonstrated that the rpoN mutant had enhanced fitness and outcompeted WBWlacZ. Mutants defective in the polar flagellum biosynthesis FliAP sigma factor also outcompeted WBWlacZ but not to the same level as the rpoN mutant, which suggested that lack of motility is not the sole cause of the fitness effect. In an in vitro growth competition assay in mouse intestinal mucus, the rpoN mutant also outcompeted the wild type and exhibited faster doubling times when grown in mucus and on individual components of mucus. Genes in the pathways for the catabolism of mucus sugars also had significantly higher expression levels in a ΔrpoN mutant than in the wild type. These data suggest that in V. parahaemolyticus, RpoN plays an important role in carbon utilization regulation, which may significantly affect host colonization. PMID:24478070

  5. Pharmacologic activation of wild-type p53 by nutlin therapy in childhood cancer.

    PubMed

    Van Maerken, Tom; Rihani, Ali; Van Goethem, Alan; De Paepe, Anne; Speleman, Frank; Vandesompele, Jo

    2014-03-28

    A peculiar feature of several types of childhood cancer is that loss-of-function mutations of the TP53 (p53) tumor suppressor gene are uncommon, in contrast to many adult tumors. As p53 needs to be inactivated in order for tumor cells to survive and thrive, pediatric tumors typically make use of other mechanisms to keep p53 in check. One of the critical negative regulators of p53 is the MDM2 oncoprotein. Many anticancer drug development efforts in the past decade have therefore been devoted to the discovery and optimization of small molecules that selectively disrupt the interaction between MDM2 and p53, which could provide, in principle, a potent means to restore p53 function in tumor cells with wild-type p53. The nutlins are the class of selective inhibitors of the p53-MDM2 interaction that are currently most advanced in their clinical development. We review here the preclinical data that support the potential therapeutic use of nutlin drugs in the treatment of various pediatric tumors, including neuroblastoma, retinoblastoma, osteosarcoma, Ewing's sarcoma, rhabdomyosarcoma, medulloblastoma, and childhood acute lymphoblastic leukemia. PMID:24262662

  6. SURVIVAL AND EFFECTS OF WILD-TYPE, MUTANT, AND RECOMBINANT STREPTOMYCES IN A SOIL ECOSYSTEM

    EPA Science Inventory

    In a laboratory simulation, selected wild-type, mutant, and recombinant Streptomyces were released into a silt loam soil. trains included genetically enhanced lignin decomposers and those expressing recombinant plasmids. heir survival and effects on soil organic carbon mineraliza...

  7. Comparison between NOx Evolution Mechanisms of Wild-Type and nr1 Mutant Soybean Leaves 1

    PubMed Central

    Klepper, Lowell

    1990-01-01

    The nr1 soybean (Glycine max [L.] Merr.) mutant does not contain the two constitutive nitrate reductases, one of which is responsible for enzymic conversion of nitrite to NOx (NO + NO2). It was tested for possible nonenzymic NOx formation and evolution because of known chemical reactions between NO2− and plant metabolites and the instability of nitrous acid. It did not evolve NOx during the in vivo NR assay, but intact leaves did evolve small amounts of NOx under dark, anaerobic conditions. Experiments were conducted to compare NO3− reduction, NO2− accumulation, and the NOx evolution processes of the wild type (cv Williams) and the nr1 mutant. In vivo NR assays showed that wild-type leaves had three times more NO3− reducing capacity than the nr1 mutant. NOx evolution from intact, anerobic nr1 leaves was approximately 10 to 20% that from wild-type leaves. Nitrite content of the nr1 mutant leaves was usually higher than wild type due to low NOx evolution. Lag times and threshold NO2− concentrations for NOx evolution were similar for the two genotypes. While only 1 to 2% of NOx from wild type is NO2, the nr1 mutant evolved 15 to 30% NO2. The kinetic patterns of NOx evolution with time weré completely different for the mutant and wild type. Comparisons of light and heat treatments also gave very different results. It is generally accepted that the NOx evolution by wild type is primarily an enzymic conversion of NO2− to NO. However, this report concludes that NOx evolution by the nr1 mutant was due to nonenzymic, chemical reactions between plant metabolites and accumulated NO2− and/or decomposition of nitrous acid. Nonenzymic NOx evolution probably also occurs in wild type to a degree but could be easily masked by high rates of the enzymic process. PMID:16667445

  8. Co-fibrillogenesis of Wild-type and D76N β2-Microglobulin

    PubMed Central

    Natalello, Antonino; Mangione, P. Patrizia; Giorgetti, Sofia; Porcari, Riccardo; Marchese, Loredana; Zorzoli, Irene; Relini, Annalisa; Ami, Diletta; Faravelli, Giulia; Valli, Maurizia; Stoppini, Monica; Doglia, Silvia M.; Bellotti, Vittorio; Raimondi, Sara

    2016-01-01

    The amyloidogenic variant of β2-microglobulin, D76N, can readily convert into genuine fibrils under physiological conditions and primes in vitro the fibrillogenesis of the wild-type β2-microglobulin. By Fourier transformed infrared spectroscopy, we have demonstrated that the amyloid transformation of wild-type β2-microglobulin can be induced by the variant only after its complete fibrillar conversion. Our current findings are consistent with preliminary data in which we have shown a seeding effect of fibrils formed from D76N or the natural truncated form of β2-microglobulin lacking the first six N-terminal residues. Interestingly, the hybrid wild-type/variant fibrillar material acquired a thermodynamic stability similar to that of homogenous D76N β2-microglobulin fibrils and significantly higher than the wild-type homogeneous fibrils prepared at neutral pH in the presence of 20% trifluoroethanol. These results suggest that the surface of D76N β2-microglobulin fibrils can favor the transition of the wild-type protein into an amyloid conformation leading to a rapid integration into fibrils. The chaperone crystallin, which is a mild modulator of the lag phase of the variant fibrillogenesis, potently inhibits fibril elongation of the wild-type even once it is absorbed on D76N β2-microglobulin fibrils. PMID:26921323

  9. Human IL-2 mutein with higher antitumor efficacy than wild type IL-2.

    PubMed

    Carmenate, Tania; Pacios, Anabel; Enamorado, Michel; Moreno, Ernesto; Garcia-Martínez, Karina; Fuente, Dasha; León, Kalet

    2013-06-15

    IL-2 has been used for the treatment of melanoma and renal cell carcinoma, but this therapy has limited efficacy and severe toxicity. Currently, it is assumed that part of the limited efficacy is due to the IL-2-driven preferential expansion of regulatory T cells, which dampen the antitumor immunity. In this study, we characterize a human IL-2 mutant with higher antitumor efficacy and lower toxicity than wild type human IL-2 (wtIL-2). The mutant differs from wtIL-2 by four mutations at the interface with the α subunit of IL-2R. The IL-2 mutant induces in vitro proliferation of CD8(+)CD44(hi) and NK1.1 cells as efficiently as does wtIL-2, but it shows a reduced capacity to induce proliferation of CD4(+)Foxp3(+) regulatory T cells. The IL-2 mutant shows a higher antimetastatic effect than does wtIL-2 in several transplantable tumor models: the experimental metastasis model of MB16F0 melanoma and the experimental and spontaneous metastasis models for the mouse pulmonary carcinoma 3LL-D1222. Relevantly, the IL-2 mutant also exhibits lower lung and liver toxicity than does wtIL-2 when used at high doses in mice. In silico simulations, using a calibrated mathematical model, predict that the properties of IL-2 mutein are a consequence of the reduction, of at least two orders of magnitude, in its affinity for the α subunit of IL-2R (CD25). The human IL-2 mutant described in the present work could be a good candidate for improving cancer therapy based on IL-2. PMID:23677467

  10. Manganese supplementation protects against diet-induced diabetes in wild type mice by enhancing insulin secretion.

    PubMed

    Lee, Soh-Hyun; Jouihan, Hani A; Cooksey, Robert C; Jones, Deborah; Kim, Hyung J; Winge, Dennis R; McClain, Donald A

    2013-03-01

    Mitochondrial dysfunction is both a contributing mechanism and complication of diabetes, and oxidative stress contributes to that dysfunction. Mitochondrial manganese-superoxide dismutase (MnSOD) is a metalloenzyme that provides antioxidant protection. We have previously shown in a mouse model of hereditary iron overload that cytosolic iron levels affected mitochondrial manganese availability, MnSOD activity, and insulin secretion. We therefore sought to determine the metallation status of MnSOD in wild-type mice and whether altering that status affected β-cell function. 129/SvEVTac mice given supplemental manganese exhibited a 73% increase in hepatic MnSOD activity and increased metallation of MnSOD. To determine whether manganese supplementation offered glucose homeostasis under a situation of β-cell stress, we challenged C57BL/6J mice, which are more susceptible to diet-induced diabetes, with a high-fat diet for 12 weeks. Manganese was supplemented or not for the final 8 weeks on that diet, after which we examined glucose tolerance and the function of isolated islets. Liver mitochondria from manganese-injected C57BL/6J mice had similar increases in MnSOD activity (81%) and metallation as were seen in 129/SvEVTac mice. The manganese-treated group fed high fat had improved glucose tolerance (24% decrease in fasting glucose and 41% decrease in area under the glucose curve), comparable with mice on normal chow and increased serum insulin levels. Isolated islets from the manganese-treated group exhibited improved insulin secretion, decreased lipid peroxidation, and improved mitochondrial function. In conclusion, MnSOD metallation and activity can be augmented with manganese supplementation in normal mice on normal chow, and manganese treatment can increase insulin secretion to improve glucose tolerance under conditions of dietary stress. PMID:23372018

  11. Internal binding sites for MSH: Analyses in wild-type and variant Cloudman melanoma cells

    SciTech Connect

    Orlow, S.J.; Hotchkiss, S.; Pawelek, J.M. )

    1990-01-01

    Cloudman S91 mouse melanoma cells express both external (plasma membrane) and internal binding sites for MSH. Using 125I-beta melanotropin (beta-MSH) as a probe, we report here an extensive series of studies on the biological relevance of these internal sites. Cells were swollen in a hypotonic buffer and lysed, and a particulate fraction was prepared by high-speed centrifugation. This fraction was incubated with 125I-beta-MSH with or without excess nonradioactive beta-MSH in the cold for 2 hours. The material was then layered onto a step-wise sucrose gradient and centrifuged; fractions were collected and counted in a gamma counter or assayed for various enzymatic activities. The following points were established: (1) Specific binding sites for MSH were observed sedimenting at an average density of 50% sucrose in amelanotic cells and at higher densities in melanotic cells. (2) These sites were similar in density to those observed when intact cells were labeled externally with 125I-beta-MSH and then warmed to promote internalization of the hormone. (3) Most of the internal binding sites were not as dense as fully melanized melanosomes. (4) In control experiments, the MSH binding sites were not found in cultured hepatoma cells. (5) Variant melanoma cells, which differed from the wild-type in their responses to MSH, had reduced expression of internal binding sites even though their ability to bind MSH to the outer cell surface appeared normal. (MSH-induced responses included changes in tyrosinase, dopa oxidase, and dopachrome conversion factor activities, melanization, proliferation, and morphology.) (6) Isobutylmethylxanthine, which enhanced cellular responsiveness to MSH, also enhanced expression of internal binding sites. The results indicate that expression of internal binding sites for MSH is an important criterion for cellular responsiveness to the hormone.

  12. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    SciTech Connect

    Su, Xin; Guan, Wuqiang; Yu, Yong-Chun; Fu, Yinghui

    2014-07-18

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1{sup +} or nestin{sup +} stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU{sup +} cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU{sup +} cells, very few are mash1{sup +} or nestin{sup +} stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1{sup +} microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.

  13. Wild-type and e antigen-minus hepatitis B viruses and course of chronic hepatitis.

    PubMed Central

    Brunetto, M R; Giarin, M M; Oliveri, F; Chiaberge, E; Baldi, M; Alfarano, A; Serra, A; Saracco, G; Verme, G; Will, H

    1991-01-01

    Using an oligonucleotide hybridization assay, we studied the clinical implication of wild-type hepatitis B virus (HBV) and a HBV mutant that is unable to secrete hepatitis B e antigen (HBeAg) because of a translational defect due to a stop codon in the pre-C region in 106 hepatitis B surface antigen-positive patients with chronic hepatitis B. Wild-type HBV was detected in 31 of 42 (73.8%) HBeAg-positive patients, whereas a mixed viral population was present in 10 (23.8%). Significant differences in the severity and outcome of liver disease were not observed in the two groups of patients. However, the emergence of HBeAg-minus HBV in wild-type HBV carriers was associated with an exacerbation of liver disease and was followed by the presence of antibodies against HBeAg (anti-HBe) in serum in 50% of the cases. In 61 of 64 (95.3%) anti-HBe-positive patients, HBeAg-minus HBV was the predominant virus: HBeAg-minus HBV was detected in 42 patients (65.6%), whereas both wild-type and HBeAg-minus HBV were present in 19 (29.7%). HBeAg-minus HBV was associated with a course of hepatitis characterized by flare-ups of liver cell necrosis interspersed with periods of asymptomatic HBV carriage (P less than 0.01). These data support the hypothesis that genetic heterogeneity of HBV significantly influences the course and outcome of chronic hepatitis B. Wild-type HBV secreting HBeAg induces immunologic tolerance and causes chronic infection. HBeAg-minus HBV might be unable to induce chronic infection without the helper function of wild-type HBV, but it appears to be more pathogenic. Once chronic infection is established, HBeAg-minus HBV variants may prevail and displace wild-type virus. Images PMID:2034663

  14. Shoaling and mate choice of wild-type Tanichthys albonubes in the presence of the red fluorescent transgenic conspecifics.

    PubMed

    Jiang, P; Bai, J J; Ye, X; Jian, Q; Chen, M; Chen, X Q

    2011-01-01

    Shoaling and sexual behaviour of wild-type male and female white cloud mountain minnow Tanichthys albonubes were measured in the presence of the red fluorescent transgenic conspecifics under laboratory conditions. Wild-type female test fish showed no significant preference, whereas wild-type male test fish preferred to be near a shoal of red transgenic fish rather than wild-type fish. When placed in a potentially reproductive context, wild-type males had a higher competitive ability over transgenic males; wild-type females spent more time with wild-type males in visually mediated experiments, but wild-type males performed more courtship displays towards transgenic females. These results suggest that the red body colouration does not appear to disturb signal communication between wild-type and transgenic T. albonubes in shoaling behaviour; transgenic males have no mating advantage over wild-type males, but the red body colouration of transgenic females may affect mate choice of wild-type males. PMID:21235550

  15. Variable stress-responsiveness in wild type and domesticated fighting fish.

    PubMed

    Verbeek, Peter; Iwamoto, Toshitaka; Murakami, Noboru

    2008-01-28

    We combined behavioral and physiological measures to compare coping style in wild-type Betta splendens and a domesticated strain selectively bred for sports fighting. We showed previously that the fighter strain is more aggressive than the wild type during experimental conditions that most closely resemble an actual fight. We predicted that compared to the wild type, the fighter strain would show a more proactive coping style, characterized by lesser cortisol and greater sympathetic responses to non-social challenges. We introduced males to an unfamiliar environment and spatial confinement as challenges that may resemble some of those that B. splendens may encounter in its natural habitat. We developed a non-invasive stress assay that enables repeated individual measures of water-borne cortisol. We estimated sympathetic activation through opercular beat rate and recorded the duration of behavioral immobility. We found that exposure to an unfamiliar environment raised cortisol levels in the wild type but not in the fighter strain and that confinement raised cortisol levels in both. In both strains opercular beat rates were significantly reduced during the latter stages of confinement compared to during the early stages. The fighter strain, but not the wild type, adopted a behavioral strategy of immobility from the very beginning of confinement. PMID:17884114

  16. A positively gravitropic mutant mirrors the wild-type protonemal response in the moss Ceratodon purpureus.

    PubMed

    Wagner, T A; Cove, D J; Sack, F D

    1997-06-01

    Wild-type Ceratodon purpureus (Hedw.) Brid. protonemata grow up in the dark by negative gravitropism. When upright wild-type protonemata are reoriented 90 degrees, they temporarily grow down soon after reorientation ("initial reversal") and also prior to cytokinesis ("mitotic reversal"). A positively gravitropic mutant designated wrong- way response (wwr-1) has been isolated by screening ultraviolet light-mutagenized Ceratodon protonemata. Protonemata of wwr-l reoriented from the vertical to the horizontal grow down with kinetics comparable to those of the wild-type. Protonemata of wwr-1 also show initial and mitotic reversals where they temporarily grow up. Thus, the direction of gravitropism, initial reversal, and mitotic reversal are coordinated though each are opposite in wwr-1 compared to the wild-type. Normal plastid zonation is still maintained in dark-grown wwr-1 apical cells, but the plastids are more numerous and plastid sedimentation is more pronounced. In addition, wwr-1 apical cells are wider and the tips greener than in the wild-type. These data suggest that a functional WWR gene product is not necessary for the establishment of some gravitropic polarity, for gravitropism, or for the coordination of the reversals. Thus, the WWR protein may normally transduce information about cell orientation. PMID:11541791

  17. A positively gravitropic mutant mirrors the wild-type protonemal response in the moss Ceratodon purpureus

    NASA Technical Reports Server (NTRS)

    Wagner, T. A.; Cove, D. J.; Sack, F. D.

    1997-01-01

    Wild-type Ceratodon purpureus (Hedw.) Brid. protonemata grow up in the dark by negative gravitropism. When upright wild-type protonemata are reoriented 90 degrees, they temporarily grow down soon after reorientation ("initial reversal") and also prior to cytokinesis ("mitotic reversal"). A positively gravitropic mutant designated wrong- way response (wwr-1) has been isolated by screening ultraviolet light-mutagenized Ceratodon protonemata. Protonemata of wwr-l reoriented from the vertical to the horizontal grow down with kinetics comparable to those of the wild-type. Protonemata of wwr-1 also show initial and mitotic reversals where they temporarily grow up. Thus, the direction of gravitropism, initial reversal, and mitotic reversal are coordinated though each are opposite in wwr-1 compared to the wild-type. Normal plastid zonation is still maintained in dark-grown wwr-1 apical cells, but the plastids are more numerous and plastid sedimentation is more pronounced. In addition, wwr-1 apical cells are wider and the tips greener than in the wild-type. These data suggest that a functional WWR gene product is not necessary for the establishment of some gravitropic polarity, for gravitropism, or for the coordination of the reversals. Thus, the WWR protein may normally transduce information about cell orientation.

  18. Orthotopic transplantation of LH receptor knockout and wild-type ovaries.

    PubMed

    Chudgar, Daksha; Lei, Zhenmin; Rao, Ch V

    2005-10-01

    Luteinizing hormone (LH) receptor knockout animals have an ovarian failure due to an arrest in folliculogenesis at the antral stage. As a result, the animals have an infertility phenotype. The present study was undertaken to determine whether this phenotype could be reversed by orthotopic transplantation of wild-type ovaries. The results revealed that transplanting wild-type ovaries into null animals did not result in resumption of estrus cycles. Although the number of different types of follicles increased, none progressed to ovulation. The serum hormone profiles improved, reflecting the ovarian changes. The wild-type animals with null ovaries also failed to cycle and their ovaries and serum hormone levels were more like null animals with their own ovaries. Although the lack of rescue of null ovaries placed into wild-type animals was predicted, the failure of wild-type ovaries placed in null animals was not, which could be due to chronic exposure of transplanted tissue to high circulating LH levels and also possibly due to altered internal milieu in null animals. These findings may have implications for potential future considerations of grafting normal donor ovaries into women who have an ovarian failure resulting from inactivating LH receptor mutations. PMID:15964032

  19. Localization of PPAR isotypes in the adult mouse and human brain

    PubMed Central

    Warden, Anna; Truitt, Jay; Merriman, Morgan; Ponomareva, Olga; Jameson, Kelly; Ferguson, Laura B.; Mayfield, R. Dayne; Harris, R. Adron

    2016-01-01

    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that act as ligand-activated transcription factors. PPAR agonists have well-documented anti-inflammatory and neuroprotective roles in the central nervous system. Recent evidence suggests that PPAR agonists are attractive therapeutic agents for treating neurodegenerative diseases as well as addiction. However, the distribution of PPAR mRNA and protein in brain regions associated with these conditions (i.e. prefrontal cortex, nucleus accumbens, amygdala, ventral tegmental area) is not well defined. Moreover, the cell type specificity of PPARs in mouse and human brain tissue has yet to be investigated. We utilized quantitative PCR and double immunofluorescence microscopy to determine that both PPAR mRNA and protein are expressed ubiquitously throughout the adult mouse brain. We found that PPARs have unique cell type specificities that are consistent between species. PPARα was the only isotype to colocalize with all cell types in both adult mouse and adult human brain tissue. Overall, we observed a strong neuronal signature, which raises the possibility that PPAR agonists may be targeting neurons rather than glia to produce neuroprotection. Our results fill critical gaps in PPAR distribution and define novel cell type specificity profiles in the adult mouse and human brain. PMID:27283430

  20. Spatiotemporal features of early neuronogenesis differ in wild-type and albino mouse retina

    NASA Technical Reports Server (NTRS)

    Rachel, Rivka A.; Dolen, Gul; Hayes, Nancy L.; Lu, Alice; Erskine, Lynda; Nowakowski, Richard S.; Mason, Carol A.

    2002-01-01

    In albino mammals, lack of pigment in the retinal pigment epithelium is associated with retinal defects, including poor visual acuity from a photoreceptor deficit in the central retina and poor depth perception from a decrease in ipsilaterally projecting retinal fibers. Possible contributors to these abnormalities are reported delays in neuronogenesis (Ilia and Jeffery, 1996) and retinal maturation (Webster and Rowe, 1991). To further determine possible perturbations in neuronogenesis and/or differentiation, we used cell-specific markers and refined birth dating methods to examine these events during retinal ganglion cell (RGC) genesis in albino and pigmented mice from embryonic day 11 (E11) to E18. Our data indicate that relative to pigmented mice, more ganglion cells are born in the early stages of neuronogenesis in the albino retina, although the initiation of RGC genesis in the albino is unchanged. The cellular organization of the albino retina is perturbed as early as E12. In addition, cell cycle kinetics and output along the nasotemporal axis differ in retinas of albino and pigmented mice, both absolutely, with the temporal aspect of the retina expanded in albino, and relative to the position of the optic nerve head. Finally, blocking melanin synthesis in pigmented eyecups in culture leads to an increase in RGC differentiation, consistent with a role for melanin formation in regulating RGC neuronogenesis. These results point to spatiotemporal defects in neuronal production in the albino retina, which could perturb expression of genes that specify cell fate, number, and/or projection phenotype.

  1. Characterizing Newly Repopulated Microglia in the Adult Mouse: Impacts on Animal Behavior, Cell Morphology, and Neuroinflammation

    PubMed Central

    Elmore, Monica R. P.; Lee, Rafael J.; West, Brian L.; Green, Kim N.

    2015-01-01

    Microglia are the primary immune cell in the brain and are postulated to play important roles outside of immunity. Administration of the dual colony-stimulating factor 1 receptor (CSF1R)/c-Kit kinase inhibitor, PLX3397, to adult mice results in the elimination of ~99% of microglia, which remain eliminated for as long as treatment continues. Upon removal of the inhibitor, microglia rapidly repopulate the entire adult brain, stemming from a central nervous system (CNS) resident progenitor cell. Using this method of microglial elimination and repopulation, the role of microglia in both healthy and diseased states can be explored. Here, we examine the responsiveness of newly repopulated microglia to an inflammatory stimulus, as well as determine the impact of these cells on behavior, cognition, and neuroinflammation. Two month-old wild-type mice were placed on either control or PLX3397 diet for 21 d to eliminate microglia. PLX3397 diet was then removed in a subset of animals to allow microglia to repopulate and behavioral testing conducted beginning at 14 d repopulation. Finally, inflammatory profiling of the microglia-repopulated brain in response to lipopolysaccharide (LPS; 0.25 mg/kg) or phosphate buffered saline (PBS) was determined 21 d after inhibitor removal using quantitative real time polymerase chain reaction (RT-PCR), as well as detailed analyses of microglial morphologies. We find mice with repopulated microglia to perform similarly to controls by measures of behavior, cognition, and motor function. Compared to control/resident microglia, repopulated microglia had larger cell bodies and less complex branching in their processes, which resolved over time after inhibitor removal. Inflammatory profiling revealed that the mRNA gene expression of repopulated microglia was similar to normal resident microglia and that these new cells appear functional and responsive to LPS. Overall, these data demonstrate that newly repopulated microglia function similarly to the

  2. Discrimination of oligonucleotides of different lengths with a wild-type aerolysin nanopore

    NASA Astrophysics Data System (ADS)

    Cao, Chan; Ying, Yi-Lun; Hu, Zheng-Li; Liao, Dong-Fang; Tian, He; Long, Yi-Tao

    2016-08-01

    Protein nanopores offer an inexpensive, label-free method of analysing single oligonucleotides. The sensitivity of the approach is largely determined by the characteristics of the pore-forming protein employed, and typically relies on nanopores that have been chemically modified or incorporate molecular motors. Effective, high-resolution discrimination of oligonucleotides using wild-type biological nanopores remains difficult to achieve. Here, we show that a wild-type aerolysin nanopore can resolve individual short oligonucleotides that are 2 to 10 bases long. The sensing capabilities are attributed to the geometry of aerolysin and the electrostatic interactions between the nanopore and the oligonucleotides. We also show that the wild-type aerolysin nanopores can distinguish individual oligonucleotides from mixtures and can monitor the stepwise cleavage of oligonucleotides by exonuclease I.

  3. Discrimination of oligonucleotides of different lengths with a wild-type aerolysin nanopore.

    PubMed

    Cao, Chan; Ying, Yi-Lun; Hu, Zheng-Li; Liao, Dong-Fang; Tian, He; Long, Yi-Tao

    2016-08-01

    Protein nanopores offer an inexpensive, label-free method of analysing single oligonucleotides. The sensitivity of the approach is largely determined by the characteristics of the pore-forming protein employed, and typically relies on nanopores that have been chemically modified or incorporate molecular motors. Effective, high-resolution discrimination of oligonucleotides using wild-type biological nanopores remains difficult to achieve. Here, we show that a wild-type aerolysin nanopore can resolve individual short oligonucleotides that are 2 to 10 bases long. The sensing capabilities are attributed to the geometry of aerolysin and the electrostatic interactions between the nanopore and the oligonucleotides. We also show that the wild-type aerolysin nanopores can distinguish individual oligonucleotides from mixtures and can monitor the stepwise cleavage of oligonucleotides by exonuclease I. PMID:27111839

  4. Transport of Wild-Type and Recombinant Nucleopolyhedroviruses by Scavenging and Predatory Arthropods.

    PubMed

    Lee; Fuxa

    2000-05-01

    Wild-type and recombinant nucleopolyhedroviruses (NPVs) were compared in their capability to be transported over limited distances by the predator Podisus maculiventris (Say) and scavengers Sarcophaga bullata (Parker) and Acheta domesticus (Linnaeus) in Trichoplusia ni (Hübner) larvae infesting collards in a greenhouse microcosm. Viruses tested were variants of Autographa californica (Speyer) NPV (AcNPV): wild-type virus (AcNPV.WT), AcNPV expressing a scorpion toxin (AcNPV.AaIT), and AcNPV expressing juvenile hormone esterase (AcJHE.SG). Podisus maculiventris transported AcNPV.WT and S. bullata transported AcNPV.WT and AcNPV.AaIT. Prevalence and transport of AcNPV.WT were greater than those of AcNPV.AaIT and AcJHE.SG, regardless of whether the nontarget organism carriers were present or absent. Podisus maculiventris and S. bullata transported recombinant and wild-type NPVs at a rate of up to 62.5 cm/day, and A. domesticus transported wild-type NPV at 125 cm/day. The infected host insects, T. ni, undoubtedly contributed to viral transport in the current research. In every experiment, both the wild-type and recombinant virus spread to some degree in the plots without predators or scavengers. The relative amounts of NPVs that accumulated in soil, as indicated by bioassay mortality percentages, generally exhibited spatial patterns similar to those of T. ni mortality due to NPV on the collards plants. Thus, the predator and scavengers in the current research demonstrated some capacity to transport wild-type as well as recombinant viruses at significant rates in a greenhouse microcosm. PMID:10882435

  5. Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes.

    PubMed Central

    Overturf, K.; al-Dhalimy, M.; Ou, C. N.; Finegold, M.; Grompe, M.

    1997-01-01

    Previous work has shown that adult mouse hepatocytes can divide at least 18 times in vivo. To test whether this represents the upper limit of their regenerative capacity, we performed serial transplantation of hepatocytes in the fumarylacetoacetate hydrolase deficiency murine model of liver repopulation. Hepatocytes from adult donors were serially transplanted in limiting numbers six times and resulted in complete repopulation during each cycle. This corresponds to a minimal number of 69 cell doublings or a 7.3 x 10(20)-fold expansion. No evidence for abnormal liver function or altered hepatic architecture was found in repopulated animals. We conclude that a fraction of adult mouse hepatocytes have growth potential similar to that of hematopoietic stem cells. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:9358753

  6. A comprehensive transcriptomic analysis of infant and adult mouse ovary.

    PubMed

    Pan, Linlin; Gong, Wei; Zhou, Yuanyuan; Li, Xiaonuan; Yu, Jun; Hu, Songnian

    2014-10-01

    Ovary development is a complex process involving numerous genes. A well-developed ovary is essential for females to keep fertility and reproduce offspring. In order to gain a better insight into the molecular mechanisms related to the process of mammalian ovary development, we performed a comparative transcriptomic analysis on ovaries isolated from infant and adult mice by using next-generation sequencing technology (SOLiD). We identified 15,454 and 16,646 transcriptionally active genes at the infant and adult stage, respectively. Among these genes, we also identified 7021 differentially expressed genes. Our analysis suggests that, in general, the adult ovary has a higher level of transcriptomic activity. However, it appears that genes related to primordial follicle development, such as those encoding Figla and Nobox, are more active in the infant ovary, whereas expression of genes vital for follicle development, such as Gdf9, Bmp4 and Bmp15, is upregulated in the adult. These data suggest a dynamic shift in gene expression during ovary development and it is apparent that these changes function to facilitate follicle maturation, when additional functional gene studies are considered. Furthermore, our investigation has also revealed several important functional pathways, such as apoptosis, MAPK and steroid biosynthesis, that appear to be much more active in the adult ovary compared to those of the infant. These findings will provide a solid foundation for future studies on ovary development in mice and other mammals and help to expand our understanding of the complex molecular and cellular events that occur during postnatal ovary development. PMID:25251848

  7. Erythritol Metabolism in Wild-Type and Mutant Strains of Schizophyllum commune

    PubMed Central

    Braun, M. L.; Niederpruem, D. J.

    1969-01-01

    Erythritol uptake and metabolism were compared in wild-type mycelium and a dome morphological mutant of the wood-rotting mushroom Schizophyllum commune. Wild-type mycelium utilized glucose, certain hexitols, and pentitols including ribitol, as well as d-erythrose, erythritol, and glycerol as sole carbon sources for growth. The dome mutant utilized all of these compounds except d-erythrose and erythritol. Erythritol- or glycerol-grown wild-type mycelium incorporated erythritol into various cellular constituents, whereas glucose-grown cells lagged considerably before initiation of erythritol uptake. This acquisition was inhibited by cycloheximide. Dome mycelium showed behavior similar to wild-type in uptake of erythritol after growth on glucose or glycerol, except that erythritol was not further catabolized. Enzymes of carbohydrate metabolism were compared in cell extracts of glucose-cultured wild-type mycelium and dome. Enzymes of hexose monophosphate catabolism, nicotinamide adenine dinucleotide (NAD)-dependent sugar alcohol dehydrogenases, and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-coupled erythrose reductase were demonstrated in both. The occurrence of erythrose reductase was unaffected by the nature of the growth carbon source, showed optimal activity at pH 7, and generated NAD phosphate and erythritol as products of the reaction. Glycerol-, d-erythrose-, or erythritol-grown wild-type mycelium contained an NAD-dependent erythritol dehydrogenase absent in glucose cells. Erythritol dehydrogenase activity was optimal at pH 8.8 and produced erythrulose during NAD reduction. Glycerol-growth of dome mycelium induced the erythritol uptake system, but a functional erythritol dehydrogenase could not be demonstrated. Neither wild-type nor dome mycelium produced erythritol dehydrogenase during growth on ribitol. Erythritol metabolism in wild-type cells of S. commune, therefore, involves an NADPH-dependent reduction of d-erythrose to produce erythritol

  8. Differential long-term effects of social stress during adolescence on anxiety in Wistar and wild-type rats.

    PubMed

    Vidal, Jose; Buwalda, Bauke; Koolhaas, Jaap M

    2011-06-01

    Severe and chronic stress may interfere with adolescent neuronal plasticity that turns the juvenile brain into an adult brain increasing the vulnerability to develop anxiety disorders. It is well-known from adult stress research that there is a large individual differentiation in stress vulnerability. The current study is aimed at the individual resilience and vulnerability to adolescent social stress. Two strains of rats that differ in social behavioral skills were subjected to social stress during adolescence. In three experiments we studied short and long term effects of adolescent social stress using a water conflict test in different contexts. Wistar rats which had been socially defeated on postnatal days 45 and 46 showed, following water deprivation, a strong decrease in the total amount of water consumed and time spent drinking when tested 2 days and 3 weeks later in the context where they received the defeat experience. Also a strong increase in drinking latency was noticed in the context of the previous defeat. No differences in these parameters were found between defeated and non-defeated wild-type rats. The results of the water conflict test in an environment where no association with the previous defeat experience was present showed that the adolescent social stress did not induce a generalized anxiety. In conclusion, the water conflict test is a useful tool to measure the influence of social defeat on the motivation to obtain resources under conditions with different stimulus properties. In addition, our data suggest the importance of the strain used in adolescent stress experiments. The fact that Wistar rats showed a strong association with the context at adulthood whereas no effect was observed in the wild-type rats shows that victim characteristics are important determining factors for the long term effects of adolescent social stress. PMID:21443935

  9. Wild-type and mutated presenilins 2 trigger p53-dependent apoptosis and down-regulate presenilin 1 expression in HEK293 human cells and in murine neurons.

    PubMed

    Alves da Costa, Cristine; Paitel, Erwan; Mattson, Mark P; Amson, Robert; Telerman, Adam; Ancolio, Karine; Checler, Frédéric; Mattson, Marc P

    2002-03-19

    Presenilins 1 and 2 are two homologous proteins that, when mutated, account for most early onset Alzheimer's disease. Several lines of evidence suggest that, among various functions, presenilins could modulate cell apoptotic responses. Here we establish that the overexpression of presenilin 2 (PS2) and its mutated form Asn-141-Ile-PS2 alters the viability of human embryonic kidney (HEK)293 cells as established by combined trypan blue exclusion, sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate assay, and propidium iodide incorporation FACS analyses. The two parent proteins increase the acetyl-DEVD-al-sensitive caspase-3-like activity in both HEK293 cells and Telencephalon specific murine neurons, modulate Bax and bcl-2 expressions, and enhance cytochrome C translocation into the cytosol. We show that overexpression of both wild-type and mutated PS2 increases p53-like immunoreactivity and transcriptional activity. We also establish that wild-type- and mutated PS2-induced caspase activation is reduced by p53 antisense approach and by pifithrin-alpha, a chemical inhibitor of p53. Furthermore, mouse fibroblasts in which the PS2 gene has been knocked out exhibited strongly reduced p53-transcriptional activity. Finally, we establish that the overexpression of both wild-type and mutated PS2 is accompanied by a drastic reduction of endogenous presenilin 1 (PS1) expression. Interestingly, pifithrin-alpha diminished endogenous PS2 immunoreactivity, whereas the inhibitor increases PS1 expression. Altogether, our data demonstrate that wild-type and familial Alzheimer's disease-linked PS2 trigger apoptosis and down-regulate PS1 expression through p53-dependent mechanisms. PMID:11904448

  10. Fertility comparison between wild type and transgenic mice by in vitro fertilization

    PubMed Central

    Vasudevan, Kuzhalini; Raber, James

    2011-01-01

    . Oocytes from superovulated females were inseminated with sperm of same background. Fertility rate was considered as the percentage of two cell embryos scored 24 h after insemination. The data collected from this study shows that the fertilization rate is affected (reduced to half fold) in some of the transgenic mice compared to the respective Wild Type (WT) mice. For the WT the average fertility rate ranged from 80% (C57BL/6), 90% (FVB/N), 45% (129Sv/J × C57Bl/6)F1 and 43% (CD1). For transgenic mice it was 52% (C57BL/6), 65% (FVB/N), 22% (129Sv/J × C57Bl/6)F1 and 25% (CD1). PMID:19844803

  11. Mouse matriptase-2: identification, characterization and comparative mRNA expression analysis with mouse hepsin in adult and embryonic tissues.

    PubMed Central

    Hooper, John D; Campagnolo, Luisa; Goodarzi, Goodarz; Truong, Tony N; Stuhlmann, Heidi; Quigley, James P

    2003-01-01

    We report the identification and characterization of mouse matriptase-2 (m-matriptase-2), an 811-amino-acid protein composed of an N-terminal cytoplasmic domain, a membrane-spanning domain, two CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains, three LDLR (low-density-lipoprotein receptor class A) domains and a C-terminal serine-protease domain. All m-matriptase-2 protein domain boundaries corresponded with intron/exon junctions of the encoding gene, which spans approx. 29 kb and comprises 18 exons. Matriptase-2 is highly conserved in human, mouse and rat, with the rat matriptase-2 gene ( r-maltriptase-2 ) predicted to encode transmembrane and soluble isoforms. Western-blot analysis indicated that m-matriptase-2 migrates close to its theoretical molecular mass of 91 kDa, and immunofluorescence analysis was consistent with the proposed surface membrane localization of this protein. Reverse-transcription PCR and in-situ -hybridization analysis indicated that m-matriptase-2 expression overlaps with the distribution of mouse hepsin (m-hepsin, a cell-surface serine protease identified in hepatoma cells) in adult tissues and during embryonic development. In adult tissues both are expressed at highest levels in liver, kidney and uterus. During embryogenesis m-matriptase-2 expression peaked between days 12.5 and 15.5. m-hepsin expression was biphasic, with peaks at day 7.5 to 8.5 and again between days 12.5 and 15.5. In situ hybridization of embryonic tissues indicated abundant expression of both m-matriptase-2 and m-hepsin in the developing liver and at lower levels in developing pharyngo-tympanic tubes. While m-hepsin was detected in the residual embryonic yolk sac and with lower intensity in lung, heart, gastrointestinal tract, developing kidney tubules and epithelium of the oral cavity, m-matriptase-2 was absent in these tissues, but strongly expressed within the nasal cavity by olfactory epithelial

  12. High-resolution gene expression atlases for adult and developing mouse brain and spinal cord.

    PubMed

    Henry, Alex M; Hohmann, John G

    2012-10-01

    Knowledge of the structure, genetics, circuits, and physiological properties of the mammalian brain in both normal and pathological states is ever increasing as research labs worldwide probe the various aspects of brain function. Until recently, however, comprehensive cataloging of gene expression across the central nervous system has been lacking. The Allen Institute for Brain Science, as part of its mission to propel neuroscience research, has completed several large gene-mapping projects in mouse, nonhuman primate, and human brain, producing informative online public resources and tools. Here we present the Allen Mouse Brain Atlas, covering ~20,000 genes throughout the adult mouse brain; the Allen Developing Mouse Brain Atlas, detailing expression of approximately 2,000 important developmental genes across seven embryonic and postnatal stages of brain growth; and the Allen Spinal Cord Atlas, revealing expression for ~20,000 genes in the adult and neonatal mouse spinal cords. Integrated data-mining tools, including reference atlases, informatics analyses, and 3-D viewers, are described. For these massive-scale projects, high-throughput industrial techniques were developed to standardize and reliably repeat experimental goals. To verify consistency and accuracy, a detailed analysis of the 1,000 most viewed genes for the adult mouse brain (according to website page views) was performed by comparing our data with peer-reviewed literature and other databases. We show that our data are highly consistent with independent sources and provide a comprehensive compendium of information and tools used by thousands of researchers each month. All data and tools are freely available via the Allen Brain Atlas portal (www.brain-map.org). PMID:22832508

  13. Unfolded protein response in hypothalamic cultures of wild-type and ATF6α-knockout mice.

    PubMed

    Lu, Wenjun; Hagiwara, Daisuke; Morishita, Yoshiaki; Tochiya, Masayoshi; Azuma, Yoshinori; Suga, Hidetaka; Goto, Motomitsu; Banno, Ryoichi; Sugimura, Yoshihisa; Oyadomari, Seiichi; Mori, Kazutoshi; Arima, Hiroshi

    2016-01-26

    Recent studies suggest that endoplasmic reticulum (ER) stress in the hypothalamus could affect systemic homeostatic regulation in areas such as energy and water balance. Activating transcription factor 6α (ATF6α) is an ER stress transducer which increases the expression of ER chaperones and ER-associated degradation (ERAD) components under ER stress. In the present study, we examined the regulation of the unfolding protein response (UPR) in mouse hypothalamic cultures of wild-type (WT) and ATF6α(-/-) mice. Thapsigargin (TG), an ER stressor, significantly increased the mRNA expression of immunoglobulin heavy chain binding protein (BiP), spliced X-box binding protein 1 (XBP1), activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), and ERAD components, in hypothalamic cultures of WT mice with the same threshold (0.1μM) and similar time courses. On the other hand, TG-induced upregulation of BiP and CHOP as well as most ERAD-related genes, but not spliced XBP1 or ATF4, was attenuated in ATF6α(-/-) mice compared with WT mice. Our data suggest that all the UPR arms are activated similarly in the mouse hypothalamus under ER stress conditions, where ATF6α regulates the expression of ER chaperones, CHOP, and ERAD components. PMID:26708632

  14. Cardiomyogenic potential of c-kit+ expressing cells derived from neonatal and adult mouse hearts

    PubMed Central

    Zaruba, Marc-Michael; Soonpaa, Mark; Reuter, Sean; Field, Loren J.

    2010-01-01

    Summary Background c-kit is a receptor tyrosine kinase family member expressed in hematopoietic stem cells. c-kit is also transiently expressed in cardiomyocyte precursors during development, and in a rare cell population in the normal adult heart. Here, the cardiomyogenic potential of c-kit+ cells isolated from normal neonatal, normal adult and infarcted adult mouse hearts was evaluated. Methods and Results Magnetic activated cell sorting (MACS) was used to prepare c-kit+ cells from the hearts of ACT-EGFP/MHC-nLAC double transgenic mice. These animals exhibit widespread enhanced green fluorescent protein (EGFP) expression and cardiomyocyte-restricted nuclear β-galactosidase activity, thus permitting simultaneous tracking of cell survival and differentiation. A subset of the c-kit+ cells from double transgenic neonatal hearts acquired a cardiomyogenic phenotype when co-cultured with fetal cardiomyocytes (2.4% of all EGFP+ cells screened), but not when cultured alone or when co-cultured with mouse fibroblasts (0.03% and 0.05% of the EGFP+ cells screened, respectively). In contrast, c-kit+ cells from normal adult double transgenic hearts failed to undergo cardiomyogenic differentiation when co-cultured with non-transgenic fetal cardiomyocytes (>18,000 EGFP+ cells screened) or when transplanted into normal or infarcted adult mouse hearts (14 EGFP+ grafts examined). A single c-kit+ cell from an infarcted double transgenic adult heart was observed to acquire a cardiomyogenic phenotype in co-culture (>37,000 EGFP+ cells screened). Conclusions These data suggest that the ability of cardiac-resident c-kit+ cells to acquire a cardiomyogenic phenotype is subject to temporal limitations, or alternatively that the cardiomyogenic population is lost. Elucidation of the underlying molecular basis may permit robust cardiomyogenic induction in adult-derived cardiac c-kit+ cells. PMID:20421520

  15. Phosphate uptake in Saccharomyces cerevisiae Hansen wild type and phenotypes exposed to space flight irradiation.

    PubMed

    Berry, D; Volz, P A

    1979-10-01

    Rates of phosphate uptake were approximately twice as great for Saccharomyces cerevisiae single-cell phenotypic isolates exposed to space parameters as for the wild-type ground control. Quantitative determination of 32P was performed by liquid scintillation spectrometry utilizing Cerenkov radiation counting techniques. PMID:395899

  16. Physical and physiological components of the graviresponses of wild-type and mutant Paramecium Tetraurelia.

    PubMed

    Nagel, U; Machemer, H

    2000-03-01

    Wild-type and the morphological mutant kin 241 of Paramecium tetraurelia showed improved orientation away from the centre of gravity (negative gravitaxis) when accelerations were increased from 1 to 7 g. Gravitaxis was more pronounced in the mutant. A correlation between the efficiency of orientation and the applied g value suggests a physical basis for gravitaxis. Transiently enhanced rates of reversal of the swimming direction coincided with transiently enhanced gravitaxis because reversals occurred more often in downward swimmers than in upward swimmers. The results provide evidence of a physiological modulation of gravitaxis by means of the randomizing effect of depolarization-dependent swimming reversals. Gravity bimodally altered propulsion rates of wild-type P. tetraurelia so that sedimentation was partly antagonized in upward and downward swimmers (negative gravikinesis). In the mutant, only increases in propulsion were observed, although the orientation-dependent sensitivity of the gravikinetic response was the same as in the wild-type population. Observed swimming speed and sedimentation rates in the wild-type and mutant cells were linearly related to acceleration, allowing the determination of gravikinesis as a linear (and so far non-saturating) function of gravity. PMID:10683165

  17. Measuring cell wall elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM

    SciTech Connect

    Beckmann, Melissa; Venkataraman, Sankar; Doktycz, Mitchel John; Nataro, James P; Sullivan, Claretta J; Morrell-Falvey, Jennifer L; Allison, David P

    2006-07-01

    Enteroaggregative Escherichia coli (EAEC) is pathogenic and produces severe diarrhea in humans. A mutant of EAEC that does not produce dispersin, a cell surface protein, is not pathogenic. It has been proposed that dispersin imparts a positive charge to the bacterial cell surface allowing the bacteria to colonize on the negatively charged intestinal mucosa. However, physical properties of the bacterial cell surface, such as rigidity, may be influenced by the presence of dispersin and may contribute to pathogenicity. Using the system developed in our laboratory for mounting and imaging bacterial cells by atomic force microscopy (AFM), in liquid, on gelatin coated mica surfaces, studies were initiated to measure cell surface elasticity. This was carried out in both wild type EAEC, that produces dispersin, and the mutant that does not produce dispersin. This was accomplished using AFM force-distance (FD) spectroscopy on the wild type and mutant grown in liquid or on solid medium. Images in liquid and in air of both the wild-type and mutant grown in liquid and on solid media are presented. This work represents an initial step in efforts to understand the pathogenic role of the dispersin protein in the wild-type bacteria.

  18. COMPARISON OF IN VITRO-CULTURED AND WILD-TYPE PERKINSUS MARINUS I: PATHOGEN VIRULENCE

    EPA Science Inventory

    Perkinsus marinus is a highly contagious pathogen of the eastern oyster Crassostrea virginica. Until recently, transmission studies have employed wild-type parasites isolated directly from infected oysters. Newly developed methods to propagate P. marinus in vitro have led to usin...

  19. Germ cell tumors of the testis overexpress wild-type p53.

    PubMed Central

    Guillou, L.; Estreicher, A.; Chaubert, P.; Hurlimann, J.; Kurt, A. M.; Metthez, G.; Iggo, R.; Gray, A. C.; Jichlinski, P.; Leisinger, H. J.; Benhattar, J.

    1996-01-01

    Several recent studies have suggested that testicular germ cell tumors express high levels of wild-type p53 protein. To clarify and confirm this unexpected result, we have investigated seminomatous and nonseminomatous germ cell tumors at the genomic, mRNA, and protein levels. Thirty-five tumors were examined for p53 overexpression using antibodies directed against the p53 (PAb1801, PAb240, and CM1), mdm2 (IF2), and p21Waf1/Clp1 (EA10) proteins. Thirty-two tumors were screened for p53 mutations by single-strand conformation polymorphism analysis. Eighteen tumors were screened with a functional assay that tests the transcriptional competence of human p53 protein expressed in yeast. On frozen sections, 100, 65, 35, 73, and 0% of tumors reacted with the CM1, PAb240, PAb1801, IF2, and EA10 antibodies, respectively. No p53 mutations were detected by single-strand conformation polymorphism or by functional assay. The fact that many tumors overexpress wild-type p53 but not mdm2 rules out mdm2 overexpression as a general explanation for the presence of wild-type p53 in these tumors. The absence of p21 overexpression suggests that p53 may be unable to activate transcription of critical target genes, which may explain why the presence of wild-type p53 is tolerated in this tumor type, although the mechanism for this transcriptional inactivity remains to be established. Images Figure 1 Figure 2 PMID:8863671

  20. ELECTROPHORETIC MOBILITIES OF ESCHERICHIA COLI 0157:H7 AND WILD-TYPE ESCHERICHIA COLI STRAINS

    EPA Science Inventory

    The electrophoretic mobility (EPM) of a number of human-virulent and "wild-type" Escherichia coli strains in phosphate buffered water was measured. The impact of pH, ionic strength, cation type (valence) and concentration, and bacterial strain on the EPM was investigated. Resul...

  1. Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea.

    PubMed

    Montgomery, Scott C; Cox, Brandon C

    2016-01-01

    The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. PMID:26779585

  2. Nutlin-3a: A Potential Therapeutic Opportunity for TP53 Wild-Type Ovarian Carcinomas

    PubMed Central

    Tsang, Yvonne T. M.; Mullany, Lisa K.; Zu, Zhifei; Richards, JoAnne S.; Gershenson, David M.; Wong, Kwong-Kwok

    2015-01-01

    Epithelial ovarian cancer is a diverse molecular and clinical disease, yet standard treatment is the same for all subtypes. TP53 mutations represent a node of divergence in epithelial ovarian cancer histologic subtypes and may represent a therapeutic opportunity in subtypes expressing wild type, including most low-grade ovarian serous carcinomas, ovarian clear cell carcinomas and ovarian endometrioid carcinomas, which represent approximately 25% of all epithelial ovarian cancer. We therefore sought to investigate Nutlin-3a—a therapeutic which inhibits MDM2, activates wild-type p53, and induces apoptosis—as a therapeutic compound for TP53 wild-type ovarian carcinomas. Fifteen epithelial ovarian cancer cell lines of varying histologic subtypes were treated with Nutlin-3a with determination of IC50 values. Western Blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses quantified MDM2, p53, and p21 expression after Nutlin-3a treatment. DNA from 15 cell lines was then sequenced for TP53 mutations in exons 2-11 including intron-exon boundaries. Responses to Nutlin-3a were dependent upon TP53 mutation status. By qRT-PCR and WB, levels of MDM2 and p21 were upregulated in wild-type TP53 sensitive cell lines, and p21 induction was reduced or absent in mutant cell lines. Annexin V assays demonstrated apoptosis in sensitive cell lines treated with Nutlin-3a. Thus, Nutlin-3a could be a potential therapeutic agent for ovarian carcinomas expressing wild-type TP53 and warrants further investigation. PMID:26248031

  3. Nutlin-3a: A Potential Therapeutic Opportunity for TP53 Wild-Type Ovarian Carcinomas.

    PubMed

    Crane, Erin K; Kwan, Suet-Yan; Izaguirre, Daisy I; Tsang, Yvonne T M; Mullany, Lisa K; Zu, Zhifei; Richards, JoAnne S; Gershenson, David M; Wong, Kwong-Kwok

    2015-01-01

    Epithelial ovarian cancer is a diverse molecular and clinical disease, yet standard treatment is the same for all subtypes. TP53 mutations represent a node of divergence in epithelial ovarian cancer histologic subtypes and may represent a therapeutic opportunity in subtypes expressing wild type, including most low-grade ovarian serous carcinomas, ovarian clear cell carcinomas and ovarian endometrioid carcinomas, which represent approximately 25% of all epithelial ovarian cancer. We therefore sought to investigate Nutlin-3a--a therapeutic which inhibits MDM2, activates wild-type p53, and induces apoptosis--as a therapeutic compound for TP53 wild-type ovarian carcinomas. Fifteen epithelial ovarian cancer cell lines of varying histologic subtypes were treated with Nutlin-3a with determination of IC50 values. Western Blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses quantified MDM2, p53, and p21 expression after Nutlin-3a treatment. DNA from 15 cell lines was then sequenced for TP53 mutations in exons 2-11 including intron-exon boundaries. Responses to Nutlin-3a were dependent upon TP53 mutation status. By qRT-PCR and WB, levels of MDM2 and p21 were upregulated in wild-type TP53 sensitive cell lines, and p21 induction was reduced or absent in mutant cell lines. Annexin V assays demonstrated apoptosis in sensitive cell lines treated with Nutlin-3a. Thus, Nutlin-3a could be a potential therapeutic agent for ovarian carcinomas expressing wild-type TP53 and warrants further investigation. PMID:26248031

  4. Adult mouse cortical cell taxonomy revealed by single cell transcriptomics.

    PubMed

    Tasic, Bosiljka; Menon, Vilas; Nguyen, Thuc Nghi; Kim, Tae Kyung; Jarsky, Tim; Yao, Zizhen; Levi, Boaz; Gray, Lucas T; Sorensen, Staci A; Dolbeare, Tim; Bertagnolli, Darren; Goldy, Jeff; Shapovalova, Nadiya; Parry, Sheana; Lee, Changkyu; Smith, Kimberly; Bernard, Amy; Madisen, Linda; Sunkin, Susan M; Hawrylycz, Michael; Koch, Christof; Zeng, Hongkui

    2016-02-01

    Nervous systems are composed of various cell types, but the extent of cell type diversity is poorly understood. We constructed a cellular taxonomy of one cortical region, primary visual cortex, in adult mice on the basis of single-cell RNA sequencing. We identified 49 transcriptomic cell types, including 23 GABAergic, 19 glutamatergic and 7 non-neuronal types. We also analyzed cell type-specific mRNA processing and characterized genetic access to these transcriptomic types by many transgenic Cre lines. Finally, we found that some of our transcriptomic cell types displayed specific and differential electrophysiological and axon projection properties, thereby confirming that the single-cell transcriptomic signatures can be associated with specific cellular properties. PMID:26727548

  5. Adult Mouse Cortical Cell Taxonomy by Single Cell Transcriptomics

    PubMed Central

    Tasic, Bosiljka; Menon, Vilas; Nguyen, Thuc Nghi; Kim, Tae Kyung; Jarsky, Tim; Yao, Zizhen; Levi, Boaz; Gray, Lucas T.; Sorensen, Staci A.; Dolbeare, Tim; Bertagnolli, Darren; Goldy, Jeff; Shapovalova, Nadiya; Parry, Sheana; Lee, Changkyu; Smith, Kimberly; Bernard, Amy; Madisen, Linda; Sunkin, Susan M.; Hawrylycz, Michael; Koch, Christof; Zeng, Hongkui

    2016-01-01

    Nervous systems are composed of various cell types, but the extent of cell type diversity is poorly understood. Here, we construct a cellular taxonomy of one cortical region, primary visual cortex, in adult mice based on single cell RNA-sequencing. We identify 49 transcriptomic cell types including 23 GABAergic, 19 glutamatergic and seven non-neuronal types. We also analyze cell-type specific mRNA processing and characterize genetic access to these transcriptomic types by many transgenic Cre lines. Finally, we show that some of our transcriptomic cell types display specific and differential electrophysiological and axon projection properties, thereby confirming that the single cell transcriptomic signatures can be associated with specific cellular properties. PMID:26727548

  6. Chronic hypoxia induces the activation of the Wnt/β-catenin signaling pathway and stimulates hippocampal neurogenesis in wild-type and APPswe-PS1ΔE9 transgenic mice in vivo

    PubMed Central

    Varela-Nallar, Lorena; Rojas-Abalos, Macarena; Abbott, Ana C.; Moya, Esteban A.; Iturriaga, Rodrigo; Inestrosa, Nibaldo C.

    2014-01-01

    Hypoxia modulates proliferation and differentiation of cultured embryonic and adult stem cells, an effect that includes β-catenin, a key component of the canonical Wnt signaling pathway. Here we studied the effect of mild hypoxia on the activity of the Wnt/β-catenin signaling pathway in the hippocampus of adult mice in vivo. The hypoxia-inducible transcription factor-1α (HIF-1α) was analyzed as a molecular control of the physiological hypoxic response. Exposure to chronic hypoxia (10% oxygen for 6–72 h) stimulated the activation of the Wnt/β-catenin signaling pathway. Because the Wnt/β-catenin pathway is a positive modulator of adult neurogenesis, we evaluated whether chronic hypoxia was able to stimulate neurogenesis in the subgranular zone (SGZ) of the hippocampal dentate gyrus. Results indicate that hypoxia increased cell proliferation and neurogenesis in adult wild-type mice as determined by Ki67 staining, Bromodeoxyuridine (BrdU) incorporation and double labeling with doublecortin (DCX). Chronic hypoxia also induced neurogenesis in a double transgenic APPswe-PS1ΔE9 mouse model of Alzheimer’s disease (AD), which shows decreased levels of neurogenesis in the SGZ. Our results show for the first time that exposure to hypoxia in vivo can induce the activation of the Wnt/β-catenin signaling cascade in the hippocampus, suggesting that mild hypoxia may have a therapeutic value in neurodegenerative disorders associated with altered Wnt signaling in the brain and also in pathological conditions in which hippocampal neurogenesis is impaired. PMID:24574965

  7. Retinoic acid receptor beta2 and neurite outgrowth in the adult mouse spinal cord in vitro.

    PubMed

    Corcoran, Jonathan; So, Po-Lin; Barber, Robert D; Vincent, Karen J; Mazarakis, Nicholas D; Mitrophanous, Kyriacos A; Kingsman, Susan M; Maden, Malcolm

    2002-10-01

    Retinoic acid, acting through the nuclear retinoic acid receptor beta2 (RARbeta2), stimulates neurite outgrowth from peripheral nervous system tissue that has the capacity to regenerate neurites, namely, embryonic and adult dorsal root ganglia. Similarly, in central nervous system tissue that can regenerate, namely, embryonic mouse spinal cord, retinoic acid also stimulates neurite outgrowth and RARbeta2 is upregulated. By contrast, in the adult mouse spinal cord, which cannot regenerate, no such upregulation of RARbeta2 by retinoic acid is observed and no neurites are extended in vitro. To test our hypothesis that the upregulation of RARbeta2 is crucial to neurite regeneration, we have transduced adult mouse or rat spinal cord in vitro with a minimal equine infectious anaemia virus vector expressing RARbeta2. After transduction, prolific neurite outgrowth occurs. Outgrowth does not occur when the cord is transduced with a different isoform of RARbeta nor does it occur following treatment with nerve growth factor. These data demonstrate that RARbeta2 is involved in neurite outgrowth, at least in vitro, and that this gene may in the future be of some therapeutic use. PMID:12235288

  8. Subretinal delivery and electroporation in pigmented and nonpigmented adult mouse eyes

    PubMed Central

    Nickerson, John M.; Goodman, Penny; Chrenek, Micah A.; Johnson, Christiana J.; Berglin, Lennart; Redmond, T. Michael.; Boatright, Jeffrey H.

    2013-01-01

    Subretinal injection offers one of the best ways to deliver many classes of drugs, reagents, cells and treatments to the photoreceptor, Müller, and retinal pigment epithelium (RPE) cells of the retina. Agents delivered to this space are placed within microns of the intended target cell, accumulating to high concentrations because there is no dilution due to transport processes or diffusion. Dilution in the interphotoreceptor space (IPS) is minimal because the IPS volume is only 10-20 microliters in the human eye and less than 1 microliter in the mouse eye. For gene delivery purposes, we wished to transfect the cells adjacent to the IPS in adult mouse eyes. Others transfect these cells in neonatal rats to study the development of the retina. In both neonates and adults, electroporation is found to be effective Here we describe the optimization of electroporation conditions for RPE cells in the adult mouse eye with naked plasmids. However, both techniques, subretinal injection and electroporation, present some technical challenges that require skill on the part of the surgeon to prevent untoward damage to the eye. Here we describe methods that we have used for the past ten years (1). PMID:22688698

  9. Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

    PubMed Central

    Li, Daxiang; Wu, Jian; Bai, Yan; Zhao, Xiaochen; Liu, Lijun

    2014-01-01

    Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes’ terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice. PMID:24894542

  10. Stem cell niches in the adult mouse heart

    PubMed Central

    Urbanek, Konrad; Cesselli, Daniela; Rota, Marcello; Nascimbene, Angelo; De Angelis, Antonella; Hosoda, Toru; Bearzi, Claudia; Boni, Alessandro; Bolli, Roberto; Kajstura, Jan; Anversa, Piero; Leri, Annarosa

    2006-01-01

    Cardiac stem cells (CSCs) have been identified in the adult heart, but the microenvironment that protects the slow-cycling, undifferentiated, and self-renewing CSCs remains to be determined. We report that the myocardium possesses interstitial structures with the architectural organization of stem cell niches that harbor long-term BrdU-retaining cells. The recognition of long-term label-retaining cells provides functional evidence of resident CSCs in the myocardium, indicating that the heart is an organ regulated by a stem cell compartment. Cardiac niches contain CSCs and lineage-committed cells, which are connected to supporting cells represented by myocytes and fibroblasts. Connexins and cadherins form gap and adherens junctions at the interface of CSCs–lineage-committed cells and supporting cells. The undifferentiated state of CSCs is coupled with the expression of α4-integrin, which colocalizes with the α2-chain of laminin and fibronectin. CSCs divide symmetrically and asymmetrically, but asymmetric division predominates, and the replicating CSC gives rise to one daughter CSC and one daughter committed cell. By this mechanism of growth kinetics, the pool of primitive CSCs is preserved, and a myocyte progeny is generated together with endothelial and smooth muscle cells. Thus, CSCs regulate myocyte turnover that is heterogeneous across the heart, faster at the apex and atria, and slower at the base–midregion of the ventricle. PMID:16754876

  11. Fusarium spp. Associated with Field-Grown Grain of Near-Isogenic Low Lignin and Wild-Type Sorghum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fusarium spp. associated with field-grown grain of near-isogenic low lignin and wild-type sorghum. Deanna Funnell-Harris and Jeff Pedersen, USDA-ARS, Lincoln, NE Previous studies indicated that low lignin brown midrib (bmr) sorghum may be more resistant to Fusarium spp. than wild-type and that phen...

  12. Mating success of wild type and sepia mutants Drosophila melanogaster in different choice.

    PubMed

    Stanić, Snezana; Pavković-Lucic, Sofija

    2005-01-01

    Mating behaviour of red-eyed (wt) and brown-eyed (sepia) Drosophila melanogaster was studied under light conditions. Mating success was directly observed in mating vials and techniques usually applied in the studies of sexual selection ("female choice" and "multiple choice"). The comparison of sexual activity of mutant and wild types clearly indicates that they are not equally successful in matings. Sepia eye colour mutation decreases sexual activity of Drosophila melanogaster males, influences the preference ability of females and decreases the number of progeny from homogamic mating of the se x se type, as well as from heterogamic copulations in which sepia females take part. Non-random mating of wild type males and sepia females (in "multiple-choice" situation), with genetically and phenotypically different individuals, could be another mechanism for conservation of genetic polymorphism in natural populations. PMID:16440285

  13. Cytochemical Analysis of Pollen Development in Wild-Type Arabidopsis and a Male-Sterile Mutant.

    PubMed Central

    Regan, SM; Moffatt, BA

    1990-01-01

    Microsporogenesis has been examined in wild-type Arabidopsis thaliana and the nuclear male-sterile mutant BM3 by cytochemical staining. The mutant lacks adenine phosphoribosyltransferase, an enzyme of the purine salvage pathway that converts adenine to AMP. Pollen development in the mutant began to diverge from wild type just after meiosis, as the tetrads of microspores were released from their callose walls. The first indication of abnormal pollen development in the mutant was a darker staining of the microspore wall due to an incomplete synthesis of the intine. Vacuole formation was delayed and irregular in the mutant, and the majority of the mutant microspores failed to undergo mitotic divisions. Enzyme activities of alcohol dehydrogenase and esterases decreased in the mutant soon after meiosis and were undetectable in mature pollen grains of the mutant. RNA accumulation was also diminished. These results are discussed in relation to the possible role(s) of adenine salvage in pollen development. PMID:12354970

  14. An emerging role for misfolded wild-type SOD1 in sporadic ALS pathogenesis

    PubMed Central

    Rotunno, Melissa S.; Bosco, Daryl A.

    2013-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that targets motor neurons, leading to paralysis and death within a few years of disease onset. While several genes have been linked to the inheritable, or familial, form of ALS, much less is known about the cause(s) of sporadic ALS, which accounts for ~90% of ALS cases. Due to the clinical similarities between familial and sporadic ALS, it is plausible that both forms of the disease converge on a common pathway and, therefore, involve common factors. Recent evidence suggests the Cu,Zn-superoxide dismutase (SOD1) protein to be one such factor that is common to both sporadic and familial ALS. In 1993, mutations were uncovered in SOD1 that represent the first known genetic cause of familial ALS. While the exact mechanism of mutant-SOD1 toxicity is still not known today, most evidence points to a gain of toxic function that stems, at least in part, from the propensity of this protein to misfold. In the wild-type SOD1 protein, non-genetic perturbations such as metal depletion, disruption of the quaternary structure, and oxidation, can also induce SOD1 to misfold. In fact, these aforementioned post-translational modifications cause wild-type SOD1 to adopt a “toxic conformation” that is similar to familial ALS-linked SOD1 variants. These observations, together with the detection of misfolded wild-type SOD1 within human post-mortem sporadic ALS samples, have been used to support the controversial hypothesis that misfolded forms of wild-type SOD1 contribute to sporadic ALS pathogenesis. In this review, we present data from the literature that both support and contradict this hypothesis. We also discuss SOD1 as a potential therapeutic target for both familial and sporadic ALS. PMID:24379756

  15. Stability of Iowa mutant and wild type Aβ-peptide aggregates

    SciTech Connect

    Alred, Erik J.; Scheele, Emily G.; Berhanu, Workalemahu M.; Hansmann, Ulrich H. E.

    2014-11-07

    Recent experiments indicate a connection between the structure of amyloid aggregates and their cytotoxicity as related to neurodegenerative diseases. Of particular interest is the Iowa Mutant, which causes early-onset of Alzheimer's disease. While wild-type Amyloid β-peptides form only parallel beta-sheet aggregates, the mutant also forms meta-stable antiparallel beta sheets. Since these structural variations may cause the difference in the pathological effects of the two Aβ-peptides, we have studied in silico the relative stability of the wild type and Iowa mutant in both parallel and antiparallel forms. We compare regular molecular dynamics simulations with such where the viscosity of the samples is reduced, which, we show, leads to higher sampling efficiency. By analyzing and comparing these four sets of all-atom molecular dynamics simulations, we probe the role of the various factors that could lead to the structural differences. Our analysis indicates that the parallel forms of both wild type and Iowa mutant aggregates are stable, while the antiparallel aggregates are meta-stable for the Iowa mutant and not stable for the wild type. The differences result from the direct alignment of hydrophobic interactions in the in-register parallel oligomers, making them more stable than the antiparallel aggregates. The slightly higher thermodynamic stability of the Iowa mutant fibril-like oligomers in its parallel organization over that in antiparallel form is supported by previous experimental measurements showing slow inter-conversion of antiparallel aggregates into parallel ones. Knowledge of the mechanism that selects between parallel and antiparallel conformations and determines their relative stability may open new avenues for the development of therapies targeting familial forms of early-onset Alzheimer's disease.

  16. Rapid and efficient gene delivery into the adult mouse brain via focal electroporation

    PubMed Central

    Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

    2016-01-01

    In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

  17. Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system.

    PubMed

    Marques, Sueli; Zeisel, Amit; Codeluppi, Simone; van Bruggen, David; Mendanha Falcão, Ana; Xiao, Lin; Li, Huiliang; Häring, Martin; Hochgerner, Hannah; Romanov, Roman A; Gyllborg, Daniel; Muñoz-Manchado, Ana B; La Manno, Gioele; Lönnerberg, Peter; Floriddia, Elisa M; Rezayee, Fatemah; Ernfors, Patrik; Arenas, Ernest; Hjerling-Leffler, Jens; Harkany, Tibor; Richardson, William D; Linnarsson, Sten; Castelo-Branco, Gonçalo

    2016-06-10

    Oligodendrocytes have been considered as a functionally homogeneous population in the central nervous system (CNS). We performed single-cell RNA sequencing on 5072 cells of the oligodendrocyte lineage from 10 regions of the mouse juvenile and adult CNS. Thirteen distinct populations were identified, 12 of which represent a continuum from Pdgfra(+) oligodendrocyte precursor cells (OPCs) to distinct mature oligodendrocytes. Initial stages of differentiation were similar across the juvenile CNS, whereas subsets of mature oligodendrocytes were enriched in specific regions in the adult brain. Newly formed oligodendrocytes were detected in the adult CNS and were responsive to complex motor learning. A second Pdgfra(+) population, distinct from OPCs, was found along vessels. Our study reveals the dynamics of oligodendrocyte differentiation and maturation, uncoupling them at a transcriptional level and highlighting oligodendrocyte heterogeneity in the CNS. PMID:27284195

  18. Neural stem/progenitor cell properties of glial cells in the adult mouse auditory nerve

    PubMed Central

    Lang, Hainan; Xing, Yazhi; Brown, LaShardai N.; Samuvel, Devadoss J.; Panganiban, Clarisse H.; Havens, Luke T.; Balasubramanian, Sundaravadivel; Wegner, Michael; Krug, Edward L.; Barth, Jeremy L.

    2015-01-01

    The auditory nerve is the primary conveyor of hearing information from sensory hair cells to the brain. It has been believed that loss of the auditory nerve is irreversible in the adult mammalian ear, resulting in sensorineural hearing loss. We examined the regenerative potential of the auditory nerve in a mouse model of auditory neuropathy. Following neuronal degeneration, quiescent glial cells converted to an activated state showing a decrease in nuclear chromatin condensation, altered histone deacetylase expression and up-regulation of numerous genes associated with neurogenesis or development. Neurosphere formation assays showed that adult auditory nerves contain neural stem/progenitor cells (NSPs) that were within a Sox2-positive glial population. Production of neurospheres from auditory nerve cells was stimulated by acute neuronal injury and hypoxic conditioning. These results demonstrate that a subset of glial cells in the adult auditory nerve exhibit several characteristics of NSPs and are therefore potential targets for promoting auditory nerve regeneration. PMID:26307538

  19. Rapid and efficient gene delivery into the adult mouse brain via focal electroporation.

    PubMed

    Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

    2016-01-01

    In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

  20. Root graviresponsiveness and cellular differentiation in wild-type and a starchless mutant of Arabidopsis thaliana

    NASA Technical Reports Server (NTRS)

    Moore, R.

    1989-01-01

    Primary roots of a starchless mutant of Arabidopsis thaliana L. are strongly graviresponsive despite lacking amyloplasts in their columella cells. The ultrastructures of calyptrogen and peripheral cells in wild-type as compared to mutant seedlings are not significantly different. The largest difference in cellular differentiation in caps of mutant and wild-type roots is the relative volume of plastids in columella cells. Plastids occupy 12.3% of the volume of columella cells in wild-type seedlings, but only 3.69% of columella cells in mutant seedlings. These results indicate that: (1) amyloplasts and starch are not necessary for root graviresponsiveness; (2) the increase in relative volume of plastids that usually accompanies differentiation of columella cells is not necessary for root graviresponsiveness; and (3) the absence of starch and amyloplasts does not affect the structure of calyptrogen (i.e. meristematic) and secretory (i.e. peripheral) cells in root caps. These results are discussed relative to proposed models for root gravitropism.

  1. A Snapshot of Histone Modifications within Transposable Elements in Drosophila Wild Type Strains

    PubMed Central

    Rebollo, Rita; Horard, Béatrice; Begeot, Flora; Delattre, Marion; Gilson, Eric; Vieira, Cristina

    2012-01-01

    Transposable elements (TEs) are a major source of genetic variability in genomes, creating genetic novelty and driving genome evolution. Analysis of sequenced genomes has revealed considerable diversity in TE families, copy number, and localization between different, closely related species. For instance, although the twin species Drosophila melanogaster and D. simulans share the same TE families, they display different amounts of TEs. Furthermore, previous analyses of wild type derived strains of D. simulans have revealed high polymorphism regarding TE copy number within this species. Several factors may influence the diversity and abundance of TEs in a genome, including molecular mechanisms such as epigenetic factors, which could be a source of variation in TE success. In this paper, we present the first analysis of the epigenetic status of four TE families (roo, tirant, 412 and F) in seven wild type strains of D. melanogaster and D. simulans. Our data shows intra- and inter-specific variations in the histone marks that adorn TE copies. Our results demonstrate that the chromatin state of common TEs varies among TE families, between closely related species and also between wild type strains. PMID:22962605

  2. Interaction of root gravitropism and phototropism in Arabidopsis wild-type and starchless mutants

    NASA Technical Reports Server (NTRS)

    Vitha, S.; Zhao, L.; Sack, F. D.

    2000-01-01

    Root gravitropism in wild-type Arabidopsis and in two starchless mutants, pgm1-1 and adg1-1, was evaluated as a function of light position to determine the relative strengths of negative phototropism and of gravitropism and how much phototropism affects gravitropic measurements. Gravitropism was stronger than phototropism in some but not all light positions in wild-type roots grown for an extended period, indicating that the relationship between the two tropisms is more complex than previously reported. Root phototropism significantly influenced the time course of gravitropic curvature and the two measures of sensitivity. Light from above during horizontal exposure overestimated all three parameters for all three genotypes except the wild-type perception time. At the irradiance used (80 micromol m(-2) s(-1)), the shortest periods of illumination found to exaggerate gravitropism were 45 min of continuous illumination and 2-min doses of intermittent illumination. By growing roots in circumlateral light or by gravistimulating in the dark, corrected values were obtained for each gravitropic parameter. Roots of both starchless mutants were determined to be about three times less sensitive than prior estimates. This study demonstrates the importance of accounting for phototropism in the design of root gravitropism experiments in Arabidopsis.

  3. Physiology and metabolic fluxes of wild-type and riboflavin-producing Bacillus subtilis.

    PubMed Central

    Sauer, U; Hatzimanikatis, V; Hohmann, H P; Manneberg, M; van Loon, A P; Bailey, J E

    1996-01-01

    Continuous cultivation in a glucose-limited chemostat was used to determine the growth parameters of wild-type Bacillus subtilis and of a recombinant, riboflavin-producing strain. Maintenance coefficients of 0.45 and 0.66 mmol of glucose g-1 h-1 were determined for the wild-type and recombinant strains, respectively. However, the maximum molar growth yield of 82 to 85 g (cell dry weight)/mol of glucose was found to be almost identical in both strains. A nonlinear relationship between the specific riboflavin production rate and the dilution rate was observed, revealing a coupling of product formation and growth under strict substrate-limited conditions. Most prominently, riboflavin formation completely ceased at specific growth rates below 0.15 h-1. For molecular characterization of B. subtilis, the total amino acid composition of the wild type was experimentally determined and the complete building block requirements for biomass formation were derived. In particular, the murein sacculus was found to constitute approximately 9% of B. subtilis biomass, three- to fivefold more than in Escherichia coli. Estimation of intracellular metabolic fluxes by a refined mass balance approach revealed a substantial, growth rate-dependent flux through the oxidative branch of the pentose phosphate pathway. Furthermore, this flux is indicated to be increased in the strain engineered for riboflavin formation. Glucose catabolism at low growth rates with reduced biomass yields was supported mainly by the tricarboxylic acid cycle. PMID:8837424

  4. In vitro Intestinal Mucosal Epithelial Responses to Wild-Type Salmonella Typhi and Attenuated Typhoid Vaccines.

    PubMed

    Fiorentino, Maria; Lammers, Karen M; Levine, Myron M; Sztein, Marcelo B; Fasano, Alessio

    2013-01-01

    Typhoid fever, caused by S. Typhi, is responsible for approximately 200,000 deaths per year worldwide. Little information is available regarding epithelium-bacterial interactions in S. Typhi infection. We have evaluated in vitro the effects of wild-type S. Typhi, the licensed Ty21a typhoid vaccine and the leading strains CVD 908-htrA and CVD 909 vaccine candidates on intestinal barrier function and immune response. Caco2 monolayers infected with wild-type S. Typhi exhibited alterations in the organization of tight junctions, increased paracellular permeability, and a rapid decrease in Trans-Epithelial Electrical Resistance as early as 4 h post-exposure. S. Typhi triggered the secretion of interleukin (IL)-8 and IL-6. Caco2 cells infected with the attenuated strains exhibited a milder pro-inflammatory response with minimal disruption of the barrier integrity. We conclude that wild-type S. Typhi causes marked transient alterations of the intestinal mucosa that are more pronounced than those observed with Ty21a or new generation attenuated typhoid vaccine candidates. PMID:23408152

  5. Comparative proteomic analysis of tobacco expressing cyanobacterial flavodoxin and its wild type under drought stress.

    PubMed

    Gharechahi, Javad; Hajirezaei, Mohammad-Reza; Salekdeh, Ghasem Hosseini

    2015-03-01

    Tobacco plants expressing cyanobacterial flavodoxin (Fld) show enhanced tolerance to a wide range of abiotic stresses including drought, temperature and UV. The mechanisms of adaptation to stress conditions under Fld expression are largely unknown. Here, we applied comparative proteomic analysis to uncover the changes in the proteome profile of Fld-expressing plants in response to drought stress. Using high-resolution two-dimensional gel electrophoresis, we were able to detect 930 protein spots and compare their abundance. We found changes up to 1.5 fold for 52 spots under drought in transgenic and/or wild type plants. Using combined MALDI-TOF/TOF and ESI-Q/TOF analysis 39 (24 in wild type, 11 in transgenic, and 4 in both) drought-responsive proteins (DRPs) could be identified. The majority of DRPs are known to be involved in photosynthesis, carbohydrate and energy metabolism, amino acid and protein synthesis and processing, and oxidative stress responses. Among candidate DRPs, the abundance of remurin, ferredoxin-NADP reductase, chloroplast manganese stabilizing protein-II, phosphoglycerate mutase, and glutathione S-transferase decreased in drought stressed Fld-tobacco while S-formylglutathione hydrolase and pyridoxine biosynthesis protein abundance increased. In wild type plants, drought caused a reduction of proteins related to carbohydrate metabolism. These results suggest that the stress tolerance conferred by Fld expression is strongly related to control mechanisms regarding carbohydrate and energy metabolism as well as oxidative stress responses. PMID:25506766

  6. Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064

    PubMed Central

    da Silva Vasconcelos, Eliton; de Lima, Vanderlei Aparecido; Goto, Leandro Seiji; Cruz-Hernández, Isara Lourdes; Hokka, Carlos Osamu

    2013-01-01

    Clavulanic acid (CA) is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064). The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant. PMID:24688492

  7. System-wide identification of wild-type SUMO-2 conjugation sites

    PubMed Central

    Hendriks, Ivo A.; D'Souza, Rochelle C.; Chang, Jer-Gung; Mann, Matthias; Vertegaal, Alfred C. O.

    2015-01-01

    SUMOylation is a reversible post-translational modification (PTM) regulating all nuclear processes. Identification of SUMOylation sites by mass spectrometry (MS) has been hampered by bulky tryptic fragments, which thus far necessitated the use of mutated SUMO. Here we present a SUMO-specific protease-based methodology which circumvents this problem, dubbed Protease-Reliant Identification of SUMO Modification (PRISM). PRISM allows for detection of SUMOylated proteins as well as identification of specific sites of SUMOylation while using wild-type SUMO. The method is generic and could be widely applied to study lysine PTMs. We employ PRISM in combination with high-resolution MS to identify SUMOylation sites from HeLa cells under standard growth conditions and in response to heat shock. We identified 751 wild-type SUMOylation sites on endogenous proteins, including 200 dynamic SUMO sites in response to heat shock. Thus, we have developed a method capable of quantitatively studying wild-type mammalian SUMO at the site-specific and system-wide level. PMID:26073453

  8. Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins.

    PubMed Central

    Chang, C P; Jacobs, Y; Nakamura, T; Jenkins, N A; Copeland, N G; Cleary, M L

    1997-01-01

    The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1. PMID:9315626

  9. Magnetic Resonance Spectroscopy discriminates the response to microglial stimulation of wild type and Alzheimer’s disease models

    PubMed Central

    Pardon, Marie-Christine; Yanez Lopez, Maria; Yuchun, Ding; Marjańska, Małgorzata; Prior, Malcolm; Brignell, Christopher; Parhizkar, Samira; Agostini, Alessandra; Bai, Li; Auer, Dorothee P.; Faas, Henryk M

    2016-01-01

    Microglia activation has emerged as a potential key factor in the pathogenesis of Alzheimer’s disease. Metabolite levels assessed by magnetic resonance spectroscopy (MRS) are used as markers of neuroinflammation in neurodegenerative diseases, but how they relate to microglial activation in health and chronic disease is incompletely understood. Using MRS, we monitored the brain metabolic response to lipopolysaccharides (LPS)-induced microglia activation in vivo in a transgenic mouse model of Alzheimer’s disease (APP/PS1) and healthy controls (wild-type (WT) littermates) over 4 hours. We assessed reactive gliosis by immunohistochemistry and correlated metabolic and histological measures. In WT mice, LPS induced a microglial phenotype consistent with activation, associated with a sustained increase in macromolecule and lipid levels (ML9). This effect was not seen in APP/PS1 mice, where LPS did not lead to a microglial response measured by histology, but induced a late increase in the putative inflammation marker myoinositol (mI) and metabolic changes in total creatine and taurine previously reported to be associated with amyloid load. We argue that ML9 and mI distinguish the response of WT and APP/PS1 mice to immune mediators. Lipid and macromolecule levels may represent a biomarker of activation of healthy microglia, while mI may not be a glial marker. PMID:26813748

  10. Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53

    SciTech Connect

    Mercer, W.E.; Shields, M.T.; Amin, M.; Sauve, G.J. ); Appella, E.; Romano, J.W.; Ullrich, S.J. )

    1990-08-01

    To investigate the effect that human wild-type p53 (wt-p53) expression has on cell proliferation the authors constructed a recombinant plasmid, pM47, in which wt-p53 cDNA is under transcriptional control of the hormone-inducible mouse mammary tumor virus promoter linked to the dominant biochemical selection marker gene Eco gpt. The pM47 plasmid was introduced into T98G cells derived from a human glioblastomas multiforme tumor, and a stable clonal cell line, GM47.23, was derived that conditionally expressed wt-p53 following exposure to dexamethasone. The authors show that induction of wt-p53 expression in exponentially growing cells inhibits cell cycle progression and that the inhibitory effect is reversible upon removal of the inducer or infection with simian virus 40. Moreover, when growth-arrested cells are stimulated to proliferate, induction of wt-p53 expression inhibits G{sub 0}/G{sub 1} progression into S phase and the cells accumulate with a DNA content equivalent to cells arrested in the G{sub 0}/G{sub 1} phase of the cell cycle. Taken together, these studies suggest that wt-p53 may play a negative role in growth regulation.

  11. Experimental research on wild-type p53 plasmid transfected into retinoblastoma cells and tissues using an ultrasound microbubble intensifier.

    PubMed

    Luo, J; Zhou, X; Diao, L; Wang, Z

    2010-01-01

    The transfection efficiency of wild-type p53 (wtp53) was investigated in retinoblastoma (RB) Y79 cells using an ultrasound microbubble technique. A human RB nude mouse xenograft tumour model was also used to investigate whether this technique could deliver wtp53 into solid tumours. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that wtp53 was successfully transfected into Y79 cells in the plasmid with microbubbles and ultrasound group and in the plasmid with liposomes group, but not in the plasmid with ultrasound group or in the untreated control group. Flow cytometry showed that apoptosis was highest in the microbubbles and ultrasound group (25.58%) compared with the plasmid with liposomes group (19.50%), and the other two groups (< 10%). RT-PCR also showed that the wtp53 gene was successfully transfected into solid tumours in the plasmid with microbubbles and ultrasound group. This study provides preliminary evidence in support of a potential new approach to RB gene therapy. PMID:20819437

  12. Spontaneous generation of rapidly transmissible prions in transgenic mice expressing wild-type bank vole prion protein.

    PubMed

    Watts, Joel C; Giles, Kurt; Stöhr, Jan; Oehler, Abby; Bhardwaj, Sumita; Grillo, Sunny K; Patel, Smita; DeArmond, Stephen J; Prusiner, Stanley B

    2012-02-28

    Currently, there are no animal models of the most common human prion disorder, sporadic Creutzfeldt-Jakob disease (CJD), in which prions are formed spontaneously from wild-type (WT) prion protein (PrP). Interestingly, bank voles (BV) exhibit an unprecedented promiscuity for diverse prion isolates, arguing that bank vole PrP (BVPrP) may be inherently prone to adopting misfolded conformations. Therefore, we constructed transgenic (Tg) mice expressing WT BVPrP. Tg(BVPrP) mice developed spontaneous CNS dysfunction between 108 and 340 d of age and recapitulated the hallmarks of prion disease, including spongiform degeneration, pronounced astrogliosis, and deposition of alternatively folded PrP in the brain. Brain homogenates of ill Tg(BVPrP) mice transmitted disease to Tg(BVPrP) mice in ∼35 d, to Tg mice overexpressing mouse PrP in under 100 d, and to WT mice in ∼185 d. Our studies demonstrate experimentally that WT PrP can spontaneously form infectious prions in vivo. Thus, Tg(BVPrP) mice may be useful for studying the spontaneous formation of prions, and thus may provide insight into the etiology of sporadic CJD. PMID:22331873

  13. Histology and Ultrastructure of Transitional Changes in Skin Morphology in the Juvenile and Adult Four-Striped Mouse (Rhabdomys pumilio)

    PubMed Central

    Stewart, Eranée; Ajao, Moyosore Salihu

    2013-01-01

    The four-striped mouse has a grey to brown coloured coat with four characteristic dark stripes interspersed with three lighter stripes running along its back. The histological differences in the skin of the juvenile and adult mouse were investigated by Haematoxylin and Eosin and Masson Trichrome staining, while melanocytes in the skin were studied through melanin-specific Ferro-ferricyanide staining. The ultrastructure of the juvenile skin, hair follicles, and melanocytes was also explored. In both the juvenile and adult four-striped mouse, pigment-containing cells were observed in the dermis and were homogeneously dispersed throughout this layer. Apart from these cells, the histology of the skin of the adult four-striped mouse was similar to normal mammalian skin. In the juvenile four-striped mouse, abundant hair follicles of varying sizes were observed in the dermis and hypodermis, while hair follicles of similar size were only present in the dermis of adult four-striped mouse. Ultrastructural analysis of juvenile hair follicles revealed that the arrangement and differentiation of cellular layers were typical of a mammal. This study therefore provides unique transition pattern in the four-striped mouse skin morphology different from the textbook description of the normal mammalian skin. PMID:24288469

  14. Kinetics and genomic profiling of adult human and mouse β-cell maturation.

    PubMed

    Szabat, Marta; Pourghaderi, Poya; Soukhatcheva, Galina; Verchere, C Bruce; Warnock, Garth L; Piret, James M; Johnson, James D

    2011-01-01

    Diabetes is a multifactorial metabolic disorder defined by the loss of functional pancreatic insulin-producing β-cells. The functional maturation and dedifferentiation of adult β-cells is central to diabetes pathogenesis and to β-cell replacement therapy for the treatment of diabetes. Despite its importance, the dynamics and mechanisms of adult β-cell maturation remain poorly understood. Using a novel Pdx1/Ins1 dual fluorescent reporter lentiviral vector, we previously found that individual adult human and mouse β-cells exist in at least two differentiation states distinguishable by the activation of the rat Ins1 promoter and performed the first real-time imaging of the maturation of individual cultured β-cells. Our previous study focused on transformed (MIN6) β-cells as a model to investigatethe kinetics of β-cell maturation. In the present study, we investigated the kinetics of the maturation process in primary human and mouse β-cells and performed gene expression profiling. Gene expression profiling of FACS purified immature Pdx1 (+) /Ins1 (low) cells and mature Pdx1 (high) /Ins1 (high ) cells from cultures of human islets, mouse islets and MIN6 cells revealed that Pdx1 (+) /Ins1 (low) cells are enriched for multiple genes associated with β-cell development/progenitor cells, proliferation, apoptosis, as well as genes coding for other islet cell hormones such as glucagon. We also demonstrated that the heterogeneity in β-cell maturation states previously observed in vitro, can also be found in vivo. Collectively, these experiments contribute to the understanding of maturation, dedifferentiation and plasticity of adult pancreatic β-cells. The results have significant implications for islet regeneration and for in vitro generation of functional β-cells to treat diabetes. PMID:21633187

  15. Sertoli Cells Maintain Leydig Cell Number and Peritubular Myoid Cell Activity in the Adult Mouse Testis

    PubMed Central

    Monteiro, Ana; Milne, Laura; Cruickshanks, Lyndsey; Jeffrey, Nathan; Guillou, Florian; Freeman, Tom C.; Mitchell, Rod T.; Smith, Lee B.

    2014-01-01

    The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health. PMID:25144714

  16. The orl rat is more responsive to methacholine challenge than wild type

    PubMed Central

    Rodriguez, Elena; Barthold, Julia S.; Kreiger, Portia A.; Armani, Milena Hirata; Wang, Jordan; Michelini, Katherine A.; Wolfson, Marla R.; Boyce, Roberta; Barone, Carol A.; Zhu, Yan; Waldman, Scott A.; Shaffer, Thomas H.

    2015-01-01

    Background This study presents an animal model of native airway hyperresponsiveness (AHR). AHR is a fundamental aspect of asthma and reflects an abnormal response characterized by airway narrowing following exposure to a wide variety of non-immunological stimuli. Undescended testis (UDT) is one of the most common male congenital anomalies. The orl rat is a Long Evans substrain with inherited UDT. Since boys born with congenital UDT are more likely to manifest asthma symptoms, the main aim in of this study was to investigate the alternative hypothesis that orl rats have greater AHR to a methacholine aerosol challenge than wild type rats. Methods Long Evans wild type (n = 9) and orl (n = 13) rats were anesthetized, tracheostomized, and mechanically ventilated at 4 weeks of age. Escalating concentrations of inhaled methacholine were delivered. The methacholine potency and efficacy in the strains were measured. Respiratory resistance was the primary endpoint. After the final methacholine aerosol challenge, the short-acting β2-adrenoceptor agonist albuterol was administered as an aerosol and lung/diaphragm tissues were assayed for interleukin (IL)-4, IL-6, and tumor necrosis factor (TNF)-α. Histological and histomorphometrical analyses were performed. Results The methacholine concentratione-response curve in the orl group indicated increased sensitivity, hyperreactivity, and exaggerated maximal response in comparison with the wild type group, indicating that orl rats had abnormally greater AHR responses to methacholine. Histological findings in orl rats showed the presence of eosinophils, unlike wild type rats. β2-Adrenoceptor agonist intervention resulted in up-regulation of IL-4 diaphragmatic levels and down-regulation of IL-4 and IL-6 in the lungs of orl rats. Conclusion orl rats had greater AHR than wild type rats during methacholine challenge, with higher IL-4 levels in diaphragmatic tissue homogenates. Positive immunostaining for IL-4 was detected in lung and

  17. Comparative effects of chlorpyrifos in wild type and cannabinoid Cb1 receptor knockout mice

    SciTech Connect

    Baireddy, Praveena; Liu, Jing; Hinsdale, Myron; Pope, Carey

    2011-11-15

    Endocannabinoids (eCBs) modulate neurotransmission by inhibiting the release of a variety of neurotransmitters. The cannabinoid receptor agonist WIN 55.212-2 (WIN) can modulate organophosphorus (OP) anticholinesterase toxicity in rats, presumably by inhibiting acetylcholine (ACh) release. Some OP anticholinesterases also inhibit eCB-degrading enzymes. We studied the effects of the OP insecticide chlorpyrifos (CPF) on cholinergic signs of toxicity, cholinesterase activity and ACh release in tissues from wild type (+/+) and cannabinoid CB1 receptor knockout (-/-) mice. Mice of both genotypes (n = 5-6/treatment group) were challenged with CPF (300 mg/kg, 2 ml/kg in peanut oil, sc) and evaluated for functional and neurochemical changes. Both genotypes exhibited similar cholinergic signs and cholinesterase inhibition (82-95% at 48 h after dosing) in cortex, cerebellum and heart. WIN reduced depolarization-induced ACh release in vitro in hippocampal slices from wild type mice, but had no effect in hippocampal slices from knockouts or in striatal slices from either genotype. Chlorpyrifos oxon (CPO, 100 {mu}M) reduced release in hippocampal slices from both genotypes in vitro, but with a greater reduction in tissues from wild types (21% vs 12%). CPO had no significant in vitro effect on ACh release in striatum. CPF reduced ACh release in hippocampus from both genotypes ex vivo, but reduction was again significantly greater in tissues from wild types (52% vs 36%). In striatum, CPF led to a similar reduction (20-23%) in tissues from both genotypes. Thus, while CB1 deletion in mice had little influence on the expression of acute toxicity following CPF, CPF- or CPO-induced changes in ACh release appeared sensitive to modulation by CB1-mediated eCB signaling in a brain-regional manner. -- Highlights: Black-Right-Pointing-Pointer C57Bl/6 mice showed dose-related cholinergic toxicity following subcutaneous chlorpyrifos exposure. Black-Right-Pointing-Pointer Wild type and

  18. Biomass Productivities in Wild Type and Pigment Mutant of Cyclotella sp. (Diatom)

    SciTech Connect

    Huesemann, Michael H.; Hausmann, Tom S.; Bartha, Richard; Aksoy, M.; Weissman, Joseph C.; Benemann, John

    2008-07-03

    Microalgae are expected to play a significant role in greenhouse gas mitigation because they can utilize CO2 from powerplant flue gases directly while producing a variety of renewable carbon-neutral biofuels. In order for such a microalgal climate change mitigation strategy to become economically feasible, it will be necessary to significantly improve biomass productivities. One approach to achieve this objective is to reduce, via mutagenesis, the number of light harvesting pigments, which, according to theory, should significantly improve the light utilization efficiency, primarily by increasing the light intensity at which photosynthesis saturates (Is). Employing chemical (ethylmethylsulfonate, EMS) and UV mutagenesis of a wild type strain of the diatom Cyclotella, approximately 10,000 pigment mutants were generated, and two of the most promising ones (CM1 and CM1-1) were subjected to further testing in both laboratory cultures and outdoor ponds. Measurements of photosynthetic oxygen production rates as a function of light intensity (i.e., P-I curves) of samples taken from laboratory batch cultures during the exponential and linear growth phase indicated that the light intensity at which photosynthesis saturates (Is) was two to three times greater in the pigment mutant CM1-1 than in the wild type, i.e., 355-443 versus 116-169 μmole/m2∙sec, respectively. While theory, i.e., the Bush equation, predicts that such a significant gain in Is should increase light utilization efficiencies and thus biomass productivities, particularly at high light intensities, no improvements in biomass productivities were observed in either semi-continuous laboratory cultures or outdoor ponds. In fact, the maximum biomass productivity in semi-continuous laboratory culture was always greater in the wild type than in the mutant, namely 883 versus 725 mg/L∙d, respectively at low light intensity (200 μmole/m2∙sec) and 1229 versus 1043 mg/L∙d, respectively at high light intensity

  19. Light Scattering Properties Vary across Different Regions of the Adult Mouse Brain

    PubMed Central

    Stubblefield, Elizabeth A.; Felsen, Gidon

    2013-01-01

    Recently developed optogenetic tools provide powerful approaches to optically excite or inhibit neural activity. In a typical in-vivo experiment, light is delivered to deep nuclei via an implanted optical fiber. Light intensity attenuates with increasing distance from the fiber tip, determining the volume of tissue in which optogenetic proteins can successfully be activated. However, whether and how this volume of effective light intensity varies as a function of brain region or wavelength has not been systematically studied. The goal of this study was to measure and compare how light scatters in different areas of the mouse brain. We delivered different wavelengths of light via optical fibers to acute slices of mouse brainstem, midbrain and forebrain tissue. We measured light intensity as a function of distance from the fiber tip, and used the data to model the spread of light in specific regions of the mouse brain. We found substantial differences in effective attenuation coefficients among different brain areas, which lead to substantial differences in light intensity demands for optogenetic experiments. The use of light of different wavelengths additionally changes how light illuminates a given brain area. We created a brain atlas of effective attenuation coefficients of the adult mouse brain, and integrated our data into an application that can be used to estimate light scattering as well as required light intensity for optogenetic manipulation within a given volume of tissue. PMID:23874433

  20. Ultrastructural analysis of adult mouse neocortex comparing aldehyde perfusion with cryo fixation

    PubMed Central

    Korogod, Natalya; Petersen, Carl CH; Knott, Graham W

    2015-01-01

    Analysis of brain ultrastructure using electron microscopy typically relies on chemical fixation. However, this is known to cause significant tissue distortion including a reduction in the extracellular space. Cryo fixation is thought to give a truer representation of biological structures, and here we use rapid, high-pressure freezing on adult mouse neocortex to quantify the extent to which these two fixation methods differ in terms of their preservation of the different cellular compartments, and the arrangement of membranes at the synapse and around blood vessels. As well as preserving a physiological extracellular space, cryo fixation reveals larger numbers of docked synaptic vesicles, a smaller glial volume, and a less intimate glial coverage of synapses and blood vessels compared to chemical fixation. The ultrastructure of mouse neocortex therefore differs significantly comparing cryo and chemical fixation conditions. DOI: http://dx.doi.org/10.7554/eLife.05793.001 PMID:26259873

  1. Ultrastructural analysis of adult mouse neocortex comparing aldehyde perfusion with cryo fixation.

    PubMed

    Korogod, Natalya; Petersen, Carl C H; Knott, Graham W

    2015-01-01

    Analysis of brain ultrastructure using electron microscopy typically relies on chemical fixation. However, this is known to cause significant tissue distortion including a reduction in the extracellular space. Cryo fixation is thought to give a truer representation of biological structures, and here we use rapid, high-pressure freezing on adult mouse neocortex to quantify the extent to which these two fixation methods differ in terms of their preservation of the different cellular compartments, and the arrangement of membranes at the synapse and around blood vessels. As well as preserving a physiological extracellular space, cryo fixation reveals larger numbers of docked synaptic vesicles, a smaller glial volume, and a less intimate glial coverage of synapses and blood vessels compared to chemical fixation. The ultrastructure of mouse neocortex therefore differs significantly comparing cryo and chemical fixation conditions. PMID:26259873

  2. ChIP-Seq analysis of the adult male mouse brain after developmental exposure to arsenic.

    PubMed

    Tyler, Christina R; Weber, Jessica A; Labrecque, Matthew; Hessinger, Justin M; Edwards, Jeremy S; Allan, Andrea M

    2015-12-01

    Exposure to the common environmental contaminant arsenic impacts the epigenetic landscape, including DNA methylation and histone modifications, of several cell types. Developmental arsenic exposure (DAE) increases acetylation and methylation of histone proteins and the protein expression of several chromatin-modifying enzymes in the dentate gyrus (DG) subregion of the adult male mouse brain [26]. To complement and support these data, ChIP-Seq analysis of DNA associated with trimethylation of histone 3 lysine 4 (H3K4me3) derived from the adult male DG after DAE was performed. DAE induced differential H3K4me3 enrichment on genes in pathways associated with cellular development and growth, cell death and survival, and neurological disorders, particularly as they relate to cancer, in the adult male brain. Comparison of H3K4me3 enrichment in controls revealed mechanisms that are potentially lacking in arsenic-exposed animals, including neurotransmission, neuronal growth and development, hormonal regulation, protein synthesis, and cellular homeostasis. New pathways impacted by arsenic include cytoskeleton organization, cell signaling, and potential disruption of immune function and warrant further investigation using this DAE paradigm in the mouse brain. PMID:26543888

  3. Identification and Comparative Profiling of miRNAs in an Early Flowering Mutant of Trifoliate Orange and Its Wild Type by Genome-Wide Deep Sequencing

    PubMed Central

    Li, Wen-Yang; Guo, Wen-Wu; Deng, Xiu-Xin; Hu, Chun-Gen; Zhang, Jin-Zhi

    2012-01-01

    MicroRNAs (miRNAs) are a new class of small, endogenous RNAs that play a regulatory role in various biological and metabolic processes by negatively affecting gene expression at the post-transcriptional level. While the number of known Arabidopsis and rice miRNAs is continuously increasing, information regarding miRNAs from woody plants such as citrus remains limited. Solexa sequencing was performed at different developmental stages on both an early flowering mutant of trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf.) and its wild-type in this study, resulting in the obtainment of 141 known miRNAs belonging to 99 families and 75 novel miRNAs in four libraries. A total of 317 potential target genes were predicted based on the 51 novel miRNAs families, GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in diverse cellular processes in plants, including development, transcription, protein degradation and cross adaptation. To characterize those miRNAs expressed at the juvenile and adult development stages of the mutant and its wild-type, further analysis on the expression profiles of several miRNAs through real-time PCR was performed. The results revealed that most miRNAs were down-regulated at adult stage compared with juvenile stage for both the mutant and its wild-type. These results indicate that both conserved and novel miRNAs may play important roles in citrus growth and development, stress responses and other physiological processes. PMID:22952759

  4. The MDM2 small-molecule inhibitor RG7388 leads to potent tumor inhibition in p53 wild-type neuroblastoma

    PubMed Central

    Lakoma, A; Barbieri, E; Agarwal, S; Jackson, J; Chen, Z; Kim, Y; McVay, M; Shohet, JM; Kim, ES

    2016-01-01

    Neuroblastoma is an aggressive pediatric malignancy which is >98% p53 wild-type at diagnosis. As a primary repressor of p53 activity and part of a p53-activated negative feedback loop, targeting of mouse double minute 2 homolog (MDM2) is an attractive therapeutic approach to reactivation of p53. Since development of the first selective MDM2 inhibitor, Nutlin-3a, newer compounds have been developed for increased potency and improved bioavailability. Herein, we sought to determine the efficacy and specificity of a second-generation MDM2 inhibitor, RG7388, in neuroblastoma cell lines and xenografts and examine its effect on the p53-independent pathway of hypoxia-inducible factor-1 alpha (HIF-1α)/vascular endothelial growth factor (VEGF). Cell viability and apoptosis studies were performed on the neuroblastoma cell lines, NGP, SH-SY5Y, LAN-5, LAN-5 si-p53 (p53 silenced), and SK-N-AS (p53 null). RG7388 potently decreased cell proliferation and activated p53-dependent apoptosis. Tumor-bearing mice treated with RG7388 demonstrated significant tumor inhibition by 59% in NGP (P = 0.003), 67% in SH-SY5Y (P = 0.006), and 75% in LAN-5 (P = 0.0019) p53 wild-type xenograft tumors, but no inhibitory effect on LAN-5 si-p53 or SK-N-AS p53-silenced/null xenograft tumors. Moreover, RG7388 was found to inhibit the p53-independent pathway of HIF-1α/VEGF with decreased gene expression and alteration of angiogenesis. Our study supports the further evaluation of RG7388 as a novel treatment option in p53 wild-type neuroblastoma at diagnosis and relapse. PMID:26998348

  5. Parallel changes in metabolite and expression profiles in crooked-tail mutant and folate-reduced wild-type mice.

    PubMed

    Ernest, Sheila; Carter, Michelle; Shao, Haifeng; Hosack, Angela; Lerner, Natalia; Colmenares, Clemencia; Rosenblatt, David S; Pao, Yoh-Han; Ross, M Elizabeth; Nadeau, Joseph H

    2006-12-01

    Anomalies in homocysteine (HCY) and folate metabolism are associated with common birth defects and adult diseases, several of which can be suppressed with dietary folate supplementation. Although supplementation reduces the occurrence and severity of neural tube defects (NTDs), many cases are resistant to these beneficial effects. The basis for variable response and biomarkers that predict responsiveness are unknown. Crooked-tail (Cd) mutant mice are an important model of folate-responsive NTDs. To identify features that are diagnostic for responsiveness versus resistance to dietary folate supplementation, we surveyed metabolite and expression levels in liver samples from folate-supplemented, folate-reduced and control diets in Cd mutant and wild-type adult females. Cd homozygotes had normal total homocysteine (tHcy) levels suggesting that folate suppresses NTDs through a mechanism that does not involve modulating serum tHcy levels. Instead, parallel changes in metabolite and expression profiles in folate-supplemented Cd/Cd homozygotes and folate-reduced+/+and Cd/+mice suggest that Crooked-tail homozygotes have a defect in the utilization of intracellular folate. Then, by combining these expression and metabolite profile results with published results for other models and their controls, two clusters were found, one of which included several folate-responsive NTD models and the other previously untested and presumably folate-resistant models. The predictive value of these profiles was verified by demonstrating that NTDs of Ski-/-mutant mice, whose profile suggested resistance to folate supplementation, were not suppressed with dietary folate supplementation. These results raise the possibility of using metabolite and expression profiles to distinguish folate-responsive and resistance adult females who are at risk for bearing fetuses with an NTD. PMID:17050573

  6. Ontogeny of SERT Expression and Antidepressant-like Response to Escitalopram in Wild-Type and SERT Mutant Mice.

    PubMed

    Mitchell, Nathan C; Gould, Georgianna G; Koek, Wouter; Daws, Lynette C

    2016-08-01

    Depression is a disabling affective disorder for which the majority of patients are not effectively treated. This problem is exacerbated in children and adolescents for whom only two antidepressants are approved, both of which are selective serotonin reuptake inhibitor (SSRIs). Unfortunately SSRIs are often less effective in juveniles than in adults; however, the mechanism(s) underlying age-dependent responses to SSRIs is unknown. To this end, we compared the antidepressant-like response to the SSRI escitalopram using the tail suspension test and saturation binding of [(3)H]citalopram to the serotonin transporter (SERT), the primary target of SSRIs, in juvenile [postnatal day (P)21], adolescent (P28), and adult (P90) wild-type (SERT+/+) mice. In addition, to model individuals carrying low-expressing SERT variants, we studied mice with reduced SERT expression (SERT+/-) or lacking SERT (SERT-/-). Maximal antidepressant-like effects were less in P21 mice relative to P90 mice. This was especially apparent in SERT+/- mice. However, the potency for escitalopram to produce antidepressant-like effects in SERT+/+ and SERT+/- mice was greater in P21 and P28 mice than in adults. SERT expression increased with age in terminal regions and decreased with age in cell body regions. Binding affinity values did not change as a function of age or genotype. As expected, in SERT-/- mice escitalopram produced no behavioral effects, and there was no specific [(3)H]citalopram binding. These data reveal age- and genotype-dependent shifts in the dose-response for escitalopram to produce antidepressant-like effects, which vary with SERT expression, and may contribute to the limited therapeutic response to SSRIs in juveniles and adolescents. PMID:27288483

  7. Regrowth of Serotonin Axons in the Adult Mouse Brain Following Injury.

    PubMed

    Jin, Yunju; Dougherty, Sarah E; Wood, Kevin; Sun, Landy; Cudmore, Robert H; Abdalla, Aya; Kannan, Geetha; Pletnikov, Mikhail; Hashemi, Parastoo; Linden, David J

    2016-08-17

    It is widely believed that damaged axons in the adult mammalian brain have little capacity to regrow, thereby impeding functional recovery after injury. Studies using fixed tissue have suggested that serotonin neurons might be a notable exception, but remain inconclusive. We have employed in vivo two-photon microscopy to produce time-lapse images of serotonin axons in the neocortex of the adult mouse. Serotonin axons undergo massive retrograde degeneration following amphetamine treatment and subsequent slow recovery of axonal density, which is dominated by new growth with little contribution from local sprouting. A stab injury that transects serotonin axons running in the neocortex is followed by local regression of cut serotonin axons and followed by regrowth from cut ends into and across the stab rift zone. Regrowing serotonin axons do not follow the pathways left by degenerated axons. The regrown axons release serotonin and their regrowth is correlated with recovery in behavioral tests. PMID:27499084

  8. De novo establishment of wild-type song culture in the zebra finch.

    PubMed

    Fehér, Olga; Wang, Haibin; Saar, Sigal; Mitra, Partha P; Tchernichovski, Ofer

    2009-05-28

    Culture is typically viewed as consisting of traits inherited epigenetically, through social learning. However, cultural diversity has species-typical constraints, presumably of genetic origin. A celebrated, if contentious, example is whether a universal grammar constrains syntactic diversity in human languages. Oscine songbirds exhibit song learning and provide biologically tractable models of culture: members of a species show individual variation in song and geographically separated groups have local song dialects. Different species exhibit distinct song cultures, suggestive of genetic constraints. Without such constraints, innovations and copying errors should cause unbounded variation over multiple generations or geographical distance, contrary to observations. Here we report an experiment designed to determine whether wild-type song culture might emerge over multiple generations in an isolated colony founded by isolates, and, if so, how this might happen and what type of social environment is required. Zebra finch isolates, unexposed to singing males during development, produce song with characteristics that differ from the wild-type song found in laboratory or natural colonies. In tutoring lineages starting from isolate founders, we quantified alterations in song across tutoring generations in two social environments: tutor-pupil pairs in sound-isolated chambers and an isolated semi-natural colony. In both settings, juveniles imitated the isolate tutors but changed certain characteristics of the songs. These alterations accumulated over learning generations. Consequently, songs evolved towards the wild-type in three to four generations. Thus, species-typical song culture can appear de novo. Our study has parallels with language change and evolution. In analogy to models in quantitative genetics, we model song culture as a multigenerational phenotype partly encoded genetically in an isolate founding population, influenced by environmental variables and taking

  9. Efavirenz concentrations in CSF exceed IC50 for wild-type HIV

    PubMed Central

    Best, Brookie M.; Koopmans, Peter P.; Letendre, Scott L.; Capparelli, Edmund V.; Rossi, Steven S.; Clifford, David B.; Collier, Ann C.; Gelman, Benjamin B.; Mbeo, Gilbert; McCutchan, J. Allen; Simpson, David M.; Haubrich, Richard; Ellis, Ronald; Grant, Igor; Grant, Igor; McCutchan, J. Allen; Ellis, Ronald J.; Marcotte, Thomas D.; Franklin, Donald; Ellis, Ronald J.; McCutchan, J. Allen; Alexander, Terry; Letendre, Scott; Capparelli, Edmund; Heaton, Robert K.; Atkinson, J. Hampton; Woods, Steven Paul; Dawson, Matthew; Wong, Joseph K.; Fennema-Notestine, Christine; Taylor, Michael J.; Theilmann, Rebecca; Gamst, Anthony C.; Cushman, Clint; Abramson, Ian; Vaida, Florin; Marcotte, Thomas D.; von Jaeger, Rodney; McArthur, Justin; Smith, Mary; Morgello, Susan; Simpson, David; Mintz, Letty; McCutchan, J. Allen; Toperoff, Will; Collier, Ann; Marra, Christina; Jones, Trudy; Gelman, Benjamin; Head, Eleanor; Clifford, David; Al-Lozi, Muhammad; Teshome, Mengesha

    2011-01-01

    Objectives HIV-associated neurocognitive disorders remain common despite use of potent antiretroviral therapy (ART). Ongoing viral replication due to poor distribution of antivirals into the CNS may increase risk for HIV-associated neurocognitive disorders. This study's objective was to determine penetration of a commonly prescribed antiretroviral drug, efavirenz, into CSF. Methods CHARTER is an ongoing, North American, multicentre, observational study to determine the effects of ART on HIV-associated neurological disease. Single random plasma and CSF samples were drawn within 1 h of each other from subjects taking efavirenz between September 2003 and July 2007. Samples were assayed by HPLC or HPLC/mass spectrometry with detection limits of 39 ng/mL (plasma) and <0.1 ng/mL (CSF). Results Eighty participants (age 44 ± 8 years; 79 ± 15 kg; 20 females) had samples drawn 12.5 ± 5.4 h post-dose. The median efavirenz concentrations after a median of 7 months [interquartile range (IQR) 2–17] of therapy were 2145 ng/mL in plasma (IQR 1384–4423) and 13.9 ng/mL in CSF (IQR 4.1–21.2). The CSF/plasma concentration ratio from paired samples drawn within 1 h of each other was 0.005 (IQR 0.0026–0.0076; n = 69). The CSF/IC50 ratio was 26 (IQR 8–41) using the published IC50 for wild-type HIV (0.51 ng/mL). Two CSF samples had concentrations below the efavirenz IC50 for wild-type HIV. Conclusions Efavirenz concentrations in the CSF are only 0.5% of plasma concentrations but exceed the wild-type IC50 in nearly all individuals. Since CSF drug concentrations reflect those in brain interstitial fluids, efavirenz reaches therapeutic concentrations in brain tissue. PMID:21098541

  10. Structural and Morphometric Comparison of Lower Incisors in PACAP-Deficient and Wild-Type Mice.

    PubMed

    Sandor, B; Fintor, K; Reglodi, D; Fulop, D B; Helyes, Z; Szanto, I; Nagy, P; Hashimoto, H; Tamas, A

    2016-06-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with widespread distribution. PACAP plays an important role in the development of the nervous system, it has a trophic and protective effect, and it is also implicated in the regulation of various physiological functions. Teeth are originated from the mesenchyme of the neural crest and the ectoderm of the first branchial arch, suggesting similarities with the development of the nervous system. Earlier PACAP-immunoreactive fibers have been found in the odontoblastic and subodontoblastic layers of the dental pulp. Our previous examinations have shown that PACAP deficiency causes alterations in the morphology and structure of the developing molars of 7-day-old mice. In our present study, morphometric and structural comparison was performed on the incisors of 1-year-old wild-type and PACAP-deficient mice. Hard tissue density measurements and morphometric comparison were carried out on the mandibles and the lower incisors with micro-CT. For structural examination, Raman microscopy was applied on frontal thin sections of the mandible. With micro-CT morphometrical measurements, the size of the incisors and the relative volume of the pulp to dentin were significantly smaller in the PACAP-deficient group compared to the wild-type animals. The density of calcium hydroxyapatite in the dentin was reduced in the PACAP-deficient mice. No structural differences could be observed in the enamel with Raman microscopy. Significant differences were found in the dentin of PACAP-deficient mice with Raman microscopy, where increased carbonate/phosphate ratio indicates higher intracrystalline disordering. The evaluation of amide III bands in the dentin revealed higher structural diversity in wild-type mice. Based upon our present and previous results, it is obvious that PACAP plays an important role in tooth development with the regulation of morphogenesis, dentin, and enamel mineralization. Further studies are

  11. Genetic variation of the transthyretin gene in wild-type transthyretin amyloidosis (ATTRwt).

    PubMed

    Sikora, Jacquelyn L; Logue, Mark W; Chan, Gloria G; Spencer, Brian H; Prokaeva, Tatiana B; Baldwin, Clinton T; Seldin, David C; Connors, Lawreen H

    2015-01-01

    Wild-type transthyretin amyloidosis (ATTRwt), typically diagnosed as congestive heart failure in elderly Caucasian men, features myocardial amyloid deposits of wild-type plasma protein transthyretin (TTR). ATTRwt is sporadic, its pathogenesis is poorly understood, and currently there are no biomarkers for diagnosis or prognosis. Genetic studies of variant-associated transthyretin amyloidosis have suggested that non-coding TTR gene variants modulate disease. We hypothesized that cis-acting regulatory elements in the TTR gene non-coding regions may modify expression, affecting ATTRwt onset and progression. We studied an ATTRwt cohort consisting of 108 Caucasian males ranging in age from 59 to 87 years with cardiomyopathy due to wild-type TTR deposition; results were compared to 118 anonymous controls matched by age, sex, and race. Four predicted non-coding regulatory regions and all exons in the TTR gene were sequenced using the Sanger method. Eleven common variants were identified; three variants were significantly associated with ATTRwt (p < 0.05), though only one, rs72922940, remained near significance (p corrected = 0.083) after multiple testing correction. Exon analyses demonstrated the occurrence of the p.G26S (G6S) polymorphism in 7 % of ATTRwt subjects and 12 % of controls; this variant was predicted to be a protective factor (p = 0.051). Four variants were significantly associated with age at onset and survival. In this first genetic study of a large, well-characterized cohort of ATTRwt, non-coding and coding variants associated with disease, age at onset, and survival were identified. Further investigation is warranted to determine the prevalence of these variants in ATTRwt, their regulatory function, and potential role in assessing disease risk. PMID:25367359

  12. Capsaicin potentiates wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride-channel currents.

    PubMed

    Ai, Tomohiko; Bompadre, Silvia G; Wang, Xiaohui; Hu, Shenghui; Li, Min; Hwang, Tzyh-Chang

    2004-06-01

    To examine the effects of capsaicin on cystic fibrosis transmembrane conductance regulator (CFTR), we recorded wild-type and mutant CFTR chloride-channel currents using patch-clamp methods. The effects of capsaicin were compared with those of genistein, a well-characterized CFTR activator. In whole-cell experiments, capsaicin potentiates cAMP-stimulated wild-type CFTR currents expressed in NIH 3T3 cells or Chinese hamster ovary cells in a dose-dependent manner with a maximal response approximately 60% of that with genistein and an apparent Kd of 48.4 +/- 6.8 microM. In cell-attached recordings, capsaicin alone fails to activate CFTR in cells that show negligible basal CFTR activity, indicating that capsaicin does not stimulate the cAMP cascade. The magnitude of potentiation with capsaicin depends on the channel activity before drug application; the lower the prestimulated Po, the higher the potentiation. Single-channel kinetic analysis shows that capsaicin potentiates CFTR by increasing the opening rate and decreasing the closing rate of the channel. Capsaicin may act as a partial agonist of genistein because the maximally enhanced wild-type CFTR currents with genistein are partially inhibited by capsaicin. Capsaicin increases DeltaR-CFTR, a protein kinase A (PKA)-independent, constitutively active channel, in cell-attached patches. In excised inside-out patches, capsaicin potentiates the PKA-phosphorylated, ATP-dependent CFTR activity. Both capsaicin and genistein potentiate the cAMP-stimulated G551D-CFTR, DeltaF508-CFTR, and 8SA mutant channel currents. The binding site for capsaicin is probably located at the cytoplasmic domain of CFTR, because pipette application of capsaicin fails to potentiate CFTR activity. In conclusion, capsaicin is a partial agonist of genistein in activation of the CFTR chloride channel. Both compounds affect ATP-dependent gating of CFTR. PMID:15155835

  13. Growth, seed development and genetic analysis in wild type and Def mutant of Pisum sativum L

    PubMed Central

    2011-01-01

    Background The def mutant pea (Pisum sativum L) showed non-abscission of seeds from the funicule. Here we present data on seed development and growth pattern and their relationship in predicting this particular trait in wild type and mutant lines as well as the inheritance pattern of the def allele in F2 and F3 populations. Findings Pod length and seed fresh weight increase with fruit maturity and this may affect the abscission event in pea seeds. However, the seed position in either the distal and proximal ends of the pod did not show any difference. The growth factors of seed fresh weight (FW), width of funicles (WFN), seed width (SW) and seed height (SH) were highly correlated and their relationships were determined in both wild type and def mutant peas. The coefficient of determination R2 values for the relationship between WFN and FW, SW and SH and their various interactions were higher for the def dwarf type. Stepwise multiple regression analysis showed that variation of WFN was associated with SH and SW. Pearson's chi square analysis revealed that the inheritance and segregation of the Def locus in 3:1 ratio was significant in two F2 populations. Structural analysis of the F3 population was used to confirm the inheritance status of the Def locus in F2 heterozygote plants. Conclusions This study investigated the inheritance of the presence or absence of the Def allele, controlling the presence of an abscission zone (AZ) or an abscission-less zone (ALZ) forming in wild type and mutant lines respectively. The single major gene (Def) controlling this phenotype was monogenic and def mutants were characterized and controlled by the homozygous recessive def allele that showed no palisade layers in the hilum region of the seed coat. PMID:22078070

  14. Expression of Quaking RNA-Binding Protein in the Adult and Developing Mouse Retina

    PubMed Central

    Aono, Kentaro; Kawashima, Togo; Inoue, Kiyoshi; Ku, Li; Feng, Yue; Koike, Chieko

    2016-01-01

    Quaking (QKI), which belongs to the STAR family of KH domain-containing RNA-binding proteins, functions in pre-mRNA splicing, microRNA regulation, and formation of circular RNA. QKI plays critical roles in myelinogenesis in the central and peripheral nervous systems and has been implicated neuron-glia fate decision in the brain; however, neither the expression nor function of QKI in the neural retina is known. Here we report the expression of QKI RNA-binding protein in the developing and mature mouse retina. QKI was strongly expressed by Müller glial cells in both the developing and adult retina. Intriguingly, during development, QKI was expressed in early differentiating neurons, such as the horizontal and amacrine cells, and subsequently in later differentiating bipolar cells, but not in photoreceptors. Neuronal expression was uniformly weak in the adult. Among QKI isoforms (5, 6, and 7), QKI-5 was the predominantly expressed isoform in the adult retina. To study the function of QKI in the mouse retina, we examined quakingviable(qkv) mice, which have a dysmyelination phenotype that results from deficiency of QKI expression and reduced numbers of mature oligodendrocytes. In homozygous qkv mutant mice (qkv/qkv), the optic nerve expression levels of QKI-6 and 7, but not QKI-5 were reduced. In the retina of the mutant homozygote, QKI-5 levels were unchanged, and QKI-6 and 7 levels, already low, were also unaffected. We conclude that QKI is expressed in developing and adult Müller glia. QKI is additionally expressed in progenitors and in differentiating neurons during retinal development, but expression weakened or diminished during maturation. Among QKI isoforms, we found that QKI-5 predominated in the adult mouse retina. Since Müller glial cells are thought to share properties with retinal progenitor cells, our data suggest that QKI may contribute to maintaining retinal progenitors prior to differentiation into neurons. On the other hand, the expression of QKI in

  15. Nutlin-3, the small-molecule inhibitor of MDM2, promotes senescence and radiosensitises laryngeal carcinoma cells harbouring wild-type p53

    PubMed Central

    Arya, A K; El-Fert, A; Devling, T; Eccles, R M; Aslam, M A; Rubbi, C P; Vlatković, N; Fenwick, J; Lloyd, B H; Sibson, D R; Jones, T M; Boyd, M T

    2010-01-01

    Background: Primary radiotherapy (RT) is a mainstay of treatment for laryngeal squamous cell carcinoma (LSCC). Although the cure rates for early (T1) vocal cord tumours are high, RT proves ineffective in up to a third of T3 carcinomas. Moreover, RT is associated with debilitating early- and late-treatment-related toxicity, thus finding means to de-escalate therapy, while retaining/augmenting therapeutic effectiveness, is highly desirable. p53 is a key mediator of radiation responses; we therefore investigated whether Nutlin-3, a small-molecule inhibitor of MDM2 (mouse double minute 2; an essential negative regulator of p53), might radiosensitise LSCC cells. Methods: We performed clonogenic assays to measure radiosensitivity in a panel of LSCC cell lines (for which we determined p53 mutational status) in the presence and absence of Nutlin-3. Results: LSCC cells harbouring wild-type p53 were significantly radiosensitised by Nutlin-3 (P<0.0001; log-rank scale), and displayed increased cell cycle arrest and significantly increased senescence (P<0.001) in the absence of increased apoptosis; thus, our data suggest that senescence may mediate this increased radiosensitivity. Conclusion: This is the first study showing Nutlin-3 as an effective radiosensitiser in LSCC cells that retain wild-type p53. The clinical application of Nutlin-3 might improve local recurrence rates or allow treatment de-escalation in these patients. PMID:20588277

  16. Cell culture (Vero) derived whole virus (H5N1) vaccine based on wild-type virus strain induces cross-protective immune responses

    PubMed Central

    Kistner, Otfried; Howard, Keith; Spruth, Martin; Wodal, Walter; Brühl, Peter; Gerencer, Marijan; Crowe, Brian A.; Savidis-Dacho, Helga; Livey, Ian; Reiter, Manfred; Mayerhofer, Ines; Tauer, Christa; Grillberger, Leopold; Mundt, Wolfgang; Falkner, Falko G.; Barrett, P. Noel

    2007-01-01

    The rapid spread and the transmission to humans of avian influenza virus (H5N1) has induced world-wide fears of a new pandemic and raised concerns over the ability of standard influenza vaccine production methods to rapidly supply sufficient amounts of an effective vaccine. We report here on a robust and flexible strategy which uses wild-type virus grown in a continuous cell culture (Vero) system to produce an inactivated whole virus vaccine. Candidate vaccines based on clade 1 and clade 2 influenza H5N1 strains were developed and demonstrated to be highly immunogenic in animal models. The vaccines induce cross-neutralising antibodies, highly cross-reactive T-cell responses and are protective in a mouse challenge model not only against the homologous virus but against other H5N1 strains, including those from another clade. These data indicate that cell culture-grown, whole virus vaccines, based on the wild-type virus, allow the rapid high yield production of a candidate pandemic vaccine. PMID:17614165

  17. Adaptive thermogenesis and thermal conductance in wild-type and UCP1-KO mice

    PubMed Central

    Willershäuser, Monja; Jastroch, Martin; Rourke, Bryan C.; Fromme, Tobias; Oelkrug, Rebecca; Heldmaier, Gerhard; Klingenspor, Martin

    2010-01-01

    We compared maximal cold-induced heat production (HPmax) and cold limits between warm (WA; 27°C), moderate cold (MCA; 18°C), or cold acclimated (CA; 5°C) wild-type and uncoupling-protein 1 knockout (UCP1-KO) mice. In wild-type mice, HPmax was successively increased after MCA and CA, and the cold limit was lowered to −8.3°C and −18.0°C, respectively. UCP1-KO mice also increased HPmax in response to MCA and CA, although to a lesser extent. Direct comparison revealed a maximal cold-induced recruitment of heat production by +473 mW and +227 mW in wild-type and UCP1-KO mice, respectively. The increase in cold tolerance of UCP1-KO mice from −0.9°C in MCA to −10.1°C in CA could not be directly related to changes in HPmax, indicating that UCP1-KO mice used the dissipated heat more efficiently than wild-type mice. As judged from respiratory quotients, acutely cold-challenged UCP1-KO mice showed a delayed transition toward lipid oxidation, and 5-h cold exposure revealed diminished physical activity and less variability in the control of metabolic rate. We conclude that BAT is required for maximal adaptive thermogenesis but also allows metabolic flexibility and a rapid switch toward sustained lipid-fuelled thermogenesis as an acute response to cold. In both CA groups, expression of contractile proteins (myosin heavy-chain isoforms) showed minor training effects in skeletal muscles, while cardiac muscle of UCP1-KO mice had novel expression of beta cardiac isoform. Neither respiration nor basal proton conductance of skeletal muscle mitochondria were different between genotypes. In subcutaneous white adipose tissue of UCP1-KO mice, cold exposure increased cytochrome-c oxidase activity and expression of the cell death-inducing DFFA-like effector A by 3.6-fold and 15-fold, respectively, indicating the recruitment of mitochondria-rich brown adipocyte-like cells. Absence of functional BAT leads to remodeling of white adipose tissue, which may significantly contribute

  18. Organophosphonate utilization by the wild-type strain of Pseudomonas fluorescens.

    PubMed

    Zboińska, E; Lejczak, B; Kafarski, P

    1992-09-01

    The wild-type strain of Pseudomonas fluorescens was found to utilize a range of structurally diverse organophosphonates as its sole carbon or nitrogen sources. Representative compounds included aminoalkylphosphonates, hydroxyalkylphosphonates, oxoalkylphosphonates, and phosphono dipeptides. Among them, amino(phenyl)methylphosphonate,2-aminoethylphosphonate, aminomethylphosphonate, diisopropyl 9-aminofluoren-9-ylphosphonate, and 2-oxoalkylphosphonates were used by P. fluorescens as its sole sources of phosphorus. Only slight growth was observed on the herbicide glyphosate (N-phosphonomethylglycine), which was metabolized to aminomethylphosphonate. Neither phosphinothricin nor its dialanyl tripeptide, bialaphos, supported growth of P. fluorescens. The possible mechanisms of organophosphonate degradation by this strain are discussed. PMID:1444412

  19. Organophosphonate utilization by the wild-type strain of Pseudomonas fluorescens.

    PubMed Central

    Zboińska, E; Lejczak, B; Kafarski, P

    1992-01-01

    The wild-type strain of Pseudomonas fluorescens was found to utilize a range of structurally diverse organophosphonates as its sole carbon or nitrogen sources. Representative compounds included aminoalkylphosphonates, hydroxyalkylphosphonates, oxoalkylphosphonates, and phosphono dipeptides. Among them, amino(phenyl)methylphosphonate,2-aminoethylphosphonate, aminomethylphosphonate, diisopropyl 9-aminofluoren-9-ylphosphonate, and 2-oxoalkylphosphonates were used by P. fluorescens as its sole sources of phosphorus. Only slight growth was observed on the herbicide glyphosate (N-phosphonomethylglycine), which was metabolized to aminomethylphosphonate. Neither phosphinothricin nor its dialanyl tripeptide, bialaphos, supported growth of P. fluorescens. The possible mechanisms of organophosphonate degradation by this strain are discussed. PMID:1444412

  20. Oxygenated cembranoids from the cultured and wild-type soft corals Sinularia flexibilis.

    PubMed

    Su, Jui-Hsin; Lin, Yu-Fang; Lu, Yi; Yeh, Hsiao-Chien; Wang, Wei-Hsien; Fan, Tung-Yung; Sheu, Jyh-Horng

    2009-11-01

    Two new cembranoids, flexibilisolide A (1) and flexibilisin A (2), along with one known combranoid 5 have been isolated from the cultured soft coral Sinularia flexibilis. Furthermore, two new cembranoids, flexibilisolide B (3) and flexibilisin B (4), along with two known combranoids (5, 6), have been isolated from the wild-type soft coral S. flexibilis. The structures of the new metabolites were determined on the basis of extensive spectroscopic analysis and by comparison of NMR data with those of known compounds. The metabolites 5 and 6 have been shown to exhibit weak cytotoxic activity against MCF-7 cancer cell line. PMID:19881265

  1. Intra-axonal calcium changes after axotomy in wild-type and slow Wallerian degeneration axons.

    PubMed

    Adalbert, R; Morreale, G; Paizs, M; Conforti, L; Walker, S A; Roderick, H L; Bootman, M D; Siklós, L; Coleman, M P

    2012-12-01

    Calcium accumulation induces the breakdown of cytoskeleton and axonal fragmentation in the late stages of Wallerian degeneration. In the early stages there is no evidence for any long-lasting, extensive increase in intra-axonal calcium but there does appear to be some redistribution. We hypothesized that changes in calcium distribution could have an early regulatory role in axonal degeneration in addition to the late executionary role of calcium. Schmidt-Lanterman clefts (SLCs), which allow exchange of metabolites and ions between the periaxonal and extracellular space, are likely to have an increased role when axon segments are separated from the cell body, so we used the oxalate-pyroantimonate method to study calcium at SLCs in distal stumps of transected wild-type and slow Wallerian degeneration (Wld(S)) mutant sciatic nerves, in which Wallerian degeneration is greatly delayed. In wild-type nerves most SLCs show a step gradient of calcium distribution, which is lost at around 20% of SLCs within 3mm of the lesion site by 4-24h after nerve transection. To investigate further the association with Wallerian degeneration, we studied nerves from Wld(S) rats. The step gradient of calcium distribution in Wld(S) is absent in around 20% of the intact nerves beneath SLCs but 4-24h following injury, calcium distribution in transected axons remained similar to that in uninjured nerves. We then used calcium indicators to study influx and buffering of calcium in injured neurites in primary culture. Calcium penetration and the early calcium increase in this system were indistinguishable between Wld(S) and wild-type axons. However, a significant difference was observed during the following hours, when calcium increased in wild-type neurites but not in Wld(S) neurites. We conclude that there is little relationship between calcium distribution and the early stages of Wallerian degeneration at the time points studied in vivo or in vitro but that Wld(S) neurites fail to show a later

  2. Differential alterations in the expression of neurotransmitter receptors in inner retina following loss of photoreceptors in rd1 mouse.

    PubMed

    Srivastava, Prerna; Sinha-Mahapatra, Sumit K; Ghosh, Abhinaba; Srivastava, Ipsit; Dhingra, Narender K

    2015-01-01

    Loss of photoreceptors leads to significant remodeling in inner retina of rd1 mouse, a widely used model of retinal degeneration. Several morphological and physiological alterations occur in the second- and third-order retinal neurons. Synaptic activity in the excitatory bipolar cells and the predominantly inhibitory amacrine cells is enhanced. Retinal ganglion cells (RGCs) exhibit hyperactivity and aberrant spiking pattern, which adversely affects the quality of signals they can carry to the brain. To further understand the pathophysiology of retinal degeneration, and how it may lead to aberrant spiking in RGCs, we asked how loss of photoreceptors affects some of the neurotransmitter receptors in rd1 mouse. Using Western blotting, we measured the levels of several neurotransmitter receptors in adult rd1 mouse retina. We found significantly higher levels of AMPA, glycine and GABAa receptors, but lower levels of GABAc receptors in rd1 mouse than in wild-type. Since GABAa receptor is expressed in several retinal layers, we employed quantitative immunohistochemistry to measure GABAa receptor levels in specific retinal layers. We found that the levels of GABAa receptors in inner plexiform layer of wild-type and rd1 mice were similar, whereas those in outer plexiform layer and inner nuclear layer combined were higher in rd1 mouse. Specifically, we found that the number of GABAa-immunoreactive somas in the inner nuclear layer of rd1 mouse retina was significantly higher than in wild-type. These findings provide further insights into neurochemical remodeling in the inner retina of rd1 mouse, and how it might lead to oscillatory activity in RGCs. PMID:25835503

  3. Distribution of EphA5 receptor protein in the developing and adult mouse nervous system

    PubMed Central

    Cooper, Margaret A.; Crockett, David P.; Nowakowski, Richard S.; Gale, Nicholas W.; Zhou, Renping

    2009-01-01

    The EphA5 receptor tyrosine kinase plays key roles in axon guidance during development. However, the presence of EphA5 protein in the nervous system has not been fully characterized. To better examine EphA5 localization, mutant mice, in which the EphA5 cytoplasmic domain was replaced with β-galactosidase, were analyzed for both temporal and regional changes in the distribution of EphA5 protein in the developing and adult nervous system. During embryonic development, high levels of EphA5 protein were found in the retina, olfactory bulb, cerebral neocortex, hippocampus, pretectum, tectum, cranial nerve nuclei, and the spinal cord. Variations in intensity were observed as development proceeded. Staining of pretectal nuclei, tectal nuclei, and other areas of the mesencephalon became more diffuse after maturity whereas the cerebral neocortex gained more robust intensity. In the adult, receptor protein continued to be detected in many areas including the olfactory nuclei, neocortex, piriform cortex, induseum griseum, hippocampus, thalamus, amygdala, hypothalamus and septum. In addition, EphA5 protein was found in the claustrum, stria terminalis, barrel cortex, striatal patches, and along discrete axon tracts within the corpus callosum of the adult. These observations suggest that EphA5 function is not limited to the developing mouse brain and may play a role in synaptic plasticity in the adult. PMID:19326470

  4. Sexually dimorphic effect of in vitro fertilization (IVF) on adult mouse fat and liver metabolomes.

    PubMed

    Feuer, Sky K; Donjacour, Annemarie; Simbulan, Rhodel K; Lin, Wingka; Liu, Xiaowei; Maltepe, Emin; Rinaudo, Paolo F

    2014-11-01

    The preimplantation embryo is particularly vulnerable to environmental perturbation, such that nutritional and in vitro stresses restricted exclusively to this stage may alter growth and affect long-term metabolic health. This is particularly relevant to the over 5 million children conceived by in vitro fertilization (IVF). We previously reported that even optimized IVF conditions reprogram mouse postnatal growth, fat deposition, and glucose homeostasis in a sexually dimorphic fashion. To more clearly interrogate the metabolic changes associated with IVF in adulthood, we used nontargeted mass spectrometry to globally profile adult IVF- and in vivo-conceived liver and gonadal adipose tissues. There was a sex- and tissue-specific effect of IVF on adult metabolite signatures indicative of metabolic reprogramming and oxidative stress and reflective of the observed phenotypes. Additionally, we observed a striking effect of IVF on adult sexual dimorphism. Male-female differences in metabolite concentration were exaggerated in hepatic IVF tissue and significantly reduced in IVF adipose tissue, with the majority of changes affecting amino acid and lipid metabolites. We also observed female-specific changes in markers of oxidative stress and adipogenesis, including reduced glutathione, cysteine glutathione disulfide, ophthalmate, urate, and corticosterone. In summary, embryo manipulation and early developmental experiences can affect adult patterns of sexual dimorphism and metabolic physiology. PMID:25211591

  5. Establishment of Leptin-Responsive Cell Lines from Adult Mouse Hypothalamus

    PubMed Central

    Iwakura, Hiroshi; Dote, Katsuko; Bando, Mika; Koyama, Hiroyuki; Hosoda, Kiminori; Kangawa, Kenji; Nakao, Kazuwa

    2016-01-01

    Leptin resistance is considered to be the primary cause of obesity. However, the cause of leptin resistance remains incompletely understood, and there is currently no cure for the leptin-resistant state. In order to identify novel drug-target molecules that could overcome leptin resistance, it would be useful to develop in vitro assay systems for evaluating leptin resistance. In this study, we established immortalized adult mouse hypothalamus—derived cell lines, termed adult mouse hypothalamus (AMH) cells, by developing transgenic mice in which SV40 Tag was overexpressed in chromogranin A—positive cells in a tamoxifen-dependent manner. In order to obtain leptin-responsive clones, we selected clones based on the phosphorylation levels of STAT3 induced by leptin. The selected clones were fairly responsive to leptin in terms of STAT3, ERK, and Akt phosphorylation and induction of c-Fos mRNA induction. Pretreatment with leptin, insulin, and palmitate attenuated the c-Fos mRNA response to leptin, suggesting that certain aspects of leptin resistance might be reconstituted in this cellular model. These cell lines are useful tools for understanding the molecular nature of the signal disturbance in the leptin-resistant state and for identifying potential target molecules for drugs that relieve leptin resistance, although they have drawbacks including de-differentiated nature and lack of long-time stability. PMID:26849804

  6. Caspase-Mediated Apoptosis in Sensory Neurons of Cultured Dorsal Root Ganglia in Adult Mouse

    PubMed Central

    Momeni, Hamid Reza; Soleimani Mehranjani, Malek; Shariatzadeh, Mohammad Ali; Haddadi, Mahnaz

    2013-01-01

    Objective: Sensory neurons in dorsal root ganglia (DRG) undergo apoptosis after peripheral nerve injury. The aim of this study was to investigate sensory neuron death and the mechanism involved in the death of these neurons in cultured DRG. Materials and Methods: In this experimental study, L5 DRG from adult mouse were dissected and incubated in culture medium for 24, 48, 72 and 96 hours. Freshly dissected and cultured DRG were then fixed and sectioned using a cryostat. Morphological and biochemical features of apoptosis were investigated using fluorescent staining (Propidium iodide and Hoechst 33342) and the terminal Deoxynucleotide transferase dUTP nick end labeling (TUNEL) method respectively. To study the role of caspases, general caspase inhibitor (Z-VAD.fmk, 100 μM) and immunohistochemistry for activated caspase-3 were used. Results: After 24, 48, 72 and 96 hours in culture, sensory neurons not only displayed morphological features of apoptosis but also they appeared TUNEL positive. The application of Z-VAD.fmk inhibited apoptosis in these neurons over the same time period. In addition, intense activated caspase-3 immunoreactivity was found both in the cytoplasm and the nuclei of these neurons after 24 and 48 hours. Conclusion: Results of the present study show caspase-dependent apoptosis in the sensory neurons of cultured DRG from adult mouse. PMID:24027661

  7. Variations in polyoma virus genotype in relation to tumor induction in mice. Characterization of wild type strains with widely differing tumor profiles.

    PubMed Central

    Dawe, C. J.; Freund, R.; Mandel, G.; Ballmer-Hofer, K.; Talmage, D. A.; Benjamin, T. L.

    1987-01-01

    The authors have explored the effects of variations in mouse polyoma virus genotype on patterns of tumor formation in the mouse. Four "wild type" virus strains were surveyed. Two were highly oncogenic, inducing multiple tumors of epithelial and mesenchymal origin, at high frequency and with short latency. The other two strains were weakly oncogenic, inducing fewer tumors, solely of mesenchymal origin, and after a long latency. These sharply contrasting tumor profiles were reproduced with virus stocks derived from molecularly cloned viral genomes. Though vastly different in their oncogenic properties, these cloned viruses proved equally effective in transforming established rat fibroblasts in culture and showed the same patterns of tumor antigen expression in cultured mouse cells. Complexes of polyoma middle T antigen and pp60c-src were demonstrated in extracts of epithelial tumors induced by a highly oncogenic virus strain. It is concluded that polyoma viral genetic determinants for tumor induction in the mouse are more complex than those previously defined by the use of cell transformation systems. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 PMID:2437801

  8. ALS mutant FUS disrupts nuclear localization and sequesters wild-type FUS within cytoplasmic stress granules

    PubMed Central

    Vance, Caroline; Scotter, Emma L.; Nishimura, Agnes L.; Troakes, Claire; Mitchell, Jacqueline C.; Kathe, Claudia; Urwin, Hazel; Manser, Catherine; Miller, Christopher C.; Hortobágyi, Tibor; Dragunow, Mike; Rogelj, Boris; Shaw, Christopher E.

    2013-01-01

    Mutations in the gene encoding Fused in Sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. FUS is a predominantly nuclear DNA- and RNA-binding protein that is involved in RNA processing. Large FUS-immunoreactive inclusions fill the perikaryon of surviving motor neurons of ALS patients carrying mutations at post-mortem. This sequestration of FUS is predicted to disrupt RNA processing and initiate neurodegeneration. Here, we demonstrate that C-terminal ALS mutations disrupt the nuclear localizing signal (NLS) of FUS resulting in cytoplasmic accumulation in transfected cells and patient fibroblasts. FUS mislocalization is rescued by the addition of the wild-type FUS NLS to mutant proteins. We also show that oxidative stress recruits mutant FUS to cytoplasmic stress granules where it is able to bind and sequester wild-type FUS. While FUS interacts with itself directly by protein–protein interaction, the recruitment of FUS to stress granules and interaction with PABP are RNA dependent. These findings support a two-hit hypothesis, whereby cytoplasmic mislocalization of FUS protein, followed by cellular stress, contributes to the formation of cytoplasmic aggregates that may sequester FUS, disrupt RNA processing and initiate motor neuron degeneration. PMID:23474818

  9. Physiological effects of fenpropimorph on wild-type Saccharomyces cerevisiae and fenpropimorph-resistant mutants.

    PubMed Central

    Lorenz, R T; Parks, L W

    1991-01-01

    Fenpropimorph-resistant mutants of Saccharomyces cerevisiae were isolated by a gradient selection procedure. The mutants were cross-resistant to other morpholines (fenpropidin, dodemorph, tridemorph) and 15-azasterol, but were susceptible to azoles (miconazole, clotrimazole, ketoconazole) and nystatin. In the absence of fenpropimorph, the major sterol produced by the mutants and the parental strain was ergosterol. In the presence of fenpropimorph, ignosterol (ergosta-8,14-dien-3 beta-ol) was the major sterol produced by the mutants and the parental strain. The resistance to fenpropimorph involves two recessive genes, each of which allows a semiresistance, when they are isolated apart from one another. Strain JR4 (erg3 erg11), which produces 14-methylfecosterol [14 alpha-methyl-ergosta-8,24(28)-dien- 3-beta-ol) as the major sterol in the presence or absence of fenpropimorph, was also found to be resistant to the drug. The growth inhibitory effect of fenpropimorph on wild-type cells appears to be linked to the production of ignosterol. The uptake of exogenous sterol by wild-type cells was greatly enhanced in the presence of fenpropimorph. The growth inhibition caused by fenpropimorph could only be overcome with bulk levels of exogenous C-5,6-unsaturated sterols. PMID:1929324

  10. ALS mutant FUS disrupts nuclear localization and sequesters wild-type FUS within cytoplasmic stress granules.

    PubMed

    Vance, Caroline; Scotter, Emma L; Nishimura, Agnes L; Troakes, Claire; Mitchell, Jacqueline C; Kathe, Claudia; Urwin, Hazel; Manser, Catherine; Miller, Christopher C; Hortobágyi, Tibor; Dragunow, Mike; Rogelj, Boris; Shaw, Christopher E

    2013-07-01

    Mutations in the gene encoding Fused in Sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. FUS is a predominantly nuclear DNA- and RNA-binding protein that is involved in RNA processing. Large FUS-immunoreactive inclusions fill the perikaryon of surviving motor neurons of ALS patients carrying mutations at post-mortem. This sequestration of FUS is predicted to disrupt RNA processing and initiate neurodegeneration. Here, we demonstrate that C-terminal ALS mutations disrupt the nuclear localizing signal (NLS) of FUS resulting in cytoplasmic accumulation in transfected cells and patient fibroblasts. FUS mislocalization is rescued by the addition of the wild-type FUS NLS to mutant proteins. We also show that oxidative stress recruits mutant FUS to cytoplasmic stress granules where it is able to bind and sequester wild-type FUS. While FUS interacts with itself directly by protein-protein interaction, the recruitment of FUS to stress granules and interaction with PABP are RNA dependent. These findings support a two-hit hypothesis, whereby cytoplasmic mislocalization of FUS protein, followed by cellular stress, contributes to the formation of cytoplasmic aggregates that may sequester FUS, disrupt RNA processing and initiate motor neuron degeneration. PMID:23474818

  11. Molecular dynamics studies on the structural stability of wild-type dog prion protein.

    PubMed

    Zhang, Jiapu; Liu, David D W

    2011-06-01

    Prion diseases such as Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob diseases, Gerstmann-Sträussler-Scheinker syndrome, Fatal Familial Insomnia, Kuru in humans, scrapie in sheep, bovine spongiform encephalopathy (or 'mad-cow' disease) and chronic wasting disease in cattle are invariably fatal and highly infectious neurodegenerative diseases affecting humans and animals. However, by now there have not been some effective therapeutic approaches to treat all these prion diseases. In 2008, canine mammals including dogs (canis familials) were the first time academically reported to be resistant to prion diseases (Vaccine 26: 2601-2614 (2008)). Thus, it is very worth studying the molecular structures of dog prion protein to obtain insights into the immunity of dogs to prion diseases. This paper studies the molecular structural dynamics of wild-type dog prion protein. The comparison analyses with rabbit prion protein show that the dog prion protein has stable molecular structures whether under neutral or low pH environments. We also find that the salt bridges such as D177-R163 contribute to the structural stability of wild-type rabbit prion protein under neutral pH environment. PMID:21469747

  12. Protein Folding Simulation of Mutant Go Models of the Wild-Type Trp-cage Protein

    NASA Astrophysics Data System (ADS)

    Linhananta, Apichart; Liu, Junmin

    2008-03-01

    For the past three decades, Go models of protein folding have played important roles in the understanding of how proteins fold from random conformations to their unique native structures. Unfortunately Go models reliance on known NMR or x-ray structures to construct Go interaction potentials severely limit their predictive powers. In this work, we introduce a novel method for constructing Go interaction potentials of mutant proteins based on Go interaction potentials of wild type proteins. As a template we employ the all-atom Go model of the 20-residue Trp-cage protein (A. Linhananta, J. Boer and I. MacKay, J. Chem. Phys., 2005, 122, 114901) as the wild type Go model. Trp-cage mutants are constructed by replacing a Trp-cage residue with a different residue. In particular the Pro-12 residue of the Trp-cage is substituted by Trp-12 to produce the Trp2-cage mutant, whose native structure is not yet known. Monte Carlo simulations, using CHARMM force fields, are performed to determine the ground-state structure mutant. The resulting mutant structures are used to construct the Go interaction potential of the Trp2-cage mutant Go model.

  13. Prolactin inhibits a major tumor-suppressive function of wild type BRCA1.

    PubMed

    Chen, Kuan-Hui Ethan; Walker, Ameae M

    2016-06-01

    Even though mutations in the tumor suppressor, BRCA1, markedly increase the risk of breast and ovarian cancer, most breast and ovarian cancers express wild type BRCA1. An important question is therefore how the tumor-suppressive function of normal BRCA1 is overcome during development of most cancers. Because prolactin promotes these and other cancers, we investigated the hypothesis that prolactin interferes with the ability of BRCA1 to inhibit the cell cycle. Examining six different cancer cell lines with wild type BRCA1, and making use of both prolactin and the growth-inhibiting selective prolactin receptor modulator, S179D PRL, we demonstrate that prolactin activation of Stat5 results in the formation of a complex between phospho-Stat5 and BRCA1. Formation of this complex does not interfere with nuclear translocation or binding of BRCA1 to the p21 promoter, but does interfere with the ability of BRCA1 to transactivate the p21 promoter. Overexpression of a dominant-negative Stat5 in prolactin-stimulated cells resulted in increased p21 expression. We conclude that prolactin inhibits a major tumor-suppressive function of BRCA1 by interfering with BRCA1's upregulation of expression of the cell cycle inhibitor, p21. PMID:26970274

  14. Global carbon utilization profiles of wild-type, mutant, and transformant strains of Hypocrea jecorina.

    PubMed

    Druzhinina, Irina S; Schmoll, Monika; Seiboth, Bernhard; Kubicek, Christian P

    2006-03-01

    The ascomycete Hypocrea jecorina (Trichoderma reesei), an industrial producer of cellulases and hemicellulases, can efficiently degrade plant polysaccharides. However, the catabolic pathways for the resulting monomers and their relationship to enzyme induction are not well known. Here we used the Biolog Phenotype MicroArrays technique to evaluate the growth of H. jecorina on 95 carbon sources. For this purpose, we compared several wild-type isolates, mutants producing different amounts of cellulases, and strains transformed with a heterologous antibiotic resistance marker gene. The wild-type isolates and transformed strains had the highest variation in growth patterns on individual carbon sources. The cellulase mutants were relatively similar to their parental strains. Both in the mutant and in the transformed strains, the most significant changes occurred in utilization of xylitol, erythritol, D-sorbitol, D-ribose, D-galactose, L-arabinose, N-acetyl-D-glucosamine, maltotriose, and beta-methyl-glucoside. Increased production of cellulases was negatively correlated with the ability to grow on gamma-aminobutyrate, adonitol, and 2-ketogluconate; and positively correlated with that on d-sorbitol and saccharic acid. The reproducibility, relative simplicity, and high resolution (+/-10% of increase in mycelial density) of the phenotypic microarrays make them a useful tool for the characterization of mutant and transformed strains and for a global analysis of gene function. PMID:16517662

  15. Wild-type macrophages reverse disease in heme oxygenase 1-deficient mice.

    PubMed

    Kovtunovych, Gennadiy; Ghosh, Manik C; Ollivierre, Wade; Weitzel, R Patrick; Eckhaus, Michael A; Tisdale, John F; Yachie, Akihiro; Rouault, Tracey A

    2014-08-28

    Loss-of-function mutation in the heme oxygenase 1 (Hmox1) gene causes a rare and lethal disease in children, characterized by severe anemia and intravascular hemolysis, with damage to endothelia and kidneys. Previously, we found that macrophages engaged in recycling of red cells were depleted from the tissues of Hmox1(-/-) mice, which resulted in intravascular hemolysis and severe damage to the endothelial system, kidneys, and other organs. Here, we report that subablative bone marrow transplantation (BMT) has a curative effect for disease in Hmox1(-/-) animals as a result of restoration of heme recycling by repopulation of the tissues with wild-type macrophages. Although engraftment was transient, BMT reversed anemia, normalized blood chemistries and iron metabolism parameters, and prevented renal damage. The largest proportion of donor-derived cells was observed in the livers of transplanted animals. These cells, identified as Kupffer cells with high levels of Hmox1 expression, persisted months after transient engraftment of the donor bone marrow and were responsible for the full restoration of heme-recycling ability in Hmox1(-/-) mice and reversing Hmox1-deficient phenotype. Our findings suggest that BMT or the development of specific cell therapies to repopulate patients' tissues with wild-type or reengineered macrophages represent promising approaches for HMOX1 deficiency treatment in humans. PMID:24963040

  16. Endocytic trafficking routes of wild type and DeltaF508 cystic fibrosis transmembrane conductance regulator.

    PubMed

    Gentzsch, Martina; Chang, Xiu-Bao; Cui, Liying; Wu, Yufeng; Ozols, Victor V; Choudhury, Amit; Pagano, Richard E; Riordan, John R

    2004-06-01

    Intracellular trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) is a focus of attention because it is defective in most patients with cystic fibrosis. DeltaF508 CFTR, which does not mature conformationally, normally does not exit the endoplasmic reticulum, but if induced to do so at reduced temperature is short-lived at the surface. We used external epitope-tagged constructs to elucidate the itinerary and kinetics of wild type and DeltaF508 CFTR in the endocytic pathway and visualized movement of CFTR from the surface to intracellular compartments. Modulation of different endocytic steps with low temperature (16 degrees C) block, protease inhibitors, and overexpression of wild type and mutant Rab GTPases revealed that surface CFTR enters several different routes, including a Rab5-dependent initial step to early endosomes, then either Rab11-dependent recycling back to the surface or Rab7-regulated movement to late endosomes or alternatively Rab9-mediated transit to the trans-Golgi network. Without any of these modulations DeltaF508 CFTR rapidly disappears from and does not return to the cell surface, confirming that its altered structure is detected in the distal as well as proximal secretory pathway. Importantly, however, the mutant protein can be rescued at the plasma membrane by Rab11 overexpression, proteasome inhibitors, or inhibition of Rab5-dependent endocytosis. PMID:15075371

  17. Gravitropism of hypocotyls of wild-type and starch-deficient Arabidopsis seedlings in spaceflight studies

    NASA Technical Reports Server (NTRS)

    Kiss, J. Z.; Edelmann, R. E.; Wood, P. C.

    1999-01-01

    The major purpose of this spaceflight project was to investigate the starch-statolith hypothesis for gravity perception, and a secondary goal was to study plant growth and development under spaceflight conditions. This research was based on our ground studies of gravity perception in the wild type and three starch-deficient (one starchless and two reduced starch) mutants of Arabidopsis thaliana (L.) Heynh. Dark-grown seedlings that developed in microgravity were given one of several (30 min, 60 min, or 90 min) 1-g stimuli by an on-board centrifuge, and additional controls for seedling development also were performed. These latter control experiments included a morphological study of plants that developed in space in microgravity (F microg), in space on a centrifuge (F 1g), on the ground (G 1g), and on a rotating clinostat on the ground. Since elevated levels of ethylene were reported in the spacecraft atmosphere, additional controls for morphology and gravitropism with added ethylene also were performed. While exogenous ethylene reduced the absolute magnitude of the response in all four strains of Arabidopsis, this gas did not appear to change the relative graviresponsiveness among the strains. The relative response of hypocotyls of microgravity-grown seedlings to the stimuli provided by the in-flight centrifuge was: wild type > starch-deficient mutants. Although the protoplast pressure model for gravity perception cannot be excluded, these results are consistent with a statolith-based model for perception in plants.

  18. Plastid sedimentation kinetics in roots of wild-type and starch-deficient mutants of Arabidopsis

    NASA Technical Reports Server (NTRS)

    MacCleery, S. A.; Kiss, J. Z.

    1999-01-01

    Sedimentation and movement of plastids in columella cells of the root cap were measured in seedlings of wild-type, a reduced starch mutant, and a starchless mutant of Arabidopsis. To assay for sedimentation, we used both linear measurements and the change of angle from the cell center as indices in vertical and reoriented plants with the aid of computer-assisted image analysis. Seedlings were fixed at short periods after reorientation, and plastid sedimentation correlated with starch content in the three strains of Arabidopsis. Amyloplasts of wild-type seedlings showed the greatest sedimentation, whereas plastids of the starchless mutant showed no significant sedimentation in the vertically grown and reoriented seedlings. Because previous research has shown that a full complement of starch is needed for full gravitropic sensitivity, this study correlates increased sensitivity with plastid sedimentation. However, although plastid sedimentation contributed to gravisensitivity, it was not required, because the gravitropic starchless mutant had plastids that did not sediment. This is the first study, to our knowledge, to measure plastid sedimentation in Arabidopsis roots after reorientation of seedlings. Taken together, the results of this study are consistent with the classic plastid-based and protoplast-based models of graviperception and suggest that multiple systems of perception exist in plant cells.

  19. Comparative metabolic profiling of mce1 operon mutant vs wild-type Mycobacterium tuberculosis strains.

    PubMed

    Queiroz, Adriano; Medina-Cleghorn, Daniel; Marjanovic, Olivera; Nomura, Daniel K; Riley, Lee W

    2015-11-01

    Mycobacterium tuberculosis disrupted in a 13-gene operon (mce1) accumulates free mycolic acids (FM) in its cell wall and causes accelerated death in mice. Here, to more comprehensively analyze differences in their cell wall lipid composition, we used an untargeted metabolomics approach to compare the lipid profiles of wild-type and mce1 operon mutant strains. By liquid chromatography-mass spectrometry, we identified >400 distinct lipids significantly altered in the mce1 mutant compared to wild type. These lipids included decreased levels of saccharolipids and glycerophospholipids, and increased levels of alpha-, methoxy- and keto mycolic acids (MA), and hydroxyphthioceranic acid. The mutant showed reduced expression of mmpL8, mmpL10, stf0, pks2 and papA2 genes involved in transport and metabolism of lipids recognized to induce proinflammatory response; these lipids were found to be decreased in the mutant. In contrast, the transcripts of mmpL3, fasI, kasA, kasB, acpM and RV3451 involved in MA transport and metabolism increased; MA inhibits inflammatory response in macrophages. Since the mce1 operon is known to be regulated in intracellular M. tuberculosis, we speculate that the differences we observed in cell wall lipid metabolism and composition may affect host response to M. tuberculosis infection and determine the clinical outcome of such an infection. PMID:26319139

  20. Wild-type Cu/Zn superoxide dismutase stabilizes mutant variants by heterodimerization.

    PubMed

    Weichert, Anna; Besemer, Anna S; Liebl, Martina; Hellmann, Nadja; Koziollek-Drechsler, Ingrid; Ip, Philbert; Decker, Heinz; Robertson, Janice; Chakrabartty, Avijit; Behl, Christian; Clement, Albrecht M

    2014-02-01

    Mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) are responsible for a subset of amyotrophic lateral sclerosis cases presumably by the acquisition of as yet unknown toxic properties. Additional overexpression of wild-type SOD1 in mutant SOD1 transgenic mice did not improve but rather accelerated the disease course. Recently, it was documented that the presence of wild-type SOD1 (SOD(WT)) reduced the aggregation propensity of mutant SOD1 by the formation of heterodimers between mutant and SOD1(WT) and that these heterodimers displayed at least a similar toxicity in cellular and animal models. In this study we investigated the biochemical and biophysical properties of obligate SOD1 dimers that were connected by a peptide linker. Circular dichroism spectra indicate an increased number of unstructured residues in SOD1 mutants. However, SOD1(WT) stabilized the folding of heterodimers compared to mutant homodimers as evidenced by an increase in resistance against proteolytic degradation. Heterodimerization also reduced the affinity of mutant SOD1 to antibodies detecting misfolded SOD1. In addition, the formation of obligate dimers resulted in a detection of substantial dismutase activity even of the relatively labile SOD1(G85R) mutant. These data indicate that soluble, dismutase-active SOD1 dimers might contribute at least partially to mutant SOD1 toxicity. PMID:24200866

  1. AGGREGATED, WILD-TYPE PRION PROTEIN CAUSES NEUROLOGICAL DYSFUNCTION AND SYNAPTIC ABNORMALITIES

    PubMed Central

    Chiesa, Roberto; Piccardo, Pedro; Biasini, Emiliano; Ghetti, Bernardino; Harris, David A.

    2008-01-01

    The neurotoxic forms of the prion protein (PrP) that cause neurodegeneration in prion diseases remain to be conclusively identified. Considerable evidence points to the importance of non-infectious oligomers of PrP in the pathogenic process. In this study, we describe lines of Tg(WT) transgenic mice that over-express wild-type PrP by either ∼5-fold or ∼10-fold (depending on whether the transgene array is, respectively, hemizygous or homozygous). Homozygous but not hemizygous Tg(WT) mice develop a spontaneous neurodegenerative illness characterized clinically by tremor and paresis. Both kinds of mice accumulate large numbers of punctate PrP deposits in the molecular layer of the cerebellum as well as in several other brain regions, and they display abnormally enlarged synaptic terminals accompanied by a dramatic proliferation of membranous structures. The over-expressed PrP in Tg(WT) mice assembles into an insoluble form that is mildly protease-resistant and is recognizable by aggregation-specific antibodies, but that is not infectious in transmission experiments. Taken together, our results demonstrate that non-infectious aggregates of wild-type PrP are neurotoxic, particularly to synapses, and they suggest common pathogenic mechanisms shared by prion diseases and non-transmissible neurodegenerative disorders associated with protein misfolding. PMID:19052217

  2. Bone turnover in wild type and pleiotrophin-transgenic mice housed for three months in the International Space Station (ISS).

    PubMed

    Tavella, Sara; Ruggiu, Alessandra; Giuliani, Alessandra; Brun, Francesco; Canciani, Barbara; Manescu, Adrian; Marozzi, Katia; Cilli, Michele; Costa, Delfina; Liu, Yi; Piccardi, Federica; Tasso, Roberta; Tromba, Giuliana; Rustichelli, Franco; Cancedda, Ranieri

    2012-01-01

    Bone is a complex dynamic tissue undergoing a continuous remodeling process. Gravity is a physical force playing a role in the remodeling and contributing to the maintenance of bone integrity. This article reports an investigation on the alterations of the bone microarchitecture that occurred in wild type (Wt) and pleiotrophin-transgenic (PTN-Tg) mice exposed to a near-zero gravity on the International Space Station (ISS) during the Mice Drawer System (MDS) mission, to date, the longest mice permanence (91 days) in space. The transgenic mouse strain over-expressing pleiotrophin (PTN) in bone was selected because of the PTN positive effects on bone turnover. Wt and PTN-Tg control animals were maintained on Earth either in a MDS payload or in a standard vivarium cage. This study revealed a bone loss during spaceflight in the weight-bearing bones of both strains. For both Tg and Wt a decrease of the trabecular number as well as an increase of the mean trabecular separation was observed after flight, whereas trabecular thickness did not show any significant change. Non weight-bearing bones were not affected. The PTN-Tg mice exposed to normal gravity presented a poorer trabecular organization than Wt mice, but interestingly, the expression of the PTN transgene during the flight resulted in some protection against microgravity's negative effects. Moreover, osteocytes of the Wt mice, but not of Tg mice, acquired a round shape, thus showing for the first time osteocyte space-related morphological alterations in vivo. The analysis of specific bone formation and resorption marker expression suggested that the microgravity-induced bone loss was due to both an increased bone resorption and a decreased bone deposition. Apparently, the PTN transgene protection was the result of a higher osteoblast activity in the flight mice. PMID:22438896

  3. AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α1-Proteinase Inhibitor

    DOE PAGESBeta

    Gaczynska, Maria; Karpowicz, Przemyslaw; Stuart, Christine E.; Norton, Malgorzata G.; Teckman, Jeffrey H.; Marszal, Ewa; Osmulski, Pawel A.

    2016-03-23

    α1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based on biochemicalmore » data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found inWT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo

  4. AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α1-Proteinase Inhibitor

    PubMed Central

    Gaczynska, Maria; Karpowicz, Przemyslaw; Stuart, Christine E.; Norton, Malgorzata G.; Teckman, Jeffrey H.; Marszal, Ewa; Osmulski, Pawel A.

    2016-01-01

    α1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based on biochemical data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found in WT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo

  5. Mitochondrial defects and neurodegeneration in mice overexpressing wild-type or G399S mutant HtrA2.

    PubMed

    Casadei, Nicolas; Sood, Poonam; Ulrich, Thomas; Fallier-Becker, Petra; Kieper, Nicole; Helling, Stefan; May, Caroline; Glaab, Enrico; Chen, Jing; Nuber, Silke; Marcus, Katrin; Rapaport, Doron; Ott, Thomas; Riess, Olaf; Krüger, Rejko; Fitzgerald, Julia C

    2016-02-01

    The protease HtrA2 has a protective role inside mitochondria, but promotes apoptosis under stress. We previously identified the G399S HtrA2 mutation in Parkinson's disease (PD) patients and reported mitochondrial dysfunction in vitro. Mitochondrial dysfunction is a common feature of PD and related to neurodegeneration. Complete loss of HtrA2 has been shown to cause neurodegeneration in mice. However, the full impact of HtrA2 overexpression or the G399S mutation is still to be determined in vivo. Here, we report the first HtrA2 G399S transgenic mouse model. Our data suggest that the mutation has a dominant-negative effect. We also describe a toxic effect of wild-type (WT) HtrA2 overexpression. Only low overexpression of the G399S mutation allowed viable animals and we suggest that the mutant protein is likely unstable. This is accompanied by reduced mitochondrial respiratory capacity and sensitivity to apoptotic cell death. Mice overexpressing WT HtrA2 were viable, yet these animals have inhibited mitochondrial respiration and significant induction of apoptosis in the brain leading to motor dysfunction, highlighting the opposing roles of HtrA2. Our data further underscore the importance of HtrA2 as a key mediator of mitochondrial function and its fine regulatory role in cell fate. The location and abundance of HtrA2 is tightly controlled and, therefore, human mutations leading to gain- or loss of function could provide significant risk for PD-related neurodegeneration. PMID:26604148

  6. Vaccine and Wild-Type Strains of Yellow Fever Virus Engage Distinct Entry Mechanisms and Differentially Stimulate Antiviral Immune Responses

    PubMed Central

    Fernandez-Garcia, Maria Dolores; Meertens, Laurent; Chazal, Maxime; Hafirassou, Mohamed Lamine; Dejarnac, Ophélie; Zamborlini, Alessia; Despres, Philippe; Sauvonnet, Nathalie; Arenzana-Seisdedos, Fernando

    2016-01-01

    ABSTRACT The live attenuated yellow fever virus (YFV) vaccine 17D stands as a “gold standard” for a successful vaccine. 17D was developed empirically by passaging the wild-type Asibi strain in mouse and chicken embryo tissues. Despite its immense success, the molecular determinants for virulence attenuation and immunogenicity of the 17D vaccine are poorly understood. 17D evolved several mutations in its genome, most of which lie within the envelope (E) protein. Given the major role played by the YFV E protein during virus entry, it has been hypothesized that the residues that diverge between the Asibi and 17D E proteins may be key determinants of attenuation. In this study, we define the process of YFV entry into target cells and investigate its implication in the activation of the antiviral cytokine response. We found that Asibi infects host cells exclusively via the classical clathrin-mediated endocytosis, while 17D exploits a clathrin-independent pathway for infectious entry. We demonstrate that the mutations in the 17D E protein acquired during the attenuation process are sufficient to explain the differential entry of Asibi versus 17D. Interestingly, we show that 17D binds to and infects host cells more efficiently than Asibi, which culminates in increased delivery of viral RNA into the cytosol and robust activation of the cytokine-mediated antiviral response. Overall, our study reveals that 17D vaccine and Asibi enter target cells through distinct mechanisms and highlights a link between 17D attenuation, virus entry, and immune activation. PMID:26861019

  7. Wild-type human TDP-43 expression causes TDP-43 phosphorylation, mitochondrial aggregation, motor deficits and early mortality in transgenic mice

    PubMed Central

    Xu, Ya-Fei; Gendron, Tania F.; Zhang, Yong-Jie; Lin, Wen-Lang; D’Alton, Simon; Sheng, Hong; Casey, Monica Castanedes; Tong, Jimei; Knight, Joshua; Yu, Xin; Rademakers, Rosa; Boylan, Kevin; Hutton, Mike; McGowan, Eileen; Dickson, Dennis W.; Lewis, Jada; Petrucelli, Leonard

    2011-01-01

    Transactivation response DNA-binding protein 43 (TDP-43) is a principal component of ubiquitinated inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis (ALS). Mutations in TARDBP, the gene encoding TDP-43, are associated with sporadic and familial ALS, yet multiple neurodegenerative diseases exhibit TDP-43 pathology without known TARDBP mutations. While TDP-43 has been ascribed a number of roles in normal biology, including mRNA splicing and transcription regulation, elucidating disease mechanisms associated with this protein is hindered by the lack of models to dissect such functions. We have generated transgenic (TDP-43PrP) mice expressing full-length human TDP-43 (hTDP-43) driven by the mouse prion promoter to provide a tool to analyze the role of wild-type hTDP-43 in the brain and spinal cord. Expression of hTDP-43 caused a dose dependant down regulation of mouse TDP-43 RNA and protein. Moderate overexpression of hTDP-43 resulted in TDP-43 truncation, increased cytoplasmic and nuclear ubiquitin levels, as well as intranuclear and cytoplasmic aggregates that were immunopositive for phosphorylated TDP-43. Of note, abnormal juxtanuclear aggregates of mitochondria were observed, accompanied by enhanced levels of Fis1 and phosphorylated DLP1, key components of the mitochondrial fission machinery. Conversely, a marked reduction in mitofusin 1 expression, which plays an essential role in mitochondrial fusion, was observed in TDP-43PrP mice. Finally, TDP-43PrP mice showed reactive gliosis, axonal and myelin degeneration, gait abnormalities and early lethality. This TDP-43 transgenic line provides a valuable tool for identifying potential roles of wild-type TDP-43 within the central nervous system and for studying TDP-43-associated neurotoxicity. PMID:20702714

  8. Mechanosensitivity of wild-type and G551D cystic fibrosis transmembrane conductance regulator (CFTR) controls regulatory volume decrease in simple epithelia.

    PubMed

    Xie, Changyan; Cao, Xu; Chen, Xibing; Wang, Dong; Zhang, Wei Kevin; Sun, Ying; Hu, Wenbao; Zhou, Zijing; Wang, Yan; Huang, Pingbo

    2016-04-01

    Mutations of cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial ligand-gated anion channel, are associated with the lethal genetic disease cystic fibrosis. The CFTR G551D mutation impairs ATP hydrolysis and thereby makes CFTR refractory to cAMP stimulation. Both wild-type (WT) and G551D CFTR have been implicated in regulatory volume decrease (RVD), but the underlying mechanism remains incompletely understood. Here, we show that the channel activity of both WT and G551D CFTR is directly stimulated by mechanical perturbation induced by cell swelling at the single-channel, cellular, and tissue levels. Hypotonicity activated CFTR single channels in cell-attached membrane patches and WT-CFTR-mediated short-circuit current (Isc) in Calu-3 cells, and this was independent of Ca(2+)and cAMP/PKA signaling. Genetic suppression and ablation but not G551D mutation of CFTR suppressed the hypotonicity- and stretch-inducedIscin Calu-3 cells and mouse duodena. Moreover, ablation but not G551D mutation of the CFTR gene inhibited the RVD of crypts isolated from mouse intestine; more importantly, CFTR-specific blockers markedly suppressed RVD in both WT- and G551D CFTR mice, demonstrating for the first time that the channel activity of both WT and G551D CFTR is required for epithelial RVD. Our findings uncover a previously unrecognized mechanism underlying CFTR involvement in epithelial RVD and suggest that the mechanosensitivity of G551D CFTR might underlie the mild phenotypes resulting from this mutation.-Xie, C., Cao, X., Chen, X, Wang, D., Zhang, W. K., Sun, Y., Hu, W., Zhou, Z., Wang, Y., Huang, P. Mechanosensitivity of wild-type and G551D cystic fibrosis transmembrane conductance regulator (CFTR) controls regulatory volume decrease in simple epithelia. PMID:26683699

  9. Impact of mTORC1 inhibition on keratinocyte proliferation during skin tumor promotion in wild-type and BK5.AktWT mice.

    PubMed

    Rho, Okkyung; Kiguchi, Kaoru; Jiang, Guiyu; DiGiovanni, John

    2014-11-01

    In this study, we examined the impact of rapamycin on mTORC1 signaling during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced keratinocyte proliferation and skin tumor promotion in both wild-type (FVB/N) and BK5.Akt(WT) mice. TPA activated mTORC1 signaling in a time-dependent manner in cultured primary mouse keratinocytes and a mouse keratinocyte cell line. Early activation (15-30 min) of mTORC1 signaling induced by TPA was mediated in part by PKC activation, whereas later activation (2-4 h) was mediated by activation of EGFR and Akt. BK5.Akt(WT) transgenic mice, where Akt1 is overexpressed in basal epidermis, are highly sensitive to TPA-induced epidermal proliferation and two-stage skin carcinogenesis. Targeting mTORC1 with rapamycin effectively inhibited TPA-induced epidermal hyperplasia and hyperproliferation as well as tumor promotion in a dose-dependent manner in both wild-type and BK5.Akt(WT) mice. A significant expansion (∼threefold) of the label retaining cell (LRC) population per hair follicle was observed in BK5.Akt(WT) mice compared to FVB/N mice. There was also a significant increase in K15 expressing cells in the hair follicle of transgenic mice that coincided with expression of phospho-Akt, phospho-S6K, and phospho-PRAS40, suggesting an important role of mTORC1 signaling in bulge-region keratinocyte stem cell (KSC) homeostasis. After 2 weeks of TPA treatment, LRCs had moved upward into the interfollicular epidermis from the bulge region of both wild-type and BK5.Akt(WT) mice. TPA-mediated LRC proliferation and migration was significantly inhibited by rapamycin. Collectively, the current data indicate that signaling through mTORC1 contributes significantly to the process of skin tumor promotion through effects on proliferation of the target cells for tumor development. PMID:24114993

  10. Localization and regulation of PML bodies in the adult mouse brain.

    PubMed

    Hall, Małgorzata H; Magalska, Adriana; Malinowska, Monika; Ruszczycki, Błażej; Czaban, Iwona; Patel, Satyam; Ambrożek-Latecka, Magdalena; Zołocińska, Ewa; Broszkiewicz, Hanna; Parobczak, Kamil; Nair, Rajeevkumar R; Rylski, Marcin; Pawlak, Robert; Bramham, Clive R; Wilczyński, Grzegorz M

    2016-06-01

    PML is a tumor suppressor protein involved in the pathogenesis of promyelocytic leukemia. In non-neuronal cells, PML is a principal component of characteristic nuclear bodies. In the brain, PML has been implicated in the control of embryonic neurogenesis, and in certain physiological and pathological phenomena in the adult brain. Yet, the cellular and subcellular localization of the PML protein in the brain, including its presence in the nuclear bodies, has not been investigated comprehensively. Because the formation of PML bodies appears to be a key aspect in the function of the PML protein, we investigated the presence of these structures and their anatomical distribution, throughout the adult mouse brain. We found that PML is broadly expressed across the gray matter, with the highest levels in the cerebral and cerebellar cortices. In the cerebral cortex PML is present exclusively in neurons, in which it forms well-defined nuclear inclusions containing SUMO-1, SUMO 2/3, but not Daxx. At the ultrastructural level, the appearance of neuronal PML bodies differs from the classic one, i.e., the solitary structure with more or less distinctive capsule. Rather, neuronal PML bodies have the form of small PML protein aggregates located in the close vicinity of chromatin threads. The number, size, and signal intensity of neuronal PML bodies are dynamically influenced by immobilization stress and seizures. Our study indicates that PML bodies are broadly involved in activity-dependent nuclear phenomena in adult neurons. PMID:25956166

  11. Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages.

    PubMed

    Seaberg, Raewyn M; Smukler, Simon R; Kieffer, Timothy J; Enikolopov, Grigori; Asghar, Zeenat; Wheeler, Michael B; Korbutt, Gregory; van der Kooy, Derek

    2004-09-01

    The clonal isolation of putative adult pancreatic precursors has been an elusive goal of researchers seeking to develop cell replacement strategies for diabetes. We report the clonal identification of multipotent precursor cells from the adult mouse pancreas. The application of a serum-free, colony-forming assay to pancreatic cells enabled the identification of precursors from pancreatic islet and ductal populations. These cells proliferate in vitro to form clonal colonies that coexpress neural and pancreatic precursor markers. Upon differentiation, individual clonal colonies produce distinct populations of neurons and glial cells, pancreatic endocrine beta-, alpha- and delta-cells, and pancreatic exocrine and stellate cells. Moreover, the newly generated beta-like cells demonstrate glucose-dependent Ca(2+) responsiveness and insulin release. Pancreas colonies do not express markers of embryonic stem cells, nor genes suggestive of mesodermal or neural crest origins. These cells represent a previously unidentified adult intrinsic pancreatic precursor population and are a promising candidate for cell-based therapeutic strategies. PMID:15322557

  12. Growth Arrest Specific 1 (GAS1) Is Abundantly Expressed in the Adult Mouse Central Nervous System

    PubMed Central

    Zarco, Natanael; Bautista, Elizabeth; Cuéllar, Manola; Vergara, Paula; Flores-Rodriguez, Paola; Aguilar-Roblero, Raúl

    2013-01-01

    Growth arrest specific 1 (GAS1) is a pleiotropic protein that induces apoptosis and cell arrest in different tumors, but it is also involved in the development of the nervous system and other tissues and organs. This dual ability is likely caused by its capacity to interact both by inhibiting the intracellular signaling cascade induced by glial cell-line derived neurotrophic factor and by facilitating the activity of the sonic hedgehog pathway. The presence of GAS1 mRNA has been described in adult mouse brain, and here we corroborated this observation. We then proceeded to determine the distribution of the protein in the adult central nervous system (CNS). We detected, by western blot analysis, expression of GAS1 in olfactory bulb, caudate-putamen, cerebral cortex, hippocampus, mesencephalon, medulla oblongata, cerebellum, and cervical spinal cord. To more carefully map the expression of GAS1, we performed double-label immunohistochemistry and noticed expression of GAS1 in neurons in all brain areas examined. We also observed expression of GAS1 in astroglial cells, albeit the pattern of expression was more restricted than that seen in neurons. Briefly, in the present article, we report the widespread distribution and cellular localization of the GAS1 native protein in adult mammalian CNS. PMID:23813868

  13. Characterization of neural stem cells and their progeny in the sensory circumventricular organs of adult mouse.

    PubMed

    Furube, Eriko; Morita, Mitsuhiro; Miyata, Seiji

    2015-11-01

    Although evidence has accumulated that neurogenesis and gliogenesis occur in the subventricular zone (SVZ) and subgranular zone (SGZ) of adult mammalian brains, recent studies indicate the presence of neural stem cells (NSCs) in adult brains, particularly the circumventricular regions. In the present study, we aimed to determine characterization of NSCs and their progenitor cells in the sensory circumventricular organs (CVOs), including organum vasculosum of the lamina terminalis, subfornical organ, and area postrema of adult mouse. There were two types of NSCs: tanycyte-like ependymal cells and astrocyte-like cells. Astrocyte-like NSCs proliferated slowly and oligodendrocyte progenitor cells (OPCs) and neural progenitor cells (NPCs) actively divided. Molecular marker protein expression of NSCs and their progenitor cells were similar to those reported in the SVZ and SGZ, except that astrocyte-like NSCs expressed S100β. These circumventricular NSCs possessed the capacity to give rise to oligodendrocytes and sparse numbers of neurons and astrocytes in the sensory CVOs and adjacent brain regions. The inhibition of vascular endothelial growth factor (VEGF) signaling by using a VEGF receptor-associated tyrosine kinase inhibitor AZD2171 largely suppressed basal proliferation of OPCs. A single systemic administration of lipopolysaccharide attenuated proliferation of OPCs and induced remarkable proliferation of microglia. The present study indicates that sensory circumventricular NSCs provide new neurons and glial cells in the sensory CVOs and adjacent brain regions. PMID:25994374

  14. Transcript profiling reveals expression differences in wild-type and glabrous soybean lines

    PubMed Central

    2011-01-01

    Background Trichome hairs affect diverse agronomic characters such as seed weight and yield, prevent insect damage and reduce loss of water but their molecular control has not been extensively studied in soybean. Several detailed models for trichome development have been proposed for Arabidopsis thaliana, but their applicability to important crops such as cotton and soybean is not fully known. Results Two high throughput transcript sequencing methods, Digital Gene Expression (DGE) Tag Profiling and RNA-Seq, were used to compare the transcriptional profiles in wild-type (cv. Clark standard, CS) and a mutant (cv. Clark glabrous, i.e., trichomeless or hairless, CG) soybean isoline that carries the dominant P1 allele. DGE data and RNA-Seq data were mapped to the cDNAs (Glyma models) predicted from the reference soybean genome, Williams 82. Extending the model length by 250 bp at both ends resulted in significantly more matches of authentic DGE tags indicating that many of the predicted gene models are prematurely truncated at the 5' and 3' UTRs. The genome-wide comparative study of the transcript profiles of the wild-type versus mutant line revealed a number of differentially expressed genes. One highly-expressed gene, Glyma04g35130, in wild-type soybean was of interest as it has high homology to the cotton gene GhRDL1 gene that has been identified as being involved in cotton fiber initiation and is a member of the BURP protein family. Sequence comparison of Glyma04g35130 among Williams 82 with our sequences derived from CS and CG isolines revealed various SNPs and indels including addition of one nucleotide C in the CG and insertion of ~60 bp in the third exon of CS that causes a frameshift mutation and premature truncation of peptides in both lines as compared to Williams 82. Conclusion Although not a candidate for the P1 locus, a BURP family member (Glyma04g35130) from soybean has been shown to be abundantly expressed in the CS line and very weakly expressed in the

  15. Comparation of enhanced green fluorescent protein gene transfected and wild-type porcine neural stem cells.

    PubMed

    Zheng, Yue-Mao; An, Zhi-Xing; Zhao, Xiao-E; Quan, Fu-Sheng; Zhao, Hui-Ying; Zhang, Ya-Rong; Liu, Jun; He, Xiao-Ying; He, Xiao-Ning

    2010-02-01

    The aim of this study was to transfect and express the enhanced green fluorescence protein (EGFP) gene into porcine neural stem cells (NSCs) to determine whether EGFP can be used as a marker to monitor NSCs. NSCs were isolated from embryonic day 30 fetal pig brain and transfected with EGFP gene using lipofection. Transfected and wild-type NSCs were induced to differentiate into cells of neuronal and myogenic lineages. Markers of passage three NSCs and their differentiated cells were tested by reverse transcription polymerase chain reaction. The results showed that EGFP could be expressed in NSCs and the differentiated cells. NSCs expressed Nestin, NogoA, DCX, Hes1, Oct4, CD-90 and Sox2. NSCs could differentiated into astrocyte (GFAP(+)), oligodendrocyte (GalC(+)), neuron (NF(+), NSE(+) and MAP2(+)) and myocyte (myf-6(+) and myoD(+)). We concluded that EGFP can be used as a marker in monitoring NSCs. PMID:19580981

  16. Purification of extrachloroplastic. beta. -amylase from leaves of starchless and wild type Arabidopsis

    SciTech Connect

    Somerville, C.; Monroe, J.; Preiss, J. )

    1989-04-01

    Amylase activity in crude leaf extracts from starchless mutants of Arabidopsis thaliana is 5 to 10 fold higher than in the wild type (WT) when plants are grown under a 12 h photoperiod. Visualized on native PAGE, the increased activity is attributed primarily to a previously characterized extrachloroplastic {beta}-(exo)amylase. The {beta}-amylases from phosoglucomutase deficient (starchless) and WT leaves were purified to homogeneity in two steps utilizing polyethylene glycol fractionation, and cyclohexaamylose affinity chromatography. The enzyme from both mutant and WT leaves had negligible activity toward either {beta}-limit dextrin or pullulan. The specific activities of both purified enzymes were similar indicating that the protein is over-expressed in the mutant. Preliminary antibody neutralization experiments suggest that the two {beta}-amylases are not different.

  17. Genome sequence of SG33 strain and recombination between wild-type and vaccine myxoma viruses.

    PubMed

    Camus-Bouclainville, Christelle; Gretillat, Magalie; Py, Robert; Gelfi, Jacqueline; Guérin, Jean Luc; Bertagnoli, Stéphane

    2011-04-01

    Myxomatosis in Europe is the result of the release of a South America strain of myxoma virus in 1952. Several attenuated strains with origins in South America or California have since been used as vaccines in the rabbit industry. We sequenced the genome of the SG33 myxoma virus vaccine strain and compared it with those of other myxoma virus strains. We show that SG33 genome carries a large deletion in its right end. Furthermore, our data strongly suggest that the virus isolate from which SG33 is derived results from an in vivo recombination between a wild-type South America (Lausanne) strain and a California MSD-derived strain. These findings raise questions about the use of insufficiently attenuated virus in vaccination. PMID:21470452

  18. The Phenotypic Effects of Royal Jelly on Wild-Type D. melanogaster Are Strain-Specific

    PubMed Central

    Morgan, Stefanie L.; Seggio, Joseph A.; Hicks, Jasmin A.; Sharp, Katherine A.; Axelrod, Jeffrey D.; Wang, Kevin C.

    2016-01-01

    The role for royal jelly (RJ) in promoting caste differentiation of honeybee larvae into queens rather than workers is well characterized. A recent study demonstrated that this poorly understood complex nutrition drives strikingly similar phenotypic effects in Drosophila melanogaster, such as increased body size and reduced developmental time, making possible the use of D. melanogaster as a model system for the genetic analysis of the cellular mechanisms underlying RJ and caste differentiation. We demonstrate here that RJ increases the body size of some wild-type strains of D. melanogaster but not others, and report significant delays in developmental time in all flies reared on RJ. These findings suggest that cryptic genetic variation may be a factor in the D. melanogaster response to RJ, and should be considered when attempting to elucidate response mechanisms to environmental changes in non-honeybee species. PMID:27486863

  19. Cholesterol Secosterol Aldehydes Induce Amyloidogenesis and Dysfunction of Wild Type Tumor Protein p53

    PubMed Central

    Nieva, Jorge; Song, Byeong-Doo; Rogel, Joseph K.; Kujawara, David; Altobel, Lawrence; Izharrudin, Alicia; Boldt, Grant E.; Grover, Rajesh K.; Wentworth, Anita D.; Wentworth, Paul

    2011-01-01

    SUMMARY Epidemiologic and clinical evidence points to an increased risk of cancer when coupled with chronic inflammation. However, the molecular mechanisms that underpin this interrelationship remain largely unresolved. Herein we show that the inflammation-derived cholesterol 5,6-secosterol aldehydes, atheronal-A (KA) and –B (ALD), but not the PUFA-derived aldehydes 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE), induce misfolding of wild-type p53 into an amyloidogenic form that binds thioflavin T and Congo Red dyes but cannot bind to a consensus DNA sequence. Treatment of lung carcinoma cells with KA and ALD leads to a loss of function of extracted p53, as determined by analysis of extracted nuclear protein and in activation of p21. Our results uncover a plausible chemical link between inflammation and cancer and expands the already pivotal role of p53 dysfunction and cancer risk. PMID:21802012

  20. The Phenotypic Effects of Royal Jelly on Wild-Type D. melanogaster Are Strain-Specific.

    PubMed

    Morgan, Stefanie L; Seggio, Joseph A; Nascimento, Nara F; Huh, Dana D; Hicks, Jasmin A; Sharp, Katherine A; Axelrod, Jeffrey D; Wang, Kevin C

    2016-01-01

    The role for royal jelly (RJ) in promoting caste differentiation of honeybee larvae into queens rather than workers is well characterized. A recent study demonstrated that this poorly understood complex nutrition drives strikingly similar phenotypic effects in Drosophila melanogaster, such as increased body size and reduced developmental time, making possible the use of D. melanogaster as a model system for the genetic analysis of the cellular mechanisms underlying RJ and caste differentiation. We demonstrate here that RJ increases the body size of some wild-type strains of D. melanogaster but not others, and report significant delays in developmental time in all flies reared on RJ. These findings suggest that cryptic genetic variation may be a factor in the D. melanogaster response to RJ, and should be considered when attempting to elucidate response mechanisms to environmental changes in non-honeybee species. PMID:27486863

  1. Predicting Gene Function from Uncontrolled Expression Variation among Individual Wild-Type Arabidopsis Plants[W

    PubMed Central

    Bhosale, Rahul; Jewell, Jeremy B.; Hollunder, Jens; Koo, Abraham J.K.; Vuylsteke, Marnik; Michoel, Tom; Hilson, Pierre; Goossens, Alain; Howe, Gregg A.; Browse, John; Maere, Steven

    2013-01-01

    Gene expression profiling studies are usually performed on pooled samples grown under tightly controlled experimental conditions to suppress variability among individuals and increase experimental reproducibility. In addition, to mask unwanted residual effects, the samples are often subjected to relatively harsh treatments that are unrealistic in a natural context. Here, we show that expression variations among individual wild-type Arabidopsis thaliana plants grown under the same macroscopic growth conditions contain as much information on the underlying gene network structure as expression profiles of pooled plant samples under controlled experimental perturbations. We advocate the use of subtle uncontrolled variations in gene expression between individuals to uncover functional links between genes and unravel regulatory influences. As a case study, we use this approach to identify ILL6 as a new regulatory component of the jasmonate response pathway. PMID:23943861

  2. Expression of Escherichia coli virulence usher protein attenuates wild-type Salmonella.

    PubMed

    Yang, Xinghong; Suo, Zhiyong; Thornburg, Theresa; Holderness, Kathryn; Cao, Ling; Lim, Timothy; Walters, Nancy; Kellerman, Laura; Loetterle, Linda; Avci, Recep; Pascual, David W

    2012-01-01

    Generation of a live attenuated vaccine for bacterial pathogens often requires prior knowledge of the pathogen's virulence factors. We hypothesized an alternative approach of heterologous gene expression would make a wild-type (wt) pathogen more susceptible to host cell killing, thus, resulting in immunization. As proof of concept, the heterologous expression of enterotoxigenic E. coli (ETEC) colonization factor antigen I (CFA/I) was tested to attenuate Salmonella. The overexpression of CFA/I resulted in significant attenuation of wt Salmonella. In-depth studies revealed the attenuation depended on the co-expression of chaperone (CfaA) and usher (CfaC) proteins. Remarkably, the CfaAC-attenuated Salmonella conferred protection against wt Salmonella challenge. Mechanistic study indicated CfaAC made Salmonella outer membranes permeable, causing Salmonella to be vulnerable to host destruction. Thus, enhancing bacterial permeability via CfaAC represents an alternative method to attenuate pathogens despite the presence of unknown virulence factors. PMID:22286706

  3. Comparative mutational analysis of wild-type and stretched tRNA3(Leu) gene promoters.

    PubMed Central

    Fabrizio, P; Coppo, A; Fruscoloni, P; Benedetti, P; Di Segni, G; Tocchini-Valentini, G P

    1987-01-01

    We demonstrate that, when the yeast tRNA(3Leu) gene is stretched so that the distance between the two portions of the intragenic promoter is increased to 365 base pairs, the A and B blocks remain functional. Mutations in the A block, which show a weak phenotype when inserted in the wild type, exert a dramatic effect when inserted into the stretched gene. Experiments with extensively purified transcription factor tau indicate that the tau B-B block interaction is not influenced by A-B distance; only the ability of tau A to interact with A block sequences is affected, possibly because of the additional free-energy cost of forming a large loop of the intervening DNA. Images PMID:3321052

  4. Proteomic characterization of a wild-type wine strain of Saccharomyces cerevisiae.

    PubMed

    Trabalzini, Lorenza; Paffetti, Alessandro; Ferro, Elisa; Scaloni, Andrea; Talamo, Fabio; Millucci, Lia; Martelli, Paola; Santucci, Annalisa

    2003-12-01

    Saccharomyces cerevisiae is the optimal eukaryotic model system to study mammalian biological responses. At the same time Saccharomyces cerevisiae is also widely utilized as a biotechnological tool in the food industry. Enological Saccharomyces cerevisiae strains have been so far routinely analyzed for their microbiological aspects. Nevertheless, wine yeasts are gaining an increasing interest in the last years since they strongly affect both the vinification process and the organoleptic properties of the final product wine. The protein repertoire is responsible of such features and, consequently, 2D-PAGE can be an useful tool to evaluate and select optimal wine yeast strains. We present here the first proteomic map of a wild-type wine Saccharomyces cerevisiae strain selected for the guided fermentation of very high quality wines. PMID:15141481

  5. Quality assessment of the blue mussel (Mytilus edulis): comparison between commercial and wild types.

    PubMed

    De Witte, B; Devriese, L; Bekaert, K; Hoffman, S; Vandermeersch, G; Cooreman, K; Robbens, J

    2014-08-15

    This study compared species identity, microplastics, chemical and microbial contamination between consumption mussels and wild type mussels, collected at Belgian department stores and Belgian groynes and quaysides, respectively. Species identification based on genetic analysis showed a high number of Mytilus (M.) edulis compared to M. galloprovincialis and M. edulis/galloprovincialis hybrid mussels. The number of total microplastics varied from 2.6 to 5.1 fibres/10 g of mussel. A higher prevalence of orange fibres at quaysides is related to fisheries activities. Chemical contamination of polycyclic aromatic hydrocarbons and polychlorobiphenyls could be related to industrial activities and water turbidity, with maximum concentrations at the quayside of port Zeebrugge. The inverse was noted for Escherichia coli contamination, which was relatively low at Zeebrugge quayside with a total count of 3.9 × 10(2)CFU/100 g tissue, due to limited agricultural effluents. Results of this complementary analysis stress the importance of integrated monitoring and quality assessment. PMID:24969855

  6. Comparative genomics of wild type yeast strains unveils important genome diversity

    PubMed Central

    Carreto, Laura; Eiriz, Maria F; Gomes, Ana C; Pereira, Patrícia M; Schuller, Dorit; Santos, Manuel AS

    2008-01-01

    Background Genome variability generates phenotypic heterogeneity and is of relevance for adaptation to environmental change, but the extent of such variability in natural populations is still poorly understood. For example, selected Saccharomyces cerevisiae strains are variable at the ploidy level, have gene amplifications, changes in chromosome copy number, and gross chromosomal rearrangements. This suggests that genome plasticity provides important genetic diversity upon which natural selection mechanisms can operate. Results In this study, we have used wild-type S. cerevisiae (yeast) strains to investigate genome variation in natural and artificial environments. We have used comparative genome hybridization on array (aCGH) to characterize the genome variability of 16 yeast strains, of laboratory and commercial origin, isolated from vineyards and wine cellars, and from opportunistic human infections. Interestingly, sub-telomeric instability was associated with the clinical phenotype, while Ty element insertion regions determined genomic differences of natural wine fermentation strains. Copy number depletion of ASP3 and YRF1 genes was found in all wild-type strains. Other gene families involved in transmembrane transport, sugar and alcohol metabolism or drug resistance had copy number changes, which also distinguished wine from clinical isolates. Conclusion We have isolated and genotyped more than 1000 yeast strains from natural environments and carried out an aCGH analysis of 16 strains representative of distinct genotype clusters. Important genomic variability was identified between these strains, in particular in sub-telomeric regions and in Ty-element insertion sites, suggesting that this type of genome variability is the main source of genetic diversity in natural populations of yeast. The data highlights the usefulness of yeast as a model system to unravel intraspecific natural genome diversity and to elucidate how natural selection shapes the yeast genome

  7. Terpenoid Metabolism in Wild-Type and Transgenic Arabidopsis PlantsW⃞

    PubMed Central

    Aharoni, Asaph; Giri, Ashok P.; Deuerlein, Stephan; Griepink, Frans; de Kogel, Willem-Jan; Verstappen, Francel W. A.; Verhoeven, Harrie A.; Jongsma, Maarten A.; Schwab, Wilfried; Bouwmeester, Harro J.

    2003-01-01

    Volatile components, such as terpenoids, are emitted from aerial parts of plants and play a major role in the interaction between plants and their environment. Analysis of the composition and emission pattern of volatiles in the model plant Arabidopsis showed that a range of volatile components are released, primarily from flowers. Most of the volatiles detected were monoterpenes and sesquiterpenes, which in contrast to other volatiles showed a diurnal emission pattern. The active terpenoid metabolism in wild-type Arabidopsis provoked us to conduct an additional set of experiments in which transgenic Arabidopsis overexpressing two different terpene synthases were generated. Leaves of transgenic plants constitutively expressing a dual linalool/nerolidol synthase in the plastids (FaNES1) produced linalool and its glycosylated and hydroxylated derivatives. The sum of glycosylated components was in some of the transgenic lines up to 40- to 60-fold higher than the sum of the corresponding free alcohols. Surprisingly, we also detected the production and emission of nerolidol, albeit at a low level, suggesting that a small pool of its precursor farnesyl diphosphate is present in the plastids. Transgenic lines with strong transgene expression showed growth retardation, possibly as a result of the depletion of isoprenoid precursors in the plastids. In dual-choice assays with Myzus persicae, the FaNES1-expressing lines significantly repelled the aphids. Overexpression of a typical cytosolic sesquiterpene synthase resulted in the production of only trace amounts of the expected sesquiterpene, suggesting tight control of the cytosolic pool of farnesyl diphosphate, the precursor for sesquiterpenoid biosynthesis. This study further demonstrates the value of Arabidopsis for studies of the biosynthesis and ecological role of terpenoids and provides new insights into their metabolism in wild-type and transgenic plants. PMID:14630967

  8. Characterization of yakju brewed from glutinous rice and wild-type yeast strains isolated from nuruks.

    PubMed

    Kim, Hye Ryun; Kim, Jae-Ho; Bae, Dong-Hoon; Ahn, Byung-Hak

    2010-12-01

    Korean traditional rice wines yakju and takju are generally brewed with nuruk as the source of the saccharogenic enzymes by natural fermentation. To improve the quality of Korean rice wine, the microorganisms in the nuruk need to be studied. The objective of this research was to improve the quality of Korean wine with the wild-type yeast strains isolated from the fermentation starter, nuruk. Only strain YA-6 showed high activity in 20% ethanol. Precipitation of Y89-5-3 was similar to that of very flocculent yeast (〉80%) at 75.95%. Using 18S rRNA sequencing, all 10 strains were identified as Saccharomyces cerevisiae. Volatile compounds present in yakju were analyzed by gas chromatography-mass selective detector. The principal component analysis (PCA) of the volatile compounds grouped long-chain esters on the right side of the first principal component, PC1; these compounds were found in yakju that was made with strains YA-6, Y89-5-3, Y89-5- 2, Y90-9, and Y89-1-1. On the other side of PC1 were short-chain esters; these compounds were found in wines that were brewed with strains Y183-2, Y268-3, Y54-3, Y98-4, and Y88-4. Overall, the results indicated that using different wild-type yeast strains in the fermentation process significantly affects the chemical characteristics of the glutinous rice wine. PMID:21193827

  9. Real-time quantification of wild-type contaminants in glyphosate tolerant soybean

    PubMed Central

    Battistini, Elena; Noli, Enrico

    2009-01-01

    Background Trait purity is a key factor for the successful utilization of biotech varieties and is currently assessed by analysis of individual seeds or plants. Here we propose a novel PCR-based approach to test trait purity that can be applied to bulk samples. To this aim the insertion site of a transgene is characterized and the corresponding sequence of the wild-type (wt) allele is used as diagnostic target for amplification. As a demonstration, we developed a real-time quantitative PCR method to test purity of glyphosate tolerant (Roundup Ready®, RR) soybean. Results The soybean wt sequence at the RR locus was characterized and found to be highly conserved among conventional genotypes, thus allowing the detection of possibly any soybean non-trait contaminant. On the other hand, no amplification product was obtained from RR soybean varieties, indicating that the wt sequence is single copy and represents a suitable marker of conventional soybean presence. In addition, results obtained from the analysis of wt-spiked RR samples demonstrate that it is possible to use the real-time PCR assay to quantify the non-trait contamination with an acceptable degree of accuracy. Conclusion In principle this approach could be successfully applied to any transgenic event, provided that the wild-type sequence is conserved and single copy. The main advantages of the assay here described derive from its applicability to bulk samples, which would allow to increase the number of single seeds or plants forming the analytical sample, thus improving accuracy and throughput while containing costs. For these reasons this application of quantitative PCR could represent a useful tool in agricultural biotechnology. PMID:19267904

  10. Phenylbutyrate Sensitizes Human Glioblastoma Cells Lacking Wild-Type P53 Function to Ionizing Radiation

    SciTech Connect

    Lopez, Carlos A. Feng, Felix Y.; Herman, Joseph M.; Nyati, Mukesh K.; Lawrence, Theodore S.; Ljungman, Mats

    2007-09-01

    Purpose: Histone deacetylase (HDAC) inhibitors induce growth arrest, differentiation, and apoptosis in cancer cells. Phenylbutyrate (PB) is a HDAC inhibitor used clinically for treatment of urea cycle disorders. Because of its low cytotoxicity, cerebrospinal fluid penetration, and high oral bioavailability, we investigated PB as a potential radiation sensitizer in human glioblastoma cell lines. Methods and Materials: Four glioblastoma cell lines were selected for this study. Phenylbutyrate was used at a concentration of 2 mM, which is achievable in humans. Western blots were used to assess levels of acetylated histone H3 in tumor cells after treatment with PB. Flow cytometry was used for cell cycle analysis. Clonogenic assays were performed to assess the effect of PB on radiation sensitivity. We used shRNA against p53 to study the role of p53 in radiosensitization. Results: Treatment with PB alone resulted in hyperacetylation of histones, confirmed by Western blot analysis. The PB alone resulted in cytostatic effects in three cell lines. There was no evidence of G{sub 1} arrest, increase in sub-G{sub 1} fraction or p21 protein induction. Clonogenic assays showed radiosensitization in two lines harboring p53 mutations, with enhancement ratios ({+-} SE) of 1.5 ({+-} 0.2) and 1.3 ({+-} 0.1), respectively. There was no radiopotentiating effect in two cell lines with wild-type p53, but knockdown of wild-type p53 resulted in radiosensitization by PB. Conclusions: Phenylbutyrate can produce p21-independent cytostasis, and enhances radiation sensitivity in p53 mutant human glioblastoma cells in vitro. This suggests the potential application of combined PB and radiotherapy in glioblastoma harboring mutant p53.

  11. LOC283731 promoter hypermethylation prognosticates survival after radiochemotherapy in IDH1 wild-type glioblastoma patients.

    PubMed

    Mock, Andreas; Geisenberger, Christoph; Orlik, Christian; Warta, Rolf; Schwager, Christian; Jungk, Christine; Dutruel, Céline; Geiselhart, Lea; Weichenhan, Dieter; Zucknick, Manuela; Nied, Ann-Katrin; Friauf, Sara; Exner, Janina; Capper, David; Hartmann, Christian; Lahrmann, Bernd; Grabe, Niels; Debus, Jürgen; von Deimling, Andreas; Popanda, Odilia; Plass, Christoph; Unterberg, Andreas; Abdollahi, Amir; Schmezer, Peter; Herold-Mende, Christel

    2016-07-15

    MGMT promoter methylation status is currently the only established molecular prognosticator in IDH wild-type glioblastoma multiforme (GBM). Therefore, we aimed to discover novel therapy-associated epigenetic biomarkers. After enrichment for hypermethylated fractions using methyl-CpG-immunoprecipitation (MCIp), we performed global DNA methylation profiling for 14 long-term (LTS; >36 months) and 15 short-term (STS; 6-10 months) surviving GBM patients. Even after exclusion of the G-CIMP phenotype, we observed marked differences between the LTS and STS methylome. A total of 1,247 probes in 706 genes were hypermethylated in LTS and 463 probes in 305 genes were found to be hypermethylated in STS patients (p values < 0.05, log2 fold change ± 0.5). We identified 13 differentially methylated regions (DMRs) with a minimum of four differentially methylated probes per gene. Indeed, we were able to validate a subset of these DMRs through a second, independent method (MassARRAY) in our LTS/STS training set (ADCY1, GPC3, LOC283731/ISLR2). These DMRs were further assessed for their prognostic capability in an independent validation cohort (n = 62) of non-G-CIMP GBMs from the TCGA. Hypermethylation of multiple CpGs mapping to the promoter region of LOC283731 correlated with improved patient outcome (p = 0.03). The prognostic performance of LOC283731 promoter hypermethylation was confirmed in a third independent study cohort (n = 89), and was independent of gender, performance (KPS) and MGMT status (p = 0.0485, HR = 0.63). Intriguingly, the prediction was most pronounced in younger GBM patients (<60 years). In conclusion, we provide compelling evidence that promoter methylation status of this novel gene is a prognostic biomarker in IDH1 wild-type/non-G-CIMP GBMs. PMID:26934681

  12. Molecular Dynamics Approach in the Comparison of Wild-Type and Mutant Paraoxonase-1 Apoenzyme Form

    PubMed Central

    Amine, Khadija; Miri, Lamia; Naimi, Adil; Saile, Rachid; El Kharrim, Abderrahmane; Mikou, Afaf; Kettani, Anass

    2015-01-01

    There is some evidence linking the mammalian paraoxonase-1 (PON1) loops (L1 and L2) to an increased flexibility and reactivity of its active site with potential substrates. The aim of this work is to study the structural, dynamical, and functional effects of the most flexible regions close to the active site and to determine the impact of mutations on the protein. For both models, wild-type (PON1wild) and PON1 mutant (PON1mut) models, the L1 loop and Q/R and L/M mutations were constructed using MODELLER software. Molecular dynamics simulations of 20 ns at 300 K on fully modeled PON1wild and PON1mut apoenzyme have been done. Detailed analyses of the root-mean-square deviation and fluctuations, H-bonding pattern, and torsion angles have been performed. The PON1wild results were then compared with those obtained for the PON1mut. Our results show that the active site in the wild-type structure is characterized by two distinct movements of opened and closed conformations of the L1 and L2 loops. The alternating and repetitive movement of loops at specific times is consistent with the presence of 11 defined hydrogen bonds. In the PON1mut, these open-closed movements are therefore totally influenced and repressed by the Q/R and L/M mutations. In fact, these mutations seem to impact the PON1mut active site by directly reducing the catalytic core flexibility, while maintaining a significant mobility of the switch regions delineated by the loops surrounding the active site. The impact of the studied mutations on structure and dynamics proprieties of the protein may subsequently contribute to the loss of both flexibility and activity of the PON1 enzyme. PMID:26417201

  13. Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli.

    PubMed

    Ho, Joanne M; Reynolds, Noah M; Rivera, Keith; Connolly, Morgan; Guo, Li-Tao; Ling, Jiqiang; Pappin, Darryl J; Church, George M; Söll, Dieter

    2016-02-19

    Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli. PMID:26544153

  14. Molecular Dynamics Approach in the Comparison of Wild-Type and Mutant Paraoxonase-1 Apoenzyme Form.

    PubMed

    Amine, Khadija; Miri, Lamia; Naimi, Adil; Saile, Rachid; El Kharrim, Abderrahmane; Mikou, Afaf; Kettani, Anass

    2015-01-01

    There is some evidence linking the mammalian paraoxonase-1 (PON1) loops (L1 and L2) to an increased flexibility and reactivity of its active site with potential substrates. The aim of this work is to study the structural, dynamical, and functional effects of the most flexible regions close to the active site and to determine the impact of mutations on the protein. For both models, wild-type (PON1wild) and PON1 mutant (PON1mut) models, the L1 loop and Q/R and L/M mutations were constructed using MODELLER software. Molecular dynamics simulations of 20 ns at 300 K on fully modeled PON1wild and PON1mut apoenzyme have been done. Detailed analyses of the root-mean-square deviation and fluctuations, H-bonding pattern, and torsion angles have been performed. The PON1wild results were then compared with those obtained for the PON1mut. Our results show that the active site in the wild-type structure is characterized by two distinct movements of opened and closed conformations of the L1 and L2 loops. The alternating and repetitive movement of loops at specific times is consistent with the presence of 11 defined hydrogen bonds. In the PON1mut, these open-closed movements are therefore totally influenced and repressed by the Q/R and L/M mutations. In fact, these mutations seem to impact the PON1mut active site by directly reducing the catalytic core flexibility, while maintaining a significant mobility of the switch regions delineated by the loops surrounding the active site. The impact of the studied mutations on structure and dynamics proprieties of the protein may subsequently contribute to the loss of both flexibility and activity of the PON1 enzyme. PMID:26417201

  15. A mouse model of adult-onset anaemia due to erythropoietin deficiency.

    PubMed

    Yamazaki, Shun; Souma, Tomokazu; Hirano, Ikuo; Pan, Xiaoqing; Minegishi, Naoko; Suzuki, Norio; Yamamoto, Masayuki

    2013-01-01

    Erythropoietin regulates erythropoiesis in a hypoxia-inducible manner. Here we generate inherited super-anaemic mice (ISAM) as a mouse model of adult-onset anaemia caused by erythropoietin deficiency. ISAM express erythropoietin in the liver but lack erythropoietin production in the kidney. Around weaning age, when the major erythropoietin-producing organ switches from the liver to the kidney, ISAM develop anaemia due to erythropoietin deficiency, which is curable by administration of recombinant erythropoietin. In ISAM severe chronic anaemia enhances transgenic green fluorescent protein and Cre expression driven by the complete erythropoietin-gene regulatory regions, which facilitates efficient labelling of renal erythropoietin-producing cells. We show that the majority of cortical and outer medullary fibroblasts have the innate potential to produce erythropoietin, and also reveal a new set of erythropoietin target genes. ISAM are a useful tool for the evaluation of erythropoiesis-stimulating agents and to trace the dynamics of erythropoietin-producing cells. PMID:23727690

  16. Brain-derived neurotrophic factor prevents dendritic retraction of adult mouse retinal ganglion cells.

    PubMed

    Binley, Kate E; Ng, Wai S; Barde, Yves-Alain; Song, Bing; Morgan, James E

    2016-08-01

    We used cultured adult mouse retinae as a model system to follow and quantify the retraction of dendrites using diolistic labelling of retinal ganglion cells (RGCs) following explantation. Cell death was monitored in parallel by nuclear staining as 'labelling' with RGC and apoptotic markers was inconsistent and exceedingly difficult to quantify reliably. Nuclear staining allowed us to delineate a lengthy time window during which dendrite retraction can be monitored in the absence of RGC death. The addition of brain-derived neurotrophic factor (BDNF) produced a marked reduction in dendritic degeneration, even when application was delayed for 3 days after retinal explantation. These results suggest that the delayed addition of trophic factors may be functionally beneficial before the loss of cell bodies in the course of conditions such as glaucoma. PMID:27285957

  17. Telomerase expression confers cardioprotection in the adult mouse heart after acute myocardial infarction

    PubMed Central

    Serrano, Rosa; Tejera, Agueda; Ayuso, Eduard; Jimenez, Veronica; Formentini, Ivan; Bobadilla, Maria; Mizrahi, Jacques; de Martino, Alba; Gomez, Gonzalo; Pisano, David; Mulero, Francisca; Wollert, Kai C.; Bosch, Fatima; Blasco, Maria A.

    2016-01-01

    Coronary heart disease is one of the main causes of death in the developed world, and treatment success remains modest, with high mortality rates within 1 year after myocardial infarction (MI). Thus, new therapeutic targets and effective treatments are necessary. Short telomeres are risk factors for age-associated diseases, including heart disease. Here we address the potential of telomerase (Tert) activation in prevention of heart failure after MI in adult mice. We use adeno-associated viruses for cardiac-specific Tert expression. We find that upon MI, hearts expressing Tert show attenuated cardiac dilation, improved ventricular function and smaller infarct scars concomitant with increased mouse survival by 17% compared with controls. Furthermore, Tert treatment results in elongated telomeres, increased numbers of Ki67 and pH3-positive cardiomyocytes and a gene expression switch towards a regeneration signature of neonatal mice. Our work suggests telomerase activation could be a therapeutic strategy to prevent heart failure after MI. PMID:25519492

  18. Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

    PubMed Central

    Chavez, Miquella G.; Hu, Jimmy; Seidel, Kerstin; Li, Chunying; Jheon, Andrew; Naveau, Adrien; Horst, Orapin; Klein, Ophir D.

    2014-01-01

    Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal's life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor. PMID:24834972

  19. Hormonal regulation of epidermal growth factor and protease in the submandibular gland of the adult mouse.

    PubMed

    Gresik, E W; Schenkein, I; van der Noen, H; Barka, T

    1981-09-01

    The structure of the granular convoluted tubules of the mouse submandibular gland is influenced by androgens, adrenal steroids, and thyroid hormones. We wished to investigate the effects of variations in hormonal status on the quantitative and qualitative distribution of two secretory products of these tubules, epidermal growth factor (EGF) and protease. The effects of the thyroid and adrenal glands on EGF content and protease activity of the submandibular glands of adult female mice were studied by RIAs (EGF), enzyme assays (protease), and immunocytochemical methods. In animals rendered chronically hypothyroid by propylthiouracil (4 months) or in animals which were adrenalectomized and ovariectomized (3 weeks), protease activity and EGF levels were reduced by 81-97%. The administration of testosterone induced these polypeptides even in hypothyroid animals. Daily administration of L-T4 (T4; 1 micrograms/g BW) for 7 days increased EGF and protease activity 3.6-fold in intact mice and reversed the effect of hypothyroidism. EGF and protease were also induced by T4 in adrenalectomized and ovariectomized mice, although to a lesser degree than in intact animals. Immunocytochemical stainings of submandibular glands indicated that the number of granular convoluted tubule cells immunoreactive for EGF correlated with the levels of EGF determined by RIAs. With respect to immunostaining for protease, such a correlation was not observed. The data indicate multihormonal regulation of EGF and protease in the mouse submandibular gland. PMID:7021131

  20. Meis1 Is Required for Adult Mouse Erythropoiesis, Megakaryopoiesis and Hematopoietic Stem Cell Expansion.

    PubMed

    Miller, Michelle Erin; Rosten, Patty; Lemieux, Madeleine E; Lai, Courteney; Humphries, R Keith

    2016-01-01

    Meis1 is recognized as an important transcriptional regulator in hematopoietic development and is strongly implicated in the pathogenesis of leukemia, both as a Hox transcription factor co-factor and independently. Despite the emerging recognition of Meis1's importance in the context of both normal and leukemic hematopoiesis, there is not yet a full understanding of Meis1's functions and the relevant pathways and genes mediating its functions. Recently, several conditional mouse models for Meis1 have been established. These models highlight a critical role for Meis1 in adult mouse hematopoietic stem cells (HSCs) and implicate reactive oxygen species (ROS) as a mediator of Meis1 function in this compartment. There are, however, several reported differences between these studies in terms of downstream progenitor populations impacted and effectors of function. In this study, we describe further characterization of a conditional knockout model based on mice carrying a loxP-flanked exon 8 of Meis1 which we crossed onto the inducible Cre localization/expression strains, B6;129-Gt(ROSA)26Sor(tm1(Cre/ERT)Nat)/J or B6.Cg-Tg(Mx1-Cre)1Cgn/J. Findings obtained from these two inducible Meis1 knockout models confirm and extend previous reports of the essential role of Meis1 in adult HSC maintenance and expansion and provide new evidence that highlights key roles of Meis1 in both megakaryopoiesis and erythropoiesis. Gene expression analyses point to a number of candidate genes involved in Meis1's role in hematopoiesis. Our data additionally support recent evidence of a role of Meis1 in ROS regulation. PMID:26986211

  1. Meis1 Is Required for Adult Mouse Erythropoiesis, Megakaryopoiesis and Hematopoietic Stem Cell Expansion

    PubMed Central

    Miller, Michelle Erin; Rosten, Patty; Lemieux, Madeleine E.; Lai, Courteney; Humphries, R. Keith

    2016-01-01

    Meis1 is recognized as an important transcriptional regulator in hematopoietic development and is strongly implicated in the pathogenesis of leukemia, both as a Hox transcription factor co-factor and independently. Despite the emerging recognition of Meis1’s importance in the context of both normal and leukemic hematopoiesis, there is not yet a full understanding of Meis1’s functions and the relevant pathways and genes mediating its functions. Recently, several conditional mouse models for Meis1 have been established. These models highlight a critical role for Meis1 in adult mouse hematopoietic stem cells (HSCs) and implicate reactive oxygen species (ROS) as a mediator of Meis1 function in this compartment. There are, however, several reported differences between these studies in terms of downstream progenitor populations impacted and effectors of function. In this study, we describe further characterization of a conditional knockout model based on mice carrying a loxP-flanked exon 8 of Meis1 which we crossed onto the inducible Cre localization/expression strains, B6;129-Gt(ROSA)26Sortm1(Cre/ERT)Nat/J or B6.Cg-Tg(Mx1-Cre)1Cgn/J. Findings obtained from these two inducible Meis1 knockout models confirm and extend previous reports of the essential role of Meis1 in adult HSC maintenance and expansion and provide new evidence that highlights key roles of Meis1 in both megakaryopoiesis and erythropoiesis. Gene expression analyses point to a number of candidate genes involved in Meis1’s role in hematopoiesis. Our data additionally support recent evidence of a role of Meis1 in ROS regulation. PMID:26986211

  2. Generation of a novel mouse model that recapitulates early and adult onset glycogenosis type IV.

    PubMed

    Akman, H Orhan; Sheiko, Tatiana; Tay, Stacey K H; Finegold, Milton J; Dimauro, Salvatore; Craigen, William J

    2011-11-15

    Glycogen storage disease type IV (GSD IV) is a rare autosomal recessive disorder caused by deficiency of the glycogen branching enzyme (GBE). The diagnostic feature of the disease is the accumulation of a poorly branched form of glycogen known as polyglucosan (PG). The disease is clinically heterogeneous, with variable tissue involvement and age of disease onset. Absence of enzyme activity is lethal in utero or in infancy affecting primarily muscle and liver. However, residual enzyme activity (5-20%) leads to juvenile or adult onset of a disorder that primarily affects muscle as well as central and peripheral nervous system. Here, we describe two mouse models of GSD IV that reflect this spectrum of disease. Homologous recombination was used to insert flippase recognition target recombination sites around exon 7 of the Gbe1 gene and a phosphoglycerate kinase-Neomycin cassette within intron 7, leading to a reduced synthesis of GBE. Mice bearing this mutation (Gbe1(neo/neo)) exhibit a phenotype similar to juvenile onset GSD IV, with wide spread accumulation of PG. Meanwhile, FLPe-mediated homozygous deletion of exon 7 completely eliminated GBE activity (Gbe1(-/-)), leading to a phenotype of lethal early onset GSD IV, with significant in utero accumulation of PG. Adult mice with residual GBE exhibit progressive neuromuscular dysfunction and die prematurely. Differently from muscle, PG in liver is a degradable source of glucose and readily depleted by fasting, emphasizing that there are structural and regulatory differences in glycogen metabolism among tissues. Both mouse models recapitulate typical histological and physiological features of two human variants of branching enzyme deficiency. PMID:21856731

  3. Poor competitive fitness of transgenically mitigated tobacco in competition with the wild type in a replacement series.

    PubMed

    Al-Ahmad, Hani; Galili, Shmuel; Gressel, Jonathan

    2005-10-01

    Transgenic crops can interbreed with other crop cultivars or with related weeds, increasing the potential of the hybrid progeny for competition. To prevent generating competitive hybrids, we previously tested tobacco (Nicotiana tabacum L.) as a model for validating the transgenic mitigation (TM) concept using tandem constructs where a gene of choice is linked to mitigating genes that are positive or neutral to the crop, but deleterious to a recipient under competition. Here, we examine the efficacy of the TM concept at various ratios of transgenically mitigated tobacco in competition with the wild type tobacco in an ecological replacement series. The dwarf/herbicide-resistant TM transgenic plants cultivated alone under self-competition grew well and formed many more flowers than the tall wild type, which is an indication of greater reproductivity. In contrast to the wild type, TM flowering was almost completely suppressed in mixed cultures at most TM/wild type ratios up to 75% transgenic, as the TM plants were extremely unfit to reproduce. In addition, homozygous TM progeny had an even lower competitive fitness against the wild type than hemizygous/homozygous TM segregants. Thus, the TM technology was effective in reducing the risk of transgene establishment of intraspecific transgenic hybrids at different competitive levels, at the close spacing typical of weed populations. PMID:15931502

  4. Mitochondrial Function in Cell Wall Glycoprotein Synthesis in Saccharomyces cerevisiae NCYC 625 (Wild Type) and [rho0] Mutants

    PubMed Central

    Iung, Annie Rakotoarivony; Coulon, Joël; Kiss, Ferenc; Ekome, Jacques Ngondi; Vallner, Judit; Bonaly, Roger

    1999-01-01

    We studied phosphopeptidomannans (PPMs) of two Saccharomyces cerevisiae NCYC 625 strains (S. diastaticus): a wild type strain grown aerobically, anaerobically, and in the presence of antimycin and a [rho0] mutant grown aerobically and anaerobically. The aerobic wild-type cultures were highly flocculent, but all others were weakly flocculent. Ligands implicated in flocculation of mutants or antimycin-treated cells were not aggregated as much by concanavalin A as were those of the wild type. The [rho0] mutants and antimycin-treated cells differ from the wild type in PPM composition and invertase, acid phosphatase, and glucoamylase activities. PPMs extracted from different cells differ in the protein but not in the glycosidic moiety. The PPMs were less stable in mitochondrion-deficient cells than in wild-type cells grown aerobically, and this difference may be attributable to defective mitochondrial function during cell wall synthesis. The reduced flocculation of cells grown in the presence of antimycin, under anaerobiosis, or carrying a [rho0] mutation may be the consequence of alterations of PPM structures which are the ligands of lectins, both involved in this cell-cell recognition phenomenon. These respiratory chain alterations also affect peripheral, biologically active glycoproteins such as extracellular enzymes and peripheral PPMs. PMID:10583995

  5. Acquired transmissibility of sheep-passaged L-type bovine spongiform encephalopathy prion to wild-type mice.

    PubMed

    Okada, Hiroyuki; Masujin, Kentaro; Miyazawa, Kohtaro; Yokoyama, Takashi

    2015-01-01

    L-type bovine spongiform encephalopathy (L-BSE) is an atypical form of BSE that is transmissible to cattle and several lines of prion protein (PrP) transgenic mice, but not to wild-type mice. In this study, we examined the transmissibility of sheep-passaged L-BSE prions to wild-type mice. Disease-associated prion protein (PrP(Sc)) was detected in the brain and/or lymphoid tissues during the lifespan of mice that were asymptomatic subclinical carriers, indicating that wild-type mice were susceptible to sheep-passaged L-BSE. The morphological characteristics of the PrP(Sc) of sheep-passaged L-BSE included florid plaques that were distributed mainly in the cerebral cortex and hippocampus of subsequent passaged mice. The PrP(Sc) glycoform profiles of wild-type mice infected with sheep-passaged L-BSE were similar to those of the original isolate. The data indicate that sheep-passaged L-BSE has an altered host range and acquired transmissibility to wild-type mice. PMID:26169916

  6. Differential proteomic and behavioral effects of long-term voluntary exercise in wild-type and APP-overexpressing transgenics.

    PubMed

    Rao, Shailaja Kishan; Ross, Jordan M; Harrison, Fiona E; Bernardo, Alexandra; Reiserer, Randall S; Reiserer, Ronald S; Mobley, James A; McDonald, Michael P

    2015-06-01

    Physical exercise may provide protection against the cognitive decline and neuropathology associated with Alzheimer's disease, although the mechanisms are not clear. In the present study, APP/PSEN1 double-transgenic and wild-type mice were allowed unlimited voluntary exercise for 7months. Consistent with previous reports, wheel-running improved cognition in the double-transgenic mice. Interestingly, the average daily distance run was strongly correlated with spatial memory in the water maze in wild-type mice (r(2)=.959), but uncorrelated in transgenics (r(2)=.013). Proteomics analysis showed that sedentary transgenic mice differed significantly from sedentary wild-types with respect to proteins involved in synaptic transmission, cytoskeletal regulation, and neurogenesis. When given an opportunity to exercise, the transgenics' deficiencies in cytoskeletal regulation and neurogenesis largely normalized, but abnormal synaptic proteins did not change. In contrast, exercise enhanced proteins associated with cytoskeletal regulation, oxidative phosphorylation, and synaptic transmission in wild-type mice. Soluble and insoluble Aβ40 and Aβ42 levels were significantly decreased in both cortex and hippocampus of active transgenics, suggesting that this may have played a role in the cognitive improvement in APP/PSEN1 mice. β-secretase was significantly reduced in active APP/PSEN1 mice compared to sedentary controls, suggesting a mechanism for reduced Aβ. Taken together, these data illustrate that exercise improves memory in wild-type and APP-overexpressing mice in fundamentally different ways. PMID:25818006

  7. Viral adaptation to an antiviral protein enhances the fitness level to above that of the uninhibited wild type.

    PubMed

    Cherwa, James E; Sanchez-Soria, Pablo; Wichman, Holly A; Fane, Bentley A

    2009-11-01

    Viruses often evolve resistance to antiviral agents. While resistant strains are able to replicate in the presence of the agent, they generally exhibit lower fitness than the wild-type strain in the absence of the inhibitor. In some cases, resistant strains become dependent on the antiviral agent. However, the agent rarely, if ever, elevates dependent strain fitness above the uninhibited wild-type level. This would require an adaptive mechanism to convert the antiviral agent into a beneficial growth factor. Using an inhibitory scaffolding protein that specifically blocks phiX174 capsid assembly, we demonstrate that such mechanisms are possible. To obtain the quintuple-mutant resistant strain, the wild-type virus was propagated for approximately 150 viral life cycles in the presence of increasing concentrations of the inhibitory protein. The expression of the inhibitory protein elevated the strain's fitness significantly above the uninhibited wild-type level. Thus, selecting for resistance coselected for dependency, which was characterized and found to operate on the level of capsid nucleation. To the best of our knowledge, this is the first report of a virus evolving a mechanism to productively utilize an antiviral agent to stimulate its fitness above the uninhibited wild-type level. The results of this study may be predictive of the types of resistant phenotypes that could be selected by antiviral agents that specifically target capsid assembly. PMID:19726521

  8. Difluoromethyl ketones: Potent inhibitors of wild type and carbamate-insensitive G119S mutant Anopheles gambiae acetylcholinesterase.

    PubMed

    Camerino, Eugene; Wong, Dawn M; Tong, Fan; Körber, Florian; Gross, Aaron D; Islam, Rafique; Viayna, Elisabet; Mutunga, James M; Li, Jianyong; Totrov, Maxim M; Bloomquist, Jeffrey R; Carlier, Paul R

    2015-10-15

    Malaria is a devastating disease in sub-Saharan Africa, and current vector control measures are threatened by emerging resistance mechanisms. With the goal of developing new, selective, resistance-breaking insecticides we explored α-fluorinated methyl ketones as reversible covalent inhibitors of Anopheles gambiae acetylcholinesterase (AgAChE). Trifluoromethyl ketones 5 demonstrated remarkable volatility in microtiter plate assays, but 5c,e-h exhibited potent (1-100 nM) inhibition of wild type (WT) AgAChE and weak inhibition of resistant mutant G119S mutant AgAChE. Fluoromethyl ketones 10c-i exhibited submicromolar to micromolar inhibition of WT AgAChE, but again only weakly inhibited G119S AgAChE. Interestingly, difluoromethyl ketone inhibitors 9c and 9g had single digit nanomolar inhibition of WT AgAChE, and 9g had excellent potency against G119S AgAChE. Approach to steady-state inhibition was quite slow, but after 23 h incubation an IC50 value of 25.1 ± 1.2 nM was measured. We attribute the slow, tight-binding G119S AgAChE inhibition of 9g to a balance of steric size and electrophilicity. However, toxicities of 5g, 9g, and 10g to adult A. gambiae in tarsal contact, fumigation, and injection assays were lower than expected based on WT AgAChE inhibition potency and volatility. Potential toxicity-limiting factors are discussed. PMID:26386602

  9. Gap Junctional Blockade Stochastically Induces Different Species-Specific Head Anatomies in Genetically Wild-Type Girardia dorotocephala Flatworms.

    PubMed

    Emmons-Bell, Maya; Durant, Fallon; Hammelman, Jennifer; Bessonov, Nicholas; Volpert, Vitaly; Morokuma, Junji; Pinet, Kaylinnette; Adams, Dany S; Pietak, Alexis; Lobo, Daniel; Levin, Michael

    2015-01-01

    The shape of an animal body plan is constructed from protein components encoded by the genome. However, bioelectric networks composed of many cell types have their own intrinsic dynamics, and can drive distinct morphological outcomes during embryogenesis and regeneration. Planarian flatworms are a popular system for exploring body plan patterning due to their regenerative capacity, but despite considerable molecular information regarding stem cell differentiation and basic axial patterning, very little is known about how distinct head shapes are produced. Here, we show that after decapitation in G. dorotocephala, a transient perturbation of physiological connectivity among cells (using the gap junction blocker octanol) can result in regenerated heads with quite different shapes, stochastically matching other known species of planaria (S. mediterranea, D. japonica, and P. felina). We use morphometric analysis to quantify the ability of physiological network perturbations to induce different species-specific head shapes from the same genome. Moreover, we present a computational agent-based model of cell and physical dynamics during regeneration that quantitatively reproduces the observed shape changes. Morphological alterations induced in a genomically wild-type G. dorotocephala during regeneration include not only the shape of the head but also the morphology of the brain, the characteristic distribution of adult stem cells (neoblasts), and the bioelectric gradients of resting potential within the anterior tissues. Interestingly, the shape change is not permanent; after regeneration is complete, intact animals remodel back to G. dorotocephala-appropriate head shape within several weeks in a secondary phase of remodeling following initial complete regeneration. We present a conceptual model to guide future work to delineate the molecular mechanisms by which bioelectric networks stochastically select among a small set of discrete head morphologies. Taken together

  10. Gap Junctional Blockade Stochastically Induces Different Species-Specific Head Anatomies in Genetically Wild-Type Girardia dorotocephala Flatworms

    PubMed Central

    Emmons-Bell, Maya; Durant, Fallon; Hammelman, Jennifer; Bessonov, Nicholas; Volpert, Vitaly; Morokuma, Junji; Pinet, Kaylinnette; Adams, Dany S.; Pietak, Alexis; Lobo, Daniel; Levin, Michael

    2015-01-01

    The shape of an animal body plan is constructed from protein components encoded by the genome. However, bioelectric networks composed of many cell types have their own intrinsic dynamics, and can drive distinct morphological outcomes during embryogenesis and regeneration. Planarian flatworms are a popular system for exploring body plan patterning due to their regenerative capacity, but despite considerable molecular information regarding stem cell differentiation and basic axial patterning, very little is known about how distinct head shapes are produced. Here, we show that after decapitation in G. dorotocephala, a transient perturbation of physiological connectivity among cells (using the gap junction blocker octanol) can result in regenerated heads with quite different shapes, stochastically matching other known species of planaria (S. mediterranea, D. japonica, and P. felina). We use morphometric analysis to quantify the ability of physiological network perturbations to induce different species-specific head shapes from the same genome. Moreover, we present a computational agent-based model of cell and physical dynamics during regeneration that quantitatively reproduces the observed shape changes. Morphological alterations induced in a genomically wild-type G. dorotocephala during regeneration include not only the shape of the head but also the morphology of the brain, the characteristic distribution of adult stem cells (neoblasts), and the bioelectric gradients of resting potential within the anterior tissues. Interestingly, the shape change is not permanent; after regeneration is complete, intact animals remodel back to G. dorotocephala-appropriate head shape within several weeks in a secondary phase of remodeling following initial complete regeneration. We present a conceptual model to guide future work to delineate the molecular mechanisms by which bioelectric networks stochastically select among a small set of discrete head morphologies. Taken together

  11. A brain-specific gene cluster isolated from the region of the mouse obesity locus is expressed in the adult hypothalamus and during mouse development

    SciTech Connect

    Laig-Webster, M.; Lim, M.E.; Chehab, F.F.

    1994-09-01

    The molecular defect underlying an autosomal recessive form of genetic obesity in a classical mouse model C57 BL/6J-ob/ob has not yet been elucidated. Whereas metabolic and physiological disturbances such as diabetes and hypertension are associated with obesity, the site of expression and the nature of the primary lesion responsible for this cascade of events remains elusive. Our efforts aimed at the positional cloning of the ob gene by YAC contig mapping and gene identification have resulted in the cloning of a brain-specific gene cluster from the ob critical region. The expression of this gene cluster is remarkably complex owing to the multitude of brain-specific mRNA transcripts detected on Northern blots. cDNA cloning of these transcripts suggests that they are expressed from different genes as well as by alternate splicing mechanisms. Furthermore, the genomic organization of the cluster appears to consist of at least two identical promoters displaying CpG islands characteristic of housekeeping genes, yet clearly involving tissue-specific expression. Sense and anti-sense synthetic RNA probes were derived from a common DNA sequence on 3 cDNA clones and hybridized to 8-16 days mouse embryonic stages and mouse adult brain sections. Expression in development was noticeable as of the 11th day of gestation and confined to the central nervous system mainly in the telencephalon and spinal cord. Coronal and sagittal sections of the adult mouse brain showed expression only in 3 different regions of the brain stem. In situ hybridization to mouse hypothalamus sections revealed the presence of a localized and specialized group of cells expressing high levels of mRNA, suggesting that this gene cluster may also be involved in the regulation of hypothalamic activities. The hypothalamus has long been hypothesized as a primary candidate tissue for the expression of the obesity gene mainly because of its well-established role in the regulation of energy metabolism and food intake.

  12. Establishment of a tamoxifen-inducible Cre-driver mouse strain for widespread and temporal genetic modification in adult mice.

    PubMed

    Ichise, Hirotake; Hori, Akiko; Shiozawa, Seiji; Kondo, Saki; Kanegae, Yumi; Saito, Izumu; Ichise, Taeko; Yoshida, Nobuaki

    2016-07-29

    Temporal genetic modification of mice using the ligand-inducible Cre/loxP system is an important technique that allows the bypass of embryonic lethal phenotypes and access to adult phenotypes. In this study, we generated a tamoxifen-inducible Cre-driver mouse strain for the purpose of widespread and temporal Cre recombination. The new line, named CM32, expresses the GFPneo-fusion gene in a wide variety of tissues before FLP recombination and tamoxifen-inducible Cre after FLP recombination. Using FLP-recombined CM32 mice (CM32Δ mice) and Cre reporter mouse lines, we evaluated the efficiency of Cre recombination with and without tamoxifen administration to adult mice, and found tamoxifen-dependent induction of Cre recombination in a variety of adult tissues. In addition, we demonstrated that conditional activation of an oncogene could be achieved in adults using CM32Δ mice. CM32Δ;T26 mice, which harbored a Cre recombination-driven, SV40 large T antigen-expressing transgene, were viable and fertile. No overt phenotype was found in the mice up to 3 months after birth. Although they displayed pineoblastomas (pinealoblastomas) and/or thymic enlargement due to background Cre recombination by 6 months after birth, they developed epidermal hyperplasia when administered tamoxifen. Collectively, our results suggest that the CM32Δ transgenic mouse line can be applied to the assessment of adult phenotypes in mice with loxP-flanked transgenes. PMID:26923756

  13. Notch2 is required for maintaining sustentacular cell function in the adult mouse main olfactory epithelium

    PubMed Central

    Rodriguez, Steve; Sickles, Heather M.; DeLeonardis, Chris; Alcaraz, Ana; Gridley, Thomas; Lin, David M.

    2008-01-01

    Notch receptors are expressed in neurons and glia in the adult nervous system, but why this expression persists is not well-understood. Here we examine the role of the Notch pathway in the postnatal mouse main olfactory system, and show evidence consistent with a model where Notch2 is required for maintaining sustentacular cell function. In the absence of Notch2, the laminar nature of these glial-like cells is disrupted. Hes1, Hey1, and Six1, which are downstream effectors of the Notch pathway, are down-regulated, and cytochrome P450 and Glutathione S-transferase (GST) expression by sustentacular cells is reduced. Functional levels of GST activity are also reduced. These disruptions are associated with increased olfactory sensory neuron degeneration. Surprisingly, expression of Notch3 is also down-regulated. This suggests the existence of a feedback loop where expression of Notch3 is initially independent of Notch2, but requires Notch2 for maintained expression. While the Notch pathway has previously been shown to be important for promoting gliogenesis during development, this is the first demonstration that the persistent expression of Notch receptors is required for maintaining glial function in adult. PMID:18155189

  14. Inhibition of Notch activity promotes nonmitotic regeneration of hair cells in the adult mouse utricles.

    PubMed

    Lin, Vincent; Golub, Justin S; Nguyen, Tot Bui; Hume, Clifford R; Oesterle, Elizabeth C; Stone, Jennifer S

    2011-10-26

    The capacity of adult mammals to regenerate sensory hair cells is not well defined. To explore early steps in this process, we examined reactivation of a transiently expressed developmental gene, Atoh1, in adult mouse utricles after neomycin-induced hair cell death in culture. Using an adenoviral reporter for Atoh1 enhancer, we found that Atoh1 transcription is activated in some hair cell progenitors (supporting cells) 3 d after neomycin treatment. By 18 d after neomycin, the number of cells with Atoh1 transcriptional activity increased significantly, but few cells acquired hair cell features (i.e., accumulated ATOH1 or myosin VIIa protein or developed stereocilia). Treatment with DAPT, an inhibitor of γ-secretase, reduced notch pathway activity, enhanced Atoh1 transcriptional activity, and dramatically increased the number of Atoh1-expressing cells with hair cell features, but only in the striolar/juxtastriolar region. Similar effects were seen with TAPI-1, an inhibitor of another enzyme required for notch activity (TACE). Division of supporting cells was rare in any control or DAPT-treated utricles. This study shows that mature mammals have a natural capacity to initiate vestibular hair cell regeneration and suggests that regional notch activity is a significant inhibitor of direct transdifferentiation of supporting cells into hair cells following damage. PMID:22031879

  15. Data for proteomic profiling of Anthers from a photosensitive male sterile mutant and wild-type cotton (Gossypium hirsutum L.).

    PubMed

    Liu, Ji; Pang, Chaoyou; Wei, Hengling; Song, Meizhen; Meng, Yanyan; Ma, Jianhui; Fan, Shuli; Yu, Shuxun

    2015-09-01

    Cotton is an important economic crop, used mainly for the production of textile fiber. Using a space mutation breeding technique, a novel photosensitive genetic male sterile mutant CCRI9106 was isolated from the wild-type upland cotton cultivar CCRI040029. To study the male sterile mechanisms of CCRI9106, histological and iTRAQ-facilitated proteomic analyses of anthers were performed. This data article contains data related to the research article titled iTRAQ-Facilitated Proteomic Profiling of Anthers From a Photosensitive Male Sterile Mutant and Wild-type Cotton (Gossypium hirsutum L.)[1]. This research article describes the iTRAQ-facilitated proteomic analysis of the wild-type and a photosensitive male sterile mutant in cotton. The report indicated that exine formation defect is the key reason for male sterility in mutant plant. The information presented here represents the tables and figures that detail the processing of the raw data obtained from iTRAQ analysis. PMID:26958592

  16. Detection and differentiation of wild-type and a vaccine strain of Streptococcus equi ssp. equi using pyrosequencing.

    PubMed

    Livengood, Julia L; Lanka, Saraswathi; Maddox, Carol; Tewari, Deepanker

    2016-07-25

    Streptococcus equi subspecies equi (S. equi), the causative agent of strangles, is an important equine pathogen. Strangles is a highly contagious disease and a commercial modified live vaccine (MLV) is used for protection, which although effective, may also result in clinical signs of the disease. A rapid means to differentiate between the MLV and wild-type infection is crucial for quarantine release and limiting the disease spread. This study describes the use of a pyrosequencing assay targeting a single nucleotide deletion upstream of the SzPSe gene to distinguish between the wild-type and vaccine strains. A set of 96 characterized clinical specimens and isolates were tested using the assay. The assay was successful in differentiating between wild-type S. equi and the vaccine strains and in discriminating S. equi from other Streptococci. The vaccine strain was identified in 61.7% (29/47) of the strangles cases in horses with a history of MLV vaccination. PMID:27317457

  17. Stroke Increases Neural Stem Cells and Angiogenesis in the Neurogenic Niche of the Adult Mouse

    PubMed Central

    Zhang, Rui Lan; Chopp, Michael; Roberts, Cynthia; Liu, Xianshuang; Wei, Min; Nejad-Davarani, Siamak P.; Wang, Xinli; Zhang, Zheng Gang

    2014-01-01

    The unique cellular and vascular architecture of the adult ventricular-subventricular zone (V/SVZ) neurogenic niche plays an important role in regulating neural stem cell function. However, the in vivo identification of neural stem cells and their relationship to blood vessels within this niche in response to stroke remain largely unknown. Using whole-mount preparation of the lateral ventricle wall, we examined the architecture of neural stem cells and blood vessels in the V/SVZ of adult mouse over the course of 3 months after onset of focal cerebral ischemia. Stroke substantially increased the number of glial fibrillary acidic protein (GFAP) positive neural stem cells that are in contact with the cerebrospinal fluid (CSF) via their apical processes at the center of pinwheel structures formed by ependymal cells residing in the lateral ventricle. Long basal processes of these cells extended to blood vessels beneath the ependymal layer. Moreover, stroke increased V/SVZ endothelial cell proliferation from 2% in non-ischemic mice to 12 and 15% at 7 and 14 days after stroke, respectively. Vascular volume in the V/SVZ was augmented from 3% of the total volume prior to stroke to 6% at 90 days after stroke. Stroke-increased angiogenesis was closely associated with neuroblasts that expanded to nearly encompass the entire lateral ventricular wall in the V/SVZ. These data indicate that stroke induces long-term alterations of the neural stem cell and vascular architecture of the adult V/SVZ neurogenic niche. These post-stroke structural changes may provide insight into neural stem cell mediation of stroke-induced neurogenesis through the interaction of neural stem cells with proteins in the CSF and their sub-ependymal neurovascular interaction. PMID:25437857

  18. Temporal profiles of synaptic plasticity-related signals in adult mouse hippocampus with methotrexate treatment.

    PubMed

    Yang, Miyoung; Kim, Juhwan; Kim, Sung-Ho; Kim, Joong-Sun; Shin, Taekyun; Moon, Changjong

    2012-07-25

    Methotrexate, which is used to treat many malignancies and autoimmune diseases, affects brain functions including hippocampal-dependent memory function. However, the precise mechanisms underlying methotrexate-induced hippocampal dysfunction are poorly understood. To evaluate temporal changes in synaptic plasticity-related signals, the expression and activity of N-methyl-D-aspartic acid receptor 1, calcium/calmodulin-dependent protein kinase II, extracellular signal-regulated kinase 1/2, cAMP responsive element-binding protein, glutamate receptor 1, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor were examined in the hippocampi of adult C57BL/6 mice after methotrexate (40 mg/kg) intraperitoneal injection. Western blot analysis showed biphasic changes in synaptic plasticity-related signals in adult hippocampi following methotrexate treatment. N-methyl-D-aspartic acid receptor 1, calcium/calmodulin-dependent protein kinase II, and glutamate receptor 1 were acutely activated during the early phase (1 day post-injection), while extracellular signal-regulated kinase 1/2 and cAMP responsive element-binding protein activation showed biphasic increases during the early (1 day post-injection) and late phases (7-14 days post-injection). Brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor expression increased significantly during the late phase (7-14 days post-injection). Therefore, methotrexate treatment affects synaptic plasticity-related signals in the adult mouse hippocampus, suggesting that changes in synaptic plasticity-related signals may be associated with neuronal survival and plasticity-related cellular remodeling. PMID:25657706

  19. Expression of Npas4 mRNA in Telencephalic Areas of Adult and Postnatal Mouse Brain

    PubMed Central

    Damborsky, Joanne C.; Slaton, G. Simona; Winzer-Serhan, Ursula H.

    2015-01-01

    The transcription factor neuronal PAS domain-containing protein 4 (Npas4) is an inducible immediate early gene which regulates the formation of inhibitory synapses, and could have a significant regulatory role during cortical circuit formation. However, little is known about basal Npas4 mRNA expression during postnatal development. Here, postnatal and adult mouse brain sections were processed for isotopic in situ hybridization using an Npas4 specific cRNA antisense probe. In adults, Npas4 mRNA was found in the telencephalon with very restricted or no expression in diencephalon or mesencephalon. In most telencephalic areas, including the anterior olfactory nucleus (AON), piriform cortex, neocortex, hippocampus, dorsal caudate putamen (CPu), septum and basolateral amygdala nucleus (BLA), basal Npas4 expression was detected in scattered cells which exhibited strong hybridization signal. In embryonic and neonatal brain sections, Npas4 mRNA expression signals were very low. Starting at postnatal day 5 (P5), transcripts for Npas4 were detected in the AON, CPu and piriform cortex. At P8, additional Npas4 hybridization was found in CA1 and CA3 pyramidal layer, and in primary motor cortex. By P13, robust mRNA expression was located in layers IV and VI of all sensory cortices, frontal cortex and cingulate cortex. After onset of expression, postnatal spatial mRNA distribution was similar to that in adults, with the exception of the CPu, where Npas4 transcripts became gradually restricted to the most dorsal part. In conclusion, the spatial distribution of Npas4 mRNA is mostly restricted to telencephalic areas, and the temporal expression increases with developmental age during postnatal development, which seem to correlate with the onset of activity-driven excitatory transmission. PMID:26633966

  20. The effect of nitric oxide synthase inhibitors nitro-L-arginine and 7-nitroindazole on spatial learning and motor functions in Lurcher mutant and wild type mice.

    PubMed

    Markvartová, V; Vozeh, F

    2008-01-01

    Nitric oxide (NO) is an intercellular messenger that, among other things, plays an important role in the nervous system as a gaseous neurotransmitter, modulating long-term potentiation (LTP) induction of synaptic transmission. LTP has been suggested to be the basis of memory formation. On the other hand NO also participates in excitotoxic processes which play an important role in many neuropathological states. The aim of this work was to observe the effect of two NO synthase (NOS) inhibitors (N omega-Nitro-L-arginine, NA; 7-nitroindazole, NI) on spontaneous behaviour, spatial learning and motor functions in Lurcher (+/Lc) and wild type (+/+) mice, derived from the B6CBA strain. Heterozygous Lurcher mutant mice represent a natural model of the olivocerebellar degeneration. They suffer from postnatal, practically total, extinction of cerebellar Purkinje cells (due to the excitotoxic apoptosis) and a partial decrease of granule cells and inferior olive neurons (ION) because of the lost target of their axons. +/+ animals are healthy littermates of +/Lc. NA is a nonselective NOS inhibitor which influences, except neuronal (n), also endothelial (e) NOS with an impact on blood pressure, NI is a selective nNOS inhibitor without any circulatory effect. The adult animals of both types (+/Lc; +/+) were influenced by acute administration of both inhibitors (25 mg/kg i.p. 30 min. before experiments) and newborns only by both acute and long-term administration of NI (1 month, starting from postnatal day 2, P2). Control solutions - saline or solvents of both NA and NI inhibitors--diluted 1M HCl and dimethyl sulfoxide (DMSO) respectively, were given at a relevant volume in the same way. The effect of both inhibitors and control solutions on motor functions was tested using four standard procedures (horizontal wire, slanting ladder, rotating cylinder, foot-bridge); in newborns at the age of 14 days. Spatial learning ability was examined in five-day long procedure in the Morris

  1. Doublecortin (DCX) is not Essential for Survival and Differentiation of Newborn Neurons in the Adult Mouse Dentate Gyrus

    PubMed Central

    Dhaliwal, Jagroop; Xi, Yanwei; Bruel-Jungerman, Elodie; Germain, Johanne; Francis, Fiona; Lagace, Diane C.

    2016-01-01

    In the adult brain, expression of the microtubule-associated protein Doublecortin (DCX) is associated with neural progenitor cells (NPCs) that give rise to new neurons in the dentate gyrus. Many studies quantify the number of DCX-expressing cells as a proxy for the level of adult neurogenesis, yet no study has determined the effect of removing DCX from adult hippocampal NPCs. Here, we use a retroviral and inducible mouse transgenic approach to either knockdown or knockout DCX from adult NPCs in the dentate gyrus and examine how this affects cell survival and neuronal maturation. Our results demonstrate that shRNA-mediated knockdown of DCX or Cre-mediated recombination in floxed DCX mice does not alter hippocampal neurogenesis and does not change the neuronal fate of the NPCs. Together these findings show that the survival and maturation of adult-generated hippocampal neurons does not require DCX. PMID:26793044

  2. High yield extraction of pure spinal motor neurons, astrocytes and microglia from single embryo and adult mouse spinal cord

    PubMed Central

    Beaudet, Marie-Josée; Yang, Qiurui; Cadau, Sébastien; Blais, Mathieu; Bellenfant, Sabrina; Gros-Louis, François; Berthod, François

    2015-01-01

    Extraction of mouse spinal motor neurons from transgenic mouse embryos recapitulating some aspects of neurodegenerative diseases like amyotrophic lateral sclerosis has met with limited success. Furthermore, extraction and long-term culture of adult mouse spinal motor neurons and glia remain also challenging. We present here a protocol designed to extract and purify high yields of motor neurons and glia from individual spinal cords collected on embryos and adult (5-month-old) normal or transgenic mice. This method is based on mild digestion of tissue followed by gradient density separation allowing to obtain two millions motor neurons over 92% pure from one E14.5 single embryo and more than 30,000 from an adult mouse. These cells can be cultured more than 14 days in vitro at a density of 100,000 cells/cm2 to maintain optimal viability. Functional astrocytes and microglia and small gamma motor neurons can be purified at the same time. This protocol will be a powerful and reliable method to obtain motor neurons and glia to better understand mechanisms underlying spinal cord diseases. PMID:26577180

  3. The mechanism of dehydration in chromophore maturation of wild-type green fluorescent protein: A theoretical study

    NASA Astrophysics Data System (ADS)

    Ma, Yingying; Yu, Jian-Guo; Sun, Qiao; Li, Zhen; Smith, Sean C.

    2015-07-01

    An interesting aspect of the green fluorescent protein (GFP) is its autocatalytic chromophore maturation. Numerous experimental studies have indicated that dehydration is the last step in the chromophore maturation process of wild-type GFP. Based on the crystal structure of wild-type GFP, the mechanism of the reverse reaction of dehydration was investigated by using density functional theory (DFT) in this study. Our results proposed that the dehydration is exothermic. Moreover, the rate-limiting step of the mechanism is the proton on guanidinium of Arg96 transferring to the β-carbon anion of Tyr66, which is consistent with the experimental observation.

  4. Different effects of progesterone and estradiol on chimeric and wild type aldosterone synthase in vitro

    PubMed Central

    2013-01-01

    Background Familial hyperaldosteronism type I (FH-I) is caused by the unequal recombination between the 11beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes, resulting in the generation of a CYP11B1/B2 chimeric gene and abnormal adrenal aldosterone production. Affected patients usually show severe hypertension and an elevated frequency of stroke at a young age. Aldosterone levels rise during pregnancy, yet in pregnant women with FH-1, their hypertensive condition either remains unchanged or may even improve. The purpose of this study was to investigate in vitro whether female sex steroids modulate the activity of chimeric (ASCE) or wild type (ASWT) aldosterone synthase enzymes. Methods We designed an in vitro assay using HEK-293 cell line transiently transfected with vectors containing the full ASCE or ASWT cDNAs. Progesterone or estradiol effects on AS enzyme activities were evaluated in transfected cells incubated with deoxycorticosterone (DOC) alone or DOC plus increasing doses of these steroids. Results In our in vitro model, both enzymes showed similar apparent kinetic parameters (Km = 1.191 microM and Vmax = 27.08 microM/24 h for ASCE and Km = 1.163 microM and Vmax = 36.98 microM/24 h for ASWT; p = ns, Mann–Whitney test). Progesterone inhibited aldosterone production by ASCE- and ASWT-transfected cells, while estradiol demonstrated no effect. Progesterone acted as a competitive inhibitor for both enzymes. Molecular modelling studies and binding affinity estimations indicate that progesterone might bind to the substrate site in both ASCE and ASWT, supporting the idea that this steroid could regulate these enzymatic activities and contribute to the decay of aldosterone synthase activity in chimeric gene-positive patients. Conclusions Our results show an inhibitory action of progesterone in the aldosterone synthesis by chimeric or wild type aldosterone synthase enzymes. This is a novel regulatory mechanism of progesterone

  5. The impact of agrin on the formation of orthogonal arrays of particles in cultured astrocytes from wild-type and agrin-null mice.

    PubMed

    Fallier-Becker, Petra; Sperveslage, Jan; Wolburg, Hartwig; Noell, Susan

    2011-01-01

    Astrocytic endfeet membranes are studded with aquaporin-4 (AQP4) containing orthogonal arrays of particles (OAP) which can be visualized exclusively by the freeze-fracturing method. They are predominantly expressed where the astroglial membrane is in contact with the superficial and perivascular basal lamina. This polarity seems to be essential for the integrity of the blood-brain barrier (BBB). The basal lamina containing many extracellular matrix (ECM) components such as collagen, laminin and heparansulfate proteoglycans like agrin is thought to influence this OAP-related polarity of astrocytes. Recently, we have shown that agrin, in particular the neuronal isoform A4B8, is capable of influencing the formation of OAPs in astrocytes when cultured in the presence of agrin-conditioned media. In this paper we wanted to investigate whether coating with exogenous agrin compared to coating with other ECM components would induce OAP formation in astrocytes of the agrin-null mouse. For this purpose, we cultured astrocytes from agrin-null and wild-type mice on agrin- or ECM-coated surfaces. Immunofluorescent cytochemical staining of AQP4 indicated a higher AQP4 expression level in cultures with agrin- or ECM-coated than in cultures with uncoated surfaces, whereas western blot analyses and PCR showed no differences. α-Dystroglycan is thought to be a potential receptor of agrin and was immunostained in wild-type as well as in agrin-null astrocytes. In freeze-fracture replicas, we observed an increase in OAP density in astrocytes when growing on agrin- and ECM-coatings. These results concurred with other experiments in which changes in volume were measured following hypotonic stress, which supported the positive influence of exogenous agrin on AQP4 insertion into the membrane, on OAP formation and on water transport. PMID:20920487

  6. Effects of Ketoconazole on the Biodistribution and Metabolism of [11C]Loperamide and [11C]N-Desmethyl-loperamide in Wild-type and P-gp Knockout Mice

    PubMed Central

    Seneca, Nicholas; Zoghbi, Sami S.; Shetty, H. Umesha; Tuan, Edward; Kannan, Pavitra; Taku, Andrew; Innis, Robert B.; Pike, Victor W.

    2010-01-01

    Introduction [11C]Loperamide and [11C]N-desmethyl-loperamide ([11C]dLop) have been proposed as radiotracers for imaging brain P-glycoprotein (P-gp) function. A major route of [11C]loperamide metabolism is N-demethylation to [11C]dLop. We aimed to test whether inhibition of CYP3A4 with ketoconazole might reduce the metabolism of [11C]loperamide and [11C]dLop in mice, and thereby improve the quality of these radiotracers. Methods Studies were performed in wild-type and P-gp knockout (mdr–1a/b −/−) mice. During each of seven study sessions, one pair of mice, comprising one wild-type and one knockout mouse, waspretreated with ketoconazole (50 mg/kg, i.p.) while another such pair was left untreated. Mice were sacrificed at 30 min after injection of [11C]loperamide or [11C]dLop. Whole brain and plasma samples were measured for radioactivity and analyzed with radio-HPLC. Results Ketoconazole increased the plasma concentrations of [11C]loperamide and its main radiometabolite, [11C]dLop, by about two-fold in both wild-type and knockout mice, whereas the most polar radiometabolite was decreased three-fold. Furthermore, ketoconazole increased the brain concentrations of [11C]loperamide and the radiometabolite [11C]dLop by about two-fold in knockout mice, and decreased the brain concentrations of the major and most polar radiometabolite in wild-type and knockout mice by 82 and 49%, respectively. In contrast, ketoconazole had no effect on plasma and brain distribution of administered [11C]dLop and its radiometabolites in either wild-type or knockout mice, except to increase the low plasma [11C]dLop concentration. The least polar radiometabolite of [11C]dLop was identified with LC-MSn as the N-hydroxymethyl analog of [11C]dLop and this also behaved as a P-gp substrate. Conclusion In this study, ketoconazole (50 mg/kg, i.p.) proved partiallyeffective for inhibiting the N-demethylation of [11C]loperamide in mouse in vivo but had relatively smaller or no effect on [11C

  7. Adult Plasticity in the Subcortical Auditory Pathway of the Maternal Mouse

    PubMed Central

    Miranda, Jason A.; Shepard, Kathryn N.; McClintock, Shannon K.; Liu, Robert C.

    2014-01-01

    Subcortical auditory nuclei were traditionally viewed as non-plastic in adulthood so that acoustic information could be stably conveyed to higher auditory areas. Studies in a variety of species, including humans, now suggest that prolonged acoustic training can drive long-lasting brainstem plasticity. The neurobiological mechanisms for such changes are not well understood in natural behavioral contexts due to a relative dearth of in vivo animal models in which to study this. Here, we demonstrate in a mouse model that a natural life experience with increased demands on the auditory system – motherhood – is associated with improved temporal processing in the subcortical auditory pathway. We measured the auditory brainstem response to test whether mothers and pup-naïve virgin mice differed in temporal responses to both broadband and tone stimuli, including ultrasonic frequencies found in mouse pup vocalizations. Mothers had shorter latencies for early ABR peaks, indicating plasticity in the auditory nerve and the cochlear nucleus. Shorter interpeak latency between waves IV and V also suggest plasticity in the inferior colliculus. Hormone manipulations revealed that these cannot be explained solely by estrogen levels experienced during pregnancy and parturition in mothers. In contrast, we found that pup-care experience, independent of pregnancy and parturition, contributes to shortening auditory brainstem response latencies. These results suggest that acoustic experience in the maternal context imparts plasticity on early auditory processing that lasts beyond pup weaning. In addition to establishing an animal model for exploring adult auditory brainstem plasticity in a neuroethological context, our results have broader implications for models of perceptual, behavioral and neural changes that arise during maternity, where subcortical sensorineural plasticity has not previously been considered. PMID:24992362

  8. Effects of major histocompatibility complex class II knockout on mouse bone mechanical properties during development.

    PubMed

    Simske, Steven J; Bateman, Ted A; Smith, Erin E; Ferguson, Virginia L; Chapes, Stephen K

    2002-01-01

    We investigated the effect of major histocompatibility complex class II (MHC II) knockout on the development of the mouse peripheral skeleton. These C2D mice had less skeletal development at 8, 12 and 16 weeks of age compared to wild-type C57BL/6J (B6) male mice. The C2D mice had decreased femur mechanical, geometric and compositional measurements compared to wild type mice at each of these ages. C2D femur stiffness (S), peak force in 3-pt bending (Pm), and mineral mass (Min-M) were 74%, 64% and 66%, respectively, of corresponding B6 values at 8 weeks of age. Similar differences were measured at 12 weeks (for which C2D femoral S, Pm and Min-M were 71%, 72% and 73%, respectively, of corresponding B6 values) and at 16 weeks (for which C2D femoral S, Pm and Min-M were 80%, 66% and 61%, respectively, of corresponding B6 values). MHC II knockout delays the development of adult bone properties and is accompanied by lower body mass compared to wild-type controls. PMID:12085652

  9. Effects of major histocompatibility complex class II knockout on mouse bone mechanical properties during development

    NASA Technical Reports Server (NTRS)

    Simske, Steven J.; Bateman, Ted A.; Smith, Erin E.; Ferguson, Virginia L.; Chapes, Stephen K.

    2002-01-01

    We investigated the effect of major histocompatibility complex class II (MHC II) knockout on the development of the mouse peripheral skeleton. These C2D mice had less skeletal development at 8, 12 and 16 weeks of age compared to wild-type C57BL/6J (B6) male mice. The C2D mice had decreased femur mechanical, geometric and compositional measurements compared to wild type mice at each of these ages. C2D femur stiffness (S), peak force in 3-pt bending (Pm), and mineral mass (Min-M) were 74%, 64% and 66%, respectively, of corresponding B6 values at 8 weeks of age. Similar differences were measured at 12 weeks (for which C2D femoral S, Pm and Min-M were 71%, 72% and 73%, respectively, of corresponding B6 values) and at 16 weeks (for which C2D femoral S, Pm and Min-M were 80%, 66% and 61%, respectively, of corresponding B6 values). MHC II knockout delays the development of adult bone properties and is accompanied by lower body mass compared to wild-type controls.

  10. Developmental phenotype of a membrane only estrogen receptor alpha (MOER) mouse.

    PubMed

    Pedram, Ali; Razandi, Mahnaz; Kim, Jin K; O'Mahony, Fiona; Lee, Eva Yhp; Luderer, Ulrike; Levin, Ellis R

    2009-02-01

    Estrogen receptors (ERs) alpha and beta exist as nuclear, cytoplasmic, and membrane cellular pools in a wide variety of organs. The relative contributions of each ERalpha pool to in vivo phenotypes resulting from estrogen signaling have not been determined. To address this, we generated a transgenic mouse expressing only a functional E domain of ERalpha at the plasma membrane (MOER). Cells isolated from many organs showed membrane only localized E domain of ERalpha and no other receptor pools. Liver cells from MOER and wild type mice responded to 17-beta-estradiol (E2) with comparable activation of ERK and phosphatidylinositol 3-kinase, not seen in cells from ERalphaKO mice. Mating the MOER female mice with proven male wild type breeders produced no pregnancies because the uterus and vagina of the MOER female mice were extremely atrophic. Ovaries of MOER and homozygous Strasbourg ERalphaKO mice showed multiple hemorrhagic cysts and no corpus luteum, and the mammary gland development in both MOER and ERalphaKO mice was rudimentary. Despite elevated serum E2 levels, serum LH was not suppressed, and prolactin levels were low in MOER mice. MOER and Strasbourg female mice showed plentiful abdominal visceral and other depots of fat and increased body weight compared to wild type mice despite comparable food consumption. These results provide strong evidence that the normal development and adult functions of important organs in female mice requires nuclear ERalpha and is not rescued by membrane ERalpha domain expression alone. PMID:19054762

  11. Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin

    PubMed Central

    Fiaschi, Luigi; Di Palo, Benedetta; Scarselli, Maria; Pozzi, Clarissa; Tomaszewski, Kelly; Galletti, Bruno; Nardi-Dei, Vincenzo; Arcidiacono, Letizia; Mishra, Ravi P. N.; Mori, Elena; Pallaoro, Michele; Falugi, Fabiana; Torre, Antonina; Fontana, Maria Rita; Soriani, Marco; Bubeck Wardenburg, Juliane; Grandi, Guido; Rappuoli, Rino

    2016-01-01

    Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus. PMID:27030589

  12. Sphingopeptides: dihydrosphingosine-based fusion inhibitors against wild-type and enfuvirtide-resistant HIV-1.

    PubMed

    Ashkenazi, Avraham; Viard, Mathias; Unger, Linor; Blumenthal, Robert; Shai, Yechiel

    2012-11-01

    Understanding the structural organization of lipids in the cell and viral membranes is essential for elucidating mechanisms of viral fusion that lead to entry of enveloped viruses into their host cells. The HIV lipidome shows a remarkable enrichment in dihydrosphingomyelin, an unusual sphingolipid formed by a dihydrosphingosine backbone. Here we investigated the ability of dihydrosphingosine to incorporate into the site of membrane fusion mediated by the HIV envelope (Env) protein. Dihydrosphingosine as well as cholesterol, fatty acid, and tocopherol was conjugated to highly conserved, short HIV-1 Env-derived peptides with no antiviral activity otherwise. We showed that dihydrosphingosine exclusively endowed nanomolar antiviral activity to the peptides (IC(50) as low as 120 nM) in HIV-1 infection on TZM-bl cells and on Jurkat T cells, as well as in the cell-cell fusion assay. These sphingopeptides were active against enfuvirtide-resistant and wild-type CXCR4 and CCR5 tropic HIV strains. The anti-HIV activity was determined by both the peptides and their dihydrosphingosine conjugate. Moreover, their mode of action involved accumulation in the cells and viruses and binding to membranes enriched in sphingomyelin and cholesterol. The data suggest that sphingopeptides are recruited to the HIV membrane fusion site and provide a general concept in developing inhibitors of sphingolipid-mediated biological systems. PMID:22872679

  13. Comparative transcriptomic analysis of silkwormBmovo-1 and wild type silkworm ovary

    PubMed Central

    Xue, Renyu; Hu, Xiaolong; Zhu, Liyuan; Cao, Guangli; Huang, Moli; Xue, Gaoxu; Song, Zuowei; Lu, Jiayu; Chen, Xueying; Gong, Chengliang

    2015-01-01

    The detailed molecular mechanism of Bmovo-1 regulation of ovary size is unclear. To uncover the mechanism of Bmovo-1 regulation of ovarian development and oogenesis using RNA-Seq, we compared the transcriptomes of wild type (WT) and Bmovo-1-overexpressing silkworm (silkworm+Bmovo-1) ovaries. Using a pair-end Illumina Solexa sequencing strategy, 5,296,942 total reads were obtained from silkworm+Bmovo-1 ovaries and 6,306,078 from WT ovaries. The average read length was about 100 bp. Clean read ratios were 98.79% for silkworm+Bmovo-1 and 98.87% for WT silkworm ovaries. Comparative transcriptome analysis showed 123 upregulated and 111 downregulated genes in silkworm+Bmovo-1 ovaries. These differentially expressed genes were enriched in the extracellular and extracellular spaces and involved in metabolism, genetic information processing, environmental information processing, cellular processes and organismal systems. Bmovo-1 overexpression in silkworm ovaries might promote anabolism for ovarian development and oogenesis and oocyte proliferation and transport of nutrients to ovaries by altering nutrient partitioning, which would support ovary development. Excessive consumption of nutrients for ovary development alters nutrient partitioning and deters silk protein synthesis. PMID:26643037

  14. Rootcap structure in wild type and in a starchless mutant of Arabidopsis

    NASA Technical Reports Server (NTRS)

    Sack, F. D.; Kiss, J. Z.

    1989-01-01

    Rootcaps of the wild type (WT) and of a starchless, gravitropic mutant (TC7) of Arabidopsis thaliana L. were examined by electron microscopy to identify cellular polarities with respect to gravity. In columella cells, nuclei are located proximally, and the nuclear envelope is continuous with endoplasmic reticulum (ER) that is in turn connected to nearby plasmodesmata. Impregnation of ER with osmium ferricyanide revealed numerous contacts between columella plastids and ER in both genotypes. ER is present mostly in the outer regions of the columella protoplast except in older columella cells that are developing into peripheral cells. In vertical roots, only columella cells that are intermediate in development (story 2 cells) have a higher surface density (S) of ER in the distal compared to proximal regions of the cell. The distal but not the proximal S of the ER is constant throughout columella development. Plastids are less sedimented in TC7 columella cells compared to those of the WT. It is hypothesized that plastid contact with the ER plays a role in gravity perception in both genotypes.

  15. Wild-Type Transthyretin Cardiac Amyloidosis: Novel Insights From Advanced Imaging.

    PubMed

    Narotsky, David L; Castano, Adam; Weinsaft, Jonathan W; Bokhari, Sabahat; Maurer, Mathew S

    2016-09-01

    Amyloidosis is caused by extracellular deposition of abnormal protein fibrils, resulting in destruction of tissue architecture and impairment of organ function. The most common forms of systemic amyloidosis are light-chain and transthyretin-related (ATTR). ATTR can result from an autosomal dominant hereditary transmission of mutated genes in the transthyretin or from a wild-type form of disease (ATTRwt), previously known as senile cardiac amyloidosis. With the aging of the worldwide population, ATTRwt will emerge as the most common type of cardiac amyloidosis that clinicians encounter. Diagnosis of systemic amyloidosis is often delayed, either because of the false assumption that it is a rare disease, or because of misdiagnosis as a result of mistaking it with other conditions. Clinicians must integrate clinical clues from history, physical examination, and common diagnostic tests to raise suspicion for ATTRwt. The historical gold standard for diagnosis of cardiac amyloid is endomyocardial biopsy analysis with pathological distinction of precursor protein type, but this method often results in delayed diagnosis because of the limited availability of expertise to perform and interpret the endomyocardial biopsy specimen. Emerging noninvasive imaging modalities provide easier, accurate screening for ATTRwt. These modalities include advanced echocardiography, using strain imaging and the myocardial contraction fraction; nuclear scintigraphy, which can differentiate between ATTR and light-chain cardiac amyloid; and cardiac magnetic resonance imaging, using extracellular volume measurement, late gadolinium enhancement, and distinct T1 mapping. These novel approaches reveal insights into the prevalence, clinical course, morphological effects, and prognosis of ATTRwt. PMID:27568874

  16. Profile of Cytokines and Chemokines Triggered by Wild-Type Strains of Rabies Virus in Mice.

    PubMed

    Appolinário, Camila Michele; Allendorf, Susan Dora; Peres, Marina Gea; Ribeiro, Bruna Devidé; Fonseca, Clóvis R; Vicente, Acácia Ferreira; Antunes, João Marcelo A de Paula; Megid, Jane

    2016-02-01

    Rabies is a lethal infectious disease that causes 55,000 human deaths per year and is transmitted by various mammalian species, such as dogs and bats. The host immune response is essential for avoiding viral progression and promoting viral clearance. Cytokines and chemokines are crucial in the development of an immediate antiviral response; the rabies virus (RABV) attempts to evade this immune response. The virus's capacity for evasion is correlated with its pathogenicity and the host's inflammatory response, with highly pathogenic strains being the most efficient at hijacking the host's defense mechanisms and thereby decreasing inflammation. The purpose of this study was to evaluate the expression of a set of cytokine and chemokine genes that are related to the immune response in the brains of mice inoculated intramuscularly or intracerebrally with two wild-type strains of RABV, one from dog and the other from vampire bat. The results demonstrated that the gene expression profile is intrinsic to the specific rabies variant. The prompt production of cytokines and chemokines seems to be more important than their levels of expression for surviving a rabies infection. PMID:26711511

  17. Evaluation of short-interfering RNAs treatment in experimental rabies due to wild-type virus.

    PubMed

    Appolinario, Camila Michele; Allendorf, Susan Dora; Peres, Marina Gea; Fonseca, Clovis Reynaldo; Vicente, Acacia Ferreira; Antunes, João Marcelo Azevedo de Paula; Pantoja, José Carlos Figueiredo; Megid, Jane

    2015-01-01

    We have evaluated the efficacy of short-interfering RNAs targeting the nucleoprotein gene and also the brain immune response in treated and non-treated infected mice. Mice were inoculated with wild-type virus, classified as dog (hv2) or vampire bat (hv3) variants and both groups were treated or left as controls. No difference was observed in the lethality rate between treated and non-treated groups, although clinical evaluation of hv2 infected mice showed differences in the severity of clinical disease (p=0.0006). Evaluation of brain immune response 5 days post-inoculation in treated hv2 group showed no difference among the analyzed genes, whereas after 10 days post-inoculation there was increased expression of 2',5'-oligoadenylate synthetase 1, tumor necrosis factor alpha, interleukin 12, interferon gamma, and C-X-C motif chemokine 10 associated with higher expression of N gene in the same period (p<0.0001). In hv2 non-treated group only higher interferon beta expression was found at day 5. The observed differences in results of the immune response genes between treated and non-treated groups is not promising as they had neither impact on mortality nor even a reduction in the expression of N gene in siRNA treated animals. This finding suggests that the use of pre-designed siRNA alone may not be useful in rabies treatment. PMID:26254692

  18. Wild Type Bone Marrow Transplant Partially Reverses Neuroinflammation in Progranulin-Deficient Mice

    PubMed Central

    Yang, Yue; Aloi, Macarena S.; Cudaback, Eiron; Josephsen, Samuel R.; Rice, Samantha J.; Jorstad, Nikolas L.; Keene, C. Dirk; Montine, Thomas J.

    2014-01-01

    Frontotemporal dementia (FTD) is a neurodegenerative disease with devastating changes in behavioral performance and social function. Mutations in the progranulin gene (GRN) are one of the most common causes of inherited FTD due to reduced progranulin expression or activity, including in brain where it is expressed primarily by neurons and microglia. Thus, efforts aimed at enhancing progranulin levels might be a promising therapeutic strategy. Bone marrow-derived cells are able to engraft in the brain and adopt a microglial phenotype under myeloablative irradiation conditioning. This ability makes bone marrow (BM)-derived cells a potential cellular vehicle for transferring therapeutic molecules to the central nervous system. Here, we utilized BM cells from Grn+/+ (wild type or wt) mice labeled with green fluorescence protein for delivery of progranulin to progranulin deficient (Grn−/−) mice. Our results showed that wt bone marrow transplantation (BMT) partially reconstituted progranulin in the periphery and in cerebral cortex of Grn−/− mice. We demonstrated a pro-inflammatory effect in vivo and in ex vivo preparations of cerebral cortex of Grn−/− mice that was partially to fully reversed five months after BMT. Our findings suggest that BMT can be administered as a stem cell-based approach to prevent or to treat neurodegenerative diseases. PMID:25199051

  19. Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin.

    PubMed

    Fiaschi, Luigi; Di Palo, Benedetta; Scarselli, Maria; Pozzi, Clarissa; Tomaszewski, Kelly; Galletti, Bruno; Nardi-Dei, Vincenzo; Arcidiacono, Letizia; Mishra, Ravi P N; Mori, Elena; Pallaoro, Michele; Falugi, Fabiana; Torre, Antonina; Fontana, Maria Rita; Soriani, Marco; Bubeck Wardenburg, Juliane; Grandi, Guido; Rappuoli, Rino; Ferlenghi, Ilaria; Bagnoli, Fabio

    2016-06-01

    Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus. PMID:27030589

  20. Extensive degradation of RNA precursors by the exosome in wild-type cells.

    PubMed

    Gudipati, Rajani Kanth; Xu, Zhenyu; Lebreton, Alice; Séraphin, Bertrand; Steinmetz, Lars M; Jacquier, Alain; Libri, Domenico

    2012-11-01

    The exosome is a complex involved in the maturation of rRNA and sn-snoRNA, in the degradation of short-lived noncoding RNAs, and in the quality control of RNAs produced in mutants. It contains two catalytic subunits, Rrp6p and Dis3p, whose specific functions are not fully understood. We analyzed the transcriptome of combinations of Rrp6p and Dis3p catalytic mutants by high-resolution tiling arrays. We show that Dis3p and Rrp6p have both overlapping and specific roles in degrading distinct classes of substrates. We found that transcripts derived from more than half of intron-containing genes are degraded before splicing. Surprisingly, we also show that the exosome degrades large amounts of tRNA precursors despite the absence of processing defects. These results underscore the notion that large amounts of RNAs produced in wild-type cells are discarded before entering functional pathways and suggest that kinetic competition with degradation proofreads the efficiency and accuracy of processing. PMID:23000176

  1. Quantification of gait parameters in freely walking wild type and sensory deprived Drosophila melanogaster

    PubMed Central

    Mendes, César S; Bartos, Imre; Akay, Turgay; Márka, Szabolcs; Mann, Richard S

    2013-01-01

    Coordinated walking in vertebrates and multi-legged invertebrates such as Drosophila melanogaster requires a complex neural network coupled to sensory feedback. An understanding of this network will benefit from systems such as Drosophila that have the ability to genetically manipulate neural activities. However, the fly's small size makes it challenging to analyze walking in this system. In order to overcome this limitation, we developed an optical method coupled with high-speed imaging that allows the tracking and quantification of gait parameters in freely walking flies with high temporal and spatial resolution. Using this method, we present a comprehensive description of many locomotion parameters, such as gait, tarsal positioning, and intersegmental and left-right coordination for wild type fruit flies. Surprisingly, we find that inactivation of sensory neurons in the fly's legs, to block proprioceptive feedback, led to deficient step precision, but interleg coordination and the ability to execute a tripod gait were unaffected. DOI: http://dx.doi.org/10.7554/eLife.00231.001 PMID:23326642

  2. Comparative whole genome sequence analysis of wild-type and cidofovir-resistant monkeypoxvirus

    PubMed Central

    2010-01-01

    We performed whole genome sequencing of a cidofovir {[(S)-1-(3-hydroxy-2-phosphonylmethoxy-propyl) cytosine] [HPMPC]}-resistant (CDV-R) strain of Monkeypoxvirus (MPV). Whole-genome comparison with the wild-type (WT) strain revealed 55 single-nucleotide polymorphisms (SNPs) and one tandem-repeat contraction. Over one-third of all identified SNPs were located within genes comprising the poxvirus replication complex, including the DNA polymerase, RNA polymerase, mRNA capping methyltransferase, DNA processivity factor, and poly-A polymerase. Four polymorphic sites were found within the DNA polymerase gene. DNA polymerase mutations observed at positions 314 and 684 in MPV were consistent with CDV-R loci previously identified in Vaccinia virus (VACV). These data suggest the mechanism of CDV resistance may be highly conserved across Orthopoxvirus (OPV) species. SNPs were also identified within virulence genes such as the A-type inclusion protein, serine protease inhibitor-like protein SPI-3, Schlafen ATPase and thymidylate kinase, among others. Aberrant chain extension induced by CDV may lead to diverse alterations in gene expression and viral replication that may result in both adaptive and attenuating mutations. Defining the potential contribution of substitutions in the replication complex and RNA processing machinery reported here may yield further insight into CDV resistance and may augment current therapeutic development strategies. PMID:20509894

  3. Differential transcription patterns in wild-type and glycoprotein G-deleted infectious laryngotracheitis viruses.

    PubMed

    Mahmoudian, Alireza; Markham, Philip F; Noormohammadi, Amir H; Devlin, Joanne M; Browning, Glenn F

    2013-01-01

    Infectious laryngotracheitis virus (ILTV) causes severe respiratory disease in poultry throughout the world. Recently the role of glycoprotein G (gG) in ILTV pathogenesis has been investigated and it has been shown to have chemokine-binding activity. An ILTV vaccine candidate deficient in gG has been developed and the deletion has been shown to alter the host's immune response to the virus. To understand the effect of the gG gene on transcription of other viral genes, the global expression profile of 72 ILTV genes in gG-deleted and wild-type ILTVs were investigated both in vivo and in vitro using quantitative reverse transcription-polymerase chain reaction. Several genes were differentially expressed in the different viruses in LMH cell cultures or in the tracheas of infected birds, and the expression of a number of genes, including ICP27, gC, gJ, Ul7 and UL40, differed significantly both in vivo and in vitro, suggesting that they had direct or indirect roles in virulence. This study has provided insights into the interactions between gG and other ILTV genes that may have a role in virulence. PMID:23611157

  4. Taste responses to sweet stimuli in alpha-gustducin knockout and wild-type mice.

    PubMed

    Danilova, Vicktoria; Damak, Sami; Margolskee, Robert F; Hellekant, Göran

    2006-07-01

    The importance of alpha-gustducin in sweet taste transduction is based on data obtained with sucrose and the artificial sweetener SC45647. Here we studied the role of alpha-gustducin in sweet taste. We compared the behavioral and electrophysiological responses of alpha-gustducin knockout (KO) and wild-type (WT) mice to 11 different sweeteners, representing carbohydrates, artificial sweeteners, and sweet amino acids. In behavioral experiments, over 48-h preference ratios were measured in two-bottle preference tests. In electrophysiological experiments, integrated responses of chorda tympani (CT) and glossopharyngeal (NG) nerves were recorded. We found that preference ratios of the KO mice were significantly lower than those of WT for acesulfame-K, dulcin, fructose, NC00174, D-phenylalanine, L-proline, D-tryptophan, saccharin, SC45647, sucrose, but not neotame. The nerve responses to all sweeteners, except neotame, were smaller in the KO mice than in the WT mice. The differences between the responses in WT and KO mice were more pronounced in the CT than in the NG. These data indicate that alpha-gustducin participates in the transduction of the sweet taste in general. PMID:16740645

  5. Overexpression of wild-type or mutants forms of CEBPA alter normal human hematopoiesis.

    PubMed

    Quintana-Bustamante, O; Lan-Lan Smith, S; Griessinger, E; Reyal, Y; Vargaftig, J; Lister, T A; Fitzgibbon, J; Bonnet, D

    2012-07-01

    CCAAT/enhancer-binding protein-α (C/EBPα/CEBPA) is mutated in approximately 8% of acute myeloid leukemia (AML) in both familial and sporadic AML and, with FLT3 and NPM1, has received most attention as a predictive marker of outcome in patients with normal karyotype disease. Mutations clustering to either the N- or C-terminal (N- and C-ter) portions of the protein have different consequences on the protein function. In familial cases, the N-ter form is inherited with patients exhibiting long latency period before the onset of overt disease, typically with the acquisition of a C-ter mutation. Despite the essential insights murine models provide the functional consequences of wild-type C/EBPα in human hematopoiesis and how different mutations are involved in AML development have received less attention. Our data underline the critical role of C/EBPα in human hematopoiesis and demonstrate that C/EBPα mutations (alone or in combination) are insufficient to convert normal human hematopoietic stem/progenitor cells into leukemic-initiating cells, although individually each altered normal hematopoiesis. It provides the first insight into the effects of N- and C-ter mutations acting alone and to the combined effects of N/C double mutants. Our results mimicked closely what happens in CEBPA mutated patients. PMID:22371011

  6. Intraperitoneal Infection of Wild-Type Mice with Synthetically Generated Mammalian Prion

    PubMed Central

    Wang, Xinhe; McGovern, Gillian; Zhang, Yi; Wang, Fei; Zha, Liang; Jeffrey, Martin; Ma, Jiyan

    2015-01-01

    The prion hypothesis postulates that the infectious agent in transmissible spongiform encephalopathies (TSEs) is an unorthodox protein conformation based agent. Recent successes in generating mammalian prions in vitro with bacterially expressed recombinant prion protein provide strong support for the hypothesis. However, whether the pathogenic properties of synthetically generated prion (rec-Prion) recapitulate those of naturally occurring prions remains unresolved. Using end-point titration assay, we showed that the in vitro prepared rec-Prions have infectious titers of around 104 LD50 / μg. In addition, intraperitoneal (i.p.) inoculation of wild-type mice with rec-Prion caused prion disease with an average survival time of 210 – 220 days post inoculation. Detailed pathological analyses revealed that the nature of rec-Prion induced lesions, including spongiform change, disease specific prion protein accumulation (PrP-d) and the PrP-d dissemination amongst lymphoid and peripheral nervous system tissues, the route and mechanisms of neuroinvasion were all typical of classical rodent prions. Our results revealed that, similar to naturally occurring prions, the rec-Prion has a titratable infectivity and is capable of causing prion disease via routes other than direct intra-cerebral challenge. More importantly, our results established that the rec-Prion caused disease is pathogenically and pathologically identical to naturally occurring contagious TSEs, supporting the concept that a conformationally altered protein agent is responsible for the infectivity in TSEs. PMID:26136122

  7. Fermentative production of 1-propanol from sugars using wild-type and recombinant Shimwellia blattae.

    PubMed

    Urano, Nobuyuki; Fujii, Misaki; Kaino, Hiroshi; Matsubara, Mitsuru; Kataoka, Michihiko

    2015-02-01

    Shimwellia blattae is an enteric bacterium and produces endogenous enzymes that convert 1,2-propanediol (1,2-PD) to 1-propanol, which is expected to be used as a fuel substitute and a precursor of polypropylene. Therefore, if S. blattae could be induced to generate its own 1,2-PD from sugars, it might be possible to produce 1-propanol from sugars with this microorganism. Here, two 1,2-PD production pathways were constructed in S. blattae, resulting in two methods for 1-propanol production with the bacterium. One method employed the L-rhamnose utilization pathway, in which L-rhamnose is split into dihydroxyacetone phosphate and 1,2-PD. When wild-type S. blattae was cultured with L-rhamnose, an accumulation of 1,2-PD was observed. The other method for producing 1,2-PD was to introduce an engineered 1,2-PD production pathway from glucose into S. blattae. In both cases, the produced 1,2-PD was then converted to 1-propanol by 1,2-PD converting enzymes, whose production was induced by the addition of glycerol. PMID:25547843

  8. Experimentally Derived Structural Constraints for Amyloid Fibrils of Wild-Type Transthyretin

    PubMed Central

    Bateman, David A.; Tycko, Robert; Wickner, Reed B.

    2011-01-01

    Transthyretin (TTR) is a largely β-sheet serum protein responsible for transporting thyroxine and vitamin A. TTR is found in amyloid deposits of patients with senile systemic amyloidosis. TTR mutants lead to familial amyloidotic polyneuropathy and familial amyloid cardiomyopathy, with an earlier age of onset. Studies of amyloid fibrils of familial amyloidotic polyneuropathy mutant TTR suggest a structure similar to the native state with only a simple opening of a β-strand-loop-strand region exposing the two main β-sheets of the protein for fibril elongation. However, we find that the wild-type TTR sequence forms amyloid fibrils that are considerably different from the previously suggested amyloid structure. Using protease digestion with mass spectrometry, we observe the amyloid core to be primarily composed of the C-terminal region, starting around residue 50. Solid-state NMR measurements prove that TTR differs from other pathological amyloids in not having an in-register parallel β-sheet architecture. We also find that the TTR amyloid is incapable of binding thyroxine as monitored by either isothermal calorimetry or 1,8-anilinonaphthalene sulfonate competition. Taken together, our experiments are consistent with a significantly different configuration of the β-sheets compared to the previously suggested structure. PMID:22098747

  9. Experimentally derived structural constraints for amyloid fibrils of wild-type transthyretin.

    PubMed

    Bateman, David A; Tycko, Robert; Wickner, Reed B

    2011-11-16

    Transthyretin (TTR) is a largely β-sheet serum protein responsible for transporting thyroxine and vitamin A. TTR is found in amyloid deposits of patients with senile systemic amyloidosis. TTR mutants lead to familial amyloidotic polyneuropathy and familial amyloid cardiomyopathy, with an earlier age of onset. Studies of amyloid fibrils of familial amyloidotic polyneuropathy mutant TTR suggest a structure similar to the native state with only a simple opening of a β-strand-loop-strand region exposing the two main β-sheets of the protein for fibril elongation. However, we find that the wild-type TTR sequence forms amyloid fibrils that are considerably different from the previously suggested amyloid structure. Using protease digestion with mass spectrometry, we observe the amyloid core to be primarily composed of the C-terminal region, starting around residue 50. Solid-state NMR measurements prove that TTR differs from other pathological amyloids in not having an in-register parallel β-sheet architecture. We also find that the TTR amyloid is incapable of binding thyroxine as monitored by either isothermal calorimetry or 1,8-anilinonaphthalene sulfonate competition. Taken together, our experiments are consistent with a significantly different configuration of the β-sheets compared to the previously suggested structure. PMID:22098747

  10. Gravitropism and development of wild-type and starch-deficient mutants of Arabidopsis during spaceflight

    NASA Technical Reports Server (NTRS)

    Kiss, J. Z.; Katembe, W. J.; Edelmann, R. E.

    1998-01-01

    The "starch-statolith" hypothesis has been used by plant physiologists to explain the gravity perception mechanism in higher plants. In order to help resolve some of the controversy associated with ground-based research that has supported this theory, we performed a spaceflight experiment during the January 1997 mission of the Space Shuttle STS-81. Seedlings of wild-type (WT) Arabidopsis, two reduced-starch strains, and a starchless mutant were grown in microgravity and then given a gravity stimulus on a centrifuge. In terms of development in space, germination was greater than 90% for seeds in microgravity, and flight seedlings were smaller (60% in total length) compared to control plants grown on the ground and to control plants on a rotating clinostat. Seedlings grown in space had two structural features that distinguished them from the controls: a greater density of root hairs and an anomalous hypocotyl hook structure. However, the slower growth and morphological changes observed in the flight seedlings may be due to the effects of ethylene present in the spacecraft. Nevertheless, during the flight hypocotyls of WT seedlings responded to a unilateral 60 min stimulus provided by a 1-g centrifuge while those of the starch-deficient strains did not. Thus the strain with the greatest amount of starch responded to the stimulus given in flight and therefore, these data support the starch-statolith model for gravity sensing.

  11. Induction of breast cancer in wild type p53 cells by BRCA1-IRIS overexpression.

    PubMed

    Elshamy, Wael M

    2010-08-01

    Cells ability to evade cell death and to proliferate post geno-/cell-toxic stresses, likely leads to formation of cancer. Activation of p38MAPK and p53 following these stresses help protect cells against cancer development by initiating apoptosis. The duration of p38MAPK and p53 activation is regulated by the WIP1 phosphatase. BRCA1-IRIS triggers WIP1 expression in p53-dependent and -independent manner. BRCA1-IRIS triggers the expression and cytoplasmic localization of the mRNA stabilization and translation inducer, HuR that binds p53 and PPM1D mRNA. Hence, BRCA1-IRIS overexpression inactivates p38MAPK and/or p53 by upregulating WIP1 expression. BRCA1-IRIS abrogation of the homeostatic balance maintained by p38MAPK-p53-WIP1 pathway suppressed cell death induced by a lethal dose of UVC, high dosages of etoposide or H2O2, and allowed cells to survive and proliferate post geno-/cell-toxic stresses. This mechanism represents a new link between geno-/cell-toxic stress and aggressive breast cancer formation in p53 wild-type cells. PMID:20845286

  12. Dietary supplementation with ipriflavone decreases hepatic iron stores in wild type mice.

    PubMed

    Patchen, Bonnie; Koppe, Tiago; Cheng, Aaron; Seo, Young Ah; Wessling-Resnick, Marianne; Fraenkel, Paula G

    2016-09-01

    Hepcidin, a peptide produced in the liver, decreases intestinal iron absorption and macrophage iron release by causing degradation of the iron exporter, ferroportin. Because its levels are inappropriately low in patients with iron overload syndromes, hepcidin is a potential drug target. We previously conducted a chemical screen that revealed ipriflavone, an orally available small molecule, as a potent inducer of hepcidin expression. To evaluate ipriflavone's effect on iron homeostasis, we placed groups of 5-week old wild type or thalassemia intermedia (Hbb(Th3+/-)) mice on a soy-free, iron-sufficient diet, AIN-93G containing 220mg iron and 0-750mgipriflavone/kg of food for 50days. Ipriflavone 500mg/kg significantly reduced liver iron stores and intestinal ferroportin expression in WT mice, while increasing the ratio of hepcidin transcript levels to liver iron stores. Ipriflavone supplementation in Hbb(Th3+/-) mice failed to alleviate iron overload and was associated with a milder reduction in intestinal ferroportin and a failure to alter the ratio of hepcidin transcript levels to liver iron stores or splenic expression of the hepcidin-regulatory hormone, erythroferrone. These data suggest that dietary supplementation with ipriflavone alone would not be sufficient to treat iron overload in thalassemia intermedia. PMID:27519943

  13. Biochemical characterization of Arabidopsis wild-type and mutant phytochrome B holoproteins.

    PubMed Central

    Elich, T D; Chory, J

    1997-01-01

    Although phytochrome B (phyB) plays a particularly important role throughout the life cycle of a plant, it has not been studied in detail at the molecular level due to its low abundance. Here, we report on the expression, assembly with chromophore, and purification of epitope-tagged Arabidopsis phyB. In addition, we have reconstructed two missense mutations, phyB-4 and phyB-101, isolated in long hypocotyl screens. We show that mutant proteins phyB-4 and phyB-101 exhibit altered spectrophotometric and biochemical properties relative to the wild-type protein. In particular, we demonstrate that phyB-101 Pfr exhibits rapid nonphotochemical (dark) reversion to Pr that results in a lower photoequilibrium level of the active Pfr form. We conclude that this occurs in vivo as well because phyB-101 mutants are shown to lack an end-of-day-far-red hypocotyl elongation response that requires a stable Pfr species. We propose that this Pfr instability may be the primary molecular mechanism underlying the phyB-101 mutant phenotype. PMID:9437866

  14. The mechanical properties of Drosophila jump muscle expressing wild-type and embryonic Myosin isoforms.

    PubMed

    Eldred, Catherine C; Simeonov, Dimitre R; Koppes, Ryan A; Yang, Chaoxing; Corr, David T; Swank, Douglas M

    2010-04-01

    Transgenic Drosophila are highly useful for structure-function studies of muscle proteins. However, our ability to mechanically analyze transgenically expressed mutant proteins in Drosophila muscles has been limited to the skinned indirect flight muscle preparation. We have developed a new muscle preparation using the Drosophila tergal depressor of the trochanter (TDT or jump) muscle that increases our experimental repertoire to include maximum shortening velocity (V(slack)), force-velocity curves and steady-state power generation; experiments not possible using indirect flight muscle fibers. When transgenically expressing its wild-type myosin isoform (Tr-WT) the TDT is equivalent to a very fast vertebrate muscle. TDT has a V(slack) equal to 6.1 +/- 0.3 ML/s at 15 degrees C, a steep tension-pCa curve, isometric tension of 37 +/- 3 mN/mm(2), and maximum power production at 26% of isometric tension. Transgenically expressing an embryonic myosin isoform in the TDT muscle increased isometric tension 1.4-fold, but decreased V(slack) 50% resulting in no significant difference in maximum power production compared to Tr-WT. Drosophila expressing embryonic myosin jumped <50% as far as Tr-WT that, along with comparisons to frog jump muscle studies, suggests fast muscle shortening velocity is relatively more important than high tension generation for Drosophila jumping. PMID:20371321

  15. Cell-autonomous alteration of dopaminergic transmission by wild type and mutant (DeltaE) TorsinA in transgenic mice.

    PubMed

    Page, Michelle E; Bao, Li; Andre, Pierrette; Pelta-Heller, Joshua; Sluzas, Emily; Gonzalez-Alegre, Pedro; Bogush, Alexey; Khan, Loren E; Iacovitti, Lorraine; Rice, Margaret E; Ehrlich, Michelle E

    2010-09-01

    Early onset torsion dystonia is an autosomal dominant movement disorder of variable penetrance caused by a glutamic acid, i.e. DeltaE, deletion in DYT1, encoding the protein TorsinA. Genetic and structural data implicate basal ganglia dysfunction in dystonia. TorsinA, however, is diffusely expressed, and therefore the primary source of dysfunction may be obscured in pan-neuronal transgenic mouse models. We utilized the tyrosine hydroxylase (TH) promoter to direct transgene expression specifically to dopaminergic neurons of the midbrain to identify cell-autonomous abnormalities. Expression of both the human wild type (hTorsinA) and mutant (DeltaE-hTorsinA) protein resulted in alterations of dopamine release as detected by microdialysis and fast cycle voltammetry. Motor abnormalities detected in these mice mimicked those noted in transgenic mice with pan-neuronal transgene expression. The locomotor response to cocaine in both TH-hTorsinA and TH-DeltaE-hTorsinA, in the face of abnormal extracellular DA levels relative to non-transgenic mice, suggests compensatory, post-synaptic alterations in striatal DA transmission. This is the first cell-subtype-specific DYT1 transgenic mouse that can serve to differentiate between primary and secondary changes in dystonia, thereby helping to target disease therapies. PMID:20460154

  16. The Resistant-Population Cutoff (RCOFF): a New Concept for Improved Characterization of Antimicrobial Susceptibility Patterns of Non-Wild-Type Bacterial Populations

    PubMed Central

    Valsesia, Giorgia; Hombach, Michael; Maurer, Florian P.; Courvalin, Patrice; Roos, Malgorzata

    2015-01-01

    This study aimed to determine resistant-population cutoffs (RCOFFs) to allow for improved characterization of antimicrobial susceptibility patterns in bacterial populations. RCOFFs can complement epidemiological cutoff (ECOFF)-based settings of clinical breakpoints (CBPs) by systematically describing the correlation between non-wild-type and wild-type populations. We illustrate this concept by describing three paradigmatic examples of wild-type and non-wild-type Escherichia coli populations from our clinical strain database of disk diffusion diameters. The statistical determination of RCOFFs and ECOFFs and their standardized applications in antimicrobial susceptibility testing (AST) facilitates the assignment of isolates to wild-type or non-wild-type populations. This should improve the correlation of in vitro AST data and distinct antibiotic resistance mechanisms with clinical outcome facilitating the setting and validation of CBPs. PMID:25762769

  17. Wild-type p53-mediated down-modulation of interleukin 15 and interleukin 15 receptors in human rhabdomyosarcoma cells.

    PubMed Central

    De Giovanni, C.; Nanni, P.; Sacchi, A.; Soddu, S.; Manni, I.; D'Orazi, G.; Bulfone-Paus, S.; Pohl, T.; Landuzzi, L.; Nicoletti, G.; Frabetti, F.; Rossi, I.; Lollini, P. L.

    1998-01-01

    We recently reported that rhabdomyosarcoma cell lines express and secrete interleukin 15 (IL-15), a tightly regulated cytokine with IL-2-like activity. To test whether the p53-impaired function that is frequently found in this tumour type could play a role in the IL-15 production, wild-type p53 gene was transduced in the human rhabdomyosarcoma cell line RD (which harbours a mutated p53 gene), and its effect on proliferation and expression of IL-15 was studied. Arrest of proliferation was induced by wild-type p53; increased proportions of G1-arrested cells and of apoptotic cells were observed. A marked down-modulation of IL-15 expression, at both the mRNA and protein level, was found in p53-transduced cells. Because a direct effect of IL-15 on normal muscle cells has been reported, the presence of IL-15 membrane receptors was studied by cytofluorometric analysis. Rhabdomyosarcoma cells showed IL-15 membrane receptors, which are down-modulated by wild-type p53 transfected gene. In conclusion, wild-type p53 transduction in human rhabdomyosarcoma cells induces the down-modulation of both IL-15 production and IL-15 receptor expression. Images Figure 3 PMID:9862562

  18. Biomarkers predicting resistance to epidermal growth factor receptor-targeted therapy in metastatic colorectal cancer with wild-type KRAS

    PubMed Central

    Liu, Jiang; Hu, Jing; Cheng, Lei; Ren, Wei; Yang, Mi; Liu, Baorui; Xie, Li; Qian, Xiaoping

    2016-01-01

    EGFR pathway is an important therapeutic target in human tumors, including metastatic colorectal cancer (mCRC). The advent of EGFR-targeted monoclonal antibodies panitumumab and cetuximab has generated promise for the treatment of mCRC and has largely improved patients’ progression-free survival (PFS) and overall survival (OS). However, treatment with anti-EGFR monoclonal antibodies is only effective in a subset of mCRC patients with wild-type KRAS. This indicates that there are other factors affecting the efficacy of anti-EGFR monoclonal antibodies. Existing studies have demonstrated that among colorectal cancer patients with wild-type KRAS, harboring mutations of BRAF, PIK3CA, NRAS, or PTEN-null may demonstrate resistance to anti-EGFR-targeted therapy, and biomarkers detection can provide better-personalized treatment for mCRC patients. How to identify and reverse the secondary resistance to anti-EGFR monoclonal antibody therapy is also another great challenge to improve the anti-EGFR efficacy in wild-type KRAS mCRC patients. Finally, both of the molecular mechanisms of response and acquired resistance would be important for the directions of future research. This review focuses on how to further improve the predictive value of anti-EGFR therapies and how to also try and avoid futile treatment for wild-type KRAS colorectal cancer patients. PMID:26869800

  19. A comparative study of cytokinins in caryopsis development in the maize miniature 1 seed mutant and its wild type

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report here a comparative developmental profile of cytokinins, both total quantity and diversity of various forms, in relation to cell size, cell number and endoreduplication in developing caryopses of a cell wall invertase-deficient miniature1 (mn1) seed mutant and its wild type, Mn1, genotype. ...

  20. Dendritic cells transduced with wild-type p53 gene elicit potent anti-tumour immune responses

    PubMed Central

    Ishida, T; Chada, S; Stipanov, M; Nadaf, S; Ciernik, F I; Gabrilovich, D I; Carbone, D P

    1999-01-01

    In this study we have tested the concept of using wild-type p53 gene for immunotherapy of cancer. Dendritic cells (DC) were transduced with a human wild-type p53 containing recombinant adenovirus (Ad-p53). About a half of DC transduced with this virus expressed p53 protein by FACS analysis 48 h after infection. Mice immunized twice with Ad-p53 DC developed substantial cytotoxic T lymphocyte (CTL) responses against tumour cells expressing wild-type and different mutant human and murine p53 genes. Very low CTL responses were observed against target cells infected with control adenovirus (Ad-c). Immunization with Ad-p53 provided complete tumour protection in 85% of mice challenged with tumour cells expressing human mutant p53 and in 72.7% of mice challenged with tumour cells with murine mutant p53. Treatment with Ad-p53-transduced DC significantly slowed the growth of established tumours. Thus, DC transduced with wild-type p53 may be a promising new tool for the immunotherapy of cancer. PMID:10444254

  1. Receptor usage and differential downregulation of CD46 by measles virus wild-type and vaccine strains.

    PubMed

    Schneider-Schaulies, J; Schnorr, J J; Brinckmann, U; Dunster, L M; Baczko, K; Liebert, U G; Schneider-Schaulies, S; ter Meulen, V

    1995-04-25

    Recently, two cell surface molecules, CD46 and moesin, have been found to be functionally associated with measles virus (MV) infectivity of cells. We investigated the receptor usage of MV wild-type, subacute sclerosing panencephalitis, and vaccine strains and their effect on the down-regulation of CD46 after infection. We found that the infection of human cell lines with all 19 MV strains tested was inhibitable with antibodies against CD46. In contrast, not all strains of MV led to the downregulation of CD46 following infection. The group of CD46 non-downregulating strains comprised four lymphotropic wild-type isolates designated AB, DF, DL, and WTF. Since the downregulation of CD46 is caused by interaction with newly synthesized MV hemagglutinin (MV-H), we tested the capability of recombinant MV-H proteins to downregulate CD46. Recombinant MV-H proteins of MV strains Edmonston, Halle, and CM led to the down-regulation of CD46, whereas those of DL and WTF did not. This observed differential downregulation by different MV strains has profound consequences, since lack of CD46 on the cell surface leads to susceptibility of cells to complement lysis. These results suggest that lymphotropic wild-type strains of MV which do not downregulate CD46 may have an advantage for replication in vivo. The relatively weak immune response against attenuated vaccine strains of MV compared with wild-type strains might be related to this phenomenon. PMID:7732009

  2. ASSESSING POTENTIAL OF COLLETOTRICHUM ACUTATUM WILD-TYPE AND AUXOTROPHIC MUTANTS AS BIOLOGICAL FRUIT THINNING AGENTS IN CITRUS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Colletotrichum acutatum, causal agent of postbloom fruit drop of citrus, and two induced C. acutatum mutants (3-3 and 3-2) were tested as potential agents for reducing fruit load on Valencia (Citrus sinensis ) and 'Temple' orange (C.reticulata x C. sinensis). Wild-type C. acutatum (RST) and a C. gl...

  3. Differences in 23S ribosomal RNA mutations between wild-type and mutant macrolide-resistant Chlamydia trachomatis isolates

    PubMed Central

    JIANG, YONG; ZHU, HUI; YANG, LI-NA; LIU, YUAN-JUN; HOU, SHU-PING; QI, MAN-LI; LIU, QUAN-ZHONG

    2015-01-01

    The aim of the present study was to determine the in vitro susceptibility of wild-type and mutant clinical isolates of Chlamydia (C.) trachomatis strains to erythromycin, azithromycin and josamycin, and to identify the resistance-conferring 23S ribosomal (r)RNA mutations in the isolates. The wild-type resistant isolates were defined as those with minimum inhibitory concentration values above the tissue concentration of the antibiotic in the urogenital system. Furthermore, all resistant C. trachomatis isolates were exposed to sub-inhibitory concentrations of macrolides, and 13 resistant mutants were selected following serial passages. Among the 8 wild-type isolates that were resistant to erythromycin, 3 isolates had a mutation at T2611C in the 23S rRNA gene while the others did not show any 23S rRNA mutations. The selected mutant isolates showed a 4- to 16-fold reduction in in vitro sensitivities. With regard to the mutant strains, the T2611C mutation was found in 10 isolates, A2057G mutation in 6 isolates, and A2059G mutation in 1 isolate. Thus, the macrolide-resistant isolates of the wild-type strain had different mutations from those selected by exposure to sub-inhibitory concentrations of macrolides. Also, since 23S rRNA mutations were not identified in certain isolates, it was considered that other molecular mechanisms may also be responsible for the macrolide resistance of C. trachomatis. PMID:26622462

  4. Quantitative Expression Profile of Distinct Functional Regions in the Adult Mouse Brain

    PubMed Central

    Nagano, Mamoru; Uno, Kenichiro D.; Tsujino, Kaori; Hanashima, Carina; Shigeyoshi, Yasufumi; Ueda, Hiroki R.

    2011-01-01

    The adult mammalian brain is composed of distinct regions with specialized roles including regulation of circadian clocks, feeding, sleep/awake, and seasonal rhythms. To find quantitative differences of expression among such various brain regions, we conducted the BrainStars (B*) project, in which we profiled the genome-wide expression of ∼50 small brain regions, including sensory centers, and centers for motion, time, memory, fear, and feeding. To avoid confounds from temporal differences in gene expression, we sampled each region every 4 hours for 24 hours, and pooled the samples for DNA-microarray assays. Therefore, we focused on spatial differences in gene expression. We used informatics to identify candidate genes with expression changes showing high or low expression in specific regions. We also identified candidate genes with stable expression across brain regions that can be used as new internal control genes, and ligand-receptor interactions of neurohormones and neurotransmitters. Through these analyses, we found 8,159 multi-state genes, 2,212 regional marker gene candidates for 44 small brain regions, 915 internal control gene candidates, and 23,864 inferred ligand-receptor interactions. We also found that these sets include well-known genes as well as novel candidate genes that might be related to specific functions in brain regions. We used our findings to develop an integrated database (http://brainstars.org/) for exploring genome-wide expression in the adult mouse brain, and have made this database openly accessible. These new resources will help accelerate the functional analysis of the mammalian brain and the elucidation of its regulatory network systems. PMID:21858037

  5. Role of oxidative stress in rabies virus infection of adult mouse dorsal root ganglion neurons.

    PubMed

    Jackson, Alan C; Kammouni, Wafa; Zherebitskaya, Elena; Fernyhough, Paul

    2010-05-01

    Rabies virus infection of dorsal root ganglia (DRG) was studied in vitro with cultured adult mouse DRG neurons. Recent in vivo studies of transgenic mice that express the yellow fluorescent protein indicate that neuronal process degeneration, involving both dendrites and axons, occurs in mice infected with the challenge virus standard (CVS) strain of rabies virus by footpad inoculation. Because of the similarities of the morphological changes in experimental rabies and in diabetic neuropathy and other diseases, we hypothesize that neuronal process degeneration occurs as a result of oxidative stress. DRG neurons were cultured from adult ICR mice. Two days after plating, they were infected with CVS. Immunostaining was evaluated with CVS- and mock-infected cultures for neuron specific beta-tubulin, rabies virus antigen, and amino acid adducts of 4-hydroxy-2-nonenal (4-HNE) (marker of lipid peroxidation and hence oxidative stress). Neuronal viability (by trypan blue exclusion), terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, and axonal growth were also assessed with the cultures. CVS infected 33 to 54% of cultured DRG neurons. Levels of neuronal viability and TUNEL staining were similar in CVS- and mock-infected DRG neurons. There were significantly more 4-HNE-labeled puncta at 2 and 3 days postinfection in CVS-infected cultures than in mock-infected cultures, and axonal outgrowth was reduced at these time points in CVS infection. Axonal swellings with 4-HNE-labeled puncta were also associated with aggregations of actively respiring mitochondria. We have found evidence that rabies virus infection in vitro causes axonal injury of DRG neurons through oxidative stress. Oxidative stress may be important in vivo in rabies and may explain previous observations of the degeneration of neuronal processes. PMID:20181692

  6. Adult mouse model of early hepatocellular carcinoma promoted by alcoholic liver disease

    PubMed Central

    Ambade, Aditya; Satishchandran, Abhishek; Gyongyosi, Benedek; Lowe, Patrick; Szabo, Gyongyi

    2016-01-01

    AIM: To establish a mouse model of alcohol-driven hepatocellular carcinoma (HCC) that develops in livers with alcoholic liver disease (ALD). METHODS: Adult C57BL/6 male mice received multiple doses of chemical carcinogen diethyl nitrosamine (DEN) followed by 7 wk of 4% Lieber-DeCarli diet. Serum alanine aminotransferase (ALT), alpha fetoprotein (AFP) and liver Cyp2e1 were assessed. Expression of F4/80, CD68 for macrophages and Ly6G, MPO, E-selectin for neutrophils was measured. Macrophage polarization was determined by IL-1β/iNOS (M1) and Arg-1/IL-10/CD163/CD206 (M2) expression. Liver steatosis and fibrosis were measured by oil-red-O and Sirius red staining respectively. HCC development was monitored by magnetic resonance imaging, confirmed by histology. Cellular proliferation was assessed by proliferating cell nuclear antigen (PCNA). RESULTS: Alcohol-DEN mice showed higher ALTs than pair fed-DEN mice throughout the alcohol feeding without weight gain. Alcohol feeding resulted in increased ALT, liver steatosis and inflammation compared to pair-fed controls. Alcohol-DEN mice had reduced steatosis and increased fibrosis indicating advanced liver disease. Molecular characterization showed highest levels of both neutrophil and macrophage markers in alcohol-DEN livers. Importantly, M2 macrophages were predominantly higher in alcohol-DEN livers. Magnetic resonance imaging revealed increased numbers of intrahepatic cysts and liver histology confirmed the presence of early HCC in alcohol-DEN mice compared to all other groups. This correlated with increased serum alpha-fetoprotein, a marker of HCC, in alcohol-DEN mice. PCNA immunostaining revealed significantly increased hepatocyte proliferation in livers from alcohol-DEN compared to pair fed-DEN or alcohol-fed mice. CONCLUSION: We describe a new 12-wk HCC model in adult mice that develops in livers with alcoholic hepatitis and defines ALD as co-factor in HCC. PMID:27122661

  7. Stimulus control by 5methoxy-N,N-dimethyltryptamine in wild-type and CYP2D6-humanized mice

    PubMed Central

    Winter, J. C.; Amorosi, D. J.; Rice, Kenner C.; Cheng, Kejun; Yu, Ai-Ming

    2011-01-01

    In previous studies we have observed that, in comparison with wild type mice, Tg-CYP2D6 mice have increased serum levels of bufotenine [5-hydroxy-N,N-dimethyltryptamine] following the administration of 5-MeO-DMT. Furthermore, following the injection of 5-MeO-DMT, harmaline was observed to increase serum levels of bufotenine and 5-MeO-DMT in both wild-type and Tg-CYP2D6 mice. In the present investigation, 5-MeO-DMT-induced stimulus control was established in wild-type and Tg-CYP2D6 mice. The two groups did not differ in their rate of acquisition of stimulus control. When tested with bufotenine, no 5-MeO-DMT-appropriate responding was observed. In contrast, the more lipid soluble analog of bufotenine, acetylbufotenine, was followed by an intermediate level of responding. The combination of harmaline with 5-MeO-DMT yielded a statistically significant increase in 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice; a comparable increase occurred in wild-type mice. In addition, it was noted that harmaline alone was followed by a significant degree of 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice. It is concluded that wild-type and Tg-CYPD2D6 mice do not differ in terms of acquisition of stimulus control by 5-MeO-DMT or in their response to bufotenine and acetylbufotenine. In both groups of mice, harmaline was found to enhance the stimulus effects of 5-MeO-DMT. PMID:21624387

  8. Functional differences in pore properties between wild-type and cysteine-less forms of the CFTR chloride channel.

    PubMed

    Holstead, Ryan G; Li, Man-Song; Linsdell, Paul

    2011-10-01

    Studies of the structure and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel have been advanced by the development of functional channel variants in which all 18 endogenous cysteine residues have been mutated ("cys-less" CFTR). However, cys-less CFTR has a slightly higher single-channel conductance than wild-type CFTR, raising questions as to the suitability of cys-less as a model of the wild-type CFTR pore. We used site-directed mutagenesis and patch-clamp recording to investigate the origin of this conductance difference and to determine the extent of functional differences between wild-type and cys-less CFTR channel permeation properties. Our results suggest that the conductance difference is the result of a single substitution, of C343: the point mutant C343S has a conductance similar to cys-less, whereas the reverse mutation, S343C in a cys-less background, restores wild-type conductance levels. Other cysteine substitutions (C128S, C225S, C376S, C866S) were without effect. Substitution of other residues for C343 suggested that conductance is dependent on amino acid side chain volume at this position. A range of other functional pore properties, including interactions with channel blockers (Au[CN] (2) (-) , 5-nitro-2-[3-phenylpropylamino]benzoic acid, suramin) and anion permeability, were not significantly different between wild-type and cys-less CFTR. Our results suggest that functional differences between these two CFTR constructs are of limited scale and scope and result from a small change in side chain volume at position 343. These results therefore support the use of cys-less as a model of the CFTR pore region. PMID:21796426

  9. Research on the ultrafast fluorescence property of thylakoid membranes of the wild-type and mutant rice

    NASA Astrophysics Data System (ADS)

    Ren, Zhao-Yu; Xu, Xiao-Ming; Wang, Shui-Cai; Xin, Yue-Yong; He, Jun-Fang; Hou, Xun

    2003-10-01

    A high yielding rice variety mutant (Oryza sativa L., Zhenhui 249) with low chlorophyll b (Chl b) has been discovered in natural fields. It has a quality character controlled by a pair of recessive genes (nuclear gene). The partial loss of Chl b in content affects the efficiency of light harvest in a light harvest complex (LHC), thus producing the difference of the exciting energy transfer and the efficiency of photochemistry conversion between the mutant and wild-type rice in photosynthetic unit. The efficiency of utilizing light energy is higher in the mutant than that in the wild-type rice relatively. For further discussion of the above-mentioned difference and learning about the mechanism of the increase in the photochemical efficiency of the mutant, the pico-second resolution fluorescence spectrum measurement with delay-frame-scanning single photon counting technique is adopted. Thylakoid membranes of the mutant and the wild-type rice are excited by an Ar+ laser with a pulse width of 120 ps, repetition rate of 4 MHz and wavelength of 514 nm. Compared with the time and spectrum property of exciting fluorescence, conclusions of those ultrafast dynamic experiments are: 1) The speeds of the exciting energy transferred in photo-system I are faster than that in photo-system II in both samples. 2) The speeds of the exciting energy transfer of mutant sample are faster than those of the wild-type. This might be one of the major reasons why the efficiency of photosynthesis is higher in mutant than that in the wild-type rice.

  10. Stimulus control by 5-methoxy-N,N-dimethyltryptamine in wild-type and CYP2D6-humanized mice.

    PubMed

    Winter, J C; Amorosi, D J; Rice, Kenner C; Cheng, Kejun; Yu, Ai-Ming

    2011-09-01

    In previous studies we have observed that, in comparison with wild type mice, Tg-CYP2D6 mice have increased serum levels of bufotenine [5-hydroxy-N,N-dimethyltryptamine] following the administration of 5-MeO-DMT. Furthermore, following the injection of 5-MeO-DMT, harmaline was observed to increase serum levels of bufotenine and 5-MeO-DMT in both wild-type and Tg-CYP2D6 mice. In the present investigation, 5-MeO-DMT-induced stimulus control was established in wild-type and Tg-CYP2D6 mice. The two groups did not differ in their rate of acquisition of stimulus control. When tested with bufotenine, no 5-MeO-DMT-appropriate responding was observed. In contrast, the more lipid soluble analog of bufotenine, acetylbufotenine, was followed by an intermediate level of responding. The combination of harmaline with 5-MeO-DMT yielded a statistically significant increase in 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice; a comparable increase occurred in wild-type mice. In addition, it was noted that harmaline alone was followed by a significant degree of 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice. It is concluded that wild-type and Tg-CYPD2D6 mice do not differ in terms of acquisition of stimulus control by 5-MeO-DMT or in their response to bufotenine and acetylbufotenine. In both groups of mice, harmaline was found to enhance the stimulus effects of 5-MeO-DMT. PMID:21624387

  11. Protein substrates for cGMP-dependent protein phosphorylation in cilia of wild type and atalanta mutants of Paramecium.

    PubMed

    Ann, K S; Nelson, D L

    1995-01-01

    In the ciliated protozoan Paramecium, swimming direction is regulated by voltage-gated Ca2+ channels in the ciliary membrane. In response to depolarizing stimuli, intraciliary Ca2+ rises, triggering reversal of the ciliary power stroke and backward swimming. One class of Ca(2+)-unresponsive behavioral mutants of Paramecium, atalanta mutants, cannot swim backward even though they have functional Ca2+ channels in their ciliary membrane. Several atalanta mutants were characterized with regard to several Ca(2+)-dependent activities, but no significant difference between wild type and the mutants was detected. However, one allelic group, atalanta A (initially characterized by Hinrichsen and Kung [1984: Genet. Res. Camb. 43:11-20]), showed a helical swimming path of opposite handedness from that of wild-type cells when detergent-permeabilized cells ("models") were reactivated with MgATP. When cGMP-dependent protein kinase purified from wild-type cells was added to atalanta A models, the handedness of the swimming path was reversed. Cyclic GMP stimulated in vitro phosphorylation of several proteins in isolated cilia, and the pattern of phosphoproteins was very similar for wild type and atalanta mutants, with one exception: a protein of 59 kDa was phosphorylated much less in the mutant ata A. When ciliary proteins were separated by gel electrophoresis and then phosphorylated "on blot" by purified cGMP-dependent protein kinase, phosphoprotein patterns were similar in wild type and ata mutants except that a 48 kDa protein (p48) from ata A3 was more heavily phosphorylated. This difference in p48 phosphorylation was also observed with cGMP-dependent protein kinase purified from ata A3 mutant cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7796456

  12. Modulation of penetrance by the wild-type allele in dominantly inherited erythropoietic protoporphyria and acute hepatic porphyrias.

    PubMed

    Gouya, Laurent; Puy, Hervé; Robreau, Anne-Marie; Lyoumi, Said; Lamoril, Jérome; Da Silva, Vasco; Grandchamp, Bernard; Deybach, Jean-Charles

    2004-02-01

    We have recently demonstrated that in an autosomal dominant porphyria, erythropoietic protoporphyria (EPP), the coinheritance of a ferrochelatase (FECH) gene defect and of a wild-type low-expressed FECH allele is generally involved in the clinical expression of EPP. This mechanism may provide a model for phenotype modulation by minor variations in the expression of the wild-type allele in the other three autosomal dominant porphyrias that exhibit incomplete penetrance: acute intermittent porphyria (AIP), variegata porphyria (VP) and hereditary coproporphyria (HC), which are caused by partial deficiencies of hydroxy-methyl bilane synthase (HMBS), protoporphyrinogen oxidase (PPOX) and coproporphyrinogen oxidase (CPO), respectively. Given the dominant mode of inheritance of EPP, VP, AIP and HC, we first confirmed that the 200 overtly porphyric subjects (55 EPP, 58 AIP, 56 VP; 31 HC) presented a single mutation restricted to one allele (20 novel mutations and 162 known mutations). We then analysed the available single-nucleotide polymorphisms (SNPs) present at high frequencies in the general population and spreading throughout the FECH, HMBS, PPOX and the CPO genes in four case-control association studies. Finally, we explored the functional consequences of polymorphisms on the abundance of wild-type RNA, and used relative allelic mRNA determinations to find out whether low-expressed HMBS, PPOX and the CPO alleles occur in the general population. We confirm that the wild-type low-expressed allele phenomenon is usually operative in the mechanism of variable penetrance in EPP, but conclude that this is not the case in AIP and VP. For HC, the CPO mRNA determinations strongly suggest that normal CPO alleles with low-expression are present, but whether this low-expression of the wild-type allele could modulate the penetrance of a CPO gene defect in HC families remains to be ascertained. PMID:14669009

  13. Isolation of high-purity myenteric plexus from adult human and mouse gastrointestinal tract

    PubMed Central

    Grundmann, David; Klotz, Markus; Rabe, Holger; Glanemann, Matthias; Schäfer, Karl-Herbert

    2015-01-01

    The enteric nervous system (ENS) orchestrates a broad range of important gastrointestinal functions such as intestinal motility and gastric secretion. The ENS can be affected by environmental factors, diet and disease. Changes due to these alterations are often hard to evaluate in detail when whole gut samples are used. Analyses based on pure ENS tissue can more effectively reflect the ongoing changes during pathological processes. Here, we present an optimized approach for the isolation of pure myenteric plexus (MP) from adult mouse and human. To do so, muscle tissue was individually digested with a purified collagenase. After incubation and a gentle mechanical disruption step, MP networks could be collected with anatomical integrity. These tissues could be stored and used either for immediate genomic, proteomic or in vitro approaches, and enteric neurospheres could be generated and differentiated. In a pilot experiment, the influence of bacterial lipopolysaccharide on human MP was analyzed using 2-dimensional gel electrophoresis. The method also allows investigation of factors that are secreted by myenteric tissue in vitro. The isolation of pure MP in large amounts allows new analytical approaches that can provide a new perspective in evaluating changes of the ENS in experimental models, human disease and aging. PMID:25791532

  14. Expression of slow skeletal TnI in adult mouse hearts confers metabolic protection to ischemia

    PubMed Central

    Pound, Kayla M.; Arteaga, Grace M.; Fasano, Mathew; Wilder, Tanganyika; Fischer, Susan K.; Warren, Chad M.; Wende, Adam R.; Farjah, Mariam; Abel, E. Dale; Solaro, R. John; Lewandowski, E. Douglas

    2011-01-01

    Changes in metabolic and myofilament phenotypes coincide in developing hearts. Posttranslational modification of sarcomere proteins influences contractility, affecting the energetic cost of contraction. However, metabolic adaptations to sarcomeric phenotypes are not well understood, particularly during pathophysiological stress. This study explored metabolic adaptations to expression of the fetal, slow skeletal muscle troponin I (ssTnI). Hearts expressing ssTnI exhibited no significant ATP loss during 5 minutes of global ischemia, while non-transgenic littermates (NTG) showed continual ATP loss. At 7 min ischemia TG-ssTnI hearts retained 80±12% of ATP vs. 49±6% in NTG (P<0.05). Hearts expressing ssTnI also had increased AMPK phosphorylation. The mechanism of ATP preservation was augmented glycolysis. Glycolytic end products (lactate and alanine) were 38% higher in TG-ssTnI than NTG at 2 min and 27% higher at 5 min. This additional glycolysis was supported exclusively by exogenous glucose, and not glycogen. Thus, expression of a fetal myofilament protein in adult mouse hearts induced elevated anaerobic ATP production during ischemia via metabolic adaptations consistent with the resistance to hypoxia of fetal hearts. The general findings hold important relevance to both our current understanding of the association between metabolic and contractile phenotypes and the potential for invoking cardioprotective mechanisms against ischemic stress. PMID:21640727

  15. MicroRNA Clusters in the Adult Mouse Heart: Age-Associated Changes

    PubMed Central

    Zhang, Xiaomin; Azhar, Gohar; Williams, Emmanuel D.; Rogers, Steven C.; Wei, Jeanne Y.

    2015-01-01

    The microRNAs and microRNA clusters have been implicated in normal cardiac development and also disease, including cardiac hypertrophy, cardiomyopathy, heart failure, and arrhythmias. Since a microRNA cluster has from two to dozens of microRNAs, the expression of a microRNA cluster could have a substantial impact on its target genes. In the present study, the configuration and distribution of microRNA clusters in the mouse genome were examined at various inter-microRNA distances. Three important microRNA clusters that are significantly impacted during adult cardiac aging, the miR-17-92, miR-106a-363, and miR-106b-25, were also examined in terms of their genomic location, RNA transcript character, sequence homology, and their relationship with the corresponding microRNA families. Multiple microRNAs derived from the three clusters potentially target various protein components of the cdc42-SRF signaling pathway, which regulates cytoskeleton dynamics associated with cardiac structure and function. The data indicate that aging impacted the expression of both guide and passenger strands of the microRNA clusters; nutrient stress also affected the expression of the three microRNA clusters. The miR-17-92, miR-106a-363, and miR-106b-25 clusters are likely to impact the Cdc42-SRF signaling pathway and thereby affect cardiac morphology and function during pathological conditions and the aging process. PMID:26221604

  16. Aryl Hydrocarbon Receptor Activity of Tryptophan Metabolites in Young Adult Mouse Colonocytes.

    PubMed

    Cheng, Yating; Jin, Un-Ho; Allred, Clint D; Jayaraman, Arul; Chapkin, Robert S; Safe, Stephen

    2015-10-01

    The tryptophan microbiota metabolites indole-3-acetate, indole-3-aldehyde, indole, and tryptamine are aryl hydrocarbon receptor (AhR) ligands, and in this study we investigated their AhR agonist and antagonist activities in nontransformed young adult mouse colonocyte (YAMC) cells. Using Cyp1a1 mRNA as an Ah-responsive end point, we observed that the tryptophan metabolites were weak AhR agonists and partial antagonists in YAMC cells, and the pattern of activity was different from that previously observed in CaCo2 colon cancer cells. However, expansion of the end points to other Ah-responsive genes including the Cyp1b1, the AhR repressor (Ahrr), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP-ribose) polymerase (TiParp) revealed a highly complex pattern of AhR agonist/antagonist activities that were both ligand- and gene-dependent. For example, the magnitude of induction of Cyp1b1 mRNA was similar for TCDD, tryptamine, and indole-3-acetate, whereas lower induction was observed for indole and indole-3-aldehyde was inactive. These results suggest that the tryptophan metabolites identified in microbiota are selective AhR modulators. PMID:25873348

  17. Neurotoxic effects of ochratoxin A on the subventricular zone of adult mouse brain.

    PubMed

    Paradells, Sara; Rocamonde, Brenda; Llinares, Cristina; Herranz-Pérez, Vicente; Jimenez, Misericordia; Garcia-Verdugo, Jose Manuel; Zipancic, Ivan; Soria, Jose Miguel; Garcia-Esparza, Ma Angeles

    2015-07-01

    Ochratoxin A (OTA), a mycotoxin that was discovered as a secondary metabolite of the fungal species Aspergillus and Penicillium, is a common contaminant in food and animal feed. This mycotoxin has been described as teratogenic, carcinogenic, genotoxic, immunotoxic and has been proven a potent neurotoxin. Other authors have previously reported the effects of OTA in different structures of the central nervous system as well as in some neurogenic regions. However, the impact of OTA exposure in the subventricular zone (SVZ) has not been assessed yet. To elucidate whether OTA affects neural precursors of the mouse SVZ we investigated, in vitro and in vivo, the effects of OTA exposure on the SVZ and on the neural precursors obtained from this neurogenic niche. In this work, we prove the cumulative effect of OTA exposure on proliferation, differentiation and depletion of neural stem cells cultured from the SVZ. In addition, we corroborated these results in vivo by immunohistochemistry and electron microscopy. As a result, we found a significant alteration in the proliferation process, which was evidenced by a decrease in the number of 5-bromo-2-deoxyuridine-positive cells and glial cells, as well as, a significant decrease in the number of neuroblasts in the SVZ. To summarize, in this study we demonstrate how OTA could be a threat to the developing and the adult SVZ through its impact in cell viability, proliferation and differentiation in a dose-dependent manner. PMID:25256750

  18. Adult pallium transcriptomes surprise in not reflecting predicted homologies across diverse chicken and mouse pallial sectors

    PubMed Central

    Belgard, T. Grant; Montiel, Juan F.; Wang, Wei Zhi; García-Moreno, Fernando; Ponting, Chris P.; Molnár, Zoltán

    2013-01-01

    The thorniest problem in comparative neurobiology is the identification of the particular brain region of birds and reptiles that corresponds to the mammalian neocortex [Butler AB, Reiner A, Karten HJ (2011) Ann N Y Acad Sci 1225:14–27; Wang Y, Brzozowska-Prechtl A, Karten HJ (2010) Proc Natl Acad Sci USA 107(28):12676–12681]. We explored which genes are actively transcribed in the regions of controversial ancestry in a representative bird (chicken) and mammal (mouse) at adult stages. We conducted four analyses comparing the expression patterns of their 5,130 most highly expressed one-to-one orthologous genes that considered global patterns of expression specificity, strong gene markers, and coexpression networks. Our study demonstrates transcriptomic divergence, plausible convergence, and, in two exceptional cases, conservation between specialized avian and mammalian telencephalic regions. This large-scale study potentially resolves the complex relationship between developmental homology and functional characteristics on the molecular level and settles long-standing evolutionary debates. PMID:23878249

  19. Adult pallium transcriptomes surprise in not reflecting predicted homologies across diverse chicken and mouse pallial sectors.

    PubMed

    Belgard, T Grant; Montiel, Juan F; Wang, Wei Zhi; García-Moreno, Fernando; Margulies, Elliott H; Ponting, Chris P; Molnár, Zoltán

    2013-08-01

    The thorniest problem in comparative neurobiology is the identification of the particular brain region of birds and reptiles that corresponds to the mammalian neocortex [Butler AB, Reiner A, Karten HJ (2011) Ann N Y Acad Sci 1225:14-27; Wang Y, Brzozowska-Prechtl A, Karten HJ (2010) Proc Natl Acad Sci USA 107(28):12676-12681]. We explored which genes are actively transcribed in the regions of controversial ancestry in a representative bird (chicken) and mammal (mouse) at adult stages. We conducted four analyses comparing the expression patterns of their 5,130 most highly expressed one-to-one orthologous genes that considered global patterns of expression specificity, strong gene markers, and coexpression networks. Our study demonstrates transcriptomic divergence, plausible convergence, and, in two exceptional cases, conservation between specialized avian and mammalian telencephalic regions. This large-scale study potentially resolves the complex relationship between developmental homology and functional characteristics on the molecular level and settles long-standing evolutionary debates. PMID:23878249

  20. Enzymatic processing by MMP-2 and MMP-9 of wild-type and mutated mouse β-dystroglycan.

    PubMed

    Sbardella, Diego; Inzitari, Rosanna; Iavarone, Federica; Gioia, Magda; Marini, Stefano; Sciandra, Francesca; Castagnola, Massimo; Van den Steen, Philippe E; Opdenakker, Ghislain; Giardina, Bruno; Brancaccio, Andrea; Coletta, Massimo; Bozzi, Manuela

    2012-12-01

    Dystroglycan (DG) is a membrane-associated protein complex formed by two noncovalently linked subunits, α-DG, a highly glycosylated extracellular protein, and β-DG, a transmembrane protein. The interface between the two DG subunits, which is crucial to maintain the integrity of the plasma membrane, involves the C-terminal domain of α-DG and the N-terminal extracellular domain of β-DG. It is well known that under both, physiological and pathological conditions, gelatinases (i.e. MMP-9 and/or MMP-2) can degrade DG, disrupting the connection between the extracellular matrix and the cytoskeleton. However, the molecular mechanisms and the exact cleavage sites underlying these events are still largely unknown. In a previous study, we have characterized the enzymatic digestion of the murine β-DG ectodomain by gelatinases, identifying a main cleavage site on the β-DG ectodomain produced by MMP-9. In this article, we have deepened the pattern of the β-DG ectodomain digestion by MMP-2 by using a combined approach based on SDS-PAGE, Orbitrap, and HPLC-ESI-IT mass spectrometry. Furthermore, we have characterized the kineticparameters of the digestion of some β-DG ectodomain mutants by gelatinases. PMID:23129308

  1. Comparative proteomic profiling of dystroglycan-associated proteins in wild type, mdx, and Galgt2 transgenic mouse skeletal muscle.

    PubMed

    Yoon, Jung Hae; Johnson, Eric; Xu, Rui; Martin, Laura T; Martin, Paul T; Montanaro, Federica

    2012-09-01

    Dystroglycan is a major cell surface glycoprotein receptor for the extracellular matrix in skeletal muscle. Defects in dystroglycan glycosylation cause muscular dystrophy and alterations in dystroglycan glycosylation can impact extracellular matrix binding. Here we describe an immunoprecipitation technique that allows isolation of beta dystroglycan with members of the dystrophin-associated protein complex (DAPC) from detergent-solubilized skeletal muscle. Immunoprecipitation, coupled with shotgun proteomics, has allowed us to identify new dystroglycan-associated proteins and define changed associations that occur within the DAPC in dystrophic skeletal muscles. In addition, we describe changes that result from overexpression of Galgt2, a normally synaptic muscle glycosyltransferase that can modify alpha dystroglycan and inhibit the development of muscular dystrophy when it is overexpressed. These studies identify new dystroglycan-associated proteins that may participate in dystroglycan's roles, both positive and negative, in muscular dystrophy. PMID:22775139

  2. Assessing benzene-induced toxicity on wild type Euglena gracilis Z and its mutant strain SMZ.

    PubMed

    Peng, Cheng; Arthur, Dionne M; Sichani, Homa Teimouri; Xia, Qing; Ng, Jack C

    2013-11-01

    Benzene is a representative member of volatile organic compounds and has been widely used as an industrial solvent. Groundwater contamination of benzene may pose risks to human health and ecosystems. Detection of benzene in the groundwater using chemical analysis is expensive and time consuming. In addition, biological responses to environmental exposures are uninformative using such analysis. Therefore, the aim of this study was to employ a microorganism, Euglena gracilis (E. gracilis) as a putative model to monitor the contamination of benzene in groundwater. To this end, we examined the wild type of E. gracilis Z and its mutant form, SMZ in their growth rate, morphology, chlorophyll content, formation of reactive oxygen species (ROS) and DNA damage in response to benzene exposure. The results showed that benzene inhibited cell growth in a dose response manner up to 48 h of exposure. SMZ showed a greater sensitivity compared to Z in response to benzene exposure. The difference was more evident at lower concentrations of benzene (0.005-5 μM) where growth inhibition occurred in SMZ but not in Z cells. We found that benzene induced morphological changes, formation of lipofuscin, and decreased chlorophyll content in Z strain in a dose response manner. No significant differences were found between the two strains in ROS formation and DNA damage by benzene at concentrations affecting cell growth. Based on these results, we conclude that E. gracilis cells were sensitive to benzene-induced toxicities for certain endpoints such as cell growth rate, morphological change, depletion of chlorophyll. Therefore, it is a potentially suitable model for monitoring the contamination of benzene and its effects in the groundwater. PMID:24034892

  3. Direct conversion of xylan to butanol by a wild-type Clostridium species strain G117.

    PubMed

    Yan, Yu; Basu, Anindya; Li, Tinggang; He, Jianzhong

    2016-08-01

    Lignocellulosic biomass has great potential for use as a carbon source for the production of second-generation biofuels by solventogenic bacteria. Here we describe the production of butanol by a newly discovered wild-type Clostridium species strain G117 with xylan as the sole carbon source for fermentation. Strain G117 produced 0.86 ± 0.07 g/L butanol and 53.4 ± 0.05 mL hydrogen directly from 60 g/L xylan provided that had undergone no prior enzymatic hydrolysis. After process optimization, the amount of butanol produced from xylan was increased to 1.24 ± 0.37 g/L. In contrast to traditional acetone-butanol-ethanol (ABE) solventogenic fermentation, xylan supported fermentation in strain G117 and negligible amount of acetone was produced. The expression of genes normally associated with acetone production (adc and ctfB2) were down-regulated compared to xylose fed cultures. This lack of acetone production may greatly simplify downstream separation process. Moreover, higher amount of butanol (2.94 g/L) was produced from 16.99 g/L xylo-oligosaccharides, suggesting a major role for strain G117 in butanol production from xylan and its oligosaccharides. The unique ability of strain G117 to produce a considerable amount of butanol directly from xylan without producing undesirable fermentation byproducts opens the door to the possibility of cost-effective biofuels production in a single step. Biotechnol. Bioeng. 2016;113: 1702-1710. © 2016 Wiley Periodicals, Inc. PMID:26803924

  4. Wild-type p53 and p73 negatively regulate expression of proliferation related genes.

    PubMed

    Scian, M J; Carchman, E H; Mohanraj, L; Stagliano, K E R; Anderson, M A E; Deb, D; Crane, B M; Kiyono, T; Windle, B; Deb, S P; Deb, S

    2008-04-17

    When normal cells come under stress, the wild-type (WT) p53 level increases resulting in the regulation of gene expression responsible for growth arrest or apoptosis. Here we show that elevated levels of WT p53 or its homologue, p73, inhibit expression of a number of cell cycle regulatory and growth promoting genes. Our analysis also identified a group of genes whose expression is differentially regulated by WT p53 and p73. We have infected p53-null H1299 human lung carcinoma cells with recombinant adenoviruses expressing WT p53, p73 or beta-galactosidase, and have undertaken microarray hybridization analyses to identify genes whose expression profile is altered by p53 or p73. Quantitative real-time PCR verified the repression of E2F-5, centromere protein A and E, minichromosome maintenance proteins (MCM)-2, -3, -5, -6 and -7 and human CDC25B after p53 expression. 5-Fluorouracil treatment of colon carcinoma HCT116 cells expressing WT p53 results in a reduction of the cyclin B2 protein level suggesting that DNA damage may indeed cause repression of these genes. Transient transcriptional assays verified that WT p53 repressed promoters of a number of these genes. Interestingly, a gain-of-function p53 mutant instead upregulated a number of these promoters in transient transfection. Using promoter deletion mutants of MCM-7 we have found that WT p53-mediated repression needs a minimal promoter that contains a single E2F site and surrounding sequences. However, a single E2F site cannot be significantly repressed by WT p53. Many of the genes identified are also repressed by p21. Thus, our work shows that WT p53 and p73 repress a number of growth-related genes and that in many instances this repression may be through the induction of p21. PMID:17982488

  5. MicroRNA-based Therapeutic Strategies for Targeting Mutant and Wild Type RAS in Cancer

    PubMed Central

    Sharma, Sriganesh B.; Ruppert, J. Michael

    2015-01-01

    MicroRNAs (miRs) have been causally implicated in the progression and development of a wide variety of cancers. miRs modulate the activity of key cell signaling networks by regulating the translation of pathway component proteins. Thus, the pharmacological targeting of miRs that regulate cancer cell signaling networks, either by promoting (using miR-supplementation) or by suppressing (using anti-sense oligonucleotide based strategies) miR activity is an area of intense research. The RAS-Extracellular signal regulated kinase (ERK) pathway represents a major miR-regulated signaling network that endows cells with some of the classical hallmarks of cancer, and is often inappropriately activated in malignancies by somatic genetic alteration through point mutation or alteration of gene copy number. In addition, recent progress indicates that many tumors may be deficient in GTPase activating proteins (GAPs) due to the collaborative action of oncogenic microRNAs. Recent studies also suggest that in tumors harboring a mutant RAS allele there is a critical role for wild type RAS proteins in determining overall RAS-ERK pathway activity. Together, these two advances comprise a new opportunity for therapeutic intervention. In this review, we evaluate miR-based therapeutic strategies for modulating RAS-ERK signaling in cancers, in particular for more direct modulation of RAS-GTP levels, with the potential to complement current strategies in order to yield more durable treatment responses. To this end, we discuss the potential for miR-based therapies focused on three prominent miRs including the pan-RAS regulator let-7 and the GAP regulator comprised of miR-206 and miR-21 (miR-206/21). PMID:26284568

  6. Genistein potentiates wild-type and delta F508-CFTR channel activity.

    PubMed

    Hwang, T C; Wang, F; Yang, I C; Reenstra, W W

    1997-09-01

    Effects of genistein on wild-type (wt) and delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) were studied in NIH/3T3 cells stably transfected with wt or mutant CFTR cDNA. As measured by I- efflux, half-maximal concentration of agonist (K1/2) for forskolin-dependent activation was greater for delta F508-CFTR than wt-CFTR. Genistein decreased the K1/2 for both forms of the channel and increased the maximal activity of delta F508-CFTR by 3.7-fold. In cell-attached patches, 10 microM forskolin induced minimal delta F508-CFTR activity with characteristic prolonged closed times (estimated time constant, > 30 s). Genistein increased the forskolin-induced macroscopic currents of wt-CFTR and delta F508-CFTR by 3- and 19-fold, respectively. Variance analysis suggested that in the presence of forskolin and genistein the open probabilities (Po) of wt- and delta F508-CFTR were identical. In single-channel studies, at maximal adenosine 3',5'-cyclic monophosphate (cAMP) stimulation, genistein increased the Po of wt-CFTR by prolonging the open time, but, at submaximal cAMP stimulation, the Po was increased by prolonging the open time and shortening the closed time. In excised patches with CFTR channels preactivated in the cell-attached mode, genistein increased ATP-dependent wt- and delta F508-CFTR current about twofold by prolonging the open time. Our results thus suggest that phosphorylation-dependent activation of delta F508-CFTR is defective and that genistein corrects this defect at least in part by binding to the CFTR protein. PMID:9316420

  7. Primary charge separation within P870* in wild type and heterodimer mutants in femtosecond time domain.

    PubMed

    Khatypov, R A; Khmelnitskiy, A Yu; Khristin, A M; Fufina, T Yu; Vasilieva, L G; Shuvalov, V A

    2012-08-01

    Primary charge separation dynamics in the reaction center (RC) of purple bacterium Rhodobacter sphaeroides and its P870 heterodimer mutants have been studied using femtosecond time-resolved spectroscopy with 20 and 40fs excitation at 870nm at 293K. Absorbance increase in the 1060-1130nm region that is presumably attributed to P(A)(δ+) cation radical molecule as a part of mixed state with a charge transfer character P*(P(A)(δ+)P(B)(δ-)) was found. This state appears at 120-180fs time delay in the wild type RC and even faster in H(L173)L and H(M202)L heterodimer mutants and precedes electron transfer (ET) to B(A) bacteriochlorophyll with absorption band at 1020nm in WT. The formation of the P(A)(δ+)B(A)(δ-) state is a result of the electron transfer from P*(P(A)(δ+)P(B)(δ-)) to the primary electron acceptor B(A) (still mixed with P*) with the apparent time delay of ~1.1ps. Next step of ET is accompanied by the 3-ps appearance of bacteriopheophytin a(-) (H(A)(-)) band at 960nm. The study of the wave packet formation upon 20-fs illumination has shown that the vibration energy of the wave packet promotes reversible overcoming of an energy barrier between two potential energy surfaces P* and P*(P(A)(δ+)B(A)(δ-)) at ~500fs. For longer excitation pulses (40fs) this promotion is absent and tunneling through an energy barrier takes about 3ps. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. PMID:22209778

  8. Profiling the RNA editomes of wild-type C. elegans and ADAR mutants.

    PubMed

    Zhao, Han-Qing; Zhang, Pan; Gao, Hua; He, Xiandong; Dou, Yanmei; Huang, August Y; Liu, Xi-Ming; Ye, Adam Y; Dong, Meng-Qiu; Wei, Liping

    2015-01-01

    RNA editing increases transcriptome diversity through post-transcriptional modifications of RNA. Adenosine deaminases that act on RNA (ADARs) catalyze the adenosine-to-inosine (A-to-I) conversion, the most common type of RNA editing in higher eukaryotes. Caenorhabditis elegans has two ADARs, ADR-1 and ADR-2, but their functions remain unclear. Here, we profiled the RNA editomes of C. elegans at different developmental stages of wild-type and ADAR mutants. We developed a new computational pipeline with a "bisulfite-seq-mapping-like" step and achieved a threefold increase in identification sensitivity. A total of 99.5% of the 47,660 A-to-I editing sites were found in clusters. Of the 3080 editing clusters, 65.7% overlapped with DNA transposons in noncoding regions and 73.7% could form hairpin structures. The numbers of editing sites and clusters were highest at the L1 and embryonic stages. The editing frequency of a cluster positively correlated with the number of editing sites within it. Intriguingly, for 80% of the clusters with 10 or more editing sites, almost all expressed transcripts were edited. Deletion of adr-1 reduced the editing frequency but not the number of editing clusters, whereas deletion of adr-2 nearly abolished RNA editing, indicating a modulating role of ADR-1 and an essential role of ADR-2 in A-to-I editing. Quantitative proteomics analysis showed that adr-2 mutant worms altered the abundance of proteins involved in aging and lifespan regulation. Consistent with this finding, we observed that worms lacking RNA editing were short-lived. Taken together, our results reveal a sophisticated landscape of RNA editing and distinct modes of action of different ADARs. PMID:25373143

  9. Targeting Mdmx to treat breast cancers with wild-type p53

    PubMed Central

    Haupt, S; Buckley, D; Pang, J-MB; Panimaya, J; Paul, P J; Gamell, C; Takano, E A; Ying Lee, Y; Hiddingh, S; Rogers, T-M; Teunisse, A F A S; Herold, M J; Marine, J-C; Fox, S B; Jochemsen, A; Haupt, Y

    2015-01-01

    The function of the tumor suppressor p53 is universally compromised in cancers. It is the most frequently mutated gene in human cancers (reviewed). In cases where p53 is not mutated, alternative regulatory pathways inactivate its tumor suppressive functions. This is primarily achieved through elevation in the expression of the key inhibitors of p53: Mdm2 or Mdmx (also called Mdm4) (reviewed). In breast cancer (BrCa), the frequency of p53 mutations varies markedly between the different subtypes, with basal-like BrCas bearing a high frequency of p53 mutations, whereas luminal BrCas generally express wild-type (wt) p53. Here we show that Mdmx is unexpectedly highly expressed in normal breast epithelial cells and its expression is further elevated in most luminal BrCas, whereas p53 expression is generally low, consistent with wt p53 status. Inducible knockdown (KD) of Mdmx in luminal BrCa MCF-7 cells impedes the growth of these cells in culture, in a p53-dependent manner. Importantly, KD of Mdmx in orthotopic xenograft transplants resulted in growth inhibition associated with prolonged survival, both in a preventative model and also in a treatment model. Growth impediment in response to Mdmx KD was associated with cellular senescence. The growth inhibitory capacity of Mdmx KD was recapitulated in an additional luminal BrCa cell line MPE600, which expresses wt p53. Further, the growth inhibitory capacity of Mdmx KD was also demonstrated in the wt p53 basal-like cell line SKBR7 line. These results identify Mdmx growth dependency in wt p53 expressing BrCas, across a range of subtypes. Based on our findings, we propose that Mdmx targeting is an attractive strategy for treating BrCas harboring wt p53. PMID:26181202

  10. Selective modulation of wild type receptor functions by mutants of G-protein-coupled receptors.

    PubMed

    Le Gouill, C; Parent, J L; Caron, C A; Gaudreau, R; Volkov, L; Rola-Pleszczynski, M; Stanková, J

    1999-04-30

    Members of the G-protein-coupled receptor (GPCR) family are involved in most aspects of higher eukaryote biology, and mutations in their coding sequence have been linked to several diseases. In the present study, we report that mutant GPCR can affect the functional properties of the co-expressed wild type (WT) receptor. Mutants of the human platelet-activating factor receptor that fail to show any detectable ligand binding (N285I and K298stop) or coupling to a G-protein (D63N, D289A, and Y293A) were co-expressed with the WT receptor in Chinese hamster ovary and COS-7 cells. In this context, N285I and K298stop mutant receptors inhibited 3H-WEB2086 binding and surface expression. Co-transfection with D63N resulted in a constitutively active receptor phenotype. Platelet-activating factor-induced inositol phosphate production in cells transfected with a 1:1 ratio of WT:D63N was higher than with the WT cDNA alone but was abolished with a 1:3 ratio. We confirmed that these findings could be extended to other GPCRs by showing that co-expression of the WT C-C chemokine receptor 2b with a carboxyl-terminal deletion mutant (K311stop), resulted in a decreased affinity and responsiveness to MCP-1. A better understanding of this phenomenon could lead to important tools for the prevention or treatment of certain diseases. PMID:10212233

  11. PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina.

    PubMed

    Hickmott, Jack W; Chen, Chih-Yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

    2016-01-01

    Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia. PMID:27556059

  12. PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina

    PubMed Central

    Hickmott, Jack W; Chen, Chih-yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

    2016-01-01

    Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia. PMID:27556059

  13. Vitamin D2-enriched button mushroom (Agaricus bisporus) improves memory in both wild type and APPswe/PS1dE9 transgenic mice.

    PubMed

    Bennett, Louise; Kersaitis, Cindy; Macaulay, Stuart Lance; Münch, Gerald; Niedermayer, Garry; Nigro, Julie; Payne, Matthew; Sheean, Paul; Vallotton, Pascal; Zabaras, Dimitrios; Bird, Michael

    2013-01-01

    Vitamin D deficiency is widespread, affecting over 30% of adult Australians, and increasing up to 80% for at-risk groups including the elderly (age>65). The role for Vitamin D in development of the central nervous system is supported by the association between Vitamin D deficiency and incidence of neurological and psychiatric disorders including Alzheimer's disease (AD). A reported positive relationship between Vitamin D status and cognitive performance suggests that restoring Vitamin D status might provide a cognitive benefit to those with Vitamin D deficiency. Mushrooms are a rich source of ergosterol, which can be converted to Vitamin D2 by treatment with UV light, presenting a new and convenient dietary source of Vitamin D2. We hypothesised that Vitamin D2-enriched mushrooms (VDM) could prevent the cognitive and pathological abnormalities associated with dementia. Two month old wild type (B6C3) and AD transgenic (APPSwe/PS1dE9) mice were fed a diet either deficient in Vitamin D2 or a diet which was supplemented with VDM, containing 1±0.2 µg/kg (∼54 IU/kg) vitamin D2, for 7 months. Effects of the dietary intervention on memory were assessed pre- and post-feeding. Brain sections were evaluated for amyloid β (Aβ) plaque loads and inflammation biomarkers using immuno-histochemical methods. Plasma vitamin D metabolites, Aβ40, Aβ42, calcium, protein and cholesterol were measured using biochemical assays. Compared with mice on the control diet, VDM-fed wild type and AD transgenic mice displayed improved learning and memory, had significantly reduced amyloid plaque load and glial fibrillary acidic protein, and elevated interleukin-10 in the brain. The results suggest that VDM might provide a dietary source of Vitamin D2 and other bioactives for preventing memory-impairment in dementia. This study supports the need for a randomised clinical trial to determine whether or not VDM consumption can benefit cognitive performance in the wider population. PMID:24204618

  14. Dynamic expression of TrkB receptor protein on proliferating and maturing cells in the adult mouse dentate gyrus

    PubMed Central

    Donovan, Michael H.; Yamaguchi, Masahiro; Eisch, Amelia J.

    2008-01-01

    Brain-derived neurotrophic factor (BDNF) is implicated in regulation of adult hippocampal neurogenesis, presumably via its primary receptor, TrkB, but controversy exists about how BDNF affects neurogenesis (e.g. proliferation vs. survival/differentiation). This controversy arises, in part, due to the lack of information about if and when TrkB is expressed on adult neural precursors in vivo. Using multiple methods to analyze proliferating and maturing cells in the adult mouse subgranular zone (SGZ), we find that the proportion of proliferating cells that are TrkB-IR is low and it remains low for at least one week following BrdU labeling, but increases as neuroblasts mature. Use of the nestin-GFP transgenic mouse revealed the likelihood of being TrkB-IR increased with presumed maturity of the cell type. Stem-like cells, which rarely divide, were likely to express TrkB. However, early progenitors and late progenitors, which are still in the cell cycle had rare TrkB expression. Immature neuroblasts, however, were more likely to express TrkB, especially as their morphology became more mature. Taken together, these findings emphasize that expression of TrkB protein is closely linked to progression towards neuronal maturity. This provides evidence that maturing cells but not proliferating cells in the adult mouse SGZ have the molecular machinery necessary to respond directly to BDNF. Furthermore, these findings lay critical groundwork for further exploration of the role of BDNF-TrkB signaling in regulation of adult hippocampal neurogenesis. PMID:18240316

  15. Basilar Membrane Measurements from Wild Type, Prestin 499, and Prestin KO mice

    NASA Astrophysics Data System (ADS)

    Weddell, Thomas; Mellado-Lagarde, Marcia; Lukashkina, Victoria; Lukashkin, Andrei; Zuo, Jian; Russell, Ian

    2011-11-01

    It has been predicted that a nonfunctional prestin in the mammalian cochlea would produce a basilar membrane response at lower characteristic frequency, as we see in the prestin knock-out mouse, but with a reduced sensitivity that would reflect an enhanced coupling between basilar membrane and reticular lamina and inner hair cell stereocilia. We demonstrate here that this is the case in measurements from the 499 mouse where prestin in the lateral membrane of the outer hair cells is present but effectively silenced.

  16. Ultrasonic vocalizations of adult male Foxp2-mutant mice: behavioral contexts of arousal and emotion.

    PubMed

    Gaub, S; Fisher, S E; Ehret, G

    2016-02-01

    Adult mouse ultrasonic vocalizations (USVs) occur in multiple behavioral and stimulus contexts associated with various levels of arousal, emotion and social interaction. Here, in three experiments of increasing stimulus intensity (water; female urine; male interacting with adult female), we tested the hypothesis that USVs of adult males express the strength of arousal and emotion via different USV parameters (18 parameters analyzed). Furthermore, we analyzed two mouse lines with heterozygous Foxp2 mutations (R552H missense, S321X nonsense), known to produce severe speech and language disorders in humans. These experiments allowed us to test whether intact Foxp2 function is necessary for developing full adult USV repertoires, and whether mutations of this gene influence instinctive vocal expressions based on arousal and emotion. The results suggest that USV calling rate characterizes the arousal level, while sound pressure and spectrotemporal call complexity (overtones/harmonics, type of frequency jumps) may provide indices of levels of positive emotion. The presence of Foxp2 mutations did not qualitatively affect the USVs; all USV types that were found in wild-type animals also occurred in heterozygous mutants. However, mice with Foxp2 mutations displayed quantitative differences in USVs as compared to wild-types, and these changes were context dependent. Compared to wild-type animals, heterozygous mutants emitted mainly longer and louder USVs at higher minimum frequencies with a higher occurrence rate of overtones/harmonics and complex frequency jump types. We discuss possible hypotheses about Foxp2 influence on emotional vocal expressions, which can be investigated in future experiments using selective knockdown of Foxp2 in specific brain circuits. PMID:26566793

  17. Hepatocyte nuclear factor 4α is required for cell differentiation and homeostasis in the adult mouse gastric epithelium.

    PubMed

    Moore, Benjamin D; Khurana, Shradha S; Huh, Won Jae; Mills, Jason C

    2016-08-01

    We have previously shown that the sequential transcription factors Xbp1→Mist1 (Bhlha15) govern the ultrastructural maturation of the secretory apparatus in enzyme-secreting zymogenic chief cells (ZCs) in the gastric unit. Here we sought to identify transcriptional regulators upstream of X-box binding protein 1 (XBP1) and MIST1. We used immunohistochemistry to characterize Hnf4α(flox/flox) adult mouse stomachs after tamoxifen-induced deletion of Hnf4α We used qRT-PCR, Western blotting, and chromatin immunoprecipitation to define the molecular interaction between hepatocyte nuclear factor 4 alpha (HNF4α) and Xbp1 in mouse stomach and human gastric cells. We show that HNF4α protein is expressed in pit (foveolar) cells, mucous neck cells, and zymogenic chief cells (ZCs) of the corpus gastric unit. Loss of HNF4α in adult mouse stomach led to reduced ZC size and ER content, phenocopying previously characterized effects of Xbp1 deletion. However, HNF4α(Δ/Δ) stomachs also exhibited additional phenotypes including increased proliferation in the isthmal stem cell zone and altered mucous neck cell migration, indicating a role of HNF4α in progenitor cells as well as in ZCs. HNF4α directly occupies the Xbp1 promoter locus in mouse stomach, and forced HNF4α expression increased abundance of XBP1 mRNA in human gastric cancer cells. Finally, as expected, loss of HNF4α caused decreased Xbp1 and Mist1 expression in mouse stomachs. We show that HNF4α regulates homeostatic proliferation in the gastric epithelium and is both necessary and sufficient for the upstream regulation of the Xbp1→Mist1 axis in maintenance of ZC secretory architecture. PMID:27340127

  18. PHEX Mimetic (SPR4-Peptide) Corrects and Improves HYP and Wild Type Mice Energy-Metabolism

    PubMed Central

    Zelenchuk, Lesya V.; Hedge, Anne-Marie; Rowe, Peter S. N.

    2014-01-01

    Context PHEX or DMP1 mutations cause hypophosphatemic-rickets and altered energy metabolism. PHEX binds to DMP1-ASARM-motif to form a complex with α5β3 integrin that suppresses FGF23 expression. ASARM-peptides increase FGF23 by disrupting the PHEX-DMP1-Integrin complex. We used a 4.2 kDa peptide (SPR4) that binds to ASARM-peptide/motif to study the DMP1-PHEX interaction and to assess SPR4 for the treatment of energy metabolism defects in HYP and potentially other bone-mineral disorders. Design Subcutaneously transplanted osmotic pumps were used to infuse SPR4-peptide or vehicle (VE) into wild-type mice (WT) and HYP-mice (PHEX mutation) for 4 weeks. Results SPR4 partially corrected HYP mice hypophosphatemia and increased serum 1.25(OH)2D3. Serum FGF23 remained high and PTH was unaffected. WT-SPR4 mice developed hypophosphatemia and hypercalcemia with increased PTH, FGF23 and 1.25(OH)2D3. SPR4 increased GAPDH HYP-bone expression 60× and corrected HYP-mice hyperglycemia and hypoinsulinemia. HYP-VE serum uric-acid (UA) levels were reduced and SPR4 infusion suppressed UA levels in WT-mice but not HYP-mice. SPR4 altered leptin, adiponectin, and sympathetic-tone and increased the fat mass/weight ratio for HYP and WT mice. Expression of perlipin-2 a gene involved in obesity was reduced in HYP-VE and WT-SPR4 mice but increased in HYP-SPR4 mice. Also, increased expression of two genes that inhibit insulin-signaling, ENPP1 and ESP, occurred with HYP-VE mice. In contrast, SPR4 reduced expression of both ENPP1 and ESP in WT mice and suppressed ENPP1 in HYP mice. Increased expression of FAM20C and sclerostin occurred with HYP-VE mice. SPR4 suppressed expression of FAM20C and sclerostin in HYP and WT mice. Conclusions ASARM peptides and motifs are physiological substrates for PHEX and modulate osteocyte PHEX-DMP1-α5β3-integrin interactions and thereby FGF23 expression. These interactions also provide a nexus that regulates bone and energy metabolism. SPR4 suppression of

  19. Wild-type phosphoribosylpyrophosphate synthase (PRS) from Mycobacterium tuberculosis: a bacterial class II PRS?

    PubMed

    Breda, Ardala; Martinelli, Leonardo K B; Bizarro, Cristiano V; Rosado, Leonardo A; Borges, Caroline B; Santos, Diógenes S; Basso, Luiz A

    2012-01-01

    The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of P(i). ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of P(i) would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of

  20. Subcellular localization of Mayven following expression of wild type and mutant EGFP tagged cDNAs

    PubMed Central

    2010-01-01

    Background Process formation by glial cells is crucial to their function. Mayven, an actin binding, multi-domain polypeptide, and member of the BTB-BACK-Kelch family have been shown to be important in oligodendrocyte process extension. To assess the role of Mayven in neural cell process extension we have tracked the subcellular distribution of exogenous Mayven following expression of a rat Mayven -EGFP cDNA in a variety of neural cell backgrounds and specifically in OEC tranfectants following drug treatment to disrupt the integrity of the cytoskeleton. A comparison was made between the subcellular localization following transient transfection of OECs with full-length Mayven cDNA and a series of mutant domain constructs. Results The subcellular location of Mayven in OEC transfectants showed a characteristic distribution with intense foci of staining towards the process tips corresponding to regions of accumulated Mayven overlapping in part with lammelipodial actin and was absent from the filipodia and the outer membrane. This signature pattern was also observed in Schwann cells, Oli-Neu cells, astrocytes and the neuroblastoma cell line B104 transfectants and resembled the exogenous and endogenous Mayven distribution in oligodendrocytes. This contrasted with the localization pattern in non-neural cells. There was a re-localization of Mayven in OEC transfectants following drug treatment to challenge the integrity of the actin cytoskeleton while breakdown of the microtubular component had no discernible impact on the accumulation of Mayven in the process tips. Deletion of the first three amino acids of the SH3 motif of the putative Fyn Kinase binding domain at the amino terminus significantly compromised this signature pattern as did the removal of the last Kelch repeat unit of six unit Kelch domain comprising the carboxyl terminus. In addition, there was a reduction in process length in mutant transfectants. Co-expression studies with a haemagglutinin (HA) tagged wild

  1. Virus-Specific Immunity in Neonatal and Adult Mouse Rotavirus Infection

    PubMed Central

    Sheridan, J. F.; Eydelloth, R. S.; Vonderfecht, S. L.; Aurelian, L.

    1983-01-01

    Mouse rotavirus (epizootic diarrhea of infant mice) was used as a model to study the role of virus-specific immunity in infection and diarrheal disease. The distribution of viral antigen in intestinal tissues was determined by immunofluorescent staining with anti-simian rotavirus (SA-11) serum. The location and proportion of antigen-positive cells appeared to vary as a function of time postinfection and age of the animal at the time of infection. In animals infected at 1 and 7 days of age, antigen-positive cells (5 to 25%) were first detected (1 day postinfection) in the proximal segment of the small intestine, and infection progressed to the middle and distal segments. At 10 days postinfection, virus-infected cells were no longer observed in the proximal segment. In animals infected at 21 days of age (disease-free), a significantly lower proportion of cells were antigen positive (2 to 5%), and they were restricted to the middle and distal segments of the small intestine. Infection, defined according to the presence of virus and viral antigens in intestinal tissues and by seroconversion in the immunoglobulin M (IgM) isotype as determined by enzyme-linked immunosorbent assay with SA-11 antigen, was observed for all age groups (neonatal to adult), even in the presence of virus-specific serum or intestinal immunoglobulins. On the other hand, diarrheal disease was not detected in neonatal mice (1 to 3 days old) positive for passively acquired virus-specific intestinal IgG. The presence of virus-specific IgA in the intestinal tract at the time of infection did not protect from subsequent diarrheal disease. Virus-specific, cell-mediated immunity, determined by a delayed-type hypersensitivity response, did not develop in neonatal mice infected at 5 and 12 days of age. Reinfection of adult mice was associated with suppression of virus-specific delayed-type hypersensitivity and a significant decrease in the titers of the virus-specific serum IgG and IgA. Images PMID:6299952

  2. Selective expression of prion protein in peripheral tissues of the adult mouse.

    PubMed

    Ford, M J; Burton, L J; Morris, R J; Hall, S M

    2002-01-01

    The level of expression of normal cellular prion protein, PrP(c) (cellular prion protein), controls both the rate and the route of neuroinvasive infection, from peripheral entry portal to the CNS. Paradoxically, an overview of the distribution of PrP(c) within tissues outside the CNS is lacking. We have used novel antibodies that recognise cellular prion protein in glutaraldehyde-fixed tissue (in order to optimise immunohistochemical labelling of this conformationally labile protein), in combination with in situ hybridisation, to examine the expression of PrP(c) in peripheral tissues of the adult mouse. We found that although prion protein is expressed in many tissues, it is expressed at high levels only in discrete subpopulations of cells. Prominent amongst these are elements of the "hardwired neuroimmune network" that integrate the body's immune defence and neuroendocrine systems under CNS control. These prion protein-expressing elements include small diameter afferent nerves in the skin and the lamina propria of the aerodigestive tract, sympathetic ganglia and nerves, antigen presenting and processing cells (both follicular and non-follicular dendritic cells) and sub-populations of lymphocytes particularly in skin, gut- and bronchus-associated lymphoid tissues. Prion protein is also expressed in the parasympathetic and enteric nervous systems, in the dispersed neuroendocrine system, and in peripheral nervous system axons and their associated Schwann cells. This selective expression of cellular prion protein provides a variety of alternative routes for the propagation and transport of prion infection entering from peripheral sites, either naturally (via the aerodigestive tract or abraded skin) or experimentally (by intraperitoneal injection) to the brain. Key regulatory cells that express prion protein, and in particular enteroendocrine cells in the mucosal wall of the gut, and dendritic cells that convey pathogens from epithelial layers to secondary lymphoid

  3. Effect of Cyanotoxins on the Hypothalamic–Pituitary–Gonadal Axis in Male Adult Mouse

    PubMed Central

    Xu, Huajun

    2014-01-01

    Background Microcystins LR (MC-LR) are hepatotoxic cyanotoxins that have been shown to induce reproductive toxicity, and Hypothalamic–Pituitary–Gonadal Axis (HPG) is responsible for the control of reproductive functions. However, few studies have been performed to evaluate the effects of MC-LR on HPG axis. This study aimed to investigate the MC-LR-induced toxicity in the reproductive system of mouse and focus on the HPG axis. Methods Adult male C57BL/6 mice were exposed to various concentrations of MC-LR (0, 3.75, 7.50, 15.00 and 30.00 µg/kg body weight per day) for 1 to 14 days, and it was found that exposure to different concentrations of MC-LR significantly disturbed sperm production in the mice testes in a dose- and time-dependent manner. To elucidate the associated possible mechanisms, the serum levels of testosterone, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were assessed. Meanwhile, PCR assays were employed to detect alterations in a series of genes involved in HPG axis, such as FSH, LH, gonadotropin-releasing hormone (GnRH) and their complement receptors. Furthermore, the effect of MC-LR on the viability and testosterone production of Leydig cells were tested in vitro. Results: MC-LR significantly impaired the spermatogenesis of mice possibly through the direct or indirect inhibition of GnRH synthesis at the hypothalamic level, which resulted in reduction of serum levels of LH that lead to suppression of testosterone production in the testis of mice. Conclusions MC-LR may be a GnRH toxin that would disrupt the reproductive system of mice. PMID:25375936

  4. Brief Isoflurane Anesthesia Produces Prominent Phosphoproteomic Changes in the Adult Mouse Hippocampus.

    PubMed

    Kohtala, Samuel; Theilmann, Wiebke; Suomi, Tomi; Wigren, Henna-Kaisa; Porkka-Heiskanen, Tarja; Elo, Laura L; Rokka, Anne; Rantamäki, Tomi

    2016-06-15

    Anesthetics are widely used in medical practice and experimental research, yet the neurobiological basis governing their effects remains obscure. We have here used quantitative phosphoproteomics to investigate the protein phosphorylation changes produced by a 30 min isoflurane anesthesia in the adult mouse hippocampus. Altogether 318 phosphorylation alterations in total of 237 proteins between sham and isoflurane anesthesia were identified. Many of the hit proteins represent primary pharmacological targets of anesthetics. However, findings also enlighten the role of several other proteins-implicated in various biological processes including neuronal excitability, brain energy homeostasis, synaptic plasticity and transmission, and microtubule function-as putative (secondary) targets of anesthetics. In particular, isoflurane increases glycogen synthase kinase-3β (GSK3β) phosphorylation at the inhibitory Ser(9) residue and regulates the phosphorylation of multiple proteins downstream and upstream of this promiscuous kinase that regulate diverse biological functions. Along with confirmatory Western blot data for GSK3β and p44/42-MAPK (mitogen-activated protein kinase; reduced phosphorylation of the activation loop), we observed increased phosphorylation of microtubule-associated protein 2 (MAP2) on residues (Thr(1620,1623)) that have been shown to render its dissociation from microtubules and alterations in microtubule stability. We further demonstrate that diverse anesthetics (sevoflurane, urethane, ketamine) produce essentially similar phosphorylation changes on GSK3β, p44/p42-MAPK, and MAP2 as observed with isoflurane. Altogether our study demonstrates the potential of quantitative phosphoproteomics to study the mechanisms of anesthetics (and other drugs) in the mammalian brain and reveals how already a relatively brief anesthesia produces pronounced phosphorylation changes in multiple proteins in the central nervous system. PMID:27074656

  5. Bergmann glia are patterned into topographic molecular zones in the developing and adult mouse cerebellum

    PubMed Central

    Reeber, Stacey L.; Arancillo, Marife K. V.; Sillitoe, Roy V.

    2015-01-01

    Cerebellar circuits are patterned into an array of topographic parasagittal domains called zones. Zones are best revealed by gene expression, circuit anatomy, and cellular degeneration patterns. Thus far, the study of zones has been focused heavily on how neurons are organized. Because of this, detailed neuronal patterning maps have been established for Purkinje cells, granule cells, Golgi cells, unipolar brush cells, and also for the terminal field organization of climbing fiber and mossy fiber afferents. In comparison, however, it remains poorly understood if glial cells are also organized into zones. We have identified an Npy-Gfp BAC transgenic mouse line (Tau-Sapphire Green fluorescent protein (Gfp) is under the control of the neuropeptide Y (Npy) gene regulatory elements) that can be used to label Bergmann glial cells with Golgi-like resolution. In these adult transgenic mice we found that Npy-Gfp expression was localized to Bergmann glia mainly in lobules VI/VII and IX/X. Using double immunofluorescence, we show that in these lobules, Npy-Gfp expression in the Bergmann glia overlaps with the pattern of the small heat shock protein HSP25, a Purkinje cell marker for zones located in lobules VI/VII and IX/X. Developmental analysis starting from the day of birth showed that HSP25 and Npy-Gfp expression follow a similar program of spatial and temporal patterning. However, loss of Npy signaling did not alter the patterning of Purkinje cell zones. We conclude that Bergmann glial cells are zonally organized and their patterns are restricted by boundaries that also confine cerebellar neurons into a topographic circuit map. PMID:24906823

  6. Designer Self-Assembling Peptide Nanofiber Scaffolds for Adult Mouse Neural Stem Cell 3-Dimensional Cultures

    PubMed Central

    Gelain, Fabrizio; Bottai, Daniele; Vescovi, Angleo; Zhang, Shuguang

    2006-01-01

    Biomedical researchers have become increasingly aware of the limitations of conventional 2-dimensional tissue cell culture systems, including coated Petri dishes, multi-well plates and slides, to fully address many critical issues in cell biology, cancer biology and neurobiology, such as the 3-D microenvironment, 3-D gradient diffusion, 3-D cell migration and 3-D cell-cell contact interactions. In order to fully understand how cells behave in the 3-D body, it is important to develop a well-controlled 3-D cell culture system where every single ingredient is known. Here we report the development of a 3-D cell culture system using a designer peptide nanofiber scaffold with mouse adult neural stem cells. We attached several functional motifs, including cell adhesion, differentiation and bone marrow homing motifs, to a self-assembling peptide RADA16 (Ac-RADARADARADARADA-COHN2). These functionalized peptides undergo self-assembly into a nanofiber structure similar to Matrigel. During cell culture, the cells were fully embedded in the 3-D environment of the scaffold. Two of the peptide scaffolds containing bone marrow homing motifs significantly enhanced the neural cell survival without extra soluble growth and neurotrophic factors to the routine cell culture media. In these designer scaffolds, the cell populations with β-Tubulin+, GFAP+ and Nestin+ markers are similar to those found in cell populations cultured on Matrigel. The gene expression profiling array experiments showed selective gene expression, possibly involved in neural stem cell adhesion and differentiation. Because the synthetic peptides are intrinsically pure and a number of desired function cellular motifs are easy to incorporate, these designer peptide nanofiber scaffolds provide a promising controlled 3-D culture system for diverse tissue cells, and are useful as well for general molecular and cell biology. PMID:17205123

  7. Reproducible expansion and characterization of mouse neural stem/progenitor cells in adherent cultures derived from the adult subventricular zone

    PubMed Central

    Theus, Michelle H.; Ricard, Jerome; Liebl, Daniel J.

    2012-01-01

    Endogenous neural stem/progenitor cells (NSPCs) residing in the subventricular zone (SVZ) of the adult mouse forebrain have been shown to enhance their neurogenic potential in response to CNS injury. Mechanisms involved in regulating adult neurogenesis under naïve or stressed conditions can be studied using a monolayer cell-culture system of the nestin-expressing NSPC lineage to analyze proliferation, survival and differentiation. Here, we describe a protocol for the expansion of NSPCs for studies aimed at understanding the functional role of NSPCs in maintaining adult neurogenic processes. In this unit, we outline in detail the procedures for: (1) isolation, maintenance and culture of the NSPC component of the SVZ niche from the lateral wall of the lateral ventricle; (2) characterization of NSPC functions by examining proliferation, survival and differentiation; and (3) efficient siRNA transfection methods in 96-well format. PMID:22415840

  8. Cadmium tolerance, cysteine and thiol peptide levels in wild type and chromium-tolerant strains of Scenedesmus acutus (Chlorophyceae).

    PubMed

    Torricelli, Elena; Gorbi, Gessica; Pawlik-Skowronska, Barbara; Di Toppi, Luigi Sanità; Corradi, Maria Grazia

    2004-07-14

    Two strains of the unicellular green alga Scenedesmus acutus with different sensitivity to hexavalent chromium were compared for their tolerance of cadmium, by means of growth and recovery tests, and determination of cysteine, reduced glutathione and phytochelatin content, after short-term exposure to various cadmium concentrations (from 1.125 to 27 microM). Growth experiments showed that, after 7-day treatments with cadmium, the chromium-tolerant strain reached a significantly higher cell density and, after 24-h exposure to Cd, was able to resume growth significantly better than the wild type. Constitutive level of cysteine was higher in the chromium-tolerant strain, while glutathione levels were similar in the two strains. The higher content of cysteine and the maintenance of both reduced glutathione and phytochelatin high levels in the presence of cadmium, support the higher cadmium co-tolerance of the chromium-tolerant strain in comparison with the wild type one. PMID:15177949

  9. Small N-terminal mutant huntingtin fragments, but not wild type, are mainly present in monomeric form: Implications for pathogenesis.

    PubMed

    Cong, Shu-Yan; Pepers, Barry A; Roos, Raymund A C; van Ommen, Gert-Jan B; Dorsman, Josephine C

    2006-06-01

    N-terminal fragments of huntingtin containing an expanded polyglutamine stretch play an important role in the molecular pathogenesis of Huntington's disease. Their ultimate accumulation in insoluble protein aggregates constitutes an important pathological hallmark of Huntington's disease. We report on systematic biochemical comparison studies of soluble wild type and mutant N-terminal huntingtin fragments. The results show that soluble wild type exon 1 fragments are predominantly present in higher molecular weight complexes with a molecular size of approximately 300 kDa, while their mutant counterparts are mainly present in their monomeric form. In contrast, longer N-terminal fragments corresponding to peptides produced by caspase cleavage do not display these differential properties. These findings suggest that especially an increased amount of monomeric form of small N-terminal mutant huntingtin fragments may facilitate aberrant interactions both with itself via the polyglutamine stretch and with other proteins and thereby contribute to molecular pathogenesis. PMID:16380118

  10. Structural variability between starch granules in wild type and in ae high-amylose mutant maize kernels.

    PubMed

    Liu, Dongli; Parker, Mary L; Wellner, Nikolaus; Kirby, Andrew R; Cross, Kathryn; Morris, Victor J; Cheng, Fang

    2013-09-12

    Starch granule structure within wild-type and ae high-amylose mutant maize kernels has been mapped in situ using light, electron and atomic force microscopy, and both Raman and infra-red spectroscopy. The population of wild-type starch granules is found to be homogenous. The ae mutant granule population is heterogeneous. Heterogeneity in chemical and physical structure is observed within individual granules, between granules within cells, and spatially within the kernel. The highest level of heterogeneity is observed in the region where starch is first deposited during kernel development. Light microscopy demonstrates structural diversity through use of potassium iodide/iodine staining and polarised microscopy. Electron and atomic force microscopy, and infra-red and Raman spectroscopy defined the nature of the structural changes within granules. The methodology provides novel information on the changes in starch structure resulting from kernel development. PMID:23911471

  11. EXAFS of Klebsiella pneumoniae nitrogenase MoFe protein from wild-type and nif V mutant strains

    SciTech Connect

    Eidsness, M.K.; Flank, A.M.; Smith, B.E.; Flood, A.C.; Garner, C.D.; Cramer. S.P.

    1986-05-14

    The enzyme nitrogenase catalyzes the biological reduction of N/sub 2/ to NH/sub 3/. In Klebsiella pneumoniae a cluster of 17 genes in seven transcriptional units has been associated with nitrogen fixation. The nitrogenase enzyme from the nif V mutants is relatively ineffective at dinitrogen reduction, is more efficient than the wild-type enzyme at HCN reduction, and has its hydrogen evolution activity inhibited up to 80% by CO. This altered substrate specificity has been shown to be associated with the iron-molybdenum cofactor, FeMo-co, of the enzyme. X-ray absorption spectroscopy has been a valuable tool for probing the molybdenum environment of wild-type nitrogenase, and the authors report here similar studies on the Nif V/sup -/ enzyme.

  12. Disruption of Ah Receptor Signaling during Mouse Development Leads to Abnormal Cardiac Structure and Function in the Adult.

    PubMed

    Carreira, Vinicius S; Fan, Yunxia; Kurita, Hisaka; Wang, Qin; Ko, Chia-I; Naticchioni, Mindi; Jiang, Min; Koch, Sheryl; Zhang, Xiang; Biesiada, Jacek; Medvedovic, Mario; Xia, Ying; Rubinstein, Jack; Puga, Alvaro

    2015-01-01

    The Developmental Origins of Health and Disease (DOHaD) Theory proposes that the environment encountered during fetal life and infancy permanently shapes tissue physiology and homeostasis such that damage resulting from maternal stress, poor nutrition or exposure to environmental agents may be at the heart of adult onset disease. Interference with endogenous developmental functions of the aryl hydrocarbon receptor (AHR), either by gene ablation or by exposure in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AHR ligand, causes structural, molecular and functional cardiac abnormalities and altered heart physiology in mouse embryos. To test if embryonic effects progress into an adult phenotype, we investigated whether Ahr ablation or TCDD exposure in utero resulted in cardiac abnormalities in adult mice long after removal of the agent. Ten-months old adult Ahr-/- and in utero TCDD-exposed Ahr+/+ mice showed sexually dimorphic abnormal cardiovascular phenotypes characterized by echocardiographic findings of hypertrophy, ventricular dilation and increased heart weight, resting heart rate and systolic and mean blood pressure, and decreased exercise tolerance. Underlying these effects, genes in signaling networks related to cardiac hypertrophy and mitochondrial function were differentially expressed. Cardiac dysfunction in mouse embryos resulting from AHR signaling disruption seems to progress into abnormal cardiac structure and function that predispose adults to cardiac disease, but while embryonic dysfunction is equally robust in males and females, the adult abnormalities are more prevalent in females, with the highest severity in Ahr-/- females. The findings reported here underscore the conclusion that AHR signaling in the developing heart is one potential target of environmental factors associated with cardiovascular disease. PMID:26555816

  13. Disruption of Ah Receptor Signaling during Mouse Development Leads to Abnormal Cardiac Structure and Function in the Adult

    PubMed Central

    Carreira, Vinicius S.; Fan, Yunxia; Kurita, Hisaka; Wang, Qin; Ko, Chia-I; Naticchioni, Mindi; Jiang, Min; Koch, Sheryl; Zhang, Xiang; Biesiada, Jacek; Medvedovic, Mario; Xia, Ying; Rubinstein, Jack; Puga, Alvaro

    2015-01-01

    The Developmental Origins of Health and Disease (DOHaD) Theory proposes that the environment encountered during fetal life and infancy permanently shapes tissue physiology and homeostasis such that damage resulting from maternal stress, poor nutrition or exposure to environmental agents may be at the heart of adult onset disease. Interference with endogenous developmental functions of the aryl hydrocarbon receptor (AHR), either by gene ablation or by exposure in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AHR ligand, causes structural, molecular and functional cardiac abnormalities and altered heart physiology in mouse embryos. To test if embryonic effects progress into an adult phenotype, we investigated whether Ahr ablation or TCDD exposure in utero resulted in cardiac abnormalities in adult mice long after removal of the agent. Ten-months old adult Ahr-/- and in utero TCDD-exposed Ahr+/+ mice showed sexually dimorphic abnormal cardiovascular phenotypes characterized by echocardiographic findings of hypertrophy, ventricular dilation and increased heart weight, resting heart rate and systolic and mean blood pressure, and decreased exercise tolerance. Underlying these effects, genes in signaling networks related to cardiac hypertrophy and mitochondrial function were differentially expressed. Cardiac dysfunction in mouse embryos resulting from AHR signaling disruption seems to progress into abnormal cardiac structure and function that predispose adults to cardiac disease, but while embryonic dysfunction is equally robust in males and females, the adult abnormalities are more prevalent in females, with the highest severity in Ahr-/- females. The findings reported here underscore the conclusion that AHR signaling in the developing heart is one potential target of environmental factors associated with cardiovascular disease. PMID:26555816

  14. Full Genome Sequence-Based Comparative Study of Wild-Type and Vaccine Strains of Infectious Laryngotracheitis Virus from Italy.

    PubMed

    Piccirillo, Alessandra; Lavezzo, Enrico; Niero, Giulia; Moreno, Ana; Massi, Paola; Franchin, Elisa; Toppo, Stefano; Salata, Cristiano; Palù, Giorgio

    2016-01-01

    Infectious laryngotracheitis (ILT) is an acute and highly contagious respiratory disease of chickens caused by an alphaherpesvirus, infectious laryngotracheitis virus (ILTV). Recently, full genome sequences of wild-type and vaccine strains have been determined worldwide, but none was from Europe. The aim of this study was to determine and analyse the complete genome sequences of five ILTV strains. Sequences were also compared to reveal the similarity of strains across time and to discriminate between wild-type and vaccine strains. Genomes of three ILTV field isolates from outbreaks occurred in Italy in 1980, 2007 and 2011, and two commercial chicken embryo origin (CEO) vaccines were sequenced using the 454 Life Sciences technology. The comparison with the Serva genome showed that 35 open reading frames (ORFs) differed across the five genomes. Overall, 54 single nucleotide polymorphisms (SNPs) and 27 amino acid differences in 19 ORFs and two insertions in the UL52 and ORFC genes were identified. Similarity among the field strains and between the field and the vaccine strains ranged from 99.96% to 99.99%. Phylogenetic analysis revealed a close relationship among them, as well. This study generated data on genomic variation among Italian ILTV strains revealing that, even though the genetic variability of the genome is well conserved across time and between wild-type and vaccine strains, some mutations may help in differentiating among them and may be involved in ILTV virulence/attenuation. The results of this study can contribute to the understanding of the molecular bases of ILTV pathogenicity and provide genetic markers to differentiate between wild-type and vaccine strains. PMID:26890525

  15. Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors.

    PubMed Central

    Moll, U M; LaQuaglia, M; Bénard, J; Riou, G

    1995-01-01

    Neuroblastoma (NB), a tumor arising from the sympathetic nervous system, is one of the most common malignancies in childhood. Several recent reports on the p53 genotype found virtually exclusive wild-type status in primary tumors, and it was postulated that p53 plays no role in the development of NB. Here, however, we report that the vast majority of undifferentiated NBs exhibit abnormal cytoplasmic sequestration of wild-type p53. This inability of p53 to translocate to the nucleus presumably prevents the protein from functioning as a suppressor. Thirty of 31 cases (96%) of undifferentiated NB showed elevated levels of wild-type p53 in the cytoplasm of all tumor cells concomittant with a lack of nuclear staining. p53 immunoprecipitation from tumor tissues showed a 4.5- to 8-fold increase over normal protein levels. All of 10 tumors analyzed harbored wild-type p53 by direct sequencing of full-length cDNA and Southern blot. In addition, no MDM-2 gene amplification was seen in all 11 tumors analyzed. In contrast, no p53 abnormality was detected in 14 differentiated ganglioneuroblastomas and 1 benign ganglioneuroma. We conclude that loss of p53 function seems to play a major role in the tumorigenesis of undifferentiated NB. This tumor might abrogate the transactivating function of p53 by inhibiting its access to the nucleus, rather than by gene mutation. Importantly, our results suggest that (i) this could be a general mechanism for p53 inactivation not limited to breast cancer (where we first described it) and that (ii) it is found in a tumor previously not thought to be affected by p53 alteration. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7753819

  16. Effect of lectin on nodulation by wild-type Bradyrhizobium japonicum and a nodulation-defective mutant.

    PubMed Central

    Halverson, L J; Stacey, G

    1986-01-01

    The nodulation characteristics of wild-type Bradyrhizobium japonicum USDA 110 and mutant strain HS111 were examined. Mutant strain HS111 exhibits a delayed-nodulation phenotype, a result of its inability to initiate successful nodulation promptly following inoculation of the soybean root. Previously, we showed that the defect in initiation of infection leading to subsequent nodulation which is found in HS111 can be phenotypically reversed by pretreatment with soybean root exudate or soybean seed lectin. This effect is not seen after pretreatment with root exudates and lectins obtained from other plant species. Treatment of strain HS111 with as little as 10 soybean seed lectin molecules per bacterium (3.3 X 10 (-12) M) resulted in enhancement of nodule formation. Pretreatment of wild-type B. japonicum USDA 110 with soybean root exudate or seed lectin increased nodule numbers twofold on 6-week-old plants. Wild-type strain USDA 110 cells inoculated at 10(4) cells per seedling exhibited a delay in initiation of infection leading to subsequent nodulation. Wild-type cells pretreated in soybean root exudates or seed lectin did not exhibit a delay in nodulation at this cell concentration. Mutant strain HS111 pretreated in seed lectin for 0 or 1 h, followed by washing with the hapten D-galactose to remove the lectin, exhibited a delay in initiation of nodulation. Phenotypic reversal of the delayed-nodulation phenotype exhibited by strain HS111 was seen if incubation was continued for an additional 71 h in plant nutrient solution following 1 h of lectin pretreatment. Reversal of the delayed-nodulation phenotype of HS111 through lectin pretreatment was prevented by chloramphenicol or rifampin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3707122

  17. Functional independence of monomeric CHIP28 water channels revealed by expression of wild-type mutant heterodimers.

    PubMed

    Shi, L B; Skach, W R; Verkman, A S

    1994-04-01

    CHIP28 is a major water transporting protein in erythrocytes and kidney which forms tetramers in membranes (Verbavatz, J. M., Brown, D., Sabolic, I., Valenti, G., Ausiello, D. A., Van Hoek, A. N., Ma, T., and Verkman, A. S. (1993) J. Cell Biol. 123, 605-618). To determine whether CHIP28 monomers function independently, chimeric cDNA dimers were constructed which contained wild-type CHIP28 in series with either wild-type CHIP28, a non-water transporting CHIP28 mutant (C189W), or a functional but mercurial-insensitive CHIP28 mutant (C189S). Transcribed cRNAs were injected in Xenopus oocytes and plasma membrane expression was assayed by quantitative immunofluorescence. Water channel function was measured by osmotically induced swelling. CHIP28 homo- and heterodimers were targeted to the oocyte plasma membrane and functioned as water channels. Relative osmotic water permeability (Pf) values (normalized for plasma membrane expression of monomeric subunits) were: 1.0 (CHIP28 monomer), 0.0 (C189W), 1.07 (C189S), 1.10 (CHIP28-CHIP28 dimer) and 0.52 (CHIP28-C189W). The increase in oocyte Pf was linearly related to plasma membrane expression of wild-type CHIP28 and C189S subunits. HgCl2 (0.3 mM) inhibited channel-mediated Pf in oocytes expressing wild-type CHIP28 monomers and dimers by 85-90%, but did not inhibit Pf in oocytes expressing C189S. HgCl2 inhibited Pf in oocytes expressing CHIP28-C189S dimers by 44 +/- 7%, consistent with one mercurial-sensitive and one insensitive subunit in the heterodimer. These results indicate that despite their assembly in tetramers, monomeric CHIP28 subunits function independently as water channels. PMID:7511600

  18. Full Genome Sequence-Based Comparative Study of Wild-Type and Vaccine Strains of Infectious Laryngotracheitis Virus from Italy

    PubMed Central

    Niero, Giulia; Moreno, Ana; Massi, Paola; Franchin, Elisa; Toppo, Stefano; Salata, Cristiano; Palù, Giorgio

    2016-01-01

    Infectious laryngotracheitis (ILT) is an acute and highly contagious respiratory disease of chickens caused by an alphaherpesvirus, infectious laryngotracheitis virus (ILTV). Recently, full genome sequences of wild-type and vaccine strains have been determined worldwide, but none was from Europe. The aim of this study was to determine and analyse the complete genome sequences of five ILTV strains. Sequences were also compared to reveal the similarity of strains across time and to discriminate between wild-type and vaccine strains. Genomes of three ILTV field isolates from outbreaks occurred in Italy in 1980, 2007 and 2011, and two commercial chicken embryo origin (CEO) vaccines were sequenced using the 454 Life Sciences technology. The comparison with the Serva genome showed that 35 open reading frames (ORFs) differed across the five genomes. Overall, 54 single nucleotide polymorphisms (SNPs) and 27 amino acid differences in 19 ORFs and two insertions in the UL52 and ORFC genes were identified. Similarity among the field strains and between the field and the vaccine strains ranged from 99.96% to 99.99%. Phylogenetic analysis revealed a close relationship among them, as well. This study generated data on genomic variation among Italian ILTV strains revealing that, even though the genetic variability of the genome is well conserved across time and between wild-type and vaccine strains, some mutations may help in differentiating among them and may be involved in ILTV virulence/attenuation. The results of this study can contribute to the understanding of the molecular bases of ILTV pathogenicity and provide genetic markers to differentiate between wild-type and vaccine strains. PMID:26890525

  19. Comprehensive Analysis of Neonatal versus Adult Unilateral Decortication in a Mouse Model Using Behavioral, Neuroanatomical, and DNA Microarray Approaches

    PubMed Central

    Yoshikawa, Akira; Nakamachi, Tomoya; Shibato, Junko; Rakwal, Randeep; Shioda, Seiji

    2014-01-01

    Previously, studying the development, especially of corticospinal neurons, it was concluded that the main compensatory mechanism after unilateral brain injury in rat at the neonatal stage was due in part to non-lesioned ipsilateral corticospinal neurons that escaped selection by axonal elimination or neuronal apoptosis. However, previous results suggesting compensatory mechanism in neonate brain were not correlated with high functional recovery. Therefore, what is the difference among neonate and adult in the context of functional recovery and potential mechanism(s) therein? Here, we utilized a brain unilateral decortication mouse model and compared motor functional recovery mechanism post-neonatal brain hemisuction (NBH) with adult brain hemisuction (ABH). Three analyses were performed: (1) Quantitative behavioral analysis of forelimb movements using ladder walking test; (2) neuroanatomical retrograde tracing analysis of unlesioned side corticospinal neurons; and (3) differential global gene expressions profiling in unlesioned-side neocortex (rostral from bregma) in NBH and ABH on a 8 × 60 K mouse whole genome Agilent DNA chip. Behavioral data confirmed higher recovery ability in NBH over ABH is related to non-lesional frontal neocortex including rostral caudal forelimb area. A first inventory of differentially expressed genes genome-wide in the NBH and ABH mouse model is provided as a resource for the scientific community. PMID:25490135

  20. Purification and some properties of wild-type and N-terminal-truncated ethanolamine ammonia-lyase of Escherichia coli.

    PubMed

    Akita, Keita; Hieda, Naoki; Baba, Nobuyuki; Kawaguchi, Satoshi; Sakamoto, Hirohisa; Nakanishi, Yuka; Yamanishi, Mamoru; Mori, Koichi; Toraya, Tetsuo

    2010-01-01

    The methods of homologous high-level expression and simple large-scale purification for coenzyme B(12)-dependent ethanolamine ammonia-lyase of Escherichia coli were developed. The eutB and eutC genes in the eut operon encoded the large and small subunits of the enzyme, respectively. The enzyme existed as the heterododecamer alpha(6)beta(6). Upon active-site titration with adeninylpentylcobalamin, a strong competitive inhibitor for coenzyme B(12), the binding of 1 mol of the inhibitor per mol of the alphabeta unit caused complete inhibition of enzyme, in consistent with its subunit structure. EPR spectra indicated the formation of substrate-derived radicals during catalysis and the binding of cobalamin in the base-on mode, i.e. with 5,6-dimethylbenzimidazole coordinating to the cobalt atom. The purified wild-type enzyme underwent aggregation and inactivation at high concentrations. Limited proteolysis with trypsin indicated that the N-terminal region is not essential for catalysis. His-tagged truncated enzymes were similar to the wild-type enzyme in catalytic properties, but more resistant to p-chloromercuribenzoate than the wild-type enzyme. A truncated enzyme was highly soluble even in the absence of detergent and resistant to aggregation and oxidative inactivation at high concentrations, indicating that a short N-terminal sequence is sufficient to change the solubility and stability of the enzyme. PMID:19762342

  1. Profile of panitumumab as first-line treatment in patients with wild-type KRAS metastatic colorectal cancer.

    PubMed

    Patel, Shiven B; Gill, David; Garrido-Laguna, Ignacio

    2016-01-01

    Targeted therapies against EGFR, vascular endothelial growth factor, and vascular endothelial growth factor receptor have expanded treatment options for patients with metastatic colorectal cancer (mCRC). Unfortunately, biomarkers to identify patients that are most likely to derive benefit from targeted therapies in this disease are still needed. Indeed, only RAS mutations have been identified as predictive of lack of benefit from monoclonal antibodies against EGFR in patients with mCRC. Panitumumab is a fully humanized monoclonal antibody against EGFR. In this study, we review data to support the use of panitumumab in combination with a chemotherapy backbone, in the first line setting in patients with RAS wild-type mCRC. Ongoing efforts are aimed at identifying smaller subsets of patients within the RAS wild-type group that will derive the largest benefit from anti-EGFR therapy. In the meantime, treatment with anti-EGFR therapy should be reserved for patients with RAS wild-type mCRC. PMID:26770060

  2. Cellular Oxidative Stress and the Control of Apoptosis by Wild-Type p53, Cytotoxic Compounds, and Cytokines

    NASA Astrophysics Data System (ADS)

    Lotem, Joseph; Peled-Kamar, Mira; Groner, Yoram; Sachs, Leo

    1996-08-01

    Apoptosis induced by wild-type p53 or cytotoxic compounds in myeloid leukemic cells can be inhibited by the cytokines interleukin 6, interleukin 3, granulocyte-macrophage colony-stimulating factor, and interferon γ and by antioxidants. The antioxidants and cytokines showed a cooperative protective effect against induction of apoptosis. Cells with a higher sensitivity to induction of apoptosis and required a higher cytokine concentration to inhibit apoptosis. Decreasing the intrinsic oxidative stress in cells by antioxidants thus inhibited apoptosis, whereas increasing this intrinsic stress by adding H2O2 enhanced apoptosis. Induction of apoptosis by wild-type p53 was not preceded by increased peroxide production or lipid peroxidation and the protective effect of cytokines was not associated with a decrease in these properties. The results indicate that the intrinsic degree of oxidative stress can regulate cell susceptibility to wild-type p53-dependent and p53-independent induction of apoptosis and the ability of cytokines to protect cells against apoptosis.

  3. Processing of CFTR bearing the P574H mutation differs from wild-type and deltaF508-CFTR.

    PubMed

    Ostedgaard, L S; Zeiher, B; Welsh, M J

    1999-07-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) containing the deltaF508 mutation is retained in the endoplasmic reticulum (ER). This defect can be partially overcome by a reduction in temperature which allows some of the deltaF508 protein to exit the ER and move to the cell surface. Earlier studies showed that the CF-associated mutants, P574H and A455E, were also misprocessed. In this study, we found that processing of P574H and A455E was also temperature-sensitive; at 26 degrees C, some of the protein matured. In contrast to other CFTR mutants, P574H accumulated in punctate cytoplasmic bodies that colocalized with endoplasmic reticulum (ER) markers. At 26 degrees C, these bodies were no longer present. P574H showed a prolonged association with Hsp70 and also colocalized with Hsp70. We used brefeldin A (BFA) to determine which processing step(s) was altered by reduced temperature. Unlike wild-type CFTR, which was converted into an intermediate that was stable in the presence of BFA at 37 degrees C, deltaF508 and P574H produced the intermediate only when the temperature was reduced to 26 degrees C. Furthermore the wild-type intermediate was not associated with Hsp70. These data suggest that formation of the stable intermediate is a key temperature-sensitive step and appears to be coincident with release of the wild-type protein from Hsp70. PMID:10362539

  4. Profile of panitumumab as first-line treatment in patients with wild-type KRAS metastatic colorectal cancer

    PubMed Central

    Patel, Shiven B; Gill, David; Garrido-Laguna, Ignacio

    2016-01-01

    Targeted therapies against EGFR, vascular endothelial growth factor, and vascular endothelial growth factor receptor have expanded treatment options for patients with metastatic colorectal cancer (mCRC). Unfortunately, biomarkers to identify patients that are most likely to derive benefit from targeted therapies in this disease are still needed. Indeed, only RAS mutations have been identified as predictive of lack of benefit from monoclonal antibodies against EGFR in patients with mCRC. Panitumumab is a fully humanized monoclonal antibody against EGFR. In this study, we review data to support the use of panitumumab in combination with a chemotherapy backbone, in the first line setting in patients with RAS wild-type mCRC. Ongoing efforts are aimed at identifying smaller subsets of patients within the RAS wild-type group that will derive the largest benefit from anti-EGFR therapy. In the meantime, treatment with anti-EGFR therapy should be reserved for patients with RAS wild-type mCRC. PMID:26770060

  5. The fusion protein of wild-type canine distemper virus is a major determinant of persistent infection

    SciTech Connect

    Plattet, Philippe; Rivals, Jean-Paul; Zuber, BenoIt; Brunner, Jean-Marc; Zurbriggen, Andreas; Wittek, Riccardo . E-mail: Riccardo.Wittek@unil.ch

    2005-07-05

    The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system but infects cell lines very inefficiently. In contrast, the genetically more distant Onderstepoort CDV vaccine strain (OP-CDV) induces extensive syncytia formation. Here, we investigated the roles of wild-type fusion (F{sub WT}) and attachment (H{sub WT}) proteins in Vero cells expressing, or not, the canine SLAM receptor by transfection experiments and by studying recombinants viruses expressing different combinations of wild-type and OP-CDV glycoproteins. We show that low fusogenicity is not due to a defect of the envelope proteins to reach the cell surface and that H{sub WT} determines persistent infection in a receptor-dependent manner, emphasizing the role of SLAM as a potent enhancer of fusogenicity. However, importantly, F{sub WT} reduced cell-to-cell fusion independently of the cell surface receptor, thus demonstrating that the fusion protein of the neurovirulent A75/17-CDV strain plays a key role in determining persistent infection.

  6. Transfection of wild-type CFTR into cystic fibrosis lymphocytes restores chloride conductance at G1 of the cell cycle.

    PubMed Central

    Krauss, R D; Bubien, J K; Drumm, M L; Zheng, T; Peiper, S C; Collins, F S; Kirk, K L; Frizzell, R A; Rado, T A

    1992-01-01

    We complemented the Cl- conductance defect in cystic fibrosis lymphocytes by transfection with wild-type cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR). Stable transfectants were selected and subjected to molecular and functional analyses. We detected expression of endogenous CFTR mRNA in several CF and non-CF lymphoid cell lines by PCR. Expression from cDNA in the transfectants was demonstrated by amplifying vector-specific sequences. Both fluorescence and patch-clamp assays showed that transfectants expressing wild-type CFTR acquired properties previously associated with Cl- conductance (GCl) regulation in non-CF lymphocytes: (i) GCl was elevated in the G1 phase of the cell cycle, (ii) cells fixed at G1 increase GCl in response to increased cellular cAMP or Ca2+, (iii) agonist-induced increases in GCl were lost as the cells progressed to the S phase of the cell cycle. The cell cycle and agonist dependent regulation of GCl was not observed in CF lymphocytes transfected with CFTR cDNA containing stop codons in all reading frames at exon 6. Our findings indicate that lymphocytes express functional CFTR since wild-type CFTR corrects the defects in Cl- conductance regulation found in CF lymphocytes. Evaluation of the mechanism of this novel, CFTR-mediated regulation of GCl during cell cycling should provide further insights into the function of CFTR. Images PMID:1372253

  7. Enhancing a search for traditional medicinal plants with anthelmintic action by using wild type and stress reporter Caenorhabditis elegans strains as screening tools.

    PubMed

    Kumarasingha, R; Palombo, E A; Bhave, M; Yeo, T C; Lim, D S L; Tu, C L; Shaw, J M; Boag, P R

    2014-04-01

    Traditional healers in Sarawak, Malaysia, use plants such as Picria fel-terrae, Linariantha bicolor and Lansium domesticum to treat gastrointestinal infections. This study aimed to test whether their nematocidal activities could be confirmed in vitro using highly standardised Caenorhabditis elegans models. We applied eight different ethanol solubilised plant extracts and two commercial anthelmintic drugs to larval and adult stages of C. elegans in vitro. Seven C. elegans strains were evaluated, one wild type and six strains with GFP-tagged stress response pathways to help characterise and compare the pathways affected by plant extracts. Our in vitro screen confirmed that both of the commercial anthelmintic drugs and five of the eight traditionally used plant extracts had significant nematocidal activity against both larval and adult C. elegans. The most effective extracts were from P. fel-terrae. The plant extracts triggered different stress response pathways from the commercial anthelmintic drugs. This study showed that using traditional knowledge of plant medicinal properties in combination with a C. elegans in vitro screen provided a rapid and economical test with a high hit rate compared with the random screening of plants for nematocidal activities. The use of transgenic C. elegans strains may allow this approach to be refined further to investigate the mode of action of active extracts. PMID:24583111

  8. CAR-Engineered NK Cells Targeting Wild-Type EGFR and EGFRvIII Enhance Killing of Glioblastoma and Patient-Derived Glioblastoma Stem Cells

    PubMed Central

    Han, Jianfeng; Chu, Jianhong; Keung Chan, Wing; Zhang, Jianying; Wang, Youwei; Cohen, Justus B.; Victor, Aaron; Meisen, Walter H.; Kim, Sung-hak; Grandi, Paola; Wang, Qi-En; He, Xiaoming; Nakano, Ichiro; Chiocca, E. Antonio; Glorioso III, Joseph C.; Kaur, Balveen; Caligiuri, Michael A.; Yu, Jianhua

    2015-01-01

    Glioblastoma (GB) remains the most aggressive primary brain malignancy. Adoptive transfer of chimeric antigen receptor (CAR)-modified immune cells has emerged as a promising anti-cancer approach, yet the potential utility of CAR-engineered natural killer (NK) cells to treat GB has not been explored. Tumors from approximately 50% of GB patients express wild-type EGFR (wtEGFR) and in fewer cases express both wtEGFR and the mutant form EGFRvIII; however, previously reported CAR T cell studies only focus on targeting EGFRvIII. Here we explore whether both wtEGFR and EGFRvIII can be effectively targeted by CAR-redirected NK cells to treat GB. We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells. EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-γ production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner. In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival. These findings support intracranial administration of NK-92-EGFR-CAR cells represents a promising clinical strategy to treat GB. PMID:26155832

  9. Pre- and postsynaptic modulations of hypoglossal motoneurons by α-adrenoceptor activation in wild-type and Mecp2−/Y mice

    PubMed Central

    Jin, Xiao-Tao; Cui, Ningren; Zhong, Weiwei; Jin, Xin; Wu, Zhongying

    2013-01-01

    Hypoglossal motoneurons (HNs) control tongue movement and play a role in maintenance of upper airway patency. Defects in these neurons may contribute to the development of sleep apnea and other cranial motor disorders including Rett syndrome (RTT). HNs are modulated by norepinephrine (NE) through α-adrenoceptors. Although postsynaptic mechanisms are known to play a role in this effect, how NE modulates the synaptic transmissions of HNs remains poorly understood. More importantly, the NE system is defective in RTT, while how the defect affects HNs is unknown. Believing that information of NE modulation of HNs may help the understanding of RTT and the design of new therapeutical interventions to motor defects in the disease, we performed these studies in which glycinergic inhibitory postsynaptic currents and intrinsic membrane properties were examined in wild-type and Mecp2−/Y mice, a mouse of model of RTT. We found that activation of α1-adrenoceptor facilitated glycinergic synaptic transmission and excited HNs. These effects were mediated by both pre- and postsynaptic mechanisms. The latter effect involved an inhibition of barium-sensitive G protein-dependent K+ currents. The pre- and postsynaptic modulations of the HNs by α1-adrenoceptors were not only retained in Mecp2-null mice but also markedly enhanced, which appears to be a compensatory mechanism for the deficiencies in NE and GABAergic synaptic transmission. The existence of the endogenous compensatory mechanism is an encouraging finding, as it may allow therapeutical modalities to alleviate motoneuronal defects in RTT. PMID:23986203

  10. Fibroblast growth factor 10 alters the balance between goblet and Paneth cells in the adult mouse small intestine.

    PubMed

    Al Alam, Denise; Danopoulos, Soula; Schall, Kathy; Sala, Frederic G; Almohazey, Dana; Fernandez, G Esteban; Georgia, Senta; Frey, Mark R; Ford, Henri R; Grikscheit, Tracy; Bellusci, Saverio

    2015-04-15

    Intestinal epithelial cell renewal relies on the right balance of epithelial cell migration, proliferation, differentiation, and apoptosis. Intestinal epithelial cells consist of absorptive and secretory lineage. The latter is comprised of goblet, Paneth, and enteroendocrine cells. Fibroblast growth factor 10 (FGF10) plays a central role in epithelial cell proliferation, survival, and differentiation in several organs. The expression pattern of FGF10 and its receptors in both human and mouse intestine and their role in small intestine have yet to be investigated. First, we analyzed the expression of FGF10, FGFR1, and FGFR2, in the human ileum and throughout the adult mouse small intestine. We found that FGF10, FGFR1b, and FGFR2b are expressed in the human ileum as well as in the mouse small intestine. We then used transgenic mouse models to overexpress Fgf10 and a soluble form of Fgfr2b, to study the impact of gain or loss of Fgf signaling in the adult small intestine. We demonstrated that overexpression of Fgf10 in vivo and in vitro induces goblet cell differentiation while decreasing Paneth cells. Moreover, FGF10 decreases stem cell markers such as Lgr5, Lrig1, Hopx, Ascl2, and Sox9. FGF10 inhibited Hes1 expression in vitro, suggesting that FGF10 induces goblet cell differentiation likely through the inhibition of Notch signaling. Interestingly, Fgf10 overexpression for 3 days in vivo and in vitro increased the number of Mmp7/Muc2 double-positive cells, suggesting that goblet cells replace Paneth cells. Further studies are needed to determine the mechanism by which Fgf10 alters cell differentiation in the small intestine. PMID:25721301

  11. Generation of Aorta Transcript Atlases of Wild-Type and Apolipoprotein E-null Mice by Laser Capture Microdissection-Based mRNA Expression Microarrays.

    PubMed

    Yin, Changjun; Mohanta, Sarajo; Ma, Zhe; Weber, Christian; Hu, Desheng; Weih, Falk; Habenicht, Andreas

    2015-01-01

    Atherosclerosis is a transmural chronic inflammatory disease of medium and large arteries. Though it is well recognized that immune responses contribute to atherosclerosis, it remains unclear whether these responses are carried out in secondary lymphoid organs such as the spleen and lymph nodes and/or within the arterial wall. Arteries are composed of three major layers, i.e., the laminae intima, media, and adventitia. However, each of these layers may play different roles in arterial wall biology and atherogenesis. We identified well-structured artery tertiary lymphoid organs (ATLOs) in the abdominal aorta adventitia but not in the intima of aged apolipoprotein E-null (ApoE(-/-)) mice. These observations suggested that disease-associated immune responses are highly territorialized within the arterial wall and that the adventitia may play distinct and hitherto unrecognized roles. Here, we set out to apply laser capture microdissection (LCM) to dissect plaque, media, adventitia, and adjacent aorta-draining lymph nodes (LN) in aged ApoE(-/-) mice in attempts to establish the territoriality of atherosclerosis immune responses. Using whole-genome mRNA expression microarrays of arterial wall tissues, we constructed robust transcript atlases of wild-type and ApoE(-/-) mouse aortas. Data were deposited in the National Center for Biotechnology Information's gene expression omnibus (GEO) and are accessible to the public through the Internet. These transcript atlases are anticipated to prove valuable to address a wide scope of issues ranging from atherosclerosis immunity and inflammation to the role of single genes in regulating arterial wall remodeling. This chapter presents protocols for LCM of mouse aorta and microarray expression analysis from LCM-isolated aorta laminae. PMID:26445797

  12. Isolation and Assessment of Single Long-Term Reconstituting Hematopoietic Stem Cells from Adult Mouse Bone Marrow.

    PubMed

    Kent, David G; Dykstra, Brad J; Eaves, Connie J

    2016-01-01

    Hematopoietic stem cells with long-term repopulating activity can now be routinely obtained at purities of 40% to 50% from suspensions of adult mouse bone marrow. Here we describe robust protocols for both their isolation as CD45(+) EPCR(+) CD150(+) CD48(-) (ESLAM) cells using multiparameter cell sorting and for tracking their clonal growth and differentiation activity in irradiated mice transplanted with single ESLAM cells. The simplicity of these procedures makes them attractive for characterizing the molecular and biological properties of individual hematopoietic stem cells with unprecedented power and precision. © 2016 by John Wiley & Sons, Inc. PMID:27532815

  13. 8-Oxoguanine accumulation in mitochondrial DNA causes mitochondrial dysfunction and impairs neuritogenesis in cultured adult mouse cortical neurons under oxidative conditions

    PubMed Central

    Leon, Julio; Sakumi, Kunihiko; Castillo, Erika; Sheng, Zijing; Oka, Sugako; Nakabeppu, Yusaku

    2016-01-01

    Oxidative stress and mitochondrial dysfunction are implicated in aging-related neurodegenerative disorders. 8-Oxoguanine (8-oxoG), a common oxidised base lesion, is often highly accumulated in brains from patients with neurodegenerative disorders. MTH1 hydrolyses 8-oxo-2′-deoxyguanosine triphosphate (8-oxo-dGTP) to 8-oxo-dGMP and pyrophosphate in nucleotide pools, while OGG1 excises 8-oxoG paired with cytosine in DNA, thereby minimising the accumulation of 8-oxoG in DNA. Mth1/Ogg1-double knockout (TO-DKO) mice are highly susceptible to neurodegeneration under oxidative conditions and show increased accumulation of 8-oxoG in mitochondrial DNA (mtDNA) in neurons, suggesting that 8-oxoG accumulation in mtDNA causes mitochondrial dysfunction. Here, we evaluated the contribution of MTH1 and OGG1 to the prevention of mitochondrial dysfunction during neuritogenesis in vitro. We isolated cortical neurons from adult wild-type and TO-DKO mice and maintained them with or without antioxidants for 2 to 5 days and then examined neuritogenesis. In the presence of antioxidants, both TO-DKO and wild-type neurons exhibited efficient neurite extension and arborisation. However, in the absence of antioxidants, the accumulation of 8-oxoG in mtDNA of TO-DKO neurons was increased resulting in mitochondrial dysfunction. Cells also exhibited poor neurite outgrowth with decreased complexity of neuritic arborisation, indicating that MTH1 and OGG1 are essential for neuritogenesis under oxidative conditions. PMID:26912170

  14. Advanced Glycation End Products Induce Obesity and Hepatosteatosis in CD-1 Wild-Type Mice

    PubMed Central

    Sayej, Wael N.; Knight III, Paul R.; Guo, Weidun Alan; Mullan, Barbara; Ohtake, Patricia J.; Davidson, Bruce A.; Khan, Abdur; Baker, Robert D.; Baker, Susan S.

    2016-01-01

    AGEs are a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with free amino groups of proteins, lipids, and/or nucleic acids. AGEs have been shown to play a role in various conditions including cardiovascular disease and diabetes. In this study, we hypothesized that AGEs play a role in the “multiple hit hypothesis” of nonalcoholic fatty liver disease (NAFLD) and contribute to the pathogenesis of hepatosteatosis. We measured the effects of various mouse chows containing high or low AGE in the presence of high or low fat content on mouse weight and epididymal fat pads. We also measured the effects of these chows on the inflammatory response by measuring cytokine levels and myeloperoxidase activity levels on liver supernatants. We observed significant differences in weight gain and epididymal fat pad weights in the high AGE-high fat (HAGE-HF) versus the other groups. Leptin, TNF-α, IL-6, and myeloperoxidase (MPO) levels were significantly higher in the HAGE-HF group. We conclude that a diet containing high AGEs in the presence of high fat induces weight gain and hepatosteatosis in CD-1 mice. This may represent a model to study the role of AGEs in the pathogenesis of hepatosteatosis and steatohepatitis. PMID:26942201

  15. Dysferlin deficiency blunts β-adrenergic-dependent lusitropic function of mouse heart.

    PubMed

    Wei, Bin; Wei, Hongguang; Jin, J-P

    2015-12-01

    Dysferlin is a cell membrane bound protein with a role in the repair of skeletal and cardiac muscle cells. Deficiency of dysferlin leads to limb-girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. In cardiac muscle, dysferlin is located at the intercalated disc and transverse tubule membranes. Loss of dysferlin causes death of cardiomyocytes, notably in ageing hearts, leading to dilated cardiomyopathy and heart failure in LGM2B patients. To understand the primary pathogenesis and pathophysiology of dysferlin cardiomyopathy, we studied cardiac phenotypes of young adult dysferlin knockout mice and found early myocardial hypertrophy with largely compensated baseline cardiac function. Cardiomyocytes isolated from dysferlin-deficient mice showed normal shortening and re-lengthening velocities in the absence of external load with normal peak systolic Ca(2+) but slower Ca(2+) re-sequestration than wild-type controls. The effects of isoproterenol on relaxation velocity, left ventricular systolic pressure and stroke volume were blunted in dysferlin-deficient mouse hearts compared with that in wild-type hearts. Young dysferlin-deficient mouse hearts expressed normal isoforms of myofilament proteins whereas the phosphorylation of ventricular myosin light chain 2 was significantly increased, implying a molecular response to the impaired lusitropic function. These early phenotypes of diastolic cardiac dysfunction and blunted lusitropic response of cardiac muscle to β-adrenergic stimulation indicate a novel pathogenic mechanism of dysferlin cardiomyopathy. PMID:26415898

  16. Morphological phenotyping of mouse hearts using optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Cua, Michelle; Lin, Eric; Lee, Ling; Sheng, Xiaoye; Wong, Kevin S. K.; Tibbits, Glen F.; Beg, Mirza Faisal; Sarunic, Marinko V.

    2014-11-01

    Transgenic mouse models have been instrumental in the elucidation of the molecular mechanisms behind many genetically based cardiovascular diseases such as Marfan syndrome (MFS). However, the characterization of their cardiac morphology has been hampered by the small size of the mouse heart. In this report, we adapted optical coherence tomography (OCT) for imaging fixed adult mouse hearts, and applied tools from computational anatomy to perform morphometric analyses. The hearts were first optically cleared and imaged from multiple perspectives. The acquired volumes were then corrected for refractive distortions, and registered and stitched together to form a single, high-resolution OCT volume of the whole heart. From this volume, various structures such as the valves and myofibril bundles were visualized. The volumetric nature of our dataset also allowed parameters such as wall thickness, ventricular wall masses, and luminal volumes to be extracted. Finally, we applied the entire acquisition and processing pipeline in a preliminary study comparing the cardiac morphology of wild-type mice and a transgenic mouse model of MFS.

  17. Blockage of VIP during mouse embryogenesis modifies adult behavior and results in permanent changes in brain chemistry.

    PubMed

    Hill, Joanna M; Hauser, Janet M; Sheppard, Lia M; Abebe, Daniel; Spivak-Pohis, Irit; Kushnir, Michal; Deitch, Iris; Gozes, Illana

    2007-01-01

    Vasoactive intestinal peptide (VIP) regulates growth and development during the early postimplantation period of mouse embryogenesis. Blockage of VIP with a VIP antagonist during this period results in growth restriction, microcephaly, and developmental delays. Similar treatment of neonatal rodents also causes developmental delays and impaired diurnal rhythms, and the adult brains of these animals exhibit neuronal dystrophy and increased VIP binding. These data suggest that blockage of VIP during the development of the nervous system can result in permanent changes to the brain. In the current study, pregnant mice were treated with a VIP antagonist during embryonic days 8 through 10. The adult male offspring were examined in tests of novelty, paired activity, and social recognition. Brain tissue was examined for several measures of chemistry and gene expression of VIP and related compounds. Glial cells from the cortex of treated newborn mice were plated with neurons and examined for VIP binding and their ability to enhance neuronal survival. Treated adult male mice exhibited increased anxiety-like behavior and deficits in social behavior. Brain tissue exhibited regionally specific changes in VIP chemistry and a trend toward increased gene expression of VIP and related compounds that reached statistical significance in the VIP receptor, VPAC-1, in the female cortex. When compared to control astrocytes, astrocytes from treated cerebral cortex produced further increases in neuronal survival with excess synaptic connections and reduced VIP binding. In conclusion, impaired VIP activity during mouse embryogenesis resulted in permanent changes to both adult brain chemistry/cell biology and behavior with aspects of autism-like social deficits. PMID:17726225

  18. Regional brain volumes changes in adult male FMR1-KO mouse on the FVB strain.

    PubMed

    Lai, J K Y; Lerch, J P; Doering, L C; Foster, J A; Ellegood, J

    2016-03-24

    Fragile X Syndrome (FXS) is the most common heritable single gene cause of autism spectrum disorder (ASD). FMR1-KO mice mimic the etiology and phenotypic manifestations of FXS. Neuroanatomical changes in specific brain regions have been reported in clinical settings and in preclinical models. FMR1-KO mice have been generated in different strains including C57Bl/6 (B6) and FVB. Mice on different genetic backgrounds have stable yet distinct behavioral phenotypes that may lead to unique gene-strain interactions on brain structure. Previous magnetic resonance imaging (MRI) studies have examined FMR1 knockout male mice on a B6 and found few differences compared to wild-type mice. Here, we examine brain volumes in FMR1 knockout male mice on a FVB background using high resolution (multi-channel 7.0Tesla) MRI. We observe multiple differences in the neuroanatomy of male FMR1-/y mice on a FVB background. Significantly larger relative volume (% total brain volume) differences were found in major white matter structures throughout the brain. In addition, there were changes in areas associated with fronto-striatal circuitry and other regions. Functional and structural connectivity differences are often seen in human ASD, and therefore, this increased white matter seen in the FMR1-KO-FVB could be highlighting a structural over-connectivity, which could lead to some of the behavioral abnormalities seen with the FMR1-KO-FVB mice. Furthermore, these results highlight the importance of genetic strain contribution to brain structure. PMID:26794591

  19. Extracellular enzyme activities during lignocellulose degradation by Streptomyces spp. : a comparative study of wild-type and genetically manipulated strains

    SciTech Connect

    Ramachandra, M.; Crawford, D.L.; Pometto, A.L. III

    1987-12-01

    The wild-type ligninolytic actinomycete Streptomyces viridosporus T7A and two genetically manipulated strains with enhanced abilities to produce a water-soluble lignin degradation intermediate, an acid-precipitable polymeric lignin (APPL), were grown on lignocellulose in solid-state fermentation cultures. Culture filtrates were periodically collected, analyzed for APPL, and assayed for extracellular lignocellulose-catabolizing enzyme activities. Two APPL-overproducing strains, UV irradiation mutant T7A-81 and protoplast fusion recombinant SR-10, had higher and longer persisting peroxidase, esterase, and endoglucanase activities than did the wild-type strain T7A. Results implicated one or more of these enzymes in lignin solubilization. Only mutant T7A-81 had higher xylanase activity than the wild type. The peroxidase was induced by both lignocellulose and APPL. This extracellular enzyme has some similarities to previously described ligninases in fungi. This is the first report of such an enzyme in Streptomyces spp. Four peroxidase isozymes were present, and all catalyzed the oxidation of 3,4-dihydroxyphenylalanine, while one also catalyzed hydrogen peroxide-dependent oxidation of homoprotocatechuic acid and caffeic acid. Three constitutive esterase isozymes were produced which differed in substrate specificity toward ..cap alpha..-naphthyl acetate and ..cap alpha..-naphthyl butyrate. Three endoglucanase bands, which also exhibited a low level of xylanase activity, were identified on polyacrylamide gels as was one xylanase-specific band. There were no major differences in the isoenzymes produced by the different strains. The probable role of each enzyme in lignocellulose degradation is discussed.

  20. Expression of mutant and wild-type TIMP3 in primary gingival fibroblasts from Sorsby's fundus dystrophy patients.

    PubMed

    Arris, Christine E; Bevitt, Debra J; Mohamed, Jeseem; Li, Zheng; Langton, Kevin P; Barker, Michael D; Clarke, Michael P; McKie, Norman

    2003-05-20

    Gingival fibroblast cell lines were derived from Sorsby's fundus dystrophy (SFD) patients carrying the S181C TIMP3 and the E139X TIMP3 mutations. These cell lines were grown in culture to study expression of the wild-type and mutant tissue inhibitor of metalloproteinase 3 (TIMP3) alleles from a normal diploid cell type. Firstly, patient cells were found to co-express the wild-type and mutant TIMP3 alleles, S181C TIMP3 or E139X TIMP3, at the mRNA level using restriction fragment length polymorphism (RFLP) analysis. A SpeI RFLP for E139X TIMP3 is described. Low levels of endogenous TIMP3 protein expression were elevated using the natural polysaccharide calcium pentosan polysulfate (CaPPs) in combination with the cytokine IL-1alpha. Immunoblotting detected protein expression from both wild-type and mutant alleles, S181C TIMP3 or E139X TIMP3. S181C TIMP3 from these cells was found to dimerise and retain MMP2 inhibitory activity. To facilitate studies of the E139X TIMP3 protein, the allele was expressed using HighFive insect cells. In this cell type, the E139X TIMP3 was synthesised as a mixture of monomer and dimer. Both monomeric and dimeric E139X TIMP3 protein retained MMP2 inhibitory activity in gelatin zymography. Expression of mutant E139X or S181C TIMP3 protein from a normal diploid patient-derived fibroblast cell had no effect on either MMP2 or MMP9 expression or activation whilst transcribed from their normal promoter context. PMID:12757930

  1. The Satellite Cell in Male and Female, Developing and Adult Mouse Muscle: Distinct Stem Cells for Growth and Regeneration

    PubMed Central

    Neal, Alice; Boldrin, Luisa; Morgan, Jennifer Elizabeth

    2012-01-01

    Satellite cells are myogenic cells found between the basal lamina and the sarcolemma of the muscle fibre. Satellite cells are the source of new myofibres; as such, satellite cell transplantation holds promise as a treatment for muscular dystrophies. We have investigated age and sex differences between mouse satellite cells in vitro and assessed the importance of these factors as mediators of donor cell engraftment in an in vivo model of satellite cell transplantation. We found that satellite cell numbers are increased in growing compared to adult and in male compared to female adult mice. We saw no difference in the expression of the myogenic regulatory factors between male and female mice, but distinct profiles were observed according to developmental stage. We show that, in contrast to adult mice, the majority of satellite cells from two week old mice are proliferating to facilitate myofibre growth; however a small proportion of these cells are quiescent and not contributing to this growth programme. Despite observed changes in satellite cell populations, there is no difference in engraftment efficiency either between satellite cells derived from adult or pre-weaned donor mice, male or female donor cells, or between male and female host muscle environments. We suggest there exist two distinct satellite cell populations: one for muscle growth and maintenance and one for muscle regeneration. PMID:22662253

  2. Chronic serotonin-norepinephrine reuptake transporter inhibition modifies basal respiratory output in adult mouse in vitro and in vivo

    PubMed Central

    Warren, Kelly A.; Solomon, Irene C.

    2012-01-01

    Respiratory disturbances are a common feature of panic disorder and present as breathing irregularity, hyperventilation, and increased sensitivity to carbon dioxide. Common therapeutic interventions, such as tricyclic (TCA) and selective serotonin reuptake inhibitor (SSRI) antidepressants, have been shown to ameliorate not only the psychological components of panic disorder but also the respiratory disturbances. These drugs are also prescribed for generalized anxiety and depressive disorders, neither of which are characterized by respiratory disturbances, and previous studies have demonstrated that TCAs and SSRIs exert effects on basal respiratory activity in animal models without panic disorder symptoms. Whether serotonin-norepinephrine reuptake inhibitors (SNRIs) have similar effects on respiratory activity remains to be determined. Therefore, the current study was designed to investigate the effects of chronic administration of the SNRI antidepressant venlafaxine (VHCL) on basal respiratory output. For these experiments, we recorded phrenic nerve discharge in an in vitro arterially-perfused adult mouse preparation and diaphragm electromyogram (EMG) activity in an in vivo urethane-anesthetized adult mouse preparation. We found that following 28-d VHCL administration, basal respiratory burst frequency was markedly reduced due to an increase in expiratory duration (TE), and the inspiratory duty cycle (TI/Ttot) was significantly shortened. In addition, post-inspiratory and spurious expiratory discharges were seen in vitro. Based on our observations, we suggest that drugs capable of simultaneously blocking both 5-HT and NE reuptake transporters have the potential to influence the respiratory control network in patients using SNRI therapy. PMID:22871263

  3. PPARγ mRNA in the adult mouse hypothalamus: distribution and regulation in response to dietary challenges

    PubMed Central

    Liu, Yang; Huang, Ying; Lee, Syann; Bookout, Angie L.; Castorena, Carlos M.; Wu, Hua; Gautron, Laurent

    2015-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor that was originally identified as a regulator of peroxisome proliferation and adipocyte differentiation. Emerging evidence suggests that functional PPARγ signaling also occurs within the hypothalamus. However, the exact distribution and identities of PPARγ-expressing hypothalamic cells remains under debate. The present study systematically mapped PPARγ mRNA expression in the adult mouse brain using in situ hybridization histochemistry. PPARγ mRNA was found to be expressed at high levels outside the hypothalamus including the neocortex, the olfactory bulb, the organ of the vasculosum of the lamina terminalis (VOLT), and the subfornical organ. Within the hypothalamus, PPARγ was present at moderate levels in the suprachiasmatic nucleus (SCh) and the ependymal of the 3rd ventricle. In all examined feeding-related hypothalamic nuclei, PPARγ was expressed at very low levels that were close to the limit of detection. Using qPCR techniques, we demonstrated that PPARγ mRNA expression was upregulated in the SCh in response to fasting. Double in situ hybridization further demonstrated that PPARγ was primarily expressed in neurons rather than glia. Collectively, our observations provide a comprehensive map of PPARγ distribution in the intact adult mouse hypothalamus. PMID:26388745

  4. Targeted deletion of Vglut2 expression in the embryonal telencephalon promotes an anxiolytic phenotype of the adult mouse

    PubMed Central

    Nordenankar, Karin; Bergfors, Assar

    2015-01-01

    Background Anxiety is a natural emotion experienced by all individuals. However, when anxiety becomes excessive, it contributes to the substantial group of anxiety disorders that affect one in three people and thus are among the most common psychiatric disorders. Anxiolysis, the reduction of anxiety, is mediated via several large groups of therapeutical compounds, but the relief is often only temporary, and increased knowledge of the neurobiology underlying anxiety is needed in order to improve future therapies. Aim We previously demonstrated that mice lacking forebrain expression of the Vesicular glutamate transporter 2 (Vglut2) from adolescence showed a strong anxiolytic behaviour as adults. In the current study, we wished to analyse if removal of Vglut2 expression already from mid-gestation of the mouse embryo would give rise to similar anxiolysis in the adult mouse. Methods We produced transgenic mice lacking Vglut2 from mid-gestation and analysed their affective behaviour, including anxiety, when they had reached adulthood. Results The transgenic mice lacking Vglut2 expression from mid-gestation showed certain signs of anxiolytic behaviour, but this phenotype was not as prominent as when Vglut2 was removed during adolescence. Conclusion Our results suggest that both embryonal and adolescent forebrain expression of Vglut2 normally contributes to balancing the level of anxiety. As the neurobiological basis for anxiety is similar across species, our results in mice may help improve the current understanding of the neurocircuitry of anxiety, and hence anxiolysis, also in humans. PMID:25857802

  5. Activation of CB1 inhibits NGF-induced sensitization of TRPV1 in adult mouse afferent neurons

    PubMed Central

    Wang, Zun-Yi; McDowell, Thomas; Wang, Peiqing; Alvarez, Roxanne; Gomez, Timothy; Bjorling, Dale E.

    2015-01-01

    Transient receptor potential vanilloid 1 (TRPV1)-containing afferent neurons convey nociceptive signals and play an essential role in pain sensation. Exposure to nerve growth factor (NGF) rapidly increases TRPV1 activity (sensitization). In the present study, we investigated whether treatment with the selective cannabinoid receptor 1 (CB1) agonist arachidonyl-2'-chloroethylamide (ACEA) affects NGF-induced sensitization of TRPV1 in adult mouse dorsal root ganglion (DRG) afferent neurons. We found that CB1, NGF receptor tyrosine kinase A (trkA), and TRPV1 are present in cultured adult mouse small- to medium-sized afferent neurons and treatment with NGF (100 ng/ml) for 30 minutes significantly increased the number of neurons that responded to capsaicin (as indicated by increased intracellular Ca2+ concentration). Pretreatment with the CB1 agonist ACEA (10 nM) inhibited the NGF-induced response, and this effect of ACEA was reversed by a selective CB1 antagonist. Further, pretreatment with ACEA inhibited NGF-induced phosphorylation of AKT. Blocking PI3 kinase activity also attenuated the NGF-induced increase in the number of neurons that responded to capsaicin. Our results indicate that the analgesic effect of CB1 activation may in part be due to inhibition of NGF-induced sensitization of TRPV1 and also that the effect of CB1 activation is at least partly mediated by attenuation of NGF-induced increased PI3 signaling. PMID:25088915

  6. A Novel Procedure for Rapid Imaging of Adult Mouse Brains with MicroCT Using Iodine-Based Contrast

    PubMed Central

    Anderson, Ryan; Maga, A. Murat

    2015-01-01

    High-resolution Magnetic Resonance Imaging (MRI) has been the primary modality for obtaining 3D cross-sectional anatomical information in animals for soft tissue, particularly brain. However, costs associated with MRI can be considerably high for large phenotypic screens for gross differences in the structure of the brain due to pathology and/or experimental manipulations. MicroCT (mCT), especially benchtop mCT, is becoming a common laboratory equipment with throughput rates equal or faster than any form of high-resolution MRI at lower costs. Here we explore adapting previously developed contrast based mCT to image adult mouse brains in-situ. We show that 2% weight per volume (w/v) iodine-potassium iodide solution can be successfully used to image adult mouse brains within 48 hours post-mortem when a structural support matrix is used. We demonstrate that hydrogel can be effectively used as a perfusant which limits the tissue shrinkage due to iodine. PMID:26571123

  7. Genetic manipulation of adult-born hippocampal neurons rescues memory in a mouse model of Alzheimer's disease.

    PubMed

    Richetin, Kevin; Leclerc, Clémence; Toni, Nicolas; Gallopin, Thierry; Pech, Stéphane; Roybon, Laurent; Rampon, Claire

    2015-02-01

    In adult mammals, neural progenitors located in the dentate gyrus retain their ability to generate neurons and glia throughout lifetime. In rodents, increased production of new granule neurons is associated with improved memory capacities, while decreased hippocampal neurogenesis results in impaired memory performance in several memory tasks. In mouse models of Alzheimer's disease, neurogenesis is impaired and the granule neurons that are generated fail to integrate existing networks. Thus, enhancing neurogenesis should improve functional plasticity in the hippocampus and restore cognitive deficits in these mice. Here, we performed a screen of transcription factors that could potentially enhance adult hippocampal neurogenesis. We identified Neurod1 as a robust neuronal determinant with the capability to direct hippocampal progenitors towards an exclusive granule neuron fate. Importantly, Neurod1 also accelerated neuronal maturation and functional integration of new neurons during the period of their maturation when they contribute to memory processes. When tested in an APPxPS1 mouse model of Alzheimer's disease, directed expression of Neurod1 in cycling hippocampal progenitors conspicuously reduced dendritic spine density deficits on new hippocampal neurons, to the same level as that observed in healthy age-matched control animals. Remarkably, this population of highly connected new neurons was sufficient to restore spatial memory in these diseased mice. Collectively our findings demonstrate that endogenous neural stem cells of the diseased brain can be manipulated to become new neurons that could allow cognitive improvement. PMID:25518958

  8. Accumulation of wild-type p53 protein in astrocytomas is not mediated by MDM2 gene amplification

    SciTech Connect

    Rubio, M.P.; Louis, D.N. Harvard Medical School, Boston, MA )

    1993-05-01

    The authors have previously described ten cases of astrocytoma (three WHO grade II, four grade III and four grade IV) with seemingly contradictory results on immunohistochemical analysis of the p53 protein and molecular genetic analysis of the p53 gene. Fixed, embedded tissues from these cases were immunohistochemically positive with the PAb 1801 antibody, which supposedly implies the presence of mutant protein. These ten cases, however, did not have mutations in exons 5 through 8 of the p53 gene, the conserved regions in which almost all human mutations have been described. The authors suggested that these cases might either represent overexpression of wild-type p53 protein (since the PAb 1801 antibody reacts with both wild-type and mutant p53 protein) or mutations in less conserved regions of the gene. To investigate these possibilities further, they performed single strand conformational polymorphism analysis and DNA sequencing on p53 exons 4, 9 and 10 in the nine cases with available DNA, since rare mutations have been noted at these loci. None of the cases showed alterations, making it highly unlikely that these tumors harbor mutations in exons of the p53 gene. They also performed immunohistochemistry on frozen sections from seven available tumors, using the mutant-specific antibody PAb 240 in addition to PAb 1801. All tumors continued to show positive staining with PAb 1801, but only one tumor reacted with PAb 240. The results support the hypothesis that the accumulated p53 protein in most cases is wild-type. Because the product of the MDM2 oncogene can bind to wild-type p53 protein, and because MDM2 amplification has recently been demonstrated in human tumors, the authors evaluated MDM2 amplification in the nine astrocytomas with available DNA. Using slot blot analysis with a 96-base pair, PCR-generated probe to the first exon of the MDM2 gene, they were unable to show MDM2 gene amplification in these tumors or in other assayed astrocytomas.

  9. The progressive nature of Wallerian degeneration in wild-type and slow Wallerian degeneration (WldS) nerves

    PubMed Central

    Beirowski, Bogdan; Adalbert, Robert; Wagner, Diana; Grumme, Daniela S; Addicks, Klaus; Ribchester, Richard R; Coleman, Michael P

    2005-01-01

    Background The progressive nature of Wallerian degeneration has long been controversial. Conflicting reports that distal stumps of injured axons degenerate anterogradely, retrogradely, or simultaneously are based on statistical observations at discontinuous locations within the nerve, without observing any single axon at two distant points. As axon degeneration is asynchronous, there are clear advantages to longitudinal studies of individual degenerating axons. We recently validated the study of Wallerian degeneration using yellow fluorescent protein (YFP) in a small, representative population of axons, which greatly improves longitudinal imaging. Here, we apply this method to study the progressive nature of Wallerian degeneration in both wild-type and slow Wallerian degeneration (WldS) mutant mice. Results In wild-type nerves, we directly observed partially fragmented axons (average 5.3%) among a majority of fully intact or degenerated axons 37–42 h after transection and 40–44 h after crush injury. Axons exist in this state only transiently, probably for less than one hour. Surprisingly, axons degenerated anterogradely after transection but retrogradely after a crush, but in both cases a sharp boundary separated intact and fragmented regions of individual axons, indicating that Wallerian degeneration progresses as a wave sequentially affecting adjacent regions of the axon. In contrast, most or all WldS axons were partially fragmented 15–25 days after nerve lesion, WldS axons degenerated anterogradely independent of lesion type, and signs of degeneration increased gradually along the nerve instead of abruptly. Furthermore, the first signs of degeneration were short constrictions, not complete breaks. Conclusions We conclude that Wallerian degeneration progresses rapidly along individual wild-type axons after a heterogeneous latent phase. The speed of progression and its ability to travel in either direction challenges earlier models in which clearance of

  10. Accumulation of a bioactive benzoisochromanequinone compound kalafungin by a wild type antitumor-medermycin-producing streptomycete strain.

    PubMed

    Lü, Jin; He, Qiang; Huang, Luyao; Cai, Xiaofeng; Guo, Wenwen; He, Jing; Zhang, Lili; Li, Aiying

    2015-01-01

    Medermycin and kalafungin, two antibacterial and antitumor antibiotics isolated from different streptomycetes, share an identical polyketide skeleton core. The present study reported the discovery of kalafungin in a medermycin-producing streptomycete strain for the first time. A mutant strain obtained through UV mutagenesis showed a 3-fold increase in the production of this antibiotic, compared to the wild type strain. Heterologous expression experiments suggested that its production was severely controlled by the gene cluster for medermycin biosynthesis. In all, these findings suggested that kalafungin and medermycin could be accumulated by the same streptomycete and share their biosynthetic pathway to some extent in this strain. PMID:25695632

  11. Accumulation of a Bioactive Benzoisochromanequinone Compound Kalafungin by a Wild Type Antitumor-Medermycin-Producing Streptomycete Strain

    PubMed Central

    Lü, Jin; He, Qiang; Huang, Luyao; Cai, Xiaofeng; Guo, Wenwen; He, Jing; Zhang, Lili; Li, Aiying

    2015-01-01

    Medermycin and kalafungin, two antibacterial and antitumor antibiotics isolated from different streptomycetes, share an identical polyketide skeleton core. The present study reported the discovery of kalafungin in a medermycin-producing streptomycete strain for the first time. A mutant strain obtained through UV mutagenesis showed a 3-fold increase in the production of this antibiotic, compared to the wild type strain. Heterologous expression experiments suggested that its production was severely controlled by the gene cluster for medermycin biosynthesis. In all, these findings suggested that kalafungin and medermycin could be accumulated by the same streptomycete and share their biosynthetic pathway to some extent in this strain. PMID:25695632

  12. Microglia replenished OHSC: A culture system to study in vivo like adult microglia.

    PubMed

    Masuch, Annette; van der Pijl, Rianne; Füner, Lisa; Wolf, Yochai; Eggen, Bart; Boddeke, Erik; Biber, Knut

    2016-08-01

    Recent data suggest that ramified microglia fulfil various tasks in the brain. However, to investigate this unique cell type cultured primary microglia are only a poor model. We here describe a method to deplete and repopulate organotypic hippocampal slice cultures (OHSC) with ramified microglia isolated from adult mouse brain creating microglia-replenished OHSC (Mrep-OHSC). Replenished microglia integrate into the tissue and ramify to a degree indistinguishable from their counterparts in the mouse brain. Moreover, wild-type slices replenished with microglia from TNFα-deficient animals provide similar results as OHSC prepared from microglia-specific TNFα-knockout mice (CX3CR1(cre) /TNFα(fl/fl) ). Furthermore, this study demonstrates that replenished microglia in OHSC maintain original functions and properties acquired in vivo. Microglia from ERCC1(Δ/ko) mice, a mouse model of accelerated aging, maintain enhanced Mac2 expression and their activated phenotype after replenishment to wild-type OHSC tissue. Thus, the present study demonstrates that Mrep-OHSC are a unique tool to construct chimeric brain slices allowing studying the function of different phenotypes of in vivo like microglia in a tissue culture setting. GLIA 2016 GLIA 2016;64:1285-1297. PMID:27145902

  13. Vitamin D2-Enriched Button Mushroom (Agaricus bisporus) Improves Memory in Both Wild Type and APPswe/PS1dE9 Transgenic Mice

    PubMed Central

    Bennett, Louise; Kersaitis, Cindy; Macaulay, Stuart Lance; Münch, Gerald; Niedermayer, Garry; Nigro, Julie; Payne, Matthew; Sheean, Paul; Vallotton, Pascal; Zabaras, Dimitrios; Bird, Michael

    2013-01-01

    Vitamin D deficiency is widespread, affecting over 30% of adult Australians, and increasing up to 80% for at-risk groups including the elderly (age>65). The role for Vitamin D in development of the central nervous system is supported by the association between Vitamin D deficiency and incidence of neurological and psychiatric disorders including Alzheimer’s disease (AD). A reported positive relationship between Vitamin D status and cognitive performance suggests that restoring Vitamin D status might provide a cognitive benefit to those with Vitamin D deficiency. Mushrooms are a rich source of ergosterol, which can be converted to Vitamin D2 by treatment with UV light, presenting a new and convenient dietary source of Vitamin D2. We hypothesised that Vitamin D2-enriched mushrooms (VDM) could prevent the cognitive and pathological abnormalities associated with dementia. Two month old wild type (B6C3) and AD transgenic (APPSwe/PS1dE9) mice were fed a diet either deficient in Vitamin D2 or a diet which was supplemented with VDM, containing 1±0.2 µg/kg (∼54 IU/kg) vitamin D2, for 7 months. Effects of the dietary intervention on memory were assessed pre- and post-feeding. Brain sections were evaluated for amyloid β (Aβ) plaque loads and inflammation biomarkers using immuno-histochemical methods. Plasma vitamin D metabolites, Aβ40, Aβ42, calcium, protein and cholesterol were measured using biochemical assays. Compared with mice on the control diet, VDM-fed wild type and AD transgenic mice displayed improved learning and memory, had significantly reduced amyloid plaque load and glial fibrillary acidic protein, and elevated interleukin-10 in the brain. The results suggest that VDM might provide a dietary source of Vitamin D2 and other bioactives for preventing memory-impairment in dementia. This study supports the need for a randomised clinical trial to determine whether or not VDM consumption can benefit cognitive performance in the wider population. PMID

  14. A physiologically based pharmacokinetic model for atrazine and its main metabolites in the adult male C57BL/6 mouse

    SciTech Connect

    Lin Zhoumeng; Fisher, Jeffrey W.; Ross, Matthew K.; Filipov, Nikolay M.

    2011-02-15

    Atrazine (ATR) is a chlorotriazine herbicide that is widely used and relatively persistent in the environment. In laboratory rodents, excessive exposure to ATR is detrimental to the reproductive, immune, and nervous systems. To better understand the toxicokinetics of ATR and to fill the need for a mouse model, a physiologically based pharmacokinetic (PBPK) model for ATR and its main chlorotriazine metabolites (Cl-TRIs) desethyl atrazine (DE), desisopropyl atrazine (DIP), and didealkyl atrazine (DACT) was developed for the adult male C57BL/6 mouse. Taking advantage of all relevant and recently made available mouse-specific data, a flow-limited PBPK model was constructed. The ATR and DACT sub-models included blood, brain, liver, kidney, richly and slowly perfused tissue compartments, as well as plasma protein binding and red blood cell binding, whereas the DE and DIP sub-models were constructed as simple five-compartment models. The model adequately simulated plasma levels of ATR and Cl-TRIs and urinary dosimetry of Cl-TRIs at four single oral dose levels (250, 125, 25, and 5 mg/kg). Additionally, the model adequately described the dose dependency of brain and liver ATR and DACT concentrations. Cumulative urinary DACT amounts were accurately predicted across a wide dose range, suggesting the model's potential use for extrapolation to human exposures by performing reverse dosimetry. The model was validated using previously reported data for plasma ATR and DACT in mice and rats. Overall, besides being the first mouse PBPK model for ATR and its Cl-TRIs, this model, by analogy, provides insights into tissue dosimetry for rats. The model could be used in tissue dosimetry prediction and as an aid in the exposure assessment to this widely used herbicide.

  15. Performance by Spring-Calving Cows Grazing Tall Fescue Pastures with Either the Wild-Type Toxic Endophyte or a Non-Toxic Novel Endophyte

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cows grazing 'Kentucky-31' tall fescue [Lolium arundinaceum (Schreb.) Darbysh.] infected with its wild-type endophyte (Neotyphodium coenophialum; E+) generally display suboptimal performance. Recently, endophyte strains that do not produce compounds toxic to cattle have been incorporated into tall ...

  16. Formation of DNA adducts in wild-type and transgenic mice expressing human sulfotransferases 1A1 and 1A2 after oral exposure to furfuryl alcohol

    PubMed Central

    Høie, Anja Hortemo; Monien, Bernhard Hans; Sakhi, Amrit Kaur; Glatt, Hansruedi; Hjertholm, Hege; Husøy, Trine

    2015-01-01

    Furfuryl alcohol (FFA) is present in many heat-treated foods as a result of its formation via dehydration of pentoses. It is also used legally as a flavouring agent. In an inhalation study conducted in the National Toxicology Program, FFA showed some evidence of carcinogenic activity in rats and mice. FFA was generally negative in conventional genotoxicity assays, which suggests that it may be a non-genotoxic carcinogen. However, it was recently found that FFA is mutagenic in Salmonella strains expressing appropriate sulfotransferases (SULTs), such as human or mouse SULT1A1. The same DNA adducts that were formed by FFA in these strains, mainly N 2-((furan-2-yl)methyl)-2′-deoxyguanosine (N 2-MF-dG), were also detected in tissues of FFA-exposed mice and even in human lung specimens. In the present study, a single oral dose of FFA (250mg/kg body weight) or saline was administered to FVB/N mice and transgenic mice expressing human SULT1A1/1A2 on the FVB/N background. The transgenic mice were used, since human and mouse SULT1A1 substantially differ in substrate specificity and tissue distribution. DNA adducts were studied in liver, kidney, proximal and distal small intestine as well as colon, using isotope-dilution ultra performance liquid chromatography (UPLC–MS/MS). Surprisingly, low levels of adducts that may represent N 2-MF-dG were detected even in tissues of untreated mice. FFA exposure enhanced the adduct levels in colon and liver, but not in the remaining investigated tissues of wild-type (wt) mice. The situation was similar in transgenic mice, except that N 2-MF-dG levels were also strongly enhanced in the proximal small intestine. These different results between wt and transgenic mice may be attributed to the fact that human SULT1A1, but not the orthologous mouse enzyme, is strongly expressed in the small intestine. PMID:25904584

  17. Hydroxyurea synergizes with valproic acid in wild-type p53 acute myeloid leukaemia

    PubMed Central

    Leitch, Calum; Osdal, Tereza; Andresen, Vibeke; Molland, Maren; Kristiansen, Silje; Nguyen, Xuan Nhi; Bruserud, Øystein; Gjertsen, Bjørn Tore; McCormack, Emmet

    2016-01-01

    Palliative care in acute myeloid leukaemia (AML) is inadequate. For elderly patients, unfit for intensive chemotherapy, median survival is 2–3 months. As such, there is urgent demand for low-toxic palliative alternatives. We have repositioned two commonly administered anti-leukaemia drugs, valproic acid (VPA) and hydroxyurea (HU), as a combination therapy in AML. The anti-leukemic effect of VPA and HU was assessed in multiple AML cell lines confirming the superior anti-leukemic effect of combination therapy. Mechanistic studies revealed that VPA amplified the ability of HU to slow S-phase progression and this correlated with significantly increased DNA damage. VPA was also shown to reduce expression of the DNA repair protein, Rad51. Interestingly, the tumour suppressor protein p53 was revealed to mitigate cell cycle recovery following combination induced arrest. The efficacy of combination therapy was validated in vivo. Combination treatment increased survival in OCI-AML3 and patient-derived xenograft mouse models of AML. Therapy response was confirmed by optical imaging with multiplexed near-infrared labelled antibodies. The combination of HU and VPA indicates significant potential in preclinical models of AML. Both compounds are widely available and well tolerated. We believe that repositioning this combination could significantly enhance the palliative care of patients unsuited to intensive chemotherapy. PMID:26812881

  18. Phenotypic expression of wild-type tomato and three wilty mutants in relation to abscisic acid accumulation in roots and leaflets of reciprocal grafts

    SciTech Connect

    Cornish, K.; Zeevaart, J.A.D. )

    1988-05-01

    Lycopersicon esculentum Mill. cv Rheinlands Ruhm (RR) and cv Moneymaker and the three wilty mutants flacca (flc), sitiens (sit), and sitiens{sup w} (sit{sup w}), together with the most reciprocal grafts, were grown in pots and in solution culture. Detached leaflets, and control and stem-girdled intact plants, were left turgid or were wilted in air. Detached leaflets and the leaflets and roots of the intact plants were analyzed for their abscisic acid (ABA) content. Turgid RR leaflets contained about 2.9 ng ABA per miligram dry weight. On average, the flc and sit leaflets contained 33 and 11% of this amount, respectively. The lack of ABA approximately correlated with the severity of the mutant phenotype. Mutant roots also contained less ABA than wild-type roots. Wild-type scions on mutant stocks (wild type/mutant) maintained the normal phenotype of ungrafted plants. Mutant scions grafted onto wild-type stocks reverted to a near wild-type phenotype. After the wild-type leaves were excised from solution culture-grown mutant/wild-type plants, the revertive morphology of the mutant scions was maintained, although endogenous ABA levels in the leaflets fell to typical mutant levels and the leaflets became wilty again. When stressed in air, both leaflets and roots of RR plants produced stress-induced ABA, but the mutant leaflets and roots did not. The roots and leaflets of the grafted plants behaved according to their own genotype, with the notable exception of mutant roots grown with wild-type scions. Roots of flc and sit{sup w} recovered the ability to accumulate stress-induced ABA when grafted with RR scions before the stress was imposed.

  19. Cellular toxicity of yeast prion protein Rnq1 can be modulated by N-terminal wild type huntingtin.

    PubMed

    Sethi, Ratnika; Patel, Vishal; Saleh, Aliabbas A; Roy, Ipsita

    2016-01-15

    Aggregation of the N-terminal human mutant huntingtin and the consequent toxicity in the yeast model of Huntington's disease (HD) requires the presence of Rnq1 protein (Rnq1p) in its prion conformation [RNQ1(+)]. The understanding of interaction of wild-type huntingtin (wt-Htt) with the amyloidogenic prion has some gaps. In this work, we show that N-terminal fragment of wt-Htt (N-wt-Htt) ameliorated the toxic effect of [RNQ1(+)] depending on expression levels of both proteins. When the expression of N-wt-Htt was high, it increased the expression and delayed the aggregation of [RNQ1(+)]. As the expression of N-wt-Htt was reduced, it formed high molecular weight aggregates along with the prion. Even when sequestered by [RNQ1(+)], the beneficial effect of N-wt-Htt on expression of Rnq1p and on cell survival was evident. Huntingtin protein ameliorated toxicity due to the prion protein [RNQ1(+)] in yeast cells in a dose-dependent manner, resulting in increase in cell survival, hinting at its probable role as a component of the proteostasis network of the cell. Taking into account the earlier reports of the beneficial effect of expression of N-wt-Htt on the aggregation of mutant huntingtin, the function of wild-type huntingtin as an inhibitor of protein aggregation in the cell needs to be explored. PMID:26628321

  20. Fructose 6-phosphate phosphoketolase activity in wild-type strains of Lactobacillus, isolated from the intestinal tract of pigs.

    PubMed

    Bolado-Martínez, E; Acedo-Félix, E; Peregrino-Uriarte, A B; Yepiz-Plascencia, G

    2012-01-01

    Phosphoketolases are key enzymes of the phosphoketolase pathway of heterofermentative lactic acid bacteria, which include lactobacilli. In heterofermentative lactobacilli xylulose 5-phosphate phosphoketolase (X5PPK) is the main enzyme of the phosphoketolase pathway. However, activity of fructose 6-phosphate phosphoketolase (F6PPK) has always been considered absent in lactic acid bacteria. In this study, the F6PPK activity was detected in 24 porcine wild-type strains of Lactobacillus reuteri and Lactobacillus mucosae, but not in the Lactobacillus salivarius or in L. reuteri ATCC strains. The activity of F6PPK increased after treatment of the culture at low-pH and diminished after porcine bile-salts stress conditions in wild-type strains of L. reuteri. Colorimetric quantification at 505 nm allowed to differentiate between microbial strains with low activity and without the activity of F6PPK. Additionally, activity of F6PPK and the X5PPK gene expression levels were evaluated by real time PCR, under stress and nonstress conditions, in 3 L. reuteri strains. Although an exact correlation, between enzyme activity and gene expression was not obtained, it remains possible that the xpk gene codes for a phosphoketolase with dual substrate, at least in the analyzed strains of L. reuteri. PMID:23101386

  1. MLL fusion proteins preferentially regulate a subset of wild-type MLL target genes in the leukemic genome

    PubMed Central

    Wang, Qian-fei; Wu, George; Mi, Shuangli; He, Fuhong; Wu, Jun; Dong, Jingfang; Luo, Roger T.; Mattison, Ryan; Kaberlein, Joseph J.; Prabhakar, Shyam; Ji, Hongkai

    2011-01-01

    MLL encodes a histone methyltransferase that is critical in maintaining gene expression during embryonic development and hematopoiesis. 11q23 translocations result in the formation of chimeric MLL fusion proteins that act as potent drivers of acute leukemia. However, it remains unclear what portion of the leukemic genome is under the direct control of MLL fusions. By comparing patient-derived leukemic cell lines, we find that MLL fusion-bound genes are a small subset of that recognized by wild-type MLL. In an inducible MLL-ENL model, MLL fusion protein binding and changes in H3K79 methylation are limited to a specific portion of the genome, whereas wild-type MLL distributes to a much larger set of gene loci. Surprisingly, among 223 MLL-ENL–bound genes, only 12 demonstrate a significant increase in mRNA expression on induction of the fusion protein. In addition to Hoxa9 and Meis1, this includes Eya1 and Six1, which comprise a heterodimeric transcription factor important in several developmental pathways. We show that Eya1 has the capacity to immortalize hematopoietic progenitor cells in vitro and collaborates with Six1 in hematopoietic transformation assays. Altogether, our data suggest that MLL fusions contribute to the development of acute leukemia through direct activation of a small set of target genes. PMID:21518926

  2. Crystal Structures and Functional Characterization of Wild Type and Active Sites Mutants of CYP101D1

    PubMed Central

    Batabyal, Dipanwita; Poulos, Thomas L.

    2014-01-01

    Although CYP101D1 and P450cam catayze the same reaction at a similar rate and share strikingly similar active site architectures, there are significance functional differences. CYP101D1 thus provides an opportunity to probe what structural and functional features must be shared and what can differ yet maintain high catalytic efficiency. Crystal structures of the cyanide complex of wild type CYP101D1 and it active site mutants, D259N and T260A, have been solved. The conformational changes in CYP101D1 upon cyanide binding are very similar to P450cam indicating a similar mechanism for proton delivery during oxygen activation using solvent assisted proton transfer. The D259N-CN− complex shows a perturbed solvent structure compared to wild type which is similar to what was observed in the oxy-complex of the corresonding D251N mutant in P450cam. As in P450cam the T260A mutant is highly uncoupled while the D259N gives barely detectable activity. Despite these similarities, CYP101D1 is able to use the P450cam redox partners while P450cam cannot use the CYP101D1 redox partners. Thus the strict requirement of P450cam for its own redox partner is relaxed in CYP101D1. Differences in the local environment of the essential Asp (Asp259 in CYP101D1) provides a strucutral basis for understanding these functional differences. PMID:24261604

  3. Nitrosative damage to free and zinc-bound cysteine thiols underlies nitric oxide toxicity in wild-type Borrelia burgdorferi

    PubMed Central

    Bourret, Travis J; Boylan, Julie A; Lawrence, Kevin A; Gherardini, Frank C

    2011-01-01

    Borrelia burgdorferi encounters potentially harmful reactive nitrogen species (RNS) throughout its infective cycle. In this study, diethylamine NONOate (DEA/NO) was used to characterize the lethal effects of RNS on B. burgdorferi. RNS produce a variety of DNA lesions in a broad spectrum of microbial pathogens; however, levels of the DNA deamination product, deoxyinosine, and the numbers of apurinic/apyrimidinic (AP) sites were identical in DNA isolated from untreated and DEA/NO-treated B. burgdorferi cells. Strains with mutations in the nucleotide excision repair (NER) pathway genes uvrC or uvrB treated with DEA/NO had significantly higher spontaneous mutation frequencies, increased numbers of AP sites in DNA and reduced survival compared with wild-type controls. Polyunsaturated fatty acids in B. burgdorferi cell membranes, which are susceptible to peroxidation by reactive oxygen species (ROS), were not sensitive to RNS-mediated lipid peroxidation. However, treatment of B. burgdorferi cells with DEA/NO resulted in nitrosative damage to several proteins, including the zinc-dependent glycolytic enzyme fructose-1,6-bisphosphate aldolase (BB0445), the Borrelia oxidative stress regulator (BosR) and neutrophil-activating protein (NapA). Collectively, these data suggested that nitrosative damage to proteins harbouring free or zinc-bound cysteine thiols, rather than DNA or membrane lipids underlies RNS toxicity in wild-type B. burgdorferi. PMID:21564333

  4. Discovery of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin in wild-type cells

    PubMed Central

    Morkunas, Bernardas; Gal, Balint; Galloway, Warren R J D; Hodgkinson, James T; Ibbeson, Brett M; Sing Tan, Yaw; Welch, Martin

    2016-01-01

    Summary Pyocyanin is a small molecule produced by Pseudomonas aeruginosa that plays a crucial role in the pathogenesis of infections by this notorious opportunistic pathogen. The inhibition of pyocyanin production has been identified as an attractive antivirulence strategy for the treatment of P. aeruginosa infections. Herein, we report the discovery of an inhibitor of pyocyanin production in cultures of wild-type P. aeruginosa which is based around a 4-alkylquinolin-2(1H)-one scaffold. To the best of our knowledge, this is the first reported example of pyocyanin inhibition by a compound based around this molecular framework. The compound may therefore be representative of a new structural sub-class of pyocyanin inhibitors, which could potentially be exploited in in a therapeutic context for the development of critically needed new antipseudomonal agents. In this context, the use of wild-type cells in this study is notable, since the data obtained are of direct relevance to native situations. The compound could also be of value in better elucidating the role of pyocyanin in P. aeruginosa infections. Evidence suggests that the active compound reduces the level of pyocyanin production by inhibiting the cell–cell signalling mechanism known as quorum sensing. This could have interesting implications; quorum sensing regulates a range of additional elements associated with the pathogenicity of P. aeruginosa and there is a wide range of other potential applications where the inhibition of quorum sensing is desirable. PMID:27559393

  5. Intra-host competition between nef-defective escape mutants and wild-type human immunodeficiency virus type 1.

    PubMed Central

    Altes, H K; Jansen, V A

    2000-01-01

    Various forms of nef genes with deletions at conserved positions along the sequence have been reported to persist in human immunodeficiency virus type 1 infected patients. We investigate the forces maintaining such variants in the proviral population. The main selection pressures are preservation of function and host immune response. The crippled Nef protein might have fewer epitopes, and as such be less visible to the specific immune response, but it will lose some function. Does a trade-off between avoidance of the immune response and loss of function explain the dynamics of the crippled virus found in the patients? To answer this question, we formulated a deterministic model of the virus-host interactions. We found that when the crippled protein presents few epitopes and suffers little loss of function, the two viral types can coexist. Otherwise, the wild-type comes to prevail. The mutant form might initially dominate, but as the selective pressure by the CD84+ T cells decreases over the course of infection, the advantage for the crippled form of losing epitopes disappears. Hence, we go from a situation of coexistence of wild-type and mutant, to a situation of only full-length nef. The results are discussed in the context of the suggested use of live attenuated vaccines having deletions in nef. PMID:10687825

  6. Characteristics of alpha/beta interferon induction after infection of murine fibroblasts with wild-type and mutant alphaviruses

    SciTech Connect

    Burke, Crystal W.; Gardner, Christina L.; Steffan, Joshua J.; Ryman, Kate D.; Klimstra, William B.

    2009-12-05

    We examined the characteristics of interferon alpha/beta (IFN-alpha/beta) induction after alphavirus or control Sendai virus (SeV) infection of murine fibroblasts (MEFs). As expected, SeV infection of wild-type (wt) MEFs resulted in strong dimerization of IRF3 and the production of high levels of IFN-alpha/beta. In contrast, infection of MEFs with multiple alphaviruses failed to elicit detectable IFN-alpha/beta. In more detailed studies, Sindbis virus (SINV) infection caused dimerization and nuclear migration of IRF3, but minimal IFN-beta promoter activity, although surprisingly, the infected cells were competent for IFN production by other stimuli early after infection. A SINV mutant defective in host macromolecular synthesis shutoff induced IFN-alpha/beta in the MEF cultures dependent upon the activities of the TBK1 IRF3 activating kinase and host pattern recognition receptors (PRRs) PKR and MDA5 but not RIG-I. These results suggest that wild-type alphaviruses antagonize IFN induction after IRF3 activation but also may avoid detection by host PRRs early after infection.

  7. Genetic background and environmental conditions drive metabolic variation in wild type and transgenic soybean (Glycine max) seeds.

    PubMed

    Cohen, Hagai; Shir, Ofer M; Yu, Yang; Hou, Wensheng; Sun, Shi; Han, Tianfu; Amir, Rachel

    2016-08-01

    The metabolic profiles and composition of storage reserves of agricultural crop seeds are strongly regulated by heritable and environmental factors. Yet, very little is known about the genetic and environmental determinants of adaptive metabolic variation amongst wild type as well as transgenic seed populations derived from the same genetic background, grown under natural field conditions. The goal of the current study was to investigate the effects of natural environmental conditions on wild type and transgenic soybean seeds expressing a feedback-insensitive form of cystathionine γ-synthase, a methionine main regulatory enzyme. The seeds were grown in four geographically distinct habitats in China and then assayed for primary metabolic profiles using gas chromatography mass spectrometry, morphological traits and storage reserve accumulation. The analyses revealed changes in the levels of primary metabolites which evidently exhibited high correlation to methionine regardless of changes in environmental conditions. The environment, however, constituted a major determinant of metabolic profiles amongst seeds, as much more metabolites were observed to be affected by this variable, particularly along the north-to-south latitudinal gradient. The observations suggest that metabolic variation amongst seeds grown under natural field conditions depends upon the complex relationships existing amongst their genetic background and the environmental conditions characterizing their cultivation areas. PMID:27038216

  8. BclxL Changes Conformation upon Binding to Wild-type but Not Mutant p53 DNA Binding Domain*

    PubMed Central

    Hagn, Franz; Klein, Christian; Demmer, Oliver; Marchenko, Natasha; Vaseva, Angelina; Moll, Ute M.; Kessler, Horst

    2010-01-01

    p53 can induce apoptosis through mitochondrial membrane permeabilization by interaction of its DNA binding region with the anti-apoptotic proteins BclxL and Bcl2. However, little is known about the action of p53 at the mitochondria in molecular detail. By using NMR spectroscopy and fluorescence polarization we characterized the binding of wild-type and mutant p53 DNA binding domains to BclxL and show that the wild-type p53 DNA binding domain leads to structural changes in the BH3 binding region of BclxL, whereas mutants fail to induce such effects due to reduced affinity. This was probed by induced chemical shift and residual dipolar coupling data. These data imply that p53 partly achieves its pro-apoptotic function at the mitochondria by facilitating interaction between BclxL and BH3-only proteins in an allosteric mode of action. Furthermore, we characterize for the first time the binding behavior of Pifithrin-μ, a specific small molecule inhibitor of the p53-BclxL interaction, and present a structural model of the protein-ligand complex. A rather unusual behavior is revealed whereby Pifithrin-μ binds to both sides of the protein-protein complex. These data should facilitate the rational design of more potent specific BclxL-p53 inhibitors. PMID:19955567

  9. Gene Expression Analysis Indicates Divergent Mechanisms in DEN-Induced Carcinogenesis in Wild Type and Bid-Deficient Livers.

    PubMed

    Yu, Changshun; Yan, Shengmin; Khambu, Bilon; Chen, Xiaoyun; Dong, Zheng; Luo, Jianhua; Michalopoulos, George K; Wu, Shangwei; Yin, Xiao-Ming

    2016-01-01

    Bid is a Bcl-2 family protein. In addition to its pro-apoptosis function, Bid can also promote cell proliferation, maintain S phase checkpoint, and facilitate inflammasome activation. Bid plays important roles in tissue injury and regeneration, hematopoietic homeostasis, and tumorigenesis. Bid participates in hepatic carcinogenesis but the mechanism is not fully understood. Deletion of Bid resulted in diminished tumor burden and delayed tumor progression in a liver cancer model. In order to better understand the Bid-regulated events during hepatic carcinogenesis we performed gene expression analysis in wild type and bid-deficient mice treated with a hepatic carcinogen, diethylnitrosamine. We found that deletion of Bid caused significantly fewer alterations in gene expression in terms of the number of genes affected and the number of pathways affected. In addition, the expression profiles were remarkably different. In the wild type mice, there was a significant increase in the expression of growth regulation-related and immune/inflammation response-related genes, and a significant decrease in the expression of metabolism-related genes, both of which were diminished in bid-deficient livers. These data suggest that Bid could promote hepatic carcinogenesis via growth control and inflammation-mediated events. PMID:27196317

  10. Gene Expression Analysis Indicates Divergent Mechanisms in DEN-Induced Carcinogenesis in Wild Type and Bid-Deficient Livers

    PubMed Central

    Yu, Changshun; Yan, Shengmin; Khambu, Bilon; Chen, Xiaoyun; Dong, Zheng; Luo, Jianhua; Michalopoulos, George K.; Wu, Shangwei; Yin, Xiao-Ming

    2016-01-01

    Bid is a Bcl-2 family protein. In addition to its pro-apoptosis function, Bid can also promote cell proliferation, maintain S phase checkpoint, and facilitate inflammasome activation. Bid plays important roles in tissue injury and regeneration, hematopoietic homeostasis, and tumorigenesis. Bid participates in hepatic carcinogenesis but the mechanism is not fully understood. Deletion of Bid resulted in diminished tumor burden and delayed tumor progression in a liver cancer model. In order to better understand the Bid-regulated events during hepatic carcinogenesis we performed gene expression analysis in wild type and bid-deficient mice treated with a hepatic carcinogen, diethylnitrosamine. We found that deletion of Bid caused significantly fewer alterations in gene expression in terms of the number of genes affected and the number of pathways affected. In addition, the expression profiles were remarkably different. In the wild type mice, there was a significant increase in the expression of growth regulation-related and immune/inflammation response-related genes, and a significant decrease in the expression of metabolism-related genes, both of which were diminished in bid-deficient livers. These data suggest that Bid could promote hepatic carcinogenesis via growth control and inflammation-mediated events. PMID:27196317

  11. Endocytic Trafficking Routes of Wild Type and ΔF508 Cystic Fibrosis Transmembrane Conductance RegulatorD⃞

    PubMed Central

    Gentzsch, Martina; Chang, Xiu-Bao; Cui, Liying; Wu, Yufeng; Ozols, Victor V.; Choudhury, Amit; Pagano, Richard E.; Riordan, John R.

    2004-01-01

    Intracellular trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) is a focus of attention because it is defective in most patients with cystic fibrosis. ΔF508 CFTR, which does not mature conformationally, normally does not exit the endoplasmic reticulum, but if induced to do so at reduced temperature is short-lived at the surface. We used external epitope-tagged constructs to elucidate the itinerary and kinetics of wild type and ΔF508 CFTR in the endocytic pathway and visualized movement of CFTR from the surface to intracellular compartments. Modulation of different endocytic steps with low temperature (16°C) block, protease inhibitors, and overexpression of wild type and mutant Rab GTPases revealed that surface CFTR enters several different routes, including a Rab5-dependent initial step to early endosomes, then either Rab11-dependent recycling back to the surface or Rab7-regulated movement to late endosomes or alternatively Rab9-mediated transit to the trans-Golgi network. Without any of these modulations ΔF508 CFTR rapidly disappears from and does not return to the cell surface, confirming that its altered structure is detected in the distal as well as proximal secretory pathway. Importantly, however, the mutant protein can be rescued at the plasma membrane by Rab11 overexpression, proteasome inhibitors, or inhibition of Rab5-dependent endocytosis. PMID:15075371

  12. A recombinant measles vaccine virus expressing wild-type glycoproteins: consequences for viral spread and cell tropism.

    PubMed

    Johnston, I C; ter Meulen, V; Schneider-Schaulies, J; Schneider-Schaulies, S

    1999-08-01

    Wild-type, lymphotropic strains of measles virus (MV) and tissue culture-adapted MV vaccine strains possess different cell tropisms. This observation has led to attempts to identify the viral receptors and to characterize the functions of the MV glycoproteins. We have functionally analyzed the interactions of MV hemagglutinin (H) and fusion (F) proteins of vaccine (Edmonston) and wild-type (WTF) strains in different combinations in transfected cells. Cell-cell fusion occurs when both Edmonston F and H proteins are expressed in HeLa or Vero cells. The expression of WTF glycoproteins in HeLa cells did not result in syncytia, yet they fused efficiently with cells of lymphocytic origin. To further investigate the role of the MV glycoproteins in virus cell entry and also the role of other viral proteins in cell tropism, we generated recombinant vaccine MVs containing one or both glycoproteins from WTF. These viruses were viable and grew similarly in lymphocytic cells. Recombinant viruses expressing the WTFH protein showed a restricted spread in HeLa cells but spread efficiently in Vero cells. Parental WTF remained restricted in both cell types. Therefore, not only differential receptor usage but also other cell-specific factors are important in determining MV cell tropism. PMID:10400788

  13. Utilization of Whole-Cell MALDI-TOF Mass Spectrometry to Differentiate Burkholderia pseudomallei Wild-Type and Constructed Mutants

    PubMed Central

    Jaresitthikunchai, Janthima; Roytrakul, Sittiruk; Tungpradabkul, Sumalee

    2015-01-01

    Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS) has been widely adopted as a useful technology in the identification and typing of microorganisms. This study employed the whole-cell MALDI-TOF MS to identify and differentiate wild-type and mutants containing constructed single gene mutations of Burkholderia pseudomallei, a pathogenic bacterium causing melioidosis disease in both humans and animals. Candidate biomarkers for the B. pseudomallei mutants, including rpoS, ppk, and bpsI isolates, were determined. Taxon-specific and clinical isolate-specific biomarkers of B. pseudomallei were consistently found and conserved across all average mass spectra. Cluster analysis of MALDI spectra of all isolates exhibited separate distribution. A total of twelve potential mass peaks discriminating between wild-type and mutant isolates were identified using ClinProTools analysis. Two peaks (m/z 2721 and 2748 Da) were specific for the rpoS isolate, three (m/z 3150, 3378, and 7994 Da) for ppk, and seven (m/z 3420, 3520, 3587, 3688, 4623, 4708, and 5450 Da) for bpsI. Our findings demonstrated that the rapid, accurate, and reproducible mass profiling technology could have new implications in laboratory-based rapid differentiation of extensive libraries of genetically altered bacteria. PMID:26656930

  14. Accelerated telomere shortening and replicative senescence in human fibroblasts overexpressing mutant and wild-type lamin A

    SciTech Connect

    Huang Shurong; Risques, Rosa Ana; Martin, George M.; Rabinovitch, Peter S.; Oshima, Junko

    2008-01-01

    LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. To our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectable WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes.

  15. BclxL changes conformation upon binding to wild-type but not mutant p53 DNA binding domain.

    PubMed

    Hagn, Franz; Klein, Christian; Demmer, Oliver; Marchenko, Natasha; Vaseva, Angelina; Moll, Ute M; Kessler, Horst

    2010-01-29

    p53 can induce apoptosis through mitochondrial membrane permeabilization by interaction of its DNA binding region with the anti-apoptotic proteins BclxL and Bcl2. However, little is known about the action of p53 at the mitochondria in molecular detail. By using NMR spectroscopy and fluorescence polarization we characterized the binding of wild-type and mutant p53 DNA binding domains to BclxL and show that the wild-type p53 DNA binding domain leads to structural changes in the BH3 binding region of BclxL, whereas mutants fail to induce such effects due to reduced affinity. This was probed by induced chemical shift and residual dipolar coupling data. These data imply that p53 partly achieves its pro-apoptotic function at the mitochondria by facilitating interaction between BclxL and BH3-only proteins in an allosteric mode of action. Furthermore, we characterize for the first time the binding behavior of Pifithrin-mu, a specific small molecule inhibitor of the p53-BclxL interaction, and present a structural model of the protein-ligand complex. A rather unusual behavior is revealed whereby Pifithrin-mu binds to both sides of the protein-protein complex. These data should facilitate the rational design of more potent specific BclxL-p53 inhibitors. PMID:19955567

  16. Ablation of the Locus Coeruleus Increases Oxidative Stress in Tg-2576 Transgenic but Not Wild-Type Mice

    PubMed Central

    Hurko, Orest; Boudonck, Kurt; Gonzales, Cathleen; Hughes, Zoe A.; Jacobsen, J. Steve; Reinhart, Peter H.; Crowther, Daniel

    2010-01-01

    Mice transgenic for production of excessive or mutant forms of beta-amyloid differ from patients with Alzheimer's disease in the degree of inflammation, oxidative damage, and alteration of intermediary metabolism, as well as the paucity or absence of neuronal atrophy and cognitive impairment. Previous observers have suggested that differences in inflammatory response reflect a discrepancy in the state of the locus coeruleus (LC), loss of which is an early change in Alzheimer's disease but which is preserved in the transgenic mice. In this paper, we extend these observations by examining the effects of the LC on markers of oxidative stress and intermediary metabolism. We compare four groups: wild-type or Tg2576 Aβ transgenic mice injected with DSP4 or vehicle. Of greatest interest were metabolites different between ablated and intact transgenics, but not between ablated and intact wild-type animals. The Tg2576_DSP4 mice were distinguished from the other three groups by oxidative stress and altered energy metabolism. These observations provide further support for the hypothesis that Tg2576 Aβ transgenic mice with this ablation may be a more congruent model of Alzheimer's disease than are transgenics with an intact LC. PMID:20981353

  17. Bronze-2 gene of maize: reconstruction of a wild-type allele and analysis of transcription and splicing.

    PubMed Central

    Nash, J; Luehrsen, K R; Walbot, V

    1990-01-01

    The maize Bronze-2 (Bz2) gene, whose product acts late in the anthocyanin biosynthetic pathway, has been cloned and its transcript has been mapped. We have developed a general procedure for reconstructing wild-type alleles from transposable element-induced mutants. An existing transposon-containing clone, bz2::mu1 [McLaughlin, M., and Walbot, V. (1987). Genetics 117, 771-776], was modified by replacing the region of bz2::mu1 containing the transposon with the corresponding polymerase chain reaction-amplified sequence from the progenitor allele that has no Mu insertion. Particle gun delivery of the reconstructed Bz2 gene to embryonic scutellar tissue lacking a functional Bz2 gene complemented the bz2 mutant phenotype, as demonstrated by the production of purple spots. Having cloned the wild-type allele, we then analyzed the Bz2 transcript, whose features include an 82-nucleotide 5'-untranslated leader, one small intron (78 base pairs) within the coding region, and multiple polyadenylation sites. Four Mutator transposon insertions that eliminate gene function were mapped within the 850-nucleotide transcription unit. We found that variable levels of unspliced Bz2 RNA are present in purple husk tissue; this finding may indicate that the expression of Bz2 is regulated in part at the level of transcript processing. PMID:1967051

  18. Resistance to cadmium ions and formation of a cadmium-binding complex in various wild-type yeasts.

    PubMed

    Inouhe, M; Sumiyoshi, M; Tohoyama, H; Joho, M

    1996-04-01

    The resistance to cadmium ions (Cd-resistance) and possible formation of cadmium-binding complexes were examined in eight different wild-type yeasts. Saccharomyces exiguus, Pichia farinosa, Torulaspora delbrueckii and Schizosaccharomyces octosporus exhibited partial Cd-resistance, as compared to the Cd-resistant strain 301N and the Cu-resistant but Cd-sensitive strain X2180-1B of Saccharomyces cerevisiae. Saccharomyces carlsbergensis, Pichia mogii, Zygosaccharomyces rouxii and Kluyveromyces lactis were all Cd-sensitive. The partially Cd-sensitive species, with the exception of S. exiguus, accumulated Cd2+ ions in the cytoplasmic fraction to varying extents. This fraction from S. octosporus included a Cd-binding complex that contained (gamma EC)nG peptides known as cadystins or phytochelatins, while P. farinosa and T. delbrueckii synthesized Cd-binding proteins that were similar to the Cd-metallothionein produced by S. cerevisiae 301N in terms of molecular weight and amino acid composition. These results suggest that such cytoplasmic molecules play a role in the Cd-tolerance of the above three species of yeast. S. exiguus retained most cadmium in the cell wall fraction and no Cd-binding complex was found in the cytoplasm, an indication of the important role of the cell wall in its Cd-tolerance. Different modes of binding of Cd2+ ions appear to be involved in the Cd-resistance of wild-type yeasts and fungi. PMID:8673342

  19. MicroRNAs and Their Targets Are Differentially Regulated in Adult and Neonatal Mouse CD8+ T Cells.

    PubMed

    Wissink, Erin M; Smith, Norah L; Spektor, Roman; Rudd, Brian D; Grimson, Andrew

    2015-11-01

    Immunological memory, which protects organisms from re-infection, is a hallmark of the mammalian adaptive immune system and the underlying principle of vaccination. In early life, however, mice and other mammals are deficient at generating memory CD8+ T cells, which protect organisms from intracellular pathogens. The molecular basis that differentiates adult and neonatal CD8+ T cells is unknown. MicroRNAs (miRNAs) are both developmentally regulated and required for normal adult CD8+ T cell functions. We used next-generation sequencing to identify mouse miRNAs that are differentially regulated in adult and neonatal CD8+ T cells, which may contribute to the impaired development of neonatal memory cells. The miRNA profiles of adult and neonatal cells were surprisingly similar during infection; however, we observed large differences prior to infection. In particular, miR-29 and miR-130 have significant differential expression between adult and neonatal cells before infection. Importantly, using RNA-Seq, we detected reciprocal changes in expression of messenger RNA targets for both miR-29 and miR-130. Moreover, targets that we validated include Eomes and Tbx21, key genes that regulate the formation of memory CD8+ T cells. Notably, age-dependent changes in miR-29 and miR-130 are conserved in human CD8+ T cells, further suggesting that these developmental differences are biologically relevant. Together, these results demonstrate that miR-29 and miR-130 are likely important regulators of memory CD8+ T cell formation and suggest that neonatal cells are committed to a short-lived effector cell fate prior to infection. PMID:26416483

  20. MicroRNAs and Their Targets Are Differentially Regulated in Adult and Neonatal Mouse CD8+ T Cells

    PubMed Central

    Wissink, Erin M.; Smith, Norah L.; Spektor, Roman; Rudd, Brian D.; Grimson, Andrew

    2015-01-01

    Immunological memory, which protects organisms from re-infection, is a hallmark of the mammalian adaptive immune system and the underlying principle of vaccination. In early life, however, mice and other mammals are deficient at generating memory CD8+ T cells, which protect organisms from intracellular pathogens. The molecular basis that differentiates adult and neonatal CD8+ T cells is unknown. MicroRNAs (miRNAs) are both developmentally regulated and required for normal adult CD8+ T cell functions. We used next-generation sequencing to identify mouse miRNAs that are differentially regulated in adult and neonatal CD8+ T cells, which may contribute to the impaired development of neonatal memory cells. The miRNA profiles of adult and neonatal cells were surprisingly similar during infection; however, we observed large differences prior to infection. In particular, miR-29 and miR-130 have significant differential expression between adult and neonatal cells before infection. Importantly, using RNA-Seq, we detected reciprocal changes in expression of messenger RNA targets for both miR-29 and miR-130. Moreover, targets that we validated include Eomes and Tbx21, key genes that regulate the formation of memory CD8+ T cells. Notably, age-dependent changes in miR-29 and miR-130 are conserved in human CD8+ T cells, further suggesting that these developmental differences are biologically relevant. Together, these results demonstrate that miR-29 and miR-130 are likely important regulators of memory CD8+ T cell formation and suggest that neonatal cells are committed to a short-lived effector cell fate prior to infection. PMID:26416483

  1. Overexpression of Galgt2 in skeletal muscle prevents injury resulting from eccentric contractions in both mdx and wild-type mice.

    PubMed

    Martin, Paul T; Xu, Rui; Rodino-Klapac, Louise R; Oglesbay, Elaine; Camboni, Marybeth; Montgomery, Chrystal L; Shontz, Kim; Chicoine, Louis G; Clark, K Reed; Sahenk, Zarife; Mendell, Jerry R; Janssen, Paul M L

    2009-03-01

    The cytotoxic T cell (CT) GalNAc transferase, or Galgt2, is a UDP-GalNAc:beta1,4-N-acetylgalactosaminyltransferase that is localized to the neuromuscular synapse in adult skeletal muscle, where it creates the synaptic CT carbohydrate antigen {GalNAcbeta1,4[NeuAc(orGc)alpha2, 3]Galbeta1,4GlcNAcbeta-}. Overexpression of Galgt2 in the skeletal muscles of transgenic mice inhibits the development of muscular dystrophy in mdx mice, a model for Duchenne muscular dystrophy. Here, we provide physiological evidence as to how Galgt2 may inhibit the development of muscle pathology in mdx animals. Both Galgt2 transgenic wild-type and mdx skeletal muscles showed a marked improvement in normalized isometric force during repetitive eccentric contractions relative to nontransgenic littermates, even using a paradigm where nontransgenic muscles had force reductions of 95% or more. Muscles from Galgt2 transgenic mice, however, showed a significant decrement in normalized specific force and in hindlimb and forelimb grip strength at some ages. Overexpression of Galgt2 in muscles of young adult mdx mice, where Galgt2 has no effect on muscle size, also caused a significant decrease in force drop during eccentric contractions and increased normalized specific force. A comparison of Galgt2 and microdystrophin overexpression using a therapeutically relevant intravascular gene delivery protocol showed Galgt2 was as effective as microdystrophin at preventing loss of force during eccentric contractions. These experiments provide a mechanism to explain why Galgt2 overexpression inhibits muscular dystrophy in mdx muscles. That overexpression also prevents loss of force in nondystrophic muscles suggests that Galgt2 is a therapeutic target with broad potential applications. PMID:19109526

  2. Chronic hemodynamic unloading regulates the morphologic development of newborn mouse hearts transplanted into the ear of isogeneic adult mice.

    PubMed Central

    Rossi, M. A.

    1992-01-01

    The morphologic development of newborn mouse hearts transplanted into the pinna of the ears of isogeneic adult mice was assessed in comparison to in situ ventricular myocardium of recipients. The grafted hearts became vascularized from the auricular artery at the base of the ear, and although these preparations appeared not to be intrinsically innervated, most of them showed grossly visible pulsatile activity. Since they were not subjected to hemodynamic load due to working against a pressure gradient, this technique provided an interesting experimental model for studies on the growth of chronically unloaded tissue. The ultrastructure of the myocardium from neonatal mouse hearts, which were fixed immediately after dissection, revealed no differences in comparison to previously published observations. By 2 months, there was virtually no change in the myocardial cell size as compared with newborn mouse cardiac tissue. The heterotopic hearts showed a mature ultrastructural appearance, with parallel bands of myofibrils alternating with rows of mitochondria and differentiated intercalated discs comparable to in situ myocardium. The interstitial space was widened due to fibrous tissue, with activated fibroblasts and a few mononuclear cells. In contrast, by 6 months after transplantation, the heterotopic myocardium showed a dispersion of the measured cell diameter of myocytes, with atrophy of a certain population of cells and hypertrophy in others; nevertheless, the mean cell diameter was similar to that observed in 2-month grafts. The myocytes showed significant dissociation from each other in fibrous tissue and a cellular infiltrate composed predominantly of mononuclear cells, and greater variability of the parallel arrangement of cells. They often contained myofibrils coursing in different directions rather than in parallel. Normal-sized or predominantly atrophic degenerated myocytes, characterized by a wide variety of ultrastructural alterations, were present. By 12

  3. Repair of liver mediated by adult mouse liver neuro-glia antigen 2-positive progenitor cell transplantation in a mouse model of cirrhosis

    PubMed Central

    Zhang, Hongyu; Siegel, Christopher T.; Shuai, Ling; Lai, Jiejuan; Zeng, Linli; Zhang, Yujun; Lai, Xiangdong; Bie, Ping; Bai, Lianhua

    2016-01-01

    NG2-expressing cells are a population of periportal vascular stem/progenitors (MLpvNG2+ cells) that were isolated from healthy adult mouse liver by using a “Percoll-Plate-Wait” procedure. We demonstrated that isolated cells are able to restore liver function after transplantation into a cirrhotic liver, and co-localized with the pericyte marker (immunohistochemistry: PDGFR-β) and CK19. Cells were positive for: stem cell (Sca-1, CD133, Dlk) and liver stem cell markers (EpCAM, CD14, CD24, CD49f); and negative for: hematopoietic (CD34, CD45) and endothelial markers (CD31, vWf, von Willebrand factor). Cells were transplanted (1 × 106 cells) in mice with diethylnitrosamine-induced cirrhosis at week 6. Cells showed increased hepatic associated gene expression of alpha-fetoprotein (AFP), Albumin (Alb), Glucose-6-phosphatase (G6Pc), SRY (sex determining region Y)-box 9 (Sox9), hepatic nuclear factors (HNF1a, HNF1β, HNF3β, HNF4α, HNF6, Epithelial cell adhesion molecule (EpCAM), Leucine-rich repeated-containing G-protein coupled receptor 5-positive (Lgr5) and Tyrosine aminotransferase (TAT). Cells showed decreased fibrogenesis, hepatic stellate cell infiltration, Kupffer cells and inflammatory cytokines. Liver function markers improved. In a cirrhotic liver environment, cells could differentiate into hepatic lineages. In addition, grafted MLpvNG2+ cells could mobilize endogenous stem/progenitors to participate in liver repair. These results suggest that MLpvNG2+ cells may be novel adult liver progenitors that participate in liver regeneration. PMID:26905303

  4. Repair of liver mediated by adult mouse liver neuro-glia antigen 2-positive progenitor cell transplantation in a mouse model of cirrhosis.

    PubMed

    Zhang, Hongyu; Siegel, Christopher T; Shuai, Ling; Lai, Jiejuan; Zeng, Linli; Zhang, Yujun; Lai, Xiangdong; Bie, Ping; Bai, Lianhua

    2016-01-01

    NG2-expressing cells are a population of periportal vascular stem/progenitors (MLpvNG2(+) cells) that were isolated from healthy adult mouse liver by using a "Percoll-Plate-Wait" procedure. We demonstrated that isolated cells are able to restore liver function after transplantation into a cirrhotic liver, and co-localized with the pericyte marker (immunohistochemistry: PDGFR-β) and CK19. Cells were positive for: stem cell (Sca-1, CD133, Dlk) and liver stem cell markers (EpCAM, CD14, CD24, CD49f); and negative for: hematopoietic (CD34, CD45) and endothelial markers (CD31, vWf, von Willebrand factor). Cells were transplanted (1 × 10(6) cells) in mice with diethylnitrosamine-induced cirrhosis at week 6. Cells showed increased hepatic associated gene expression of alpha-fetoprotein (AFP), Albumin (Alb), Glucose-6-phosphatase (G6Pc), SRY (sex determining region Y)-box 9 (Sox9), hepatic nuclear factors (HNF1a, HNF1β, HNF3β, HNF4α, HNF6, Epithelial cell adhesion molecule (EpCAM), Leucine-rich repeated-containing G-protein coupled receptor 5-positive (Lgr5) and Tyrosine aminotransferase (TAT). Cells showed decreased fibrogenesis, hepatic stellate cell infiltration, Kupffer cells and inflammatory cytokines. Liver function markers improved. In a cirrhotic liver environment, cells could differentiate into hepatic lineages. In addition, grafted MLpvNG2(+) cells could mobilize endogenous stem/progenitors to participate in liver repair. These results suggest that MLpvNG2(+) cells may be novel adult liver progenitors that participate in liver regeneration. PMID:26905303

  5. Villin Promoter-Mediated Transgenic Expression of TRPV6 Increases Intestinal Calcium Absorption in Wild-type and VDR Knockout Mice

    PubMed Central

    Cui, Min; Li, Qiang; Johnson, Robert; Fleet, James C.

    2012-01-01

    Transient receptor potential cation channel, subfamily V, member 6 (TRPV6) is an apical membrane calcium (Ca) channel in the small intestine proposed to be essential for vitamin D regulated intestinal Ca absorption. Recent studies have challenged the proposed role for TRPV6 in Ca absorption. We directly tested intestinal TRPV6 function in Ca and bone metabolism in wild-type (WT) and vitamin D receptor knockout (VDRKO) mice. Transgenic mice (TG) were made with intestinal epithelium-specific expression of a 3X flag-tagged human TRPV6 protein. TG and VDRKO mice were crossed to make TG-VDRKO mice. Ca and bone metabolism was examined in WT, TG, VDRKO, and TG-VDRKO mice. TG mice developed hypercalcemia and soft tissue calcification on a chow diet. In TG mice fed a 0.25% Ca diet, Ca absorption was >3 fold higher and femur bone mineral density (BMD) was 26% higher than WT. Renal CYP27B1 mRNA and intestinal expression of the natural mouse TRPV6 gene were reduced to <10% of WT but small intestine calbindin-D9k expression was elevated >15X in TG mice. TG-VDRKO mice had high Ca absorption that prevented the low serum Ca, high renal CYP27B1 mRNA, and low BMD and abnormal bone microarchitecture seen in VDRKO mice. In addition, small intestinal calbindin D9K mRNA and protein levels were elevated in TG-VDRKO. Transgenic TRPV6 expression in intestine is sufficient to increase Ca absorption and bone density, even in VDRKO mice. VDR independent up-regulation of intestinal calbindin D9k in TG-VDRKO suggests this protein may buffer intracellular Ca during Ca absorption. PMID:22589201

  6. Hepatic effects of repeated oral administration of diclofenac to hepatic cytochrome P450 reductase null (HRN™) and wild-type mice.

    PubMed

    Akingbasote, James A; Foster, Alison J; Wilson, Ian; Sarda, Sunil; Jones, Huw B; Kenna, J Gerry

    2016-04-01

    Hepatic NADPH-cytochrome P450 oxidoreductase null (HRN™) mice exhibit normal hepatic and extrahepatic biotransformation enzyme activities when compared to wild-type (WT) mice, but express no functional hepatic cytochrome P450 activities. When incubated in vitro with [(14)C]-diclofenac, liver microsomes from WT mice exhibited extensive biotransformation to oxidative and glucuronide metabolites and covalent binding to proteins was also observed. In contrast, whereas glucuronide conjugates and a quinone-imine metabolite were formed when [(14)C]-diclofenac was incubated with HRN™ mouse liver, only small quantities of P450-derived oxidative metabolites were produced in these samples and covalent binding to proteins was not observed. Livers from vehicle-treated HRN™ mice exhibited enhanced lipid accumulation, bile duct proliferation, hepatocellular degeneration and necrosis and inflammatory cell infiltration, which were not present in livers from WT mice. Elevated liver-derived alanine aminotransferase, glutamate dehydrogenase and alkaline phosphatase activities were also observed in plasma from HRN™ mice. When treated orally with diclofenac for 7 days, at 30 mg/kg/day, the severities of the abnormal liver histopathology and plasma liver enzyme findings in HRN™ mice were reduced markedly. Oral diclofenac administration did not alter the liver histopathology or elevate plasma enzyme activities of WT mice. These findings indicate that HRN™ mice are valuable for exploration of the role played by hepatic P450s in drug biotransformation, but poorly suited to investigations of drug-induced liver toxicity. Nevertheless, studies in HRN™ mice could provide novel insights into the role played by inflammation in liver injury and may aid the evaluation of new strategies for its treatment. PMID:25820915

  7. Bone Turnover in Wild Type and Pleiotrophin-Transgenic Mice Housed for Three Months in the International Space Station (ISS)

    PubMed Central

    Brun, Francesco; Canciani, Barbara; Manescu, Adrian; Marozzi, Katia; Cilli, Michele; Costa, Delfina; Liu, Yi; Piccardi, Federica; Tasso, Roberta; Tromba, Giuliana; Rustichelli, Franco; Cancedda, Ranieri

    2012-01-01

    Bone is a complex dynamic tissue undergoing a continuous remodeling process. Gravity is a physical force playing a role in the remodeling and contributing to the maintenance of bone integrity. This article reports an investigation on the alterations of the bone microarchitecture that occurred in wild type (Wt) and pleiotrophin-transgenic (PTN-Tg) mice exposed to a near-zero gravity on the International Space Station (ISS) during the Mice Drawer System (MDS) mission, to date, the longest mice permanence (91 days) in space. The transgenic mouse strain over-expressing pleiotrophin (PTN) in bone was selected because of the PTN positive effects on bone turnover. Wt and PTN-Tg control animals were maintained on Earth either in a MDS payload or in a standard vivarium cage. This study revealed a bone loss during spaceflight in the weight-bearing bones of both strains. For both Tg and Wt a decrease of the trabecular number as well as an increase of the mean trabecular separation was observed after flight, whereas trabecular thickness did not show any significant change. Non weight-bearing bones were not affected. The PTN-Tg mice exposed to normal gravity presented a poorer trabecular organization than Wt mice, but interestingly, the expression of the PTN transgene during the flight resulted in some protection against microgravity’s negative effects. Moreover, osteocytes of the Wt mice, but not of Tg mice, acquired a round shape, thus showing for the first time osteocyte space-related morphological alterations in vivo. The analysis of specific bone formation and resorption marker expression suggested that the microgravity-induced bone loss was due to both an increased bone resorption and a decreased bone deposition. Apparently, the PTN transgene protection was the result of a higher osteoblast activity in the flight mice. PMID:22438896

  8. Reduced number of axonal mitochondria and tau hypophosphorylation in mouse P301L tau knockin neurons

    PubMed Central

    Rodríguez-Martín, Teresa; Pooler, Amy M.; Lau, Dawn H.W.; Mórotz, Gábor M.; De Vos, Kurt J.; Gilley, Jonathan; Coleman, Michael P.; Hanger, Diane P.

    2016-01-01

    Expression of the frontotemporal dementia-related tau mutation, P301L, at physiological levels in adult mouse brain (KI-P301L mice) results in overt hypophosphorylation of tau and age-dependent alterations in axonal mitochondrial transport in peripheral nerves. To determine the effects of P301L tau expression in the central nervous system, we examined the kinetics of mitochondrial axonal transport and tau phosphorylation in primary cortical neurons from P301L knock-in (KI-P301L) mice. We observed a significant 50% reduction in the number of mitochondria in the axons of cortical neurons cultured from KI-P301L mice compared to wild-type neurons. Expression of murine P301L tau did not change the speed, direction of travel or likelihood of movement of mitochondria. Notably, the angle that defines the orientation of the mitochondria in the axon, and the volume of individual moving mitochondria, were significantly increased in neurons expressing P301L tau. We found that murine tau phosphorylation in KI-P301L mouse neurons was diminished and the ability of P301L tau to bind to microtubules was also reduced compared to tau in wild-type neurons. The P301L mutation did not influence the ability of murine tau to associate with membranes in cortical neurons or in adult mouse brain. We conclude that P301L tau is associated with mitochondrial changes and causes an early reduction in murine tau phosphorylation in neurons coupled with impaired microtubule binding of tau. These results support the association of mutant tau with detrimental effects on mitochondria and will be of significance for the pathogenesis of tauopathies. PMID:26459111

  9. Daily rhythms of core temperature and locomotor activity indicate different adaptive strategies to cold exposure in adult and aged mouse lemurs acclimated to a summer-like photoperiod.

    PubMed

    Terrien, Jeremy; Zizzari, Philippe; Epelbaum, Jacques; Perret, Martine; Aujard, Fabienne

    2009-07-01

    Daily variations in core temperature (Tc) within the normothermic range imply thermoregulatory processes that are essential for optimal function and survival. Higher susceptibility towards cold exposure in older animals suggests that these processes are disturbed with age. In the mouse lemur, a long-day breeder, we tested whether aging affected circadian rhythmicity of Tc, locomotor activity (LA), and energy balance under long-day conditions when exposed to cold. Adult (N = 7) and aged (N = 5) mouse lemurs acclimated to LD14/10 were exposed to 10-day periods at 25 and 12 degrees C. Tc and LA rhythms were recorded by telemetry, and caloric intake (CI), body mass changes, and plasma IGF-1 were measured. During exposure to 25 degrees C, both adult and aged mouse lemurs exhibited strong daily variations in Tc. Aged animals exhibited lower levels of nocturnal LA and nocturnal and diurnal Tc levels in comparison to adults. Body mass and IGF-1 levels remained unchanged with aging. Under cold exposure, torpor bout occurrence was never observed whatever the age category. Adult and aged mouse lemurs maintained their Tc in the normothermic range and a positive energy balance. All animals exhibited increase in CI and decrease in IGF-1 in response to cold. The decrease in IGF-1 was delayed in aged mouse lemurs compared to adults. Moreover, both adult and aged animals responded to cold exposure by increasing their diurnal LA compared to those under Ta = 25 degrees C. However, aged animals exhibited a strong decrease in nocturnal LA and Tc, whereas cold effects were only slight in adults. The temporal organization and amplitude of the daily phase of low Tc were particularly well preserved under cold exposure in both age groups. Sexually active mouse lemurs exposed to cold thus seemed to prevent torpor exhibition and temporal disorganization of daily rhythms of Tc, even during aging. However, although energy balance was not impaired with age in mouse lemurs after cold exposure

  10. CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections.

    PubMed

    Cronan, Mark R; Rosenberg, Allison F; Oehlers, Stefan H; Saelens, Joseph W; Sisk, Dana M; Jurcic Smith, Kristen L; Lee, Sunhee; Tobin, David M

    2015-12-01

    Visualization of infection and the associated host response has been challenging in adult vertebrates. Owing to their transparency, zebrafish larvae have been used to directly observe infection in vivo; however, such larvae have not yet developed a functional adaptive immune system. Cells involved in adaptive immunity mature later and have therefore been difficult to access optically in intact animals. Thus, the study of many aspects of vertebrate infection requires dissection of adult organs or ex vivo isolation of immune cells. Recently, CLARITY and PACT (passive clarity technique) methodologies have enabled clearing and direct visualization of dissected organs. Here, we show that these techniques can be applied to image host-pathogen interactions directly in whole animals. CLARITY and PACT-based clearing of whole adult zebrafish and Mycobacterium tuberculosis-infected mouse lungs enables imaging of mycobacterial granulomas deep within tissue to a depth of more than 1 mm. Using established transgenic lines, we were able to image normal and pathogenic structures and their surrounding host context at high resolution. We identified the three-dimensional organization of granuloma-associated angiogenesis, an important feature of mycobacterial infection, and characterized the induction of the cytokine tumor necrosis factor (TNF) within the granuloma using an established fluorescent reporter line. We observed heterogeneity in TNF induction within granuloma macrophages, consistent with an evolving view of the tuberculous granuloma as a non-uniform, heterogeneous structure. Broad application of this technique will enable new understanding of host-pathogen interactions in situ. PMID:26449262

  11. CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections