Science.gov

Sample records for mutant mouse strain

  1. Genetics of Peripheral Vestibular Dysfunction: Lessons from Mutant Mouse Strains

    PubMed Central

    Jones, Sherri M.; Jones, Timothy A.

    2015-01-01

    Background A considerable amount of research has been published about genetic hearing impairment. Fifty to sixty percent of hearing loss is thought to have a genetic cause. Genes may also play a significant role in acquired hearing loss due to aging, noise exposure, or ototoxic medications. Between 1995 and 2012, over 100 causative genes have been identified for syndromic and nonsyndromic forms of hereditary hearing loss (see Hereditary Hearing Loss Homepage http://hereditaryhearingloss.org). Mouse models have been extremely valuable in facilitating the discovery of hearing loss genes, and in understanding inner ear pathology due to genetic mutations or elucidating fundamental mechanisms of inner ear development. Purpose Whereas much is being learned about hereditary hearing loss and the genetics of cochlear disorders, relatively little is known about the role genes may play in peripheral vestibular impairment. Here we review the literature with regard to genetics of vestibular dysfunction and discuss what we have learned from studies using mutant mouse models and direct measures of peripheral vestibular neural function. Results Several genes are considered that when mutated lead to varying degrees of inner ear vestibular dysfunction due to deficits in otoconia, stereocilia, hair cells, or neurons. Behavior often does not reveal the inner ear deficit. Many of the examples presented are also known to cause human disorders. Conclusions Knowledge regarding the roles of particular genes in the operation of the vestibular sensory apparatus is growing and it is clear that gene products co-expressed in the cochlea and vestibule may play different roles in the respective end organs. The discovery of new genes mediating critical inner ear vestibular function carries the promise of new strategies in diagnosing, treating and managing patients as well as predicting the course and level of morbidity in human vestibular disease. PMID:25032973

  2. A phenotype survey of 36 mutant mouse strains with gene-targeted defects in glycosyltransferases or glycan-binding proteins

    PubMed Central

    Orr, Sally L; Le, Dzung; Long, Jeffrey M; Sobieszczuk, Peter; Ma, Bo; Tian, Hua; Fang, Xiaoqun; Paulson, James C; Marth, Jamey D; Varki, Nissi

    2013-01-01

    The consortium for functional glycomics (CFG) was a large research initiative providing networking and resources for investigators studying the role of glycans and glycan-binding proteins in health and disease. Starting in 2001, six scientific cores were established to generate data, materials and new technologies. By the end of funding in 2011, the mouse phenotype core (MPC) submitted data to a website from the phenotype screen of 36 mutant mouse strains deficient in a gene for either a glycan-binding protein (GBP) or glycosyltransferase (GT). Each mutant strain was allotted three months for analysis and screened by standard phenotype assays used in the fields of immunology, histology, hematology, coagulation, serum chemistry, metabolism and behavior. Twenty of the deficient mouse strains had been studied in other laboratories, and additional tests were performed on these strains to confirm previous observations and discover new data. The CFG constructed 16 new homozygous mutant mouse strains and completed the initial phenotype screen of the majority of these new mutant strains. In total, >300 phenotype changes were observed, but considering the over 100 assays performed on each strain, most of the phenotypes were unchanged. Phenotype differences include abnormal testis morphology in GlcNAcT9- and Siglec-H-deficient mice and lethality in Pomgnt1-deficient mice. The numerous altered phenotypes discovered, along with the consideration of the significant findings of normality, will provide a platform for future characterization to understand the important roles of glycans and GBPs in the mechanisms of health and disease. PMID:23118208

  3. Safety, efficacy and efficiency of laser-assisted IVF in subfertile mutant mouse strains

    PubMed Central

    Li, Ming-Wen; Kinchen, Kristy L; Vallelunga, Jadine M; Young, Diana L; Wright, Kaleb D K; Gorano, Lisa N; Wasson, Katherine; Lloyd, K C Kent

    2013-01-01

    In the present report we studied the safety, efficacy and efficiency of using an infrared laser to facilitate IVF by assessing fertilization, development and birth rates after laser-zona drilling (LZD) in 30 subfertile genetically modified (GM) mouse lines. We determined that LZD increased the fertilization rate four to ten times that of regular IVF, thus facilitating the derivation of 26 of 30 (86.7%) GM mouse lines. Cryopreserved two-cell stage embryos derived by LZD-assisted IVF were recovered and developed to blastocysts in vitro at the same rate as frozen–thawed embryos derived by regular IVF. Surprisingly after surgical transfer to pseudopregnant recipients the birth rate of embryos derived by LZD-assisted IVF was significantly lower than that of embryos derived by regular IVF. However this result could be completely mitigated by the addition of 0.25 M sucrose to the culture medium during LZD which caused the oocyte to shrink in volume relative to the perivitelline space. By increasing the distance from the laser target site on the zona pellucida, we hypothesize that the hyperosmotic effect of sucrose reduced the potential for laser-induced cytotoxic thermal damage to the underlying oocytes. With appropriate preparation and cautious application, our results indicate that LZD-assisted IVF is a safe, efficacious and efficient assisted reproductive technology for deriving mutant mouse lines with male factor infertility and subfertility caused by sperm–zona penetration defects. PMID:23315689

  4. Construction of mouse adenovirus type 1 mutants.

    PubMed

    Cauthen, Angela N; Welton, Amanda R; Spindler, Katherine R

    2007-01-01

    Mouse adenovirus provides a model for studying adenovirus pathogenesis in the natural host. The ability to make viral mutants allows the investigation of specific mouse adenoviral gene contributions to virus-host interactions. Methods for propagation and titration of wild-type mouse adenovirus, production of viral DNA and viral DNA-protein complex, and transfection of mouse cells to obtain mouse adenovirus mutants are described in this chapter. Plaque purification, propagation, and titration of the mutant viruses are also presented.

  5. Traumatic brain injury causes delayed motor and cognitive impairment in a mutant mouse strain known to exhibit delayed Wallerian degeneration.

    PubMed

    Fox, G B; Faden, A I

    1998-09-15

    Delayed Wallerian degeneration after neuronal injury is a feature of the C57BL/Wld(s) mouse mutant. In the present study, we examined the effect of unilateral controlled cortical impact (CCI) on motor and cognitive performance in C57BL/6 and C57BL/Wld(s) mice. Performance on a beam-walking task was impaired in both injured groups over the first 3 weeks; however, between 28 and 35 days post injury, C57BL/6 mice continued to improve whereas C57BL/Wld(s) mice showed increased footfaults. In a spatial learning task, C57BL/Wld(s) animals performed consistently better than C57BL/6 mice when tested 7-10 days and 14-17 days following CCI. C57BL/Wld(s) mice also demonstrated improved working memory performance as compared with C57BL/6 mice when trained on days 21-22 after injury; this effect was lost on days 23 and 24, and was not evident in other animals tested in the same task at 28-31 days following injury. These results indicate a marked delay in motor and cognitive impairment following CCI in C57BL/Wld(s) mice compared with injured C57BL/6 controls. This is consistent with previous work showing delayed temporal evolution of neuronal degeneration in C57BL/Wld(s) mice and suggests CCI may be a suitable model for examining the functional consequences of traumatic brain injury (TBI) in genetically altered mice.

  6. Genealogies of mouse inbred strains.

    PubMed

    Beck, J A; Lloyd, S; Hafezparast, M; Lennon-Pierce, M; Eppig, J T; Festing, M F; Fisher, E M

    2000-01-01

    The mouse is a prime organism of choice for modelling human disease. Over 450 inbred strains of mice have been described, providing a wealth of different genotypes and phenotypes for genetic and other studies. As new strains are generated and others become extinct, it is useful to review periodically what strains are available and how they are related to each other, particularly in the light of available DNA polymorphism data from microsatellite and other markers. We describe the origins and relationships of inbred mouse strains, 90 years after the generation of the first inbred strain. Given the large collection of inbred strains available, and that published information on these strains is incomplete, we propose that all genealogical and genetic data on inbred strains be submitted to a common electronic database to ensure this valuable information resource is preserved and used efficiently.

  7. Isolation of mouse cell proteoglycan mutants

    SciTech Connect

    Keller, K.M.; Keller, J.M.

    1986-05-01

    The sulfated proteoglycans on the surface of cultured mammalian cells have been implicated in a variety of phenomena. To obtain more direct evidence for the role of these molecules in specific cellular functions, they are isolating mutants that produce altered sulfated proteoglycans from a cloned line of Swiss mouse 3T3 cells. This cell type was selected because it exhibits contact inhibition of growth and there is extensive information on its' cell surface and extracellular proteoglycans and other glycoproteins. Cells were chemically mutagenized and subjected to one or more cycles of radiation suicide in the presence of /sup 35/S-sulfate. By replica plating, 150 clones, which appear to incorporate abnormal amounts of /sup 35/S-sulfate, have been selected. After recloning three times via the replica plating technique, the proteoglycans of 29 clones have thus far been analyzed. They have identified four clones which appear to make altered amounts of either cell surface heparan sulfate or chondroitin sulfate. The biochemical bases for the altered levels of the proteoglycans are under study. Of particular interest, however, is the fact that in this limited collection of mutants the chemical alterations correlate with specific altered cellular morphologies.

  8. Reeler: new tales on an old mutant mouse.

    PubMed

    D'Arcangelo, G; Curran, T

    1998-03-01

    Neurological mouse mutants provide an opportunity to dissect the complex mechanisms that underlie vertebrate brain development. Advances in genetic technologies have permitted the identification of genes disrupted in many mutants, allowing a molecular interpretation of the phenotypes. For several decades, the spontaneous mutant mouse reeler has been used as a model for the analysis of the development of laminated brain structures. In this ataxic mutant, the migration of many neurons is aberrant, resulting in disrupted cellular organization. Recently, reelin, the gene disrupted in the reeler mouse, has been identified, reelin encodes a novel extracellular molecule that controls neural cell positioning through mechanisms that are not yet completely understood. Analysis of the expression pattern and the properties of the reelin gene product (Reelin) suggests models for its function during brain development. Furthermore, the recent identification of genes that may function in the Reelin signaling pathway advances our knowledge of the molecular basis of neuronal migration. PMID:9631651

  9. Formation of a host range mutant of the lymphotropic strain of minute virus of mice during persistent infection in mouse L cells.

    PubMed Central

    Ron, D; Tattersall, P; Tal, J

    1984-01-01

    Minute virus of mice (i), the lymphotropic strain of minute virus of mice, established a persistent infection in normally restrictive L cells. The carrier state, which lasted 150 days, exhibited three clearly distinguishable stages. During the early stage (days 1 to 10 postinfection), small amounts of virus were formed. A "crisis" then developed that lasted 50 to 60 days and was characterized by massive cell lysis and high titers of virus. This was followed by a 70- to 80-day period in which small but stable quantities of virus were produced. Virus shed by the carrier culture during the latter phase had acquired an altered host range, namely, it had lost its ability to replicate in T-lymphocyte cell lines and had adapted to growth in L cells. Virus isolated at this time from a single plaque in L cells, designated hr301, was shown to possess similar host range properties. No differences, however, could be found between the DNAs of minute virus of mice (i) and of hr301 by restriction enzyme analysis, suggesting that the mutation that affected the viral host range did not involve an extensive region of the viral genome. Images PMID:6090711

  10. Blastocyst genotyping for quality control of mouse mutant archives: an ethical and economical approach.

    PubMed

    Scavizzi, Ferdinando; Ryder, Edward; Newman, Stuart; Raspa, Marcello; Gleeson, Diane; Wardle-Jones, Hannah; Montoliu, Lluis; Fernandez, Almudena; Dessain, Marie-Laure; Larrigaldie, Vanessa; Khorshidi, Zuzana; Vuolteenaho, Reetta; Soininen, Raija; André, Philippe; Jacquot, Sylvie; Hong, Yi; de Angelis, Martin Hrabe; Ramirez-Solis, Ramiro; Doe, Brendan

    2015-10-01

    With the advent of modern developmental biology and molecular genetics, the scientific community has generated thousands of newly genetically altered strains of laboratory mice with the aim of elucidating gene function. To this end, a large group of Institutions which form the International Mouse Phenotyping Consortium is generating and phenotyping a knockout mouse strain for each of the ~20,000 protein-coding genes using the mutant ES cell resource produced by the International Knockout Mouse Consortium. These strains are made available to the research community via public repositories, mostly as cryopreserved sperm or embryos. To ensure the quality of this frozen resource there is a requirement that for each strain the frozen sperm/embryos are proven able to produce viable mutant progeny, before the live animal resource is removed from cages. Given the current requirement to generate live pups to demonstrate their mutant genotype, this quality control check necessitates the use and generation of many animals and requires considerable time, cage space, technical and economic resources. Here, we describe a simple and efficient method of genotyping pre-implantation stage blastocysts with significant ethical and economic advantages especially beneficial for current and future large-scale mouse mutagenesis projects. PMID:26178246

  11. Blastocyst genotyping for quality control of mouse mutant archives: an ethical and economical approach.

    PubMed

    Scavizzi, Ferdinando; Ryder, Edward; Newman, Stuart; Raspa, Marcello; Gleeson, Diane; Wardle-Jones, Hannah; Montoliu, Lluis; Fernandez, Almudena; Dessain, Marie-Laure; Larrigaldie, Vanessa; Khorshidi, Zuzana; Vuolteenaho, Reetta; Soininen, Raija; André, Philippe; Jacquot, Sylvie; Hong, Yi; de Angelis, Martin Hrabe; Ramirez-Solis, Ramiro; Doe, Brendan

    2015-10-01

    With the advent of modern developmental biology and molecular genetics, the scientific community has generated thousands of newly genetically altered strains of laboratory mice with the aim of elucidating gene function. To this end, a large group of Institutions which form the International Mouse Phenotyping Consortium is generating and phenotyping a knockout mouse strain for each of the ~20,000 protein-coding genes using the mutant ES cell resource produced by the International Knockout Mouse Consortium. These strains are made available to the research community via public repositories, mostly as cryopreserved sperm or embryos. To ensure the quality of this frozen resource there is a requirement that for each strain the frozen sperm/embryos are proven able to produce viable mutant progeny, before the live animal resource is removed from cages. Given the current requirement to generate live pups to demonstrate their mutant genotype, this quality control check necessitates the use and generation of many animals and requires considerable time, cage space, technical and economic resources. Here, we describe a simple and efficient method of genotyping pre-implantation stage blastocysts with significant ethical and economic advantages especially beneficial for current and future large-scale mouse mutagenesis projects.

  12. Human-mouse interspecies collagen I heterotrimer is functional during embryonic development of Mov13 mutant mouse embryos.

    PubMed

    Wu, H; Bateman, J F; Schnieke, A; Sharpe, A; Barker, D; Mascara, T; Eyre, D; Bruns, R; Krimpenfort, P; Berns, A

    1990-04-01

    To investigate whether the human pro alpha 1(I) collagen chain could form an in vivo functional interspecies heterotrimer with the mouse pro alpha 2(I) collagen chain, we introduced the human COL1A1 gene into Mov13 mice which have a functional deletion of the endogenous COL1A1 gene. Transgenic mouse strains (HucI and HucII) carrying the human COL1A1 gene were first generated by microinjecting the COL1A1 gene into wild-type mouse embryos. Genetic evidence indicated that the transgene in the HucI strain was closely linked to the endogenous mouse COL1A1 gene and was X linked in the HucII transgenic strain. Northern (RNA) blot and S1 protection analyses showed that the transgene was expressed in the appropriate tissue-specific manner and as efficiently as the endogenous COL1A1 gene. HucII mice were crossed with Mov13 mice to transfer the human transgene into the mutant strain. Whereas homozygous Mov13 embryos die between days 13 and 14 of gestation, the presence of the transgene permitted apparently normal development of the mutant embryos to birth. This indicated that the mouse-human interspecies collagen I heterotrimer was functional in the animal. The rescue was, however, only partial, as all homozygotes died within 36 h after delivery, with signs of internal bleeding. This could have been due to a functional defect in the interspecies hybrid collagen. Extensive analysis failed to reveal any biochemical or morphological abnormalities of the collagen I molecules in Mov13-HucII embryos. This may indicate that there was a subtle functional defect of the interspecies hybrid protein which was not revealed by our analysis or that another gene has been mutated by the retroviral insertion in the Mov13 mutant strain.

  13. Auditory development in progressive motor neuronopathy mouse mutants.

    PubMed

    Volkenstein, Stefan; Brors, Dominik; Hansen, Stefan; Berend, Achim; Mlynski, Robert; Aletsee, Christoph; Dazert, Stefan

    2009-11-01

    The present study was performed to elucidate the hearing development in the progressive motor neuronopathy (pmn) mouse mutant. This mouse has been used as a model for human motoneuron disease. A missense mutation in the tubulin-specific chaperon E (Tbce) gene on mouse chromosome 13 was localized as the underlying genetic defect. The protein encoded by the Tbce gene is essential for the formation of primary tubulin complexes. Studies on motoneurons show disorganization in microtubules and disturbed axonal transport, followed by retrograde degeneration of the motoneurons. A similar pathomechanism is also possible for hearing disorders where disrupted microtubules could cause functional deficits in spiral ganglion neurons or in cochlear hair cells. Click auditory brainstem response (ABR) audiometry in homozygous pmn mutants showed a normal onset of hearing, but an increasing hearing threshold from postnatal day 26 (P26) on to death, compared to heterozygous mutants and wild-type mice. Histological sections of the cochlea at different ages showed a regular morphology. Additionally, spiral ganglion explants from mutant and wild-type mice were cultured. The neurite length from pmn mutants was shorter than in wild-type mice, and the neurite number/explant was significantly decreased in pmn mutants. We show that the pmn mouse mutant is a model for a progressive rapid hearing loss from P26 on, after initially normal hearing development. Heterozygous mice are not affected by this defect. With the knowledge of the well-known pathomechanism of this defect in motoneurons, a dysfunction of cellular mechanisms regulating tubulin assembling suggests that tubulin assembling plays an essential role in hearing function and maintenance.

  14. Auditory development in progressive motor neuronopathy mouse mutants.

    PubMed

    Volkenstein, Stefan; Brors, Dominik; Hansen, Stefan; Berend, Achim; Mlynski, Robert; Aletsee, Christoph; Dazert, Stefan

    2009-11-01

    The present study was performed to elucidate the hearing development in the progressive motor neuronopathy (pmn) mouse mutant. This mouse has been used as a model for human motoneuron disease. A missense mutation in the tubulin-specific chaperon E (Tbce) gene on mouse chromosome 13 was localized as the underlying genetic defect. The protein encoded by the Tbce gene is essential for the formation of primary tubulin complexes. Studies on motoneurons show disorganization in microtubules and disturbed axonal transport, followed by retrograde degeneration of the motoneurons. A similar pathomechanism is also possible for hearing disorders where disrupted microtubules could cause functional deficits in spiral ganglion neurons or in cochlear hair cells. Click auditory brainstem response (ABR) audiometry in homozygous pmn mutants showed a normal onset of hearing, but an increasing hearing threshold from postnatal day 26 (P26) on to death, compared to heterozygous mutants and wild-type mice. Histological sections of the cochlea at different ages showed a regular morphology. Additionally, spiral ganglion explants from mutant and wild-type mice were cultured. The neurite length from pmn mutants was shorter than in wild-type mice, and the neurite number/explant was significantly decreased in pmn mutants. We show that the pmn mouse mutant is a model for a progressive rapid hearing loss from P26 on, after initially normal hearing development. Heterozygous mice are not affected by this defect. With the knowledge of the well-known pathomechanism of this defect in motoneurons, a dysfunction of cellular mechanisms regulating tubulin assembling suggests that tubulin assembling plays an essential role in hearing function and maintenance. PMID:19735697

  15. Developmental mechanisms underlying polydactyly in the mouse mutant Doublefoot

    PubMed Central

    Crick, Alexandra P; Babbs, Christian; Brown, Jennifer M; Morriss-Kay, Gillian M

    2003-01-01

    The pre-axial polydactylous mouse mutant Doublefoot has 6–9 digits per limb but lacks anteroposterior polarity (there is no biphalangeal digit 1). It differs from other polydactylous mutants in showing normal Shh expression, but polarizing activity (shown by mouse-chick grafting experiments) and hedgehog signalling activity (shown by expression of Ptc1) are present throughout the distal mesenchyme. The Dbf mutation has not yet been identified. Here we review current understanding of this mutant, and briefly report new results indicating (1) that limb bud expansion is concomitant with ectopic Ihh expression and with extension of the posterior high cell proliferation rate into the anterior region, and (2) that the Dbf mutation is epistatic to Shh in the limb. PMID:12587916

  16. Two-Pore Channels: Lessons from Mutant Mouse Models

    PubMed Central

    Ruas, Margarida; Galione, Antony; Parrington, John

    2016-01-01

    Recent interest in two-pore channels (TPCs) has resulted in a variety of studies dealing with the functional role and mechanism of action of these endo-lysosomal proteins in diverse physiological processes. With the availability of mouse lines harbouring mutant alleles for Tpcnl and/or Tpcn2 genes, several studies have made use of them to validate, consolidate and discover new roles for these channels not only at the cellular level but, importantly, also at the level of the whole organism. The different mutant mouse lines that have been used were derived from distinct genetic manipulation strategies, with the aim of knocking out expression of TPC proteins. However, the expression of different residual TPC sequences predicted to occur in these mutant mouse lines, together with the varied degree to which the effects on Tpcn expression have been studied, makes it important to assess the true knockout status of some of the lines. In this review we summarize these Tpcn mutant mouse lines with regard to their predicted effect on Tpcn expression and the extent to which they have been characterized. Additionally, we discuss how results derived from studies using these Tpcn mutant mouse lines have consolidated previously proposed roles for TPCs, such as mediators of NAADP signalling, endo-lysosomal functions, and pancreatic β cell physiology. We will also review how they have been instrumental in the assignment of new physiological roles for these cation channels in processes such as membrane electrical excitability, neoangiogenesis, viral infection and brown adipose tissue and heart function, revealing, in some cases, a specific contribution of a particular TPC isoform. PMID:27330869

  17. Bovine and rabbit models for the study of a Staphylococcus aureus avirulent mutant strain, RC122

    PubMed Central

    Reinoso, Elina; Magnano, Gabriel; Giraudo, Jose; Calzolari, Aldo; Bogni, Cristina

    2002-01-01

    Staphylococcus aureus is the main etiological agent of bovine mastitis. Intramammary infections are difficult to cure and vaccination appears to be an alternative to prevent the disease. Research has focused on the development of mutants affected in the synthesis of pathogenicity determinants. We constructed a mutant strain (RC122) after chemical mutagenesis. In a mouse model, the strain was shown to be 1500 times less virulent, showed similar kinetics of disappearance in the kidney as its parental strain, and a good degree of protection against a challenge from homologous and heterologous strains. The objective of the present report was to study the avirulent RC122 S. aureus mutant strain in rabbit and bovine infection models. The results clearly show that RC122 was less virulent than its parental strain in a rabbit skin model, and was also correlated with its avirulence as an udder pathogen. These traits make the RC122 mutant strain interesting as a potential strain for an experimental vaccine trial in dairy herds. PMID:12418786

  18. Human-mouse interspecies collagen I heterotrimer is functional during embryonic development of Mov13 mutant mouse embryos.

    PubMed Central

    Wu, H; Bateman, J F; Schnieke, A; Sharpe, A; Barker, D; Mascara, T; Eyre, D; Bruns, R; Krimpenfort, P; Berns, A

    1990-01-01

    To investigate whether the human pro alpha 1(I) collagen chain could form an in vivo functional interspecies heterotrimer with the mouse pro alpha 2(I) collagen chain, we introduced the human COL1A1 gene into Mov13 mice which have a functional deletion of the endogenous COL1A1 gene. Transgenic mouse strains (HucI and HucII) carrying the human COL1A1 gene were first generated by microinjecting the COL1A1 gene into wild-type mouse embryos. Genetic evidence indicated that the transgene in the HucI strain was closely linked to the endogenous mouse COL1A1 gene and was X linked in the HucII transgenic strain. Northern (RNA) blot and S1 protection analyses showed that the transgene was expressed in the appropriate tissue-specific manner and as efficiently as the endogenous COL1A1 gene. HucII mice were crossed with Mov13 mice to transfer the human transgene into the mutant strain. Whereas homozygous Mov13 embryos die between days 13 and 14 of gestation, the presence of the transgene permitted apparently normal development of the mutant embryos to birth. This indicated that the mouse-human interspecies collagen I heterotrimer was functional in the animal. The rescue was, however, only partial, as all homozygotes died within 36 h after delivery, with signs of internal bleeding. This could have been due to a functional defect in the interspecies hybrid collagen. Extensive analysis failed to reveal any biochemical or morphological abnormalities of the collagen I molecules in Mov13-HucII embryos.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:1690840

  19. Mutant strain of C. acetobutylicum and process for making butanol

    DOEpatents

    Jain, Mahendra K.; Beacom, Daniel; Datta, Rathin

    1993-01-01

    A biologically pure asporogenic mutant of Clostridium acetobutylicum is produced by growing sporogenic C. acetobutylicum ATCC 4259 and treating the parent strain with ethane methane sulfonate. The mutant which as been designated C. acetobutylicum ATCC 55025 is useful in an improved ABE fermentation process, and produces high concentrations of butanol and total solvents.

  20. Insights into prevention of human neural tube defects by folic acid arising from consideration of mouse mutants.

    PubMed

    Harris, Muriel J

    2009-04-01

    Almost 30 years after the initial study by Richard W. Smithells and coworkers, it is still unknown how maternal periconceptional folic acid supplementation prevents human neural tube defects (NTDs). In this article, questions about human NTD prevention are considered in relation to three groups of mouse models: NTD mutants that respond to folate, NTD mutants and strains that do not respond to folate, and mutants involving folate-pathway genes. Of the 200 mouse NTD mutants, only a few have been tested with folate; half respond and half do not. Among responsive mutants, folic acid supplementation reduces exencephaly and/or spina bifida aperta frequency in the Sp(2H), Sp, Cd, Cited2, Cart1, and Gcn5 mutants. Prevention ranges from 35 to 85%. The responsive Sp(2H) (Pax3) mutant has abnormal folate metabolism, but the responsive Cited2 mutant does not. Neither folic nor folinic acid reduces NTD frequency in Axd, Grhl3, Fkbp8, Map3k4, or Nog mutants or in the curly tail or SELH/Bc strains. Spina bifida frequency is reduced in Axd by methionine and in curly tail by inositol. Exencephaly frequency is reduced in SELH/Bc by an alternative commercial ration. Mutations in folate-pathway genes do not cause NTDs, except for 30% exencephaly in folate-treated Folr1. Among folate-pathway mutants, neural tube closure is normal in Cbs, Folr2, Mthfd1, Mthfd2, Mthfr, and Shmt1 mutants. Embryos die by midgestation in Folr1, Mtr, Mtrr, and RFC1 mutants. The mouse models point to genetic heterogeneity in the ability to respond to folic acid and also to heterogeneity in genetic cause of NTDs that can be prevented by folic acid.

  1. The Tennessee Mouse Genome Consortium: Identification of ocular mutants

    SciTech Connect

    Jablonski, Monica M.; Wang, Xiaofei; Lu, Lu; Miller, Darla R; Rinchik, Eugene M; Williams, Robert; Goldowitz, Daniel

    2005-06-01

    The Tennessee Mouse Genome Consortium (TMGC) is in its fifth year of a ethylnitrosourea (ENU)-based mutagenesis screen to detect recessive mutations that affect the eye and brain. Each pedigree is tested by various phenotyping domains including the eye, neurohistology, behavior, aging, ethanol, drug, social behavior, auditory, and epilepsy domains. The utilization of a highly efficient breeding protocol and coordination of various universities across Tennessee makes it possible for mice with ENU-induced mutations to be evaluated by nine distinct phenotyping domains within this large-scale project known as the TMGC. Our goal is to create mutant lines that model human diseases and disease syndromes and to make the mutant mice available to the scientific research community. Within the eye domain, mice are screened for anterior and posterior segment abnormalities using slit-lamp biomicroscopy, indirect ophthalmoscopy, fundus photography, eye weight, histology, and immunohistochemistry. As of January 2005, we have screened 958 pedigrees and 4800 mice, excluding those used in mapping studies. We have thus far identified seven pedigrees with primary ocular abnormalities. Six of the mutant pedigrees have retinal or subretinal aberrations, while the remaining pedigree presents with an abnormal eye size. Continued characterization of these mutant mice should in most cases lead to the identification of the mutated gene, as well as provide insight into the function of each gene. Mice from each of these pedigrees of mutant mice are available for distribution to researchers for independent study.

  2. Applications of mutant yeast strains with low glycogen storage capability

    NASA Technical Reports Server (NTRS)

    Petersen, G. R.; Schubert, W. W.; Stokes, B. O.

    1981-01-01

    Several strains of Hansenula polymorpha were selected for possible low glycogen storage characteristics based on a selective I2 staining procedure. The levels of storage carbohydrates in the mutant strains were found to be 44-70% of the levels in the parent strain for cultures harvested in stationary phase. Similar differences generally were not found for cells harvested in exponential phase. Yeast strains deficient in glycogen storage capability are valuable in increasing the relative protein value of microbial biomass and also may provide significant cost savings in substrate utilization in fermentative processes.

  3. Hypothalamic-pituitary-gonadal axis in the mutant weaver mouse.

    PubMed

    Schwartz, N B; Szabo, M; Verina, T; Wei, J; Dlouhy, S R; Won, L; Heller, A; Hodes, M E; Ghetti, B

    1998-12-01

    The weaver (wv) mutant mouse manifests severe locomotor defects, a deficiency in granule cells of the cerebellum, and cellular deficits in the midbrain dopaminergic system. The wv phenotype is associated with a missense mutation in the pore region of the G-protein-gated inwardly rectifying potassium channel, GIRK2. The homozygous male wv mouse is essentially infertile due to an inadequate level of sperm production. Females are fertile although they also manifest the neurological phenotype. Homozygotes of both sexes have reduced body weight. We have evaluated the hypothalamic-pituitary-gonadal axis in heterozygote and homozygote male and female wv mutants in comparison with wild-type controls. Testicular weight was significantly reduced in the homozygous males, due to degenerative changes of seminiferous epithelium. Serum and pituitary content of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin were normal in all groups, and the normal sex differences were noted (FSH and LH higher in males, prolactin higher in females). Pituitary growth hormone (GH) concentration was normal, with control and mutant males showing higher GH than females. Serum testosterone levels were normal in the mutants, as was testicular testosterone. Testicular alpha-inhibin content was mildly reduced, but high in proportion to testicular weight. The defect in spermatogenesis appeared predominantly in the postmeiotic stages. In situ hybridization was consistent with expression of some GIRK2 mRNA isoforms in seminiferous epithelium. There were no significant differences between genotypes in the levels of dopamine, dihydroxyphenylacetic acid, serotonin and 5-hydroxyindoleacetic acid in the mediobasal and preoptic hypothalamic regions. Homovanillic acid levels in these two areas were, however, reduced in wv homozygotes compared to wild-type animals. In the light of normal pituitary hormone levels, normal hypothalamic monoamine concentrations and normal sex differences in

  4. Glycine receptor mouse mutants: model systems for human hyperekplexia

    PubMed Central

    Schaefer, Natascha; Langlhofer, Georg; Kluck, Christoph J; Villmann, Carmen

    2013-01-01

    Human hyperekplexia is a neuromotor disorder caused by disturbances in inhibitory glycine-mediated neurotransmission. Mutations in genes encoding for glycine receptor subunits or associated proteins, such as GLRA1, GLRB, GPHN and ARHGEF9, have been detected in patients suffering from hyperekplexia. Classical symptoms are exaggerated startle attacks upon unexpected acoustic or tactile stimuli, massive tremor, loss of postural control during startle and apnoea. Usually patients are treated with clonazepam, this helps to dampen the severe symptoms most probably by up-regulating GABAergic responses. However, the mechanism is not completely understood. Similar neuromotor phenotypes have been observed in mouse models that carry glycine receptor mutations. These mouse models serve as excellent tools for analysing the underlying pathomechanisms. Yet, studies in mutant mice looking for postsynaptic compensation of glycinergic dysfunction via an up-regulation in GABAA receptor numbers have failed, as expression levels were similar to those in wild-type mice. However, presynaptic adaptation mechanisms with an unusual switch from mixed GABA/glycinergic to GABAergic presynaptic terminals have been observed. Whether this presynaptic adaptation explains the improvement in symptoms or other compensation mechanisms exist is still under investigation. With the help of spontaneous glycine receptor mouse mutants, knock-in and knock-out studies, it is possible to associate behavioural changes with pharmacological differences in glycinergic inhibition. This review focuses on the structural and functional characteristics of the various mouse models used to elucidate the underlying signal transduction pathways and adaptation processes and describes a novel route that uses gene-therapeutic modulation of mutated receptors to overcome loss of function mutations. PMID:23941355

  5. Lethal infection by Bordetella pertussis mutants in the infant mouse model.

    PubMed Central

    Weiss, A A; Goodwin, M S

    1989-01-01

    Different aspects of lethal infection of infant mice with Bordetella pertussis were examined. Mutants deficient in vir-regulated genes were tested for the ability to cause a lethal infection in the infant mouse model. Adenylate cyclase toxin-hemolysin and pertussis toxin were required to cause a lethal infection at low doses. Mixed infection caused by challenging the mice with an equal number of pertussis toxin and adenylate cyclase toxin-hemolysin mutants at a dose at which neither alone was lethal was also unable to cause a lethal infection. Production of the filamentous hemagglutinin and the dermonecrotic toxin was not required to cause a lethal infection. Nine other mutants in vir-regulated genes whose phenotypes have yet to be determined were also tested. Only two of these mutants were impaired in the ability to cause a lethal infection. Expression of fimbriae does not appear to affect the dose required to cause a lethal infection; however, fimbrial expression was correlated with the later stages of a nonlethal, persistent infection. Growth of the bacteria in MgSO4, a condition which reversibly suppresses expression of the genes required for virulence, did not alter the ability of the bacteria to cause a lethal infection. Auxotrophic mutants deficient in leucine biosynthesis were as virulent as the parental strain; however, mutants deficient in methionine biosynthesis were less virulent. A B. parapertussis strain was much less effective in promoting a lethal infection than any of the wild-type B. pertussis strains examined. A persistent infection in the lungs was observed for weeks after challenge for mice given a sublethal dose of B. pertussis, and transmission from infected infants to the mother was never observed. PMID:2572561

  6. Development of amnesia in different mouse strains.

    PubMed

    Sinovyev, D R; Dubrovina, N I; Kulikov, A V

    2009-05-01

    We studied passive avoidance retrieval after amnestic stimulation (arrest in unsafe section of the experimental setup) in C57Bl/6J, BALB/c, CBA/Lac, AKR/J, DBA/2J, C3H/HeJ, and ASC/Icg mice. We demonstrated resistance to amnestic stimulation in mice with high predisposition to freezing reaction (ASC/Icg) and memory deficit in other mouse strains.

  7. A rat homolog of the mouse deafness mutant jerker (je).

    PubMed

    Truett, G E; Walker, J A; Brock, J W

    1996-05-01

    An autosomal recessive deafness mutant was discovered in our colony of Zucker (ZUC) rats. These mutants behave like shaker-waltzer deafness mutants, and their inner ear pathology classifies them among neuroepithelial degeneration type of deafness mutants. To determine whether this rat deafness mutation (-) defines a unique locus or one that has been previously described, we mapped its chromosomal location. F2 progeny of (Pbrc:ZUC x BN/Crl) A/a B/b H/h +/- F1 rats were scored for coat color and behavioral phenotypes. Segregation analysis indicated that the deafness locus might be loosely linked with B on rat Chromosome (Chr) 5 (RNO5). Therefore, 40 -/- rats were scored for BN and ZUC alleles at four additional loci, D5Mit11, D5Mit13, Oprd1, and Gnb1, known to map to RNO5 or its homolog, mouse Chr 4 (MMU4). Linkage analysis established the gene order (cM distance) as D5Mit11-(19.3)-B-(17.9)-D5Mit13-(19. 2)-Oprd1-(21.5) - (1.2) Gnb1, placing the deafness locus on distal RNO5. The position of the deafness locus on RNO5 is similar to that ofjerker (je) on MMU4; the phenotypes and patterns of inheritance of the deafness mutation and je are also similar. It seems likely that the mutation affects the rat homolog of je. The rat deafness locus should, therefore, be named jerker and assigned the gene symbol Je. PMID:8661723

  8. Behavioural performance in three substrains of mouse strain 129.

    PubMed

    Montkowski, A; Poettig, M; Mederer, A; Holsboer, F

    1997-07-11

    Recently, the possibility has been raised that the behavioural abnormalities seen in null-mutant mice might be determined by their genetic background rather than by loss of gene function, especially when the 129 mouse strain is used as supplier for embryonic stem (ES) cells. To examine this issue we tested three 129 mouse substrains (129/J, 129/Ola, 129/Sv-ter/+) and C57BL/6 (B6) in the Morris water maze, the open field, the plus maze and two tests assessing motor co-ordination. We identified only for the 129/J substrain substantial behavioural deficits. These mice are albinos and carry the pink-eyed dilution allele and differed in their basal anxiety level as assessed in the open-field test. They were severely impaired in spatial learning and memory (Morris water maze test), in the Porsolt swim test, which also measures learning and in motor co-ordination. However, the 129/J substrain has not been used as ES cell donor in null-mutant mice where behavioural abnormalities were observed. Instead, mice from 129/Ola and 129/Sv-ter/+ substrains have been commonly used as suppliers for ES cells. These performed normally in most of the tests, including Morris water maze test.

  9. An Escherichia coli MG1655 Lipopolysaccharide Deep-Rough Core Mutant Grows and Survives in Mouse Cecal Mucus but Fails To Colonize the Mouse Large Intestine

    PubMed Central

    Møller, Annette K.; Leatham, Mary P.; Conway, Tyrrell; Nuijten, Piet J. M.; de Haan, Louise A. M.; Krogfelt, Karen A.; Cohen, Paul S.

    2003-01-01

    The ability of E. coli strains to colonize the mouse large intestine has been correlated with their ability to grow in cecal and colonic mucus. In the present study, an E. coli MG1655 strain was mutagenized with a mini-Tn5 Km (kanamycin) transposon, and mutants were tested for the ability to grow on agar plates with mouse cecal mucus as the sole source of carbon and nitrogen. One mutant, designated MD42 (for mucus defective), grew poorly on cecal-mucus agar plates but grew well on Luria agar plates and on glucose minimal-agar plates. Sequencing revealed that the insertion in MD42 was in the waaQ gene, which is involved in lipopolysaccharide (LPS) core biosynthesis. Like “deep-rough” E. coli mutants, MD42 was hypersensitive to sodium dodecyl sulfate (SDS), bile salts, and the hydrophobic antibiotic novobiocin. Furthermore, its LPS core oligosaccharide was truncated, like that of a deep-rough mutant. MD42 initially grew in the large intestines of streptomycin-treated mice but then failed to colonize (<102 CFU per g of feces), whereas its parent colonized at levels between 107 and 108 CFU per g of feces. When mouse cecal mucosal sections were hybridized with an E. coli-specific rRNA probe, MD42 was observed in cecal mucus as clumps 24 h postfeeding, whereas its parent was present almost exclusively as single cells, suggesting that clumping may play a role in preventing MD42 colonization. Surprisingly, MD42 grew nearly as well as its parent during growth in undiluted, highly viscous cecal mucus isolated directly from the mouse cecum and, like its parent, survived well after reaching stationary phase, suggesting that there are no antimicrobials in mucus that prevent MD42 colonization. After mini-mariner transposon mutagenesis, an SDS-resistant suppressor mutant of MD42 was isolated. The mini-mariner insertion was shown to be in the bipA gene, a known regulator of E. coli surface components. When grown in Luria broth, the LPS core of the suppressor mutant remained

  10. A new inbred strain JF1 established from Japanese fancy mouse carrying the classic piebald allele.

    PubMed

    Koide, T; Moriwaki, K; Uchida, K; Mita, A; Sagai, T; Yonekawa, H; Katoh, H; Miyashita, N; Tsuchiya, K; Nielsen, T J; Shiroishi, T

    1998-01-01

    A new inbred strain JF1 (Japanese Fancy Mouse 1) was established from a strain of fancy mouse. Morphological and genetical analysis indicated that the mouse originated from the Japanese wild mouse, Mus musculus molossinus. JF1 has characteristic coat color, black spots on the white coat, with black eyes. The mutation appeared to be linked to an old mutation piebald (s). Characterization of the causative gene for piebald, endothelin receptor type B (ednrb), demonstrated that the allele in JF1 is same as that of classic piebald allele, suggesting an identical origin of these two mutants. Possibly, classic piebald mutation was introduced from the Japanese tame mouse, which was already reported at the end of the 1700s. We showed that JF1 is a useful strain for mapping of mutant genes on laboratory strains owing to a high level of polymorphisms in microsatellite markers between JF1 and laboratory strains. The clarified genotypes of JF1 for coat color are "aa BB CC DD ss".

  11. Live Attenuated Borrelia burgdorferi Targeted Mutants in an Infectious Strain Background Protect Mice from Challenge Infection.

    PubMed

    Hahn, Beth L; Padmore, Lavinia J; Ristow, Laura C; Curtis, Michael W; Coburn, Jenifer

    2016-08-01

    Borrelia burgdorferi, B. garinii, and B. afzelii are all agents of Lyme disease in different geographic locations. If left untreated, Lyme disease can cause significant and long-term morbidity, which may continue after appropriate antibiotic therapy has been administered and live bacteria are no longer detectable. The increasing incidence and geographic spread of Lyme disease are renewing interest in the vaccination of at-risk populations. We took the approach of vaccinating mice with two targeted mutant strains of B. burgdorferi that, unlike the parental strain, are avirulent in mice. Mice vaccinated with both strains were protected against a challenge with the parental strain and a heterologous B. burgdorferi strain by either needle inoculation or tick bite. In ticks, the homologous strain was eliminated but the heterologous strain was not, suggesting that the vaccines generated a response to antigens that are produced by the bacteria both early in mammalian infection and in the tick. Partial protection against B. garinii infection was also conferred. Protection was antibody mediated, and reactivity to a variety of proteins was observed. These experiments suggest that live attenuated B. burgdorferi strains may be informative regarding the identification of protective antigens produced by the bacteria and recognized by the mouse immune system in vivo Further work may illuminate new candidates that are effective and safe for the development of Lyme disease vaccines. PMID:27335385

  12. Dystonia and Cerebellar Degeneration in the Leaner Mouse Mutant

    PubMed Central

    Raike, Robert S.; Hess, Ellen J.; Jinnah, H.A.

    2015-01-01

    Cerebellar degeneration is traditionally associated with ataxia. Yet, there are examples of both ataxia and dystonia occurring in individuals with cerebellar degeneration. There is also substantial evidence suggesting that cerebellar dysfunction alone may cause dystonia. The types of cerebellar defects that may cause ataxia, dystonia, or both have not been delineated. In the current study, we explored the relationship between cerebellar degeneration and dystonia using the leaner mouse mutant. Leaner mice have severe dystonia that is associated with dysfunctional and degenerating cerebellar Purkinje cells. Whereas the density of Purkinje cells was not significantly reduced in 4 week-old leaner mice, approximately 50% of the neurons were lost by 34 weeks of age. On the other hand, the dystonia and associated functional disability became significantly less severe during this same interval. In other words, dystonia improved as Purkinje cells were lost, suggesting that dysfunctional Purkinje cells, rather than Purkinje cell loss, contribute to the dystonia. These results provide evidence that distorted cerebellar function may cause dystonia and support the concept that different types of cerebellar defects can have different functional consequences. PMID:25791619

  13. Identification of 17 hearing impaired mouse strains in the TMGC ENU-mutagenesis screen

    SciTech Connect

    Kermany, Mohammad; Parker, Lisan; Guo, Yun-Kai; Miller, Darla R; Swanson, Douglas J; Yoo, Tai-June; Goldowitz, Daniel; Zuo, Jian

    2006-01-01

    The Tennessee Mouse Genome Consortium (TMGC) employed an N-ethyl-N-nitrosourea (ENU)-mutagenesis scheme to identify mouse recessive mutants with hearing phenotypes. We employed auditory brainstem responses (ABR) to click and 8, 16, and 32 kHz stimuli and screened 285 pedigrees (1819 mice of 8-11 weeks old in various mixed genetic backgrounds) each bred to carry a homozygous ENU-induced mutation. To define mutant pedigrees, we measured P12 mice per pedigree in P2 generations and used a criterion where the mean ABR threshold per pedigree was two standard deviations above the mean of all offspring from the same parental strain. We thus identified 17 mutant pedigrees (6%), all exhibiting hearing loss at high frequencies (P16 kHz) with an average threshold elevation of 30-35 dB SPL. Interestingly, four mutants showed sex-biased hearing loss and six mutants displayed wide range frequency hearing loss. Temporal bone histology revealed that six of the first nine mutants displayed cochlear morphological defects: degeneration of spiral ganglia, spiral ligament fibrocytes or inner hair cells (but not outer hair cells) mostly in basal turns. In contrast to other ENU-mutagenesis auditory screens, our screen identified high-frequency, mild and sex-biased hearing defects. Further characterization of these 17 mouse models will advance our understanding of presbycusis and noise-induced hearing loss in humans.

  14. Revealing Differences in Metabolic Flux Distributions between a Mutant Strain and Its Parent Strain Gluconacetobacter xylinus CGMCC 2955

    PubMed Central

    Liu, Miao; Yang, Xiao-Ning; Zhu, Hui-Xia; Jia, Yuan-Yuan; Jia, Shi-Ru; Piergiovanni, Luciano

    2014-01-01

    A better understanding of metabolic fluxes is important for manipulating microbial metabolism toward desired end products, or away from undesirable by-products. A mutant strain, Gluconacetobacter xylinus AX2-16, was obtained by combined chemical mutation of the parent strain (G. xylinus CGMCC 2955) using DEC (diethyl sulfate) and LiCl. The highest bacterial cellulose production for this mutant was obtained at about 11.75 g/L, which was an increase of 62% compared with that by the parent strain. In contrast, gluconic acid (the main byproduct) concentration was only 5.71 g/L for mutant strain, which was 55.7% lower than that of parent strain. Metabolic flux analysis indicated that 40.1% of the carbon source was transformed to bacterial cellulose in mutant strain, compared with 24.2% for parent strain. Only 32.7% and 4.0% of the carbon source were converted into gluconic acid and acetic acid in mutant strain, compared with 58.5% and 9.5% of that in parent strain. In addition, a higher flux of tricarboxylic acid (TCA) cycle was obtained in mutant strain (57.0%) compared with parent strain (17.0%). It was also indicated from the flux analysis that more ATP was produced in mutant strain from pentose phosphate pathway (PPP) and TCA cycle. The enzymatic activity of succinate dehydrogenase (SDH), which is one of the key enzymes in TCA cycle, was 1.65-fold higher in mutant strain than that in parent strain at the end of culture. It was further validated by the measurement of ATPase that 3.53–6.41 fold higher enzymatic activity was obtained from mutant strain compared with parent strain. PMID:24901455

  15. Live four-dimensional optical coherence tomography reveals embryonic cardiac phenotype in mouse mutant

    NASA Astrophysics Data System (ADS)

    Lopez, Andrew L., III; Wang, Shang; Larin, Kirill V.; Overbeek, Paul A.; Larina, Irina V.

    2015-09-01

    Efficient phenotyping of developmental defects in model organisms is critical for understanding the genetic specification of normal development and congenital abnormalities in humans. We previously reported that optical coherence tomography (OCT) combined with live embryo culture is a valuable tool for mouse embryo imaging and four-dimensional (4-D) cardiodynamic analysis; however, its capability for analysis of mouse mutants with cardiac phenotypes has not been previously explored. Here, we report 4-D (three-dimensional+time) OCT imaging and analysis of the embryonic heart in a Wdr19 mouse mutant, revealing a heart looping defect. Quantitative analysis of cardiac looping revealed a statistically significant difference between mutant and control embryos. Our results indicate that live 4-D OCT imaging provides a powerful phenotyping approach to characterize embryonic cardiac function in mouse models.

  16. Rhizobium japonicum mutant strains unable to grow chemoautotrophically with H2.

    PubMed Central

    Maier, R J

    1981-01-01

    Rhizobium japonicum strain SR grows chemoautotrophically on a mineral salts medium when incubated in an H2- and CO2-containing atmosphere. Mutant strains unable to grow or that grow very poorly chemoautotrophically with H2 have been isolated from strain SR. The mutant isolation procedure involved mutagenesis with ethyl methane sulfonate, penicillin selection under chemoautotrophic growth conditions, and plating of the survivors onto medium containing carbon. The resulting colonies were replica plated onto medium that did not contain carbon, and the plates were incubated in an H2- and CO2-containing atmosphere. Mutant strains unable to grow under these conditions were chosen. Over 100 mutant strains with defects in chemoautotrophic metabolism were obtained. The phenotypes of the mutants fall into various classes. These include strains unable to oxidize H2 and strains deficient in CO2 uptake. Some of the mutant strains were capable of oxidizing H2 only when artificial electron acceptors were provided. Two mutant strains specifically lack activity of the key CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase. Other mutant strains lack both H2-oxidizing ability and ribulose 1,5-bisphosphate carboxylase activity. PMID:6780521

  17. Gastrointestinal Colonization by Candida albicans Mutant Strains in Antibiotic-Treated Mice

    PubMed Central

    Wiesner, Stephen M.; Jechorek, Robert P.; Garni, Robb M.; Bendel, Catherine M.; Wells, Carol L.

    2001-01-01

    Antibiotic-treated mice orally inoculated with one of three Candida albicans strains (including two mutant strains) or indigenous Candida pelliculosa showed levels of candidal gastrointestinal colonization that were strain specific. However, regardless of strain, the numbers of viable candida were intermediate to high in the stomach, were consistently lowest in the upper small intestine, and increased progressively down the intestinal tract. PMID:11139219

  18. Increased riboflavin production from activated bleaching earth by a mutant strain of Ashbya gossypii.

    PubMed

    Tajima, Satoshi; Itoh, Yoko; Sugimoto, Takashi; Kato, Tatsuya; Park, Enoch Y

    2009-10-01

    The production of riboflavin from vegetable oil was increased using a mutant strain of Ashbya gossypii. This mutant was generated by treating the wild-type strain with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Riboflavin production was 10-fold higher in the mutant compared to the wild-type strain. The specific intracellular catalase activity after 3 d of culture was 6-fold higher in the mutant than in the wild-type strain. For the mutant, riboflavin production in the presence of 40 mM hydrogen peroxide was 16% less than that in the absence of hydrogen peroxide, whereas it was 56% less for the wild-type strain. The isocitrate lyase (ICL) activity of the mutant was 0.26 mU/mg of protein during the active riboflavin production phase, which was 2.6-fold higher than the wild-type strain. These data indicate that the mutant utilizes the carbon flux from the TCA cycle to the glyoxylate cycle more efficiently than the wild-type strain, resulting in enhanced riboflavin production. This novel mutant has the potential to be of use for industrial-scale riboflavin production from waste-activated bleaching earth (ABE), thereby transforming a useless material into a valuable bioproduct. PMID:19716523

  19. Generation of mouse mutants as tools in dissecting the molecular clock.

    PubMed

    Anand, Sneha N; Edwards, Jessica K; Nolan, Patrick M

    2012-01-01

    Elucidation of the molecular basis of mammalian circadian rhythms has progressed dramatically in recent years through the characterization of mouse mutants. With the implementation of numerous mouse genetics programs, comprehensive sets of mutations in genes affecting circadian output measures have been generated. Although incomplete, existing arrays of mutants have been instrumental in our understanding of how the internal SCN clock interacts with the environment and how it conveys its rhythm to remote oscillators. The use of ENU mutagenesis has proven to be a significant contributor, generating mutations leading to subtle and distinct alterations in circadian protein function. In parallel, progress with mouse gene targeting allows one to study gene function in depth by ablating it entirely, in specific tissues at specific times, or by targeting specific functional domains. This has culminated in worldwide efforts to target every gene in the mouse genome allowing researchers to study multiple gene targeting effects systematically.

  20. Identification of a Van der Woude syndrome mutation in the cleft palate 1 mutant mouse.

    PubMed

    Stottmann, R W; Bjork, B C; Doyle, J B; Beier, D R

    2010-05-01

    Mutations in Interferon Regulatory Factor 6 (IRF6) have been identified in two human allelic syndromes with cleft lip and/or palate: Van der Woude (VWS) and Popliteal Pterygium syndromes (PPS). Furthermore, common IRF6 haplotypes and single nucleotide polymorphisms (SNP) alleles are strongly associated with nonsyndromic clefting defects in multiple ethnic populations. Mutations in the mouse often provide good models for the study of human diseases and developmental processes. We identified the cleft palate 1 (clft1) mouse mutant in a forward genetic screen for phenotypes modeling human congenital disease. In the clft1 mutant, we have identified a novel missense point mutation in the mouse Irf6 gene, which confers an amino acid alteration that has been found in a VWS family. Phenotypic comparison of clft1 mutants to previously reported Irf6 mutant alleles demonstrates the Irf6(clft1) allele is a hypomorphic allele. The cleft palate seen in these mutants appears to be due to abnormal adhesion between the palate and tongue. The Irf6(clft1) allele provides the first mouse model for the study of an etiologic IRF6 missense mutation observed in a human VWS family. PMID:20196077

  1. Survival of smooth, rough and transposon mutant strains of Brucella abortus in bovine mammary macrophages.

    PubMed

    Price, R E; Templeton, J W; Adams, L G

    1990-12-01

    Transposon mutants offer a unique way to evaluate the role of lipopolysaccharide (LPS) by producing a theoretical single-gene difference between the original strain and the transposon mutant strain. Comparative survival of Brucella abortus smooth strain 2308, rough RB51, smooth strain 19, and two transposon mutant strains (rough strain 2308::Tn5 Lac Z [m106] and rough strain 19::Tn5 Lac Z [m3], was tested in restrictive bovine mammary macrophages that were able to effectively reduce the percentage of intracellular bacterial survival and permissive bovine mammary macrophages that were unable to control the intracellular replication of B. abortus. The theoretical single-gene difference between strain 19 and strain 19::Tn5 lac Z [m3] and between smooth virulent strain 2308 and rough transposon mutant 2308::Tn5 lacZ [m106] is likely related to differences in LPS content or structure. Significant (P less than 0.05) reduction in the survival of rough strain 19::Tn5 Lac Z [m3] with no significant reduction in the rough transposon mutant strain 2308::Tn5 lacZ [m106] indicated that at least one factor other than LPS contributes to the intracellular survival of B. abortus in bovine macrophages.

  2. Factors influencing maternal behavior in the hubb/hubb mutant mouse.

    PubMed

    Alston-Mills, B; Parker, A C; Eisen, E J; Wilson, R; Fletcher, S

    We examined the maternal behavior of hubb/hubb mutant mice and normal control (+/hubb) siblings. From previous observations we noted that mutants groom their pups less, suckle less than normal, and often cannibalize the young. To date, these observations had not been quantified. Although prolactin (PRL) is linked to maternal behavior, it was difficult to measure because of the hyperirratibility of the mutant mice. Consequently, dopamine (DA) and its metabolite, dihydroxyphenylacetic acid (DOPAC), were measured in the median eminence in brains of both normal and mutant mice. Tyrosine hydroxylase, the rate-determining step in dopamine synthesis, was localized in the brain by immunohistochemistry. Five mutant and nine normal dams were observed for pup retrieval and crouching. Mean time for pup retrieval was slower (p < 0.06) for mutants (28.09 s) than for normal dams (18.49 s). Crouching was the same for both strains. Mutant pups were cold to the touch, and not well groomed. Brains from both strains were examined at Day 11 and Day 18 of gestation and Day 2 and Day 11 of lactation. Qualitatively, tyrosine hydroxylase localization in the arcuate nucleus and median eminence was the same in both strains for the gestation samples. The decrease in staining observed from gestation to lactation in the normal mice was increased in the mutants. Dopamine was similar in both strains at all stages, but DOPAC was significantly higher at early lactation in the mutants. We do not assume an absolute inverse relationship between dopaminergic activities and prolactin, but it is likely that the increase in DOPAC in the mutant reflects a decrease in prolactin, which could contribute to the diminished maternal care in the mutants. PMID:10627055

  3. Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment.

    PubMed

    Weitzner, Daniel S; Engler-Chiurazzi, Elizabeth B; Kotilinek, Linda A; Ashe, Karen Hsiao; Reed, Miranda Nicole

    2015-06-22

    The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer's disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer's disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

  4. Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

    PubMed Central

    Weitzner, Daniel S.; Engler-Chiurazzi, Elizabeth B.; Kotilinek, Linda A.; Ashe, Karen Hsiao; Reed, Miranda Nicole

    2015-01-01

    The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer’s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer’s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized. PMID:26132096

  5. Establishment of permanent chimerism in a lactate dehydrogenase-deficient mouse mutant with hemolytic anemia

    SciTech Connect

    Datta, T.; Doermer, P.

    1987-12-01

    Pluripotent hemopoietic stem cell function was investigated in the homozygous muscle type lactate dehydrogenase (LDH-A) mutant mouse using bone marrow transplantation experiments. Hemopoietic tissues of LDH-A mutants showed a marked decreased in enzyme activity that was associated with severe hemolytic anemia. This condition proved to be transplantable into wild type mice (+/+) through total body irradiation (TBI) at a lethal dose of 8.0 Gy followed by engraftment of mutant bone marrow cells. Since the mutants are extremely radiosensitive (lethal dose50/30 4.4 Gy vs 7.3 Gy in +/+ mice), 8.0-Gy TBI followed by injection of even high numbers of normal bone marrow cells did not prevent death within 5-6 days. After a nonlethal dose of 4.0 Gy and grafting of normal bone marrow cells, a transient chimerism showing peripheral blood characteristics of the wild type was produced that returned to the mutant condition within 12 weeks. The transfusion of wild type red blood cells prior to and following 8.0-Gy TBI and reconstitution with wild type bone marrow cells prevented the early death of the mutants and permanent chimerism was achieved. The chimeras showed all hematological parameters of wild type mice, and radiosensitivity returned to normal. It is concluded that the mutant pluripotent stem cells are functionally comparable to normal stem cells, emphasizing the significance of this mouse model for studies of stem cell regulation.

  6. Quadruple zebrafish mutant reveals different roles of Mesp genes in somite segmentation between mouse and zebrafish.

    PubMed

    Yabe, Taijiro; Hoshijima, Kazuyuki; Yamamoto, Takashi; Takada, Shinji

    2016-08-01

    The segmental pattern of somites is generated by sequential conversion of the temporal periodicity provided by the molecular clock. Whereas the basic structure of this clock is conserved among different species, diversity also exists, especially in terms of the molecular network. The temporal periodicity is subsequently converted into the spatial pattern of somites, and Mesp2 plays crucial roles in this conversion in the mouse. However, it remains unclear whether Mesp genes play similar roles in other vertebrates. In this study, we generated zebrafish mutants lacking all four zebrafish Mesp genes by using TALEN-mediated genome editing. Contrary to the situation in the mouse Mesp2 mutant, in the zebrafish Mesp quadruple mutant embryos the positions of somite boundaries were clearly determined and morphological boundaries were formed, although their formation was not completely normal. However, each somite was caudalized in a similar manner to the mouse Mesp2 mutant, and the superficial horizontal myoseptum and lateral line primordia were not properly formed in the quadruple mutants. These results clarify the conserved and species-specific roles of Mesp in the link between the molecular clock and somite morphogenesis. PMID:27385009

  7. Mutant strains of Tetrahymena thermophila defective in thymidine kinase activity: Biochemical and genetic characterization

    SciTech Connect

    Cornish, K.V.; Pearlman, R.E.

    1982-08-01

    Three mutant strains, one conditional, of Tetrahymena thermophila were defective in thymidine phosphorylating activity in vivo and in thymidine kinase activity in vitro. Nucleoside phosphotransferase activity in mutant cell extracts approached wild-type levels, suggesting that thymidine kinase is responsible for most, if not all, thymidine phosphorylation in vivo. Thymidine kinase activity in extracts of the conditional mutant strain was deficient when the cells were grown or assayed or both at the permissive temperature, implying a structural enzyme defect. Analysis of the reaction products from in vitro assays with partially purified enzymes showed that phosphorylation by thymidine kinase and nucleoside phosphotransferase occurred at the 5' position. Genetic analyses showed that the mutant phenotype was recessive and that mutations in each of the three mutant strains did not complement, suggesting allelism.

  8. Properties of Mutants of Synechocystis sp. Strain PCC 6803 Lacking Inorganic Carbon Sequestration Systems

    SciTech Connect

    Xu, Min; Bernat, Gabor; Singh, Abhay K.; Mi, Hualing; Rogner, Matthias; Pakrasi, Himadri B.; Ogawa, Teruo

    2008-09-10

    A mutant ( 5) of Synechocystis sp. strain PCC 6803 constructed by inactivating five inorganic carbon sequestration systems did not take up CO2 or HCO3– and was unable to grow in air with or without glucose. The 4 mutant in which BicA is the only active inorganic carbon sequestration system showed low activity of HCO3– uptake and grew under these conditions but more slowly than the wild-type strain. The 5 mutant required 1.7% CO2 to attain half the maximal growth rate. Electron transport activity of the mutants was strongly inhibited under high light intensities, with the 5 mutant more susceptible to high light than the 4 mutant. The results implicated the significance of carbon sequestration in dissipating excess light energy.

  9. Development of a mutant strain of Escherichia coli for molecular cloning of highly methylated DNA

    SciTech Connect

    Bishr, M.A.

    1991-01-01

    A mutant strain of Escherichia coli designated as GR219 that allows efficient molecular cloning of highly methylated bean DNA has been developed by UV light mutation of the parent LE392 str[sup r] strain. This mutant strain, like the parent, is streptomycin resistant and is biologically contained, because it requires thymidine for growth. Both the wild type and the mutant strain have lambda phage receptors so both can be utilized for construction of genomic libraries using the phase as a vector. The efficiency of transformation of the parent and the mutant strain with a recombinant plasmid containing bean DNA was compared to the efficiency of transformation of the PLK-F[prime] strain, which has a deletion of mcrA and mcrB genes and, therefore, allows transformation with methylated bean DNA. It has been found that the GR219 strain has the highest efficiency of transformation, while the PLK-F[prime] strain shows less, and the parent LE392 str[sup r] strain the least efficiency of transformation. These results indicate that strains of E. coli with mcrA and mcrB genes can recognize and degrade highly methylated DNA. However, other undefined factors affected by the altered gene(s) in the GR219 strain are also involved in the recognition and degradation of any cloned foreign DNA.

  10. [Composition of cell walls of 2 mutant strains of Streptomyces chrysomallus].

    PubMed

    Zaretskaia, M Sh; Nefelova, M V; Baratova, L A; Polin, A N

    1984-12-01

    The cell walls and peptidoglycans of two mutant strains, Streptomyces chrysomallus var. carotenoides and Streptomyces chrysomallus var. macrotetrolidi, were studied. The strains are organisms producing carotenes and antibiotics of the macrotetrolide group. By the qualitative composition of the peptidoglycans the mutants belong to Streptomyces and are similar. Their glycan portion consists of equimolar quantities of N-acetyl glucosamine and muramic acid. The peptide subunit is presented by glutamic acid, L, L-diaminopimelic acid, glycine and alanine. The molar ratio of alanine is 1.2-1.3. The mutant strains differ in the content of carbohydrates, total phosphorus and phosphorus belonging to teichoic acids. Teichoic acids of the cell walls of the both strains are of the ribitolhosphate nature. The cell walls of the mutants contain polysaccharides differing from teichoic acids and consisting of glucose, galactose, arabinose and fucose. The influence of the cell wall composition of the mutant strains on their morphology and metabolism and comparison of the data relative to the mutant strains with those relative to the starting strain are discussed.

  11. Induction, isolation, and characterization of aspergillus niger mutant strains producing elevated levels of beta-galactosidase.

    PubMed Central

    Nevalainen, K M

    1981-01-01

    An Aspergillus niger mutant strain, VTT-D-80144, with an improvement of three- to fourfold in the production of extracellular beta-galactosidase was isolated after mutagenesis. The production of beta-galactosidase by this mutant was unaffected by fermentor size, and the enzyme was also suitable for immobilization. PMID:6784672

  12. 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

  13. 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.

  14. Mouse model of Sanfilippo syndrome type B: relation of phenotypic features to background strain.

    PubMed

    Gografe, Sylvia I; Garbuzova-Davis, Svitlana; Willing, Alison E; Haas, Ken; Chamizo, Wilfredo; Sanberg, Paul R

    2003-12-01

    Sanfilippo syndrome type B or mucopolysaccharidosis type III B (MPS IIIB) is a lysosomal storage disorder that is inherited in autosomal recessive manner. It is characterized by systemic heparan sulfate accumulation in lysosomes due to deficiency of the enzyme alpha-N-acetylglucosaminidase (Naglu). Devastating clinical abnormalities with severe central nervous system involvement and somatic disease lead to premature death. A mouse model of Sanfilippo syndrome type B was created by targeted disruption of the gene encoding Naglu, providing a powerful tool for understanding pathogenesis and developing novel therapeutic strategies. However, the JAX GEMM Strain B6.129S6-Naglutm1Efn mouse, although showing biochemical similarities to humans with Sanfilippo syndrome, exhibits aging and behavioral differences. We observed idiosyncrasies, such as skeletal dysmorphism, hydrocephalus, ocular abnormalities, organomegaly, growth retardation, and anomalies of the integument, in our breeding colony of Naglu mutant mice and determined that several of them were at least partially related to the background strain C57BL/6. These background strain abnormalities, therefore, potentially mimic or overlap signs of the induced syndrome in our mice. Our observations may prove useful in studies of Naglu mutant mice. The necessity for distinguishing background anomalies from signs of the modeled disease is apparent. PMID:14727810

  15. Strain preservation of experimental animals: vitrification of two-cell stage embryos for multiple mouse strains.

    PubMed

    Eto, Tomoo; Takahashi, Riichi; Kamisako, Tsutomu

    2015-04-01

    Strain preservation of experimental animals is crucial for experimental reproducibility. Maintaining complete animal strains, however, is costly and there is a risk for genetic mutations as well as complete loss due to disasters or illness. Therefore, the development of effective vitrification techniques for cryopreservation of multiple experimental animal strains is important. We examined whether a vitrification method using cryoprotectant solutions, P10 and PEPeS, is suitable for preservation of multiple inbred and outbred mouse strains. First, we investigated whether our vitrification method using cryoprotectant solutions was suitable for two-cell stage mouse embryos. In vitro development of embryos exposed to the cryoprotectant solutions was similar to that of fresh controls. Further, the survival rate of the vitrified embryos was extremely high (98.1%). Next, we collected and vitrified two-cell stage embryos of 14 mouse strains. The average number of embryos obtained from one female was 7.3-33.3. The survival rate of vitrified embryos ranged from 92.8% to 99.1%, with no significant differences among mouse strains. In vivo development did not differ significantly between fresh controls and vitrified embryos of each strain. For strain preservation using cryopreserved embryos, two offspring for inbred lines and one offspring for outbred lines must be produced from two-cell stage embryos collected from one female. The expected number of surviving fetuses obtained from embryos collected from one female of either the inbred or outbred strains ranged from 2.9 to 19.5. The findings of the present study indicated that this vitrification method is suitable for strain preservation of multiple mouse strains.

  16. Kiss1 mutant placentas show normal structure and function in the mouse

    PubMed Central

    Herreboudt, A.M.; Kyle, V.R.L.; Lawrence, J.; Doran, J.; Colledge, W.H.

    2015-01-01

    Introduction Kisspeptins, encoded by the Kiss1 gene, are a set of related neuropeptides that are required for activation of the mammalian reproductive axis at puberty and to maintain fertility. In addition, kisspeptin signaling via the G-protein coupled receptor GPR54 (KISS1R) has been suggested to regulate human placental formation and correlations have been found between altered kisspeptin levels in the maternal blood and the development of pre-eclampsia. Methods We have used Kiss1 and Gpr54 mutant mice to investigate the role of kisspeptin signaling in the structure and function of the mouse placenta. Results Expression of Kiss1 and Gpr54 was confirmed in the mouse placenta but no differences in birth weight were found in mice that had been supported by a mutant placenta during fetal development. Stereological measurements found no differences between Kiss1 mutant and wild-type placentas. Measurement of amino-acid and glucose transport across the Kiss1 mutant placentas at E15.5 days did not reveal any functional defects. Discussion These data indicate that mouse placentas can develop a normal structure and function without kisspeptin signaling and can support normal fetal development and growth. PMID:25468546

  17. Pathogenicity and immunogenicity of a mutant strain of Listeria monocytogenes in the chicken infection model.

    PubMed

    Yin, Yuelan; Tian, Debin; Jiao, Hongmei; Zhang, Chenju; Pan, Zhiming; Zhang, Xiaoming; Wang, Xiaobo; Jiao, Xinan

    2011-03-01

    Listeria monocytogenes has been exploited as a vaccine carrier based upon its ability to induce a strong cell-mediated immune response. At present, the safety of live, attenuated L. monocytogenes vaccines in patients is being studied in clinical trials. L. monocytogenes is also an attractive vaccine vector for use in poultry; however, the pathogenicity and immunogenicity of this organism in poultry remain to be fully elucidated. In this study, we investigated the pathogenicity and immunogenicity of an actA- and plcB-deficient L. monocytogenes strain, yzuLM4ΔactA/plcB, and its wild-type parent strain, yzuLM4, in an avian infection model. The results showed that the wild-type strain could infect ISA brown chickens, causing serious tissue disruptions, including various degrees of degeneration, necrotic lesions, and inflammatory cell infiltration in the liver, spleen, heart, and kidney. However, the mutant strain showed reduced virulence in embryonated eggs compared with that of the parent strain (the 50% lethal dose [LD(50)] was 3 logs higher). The mutant strain also showed low virulence in chickens and was rapidly eliminated by the host. There were no obvious pathological changes in tissue sections, but the mutant strain still retained the ability to stimulate high levels of antibody against the protein listeriolysin O (LLO). Booster immunization with the mutant strain led to rapid bacterial clearance from the livers and spleens of chickens challenged by the intramuscular route or the oral route. Collectively, our data suggest that the wild-type serotype 1/2a L. monocytogenes strain can cause serious disease in chickens but the mutant strain with a deletion of the actA and plcB genes is less virulent but induces a strong immune response. This mutant strain of L. monocytogenes is therefore a promising candidate as a safe and effective vector for the delivery of heterologous antigens to prevent zoonosis and infectious disease in poultry.

  18. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, M.; Millard, C.S.; Stols, L.

    1998-06-23

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria. 2 figs.

  19. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    2002-01-01

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  20. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    2001-09-25

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  1. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    1998-01-01

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  2. Weaver mutant mouse cerebellar granule cells respond normally to chronic depolarization.

    PubMed

    Bjerregaard, A; Mogensen, H S; Hack, N; Balázs, R; Jørgensen, O S

    1997-04-01

    We studied the effects of chronic K(+)-induced membrane depolarization and treatment with N-methyl-D-aspartate (NMDA) on cerebellar granule cells (CGCs) from weaver mutant mice and non-weaver litter-mates. The weaver mutation is a Gly-to-Ser substitution in a conserved region of the Girk2 G protein-coupled inward rectifying potassium channel [Patil N., Cox D. R., Bhat D., Faham M., Myers R. M. and Peterson A. S. (1995) Nature Genet. 11, 126-129] which induces early death of CGCs. The biochemical differentiation of CGCs was estimated as the rate of 2-deoxy-D-glucose accumulation and the expression of neural cell adhesion molecule (NCAM). High (25 mM) K+ ion concentration or treatment with NMDA greatly promoted the biochemical differentiation of both weaver mutant and non-weaver litter-mate mouse CGCs. In contrast to the marked effect on biochemical differentiation in both weaver and non-weaver mice CGSs, chronic high K+ treatment only had limited effect on survival. The survival of weaver mutant mouse CGCs in medium containing 5 mM K+ ions was very low, only 20% of the plated cells surviving at 7 days after plating, as opposed to the 50% for non-weaver CGCs. Chronic high K+ treatment improved the relative survival of weaver mutant mouse CGCs 1.6 2.2-fold and that of non-weaver CGCs 1.2-1.4-fold; the same number of CGCs (about 20% of the plated cells) were rescued by high K+ in both types of culture. The findings indicate that, in culture weaver mutant mouse, CGCs have a normal response to membrane depolarization and that the normal function of the Girk2 potassium channel is not critical for the survival of differentiated CGCs.

  3. Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation

    PubMed Central

    Kirov, Julia V.; Adkisson, Michael; Nava, A. J.; Cipollone, Andreana; Willis, Brandon; Engelhard, Eric K.; Lloyd, K. C. Kent; de Jong, Pieter; West, David B.

    2015-01-01

    Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown. PMID:26275310

  4. Isolation and characterization of nitrogenase-derepressed mutant strains of cyanobacterium Anabaena variabilis.

    PubMed Central

    Spiller, H; Latorre, C; Hassan, M E; Shanmugam, K T

    1986-01-01

    A positive selection method for isolation of nitrogenase-derepressed mutant strains of a filamentous cyanobacterium, Anabaena variabilis, is described. Mutant strains that are resistant to a glutamate analog, L-methionine-D,L-sulfoximine, were screened for their ability to produce and excrete NH4+ into medium. Mutant strains capable of producing nitrogenase in the presence of NH4+ were selected from a population of NH4+-excreting mutants. One of the mutant strains (SA-1) studied in detail was found to be a conditional glutamine auxotroph requiring glutamine for growth in media containing N2, NO3-, or low concentrations of NH4+ (less than 0.5 mM). This glutamine requirement is a consequence of a block in the assimilation of NH4+ produced by an enzyme system like nitrogenase. Glutamate and aspartate failed to substitute for glutamine because of a defect in the transport and utilization of these amino acids. Strain SA-1 assimilated NH4+ when the concentration in the medium reached about 0.5 mM, and under these conditions the growth rate was similar to that of the parent. Mutant strain SA-1 produced L-methionine-D,L-sulfoximine-resistant glutamine synthetase activity. Kinetic properties of the enzyme from the parent and mutant were similar. Mutant strain SA-1 can potentially serve as a source of fertilizer nitrogen to support growth of crop plants, since the NH4+ produced by nitrogenase, utilizing sunlight and water as sources of energy and reductant, respectively, is excreted into the environment. PMID:2867990

  5. Enhanced cellulase producing mutants developed from heterokaryotic Aspergillus strain.

    PubMed

    Kaur, Baljit; Oberoi, H S; Chadha, B S

    2014-03-01

    A heterokaryon 28, derived through protoplast fusion between Aspergillus nidulans and Aspergillus tubingensis (Dal8), was subjected cyclic mutagenesis followed by selection on increasing levels of 2-deoxy glucose (2-DG) as selection marker. The derived deregulated cellulase hyper producing mutant '64', when compared to fusant 28, produced 9.83, 7.8, 3.2, 4.2 and 19.74 folds higher endoglucanase, β-glucosidase, cellobiohydrolase, FPase and xylanase, respectively, under shake cultures. The sequence analysis of PCR amplified β-glucosidase gene from wild and mutant showed nucleotide deletion/substitution. The mutants showed highly catalytic efficient β-glucosidase as evident from low Km and high Vmax values. The expression profiling through zymogram analysis also indicated towards over-expression of cellulases. The up/down regulated expressed proteins observed through SDS-PAGE were identified by Peptide mass fingerprinting The cellulase produced by mutants in conjunction with cellulase free xylanase derived from Thermomyces lanuginosus was used for efficient utilization of alkali treated rice straw for obtaining xylo-oligosaccharides and ethanol.

  6. Impaired cholesterol esterification in primary brain cultures of the lysosomal cholesterol storage disorder (LCSD) mouse mutant

    SciTech Connect

    Patel, S.C.; Suresh, S.; Weintroub, H.; Brady, R.O.; Pentchev, P.G.

    1987-02-27

    Esterification of cholesterol was investigated in primary neuroglial cultures obtained from newborn lysosomal cholesterol storage disorder (LCSD) mouse mutants. An impairment in /sup 3/H-oleic acid incorporation into cholesteryl esters was demonstrated in cultures of homozygous LCSD brain. Primary cultures derived from other phenotypically normal pups of the carrier breeders esterified cholesterol at normal levels or at levels which were intermediary between normal and deficient indicating a phenotypic expression of the LCSD heterozygote genotype. These observations on LCSD mutant brain cells indicate that the defect in cholesterol esterification is closely related to the primary genetic defect and is expressed in neuroglial cells in culture.

  7. Mutant breeding of Serratia marcescens strain for enhancing prodigiosin production and application to textiles.

    PubMed

    Liu, Xiaoxia; Wang, Yujie; Sun, Shiqing; Zhu, Changjun; Xu, Wei; Park, Yongdoo; Zhou, Haimeng

    2013-01-01

    Microwaves have been used as a mutant agent to select mutant strains with high-yield and high-purity pigment. Mass spectrometry and nuclear magnetic resonance spectroscopic techniques were used to elucidate the structures of the pigment. High-performance liquid chromatography was used to measure pigment purity. The analysis of the mutant strain showed that pigment yield increased by 109% and was 98% pure. Prodigiosin in ethanol solution had good stability under ambient temperature and natural indoor light. However, prodigiosin rapidly decomposed under intense sunlight. Prodigiosin is an ecological colorant to dye fabrics, including synthetic and natural fibers. Synthetic fabrics dyed with prodigiosin, such as polyamide and acrylic, have high colorfastness to washing (≥4th grade) and antimicrobial properties (>90%) against Escherichia coli and Staphylococcus aureus. Antimicrobial properties were significantly different between synthetic and natural fabrics. The mutant strain Serratia marcescens jx1-1, with high prodigiosin yield and purity, has promising prospects in food, cosmetic, and textile industries.

  8. Dopaminergic function in relation to genes associated with risk for schizophrenia: translational mutant mouse models.

    PubMed

    Moran, Paula M; O'Tuathaigh, Colm M P; Papaleo, Francesco; Waddington, John L

    2014-01-01

    Mutant mice play an increasingly important role in understanding disease processes at multiple levels. In particular, they illuminate the impact of risk genes for disease on such processes. This article reviews recent advances in the application of mutant mice to study the intricacies of dopaminergic (DAergic) function in relation to the putative pathophysiology of psychotic illness, particularly schizophrenia, and antipsychotic drug action. It considers models for understanding the role(s) of risk genes, with a particular focus on DTNBP1 and NRG1, their interactions with environmental factors, and with each other (epistasis). In overview, it considers new schemas for understanding psychotic illness that integrate DAergic pathophysiology with developmental, social, and cognitive processes, and how mutant mouse models can reflect and inform on such schemas.

  9. The Mouse MC13 Mutant Is a Novel ENU Mutation in Collagen Type II, Alpha 1

    PubMed Central

    Cionni, Megan; Menke, Chelsea; Stottmann, Rolf W.

    2014-01-01

    Phenotype-driven mutagenesis experiments are a powerful approach to identifying novel alleles in a variety of contexts. The traditional disadvantage of this approach has been the subsequent task of identifying the affected locus in the mutants of interest. Recent advances in bioinformatics and sequencing have reduced the burden of cloning these ENU mutants. Here we report our experience with an ENU mutagenesis experiment and the rapid identification of a mutation in a previously known gene. A combination of mapping the mutation with a high-density SNP panel and a candidate gene approach has identified a mutation in collagen type II, alpha I (Col2a1). Col2a1 has previously been studied in the mouse and our mutant phenotype closely resembles mutations made in the Col2a1 locus. PMID:25541700

  10. Silencing neuronal mutant androgen receptor in a mouse model of spinal and bulbar muscular atrophy.

    PubMed

    Sahashi, Kentaro; Katsuno, Masahisa; Hung, Gene; Adachi, Hiroaki; Kondo, Naohide; Nakatsuji, Hideaki; Tohnai, Genki; Iida, Madoka; Bennett, C Frank; Sobue, Gen

    2015-11-01

    Spinal and bulbar muscular atrophy (SBMA), an adult-onset neurodegenerative disease that affects males, results from a CAG triplet repeat/polyglutamine expansions in the androgen receptor (AR) gene. Patients develop progressive muscular weakness and atrophy, and no effective therapy is currently available. The tissue-specific pathogenesis, especially relative pathological contributions between degenerative motor neurons and muscles, remains inconclusive. Though peripheral pathology in skeletal muscle caused by toxic AR protein has been recently reported to play a pivotal role in the pathogenesis of SBMA using mouse models, the role of motor neuron degeneration in SBMA has not been rigorously investigated. Here, we exploited synthetic antisense oligonucleotides to inhibit the RNA levels of mutant AR in the central nervous system (CNS) and explore its therapeutic effects in our SBMA mouse model that harbors a mutant AR gene with 97 CAG expansions and characteristic SBMA-like neurogenic phenotypes. A single intracerebroventricular administration of the antisense oligonucleotides in the presymptomatic phase efficiently suppressed the mutant gene expression in the CNS, and delayed the onset and progression of motor dysfunction, improved body weight gain and survival with the amelioration of neuronal histopathology in motor units such as spinal motor neurons, neuromuscular junctions and skeletal muscle. These findings highlight the importance of the neurotoxicity of mutant AR protein in motor neurons as a therapeutic target.

  11. Genomic landscapes of endogenous retroviruses unveil intricate genetics of conventional and genetically-engineered laboratory mouse strains.

    PubMed

    Lee, Kang-Hoon; Lim, Debora; Chiu, Sophia; Greenhalgh, David; Cho, Kiho

    2016-04-01

    Laboratory strains of mice, both conventional and genetically engineered, have been introduced as critical components of a broad range of studies investigating normal and disease biology. Currently, the genetic identity of laboratory mice is primarily confirmed by surveying polymorphisms in selected sets of "conventional" genes and/or microsatellites in the absence of a single completely sequenced mouse genome. First, we examined variations in the genomic landscapes of transposable repetitive elements, named the TREome, in conventional and genetically engineered mouse strains using murine leukemia virus-type endogenous retroviruses (MLV-ERVs) as a probe. A survey of the genomes from 56 conventional strains revealed strain-specific TREome landscapes, and certain families (e.g., C57BL) of strains were discernible with defined patterns. Interestingly, the TREome landscapes of C3H/HeJ (toll-like receptor-4 [TLR4] mutant) inbred mice were different from its control C3H/HeOuJ (TLR4 wild-type) strain. In addition, a CD14 knock-out strain had a distinct TREome landscape compared to its control/backcross C57BL/6J strain. Second, an examination of superantigen (SAg, a "TREome gene") coding sequences of mouse mammary tumor virus-type ERVs in the genomes of the 46 conventional strains revealed a high diversity, suggesting a potential role of SAgs in strain-specific immune phenotypes. The findings from this study indicate that unexplored and intricate genomic variations exist in laboratory mouse strains, both conventional and genetically engineered. The TREome-based high-resolution genetics surveillance system for laboratory mice would contribute to efficient study design with quality control and accurate data interpretation. This genetics system can be easily adapted to other species ranging from plants to humans.

  12. Genomic landscapes of endogenous retroviruses unveil intricate genetics of conventional and genetically-engineered laboratory mouse strains.

    PubMed

    Lee, Kang-Hoon; Lim, Debora; Chiu, Sophia; Greenhalgh, David; Cho, Kiho

    2016-04-01

    Laboratory strains of mice, both conventional and genetically engineered, have been introduced as critical components of a broad range of studies investigating normal and disease biology. Currently, the genetic identity of laboratory mice is primarily confirmed by surveying polymorphisms in selected sets of "conventional" genes and/or microsatellites in the absence of a single completely sequenced mouse genome. First, we examined variations in the genomic landscapes of transposable repetitive elements, named the TREome, in conventional and genetically engineered mouse strains using murine leukemia virus-type endogenous retroviruses (MLV-ERVs) as a probe. A survey of the genomes from 56 conventional strains revealed strain-specific TREome landscapes, and certain families (e.g., C57BL) of strains were discernible with defined patterns. Interestingly, the TREome landscapes of C3H/HeJ (toll-like receptor-4 [TLR4] mutant) inbred mice were different from its control C3H/HeOuJ (TLR4 wild-type) strain. In addition, a CD14 knock-out strain had a distinct TREome landscape compared to its control/backcross C57BL/6J strain. Second, an examination of superantigen (SAg, a "TREome gene") coding sequences of mouse mammary tumor virus-type ERVs in the genomes of the 46 conventional strains revealed a high diversity, suggesting a potential role of SAgs in strain-specific immune phenotypes. The findings from this study indicate that unexplored and intricate genomic variations exist in laboratory mouse strains, both conventional and genetically engineered. The TREome-based high-resolution genetics surveillance system for laboratory mice would contribute to efficient study design with quality control and accurate data interpretation. This genetics system can be easily adapted to other species ranging from plants to humans. PMID:26779669

  13. A γA-Crystallin Mouse Mutant Secc with Small Eye, Cataract and Closed Eyelid

    PubMed Central

    Cheng, Man Hei; Tam, Chung Nga; Choy, Kwong Wai; Tsang, Wai Hung; Tsang, Sze Lan; Pang, Chi Pui; Song, You Qiang; Sham, Mai Har

    2016-01-01

    Cataract is the most common cause of visual loss in humans. A spontaneously occurred, autosomal dominant mouse mutant Secc, which displayed combined features of small eye, cataract and closed eyelid was discovered in our laboratory. In this study, we identified the mutation and characterized the cataract phenotype of this novel Secc mutant. The Secc mutant mice have eyelids that remain half-closed throughout their life. The mutant lens has a significant reduction in size and with opaque spots clustered in the centre. Histological analysis showed that in the core region of the mutant lens, the fiber cells were disorganized and clefts and vacuoles were observed. The cataract phenotype was evident from new born stage. We identified the Secc mutation by linkage analysis using whole genome microsatellite markers and SNP markers. The Secc locus was mapped at chromosome 1 flanked by SNPs rs3158129 and rs13475900. Based on the chromosomal position, the candidate cataract locus γ-crystallin gene cluster (Cryg) was investigated by sequencing. A single base deletion (299delG) in exon 3 of Cryga which led to a frame-shift of amino acid sequence from position 91 was identified. As a result of this mutation, the sequences of the 3rd and 4th Greek-key motifs of the γA-crystallin are replaced with an unrelated C-terminal peptide of 75 residues long. Coincidentally, the point mutation generated a HindIII restriction site, allowing the identification of the CrygaSecc mutant allele by RFLP. Western blot analysis of 3-week old lenses showed that the expression of γ-crystallins was reduced in the CrygaSecc mutant. Furthermore, in cell transfection assays using CrygaSecc mutant cDNA expression constructs in 293T, COS-7 and human lens epithelial B3 cell lines, the mutant γA-crystallins were enriched in the insoluble fractions and appeared as insoluble aggregates in the transfected cells. In conclusion, we have demonstrated that the Secc mutation leads to the generation of Cryga

  14. A γA-Crystallin Mouse Mutant Secc with Small Eye, Cataract and Closed Eyelid.

    PubMed

    Cheng, Man Hei; Tam, Chung Nga; Choy, Kwong Wai; Tsang, Wai Hung; Tsang, Sze Lan; Pang, Chi Pui; Song, You Qiang; Sham, Mai Har

    2016-01-01

    Cataract is the most common cause of visual loss in humans. A spontaneously occurred, autosomal dominant mouse mutant Secc, which displayed combined features of small eye, cataract and closed eyelid was discovered in our laboratory. In this study, we identified the mutation and characterized the cataract phenotype of this novel Secc mutant. The Secc mutant mice have eyelids that remain half-closed throughout their life. The mutant lens has a significant reduction in size and with opaque spots clustered in the centre. Histological analysis showed that in the core region of the mutant lens, the fiber cells were disorganized and clefts and vacuoles were observed. The cataract phenotype was evident from new born stage. We identified the Secc mutation by linkage analysis using whole genome microsatellite markers and SNP markers. The Secc locus was mapped at chromosome 1 flanked by SNPs rs3158129 and rs13475900. Based on the chromosomal position, the candidate cataract locus γ-crystallin gene cluster (Cryg) was investigated by sequencing. A single base deletion (299delG) in exon 3 of Cryga which led to a frame-shift of amino acid sequence from position 91 was identified. As a result of this mutation, the sequences of the 3rd and 4th Greek-key motifs of the γA-crystallin are replaced with an unrelated C-terminal peptide of 75 residues long. Coincidentally, the point mutation generated a HindIII restriction site, allowing the identification of the CrygaSecc mutant allele by RFLP. Western blot analysis of 3-week old lenses showed that the expression of γ-crystallins was reduced in the CrygaSecc mutant. Furthermore, in cell transfection assays using CrygaSecc mutant cDNA expression constructs in 293T, COS-7 and human lens epithelial B3 cell lines, the mutant γA-crystallins were enriched in the insoluble fractions and appeared as insoluble aggregates in the transfected cells. In conclusion, we have demonstrated that the Secc mutation leads to the generation of Cryga

  15. DNA binding properties of dioxin receptors in wild-type and mutant mouse hepatoma cells

    SciTech Connect

    Cuthill, S.; Poellinger, L.

    1988-04-19

    The current model of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) entails stimulation of target gene transcription via the formation of dioxin-receptor complexes and subsequent accumulation of the complexes within the cell nucleus. Here, the authors have analyzed the DNA binding properties of the dioxin receptor in wild-type mouse hepatoma (Hepa 1c1c7) cells and a class of nonresponsive mutant cells which fail to accumulate dioxin-receptor complexes within the nucleus in vivo. In vitro, both the wild-type and mutant (/sup 3/H)dioxin-receptor complexes exhibited low affinity for DNA-cellulose (5-8% and around 4% retention, respectively) in the absence of prior biochemical manipulations. However, following chromatography on heparin-Sepharose, the wild-type but not the mutant dioxin receptor was transformed to a species with an increased affinity for DNA (40-50% retention on DNA-cellulose). The gross molecular structure of the mutant, non DNA binding dioxin receptor did not appear to be altered as compared to that of the wild-type receptor. These results imply that the primary deficiency in the mutant dioxin receptor form may reside at the DNA binding level and that, in analogy to steroid hormone receptors, DNA binding of the receptor may be an essential step in the regulation of target gene transcription by dioxin.

  16. Link between reduced nephron number and hypertension: studies in a mutant mouse model.

    PubMed

    Poladia, Deepali Pitre; Kish, Kayle; Kutay, Benjamin; Bauer, John; Baum, Michel; Bates, Carlton M

    2006-04-01

    Low birth weight (LBW) infants with reduced nephron numbers have significantly increased risk for hypertension later in life, which is a devastating health problem. The risk from a reduction in nephron number alone is not clear. Recently, using conditional knock-out approach, we have developed a mutant mouse with reduced nephron number in utero and no change in birth weight, by deleting fibroblast growth factor receptor 2 (fgfr2) in the ureteric bud. Our purpose was to investigate the role of in utero reduced nephron number alone in absence of LBW as a risk for developing hypertension in adulthood. Using tail cuff blood pressure measurements we observed significant increases in systolic blood pressure in one year old mutant mice versus controls. We also detected cardiac end-organ injury from hypertension as shown by significant increases in normalized heart weights, left ventricular (LV) wall thickness, and LV tissue area. Two-dimensional echocardiography revealed no changes in cardiac output and therefore significant increases in systemic vascular resistance in mutants versus controls. We also observed increases in serum blood urea nitrogen (BUN) levels and histologic evidence of glomerular and renal tubular injury in mutant mice versus controls. Thus, these studies suggest that our mutant mice may serve as a relevant model to study the link between reduction of nephron number in utero and the risk of hypertension and chronic renal failure in adulthood.

  17. Phosphoregulation of an Inner Dynein Arm Complex in Chlamydomonas reinhardtii Is Altered in Phototactic Mutant Strains

    PubMed Central

    King, Stephen J.; Dutcher, Susan K.

    1997-01-01

    To gain a further understanding of axonemal dynein regulation, mutant strains of Chlamydomonas reinhardtii that had defects in both phototactic behavior and flagellar motility were identified and characterized. ptm1, ptm2, and ptm3 mutant strains exhibited motility phenotypes that resembled those of known inner dynein arm region mutant strains, but did not have biochemical or genetic phenotypes characteristic of other inner dynein arm mutations. Three other mutant strains had defects in the f class of inner dynein arms. Dynein extracts from the pf9-4 strain were missing the entire f complex. Strains with mutations in pf9/ida1, ida2, or ida3 failed to assemble the f dynein complex and did not exhibit phototactic behavior. Fractionated dynein from mia1-1 and mia2-1 axonemes exhibited a novel f class inner dynein arm biochemical phenotype; the 138-kD f intermediate chain was present in altered phosphorylation forms. In vitro axonemal dynein activity was reduced by the mia1-1 and mia2-1 mutations. The addition of kinase inhibitor restored axonemal dynein activity concomitant with the dephosphorylation of the 138-kD f intermediate chain. Dynein extracts from uni1-1 axonemes, which specifically assemble only one of the two flagella, contained relatively high levels of the altered phosphorylation forms of the 138-kD intermediate chain. We suggest that the f dynein complex may be phosphoregulated asymmetrically between the two flagella to achieve phototactic turning. PMID:9008712

  18. [Generation of nalidixic acid-resistant strains and signature-tagged mutants of Actinobacillus pleuropneumoniae].

    PubMed

    Shang, Lin; Li, Wei; Li, Liangjun; Li, Lu; Zhang, Sihua; Li, Tingting; Li, Yaokun; Liu, Lei; Guo, Zhiwei; Zhou, Rui; Chen, Huanchun

    2008-01-01

    Actinobacillus pleuropneumoniae is a very important respiratory pathogen for swine and causes great economic losses in pig industry worldwide. Signature-tagged mutagenesis (STM) is an effective method to identify virulence genes in bacteria. In this study, we selected nalidixic acid-resistant strains of APP serotypes 1 and 3 by in vitro cultivation, and used as receipt strains for constructing transposon mutants by mating with E. coli CC 118 lambdapir or S17-1 lambdapir containing mini-Tn10 tag plasmids pLOF/TAG1-48, with or without the help of E. coli DH5alpha (pRK2073). We screened mutant strains by antibiotics selection, PCR and Southern blot identification. Our data revealed that nalidixic acid-resistance of APP strains could easily be induced in vitro and the resistance was due to the mutation in the DNA gyrase A subunit gene gyrA. In the mating experiments, the bi-parental mating was more effective and easier than tri-parental mating. Different APP strains showed a different mating and transposon efficiency in the bi-parental mating, with the strains of serotype 1 much higher than serotype 3 and the reference strain of serotype 3 higher than the field strains. These data were helpful for the construction of STM mutants and pickup of virulence genes of APP. PMID:18338580

  19. Dorsoventral patterning of the telencephalon is disrupted in the mouse mutant extra-toes(J).

    PubMed

    Tole, S; Ragsdale, C W; Grove, E A

    2000-01-15

    Little is known about the mechanisms that control the development of regional identity in the mammalian telencephalon. The Gli family of transcription factor genes is involved in the regulation of pattern at many sites in the embryo and is expressed in the embryonic mouse telencephalon. We have analyzed telencephalic patterning in the extra-toes (J) (Xt(J)) mouse mutant, which carries a deletion in the Gli family member Gli3. We report that dorsoventral patterning of the telencephalon is dramatically disrupted in the Xt(J) mutant. Specific dorsal telencephalic cell types and gene expression patterns are lost in homozygous Xt(J) mutants, and features of ventral telencephalic identity develop ectopically in the dorsal telencephalon. This partial ventralization of the dorsal telencephalon does not appear to be induced by an expansion of Sonic hedgehog expression in the telencephalon, but may be due to a loss of Bmp and Wnt gene expression in a putative dorsal telencephalic signaling center, the cortical hem. Our findings suggest that in dorsal telencephalon Gli3 is needed to repress ventral telencephalic identity.

  20. Disparate metabolic response to fructose feeding between different mouse strains.

    PubMed

    Montgomery, M K; Fiveash, C E; Braude, J P; Osborne, B; Brown, S H J; Mitchell, T W; Turner, N

    2015-12-22

    Diets enriched in fructose (FR) increase lipogenesis in the liver, leading to hepatic lipid accumulation and the development of insulin resistance. Previously, we have shown that in contrast to other mouse strains, BALB/c mice are resistant to high fat diet-induced metabolic deterioration, potentially due to a lack of ectopic lipid accumulation in the liver. In this study we have compared the metabolic response of BALB/c and C57BL/6 (BL6) mice to a fructose-enriched diet. Both strains of mice increased adiposity in response to FR-feeding, while only BL6 mice displayed elevated hepatic triglyceride (TAG) accumulation and glucose intolerance. The lack of hepatic TAG accumulation in BALB/c mice appeared to be linked to an altered balance between lipogenic and lipolytic pathways, while the protection from fructose-induced glucose intolerance in this strain was likely related to low levels of ER stress, a slight elevation in insulin levels and an altered profile of diacylglycerol species in the liver. Collectively these findings highlight the multifactorial nature of metabolic defects that develop in response to changes in the intake of specific nutrients and the divergent response of different mouse strains to dietary challenges.

  1. Disparate metabolic response to fructose feeding between different mouse strains

    PubMed Central

    Montgomery, M. K.; Fiveash, C. E.; Braude, J. P.; Osborne, B.; Brown, S. H. J.; Mitchell, T. W.; Turner, N.

    2015-01-01

    Diets enriched in fructose (FR) increase lipogenesis in the liver, leading to hepatic lipid accumulation and the development of insulin resistance. Previously, we have shown that in contrast to other mouse strains, BALB/c mice are resistant to high fat diet-induced metabolic deterioration, potentially due to a lack of ectopic lipid accumulation in the liver. In this study we have compared the metabolic response of BALB/c and C57BL/6 (BL6) mice to a fructose-enriched diet. Both strains of mice increased adiposity in response to FR-feeding, while only BL6 mice displayed elevated hepatic triglyceride (TAG) accumulation and glucose intolerance. The lack of hepatic TAG accumulation in BALB/c mice appeared to be linked to an altered balance between lipogenic and lipolytic pathways, while the protection from fructose-induced glucose intolerance in this strain was likely related to low levels of ER stress, a slight elevation in insulin levels and an altered profile of diacylglycerol species in the liver. Collectively these findings highlight the multifactorial nature of metabolic defects that develop in response to changes in the intake of specific nutrients and the divergent response of different mouse strains to dietary challenges. PMID:26690387

  2. Mouse and hamster mutants as models for Waardenburg syndromes in humans.

    PubMed Central

    Asher, J H; Friedman, T B

    1990-01-01

    Four different Waardenburg syndromes have been defined based upon observed phenotypes. These syndromes are responsible for approximately 2% of subjects with profound congenital hearing loss. At present, Waardenburg syndromes have not been mapped to particular human chromosomes. One or more of the mouse mutant alleles, Ph (patch), s (piebald), Sp (splotch), and Mior (microphthalmia-Oak Ridge) and the hamster mutation Wh (anophthalmic white) may be homologous to mutations causing Waardenburg syndromes. In heterozygotes, phenotypic effects of these four mouse mutations and the hamster mutation are similar to the phenotypes produced by different Waardenburg syndrome mutations. The chromosomal locations and syntenic relationships associated with three of the four mouse mutant genes have been used to predict human chromosomal locations for Waardenburg syndromes: (1) on chromosome 2q near FN1 (fibronectin 1), (2) on chromosome 3p near the proto-oncogene RAF1 or 3q near RHO (rhodopsin), and (3) on chromosome 4p near the proto-oncogene KIT. Waardenburg syndromes show extensive intrafamilial phenotypic variability. Results of our studies with the hamster mutation Wh suggest that this variability may be explained in part by modifier genes segregating within families. Images PMID:2246770

  3. 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.

  4. Characterization of a Mouse-Adapted Staphylococcus aureus Strain

    PubMed Central

    Holtfreter, Silva; Radcliff, Fiona J.; Grumann, Dorothee; Read, Hannah; Johnson, Sarah; Monecke, Stefan; Ritchie, Stephen; Clow, Fiona; Goerke, Christiane; Bröker, Barbara M.; Fraser, John D.; Wiles, Siouxsie

    2013-01-01

    More effective antibiotics and a protective vaccine are desperately needed to combat the ‘superbug’ Staphylococcus aureus. While in vivo pathogenicity studies routinely involve infection of mice with human S. aureus isolates, recent genetic studies have demonstrated that S. aureus lineages are largely host-specific. The use of such animal-adapted S. aureus strains may therefore be a promising approach for developing more clinically relevant animal infection models. We have isolated a mouse-adapted S. aureus strain (JSNZ) which caused a severe outbreak of preputial gland abscesses among male C57BL/6J mice. We aimed to extensively characterize this strain on a genomic level and determine its virulence potential in murine colonization and infection models. JSNZ belongs to the MLST type ST88, rare among human isolates, and lacks an hlb-converting phage encoding human-specific immune evasion factors. Naive mice were found to be more susceptible to nasal and gastrointestinal colonization with JSNZ than with the human-derived Newman strain. Furthermore, naïve mice required antibiotic pre-treatment to become colonized with Newman. In contrast, JSNZ was able to colonize mice in the absence of antibiotic treatment suggesting that this strain can compete with the natural flora for space and nutrients. In a renal abscess model, JSNZ caused more severe disease than Newman with greater weight loss and bacterial burden. In contrast to most other clinical isolates, JSNZ can also be readily genetically modified by phage transduction and electroporation. In conclusion, the mouse-adapted strain JSNZ may represent a valuable tool for studying aspects of mucosal colonization and for screening novel vaccines and therapies directed at preventing colonization. PMID:24023720

  5. Frequency-dependent viability in mutant strains of Drosophila melanogaster.

    PubMed

    Curtsinger, J W; Sheen, F M

    1991-01-01

    We investigated the effects of genotypic frequencies on egg-to-adult viabilities in pairwise combinations of four strains of Drosophila melanogaster. The experiments involved mixture of a total of 42,000 eggs in varying proportions under controlled densities and observation of surviving adults. Viabilities were found to depend on frequencies in several genotypic combinations. In the most extreme case, the absolute viability of cn;bw females increased monotonically from 54% when common to 70% when rare. The results illustrate several statistical and methodological problems that might explain why some experiments have failed to detect frequency-dependent viabilities. These problems include heterogeneity between replications, sex differences in susceptibility to competition, and strong dependence of the experimental outcome on the choice of competitor genotypes. PMID:1901577

  6. Characteristics of stria vascularis melanocytes of viable dominant spotting (Wv/Wv) mouse mutants.

    PubMed

    Cable, J; Barkway, C; Steel, K P

    1992-12-01

    The Wv mutation lies in the kinase domain of the proto-oncogene c-kit which is expressed in a variety of cells including neural crest derived melanoblasts. The mutation results in the abnormal migration, proliferation, survival and/or differentiation of melanoblasts. Viable Dominant Spotting (Wv/Wv) mouse mutants have a white coat due to the absence of melanocytes. The majority of these animals have no melanocytes within the stria vascularis and no endocochlear potential (EP). A proportion of homozygous mutants partially escape the effects of the mutation: 47.2% of pinnae and 21% of vestibular regions were pigmented and 10.8% of ears had an EP. All ears with an EP that were available for histology had some pigmentation of the stria. There was no obvious correlation between external and internal spotting in Wv/Wv mice, and asymmetrical pigmentation of the ears was common. Both light and dark intermediate cells (which are derived from melanocytes) were present in the middle and/or basal turns of these cochlear ducts and they appeared to function normally in enabling the stria to produce an EP (although the EP was usually lower than normal). This suggests that the c-kit gene product is needed only during development of the stria, and not for mature melanocyte function because the melanocytes present in the mutant strias were carrying the mutant version of the c-kit gene. Melanocytes were similar in appearance in controls and mutants, except that fewer melanin granules were observed in the strias of Wv/Wv mice. The observations that strial melanocytes with very few melanin granules in Wv/Wv mutants are able to support EP production, together with previous observations that albino animals with strial melanocytes but no melanin have a normal EP, suggest that melanocytes but not melanin are essential for normal strial function.

  7. Selection and Characterization of Phage-Resistant Mutant Strains of Listeria monocytogenes Reveal Host Genes Linked to Phage Adsorption

    PubMed Central

    Denes, Thomas; den Bakker, Henk C.; Tokman, Jeffrey I.; Guldimann, Claudia

    2015-01-01

    Listeria-infecting phages are readily isolated from Listeria-containing environments, yet little is known about the selective forces they exert on their host. Here, we identified that two virulent phages, LP-048 and LP-125, adsorb to the surface of Listeria monocytogenes strain 10403S through different mechanisms. We isolated and sequenced, using whole-genome sequencing, 69 spontaneous mutant strains of 10403S that were resistant to either one or both phages. Mutations from 56 phage-resistant mutant strains with only a single mutation mapped to 10 genes representing five loci on the 10403S chromosome. An additional 12 mutant strains showed two mutations, and one mutant strain showed three mutations. Two of the loci, containing seven of the genes, accumulated the majority (n = 64) of the mutations. A representative mutant strain for each of the 10 genes was shown to resist phage infection through mechanisms of adsorption inhibition. Complementation of mutant strains with the associated wild-type allele was able to rescue phage susceptibility for 6 out of the 10 representative mutant strains. Wheat germ agglutinin, which specifically binds to N-acetylglucosamine, bound to 10403S and mutant strains resistant to LP-048 but did not bind to mutant strains resistant to only LP-125. We conclude that mutant strains resistant to only LP-125 lack terminal N-acetylglucosamine in their wall teichoic acid (WTA), whereas mutant strains resistant to both phages have disruptive mutations in their rhamnose biosynthesis operon but still possess N-acetylglucosamine in their WTA. PMID:25888172

  8. Mutant Mouse Models: Genotype-Phenotype Relationships to Negative Symptoms in Schizophrenia

    PubMed Central

    O'Tuathaigh, Colm M. P.; Kirby, Brian P.; Moran, Paula M.; Waddington, John L.

    2010-01-01

    Negative symptoms encompass diminution in emotional expression and motivation, some of which relate to human attributes that may not be accessible readily in animals. Additionally, their refractoriness to treatment precludes therapeutic validation of putative models. This review considers critically the application of mutant mouse models to the study of the pathobiology of negative symptoms. It focuses on 4 main approaches: genes related to the pathobiology of schizophrenia, genes associated with risk for schizophrenia, neurodevelopmental-synaptic genes, and variant approaches from other areas of neurobiology. Despite rapid advances over the past several years, it is clear that we continue to face substantive challenges in applying mutant models to better understand the pathobiology of negative symptoms: the majority of evidence relates to impairments in social behavior, with only limited data relating to anhedonia and negligible data concerning avolition and other features; even for the most widely examined feature, social behavior, studies have used diverse assessments thereof; modelling must proceed in cognizance of increasing evidence that genes and pathobiologies implicated in schizophrenia overlap with other psychotic disorders, particularly bipolar disorder. Despite the caveats and challenges, several mutant lines evidence a phenotype for at least one index of social behavior. Though this may suggest superficially some shared relationship to negative symptoms, it is not yet possible to specify either the scope or the pathobiology of that relationship for any given gene. The breadth and depth of ongoing studies in mutants hold the prospect of addressing these shortcomings. PMID:19934211

  9. An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants

    PubMed Central

    Liberati, Nicole T.; Urbach, Jonathan M.; Miyata, Sachiko; Lee, Daniel G.; Drenkard, Eliana; Wu, Gang; Villanueva, Jacinto; Wei, Tao; Ausubel, Frederick M.

    2006-01-01

    Random transposon insertion libraries have proven invaluable in studying bacterial genomes. Libraries that approach saturation must be large, with multiple insertions per gene, making comprehensive genome-wide scanning difficult. To facilitate genome-scale study of the opportunistic human pathogen Pseudomonas aeruginosa strain PA14, we constructed a nonredundant library of PA14 transposon mutants (the PA14NR Set) in which nonessential PA14 genes are represented by a single transposon insertion chosen from a comprehensive library of insertion mutants. The parental library of PA14 transposon insertion mutants was generated by using MAR2xT7, a transposon compatible with transposon-site hybridization and based on mariner. The transposon-site hybridization genetic footprinting feature broadens the utility of the library by allowing pooled MAR2xT7 mutants to be individually tracked under different experimental conditions. A public, internet-accessible database (the PA14 Transposon Insertion Mutant Database, http://ausubellab.mgh.harvard.edu/cgi-bin/pa14/home.cgi) was developed to facilitate construction, distribution, and use of the PA14NR Set. The usefulness of the PA14NR Set in genome-wide scanning for phenotypic mutants was validated in a screen for attachment to abiotic surfaces. Comparison of the genes disrupted in the PA14 transposon insertion library with an independently constructed insertion library in P. aeruginosa strain PAO1 provides an estimate of the number of P. aeruginosa essential genes. PMID:16477005

  10. An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants.

    PubMed

    Liberati, Nicole T; Urbach, Jonathan M; Miyata, Sachiko; Lee, Daniel G; Drenkard, Eliana; Wu, Gang; Villanueva, Jacinto; Wei, Tao; Ausubel, Frederick M

    2006-02-21

    Random transposon insertion libraries have proven invaluable in studying bacterial genomes. Libraries that approach saturation must be large, with multiple insertions per gene, making comprehensive genome-wide scanning difficult. To facilitate genome-scale study of the opportunistic human pathogen Pseudomonas aeruginosa strain PA14, we constructed a nonredundant library of PA14 transposon mutants (the PA14NR Set) in which nonessential PA14 genes are represented by a single transposon insertion chosen from a comprehensive library of insertion mutants. The parental library of PA14 transposon insertion mutants was generated by using MAR2xT7, a transposon compatible with transposon-site hybridization and based on mariner. The transposon-site hybridization genetic footprinting feature broadens the utility of the library by allowing pooled MAR2xT7 mutants to be individually tracked under different experimental conditions. A public, internet-accessible database (the PA14 Transposon Insertion Mutant Database, http://ausubellab.mgh.harvard.edu/cgi-bin/pa14/home.cgi) was developed to facilitate construction, distribution, and use of the PA14NR Set. The usefulness of the PA14NR Set in genome-wide scanning for phenotypic mutants was validated in a screen for attachment to abiotic surfaces. Comparison of the genes disrupted in the PA14 transposon insertion library with an independently constructed insertion library in P. aeruginosa strain PAO1 provides an estimate of the number of P. aeruginosa essential genes.

  11. 129-Derived Mouse Strains Express an Unstable but Catalytically Active DNA Polymerase Iota Variant.

    PubMed

    Aoufouchi, Said; De Smet, Annie; Delbos, Frédéric; Gelot, Camille; Guerrera, Ida Chiara; Weill, Jean-Claude; Reynaud, Claude-Agnès

    2015-09-01

    Mice derived from the 129 strain have a nonsense codon mutation in exon 2 of the polymerase iota (Polι) gene and are therefore considered Polι deficient. When we amplified Polι mRNA from 129/SvJ or 129/Ola testes, only a small fraction of the full-length cDNA contained the nonsense mutation; the major fraction corresponded to a variant Polι isoform lacking exon 2. Polι mRNA lacking exon 2 contains an open reading frame, and the corresponding protein was detected using a polyclonal antibody raised against the C terminus of the murine Polι protein. The identity of the corresponding protein was further confirmed by mass spectrometry. Although the variant protein was expressed at only 5 to 10% of the level of wild-type Polι, it retained de novo DNA synthesis activity, the capacity to form replication foci following UV irradiation, and the ability to rescue UV light sensitivity in Polι(-/-) embryonic fibroblasts derived from a new, fully deficient Polι knockout (KO) mouse line. Furthermore, in vivo treatment of 129-derived male mice with Velcade, a drug that inhibits proteasome function, stabilized and restored a substantial amount of the variant Polι in these animals, indicating that its turnover is controlled by the proteasome. An analysis of two xeroderma pigmentosum-variant (XPV) cases corresponding to missense mutants of Polη, a related translesion synthesis (TLS) polymerase in the same family, similarly showed a destabilization of the catalytically active mutant protein by the proteasome. Collectively, these data challenge the prevailing hypothesis that 129-derived strains of mice are completely deficient in Polι activity. The data also document, both for 129-derived mouse strains and for some XPV patients, new cases of genetic defects corresponding to the destabilization of an otherwise functional protein, the phenotype of which is reversible by proteasome inhibition.

  12. Bone defects and future regenerative nanomedicine approach using stem cells in the mutant Tabby mouse model.

    PubMed

    Noordijk, Mélanie; Davideau, Jean-Luc; Eap, Sandy; Huck, Olivier; Fioretti, Florence; Stoltz, Jean-François; Bacon, William; Benkirane-Jessel, Nadia; Clauss, François

    2015-01-01

    X-linked Hypohidrotic Ectodermal Dysplasia (XLHED) is associated to a large spectrum of ectodermal and extra-ectodermal symptoms, especially craniofacial bone morphological, structural and metabolic anomalies. This skeletal phenotype described in affected patients and in the Ta mutant mouse model leads to craniofacial dysmorphies, endosseous implants and jaw bone grafts complications. Bone tissue bioengineering based on the use of PCL synthetic nanofibrous membrane and BMP nanoreservoirs appears as an original and promising approach to prevent such complications in the context of dysfunctional bone. Use of osteoblasts or stem cells seeded biomembranes appears as another strategy developed on the Tabby (Ta) model of XLHED. The Ta mouse experimental model is used to study the jaw bone response during the post-operative period after bone lesion and placement of synthetic PCL membrane functionalized with nanoreservoirs embedding different BMPs dimers or seeded with living cells. PMID:25538062

  13. A diastrophic dysplasia sulfate transporter (SLC26A2) mutant mouse: morphological and biochemical characterization of the resulting chondrodysplasia phenotype.

    PubMed

    Forlino, Antonella; Piazza, Rocco; Tiveron, Cecilia; Della Torre, Sara; Tatangelo, Laura; Bonafè, Luisa; Gualeni, Benedetta; Romano, Assunta; Pecora, Fabio; Superti-Furga, Andrea; Cetta, Giuseppe; Rossi, Antonio

    2005-03-15

    Mutations in the diastrophic dysplasia sulfate transporter (DTDST or SLC26A2) cause a family of recessively inherited chondrodysplasias including, in order of decreasing severity, achondrogenesis 1B, atelosteogenesis 2, diastrophic dysplasia (DTD) and recessive multiple epiphyseal dysplasia. The gene encodes a widely distributed sulfate/chloride antiporter of the cell membrane whose function is crucial for the uptake of inorganic sulfate, which is needed for proteoglycan sulfation. To provide new insights in the pathogenetic mechanisms leading to skeletal and connective tissue dysplasia and to obtain an in vivo model for therapeutic approaches to DTD, we generated a Dtdst knock-in mouse with a partial loss of function of the sulfate transporter. In addition, the intronic neomycine cassette in the mutant allele contributed to the hypomorphic phenotype by inducing abnormal splicing. Homozygous mutant mice were characterized by growth retardation, skeletal dysplasia and joint contractures, thereby recapitulating essential aspects of the DTD phenotype in man. The skeletal phenotype included reduced toluidine blue staining of cartilage, chondrocytes of irregular size, delay in the formation of the secondary ossification center and osteoporosis of long bones. Impaired sulfate uptake was demonstrated in chondrocytes, osteoblasts and fibroblasts. In spite of the generalized nature of the sulfate uptake defect, significant proteoglycan undersulfation was detected only in cartilage. Chondrocyte proliferation and apoptosis studies suggested that reduced proliferation and/or lack of terminal chondrocyte differentiation might contribute to reduced bone growth. The similarity with human DTD makes this mouse strain a useful model to explore pathogenetic and therapeutic aspects of DTDST-related disorders. PMID:15703192

  14. Derepression of colicin E1 synthesis in the constitutive tif mutant strain (spr tif sfi) and in a tif sfi mutant strain of Escherichia coli K-12.

    PubMed Central

    Tessman, E S; Gritzmacher, C A; Peterson, P K

    1978-01-01

    We show here that expression of the colicin gene of the ColE1 plasmid is greatly derepressed in Escherichia coli K-12 strain DM1187 spr tif sfi, which is a constitutive tif mutant, altered in the lexA gene, and which shows constitutive expression of various pathways of the recA-dependent, lexA-blocked (SOS) repair system. In this strain colicin E1 synthesis is at least 100-fold greater than that observed in uninduced control strains (spr+ tif sfi and spr+ tif+ sfi). This result confirms the regulatory role of the lexA product in colicin E1 synthesis. Colicin yields by the uninduced strain DM1187 are as high as the maximum yields from mitomycin-induced control strains and often are several-fold higher. When the nonconstitutive tif sfi strain GC467 is raised to 43 degrees C to induce the SOS system, a low level of colicin synthesis is observed which is less than one-tenth of the yield obtained by induction with mitomycin C. Addition of adenine at the time of shift-up can increase the colicin yield of tif sfi to about one-third of the yield obtained with mitomycin C. We have also found that colicin overproduction can be detected by altered colony appearance in an overlay assay with colicin-sensitive bacteria. In addition, the lethality of the process of colicin synthesis is observed here without the use of bacteriostatic inducing agents. Images PMID:353034

  15. Autosomal dominant frontonasal dysplasia (atypical Greig syndrome): Lessons from the Xt mutant mouse

    SciTech Connect

    Cunningham, M.L.; Nunes, M.E.

    1994-09-01

    Greig syndrome is the autosomal dominant association of mild hypertelorism, variable polysyndactyly, and normal intelligence. Several families have been found to have translocations or deletions of 7p13 interrupting the normal expression of GLI3 (a zinc finger, DNA binding, transcription repressor). Recently, a mutation in the mouse homologue of GLI3 was found in the extra-toes mutant mouse (Xt). The phenotypic features of this mouse model include mild hypertelorism, postaxial polydactyly of the forelimbs, preaxial polydactyly of the hindlimbs, and variable tibial hemimelia. The homozygous mutant Xt/Xt have severe frontonasal dysplasia (FND), polysyndactyly of fore-and hindlimbs and invariable tibial hemimelia. We have recently evaluated a child with severe (type D) frontonasal dysplasia, fifth finger camptodactyly, preaxial polydactyly of one foot, and ispilateral tibial hemimelia. His father was born with a bifid nose, broad columnella, broad feet, and a two centimeter leg length discrepancy. The paternal grandmother of the proband is phenotypically normal; however, her fraternal twin died at birth with severe facial anomalies. The paternal great-grandmother of the proband is phenotypically normal however her niece was born with moderate ocular hypertelorism. This pedigree is suggestive of an autosomal dominant form of frontonasal dysplasia with variable expressivity. The phenotypic features of our case more closely resemble the Xt mouse than the previously defined features of Greig syndrome in humans. This suggests that a mutation in GLI3 may be responsible for FND in this family. We are currently using polymorphic dinucleotide repeat markers flanking GLI3 in a attempt to demonstrate linkage in this pedigree. Demonstration of a GLI3 mutation in this family would broaden our view of the spectrum of phenotypes possible in Greig syndrome and could provide insight into genotype/phenotype correlation in FND.

  16. Neuregulin 1 Expression and Electrophysiological Abnormalities in the Neuregulin 1 Transmembrane Domain Heterozygous Mutant Mouse

    PubMed Central

    Frank, Elisabeth; Shaw, Alex; Liu, Shijie; Huang, Xu-Feng; Pinault, Didier; Karl, Tim; O’Brien, Terence J.; Shannon Weickert, Cynthia; Jones, Nigel C.

    2015-01-01

    Background The Neuregulin 1 transmembrane domain heterozygous mutant (Nrg1 TM HET) mouse is used to investigate the role of Nrg1 in brain function and schizophrenia-like behavioural phenotypes. However, the molecular alterations in brain Nrg1 expression that underpin the behavioural observations have been assumed, but not directly determined. Here we comprehensively characterise mRNA Nrg1 transcripts throughout development of the Nrg1 TM HET mouse. In addition, we investigate the regulation of high-frequency (gamma) electrophysiological oscillations in this mutant mouse to associate molecular changes in Nrg1 with a schizophrenia-relevant neurophysiological profile. Methods Using exonic probes spanning the cysteine-rich, epidermal growth factor (EGF)-like, transmembrane and intracellular domain encoding regions of Nrg1, mRNA levels were measured using qPCR in hippocampus and frontal cortex from male and female Nrg1 TM HET and wild type-like (WT) mice throughout development. We also performed electrophysiological recordings in adult mice and analysed gamma oscillatory at baseline, in responses to auditory stimuli and to ketamine. Results In both hippocampus and cortex, Nrg1 TM HET mice show significantly reduced expression of the exon encoding the transmembrane domain of Nrg1 compared with WT, but unaltered mRNA expression encoding the extracellular bioactive EGF-like and the cysteine-rich (type III) domains, and development-specific and region-specific reductions in the mRNA encoding the intracellular domain. Hippocampal Nrg1 protein expression was not altered, but NMDA receptor NR2B subunit phosphorylation was lower in Nrg1 TM HET mice. We identified elevated ongoing and reduced sensory-evoked gamma power in Nrg1 TM HET mice. Interpretation We found no evidence to support the claim that the Nrg1 TM HET mouse represents a simple haploinsufficient model. Further research is required to explore the possibility that mutation results in a gain of Nrg1 function. PMID

  17. Mechanical properties of elytra from Tribolium castaneum wild-type and body color mutant strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cuticle tanning in insects involves simultaneous cuticular hardening and pigmentation. The dynamic mechanical properties of the highly modified and cuticle-rich forewings (elytra) from Tribolium castaneum (red flour beetle) body color mutant strains were investigated to determine the relationship b...

  18. Reselection of a Genomic Upstream Open Reading Frame in Mouse Hepatitis Coronavirus 5′-Untranslated-Region Mutants

    PubMed Central

    Wu, Hung-Yi; Guan, Bo-Jhih; Su, Yu-Pin; Fan, Yi-Hsin

    2014-01-01

    An AUG-initiated upstream open reading frame (uORF) encoding a potential polypeptide of 3 to 13 amino acids (aa) is found within the 5′ untranslated region (UTR) of >75% of coronavirus genomes based on 38 reference strains. Potential CUG-initiated uORFs are also found in many strains. The AUG-initiated uORF is presumably translated following genomic 5′-end cap-dependent ribosomal scanning, but its function is unknown. Here, in a reverse-genetics study with mouse hepatitis coronavirus, the following were observed. (i) When the uORF AUG-initiating codon was replaced with a UAG stop codon along with a U112A mutation to maintain a uORF-harboring stem-loop 4 structure, an unimpaired virus with wild-type (WT) growth kinetics was recovered. However, reversion was found at all mutated sites within five virus passages. (ii) When the uORF was fused with genomic (main) ORF1 by converting three in-frame stop codons to nonstop codons, a uORF-ORF1 fusion protein was made, and virus replicated at WT levels. However, a frameshifting G insertion at virus passage 7 established a slightly 5′-extended original uORF. (iii) When uAUG-eliminating deletions of 20, 30, or 51 nucleotides (nt) were made within stem-loop 4, viable but debilitated virus was recovered. However, a C80U mutation in the first mutant and an A77G mutation in the second appeared by passage 10, which generated alternate uORFs that correlated with restored WT growth kinetics. In vitro, the uORF-disrupting nondeletion mutants showed enhanced translation of the downstream ORF1 compared with the WT. These results together suggest that the uORF represses ORF1 translation yet plays a beneficial but nonessential role in coronavirus replication in cell culture. PMID:24173235

  19. Mutant strains of Pichia pastoris with enhanced secretion of recombinant proteins.

    PubMed

    Larsen, Sasha; Weaver, Jun; de Sa Campos, Katherine; Bulahan, Rhobe; Nguyen, Jackson; Grove, Heather; Huang, Amy; Low, Lauren; Tran, Namphuong; Gomez, Seth; Yau, Jennifer; Ilustrisimo, Thomas; Kawilarang, Jessica; Lau, Jonathan; Tranphung, Maivi; Chen, Irene; Tran, Christina; Fox, Marcia; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P

    2013-11-01

    Although Pichia pastoris is a popular protein expression system, it exhibits limitations in its ability to secrete heterologous proteins. Therefore, a REMI (restriction enzyme mediated insertion) strategy was utilized to select mutant beta-g alactosidase s upersecretion (bgs) strains that secreted increased levels of a β-galactosidase reporter. Many of the twelve BGS genes may have functions in intracellular signaling or vesicle transport. Several of these strains also appeared to contain a more permeable cell wall. Preliminary characterization of four bgs mutants showed that they differed in the ability to enhance the export of other reporter proteins. bgs13, which has a disruption in a gene homologous to Saccharomyces cerevisiae protein kinase C (PKC1), gave enhanced secretion of most recombinant proteins that were tested, raising the possibility that it has the universal super-secreter phenotype needed in an industrial production strain of P. pastoris. PMID:23881328

  20. Construction and characterization of a H19 epitope point mutant of MDV CVI988/Rispens strain.

    PubMed

    Cui, Z; Qin, A; Lee, L F; Wu, P; Kung, H J

    1999-01-01

    A recombinant virus, CVI/rpp38, was developed from the Marek's disease virus (MDV) CVI988/Rispens vaccine strain. This recombinant was obtained by transfection of CVI988/Rispens-infected chick embryo fibroblasts (CEFs) with plasmid pHA25 DNA containing pp38 gene from GA strain of MDV. Monoclonal antibody (MAb) H19 which reacts with pp38 from GA but not with that from CVI988 was used to screen for recombinant viruses in transfected cell culture plates by immunofluorescent assay (IFA). A positive plaque was isolated, propagated, and purified from cell-free virus particles after sonication of infected CEFs. The mutant CVI/rpp38 was not only reactive with MAb H19 in IFA but also in immunoprecipitation. A 38 kDa protein was immunoprecipitated from the CVI/rpp38 mutant virus but not from parental CVI988 virus. DNA sequence of the mutant virus showed a substitution of G at position 320 by a resulting in a change of an amino acid residue from arginine to glutamine. Comparison of nucleotide sequence of pp38 from strains GA, Md5 and Md11/75c/R2 and CVI988 revealed change to glutamine in this position. The result of this study provides a direct evidence for the location of the identified H19 epitope in pp38. This mutant is potentially useful to further explore the biological function of pp38 and its H19 epitope.

  1. A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines

    PubMed Central

    Pasumarthi, Ravi K.; Baridon, Brian; Djan, Esi; Trainor, Amanda; Griffey, Stephen M.; Engelhard, Eric K.; Rapp, Jared; Li, Bowen; de Jong, Pieter J.; Lloyd, K.C. Kent

    2015-01-01

    Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, ∼80% of mutants showed specific staining in one or more tissues, while ∼20% showed no specific staining, ∼13% had staining in only one tissue, and ∼25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (∼50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had >90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known. PMID:25591789

  2. Identification of sonic hedgehog as a candidate gene responsible for the polydactylous mouse mutant Sasquatch.

    PubMed

    Sharpe, J; Lettice, L; Hecksher-Sorensen, J; Fox, M; Hill, R; Krumlauf, R

    1999-01-28

    The mouse mutants of the hemimelia-luxate group (lx, lu, lst, Dh, Xt, and the more recently identified Hx, Xpl and Rim4; [1] [2] [3] [4] [5]) have in common preaxial polydactyly and longbone abnormalities. Associated with the duplication of digits are changes in the regulation of development of the anterior limb bud resulting in ectopic expression of signalling components such as Sonic hedgehog (Shh) and fibroblast growth factor-4 (Fgf4), but little is known about the molecular causes of this misregulation. We generated, by a transgene insertion event, a new member of this group of mutants, Sasquatch (Ssq), which disrupted aspects of both anteroposterior (AP) and dorsoventral (DV) patterning. The mutant displayed preaxial polydactyly in the hindlimbs of heterozygous embryos, and in both hindlimbs and forelimbs of homozygotes. The Shh, Fgf4, Fgf8, Hoxd12 and Hoxd13 genes were all ectopically expressed in the anterior region of affected limb buds. The insertion site was found to lie close to the Shh locus. Furthermore, expression from the transgene reporter has come under the control of a regulatory element that directs a pattern mirroring the endogenous expression pattern of Shh in limbs. In abnormal limbs, both Shh and the reporter were ectopically induced in the anterior region, whereas in normal limbs the reporter and Shh were restricted to the zone of polarising activity (ZPA). These data strongly suggest that Ssq is caused by direct interference with the cis regulation of the Shh gene.

  3. Biotransformation of ethanol to acetaldehyde by wild and mutant strains of methylotrophic yeast

    SciTech Connect

    Moroz, O.M.; Sibirnyi, A.A.; Ksheminskaya, G.P. |

    1995-05-01

    The conversion of ethanol to acetaldehyde by intact cells of wild and mutant strains of methylotrophic yeast Hansenula polymorpha was studied. It was established that mutations that lower the activity of aldehyde reductase and acetaldehyde dehydrogenase stimulate acetaldehyde accumulation. The highest accumulation of acetaldehyde was found in a mutant that possessed increased alcohol oxidase activity in growth on a medium with glucose. A decrease in formaldehyde dehydrogenase did not stimulate acetaldehyde accumulation. Bioconversion of ethanol to acetaldehyde was most effective at lowered temperatures due to marked suppression of catabolic alcohol oxidase inactivation, but not to the activity of this enzyme under indicated conditions. 27 refs., 4 figs., 3 tabs.

  4. Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum

    SciTech Connect

    Shao, Xiongjun; Raman, Babu; Zhu, Mingjun; Mielenz, Jonathan R; Brown, Steven D; Guss, Adam M; Lynd, Lee R

    2011-01-01

    Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1-2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed.

  5. Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum

    SciTech Connect

    Lynd, Lee R; Shao, Xiongjun; Raman, Babu; Mielenz, Jonathan R; Brown, Steven D; Guss, Adam M; Zhu, Mingjun

    2011-01-01

    Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1 2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed.

  6. Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum.

    PubMed

    Shao, Xiongjun; Raman, Babu; Zhu, Mingjun; Mielenz, Jonathan R; Brown, Steven D; Guss, Adam M; Lynd, Lee R

    2011-11-01

    Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1-2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed. PMID:21874277

  7. Novel DNA Motif Binding Activity Observed In Vivo With an Estrogen Receptor α Mutant Mouse

    PubMed Central

    Li, Leping; Grimm, Sara A.; Winuthayanon, Wipawee; Hamilton, Katherine J.; Pockette, Brianna; Rubel, Cory A.; Pedersen, Lars C.; Fargo, David; Lanz, Rainer B.; DeMayo, Francesco J.; Schütz, Günther; Korach, Kenneth S.

    2014-01-01

    Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as “tethering.” Evidence for tethering is based on in vitro studies and a widely used “KIKO” mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the “EAAE” ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null–like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo. PMID:24713037

  8. A novel spontaneous mutation of BCAR3 results in extrusion cataracts in CF#1 mouse strain.

    PubMed

    Kondo, Tomohiro; Nakamori, Taketo; Nagai, Hiroaki; Takeshita, Ai; Kusakabe, Ken-Takeshi; Okada, Toshiya

    2016-10-01

    A substrain of mice originating from the CF#1 strain (an outbred colony) reared at Osaka Prefecture University (CF#1/lr mice) develops cataracts beginning at 4 weeks of age. Affected mice were fully viable and fertile and developed cataracts by 14 weeks of age. Histologically, CF#1/lr mice showed vacuolation of the lens cortex, swollen lens fibers, lens rupture and nuclear extrusion. To elucidate the mode of inheritance, we analyzed heterozygous mutant hybrids generated from CF#1/lr mice and wild-type BALB/c mice. None of the heterozygous mutants were affected, and the ratio of affected to unaffected mice was 1:3 among the offspring of the heterozygous mutants. For the initial genome-wide screening and further mapping, we used affected progeny of CF#1/lr × (CF#1/lr × BALB/c) mice. We concluded that the cataracts in CF#1/lr mice are inherited through an autosomal recessive mutation and that the mutant gene is located on mouse chromosome 3 between D3Mit79 and D3Mit216. In this region, we identified 8 genes associated with ocular disease. All 8 genes were sequenced and a novel point mutation (1 bp insertion of cytosine) in exon 7 of the Bcar3 gene was identified. This mutation produced a premature stop codon and a truncated protein. In conclusion, we have identified the first spontaneous mutation in the Bcar3 gene associated with lens extrusion cataracts. This novel cataract model may provide further knowledge of the molecular biology of cataractogenesis and the function of the BCAR3 protein. PMID:27364350

  9. Development of a Mutant Strain of Bacillus polymyxa Showing Enhanced Production of 2,3-Butanediol

    PubMed Central

    Mallonee, D. H.; Speckman, R. A.

    1988-01-01

    2,3-Butanediol is a feedstock chemical of potential industrial importance. It can serve as a monomer for many polymers of consumer interest that are currently supplied by the fossil fuel industry. Bacillus polymyxa can grow on inexpensive waste products of the food-processing industry and produce this glycol. This paper describes a mutant strain of B. polymyxa which displays constitutive production of catabolic α-acetolactate synthase, an enzyme in the 2,3-butanediol pathway which is normally produced only in the late log or stationary phase of growth. The mutant was obtained by treating the wild type with nitrosoguanidine and subjecting it to a penicillin counterselection procedure. One of the selected mutant strains produced four times as much of the glycol as the wild type and utilized approximately 25% of the energy source, compared with essentially complete utilization of the energy source by the wild type. Studies are under way to optimize the production of the glycol by the mutant. PMID:16347522

  10. Development of a mutant strain of Bacillus polymyxa showing enhanced production of 2,3-butanediol

    SciTech Connect

    Mallonee, D.H.; Speckman, R.A.

    1988-01-01

    2,3-Butanediol is a feedstock chemical of potential industrial importance. It can serve as a monomer for many polymers of consumer interest that are currently supplied by the fossil fuel industry. Bacillus polymyxa can grow on inexpensive waste products of the food-processing industry and produce this glycol. This paper describes a mutant strain of B. polymyxa which displays constitutive production of catabolic ..cap alpha..-acetolactate synthase, an enzyme in the 2,3-butanediol pathway which is normally produced only in the late log or stationary phase of growth. The mutant was obtained by treating the wild type with nitrosoguanidine and subjecting it to a penicillin counterselection procedure. One of the selected mutant strains produced four times as much of the glycol as the wild type and utilized approximately 25% of the energy source, compared with essentially complete utilization of the energy source by the wild type. Studies are under way to optimize the production of the glycol by the mutant.

  11. Deciphering the mechanisms of developmental disorders: phenotype analysis of embryos from mutant mouse lines

    PubMed Central

    Wilson, Robert; McGuire, Christina; Mohun, Timothy

    2016-01-01

    The Deciphering the Mechanisms of Developmental Disorders (DMDD) consortium is a research programme set up to identify genes in the mouse, which if mutated (or knocked-out) result in embryonic lethality when homozygous, and initiate the study of why disruption of their function has such profound effects on embryo development and survival. The project uses a combination of comprehensive high resolution 3D imaging and tissue histology to identify abnormalities in embryo and placental structures of embryonic lethal lines. The image data we have collected and the phenotypes scored are freely available through the project website (http://dmdd.org.uk). In this article we describe the web interface to the images that allows the embryo data to be viewed at full resolution in different planes, discuss how to search the database for a phenotype, and our approach to organising the data for an embryo and a mutant line so it is easy to comprehend and intuitive to navigate. PMID:26519470

  12. Deciphering the mechanisms of developmental disorders: phenotype analysis of embryos from mutant mouse lines.

    PubMed

    Wilson, Robert; McGuire, Christina; Mohun, Timothy

    2016-01-01

    The Deciphering the Mechanisms of Developmental Disorders (DMDD) consortium is a research programme set up to identify genes in the mouse, which if mutated (or knocked-out) result in embryonic lethality when homozygous, and initiate the study of why disruption of their function has such profound effects on embryo development and survival. The project uses a combination of comprehensive high resolution 3D imaging and tissue histology to identify abnormalities in embryo and placental structures of embryonic lethal lines. The image data we have collected and the phenotypes scored are freely available through the project website (http://dmdd.org.uk). In this article we describe the web interface to the images that allows the embryo data to be viewed at full resolution in different planes, discuss how to search the database for a phenotype, and our approach to organising the data for an embryo and a mutant line so it is easy to comprehend and intuitive to navigate.

  13. Pseudomonas mutant strains that accumulate androstane and seco-androstane intermediates from bile acids.

    PubMed Central

    Leppik, R A; Sinden, D J

    1987-01-01

    Transposon mutant strains which were affected in bile acid catabolism were isolated from four Pseudomonas spp. Two of the mutant groups isolated were found to accumulate 12 alpha-hydroxyandrosta-1,4-diene-3,17-dione as the major product from deoxycholic acid. Strains in one of these two groups were able to grow on steroids such as chenodeoxycholic acid, which lacks a 12 alpha-hydroxy function, whereas the one member of the second group could not. With chenodeoxycholic acid, this latter strain accumulated a yellow muconic-like derivative, tentatively identified as 3,7-dihydroxy-5,9,17-trioxo-4(5),9(10)-disecoandrosta-1(10)2 -dien-4-oic acid. Members of two further mutant groups accumulated either 12 beta-hydroxyandrosta-1,4-diene-3,17-dione or 3,12 beta-dihydroxy-9(10)-secoandrosta-1,3,5(10)-triene-9,17-dione as the major product from deoxycholic acid. The relationship between the catabolism of m- and p-cresol, 3-ethylphenol and the bile acids was also examined. PMID:3038076

  14. Guanabenz Treatment Accelerates Disease in a Mutant SOD1 Mouse Model of ALS.

    PubMed

    Vieira, Fernando G; Ping, Qinggong; Moreno, Andy J; Kidd, Joshua D; Thompson, Kenneth; Jiang, Bingbing; Lincecum, John M; Wang, Monica Z; De Zutter, Gerard S; Tassinari, Valerie R; Levine, Beth; Hatzipetros, Theo; Gill, Alan; Perrin, Steven

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor neurons. The mechanisms leading to motor neuron degeneration in ALS are unclear. However, there is evidence for involvement of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in ALS, notably in mutant SOD1 mediated models of ALS. Stress induced phosphorylation of the eIF2 alpha subunit by eukaryotic translation initiation factor 2-alpha kinase 3 Perk activates the UPR. Guanabenz is a centrally acting alpha2 adrenergic receptor agonist shown to interact with a regulatory subunit of the protein phosphatase, Pp1/Gadd34, and selectively disrupt the dephosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eif2alpha). Here we demonstrate that guanabenz is protective in fibroblasts expressing G93A mutant SOD1 when they are exposed to tunicamycin mediated ER stress. However, in contrast to other reports, guanabenz treatment accelerated ALS-like disease progression in a strain of mutant SOD1 transgenic ALS mice. This study highlights challenges of pharmacological interventions of cellular stress responses in whole animal models of ALS.

  15. Guanabenz Treatment Accelerates Disease in a Mutant SOD1 Mouse Model of ALS

    PubMed Central

    Vieira, Fernando G.; Ping, Qinggong; Moreno, Andy J.; Kidd, Joshua D.; Thompson, Kenneth; Jiang, Bingbing; Lincecum, John M.; Wang, Monica Z.; De Zutter, Gerard S.; Tassinari, Valerie R.; Levine, Beth; Hatzipetros, Theo; Gill, Alan; Perrin, Steven

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor neurons. The mechanisms leading to motor neuron degeneration in ALS are unclear. However, there is evidence for involvement of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in ALS, notably in mutant SOD1 mediated models of ALS. Stress induced phosphorylation of the eIF2 alpha subunit by eukaryotic translation initiation factor 2-alpha kinase 3 Perk activates the UPR. Guanabenz is a centrally acting alpha2 adrenergic receptor agonist shown to interact with a regulatory subunit of the protein phosphatase, Pp1/Gadd34, and selectively disrupt the dephosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eif2alpha). Here we demonstrate that guanabenz is protective in fibroblasts expressing G93A mutant SOD1 when they are exposed to tunicamycin mediated ER stress. However, in contrast to other reports, guanabenz treatment accelerated ALS-like disease progression in a strain of mutant SOD1 transgenic ALS mice. This study highlights challenges of pharmacological interventions of cellular stress responses in whole animal models of ALS. PMID:26288094

  16. A live attenuated strain of Yersinia pestis ΔyscB provides protection against bubonic and pneumonic plagues in mouse model.

    PubMed

    Zhang, Xuecan; Qi, Zhizhen; Du, Zongmin; Bi, Yujing; Zhang, Qingwen; Tan, Yafang; Yang, Huiying; Xin, Youquan; Yang, Ruifu; Wang, Xiaoyi

    2013-05-24

    To develop a safe and effective live plague vaccine, the ΔyscB mutant was constructed based on Yersinia pestis biovar Microtus strain 201 that is avirulent to humans, but virulent to mice. The virulence, immunogenicity and protective efficacy of the ΔyscB mutant were evaluated in this study. The results showed that the ΔyscB mutant was severely attenuated, elicited a higher F1-specific antibody titer and provided protective efficacy against bubonic and pneumonic plague in mouse model. The ΔyscB mutant could induce the secretion of both Th1-associated cytokines (IFN-γ, IL-2 and TNF-α) and Th2-associated cytokines (IL-4 and IL-10). Taken together, the ΔyscB mutant represented a potential vaccine candidate based on its ability to generate strong humoral and cell-mediated immune responses and to provide good protection against both subcutaneous and intranasal Y. pestis challenge.

  17. Characterization of a mutant strain of a filamentous fungus Cladosporium phlei for the mass production of the secondary metabolite phleichrome.

    PubMed

    Yi, Min-Hee; Kim, Jung-Ae; Kim, Jung-Mi; Park, Jin-Ah; Kim, Beom-Tae; Park, Seung-Moon; Yang, Moon-Sik; Hwang, Ki-Jun; Kim, Dae-Hyuk

    2011-08-01

    UV-mutagenesis was performed to obtain mutant strains that demonstrate altered production of phleichrome, a secondary metabolite of Cladosporium phlei. Among fifty mutants selected, based on the increased area and intensity of the purple pigment surrounding the colonies, the strain M0035 showed the highest production of phleichrome, more than seven fold over wild type. Plate cultures of the M0035 strain resulted in a total of 592 mg phleichrome consisting of 146 mg and 446 mg from the mycelia and agar media, respectively. The M0035 strain displayed a growth rate and a mycelial mass comparable to the parental strain but had significantly reduced asexual sporulation.

  18. Isolation and characterization of symbiotic mutants of bradyrhizobium sp. (Arachis) strain NC92: mutants with host-specific defects in nodulation and nitrogen fixation.

    PubMed Central

    Wilson, K J; Anjaiah, V; Nambiar, P T; Ausubel, F M

    1987-01-01

    Random transposon Tn5 mutagenesis of Bradyrhizobium sp. (Arachis) strain NC92, a member of the cowpea cross-inoculation group, was carried out, and kanamycin-resistant transconjugants were tested for their symbiotic phenotype on three host plants: groundnut, siratro, and pigeonpea. Two nodulation (Nod- phenotype) mutants were isolated. One is unable to nodulate all three hosts and appears to contain an insertion in one of the common nodulation genes (nodABCD); the other is a host-specific nodulation mutant that fails to nodulate pigeonpea, elicits uninvaded nodules on siratro, and elicits normal, nitrogen-fixing nodules on groundnut. In addition, nine mutants defective in nitrogen fixation (Fix- phenotype) were isolated. Three fail to supply symbiotically fixed nitrogen to all three host plants. Surprisingly, nodules elicited by one of these mutants exhibit high levels of acetylene reduction activity, demonstrating the presence of the enzyme nitrogenase. Three more mutants have partially effective phenotypes (Fix +/-) in symbiosis with all three host plants. The remaining three mutants fail to supply fixed nitrogen to one of the host plants tested while remaining partially or fully effective on the other two hosts; two of these mutants are Fix- in pigeonpea and Fix +/- on groundnut and on siratro, whereas the other one is Fix- on groundnut but Fix+ on siratro and on pigeonpea. These latter mutants also retain significant nodule acetylene reduction activity, even in the ineffective symbioses. Such bacterial host-specific fixation (Hsf) mutants have not previously been reported. Images PMID:3032910

  19. Identification of two new Pmp22 mouse mutants using large-scale mutagenesis and a novel rapid mapping strategy.

    PubMed

    Isaacs, A M; Davies, K E; Hunter, A J; Nolan, P M; Vizor, L; Peters, J; Gale, D G; Kelsell, D P; Latham, I D; Chase, J M; Fisher, E M; Bouzyk, M M; Potter, A; Masih, M; Walsh, F S; Sims, M A; Doncaster, K E; Parsons, C A; Martin, J; Brown, S D; Rastan, S; Spurr, N K; Gray, I C

    2000-07-22

    Mouse mutants have a key role in discerning mammalian gene function and modelling human disease; however, at present mutants exist for only 1-2% of all mouse genes. In order to address this phenotype gap, we have embarked on a genome-wide, phenotype-driven, large-scale N-ethyl-N--nitrosourea (ENU) mutagenesis screen for dominant mutations of clinical and pharmacological interest in the mouse. Here we describe the identification of two similar neurological phenotypes and determination of the underlying mutations using a novel rapid mapping strategy incorporating speed back-crosses and high throughput genotyping. Two mutant mice were identified with marked resting tremor and further characterized using the SHIRPA behavioural and functional assessment protocol. Back-cross animals were generated using in vitro fertilization and genome scans performed utilizing DNA pools derived from multiple mutant mice. Both mutants were mapped to a region on chromosome 11 containing the peripheral myelin protein 22 gene (Pmp22). Sequence analysis revealed novel point mutations in Pmp22 in both lines. The first mutation, H12R, alters the same amino acid as in the severe human peripheral neuropathy Dejerine Sottas syndrome and Y153TER in the other mutant truncates the Pmp22 protein by seven amino acids. Histological analysis of both lines revealed hypo-myelination of peripheral nerves. This is the first report of the generation of a clinically relevant neurological mutant and its rapid genetic characterization from a large-scale mutagenesis screen for dominant phenotypes in the mouse, and validates the use of large-scale screens to generate desired clinical phenotypes in mice. PMID:10915775

  20. Draft Genome Sequences for Clostridium thermocellum Wild-Type Strain YS and Derived Cellulose Adhesion-Defective Mutant Strain AD2

    SciTech Connect

    Brown, Steven D; Lamed, Raphael; Morag, Ely; Borovok, Ilya; Shoham, Yuval; Klingeman, Dawn Marie; Johnson, Courtney M; Yang, Zamin; Land, Miriam L; Utturkar, Sagar M; Keller, Martin; Bayer, Edward A

    2012-01-01

    Clostridium thermocellum wild-type strain YS is an anaerobic, thermophilic, cellulolytic bacterium capable of directly converting cellulosic substrates into ethanol. Strain YS and a derived cellulose adhesion-defective mutant strain AD2 played pivotal roles in describing the original cellulosome concept. We present their draft genome sequences.

  1. Mutation induction by different dose rates of gamma rays in radiation-sensitive mutants of mouse leukemia cells

    SciTech Connect

    Furuno-Fukushi, I.; Matsudaira, H. )

    1989-11-01

    Induction of cell killing and mutation to 6-thioguanine resistance was examined in a radiation-sensitive mutant strain LX830 of mouse leukemia cells following gamma irradiation at dose rates of 30 Gy/h (acute), 20 cGy/h (low dose rate), and 6.2 mGy/h (very low dose rate). LX830 cells were hypersensitive to killing by acute gamma rays. A slight but significant increase was observed in cell survival with decreasing dose rate down to 6.2 mGy/h, where the survival leveled off above certain total doses. The cells were also hypersensitive to mutation induction compared to the wild type. The mutation frequency increased linearly with increasing dose for all dose rates. No significant difference was observed in the frequency of induced mutations versus total dose at the three different dose rates so that the mutation frequency in LX830 cells at 6.2 mGy/h was not significantly different from that for moderate or acute irradiation.

  2. Lithium prevents parkinsonian behavioral and striatal phenotypes in an aged parkin mutant transgenic mouse model.

    PubMed

    Lieu, Christopher A; Dewey, Colleen M; Chinta, Shankar J; Rane, Anand; Rajagopalan, Subramanian; Batir, Sean; Kim, Yong-Hwan; Andersen, Julie K

    2014-12-01

    Lithium has long been used as a treatment for the psychiatric disease bipolar disorder. However, previous studies suggest that lithium provides neuroprotective effects in neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease. The exact mechanism by which lithium exerts these effects still remains unclear. In the present study, we evaluated the effects of low-dose lithium treatment in an aged mouse model expressing a parkin mutation within dopaminergic neurons. We found that low-dose lithium treatment prevented motor impairment as demonstrated by the open field test, pole test, and rearing behavior. Furthermore, lithium prevented dopaminergic striatal degeneration in parkin animals. We also found that parkin-induced striatal astrogliosis and microglial activation were prevented by lithium treatment. Our results further corroborate the use of this parkin mutant transgenic mouse line as a model for PD for testing novel therapeutics. The findings of the present study also provide further validation that lithium could be re-purposed as a therapy for PD and suggest that anti-inflammatory effects may contribute to its neuroprotective mechanisms.

  3. In vitro characterization of Salmonella typhi mutant strains for live oral vaccines.

    PubMed

    Dragunsky, E M; Rivera, E; Hochstein, H D; Levenbook, I S

    1990-06-01

    Several Salmonella typhi attenuated mutant strains, suggested as candidates for live oral vaccine, were examined for their characteristics in vitro in comparison with parental strains Ty2 and CDC10-80. Three methods were used: interaction of bacteria with the human monocyte-macrophage U937 cell line evaluated by microscopic examination, bacterial growth in the cell culture medium estimated by absorbance and bacterial resistance to human plasma assessed by the viable count technique. The most informative data were obtained in the test with U937 cells. Ty2 penetrated almost 100% of the cells, multiplied rapidly and caused death of the cells. CDC10-80 infected about 30% of the cells, multiplied slightly and did not kill the cells. The Ty2 mutant galE via EX462 behaved like CDC10-80. Bacteria of the galE Ty21a, Vi + Ty21a, 541 Ty and 543 Ty, found in only 3-4% of the cells, did not multiply within the cells and decreased in number with time. These findings correlate with the reported virulence of these strains for humans. With the second method, the rate of bacterial growth in cell culture medium did not differentiate Ty2, CDC10-80 and EX462. They grew at the same rate and faster than the remaining mutants. The plasma resistance test did not discriminate between EX462 and other mutants. These tests did not reveal any difference between Vi + Ty21a and Vi-Ty21a.

  4. A Small Indel Mutant Mouse Model of Epidermolytic Palmoplantar Keratoderma and Its Application to Mutant-specific shRNA Therapy.

    PubMed

    Lyu, Ya-Su; Shi, Pei-Liang; Chen, Xiao-Ling; Tang, Yue-Xiao; Wang, Yan-Fang; Liu, Rong-Rong; Luan, Xiao-Rui; Fang, Yu; Mei, Ru-Huan; Du, Zhen-Fang; Ke, Hai-Ping; Matro, Erik; Li, Ling-En; Lin, Zhao-Yu; Zhao, Jing; Gao, Xiang; Zhang, Xian-Ning

    2016-03-22

    Epidermolytic palmoplantar keratoderma (EPPK) is a relatively common autosomal-dominant skin disorder caused by mutations in the keratin 9 gene (KRT9), with few therapeutic options for the affected so far. Here, we report a knock-in transgenic mouse model that carried a small insertion-deletion (indel) mutant of Krt9, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), corresponding to the human mutation KRT9/c.500delAinsGGCT (p.Tyr167delinsTrpLeu), which resulted in a human EPPK-like phenotype in the weight-stress areas of the fore- and hind-paws of both Krt9(+/mut) and Krt9(mut/mut) mice. The phenotype confirmed that EPPK is a dominant-negative condition, such that mice heterozygotic for the K9-mutant allele (Krt9(+/mut)) showed a clear EPPK-like phenotype. Then, we developed a mutant-specific short hairpin RNA (shRNA) therapy for EPPK mice. Mutant-specific shRNAs were systematically identified in vitro using a luciferase reporter gene assay and delivered into Krt9(+/mut) mice. shRNA-mediated knockdown of mutant protein resulted in almost normal morphology and functions of the skin, whereas the same shRNA had a negligible effect in wild-type K9 mice. Our results suggest that EPPK can be treated by gene therapy, and this has significant implications for future clinical application.

  5. A Small Indel Mutant Mouse Model of Epidermolytic Palmoplantar Keratoderma and Its Application to Mutant-specific shRNA Therapy

    PubMed Central

    Lyu, Ya-Su; Shi, Pei-liang; Chen, Xiao-Ling; Tang, Yue-Xiao; Wang, Yan-Fang; Liu, Rong-Rong; Luan, Xiao-Rui; Fang, Yu; Mei, Ru-Huan; Du, Zhen-Fang; Ke, Hai-Ping; Matro, Erik; Li, Ling-En; Lin, Zhao-Yu; Zhao, Jing; Gao, Xiang; Zhang, Xian-Ning

    2016-01-01

    Epidermolytic palmoplantar keratoderma (EPPK) is a relatively common autosomal-dominant skin disorder caused by mutations in the keratin 9 gene (KRT9), with few therapeutic options for the affected so far. Here, we report a knock-in transgenic mouse model that carried a small insertion–deletion (indel) mutant of Krt9, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), corresponding to the human mutation KRT9/c.500delAinsGGCT (p.Tyr167delinsTrpLeu), which resulted in a human EPPK-like phenotype in the weight-stress areas of the fore- and hind-paws of both Krt9+/mut and Krt9mut/mut mice. The phenotype confirmed that EPPK is a dominant-negative condition, such that mice heterozygotic for the K9-mutant allele (Krt9+/mut) showed a clear EPPK-like phenotype. Then, we developed a mutant-specific short hairpin RNA (shRNA) therapy for EPPK mice. Mutant-specific shRNAs were systematically identified in vitro using a luciferase reporter gene assay and delivered into Krt9+/mut mice. shRNA-mediated knockdown of mutant protein resulted in almost normal morphology and functions of the skin, whereas the same shRNA had a negligible effect in wild-type K9 mice. Our results suggest that EPPK can be treated by gene therapy, and this has significant implications for future clinical application. PMID:27003758

  6. Asexual development is increased in Neurospora crassa cat-3-null mutant strains.

    PubMed

    Michán, Shaday; Lledías, Fernando; Hansberg, Wilhelm

    2003-08-01

    We use asexual development of Neurospora crassa as a model system with which to determine the causes of cell differentiation. Air exposure of a mycelial mat induces hyphal adhesion, and adherent hyphae grow aerial hyphae that, in turn, form conidia. Previous work indicated the development of a hyperoxidant state at the start of these morphogenetic transitions and a large increase in catalase activity during conidiation. Catalase 3 (CAT-3) increases at the end of exponential growth and is induced by different stress conditions. Here we analyzed the effects of cat-3-null strains on growth and asexual development. The lack of CAT-3 was not compensated by other catalases, even under oxidative stress conditions, and cat-3(RIP) colonies were sensitive to H(2)O(2), indicating that wild-type (Wt) resistance to external H(2)O(2) was due to CAT-3. cat-3(RIP) colonies grown in the dark produced high levels of carotenes as a consequence of oxidative stress. Light exacerbated oxidative stress and further increased carotene synthesis. In the cat-3(RIP) mutant strain, increased aeration in liquid cultures led to increased hyphal adhesion and protein oxidation. Compared to the Wt, the cat-3(RIP) mutant strain produced six times more aerial hyphae and conidia in air-exposed mycelial mats, as a result of longer and more densely packed aerial hyphae. Protein oxidation in colonies was threefold higher and showed more aerial hyphae and conidia in mutant strains than did the Wt. Results indicate that oxidative stress due to lack of CAT-3 induces carotene synthesis, hyphal adhesion, and more aerial hyphae and conidia.

  7. 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

  8. 129-Derived Mouse Strains Express an Unstable but Catalytically Active DNA Polymerase Iota Variant

    PubMed Central

    De Smet, Annie; Delbos, Frédéric; Gelot, Camille; Guerrera, Ida Chiara; Weill, Jean-Claude; Reynaud, Claude-Agnès

    2015-01-01

    Mice derived from the 129 strain have a nonsense codon mutation in exon 2 of the polymerase iota (Polι) gene and are therefore considered Polι deficient. When we amplified Polι mRNA from 129/SvJ or 129/Ola testes, only a small fraction of the full-length cDNA contained the nonsense mutation; the major fraction corresponded to a variant Polι isoform lacking exon 2. Polι mRNA lacking exon 2 contains an open reading frame, and the corresponding protein was detected using a polyclonal antibody raised against the C terminus of the murine Polι protein. The identity of the corresponding protein was further confirmed by mass spectrometry. Although the variant protein was expressed at only 5 to 10% of the level of wild-type Polι, it retained de novo DNA synthesis activity, the capacity to form replication foci following UV irradiation, and the ability to rescue UV light sensitivity in Polι−/− embryonic fibroblasts derived from a new, fully deficient Polι knockout (KO) mouse line. Furthermore, in vivo treatment of 129-derived male mice with Velcade, a drug that inhibits proteasome function, stabilized and restored a substantial amount of the variant Polι in these animals, indicating that its turnover is controlled by the proteasome. An analysis of two xeroderma pigmentosum-variant (XPV) cases corresponding to missense mutants of Polη, a related translesion synthesis (TLS) polymerase in the same family, similarly showed a destabilization of the catalytically active mutant protein by the proteasome. Collectively, these data challenge the prevailing hypothesis that 129-derived strains of mice are completely deficient in Polι activity. The data also document, both for 129-derived mouse strains and for some XPV patients, new cases of genetic defects corresponding to the destabilization of an otherwise functional protein, the phenotype of which is reversible by proteasome inhibition. PMID:26124279

  9. Cutaneous delayed hypersensitivity reactions of cattle vaccinated with mutant strains of Brucella abortus, using brucellins prepared from various brucellar strains.

    PubMed

    Cheville, N F; Jensen, A E; Morfitt, D C; Stabel, T J

    1994-09-01

    Cutaneous reactivity to brucellin was evaluated in 10-month-old heifers vaccinated with low-virulence mutant strains of Brucella abortus and was compared with brucellin reactions in postparturient cows with active brucellosis. In the cows, the cutaneous lesion was characterized microscopically as severe, acute, serofibrinous vasculitis; dermal lesions at 6, 12, 25, and 48 hours after brucellin injection consisted of endothelial activation and perivascular exudation that led to progressive accumulation of fibrin, monocytes, macrophages, and lymphocytes. In vaccinated heifers, cutaneous tests were done, using standard brucellin, brucellin prepared from strain RB51, and the purified brucellar proteins-31K and superoxide dismutase. Negative-control cattle given saline solution, did not have cutaneous reactions. Standard brucellin induced the most marked reactions in vaccinated heifers. Brucellin from rough strain RB51 caused positive reactions in heifers vaccinated with strain 19, but reactions were variable in other groups. Skin lesions induced by purified superoxide dismutase and 31-kd proteins in vaccinated cattle were not acceptable for diagnosis. Marked variability of test responses in vaccinated cattle precludes field use of this test to determine vaccination status.

  10. Regulation of allantoate transport in wild-type and mutant strains of Saccharomyces cerevisiae.

    PubMed Central

    Chisholm, V T; Lea, H Z; Rai, R; Cooper, T G

    1987-01-01

    Accumulation of intracellular allantoin and allantoate is mediated by two distinct active transport systems in Saccharomyces cerevisiae. Allantoin transport (DAL4 gene) is inducible, while allantoate uptake is constitutive (it occurs at full levels in the absence of any allantoate-related compounds from the culture medium). Both systems appear to be sensitive to nitrogen catabolite repression, feedback inhibition, and trans-inhibition. Mutants (dal5) that lack allantoate transport have been isolated. These strains also exhibit a 60% loss of allantoin transport capability. Conversely, dal4 mutants previously described are unable to transport allantoin and exhibit a 50% loss of allantoate transport. We interpret the pleiotropic behavior of the dal4 and dal5 mutations as deriving from a functional interaction between elements of the two transport systems. PMID:3549700

  11. Production and downstream processing of (1→3)-β-D-glucan from mutant strain of Agrobacterium sp. ATCC 31750

    PubMed Central

    2012-01-01

    We isolated a mutant that produced higher levels of curdlan than the wild strain Agrobacterium sp. ATCC 31750 by chemical mutagenesis using N-methyl-N-nitro-nitrosoguanidine. The mutant strain produced 66 g/L of curdlan in 120 h with a yield of (0.88) while, the wild strain produced 41 g/L in 120 h with a yield of (0.62) in a stirred bioreactor. The mutant could not produce curdlan when the pH was shifted from 7.0 to 5.5 after nitrogen depletion as followed for wild strain. In contrast, pH optimum for cell growth and curdlan production for mutant was found to be 7.0. We optimized the downstream processing of curdlan by varying different volumes of NaOH and HCl for extraction and precipitation of curdlan. The molecular weight of the purified curdlan from the wild and mutant strain was 6.6 × 105 Da and 5.8 × 105 Da respectively. The monosaccharide analyses confirm that curdlan from both wild and mutant strain contains only glucose units. From the NMR and FTIR data, it has been confirmed that curdlan was exclusively composed of β (1 → 3)-D-glucan residues. PMID:22681895

  12. HIF-VEGF pathways are critical for chronic otitis media in Junbo and Jeff mouse mutants.

    PubMed

    Cheeseman, Michael T; Tyrer, Hayley E; Williams, Debbie; Hough, Tertius A; Pathak, Paras; Romero, Maria R; Hilton, Helen; Bali, Sulzhan; Parker, Andrew; Vizor, Lucie; Purnell, Tom; Vowell, Kate; Wells, Sara; Bhutta, Mahmood F; Potter, Paul K; Brown, Steve D M

    2011-10-01

    Otitis media with effusion (OME) is the commonest cause of hearing loss in children, yet the underlying genetic pathways and mechanisms involved are incompletely understood. Ventilation of the middle ear with tympanostomy tubes is the commonest surgical procedure in children and the best treatment for chronic OME, but the mechanism by which they work remains uncertain. As hypoxia is a common feature of inflamed microenvironments, moderation of hypoxia may be a significant contributory mechanism. We have investigated the occurrence of hypoxia and hypoxia-inducible factor (HIF) mediated responses in Junbo and Jeff mouse mutant models, which develop spontaneous chronic otitis media. We found that Jeff and Junbo mice labeled in vivo with pimonidazole showed cellular hypoxia in inflammatory cells in the bulla lumen, and in Junbo the middle ear mucosa was also hypoxic. The bulla fluid inflammatory cell numbers were greater and the upregulation of inflammatory gene networks were more pronounced in Junbo than Jeff. Hif-1α gene expression was elevated in bulla fluid inflammatory cells, and there was upregulation of its target genes including Vegfa in Junbo and Jeff. We therefore investigated the effects in Junbo of small-molecule inhibitors of VEGFR signaling (PTK787, SU-11248, and BAY 43-9006) and destabilizing HIF by inhibiting its chaperone HSP90 with 17-DMAG. We found that both classes of inhibitor significantly reduced hearing loss and the occurrence of bulla fluid and that VEGFR inhibitors moderated angiogenesis and lymphangiogenesis in the inflamed middle ear mucosa. The effectiveness of HSP90 and VEGFR signaling inhibitors in suppressing OM in the Junbo model implicates HIF-mediated VEGF as playing a pivotal role in OM pathogenesis. Our analysis of the Junbo and Jeff mutants highlights the role of hypoxia and HIF-mediated pathways, and we conclude that targeting molecules in HIF-VEGF signaling pathways has therapeutic potential in the treatment of chronic OM. PMID

  13. Human-to-mouse prion-like propagation of mutant huntingtin protein.

    PubMed

    Jeon, Iksoo; Cicchetti, Francesca; Cisbani, Giulia; Lee, Suji; Li, Endan; Bae, Jiwoo; Lee, Nayeon; Li, Ling; Im, Wooseok; Kim, Manho; Kim, Hyun Sook; Oh, Seung-Hun; Kim, Tae-Aug; Ko, Jung Jae; Aubé, Benoit; Oueslati, Abid; Kim, Yun Joong; Song, Jihwan

    2016-10-01

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder of the central nervous system (CNS) that is defined by a CAG expansion in exon 1 of the huntingtin gene leading to the production of mutant huntingtin (mHtt). To date, the disease pathophysiology has been thought to be primarily driven by cell-autonomous mechanisms, but, here, we demonstrate that fibroblasts derived from HD patients carrying either 72, 143 and 180 CAG repeats as well as induced pluripotent stem cells (iPSCs) also characterized by 143 CAG repeats can transmit protein aggregates to genetically unrelated and healthy host tissue following implantation into the cerebral ventricles of neonatal mice in a non-cell-autonomous fashion. Transmitted mHtt aggregates gave rise to both motor and cognitive impairments, loss of striatal medium spiny neurons, increased inflammation and gliosis in associated brain regions, thereby recapitulating the behavioural and pathological phenotypes which characterizes HD. In addition, both in vitro work using co-cultures of mouse neural stem cells with 143 CAG fibroblasts and the SH-SY5Y human neuroblastoma cell line as well as in vivo experiments conducted in newborn wild-type mice suggest that exosomes can cargo mHtt between cells triggering the manifestation of HD-related behaviour and pathology. This is the first evidence of human-to-mouse prion-like propagation of mHtt in the mammalian brain; a finding which will help unravel the molecular bases of HD pathology as well as to lead to the development of a whole new range of therapies for neurodegenerative diseases of the CNS. PMID:27221146

  14. Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse.

    PubMed

    Hashimoto, Masakazu; Yamashita, Yukiko; Takemoto, Tatsuya

    2016-10-01

    The CRISPR/Cas9 system is a powerful tool for elucidating the roles of genes in a wide variety of organisms including mice. To obtain genetically modified embryos or mice by this method, Cas9 mRNA and sgRNA are usually introduced into zygotes by microinjection or electroporation. However, most mutants generated with this method are genetically mosaic, composed of several types of cells carrying different mutations, which complicates phenotype analysis in founder embryos or mice. To simplify the analysis and to elucidate the roles of genes involved in developmental processes, a method for producing non-mosaic mutants is needed. Here, we established a method for generating non-mosaic mouse mutant embryos. We introduced Cas9 protein and sgRNA into in vitro fertilized (IVF) zygotes by electroporation, which enabled the genome editing to occur before the first replication of the mouse genome. As a result, all of the cells in the mutant carried the same set of mutations. This method solves the problem of mosaicism/allele complexity in founder mutant embryos or mice generated by the CRIPSR/Cas9 system.

  15. CHLOROPLAST STRUCTURE AND FUNCTION IN ac-20, A MUTANT STRAIN OF CHLAMYDOMONAS REINHARDI

    PubMed Central

    Goodenough, Ursula W.; Levine, R. P.

    1970-01-01

    The fine structure of the ac-20 strain of Chlamydomonas reinhardi is described. Cells grown mixotrophically in the presence of acetate have a highly disordered chloroplast membrane organization and usually lack pyrenoids. Chloroplast ribosome levels are only 5–10% of wild-type levels. Cells grown phototrophically without acetate possess more chloroplast ribosomes and have more normal membrane and pyrenoid organization. Chloroplast ribosome levels rise rapidly when cells are transferred from acetate to minimal medium, whereas membrane reorganization occurs only after a lag. These results, combined with earlier studies of the photosynthetic properties of the mutant strain, suggest that proper membrane organization, Photosystem II activity, and ribulose-1,5-diphosphate carboxylase formation are dependent on the presence of chloroplast ribosomes. Other chloroplast components tested are unaffected by a 10-fold reduction in levels of chloroplast ribosomes. PMID:5415236

  16. Symbiotic effectiveness of antibiotic-resistant mutants of fast- and slow-growing strains of Rhizobium nodulating Lotus species.

    PubMed

    Pankhurst, C E

    1977-08-01

    Mutants resistant ot 16 individual antibiotics were isolated from two fast-growing and two slow-growing strains of Lotus rhizobia and their symbiotic effectiveness on Lotus pedunculatus evaluated. Resistance to streptomycin, spectinomycin, chloramphenicol, and tetracycline (inhibitors of protein synthesis) was associated with little or no loss of effectiveness with all four strains but resistance to nalidixic acid and rifampicin (inhibitors of nucleic acid synthesis), and to D-cycloserine, novobiocin, and penicillin (inhibitors of cell wall-cell membrane synthesis) was associated with significant loss of effectiveness in 20-100% of the mutants. Resistance to viomycin, neomycin, kanamycin, and vibramycin was associated with loss of effectiveness with mutants of the two fast-growing strains but not with mutants of the two slow-growing strains. When tested on four alternate host legumes individual mutants of a slow-growing strain showed significantly different levels of effectiveness. The results suggest that both the inherent characteristics of the bacterium and of the host plant will influence the symbiotic effectiveness of antibiotic-resistant mutants of Rhizobium. PMID:890601

  17. Juvenile manifestation of ultrasound communication deficits in the neuroligin-4 null mutant mouse model of autism.

    PubMed

    Ju, Anes; Hammerschmidt, Kurt; Tantra, Martesa; Krueger, Dilja; Brose, Nils; Ehrenreich, Hannelore

    2014-08-15

    Neuroligin-4 (Nlgn4) is a member of the neuroligin family of postsynaptic cell adhesion molecules. Loss-of-function mutations of NLGN4 are among the most frequent, known genetic causes of heritable autism. Adult Nlgn4 null mutant (Nlgn4(-/-)) mice are a construct valid model of human autism, with both genders displaying a remarkable autistic phenotype, including deficits in social interaction and communication as well as restricted and repetitive behaviors. In contrast to adults, autism-related abnormalities in neonatal and juvenile Nlgn4(-/-) mice have not been reported yet. The present study has been designed to systematically investigate in male and female Nlgn4(-/-) pups versus wildtype littermates (WT, Nlgn4(+/+)) developmental milestones and stimulus-induced ultrasound vocalization (USV). Neonatal development, followed daily from postnatal days (PND) 4 to 21, including physical development, neurological reflexes and neuromotor coordination, did not yield any differences between Nlgn4(-/-) and their WT littermates. USV in pups (PND8-9) in response to brief separation from their mothers revealed remarkable gender effects, and a genotype influence in females regarding latency to first call. In juveniles (PND22-23), USV monitoring upon exposure to an anesthetized female intruder mouse uncovered a clear genotype effect with reduced USV in Nlgn4(-/-) mice, and again a more prominent phenotype in females. Together, these data support an early manifestation of communication deficits in Nlgn4(-/-) mice that appear more pronounced in immature females with their overall stronger USV as compared to males.

  18. Low-proline environments impair growth, proline transport and in vivo survival of Staphylococcus aureus strain-specific putP mutants.

    PubMed

    Schwan, William R; Wetzel, Keith J; Gomez, Timothy S; Stiles, Melissa A; Beitlich, Brian D; Grunwald, Sandra

    2004-04-01

    Staphylococcus aureus is a common cause of disease in humans, particularly in hospitalized patients. This species needs to import several amino acids to survive, including proline. Previously, it was shown that an insertion mutation in the high-affinity proline uptake gene putP in strain RN6390 affected proline uptake by the bacteria as well as reducing their ability to survive in vivo. To further delineate the effect of the putP mutation on growth of S. aureus strain RN6390, a proline uptake assay that spanned less than 1 min was done to measure transport. An eightfold difference in proline levels was observed between the wild-type strain and the high-affinity proline transport mutant strain after 15 s, indicating that the defect was only in proline transport and not a combination of proline transport, metabolism and accumulation that would have been assessed with longer assays. A putP mutant of S. aureus strain RN4220 was then grown in minimal medium with different concentrations of proline. When compared to the wild-type strain, the putP mutant strain was significantly growth impaired when the level of proline was decreased to 1.74 microM. An assessment of proline concentrations in mouse livers and spleens showed proline concentrations of 7.5 micromol per spleen and 88.4 micromol per liver. To verify that the effects on proline transport and bacterial survival were indeed caused solely by a mutation in putP, the putP mutation was complemented by cloning a full-length putP gene on a plasmid that replicates in S. aureus. Complementation of the putP mutant strains restored proline transport, in vitro growth in low-proline medium, and in vivo survival within mice. These results show that the mutation in putP led to attenuated growth in low-proline media and by corollary low-proline murine organ tissues due to less efficient transport of proline into the bacteria.

  19. A murine model for type III tyrosinemia: lack of immunologically detectable 4-hydroxyphenylpyruvic acid dioxygenase enzyme protein in a novel mouse strain with hypertyrosinemia.

    PubMed

    Endo, F; Katoh, H; Yamamoto, S; Matsuda, I

    1991-04-01

    We have characterized a new mutant strain of mouse that has hypertyrosinemia. The blood tyrosine level was persistently high, and increased amounts of 4-hydroxyphenylpyruvic acid and its derivatives were excreted into the urine. Succinylacetone was not detected in urine samples from these mice. All the animals were apparently healthy, and there was no evidence of hepatorenal dysfunction. The hypertyrosinemia was transmitted through an autosomal recessive inheritance. Analyses of hepatic enzymes related to tyrosine metabolism revealed that 4-hydroxyphenylpyruvic acid dioxygenase activity was virtually absent, while fumarylacetoacetase and tyrosine aminotransferases (cytosolic and mitochondrial forms) were normal in these mutant mice. Immunoblot analysis of 4-hydroxyphenylpyruvic acid dioxygenase protein in the liver indicated that the subunit protein of the enzyme was absent. It would appear that hypertyrosinemia in this mutant strain was caused by a genetic defect in 4-hydroxyphenylpyruvic acid dioxygenase. These features are similar to type III tyrosinemia in humans. Analysis of this mutant strain of mouse is expected to provide valuable information on the pathogenesis of human type III tyrosinemia and can also serve as a useful system for studies on tyrosine metabolism.

  20. Systematic enhancement of polymerization of recombinant sickle hemoglobin mutants: implications for transgenic mouse model for sickle cell anemia.

    PubMed

    Li, X; Mirza, U A; Chait, B T; Manning, J M

    1997-12-01

    To provide quantitative information on the sites that promote polymerization of sickle hemoglobin (HbS) after formation of the initial hydrophobic bond involving Val-6(beta) [E6V(beta)] and also to provide hemoglobins with an enhanced polymerization that could be used in a mouse model for sickle cell anemia, we have expressed recombinant double, triple, and quadruple HbS mutants with substitutions on both the alpha- and beta-chains, E6V(beta)/E121R(beta), D75Y(alpha)/E6V(beta)/E121R(beta) and D6A(alpha)/D75Y(alpha)/E6V(beta)/E121R(beta). These recombinant hemoglobins were extensively characterized by high-performance liquid chromatography analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, amino acid analysis, and mass spectroscopy. They retained the functional properties of the Hb tetramer and polymerized in a linear manner at progressively lower Hb concentration as a function of the degree of substitution, suggesting that these remote sites (alphaD6A, alphaD75Y, and betaE121R) on the alpha- and beta-chains exhibit additive, enhanced polymerization properties. The quadruple mutant has a polymerization concentration close to that of the purified SAD hemoglobin from transgenic mouse red blood cells consisting of HbS, Hb Antilles, and Hb D-Punjab. Normal mouse Hb increases the polymerization concentration of each mutant. Thus, the general approach of using recombinant Hbs as described here should prove useful in elucidating the quantitative aspects of the mechanism of HbS polymerization and in identifying the contribution of individual sites to the overall process. The strategy described here demonstrates the feasibility of a systematic approach to achieve future recombinant HbS mutants that could provide a new generation of the transgenic mouse model for sickle cell anemia. PMID:9373274

  1. Activation of dormant bacterial genes by Nonomuraea sp. strain ATCC 39727 mutant-type RNA polymerase.

    PubMed

    Talà, Adelfia; Wang, Guojun; Zemanova, Martina; Okamoto, Susumu; Ochi, Kozo; Alifano, Pietro

    2009-02-01

    There is accumulating evidence that the ability of actinomycetes to produce antibiotics and other bioactive secondary metabolites has been underestimated due to the presence of cryptic gene clusters. The activation of dormant genes is therefore one of the most important areas of experimental research for the discovery of drugs in these organisms. The recent observation that several actinomycetes possess two RNA polymerase beta-chain genes (rpoB) has opened up the possibility, explored in this study, of developing a new strategy to activate dormant gene expression in bacteria. Two rpoB paralogs, rpoB(S) and rpoB(R), provide Nonomuraea sp. strain ATCC 39727 with two functionally distinct and developmentally regulated RNA polymerases. The product of rpoB(R), the expression of which increases after transition to stationary phase, is characterized by five amino acid substitutions located within or close to the so-called rifampin resistance clusters that play a key role in fundamental activities of RNA polymerase. Here, we report that rpoB(R) markedly activated antibiotic biosynthesis in the wild-type Streptomyces lividans strain 1326 and also in strain KO-421, a relaxed (rel) mutant unable to produce ppGpp. Site-directed mutagenesis demonstrated that the rpoB(R)-specific missense H426N mutation was essential for the activation of secondary metabolism. Our observations also indicated that mutant-type or duplicated, rpoB often exists in nature among rare actinomycetes and will thus provide a basis for further basic and applied research.

  2. Recovery of Nonpathogenic Mutant Bacteria from Tumors Caused by Several Agrobacterium tumefaciens Strains: a Frequent Event?▿

    PubMed Central

    Llop, Pablo; Murillo, Jesús; Lastra, Beatriz; López, María M.

    2009-01-01

    We have evaluated the interaction that bacterial genotypes and plant hosts have with the loss of pathogenicity in tumors, using seven Agrobacterium tumefaciens strains inoculated on 12 herbaceous and woody hosts. We performed a screening of the agrobacteria present inside the tumors, looking for nonpathogenic strains, and found a high variability of those strains in this niche. To verify the origin of the putative nonpathogenic mutant bacteria, we applied an efficient, reproducible, and specific randomly amplified polymorphic DNA analysis method. In contrast with previous studies, we recovered a very small percentage (0.01%) of nonpathogenic strains that can be considered true mutants. Of 5,419 agrobacterial isolates examined, 662 were nonpathogenic in tomato, although only 7 (from pepper and tomato tumors induced by two A. tumefaciens strains) could be considered to derive from the inoculated strain. Six mutants were affected in the transferred DNA (T-DNA) region; one of them contained IS426 inserted into the iaaM gene, whereas the whole T-DNA region was apparently deleted in three other mutants, and the virulence of the remaining two mutants was fully restored with the T-DNA genes as well. The plasmid profile was altered in six of the mutants, with changes in the size of the Ti plasmid or other plasmids and/or the acquisition of new plasmids. Our results also suggest that the frequent occurrence of nonpathogenic clones in the tumors is probably due to the preferential growth of nonpathogenic agrobacteria, of either endophytic or environmental origin, but different from the bacterial strain inducing the tumor. PMID:19700547

  3. Identification of sdiA-regulated genes in a mouse commensal strain of Enterobacter cloacae

    PubMed Central

    Sabag-Daigle, Anice; Dyszel, Jessica L.; Gonzalez, Juan F.; Ali, Mohamed M.; Ahmer, Brian M. M.

    2015-01-01

    Many bacteria determine their population density using quorum sensing. The most intensively studied mechanism of quorum sensing utilizes proteins of the LuxI family to synthesize a signaling molecule of the acylhomoserine lactone (AHL) type, and a protein of the LuxR family to bind AHL and regulate transcription. Genes regulated by quorum sensing often encode functions that are most effective when a group of bacteria are working cooperatively (e.g., luminescence, biofilm formation, host interactions). Bacteria in the Escherichia, Salmonella, Klebsiella, and Enterobacter genera do not encode an AHL synthase but they do encode an AHL receptor of the LuxR family, SdiA. Instead of detecting their own AHL synthesis, these organisms use SdiA to detect the AHLs synthesized by other bacterial species. In this study, we used a genetic screen to identify AHL-responsive genes in a commensal Enterobacter cloacae strain that was isolated from a laboratory mouse. The genes include a putative type VI secretion system, copA (a copper transporter), and fepE (extends O-antigen chain length). A new transposon mutagenesis strategy and suicide vectors were used to construct an sdiA mutant of E. cloacae. The AHL-responsiveness of all fusions was entirely sdiA-dependent, although some genes were regulated by sdiA in the absence of AHL. PMID:26075189

  4. Synechococcus sp. strain PCC 7002 nifJ mutant lacking pyruvate:ferredoxin oxidoreductase.

    PubMed

    McNeely, Kelsey; Xu, Yu; Ananyev, Gennady; Bennette, Nicholas; Bryant, Donald A; Dismukes, G Charles

    2011-04-01

    The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. strain PCC 7002. Remarkably, the photoautotrophic growth rate of this mutant increased by 20% relative to the wild-type (WT) rate under conditions of light-dark cycling. This result is attributed to an increase in the quantum yield of photosystem II (PSII) charge separation as measured by photosynthetic electron turnover efficiency determined using fast-repetition-rate fluorometry (F(v)/F(m)). During autofermentation, the excretion of acetate and lactate products by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT cells, H(2) production in vivo was 1.3-fold lower than the WT level. Inhibition of acetate-CoA ligase and pyruvate dehydrogenase complex by glycerol eliminated acetate production, with a resulting loss of reductant and a 3-fold decrease in H(2) production by nifJ cells compared to WT cells. Continuous electrochemical detection of dissolved H(2) revealed two temporally resolved phases of H(2) production during autofermentation, a minor first phase and a major second phase. The first phase was attributed to reduction of ferredoxin, because its level decreased 2-fold in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD(+) ratio revealed that the reductant generated by PFOR contributing to the first phase of H(2) production was not in equilibrium with bulk NADH/NAD(+) and that the second phase corresponded to the equilibrium NADH-mediated process. PMID:21317262

  5. Biosynthesis of a substituted cellulose from a mutant strain of Xanthomonas campestris.

    PubMed

    Vojnov, Adrián A; Bassi, Daniel E; Daniels, Michael J; Dankert, Marcelo A

    2002-02-18

    In Xanthomonas campestris the genes involved in polysaccharide (xanthan) biosynthesis are located in a gene cluster (gum) of 16 kb. A Tn5 insertion mutant with a reduced slimy phenotype has been characterized. This mutant failed to produce the pentasaccharide repeating-unit of xanthan. Only three sugars were transferred to the prenyl phosphate intermediate. Several lines of evidence suggested that the lipid-associated saccharide was the trisaccharide reducing end of the pentasaccharide from the wild-type strain. This trisaccharide was built up from UDP-Glc and GDP-Man, and a glucose residue was at the reducing end, linked to an allylic prenol through a diphosphate bridge. Results from one- or two-stage reactions showed that the trisaccharide-P-P-polyprenol was the precursor of the polymer. This new polymer, a polytrisaccharide, was detected also in vivo. The transposon responsible for the mutation was located within gumK gene. Therefore, this gene encodes for the glycosyltransferase IV, which catalyses the transfer of glucuronic acid to the lipid-linked beta-D-Manp-(1-->3)-beta-D-Glcp-(1-->4)-beta-D-Glcp trisaccharide. A recombinant plasmid with the whole gum cluster restored the wild type phenotype.

  6. Comparison of three methods of calculating strain in the mouse ulna in exogenous loading studies.

    PubMed

    Norman, Stephanie C; Wagner, David W; Beaupre, Gary S; Castillo, Alesha B

    2015-01-01

    Axial compression of mouse limbs is commonly used to induce bone formation in a controlled, non-invasive manner. Determination of peak strains caused by loading is central to interpreting results. Load-strain calibration is typically performed using uniaxial strain gauges attached to the diaphyseal, periosteal surface of a small number of sacrificed animals. Strain is measured as the limb is loaded to a range of physiological loads known to be anabolic to bone. The load-strain relationship determined by this subgroup is then extrapolated to a larger group of experimental mice. This method of strain calculation requires the challenging process of strain gauging very small bones which is subject to variability in placement of the strain gauge. We previously developed a method to estimate animal-specific periosteal strain during axial ulnar loading using an image-based computational approach that does not require strain gauges. The purpose of this study was to compare the relationship between load-induced bone formation rates and periosteal strain at ulnar midshaft using three different methods to estimate strain: (A) Nominal strain values based solely on load-strain calibration; (B) Strains calculated from load-strain calibration, but scaled for differences in mid-shaft cross-sectional geometry among animals; and (C) An alternative image-based computational method for calculating strains based on beam theory and animal-specific bone geometry. Our results show that the alternative method (C) provides comparable correlation between strain and bone formation rates in the mouse ulna relative to the strain gauge-dependent methods (A and B), while avoiding the need to use strain gauges. PMID:25443882

  7. Alteration of medial-edge epithelium cell adhesion in two Tgf-β3 null mouse strains

    PubMed Central

    Martínez-Sanz, Elena; Del Río, Aurora; Barrio, Carmen; Murillo, Jorge; Maldonado, Estela; Garcillán, Beatriz; Amorós, María; Fuerte, Tamara; Fernández, Álvaro; Trinidad, Eva; Rabadán, M Ángeles; López, Yamila; Martínez, M Luisa; Martínez-Álvarez, Concepción

    2008-01-01

    Although palatal shelf adhesion is a crucial event during palate development, little work has been carried out to determine which molecules are responsible for this process. Furthermore, whether altered palatal shelf adhesion causes the cleft palate presented by Tgf-β3 null mutant mice has not yet been clarified. Here, we study the presence/distribution of some extracellular matrix and cell adhesion molecules at the time of the contact of palatal shelves in both wild-type and Tgf-β3 null mutant palates of two strains of mice (C57/BL/6J (C57), and MF1) that develop cleft palates of different severity. We have performed immunohistochemistry with antibodies against collagens IV and IX, laminin, fibronectin, the α5- and β1-integrins, and ICAM-1; in situ hybridization with a Nectin-1 riboprobe; and palatal shelf cultures treated or untreated with TGF-β3 or neutralizing antibodies against fibronectin or the α5-integrin. Our results show the location of these molecules in the wild-type mouse medial edge epithelium (MEE) of both strains at the time of the contact of palatal shelves; the heavier (C57) and milder (MF1) alteration of their presence in the Tgf-β3 null mutants; the importance of TGF-β3 to restore their normal pattern of expression; and the crucial role of fibronectin and the α5-integrin in palatal shelf adhesion. We thus provide insight into the molecular bases of this important process and the cleft palate presented by Tgf-β3 null mutant mice. PMID:18431835

  8. Alteration of medial-edge epithelium cell adhesion in two Tgf-beta3 null mouse strains.

    PubMed

    Martínez-Sanz, Elena; Del Río, Aurora; Barrio, Carmen; Murillo, Jorge; Maldonado, Estela; Garcillán, Beatriz; Amorós, María; Fuerte, Tamara; Fernández, Alvaro; Trinidad, Eva; Rabadán, María Angeles; López, Yamila; Martínez, María Luisa; Martínez-Alvarez, Concepción

    2008-04-01

    Although palatal shelf adhesion is a crucial event during palate development, little work has been carried out to determine which molecules are responsible for this process. Furthermore, whether altered palatal shelf adhesion causes the cleft palate presented by Tgf-beta3 null mutant mice has not yet been clarified. Here, we study the presence/distribution of some extracellular matrix and cell adhesion molecules at the time of the contact of palatal shelves in both wild-type and Tgf-beta3 null mutant palates of two strains of mice (C57/BL/6J (C57), and MF1) that develop cleft palates of different severity. We have performed immunohistochemistry with antibodies against collagens IV and IX, laminin, fibronectin, the alpha5- and beta1-integrins, and ICAM-1; in situ hybridization with a Nectin-1 riboprobe; and palatal shelf cultures treated or untreated with TGF-beta3 or neutralizing antibodies against fibronectin or the alpha5-integrin. Our results show the location of these molecules in the wild-type mouse medial edge epithelium (MEE) of both strains at the time of the contact of palatal shelves; the heavier (C57) and milder (MF1) alteration of their presence in the Tgf-beta3 null mutants; the importance of TGF-beta3 to restore their normal pattern of expression; and the crucial role of fibronectin and the alpha5-integrin in palatal shelf adhesion. We thus provide insight into the molecular bases of this important process and the cleft palate presented by Tgf-beta3 null mutant mice.

  9. Degradation of p-chlorotoluene by a mutant of Pseudomonas sp. strain JS6

    SciTech Connect

    Haigler, B.E.; Spain, J.C. )

    1989-02-01

    Pseudomonas sp. strain JS6 grows on chlorobenzene, p-dichlorobenzene, or toluene as a sole source of carbon and energy. It does not grow on p-chlorotoluene (p-CT). Growth on glucose in the presence of p-CT resulted in the accumulation of 4-chloro-2,3- dihydroxy-1-methyl benzene (3-chloro-6- methylcatechol), 4-chloro- 2,3-dihydroxy-1-methyl cyclohexa- 4,6-diene (p-CT dihydrodoil), and 2-methyl-4-carboxy methylenebut-2-en-4-olide (2-methyl dienelactone). Strain JS21, a spontaneous mutant capable of growth on p-CT, was isolated from cultures of strain JS6 after extended exposure to p-CT. In addition to growing on p-CT, JS21 grew on all of the substrates that supported growth of the parent strain, including p-dichlorobenzene, chlorobenzene, benzene, toluene, benzoate, p-hydroxybenzoate, phenol, and ethylbenzene. The pathway for degradation of p-CT by JS21 was investigated by respirometry, isolation of intermediates, and assay of enzymes in cell extracts. p-CT was converted to 3-chloro-6-methylcatechol by dioxygenase and dihydrodiol dehydrogenase enzymes. 3-Chloro-6-methylcatechol underwent ortho ring cleavage catalyzed by a catechol 1,2-dioxygenase to form 2-chloro-5-methyl-cis,cis-muconate, which was converted to 2-methyl dienelactone. A dienelactone hydrolase converted 2-methyl dienelactone to 2-methylmaleylacetic acid. Preliminary results indicate that a change in wild-type induction patterns allows JS21 to grow on p-CT.

  10. Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant.

    PubMed

    Kramer, Annemarie; Beck, Hans Christian; Kumar, Abhishek; Kristensen, Lars Peter; Imhoff, Johannes F; Labes, Antje

    2015-01-01

    The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum. PMID:26460745

  11. Biotransformation of L-tyrosine to Dopamine by a Calcium Alginate Immobilized Mutant Strain of Aspergillus oryzae.

    PubMed

    Ali, Sikander; Nawaz, Wajeeha

    2016-08-01

    The present research work is concerned with the biotransformation of L-tyrosine to dopamine (DA) by calcium alginate entrapped conidiospores of a mutant strain of Aspergillus oryzae. Different strains of A. oryzae were isolated from soil. Out of 13 isolated strains, isolate-2 (I-2) was found to be a better DA producer. The wild-type I-2 was chemically improved by treating it with different concentrations of ethyl methyl sulfonate (EMS). Among seven mutant variants, EMS-6 exhibiting maximal DA activity of 43 μg/ml was selected. The strain was further exposed with L-cysteine HCl to make it resistant against diversion and environmental stress. The conidiospores of selected mutant variant A. oryzae EMS-6 strain were entrapped in calcium alginate beads. Different parameters for immobilization were investigated. The activity was further improved from 44 to 62 μg/ml under optimized conditions (1.5 % sodium alginate, 2 ml inoculum, and 2 mm bead size). The best resistant mutant variable exhibited over threefold increase in DA activity (62 μg/ml) than did wild-type I-2 (21 μg/ml) in the reaction mixture. From the results presented in the study, it was observed that high titers of DA activity in vitro could effectively be achieved by the EMS-induced mutagenesis of filamentous fungus culture used.

  12. Induced drought tolerance through wild and mutant bacterial strain Pseudomonas simiae in mung bean (Vigna radiata L.).

    PubMed

    Kumari, Sarita; Vaishnav, Anukool; Jain, Shekhar; Varma, Ajit; Choudhary, Devendra Kumar

    2016-01-01

    The present study focused on the overproducing mutant of a plant growth promoting rhizobacterium (PGPR) Pseudomonas simiae strain AU (MTCC-12057) for significant drought tolerance in mung bean plants. Five mutants namely AU-M1, AU-M2, AU-M3, AU-M4 and AU-M5 were made after treatment of wild type strain with N-methyl-N-nitro-N-nitrosoguanidine. Mutant strain AU-M4 was recorded for enhanced ACC deaminase (ACC-D) activity, indole acetic acid (IAA) production and inorganic phosphate (Pi) solubilization compared to wild strain and other four mutant strains under drought condition. AU-M4 showed higher phosphate solubilization index (8.17) together with higher ACC-D activity (98 nmol/mg/h) and IAA concentration (69.35 µg/ml) compared with the wild type P. simiae strain AU ACC-D activity (79 nmol/mg/h) and IAA concentration (38.98 µg/ml) respectively. In this report, we investigated the effect of both wild and mutant type bacterial strain on mung bean plants under drought stress. Results showed that mutant AU-M4 and wild type strain AU inoculated plants exhibited superior tolerance against drought stress, as shown by their enhanced plant biomass (fresh weight), higher water content, higher proline accumulation and lower osmotic stress injury. Mutant AU-M4 and wild strain AU inoculated plants reduced the ethylene level by 59 and 45% respectively, compared to the control under stress condition. Furthermore, bacterial inoculated plants showed enhanced induced systemic drought tolerance by reducing stomata size and net photosynthesis resulting higher water content in mung bean plants that may help in survival of plants during drought condition. To mitigate the effects of drought stress, use of PGPR will be needed to ensure sufficient production of food from crop plants. Taking current leads available, concerted future research is needed in this area, particularly on field evaluation with application of potential microorganisms.

  13. Induced drought tolerance through wild and mutant bacterial strain Pseudomonas simiae in mung bean (Vigna radiata L.).

    PubMed

    Kumari, Sarita; Vaishnav, Anukool; Jain, Shekhar; Varma, Ajit; Choudhary, Devendra Kumar

    2016-01-01

    The present study focused on the overproducing mutant of a plant growth promoting rhizobacterium (PGPR) Pseudomonas simiae strain AU (MTCC-12057) for significant drought tolerance in mung bean plants. Five mutants namely AU-M1, AU-M2, AU-M3, AU-M4 and AU-M5 were made after treatment of wild type strain with N-methyl-N-nitro-N-nitrosoguanidine. Mutant strain AU-M4 was recorded for enhanced ACC deaminase (ACC-D) activity, indole acetic acid (IAA) production and inorganic phosphate (Pi) solubilization compared to wild strain and other four mutant strains under drought condition. AU-M4 showed higher phosphate solubilization index (8.17) together with higher ACC-D activity (98 nmol/mg/h) and IAA concentration (69.35 µg/ml) compared with the wild type P. simiae strain AU ACC-D activity (79 nmol/mg/h) and IAA concentration (38.98 µg/ml) respectively. In this report, we investigated the effect of both wild and mutant type bacterial strain on mung bean plants under drought stress. Results showed that mutant AU-M4 and wild type strain AU inoculated plants exhibited superior tolerance against drought stress, as shown by their enhanced plant biomass (fresh weight), higher water content, higher proline accumulation and lower osmotic stress injury. Mutant AU-M4 and wild strain AU inoculated plants reduced the ethylene level by 59 and 45% respectively, compared to the control under stress condition. Furthermore, bacterial inoculated plants showed enhanced induced systemic drought tolerance by reducing stomata size and net photosynthesis resulting higher water content in mung bean plants that may help in survival of plants during drought condition. To mitigate the effects of drought stress, use of PGPR will be needed to ensure sufficient production of food from crop plants. Taking current leads available, concerted future research is needed in this area, particularly on field evaluation with application of potential microorganisms. PMID:26712619

  14. Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain.

    PubMed

    Okuda, Naoyuki; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Hoshino, Tamotsu

    2016-10-01

    We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression. PMID:27309759

  15. Isolation of homozygous mutant mouse embryonic stem cells using a dual selection system

    PubMed Central

    Huang, Yue; Pettitt, Stephen J.; Guo, Ge; Liu, Guang; Li, Meng Amy; Yang, Fengtang; Bradley, Allan

    2012-01-01

    Obtaining random homozygous mutants in mammalian cells for forward genetic studies has always been problematic due to the diploid genome. With one mutation per cell, only one allele of an autosomal gene can be disrupted, and the resulting heterozygous mutant is unlikely to display a phenotype. In cells with a genetic background deficient for the Bloom's syndrome helicase, such heterozygous mutants segregate homozygous daughter cells at a low frequency due to an elevated rate of crossover following mitotic recombination between homologous chromosomes. We constructed DNA vectors that are selectable based on their copy number and used these to isolate these rare homozygous mutant cells independent of their phenotype. We use the piggyBac transposon to limit the initial mutagenesis to one copy per cell, and select for cells that have increased the transposon copy number to two or more. This yields homozygous mutants with two allelic mutations, but also cells that have duplicated the mutant chromosome and become aneuploid during culture. On average, 26% of the copy number gain events occur by the mitotic recombination pathway. We obtained homozygous cells from 40% of the heterozygous mutants tested. This method can provide homozygous mammalian loss-of-function mutants for forward genetic applications. PMID:22127858

  16. Maltodextrin Acceptance and Preference in Eight Mouse Strains.

    PubMed

    Poole, Rachel L; Aleman, Tiffany R; Ellis, Hillary T; Tordoff, Michael G

    2016-01-01

    Rodents are strongly attracted to the taste(s) of maltodextrins. A first step toward discovery of the underlying genes involves identifying phenotypic differences among inbred strains of mice. To do this, we used 5-s brief-access tests and 48-h 2-bottle choice tests to survey the avidity for the maltodextrin, Maltrin M040, of mice from 8 inbred strains (129S1/SvImJ, A/J, CAST/EiJ, C57BL/6J, NOD/ShiLTJ, NZO/HlLtJ, PWK/PhJ, and WSB/EiJ). In brief-access tests, the CAST and PWK strains licked significantly less maltodextrin than equivalent concentrations of sucrose, whereas the other strains generally licked the 2 carbohydrates equally. Similarly, in 2-bottle choice tests, the CAST and PWK strains drank less 4% maltodextrin than 4% sucrose, whereas the other strains had similar intakes of these 2 solutions; the CAST and PWK strains did not differ from the C57, NOD, or NZO strains in 4% sucrose intake. In sum, we have identified strain variation in maltodextrin perception that is distinct from variation in sucrose perception. The phenotypic variation characterized here will aid in identifying genes responsible for maltodextrin acceptance. Our results identify C57 × PWK mice or NZO × CAST mice as informative crosses to produce segregating hybrids that will expose quantitative trait loci underlying maltodextrin acceptance and preference.

  17. The spontaneous ataxic mouse mutant tippy is characterized by a novel Purkinje cell morphogenesis and degeneration phenotype

    PubMed Central

    Shih, Evelyn K.; Sekerková, Gabriella; Ohtsuki, Gen; Aldinger, Kimberly A.; Chizhikov, Victor V.; Hansel, Christian; Mugnaini, Enrico; Millen, Kathleen J.

    2015-01-01

    This study represents the first detailed analysis of the spontaneous neurological mouse mutant, tippy, uncovering its unique cerebellar phenotype. Homozygous tippy mutant mice are small, ataxic and die around weaning. Although the cerebellum shows grossly normal foliation, tippy mutants display a complex cerebellar Purkinje cell phenotype consisting of abnormal dendritic branching with immature spine features and patchy, non-apoptotic cell death that is associated with widespread dystrophy and degeneration of the Purkinje cell axons throughout the white matter, the cerebellar nuclei and the vestibular nuclei. Moderate anatomical abnormalities of climbing fiber innervation of tippy mutant Purkinje cells were not associated with changes in climbing fiber-EPSC amplitudes. However, decreased ESPC amplitudes were observed in response to parallel fiber stimulation and correlated well with anatomical evidence for patchy dark cell degeneration of Purkinje cell dendrites in the molecular layer. The data suggest that the Purkinje neurons are a primary target of the tippy mutation. Furthermore, we hypothesize that the Purkinje cell axonal pathology together with disruptions in the balance of climbing fiber and parallel fiber Purkinje cell input in the cerebellar cortex underlie the ataxic phenotype in these mice. The constellation of Purkinje cell dendritic malformation and degeneration phenotypes in tippy mutants is unique and has not been reported in any other neurologic mutant. Fine mapping of the tippy mutation to a 2.1MB region of distal chromosome 9, which does not encompass any gene previously implicated in cerebellar development or neuronal degeneration, confirms that the tippy mutation identifies novel biology and gene function. PMID:25626522

  18. A short-filament mutant of Anabaena sp. strain PCC 7120 that fragments in nitrogen-deficient medium.

    PubMed Central

    Bauer, C C; Buikema, W J; Black, K; Haselkorn, R

    1995-01-01

    Strain 129 is a fragmentation mutant of the filamentous cyanobacterium Anabaena sp. strain PCC 7120. Growing with fixed nitrogen, this mutant forms filaments that are much shorter than wild-type filaments. Following starvation for fixed nitrogen, strain 129 becomes nearly unicellular and forms few heterocysts, although electron microscopy suggests that proheterocysts form while fragmentation occurs. Starvation for sulfate, phosphate, iron, and calcium does not cause this fragmentation. The affected gene in strain 129, fraC, was cloned by complementation and characterized. It encodes a unique 179-amino-acid protein rich in phenylalanine. Insertional inactivation of the chromosomal copy of fraC results in a phenotype identical to that of strain 129, while complementation using a truncated version of FraC results in only partial complementation of the original mutant. Heterocysts could be induced to form in N-replete cultures of strain 129, as in wild-type cells, by supplying extra copies of the hetR gene on a plasmid. Thus, FraC is required for the integrity of cell junctions in general but is apparently not directly involved in normal differentiation and nitrogen fixation. PMID:7883709

  19. Expression of chicken vinculin complements the adhesion-defective phenotype of a mutant mouse F9 embryonal carcinoma cell

    PubMed Central

    1993-01-01

    A mutant cell line, derived from the mouse embryonal carcinoma cell line F9, is defective in cell-cell adhesion (compaction) and in cell- substrate adhesion. We have previously shown that neither uvomorulin (E- cadherin) nor integrins are responsible for the mutant phenotype (Calogero, A., M. Samuels, T. Darland, S. A. Edwards, R. Kemler, and E. D. Adamson. 1991. Dev. Biol. 146:499-508). Several cytoskeleton proteins were assayed and only vinculin was found to be absent in mutant (5.51) cells. A chicken vinculin expression vector was transfected into the 5.51 cells together with a neomycin-resistance vector. Clones that were adherent to the substrate were selected in medium containing G418. Two clones, 5.51Vin3 and Vin4, were analyzed by Nomarski differential interference contrast and laser confocal microscopy as well as by biochemical and molecular biological techniques. Both clones adhered well to substrates and both exhibited F- actin stress fibers with vinculin localized at stress fiber tips in focal contacts. This was in marked contrast to 5.51 parental cells, which had no stress fibers and no vinculin. The mutant and complemented F9 cell lines will be useful models for examining the complex interactions between cytoskeletal and cell adhesion proteins. PMID:8491782

  20. Sensorimotor enhancement in mouse mutants lacking the Purkinje cell-specific Gi/o modulator, Pcp2(L7)

    PubMed Central

    Iscru, Emilia; Serinagaoglu, Yelda; Schilling, Karl; Tian, Jinbin; Bowers-Kidder, Stephanie L.; Zhang, Rui; Morgan, James I.; DeVries, A. Courtney; Nelson, Randy J.; Zhu, Michael X.; Oberdick, John

    2009-01-01

    Pcp2(L7) is a GoLoco domain protein specifically and abundantly expressed in cerebellar Purkinje cells. It has been hypothesized to “tune” Gi/o-coupled receptor modulation of physiological effectors, including the P-type Ca2+ channel. We have analyzed a mouse mutant in which the Pcp2(L7) gene was inactivated and find significant anatomical, behavioral and electrophysiological changes. Anatomically, we observed mild cerebellar hypoplasia. Behaviorally, the mutants were altered in modalities atypical for a traditional cerebellar mutant, and oddly, all of these changes could be considered functional enhancements. This includes increased asymptotic performance in gross motor learning, increased rate of acquisition in tone-conditioned fear, and enhanced pre-pulse inhibition of the acoustic startle response. Electrophysiological analysis of Purkinje cells in the mutants reveals depression of the complex spike waveform that may underlie the behavioral changes. Based on these observations we suggest that the Pcp2(L7) protein acts as a sensorimotor damper that modulates time- and sense-dependent changes in motor responses. PMID:18930827

  1. Draft Genome Sequence of the Intermediate Rough Vaccine Strain Brucella abortus S19Δper Mutant.

    PubMed

    Chaudhuri, Pallab; Goswami T, Tapas K; Lalsiamthara, Jonathan; Kaur, Gurpreet; Vishnu, Udayakumar S; Sankarasubramanian, Jagadesan; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

    2015-11-12

    Here, we report the genome sequence of the intermediate rough vaccine strain mutant, Brucella abortus S19Δper. The length of the draft genome was 3,271,238 bp, with 57.2% G+C content. A total of 3,204 protein-coding genes and 56 RNA genes were predicted.

  2. Draft Genome Sequence of the Intermediate Rough Vaccine Strain Brucella abortus S19Δper Mutant

    PubMed Central

    Chaudhuri, Pallab; Goswami T, Tapas K.; Lalsiamthara, Jonathan; Kaur, Gurpreet; Vishnu, Udayakumar S.; Sankarasubramanian, Jagadesan; Gunasekaran, Paramasamy

    2015-01-01

    Here, we report the genome sequence of the intermediate rough vaccine strain mutant, Brucella abortus S19Δper. The length of the draft genome was 3,271,238 bp, with 57.2% G+C content. A total of 3,204 protein-coding genes and 56 RNA genes were predicted. PMID:26564050

  3. Altered calmodulin activity in fluphenazine-resistant mutant strains. Pleiotropic effect on development and cellular organization in Volvox carteri.

    PubMed

    Kurn, N; Sela, B A

    1981-12-01

    Genetically altered calmodulin activity in spontaneously derived mutant strains, which were selected for resistance to the toxic effect of a specific inhibitor, the phenothiazine drug fluphenazine, is demonstrated. Partially purified calmodulin preparations from wild-type and fluphenazine-resistant strains of the multicellular alga Volvox carteri, were tested for the ability to activate Ca2+-ATPase of the erythrocyte membranes, and the inhibition of this stimulatory activity by fluphenazine. Unlike the preparation obtained from wild-type cells, mutant calmodulin is shown to be insensitive to fluphenazine inhibition, in one case, and calmodulin from another strain was found to be inactive in vitro, i.e. it did not activate Ca2+-ATPase. The pleiotropic phenotype of the spontaneously derived mutant strains involved aberrant multicellular organization and hormone-independent commitment of the multipotent asexual reproductive cells, gonodia, to sexual development. These results clearly implicate calmodulin in the control of development and morphogenesis in this simple multicellular eukaryote. In addition, intracellular inhibition of calmodulin in wild-type cells is shown to block the morphogenic process of embryo inversion and to arrest motility. The availability of mutant calmodulin will facilitate further investigation of the role of this ubiquitous regulatory protein in the control of development and differentiation in multicellular eukarytes, as well as the fine structure/function relationship with regard to calmodulin modulation of a wide variety of cellular processes. PMID:6459931

  4. Immunological variation between inbred laboratory mouse strains: points to consider in phenotyping genetically immunomodified mice.

    PubMed

    Sellers, R S; Clifford, C B; Treuting, P M; Brayton, C

    2012-01-01

    Inbred laboratory mouse strains are highly divergent in their immune response patterns as a result of genetic mutations and polymorphisms. The generation of genetically engineered mice (GEM) has, in the past, used embryonic stem (ES) cells for gene targeting from various 129 substrains followed by backcrossing into more fecund mouse strains. Although common inbred mice are considered "immune competent," many have variations in their immune system-some of which have been described-that may affect the phenotype. Recognition of these immune variations among commonly used inbred mouse strains is essential for the accurate interpretation of expected phenotypes or those that may arise unexpectedly. In GEM developed to study specific components of the immune system, accurate evaluation of immune responses must take into consideration not only the gene of interest but also how the background strain and microbial milieu contribute to the manifestation of findings in these mice. This article discusses points to consider regarding immunological differences between the common inbred laboratory mouse strains, particularly in their use as background strains in GEM.

  5. Enhanced production of thrombinase by Streptomyces venezuelae: kinetic studies on growth and enzyme production of mutant strain.

    PubMed

    Naveena, Balakrishnan; Gopinath, Kannapan Panchamoorthy; Sakthiselvan, Punniavan; Partha, Nagarajan

    2012-05-01

    This investigation provides the enhanced production of thrombinase, a fibrinolytic enzyme using mutant Streptomyces venezuelae. Initially the mutagenesis of the marine isolate was done by UV and Ethyl methane sulfonate (EMS) and their mutational efficiencies were compared. The mutants were selected based on their high thrombinase activity and used for further studies. The mutant was found to be more halo and thermo tolerant comparing to wild. The effect of Dissolved oxygen level was also determined and the mutant offered the maximum specific growth rate as 0.2404 (h(-1)). The mutant showed high resistance to higher initial lactose concentration and the inhibition concentration was found to be 155.1mg/mL. The effect of S(0)/X(0) ratio on specific substrate consumption and production rate were also investigated. Both mutant and wild showed increase in specific substrate consumption and production rate at higher S(0)/X(0) ratio but the mutant showed better values than the wild strain. PMID:22401714

  6. Enlargement of the Axial Length and Altered Ultrastructural Features of the Sclera in a Mutant Lumican Transgenic Mouse Model

    PubMed Central

    Song, Yanzheng; Zhang, Fengju; Zhao, Yanyan; Sun, Mingshen; Tao, Jun; Liang, Yanchuang; Ma, Ling; Yu, Yanqiu; Wang, Jianhua; Hao, Junfeng

    2016-01-01

    Lumican (LUM) is a candidate gene for myopia in the MYP3 locus. In this study, a mutant lumican (L199P) transgenic mouse model was established to investigate the axial length changes and ultrastructural features of the sclera. The mouse model was established by pronuclear microinjection. Transgenic mice and wild-type B6 mice were killed at eight weeks of age. Gene expression levels of LUM and collagen type I (COL1) in the sclera were analyzed by quantitative real-time polymerase chain reaction (qPCR), and the protein levels were assessed by Western blot analysis. Ocular axial lengths were measured on the enucleated whole eye under a dissecting microscope. Ultrastructural features of collagen fibrils in the sclera were examined with transmission electron microscopy (TEM). Lumican and collagen type I were both elevated at the transcriptional and protein levels. The mean axial length of eyes in the transgenic mice was significantly longer than that in the wild-type mice (3,231.0 ± 11.2 μm (transgenic group) vs 3,199.7 ± 11.1 μm (controls), p<0.05 =). Some ultrastructural changes were observed in the sclera of the transgenic mice under TEM, such as evident lamellar disorganizations and abnormal inter-fibril spacing. The average collagen fibril diameter was smaller than that in their wild-type counterparts. These results indicate that the ectopic mutant lumican (L199P) may induce enlargement of axial lengths and abnormal structures and distributions of collagen fibrils in mouse sclera. This transgenic mouse model can be used for the mechanistic study of myopia. PMID:27711221

  7. Enhanced bioethanol production from wheat straw hemicellulose by mutant strains of pentose fermenting organisms Pichia stipitis and Candida shehatae.

    PubMed

    Koti, Sravanthi; Govumoni, Sai Prashanthi; Gentela, Jahnavi; Venkateswar Rao, L

    2016-01-01

    The main aim of the present study was to mutate yeast strains, Pichia stipitis NCIM 3498 and Candida shehatae NCIM 3501 and assess the mutant's ability to utilize, ferment wheat straw hemicellulose with enhanced ethanol yield. The organisms were subjected to random mutagenesis using physical (ultraviolet radiation) and chemical (ethidium bromide) mutagens. The mutant and wild strains were used to ferment the hemicellulosic hydrolysates of wheat straw obtained by 2 % dilute sulphuric acid and enzymatic hydrolysis by crude xylanase separately. Among all the mutant strains, PSUV9 and CSEB7 showed enhanced ethanol production (12.15 ± 0.57, 9.55 ± 0.47 g/L and yield 0.450 ± 0.009, 0.440 ± 0.001 g/g) as compared to the wild strains (8.28 ± 0.54, 7.92 ± 0.89 g/L and yield 0.380 ± 0.006 and 0.370 ± 0.002 g/g) in both the hydrolysates. The mutant strains were also checked for their consistency in ethanol production and found stable for 19 cycles in hemicellulosic hydrolysates of wheat straw. A novel element in the present study was introduction of chemical mutagenesis in wild type as well as UV induced mutants. This combination of treatments i.e., UV followed by chemical mutagenesis was practically successful. PMID:27652118

  8. Effects of cold acclimation on the energetic metabolism of the staggerer mutant mouse.

    PubMed

    Bertin, R; Guastavino, J M; Portet, R

    1990-02-01

    Staggerer mutant mice are lean despite their hyperphagia. Brown adipose tissue activity may be implicated in this phenomenon. The aim of this work is to determine the energetic metabolism and to detail some characteristics of the brown adipose tissue of Staggerer mutant mice born and reared either at 28 degrees C (within the thermoneutral zone) or 22 degrees C (cold temperature) compared to nonmutant control mice. In mutant mice reared at thermoneutrality the resting metabolism was found to be higher than that of controls, and further the activity of the brown adipose tissue increased as indicated in relative mass, composition and cytochrome oxydase activity. A stimulatory effect of cold exposure was observed in both mutant and nonmutant mice. It is suggested that Staggerer mice may provide a good model for the study of the cold-induced or diet-induced mechanisms of brown fat stimulation.

  9. Genetic typing of the senescence-accelerated mouse (SAM) strains with microsatellite markers.

    PubMed

    Xia, C; Higuchi, K; Shimizu, M; Matsushita, T; Kogishi, K; Wang, J; Chiba, T; Festing, M F; Hosokawa, M

    1999-03-01

    The Senescence-Accelerated Mouse (SAM) strains constitute a murine model of accelerated senescence originating from the ancestral AKR/J strains and consist of nine senescence-prone (SAMP) strains and four senescence-resistant (SAMR) strains. The chromosomes (Chrs) of the SAM strains were typed with 581 microsatellite markers amplified by PCR, and the fundamental genetic information of the SAM strains was obtained. One-third of the examined markers displayed polymorphism among the strains, and only two alleles were detected in almost all loci among the SAM and AKR/J strains. However, in 12 loci (5.6% of total 215 polymorphic markers), the third allele was detected among the SAM strains. The genetic typing and developmental history suggested that the SAM strains were related inbred strains developed by the accidental crossing between the AKR/J strain and other unknown strain(s). Comparison of the distribution of the loci in the SAMP and the SAMR series revealed notable differences in the four regions on Chrs 4, 14, 16, and 17. This indicated that some of these chromosomal sites might contain the genes responsible for accelerated senescence in the SAMP series. PMID:10051317

  10. Non-contact strain measurement in the mouse forearm loading model using digital image correlation (DIC).

    PubMed

    Begonia, Mark T; Dallas, Mark; Vizcarra, Bruno; Liu, Ying; Johnson, Mark L; Thiagarajan, Ganesh

    2015-12-01

    This study investigates the use of a non-contact method known as digital image correlation (DIC) to measure strains in the mouse forearm during axial compressive loading. A two camera system was adapted to analyze the medial and lateral forearm displacements simultaneously, and the derived DIC strain measurements were compared to strain gage readings from both the ulna and radius. Factors such as region-of-interest (ROI) location, lens magnification, noise, and out-of-plane motion were examined to determine their influence on the DIC strain measurements. We confirmed that our DIC system can differentiate ROI locations since it detected higher average strains in the ulna compared to the radius and detected compressive strains on medial bone surfaces vs. tensile strains on lateral bone surfaces. Interestingly, the DIC method also captured heterogeneity in surface strain fields which are not detectable by strain gage based methods. A separate analysis of the noise intrinsic to the DIC system also revealed that the noise constituted less than 4.5% of all DIC strain measurements. Furthermore, finite element (FE) simulations of the forearm showed that out-of-plane motion was not a significant factor that influenced DIC measurements. Finally, we observed that average DIC strain measurements can be up to 1.5-2 times greater than average strain gage readings on the medial bone surfaces. These findings suggest that strain experienced in the mouse forearm model by loading is better captured through DIC as opposed to strain gages, which as a result of being glued to the bone surface artificially stiffen the bone and lead to an underestimation of the strain response. PMID:26388521

  11. Non-contact strain measurement in the mouse forearm loading model using digital image correlation (DIC).

    PubMed

    Begonia, Mark T; Dallas, Mark; Vizcarra, Bruno; Liu, Ying; Johnson, Mark L; Thiagarajan, Ganesh

    2015-12-01

    This study investigates the use of a non-contact method known as digital image correlation (DIC) to measure strains in the mouse forearm during axial compressive loading. A two camera system was adapted to analyze the medial and lateral forearm displacements simultaneously, and the derived DIC strain measurements were compared to strain gage readings from both the ulna and radius. Factors such as region-of-interest (ROI) location, lens magnification, noise, and out-of-plane motion were examined to determine their influence on the DIC strain measurements. We confirmed that our DIC system can differentiate ROI locations since it detected higher average strains in the ulna compared to the radius and detected compressive strains on medial bone surfaces vs. tensile strains on lateral bone surfaces. Interestingly, the DIC method also captured heterogeneity in surface strain fields which are not detectable by strain gage based methods. A separate analysis of the noise intrinsic to the DIC system also revealed that the noise constituted less than 4.5% of all DIC strain measurements. Furthermore, finite element (FE) simulations of the forearm showed that out-of-plane motion was not a significant factor that influenced DIC measurements. Finally, we observed that average DIC strain measurements can be up to 1.5-2 times greater than average strain gage readings on the medial bone surfaces. These findings suggest that strain experienced in the mouse forearm model by loading is better captured through DIC as opposed to strain gages, which as a result of being glued to the bone surface artificially stiffen the bone and lead to an underestimation of the strain response.

  12. 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.

  13. Salmonella enterica serovar typhimurium trxA mutants are protective against virulent challenge and induce less inflammation than the live-attenuated vaccine strain SL3261.

    PubMed

    Peters, S E; Paterson, G K; Bandularatne, E S D; Northen, H C; Pleasance, S; Willers, C; Wang, J; Foote, A K; Constantino-Casas, F; Scase, T J; Blacklaws, B A; Bryant, C E; Mastroeni, P; Charles, I G; Maskell, D J

    2010-01-01

    In Salmonella enterica serovar Typhimurium, trxA encodes thioredoxin 1, a small, soluble protein with disulfide reductase activity, which catalyzes thiol disulfide redox reactions in a variety of substrate proteins. Thioredoxins are involved as antioxidants in defense against oxidative stresses, such as exposure to hydrogen peroxide and hydroxyl radicals. We have made a defined, complete deletion of trxA in the mouse-virulent S. Typhimurium strain SL1344 (SL1344 trxA), replacing the gene with a kanamycin resistance gene cassette. SL1344 trxA was attenuated for virulence in BALB/c mice by the oral and intravenous routes and when used in immunization experiments provided protection against challenge with the virulent parent strain. SL1344 trxA induced less inflammation in murine spleens and livers than SL3261, the aroA mutant, live attenuated vaccine strain. The reduced splenomegaly observed following infection with SL1344 trxA was partially attributed to a reduction in the number of both CD4(+) and CD8(+) T cells and B lymphocytes in the spleen and reduced infiltration by CD11b(+) cells into the spleen compared with spleens from mice infected with SL3261. This less severe pathological response indicates that a trxA mutation might be used to reduce reactogenicity of live attenuated vaccine strains. We tested this by deleting trxA in SL3261. SL3261 trxA was also less inflammatory than SL3261 but was slightly less effective as a vaccine strain than either the SL3261 parent strain or SL1344 trxA.

  14. A study of gender, strain and age differences in mouse liver glutathione-S-transferase.

    PubMed

    Egaas, E; Falls, J G; Dauterman, W C

    1995-01-01

    The hepatic cytosolic glutathione S-transferase (GST) activity in four strains of the mouse and one strain of the rat was studied with the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethachrynic acid (ETHA), cumene hydroperoxide (CU) and atrazine as the in vitro substrates. In the mouse, significant gender, strain and age-related differences in the GST activity towards CDNB and atrazine were found between adolescent and sexually mature males and females of the CD-1, C57BL/6, DBA/2 and Swiss-Webster strains, and the differences were larger with atrazine as the substrate. With DCNB and CU a similar tendency was observed, however not significant for all strains. The GST activity towards ETHA was also gender and strain specific, but revealed no age-related differences. The herbicide atrazine seems to be a useful substrate in the study of strain and age-related differences in the mouse GST class Pi.

  15. Cellulase production and saccharification of rice straw by the mutant strain Hypocrea koningii RSC1.

    PubMed

    Palaniyandi, Sasikumar Arunachalam; Yang, Seung Hwan; Suh, Joo-Won

    2014-01-01

    The production of cellulase using solid-state fermentation of rice straw by the mutant strain Hypocrea koningii RSC1 was studied. Optimization of culture conditions, such as the nitrogen source, pH, and temperature, resulted in a maximum filter paper cellulase activity of 44.15 U g(-1) substrate, a carboxymethylcellulase activity of 324.6 U g(-1) substrate, and a β-glucosidase activity of 7.45 U g(-1) substrate. Saccharification of untreated, 1% H(2)SO(4)-treated, and 2.5% NaOH-treated rice straw using the RSC1 cellulase resulted in 19, 17, and 34 g L(-1) of reducing sugar, respectively. Further studies on the morphological and compositional changes of rice straw upon treatment with the cellulase by scanning electron microscopy analysis and Fourier transform infrared spectroscopy revealed the disruption of the arrangement of fibers and changes in the functional groups that occur in cellulose. X-ray diffraction analysis revealed a reduction in crystallinity of the rice straw upon treatment with the cellulase. Our study shows that H. koningii RSC1 could be a good choice for the production of cellulase and reducing sugars from rice straw.

  16. Pilin regulation in the pilT mutant of Neisseria gonorrhoeae strain MS11

    PubMed Central

    Dietrich, Manuela; Mollenkopf, Hans; So, Magdalene; Friedrich, Alexandra

    2009-01-01

    The ATPase protein PilT mediates retraction of type IV pili (Tfp). Tfp retraction of Neisseria gonorrhoeae causes many signal transduction events and changes in gene expression in infected epithelial cells. To find out whether a pilT mutation and lack of Tfp retraction, respectively, lead also to gene regulation in bacteria we performed microarrays comparing the transcriptional profiles of the N. gonorrhoeae parent strain MS11 and its isogenic pilT mutant during growth in vitro. A loss-of-function-mutation in pilT led to altered transcript levels of 63 open reading frames. Levels of pilE transcripts and its deduced protein the major Tfp subunit pilin, were increased most markedly by a mutation in pilT. Further studies revealed that pilE expression was also controlled by two other genes encoding Tfp biogenesis proteins, pilD and pilF. Our studies strongly suggest that pilE expression is a finely-tuned process. PMID:19486161

  17. Shedding of Brucella abortus rough mutant strain RB51 in milk of water buffalo (Bubalus bubalis).

    PubMed

    Longo, Mariangela; Mallardo, Karina; Montagnaro, Serena; De Martino, Luisa; Gallo, Sergio; Fusco, Giovanna; Galiero, Giorgio; Guarino, Achille; Pagnini, Ugo; Iovane, Giuseppe

    2009-07-01

    The objective of this study was to determine if Brucella abortus rough mutant strain RB51 (SRB51) is eliminated in buffalo milk. Thirty Brucella-free female buffaloes were used in this study: ten 4-5 years old were inoculated with the triple of the recommended calfhood dose of SRB51 by subcutaneous route, ten 2-3 years old at the first lactation were previously vaccinated twice as calves with triple the recommended calf dose of RB51, while five 4-5 years old and five 2-3 years old not vaccinated Brucella-free female buffaloes served as controls. Milk samples were taken aseptically on a daily basis for the first 30 days and weekly for the second and third months. The samples were inoculated on selective media for isolation of SRB51 and incubated for 11 days. Moreover, PCR analysis was also performed directly on milk samples. SRB51 was isolated from milk samples only during the first week post-vaccination while RB51 DNA was detected during the first week till the fourth week post-vaccination only in water buffaloes vaccinated as adults. The identification of Brucella RB51 in milk samples, strongly suggests that this Brucella vaccine could be excreted in milk of buffalo cows vaccinated as adults, while our data demonstrate that the vaccine is safe for use in buffaloes vaccinated as calves in which it was not excreted in milk.

  18. Isolation and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen

    SciTech Connect

    Wolk, C.P.; Cai, Y.; Cardemil, L.; Flores, E.; Hohn, B.; Murry, M.; Schmetterer, G.; Schrautemeier, B.; Wilson, R.

    1988-03-01

    Mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen were isolated by mutagenesis with UV irradiation, followed by a period of incubation in yellow light and then by penicillin enrichment. A cosmid vector, pRL25C, containing replicons functional in Escherichia coli and in Anabaena species was constructed. DNA from wild-type Anabaena sp. strain PCC 7120 was partially digested with Sau3AI, and size-fractionated fragments about 40 kilobases (kb) in length were ligated into the phosphatase-treated unique BamHI site of pRL25C. A library of 1054 cosmid clones was generated in E. coli DH1 bearing helper plasmid pDS4101. A derivative of conjugative plasmid RP-4 was transferred to this library by conjugation, and the library was replicated to lawns of mutant Anabaena strains with defects in the polysaccharide layer of the envelopes of the heterocysts. Mutant EF116 was complemented by five cosmids, three of which were subjected to detailed restriction mapping; a 2.8-kb fragment of DNA derived from one of the cosmids was found to complement EF116. Mutant EF113 was complemented by a single cosmid, which was also restriction mapped, and was shown to be complemented by a 4.8-kb fragment of DNA derived from this cosmid.

  19. Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

    PubMed Central

    Verissimo-Villela, Erika; Kitahara-Oliveira, Milene Yoko; dos Reis, Ana Beatriz de Bragança; Albano, Rodolpho Mattos; Da-Cruz, Alda Maria; Bello, Alexandre Ribeiro

    2016-01-01

    During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins. PMID:27223868

  20. Mouse Slc9a8 Mutants Exhibit Retinal Defects Due to Retinal Pigmented Epithelium Dysfunction

    PubMed Central

    Jadeja, Shalini; Barnard, Alun R.; McKie, Lisa; Cross, Sally H.; White, Jacqueline K.; Robertson, Morag; Budd, Peter S.; MacLaren, Robert E.; Jackson, Ian J.

    2015-01-01

    Purpose. As part of a large scale systematic screen to determine the effects of gene knockout mutations in mice, a retinal phenotype was found in mice lacking the Slc9a8 gene, encoding the sodium/hydrogen ion exchange protein NHE8. We aimed to characterize the mutant phenotype and the role of sodium/hydrogen ion exchange in retinal function. Methods. Detailed histology characterized the pathological consequences of Slc9a8 mutation, and retinal function was assessed by electroretinography (ERG). A conditional allele was used to identify the cells in which NHE8 function is critical for retinal function, and mutant cells analyzed for the effect of the mutation on endosomes. Results. Histology of mutant retinas reveals a separation of photoreceptors from the RPE and infiltration by macrophages. There is a small reduction in photoreceptor length and a mislocalization of visual pigments. The ERG testing reveals a deficit in rod and cone pathway function. The RPE shows abnormal morphology, and mutation of Slc9a8 in only RPE cells recapitulates the mutant phenotype. The NHE8 protein localizes to endosomes, and mutant cells have much smaller recycling endosomes. Conclusions. The NHE8 protein is required in the RPE to maintain correct regulation of endosomal volume and/or pH which is essential for the cellular integrity and subsequent function of RPE. PMID:25736793

  1. Dynamics of Photosynthesis in a Glycogen-Deficient glgC Mutant of Synechococcus sp. Strain PCC 7002

    PubMed Central

    Jackson, Simon A.; Eaton-Rye, Julian J.; Bryant, Donald A.; Posewitz, Matthew C.

    2015-01-01

    Cyanobacterial glycogen-deficient mutants display impaired degradation of light-harvesting phycobilisomes under nitrogen-limiting growth conditions and secrete a suite of organic acids as a putative reductant-spilling mechanism. This genetic background, therefore, represents an important platform to better understand the complex relationships between light harvesting, photosynthetic electron transport, carbon fixation, and carbon/nitrogen metabolisms. In this study, we conducted a comprehensive analysis of the dynamics of photosynthesis as a function of reductant sink manipulation in a glycogen-deficient glgC mutant of Synechococcus sp. strain PCC 7002. The glgC mutant showed increased susceptibility to photoinhibition during the initial phase of nitrogen deprivation. However, after extended periods of nitrogen deprivation, glgC mutant cells maintained higher levels of photosynthetic activity than the wild type, supporting continuous organic acid secretion in the absence of biomass accumulation. In contrast to the wild type, the glgC mutant maintained efficient energy transfer from phycobilisomes to photosystem II (PSII) reaction centers, had an elevated PSII/PSI ratio as a result of reduced PSII degradation, and retained a nitrogen-replete-type ultrastructure, including an extensive thylakoid membrane network, after prolonged nitrogen deprivation. Together, these results suggest that multiple global signals for nitrogen deprivation are not activated in the glgC mutant, allowing the maintenance of active photosynthetic complexes under conditions where photosynthesis would normally be abolished. PMID:26150450

  2. Mutant strains of Spirulina (Arthrospira) platensis to increase the efficiency of micro-ecological life support systems

    NASA Astrophysics Data System (ADS)

    Brown, Igor

    The European Micro-Ecological Life Support System Alternative (MELiSSA) is an advanced idea for organizing a bioregenerative system for long term space flights and extraterrestrial settlements (Hendrickx, De Wever et al., 2005). Despite the hostility of both lunar and Martian environments to unprotected life, it seems possible to cultivate photosynthetic bacteria using closed bioreactors illuminated and heated by solar energy. Such reactors might be employed in critical processes, e.g. air revitalization, foodcaloric and protein source, as well as an immunomodulators production. The MELiSSA team suggested cyanobacterium Spirulina as most appropriate agent to revitalize air and produce a simple "fast" food. This is right suggestion because Spirulina was recently shown to be an oxygenic organism with the highest level of O2 production per unit mass (Ananyev et al., 2005). Chemical composition of Spirulina includes proteins (55Aiming to make Spirulina cultivation in life support systems like MELiSSA more efficient, we selected Spirulina mutant strains with increased fraction of methionine in the biomass of this cyanobacterium and compared the effect of parental wild strain of Spirulina and its mutants on the tendency of such experimental illnesses as radiationinduced lesions and hemolythic anemia. Results: It was found that mutant strains 198B and 27G contain higher quantities of total protein, essential amino acids, c-phycocyanin, allophycocyanin and chlorophyll a than parental wild strain of S. platensis. The strain 198B is also characterized with increased content of carotenoids. Revealed biochemical peculiarities of mutant strains suggest that these strains can serve as an additional source of essential amino acids as well as phycobiliproteins and carotenoids for the astronauts. Feeding animals suffering from radiation-induced lesions, c-phycocyanin, extracted from strain 27G, led to a correction in deficient dehydrogenase activity and energy-rich phosphate levels

  3. A quantitative method for defining high-arched palate using the Tcof1(+/-) mutant mouse as a model.

    PubMed

    Conley, Zachary R; Hague, Molly; Kurosaka, Hiroshi; Dixon, Jill; Dixon, Michael J; Trainor, Paul A

    2016-07-15

    The palate functions as the roof of the mouth in mammals, separating the oral and nasal cavities. Its complex embryonic development and assembly poses unique susceptibilities to intrinsic and extrinsic disruptions. Such disruptions may cause failure of the developing palatal shelves to fuse along the midline resulting in a cleft. In other cases the palate may fuse at an arch, resulting in a vaulted oral cavity, termed high-arched palate. There are many models available for studying the pathogenesis of cleft palate but a relative paucity for high-arched palate. One condition exhibiting either cleft palate or high-arched palate is Treacher Collins syndrome, a congenital disorder characterized by numerous craniofacial anomalies. We quantitatively analyzed palatal perturbations in the Tcof1(+/-) mouse model of Treacher Collins syndrome, which phenocopies the condition in humans. We discovered that 46% of Tcof1(+/-) mutant embryos and new born pups exhibit either soft clefts or full clefts. In addition, 17% of Tcof1(+/-) mutants were found to exhibit high-arched palate, defined as two sigma above the corresponding wild-type population mean for height and angular based arch measurements. Furthermore, palatal shelf length and shelf width were decreased in all Tcof1(+/-) mutant embryos and pups compared to controls. Interestingly, these phenotypes were subsequently ameliorated through genetic inhibition of p53. The results of our study therefore provide a simple, reproducible and quantitative method for investigating models of high-arched palate.

  4. A mouse renin distal enhancer is essential for blood pressure homeostasis in BAC-rescued renin-null mutant mice.

    PubMed

    Tanimoto, Keiji; Kanafusa, Sumiyo; Ushiki, Aki; Matsuzaki, Hitomi; Ishida, Junji; Sugiyama, Fumihiro; Fukamizu, Akiyoshi

    2014-10-01

    Renin is predominantly expressed in juxtaglomerular cells in the kidney and regulates blood pressure homeostasis. To examine possible in vivo functions of a mouse distal enhancer (mdE), we generated transgenic mice (TgM) carrying either wild-type or mdE-deficient renin BACs (bacterial artificial chromosome), integrated at the identical chromosomal site. In the kidneys of the TgM, the mdE contributed 80% to basal renin promoter activity. To test for possible physiological roles for the mdE, renin BAC transgenes were used to rescue the hypotensive renin-null mice. Interestingly, renal renin expression in the Tg(BAC):renin-null compound mice was indistinguishable between the wild-type and mutant BAC carriers. Surprisingly, however, the plasma renin activity and angiotensin I concentration in the mdE compound mutant mice were significantly lower than the same parameters in the control mice, and the mutants were consistently hypotensive, demonstrating that blood pressure homeostasis is regulated through transcriptional cis elements controlling renin activity.

  5. A Novel Bmal1 Mutant Mouse Reveals Essential Roles of the C-Terminal Domain on Circadian Rhythms

    PubMed Central

    Cheon, Solmi; Row, Hansang; Lee, Jiyeon; Han, Dong-Hee; Cho, Sehyung; Kim, Kyungjin

    2015-01-01

    The mammalian circadian clock is an endogenous biological timer comprised of transcriptional/translational feedback loops of clock genes. Bmal1 encodes an indispensable transcription factor for the generation of circadian rhythms. Here, we report a new circadian mutant mouse from gene-trapped embryonic stem cells harboring a C-terminus truncated Bmal1 (Bmal1GTΔC) allele. The homozygous mutant (Bmal1GTΔC/GTΔC) mice immediately lost circadian behavioral rhythms under constant darkness. The heterozygous (Bmal1+/GTΔC) mice displayed a gradual loss of rhythms, in contrast to Bmal1+/- mice where rhythms were sustained. Bmal1GTΔC/GTΔC mice also showed arrhythmic mRNA and protein expression in the SCN and liver. Lack of circadian reporter oscillation was also observed in cultured fibroblast cells, indicating that the arrhythmicity of Bmal1GTΔC/GTΔC mice resulted from impaired molecular clock machinery. Expression of clock genes exhibited distinct responses to the mutant allele in Bmal1+/GTΔC and Bmal1GTΔC/GTΔC mice. Despite normal cellular localization and heterodimerization with CLOCK, overexpressed BMAL1GTΔC was unable to activate transcription of Per1 promoter and BMAL1-dependent CLOCK degradation. These results indicate that the C-terminal region of Bmal1 has pivotal roles in the regulation of circadian rhythms and the Bmal1GTΔC mice constitute a novel model system to evaluate circadian functional mechanism of BMAL1. PMID:26394143

  6. Transcriptome Profiling of Wild-Type and pga-Knockout Mutant Strains Reveal the Role of Exopolysaccharide in Aggregatibacter actinomycetemcomitans

    PubMed Central

    Shanmugam, Mayilvahanan; El Abbar, Faiha; Ramasubbu, Narayanan

    2015-01-01

    Exopolysaccharides have a diverse set of functions in most bacteria including a mechanistic role in protecting bacteria against environmental stresses. Among the many functions attributed to the exopolysaccharides, biofilm formation, antibiotic resistance, immune evasion and colonization have been studied most extensively. The exopolysaccharide produced by many Gram positive as well as Gram negative bacteria including the oral pathogen Aggregatibacter actinomycetemcomitans is the homopolymer of β(1,6)-linked N-acetylglucosamine. Recently, we reported that the PGA-deficient mutant of A. actinomycetemcomitans failed to colonize or induce bone resorption in a rat model of periodontal disease, and the colonization genes, apiA and aae, were significantly down regulated in the mutant strain. To understand the role of exopolysaccharide and the pga locus in the global expression of A. actinomycetemcomitans, we have used comparative transcriptome profiling to identify differentially expressed genes in the wild-type strain in relation to the PGA-deficient strain. Transcriptome analysis revealed that about 50% of the genes are differently expressed (P < 0.05 and fold change >1.5). Our study demonstrated that the absence of the pga locus affects the genes involved in peptidoglycan recycling, glycogen storage, and virulence. Further, using confocal microscopy and plating assays, we show that the viability of pga mutant strain is significantly reduced during biofilm growth. Thus, this study highlights the importance of pga genes and the exopolysaccharide in the virulence of A. actinomycetemcomitans. PMID:26221956

  7. Individual strains of Lactobacillus paracasei differentially inhibit human basophil and mouse mast cell activation

    PubMed Central

    Cassard, Lydie; Lalanne, Ana Inés; Garault, Peggy; Cotillard, Aurélie; Chervaux, Christian; Wels, Michiel; Smokvina, Tamara

    2016-01-01

    Abstract Introduction The microbiota controls a variety of biological functions, including immunity, and alterations of the microbiota in early life are associated with a higher risk of developing allergies later in life. Several probiotic bacteria, and particularly lactic acid bacteria, were described to reduce both the induction of allergic responses and allergic manifestations. Although specific probiotic strains were used in these studies, their protective effects on allergic responses also might be common for all lactobacilli. Methods To determine whether allergic effector cells inhibition is a common feature of lactobacilli or whether it varies among lactobacilli strains, we compared the ability of 40 strains of the same Lactobacillus paracasei species to inhibit IgE‐dependent mouse mast cell and human basophil activation. Results We uncovered a marked heterogeneity in the inhibitory properties of the 40 Lactobacillus strains tested. These segregated into three to four clusters depending on the intensity of inhibition. Some strains inhibited both mouse mast cell and human basophil activation, others strains inhibited only one cell type and another group induced no inhibition of activation for either cell type. Conclusions Individual Lactobacillus strains of the same species differentially inhibit IgE‐dependent activation of mouse mast cells and human basophils, two cell types that are critical in the onset of allergic manifestations. Although we failed to identify specific bacterial genes associated with inhibition by gene‐trait matching analysis, our findings demonstrate the complexity of the interactions between the microbiota and the host. These results suggest that some L. paracasei strains might be more beneficial in allergies than others strains and provide the bases for a rational screening of lactic acid bacteria strains as next‐generation probiotics in the field of allergy.

  8. Individual strains of Lactobacillus paracasei differentially inhibit human basophil and mouse mast cell activation

    PubMed Central

    Cassard, Lydie; Lalanne, Ana Inés; Garault, Peggy; Cotillard, Aurélie; Chervaux, Christian; Wels, Michiel; Smokvina, Tamara

    2016-01-01

    Abstract Introduction The microbiota controls a variety of biological functions, including immunity, and alterations of the microbiota in early life are associated with a higher risk of developing allergies later in life. Several probiotic bacteria, and particularly lactic acid bacteria, were described to reduce both the induction of allergic responses and allergic manifestations. Although specific probiotic strains were used in these studies, their protective effects on allergic responses also might be common for all lactobacilli. Methods To determine whether allergic effector cells inhibition is a common feature of lactobacilli or whether it varies among lactobacilli strains, we compared the ability of 40 strains of the same Lactobacillus paracasei species to inhibit IgE‐dependent mouse mast cell and human basophil activation. Results We uncovered a marked heterogeneity in the inhibitory properties of the 40 Lactobacillus strains tested. These segregated into three to four clusters depending on the intensity of inhibition. Some strains inhibited both mouse mast cell and human basophil activation, others strains inhibited only one cell type and another group induced no inhibition of activation for either cell type. Conclusions Individual Lactobacillus strains of the same species differentially inhibit IgE‐dependent activation of mouse mast cells and human basophils, two cell types that are critical in the onset of allergic manifestations. Although we failed to identify specific bacterial genes associated with inhibition by gene‐trait matching analysis, our findings demonstrate the complexity of the interactions between the microbiota and the host. These results suggest that some L. paracasei strains might be more beneficial in allergies than others strains and provide the bases for a rational screening of lactic acid bacteria strains as next‐generation probiotics in the field of allergy. PMID:27621812

  9. What makes a good mother? Implication of inter-, and intrastrain strain "cross fostering" for emotional changes in mouse offspring.

    PubMed

    Lerch, Sandra; Brandwein, Christiane; Dormann, Christof; Gass, Peter; Chourbaji, Sabine

    2014-11-01

    Currently, the mouse represents the preferred model organism among mammals used for animal studies. Due to a great availability of mutant strains it represents a standard method to analyze in vivo the effects of targeted gene manipulations. While this - at least in theory - represents a valuable tool to elucidate the pathophysiology of certain human diseases, there are several caveats which need to be considered working with animals. In our study we aimed at elucidating, how a widely established breeding strategy, i.e. the use of "foster mothers" to save the survival of compromised mouse pups for ongoing experiments, per se, affects the emotional phenotype of the fostered offspring. Since it is a popular method to use outbred strains like NMRI to do this job, we sought to evaluate the potential effects of such an artificial postnatal condition and compare either offspring nurtured by their biological mothers or two different strains of foster mothers. Hence we analysed changes in maternal care and later on the emotional behaviour of male and female C57BL/6 mice reared by (i) their biological C57BL/6 mothers, (ii) C57BL/6 foster mothers and (iii) NMRI foster mothers in a behavioural test battery. In addition we assessed corticosterone levels as indicator for stress-physiological changes. Besides clear differences in maternal behaviour, our study indicates an altered emotional state (i.e. differences in anxiety and depressive-like features) in mice reared by different "categories" of mothers, which emphasizes the importance to embed such perinatal conditions in the evaluation of animal-deriving data.

  10. Murine leukemia virus in organs of senescence-prone and -resistant mouse strains.

    PubMed

    Carp, R I; Meeker, H C; Chung, R; Kozak, C A; Hosokawa, M; Fujisawa, H

    2002-03-31

    A series of inbred strains of mice have been developed that are either prone (SAMP) or resistant (SAMR) to accelerated senescence. All of these strains originated from an inadvertent cross or crosses between the AKR/J mouse strain and an unknown strain(s). The characteristics of the nine senescence-prone lines differ, with all strains showing generalized aspects of accelerated aging but with each line having a specific aging-related change that is emphasized, e.g. learning and memory deficits, osteoporosis and senile amyloidosis. The senescence-resistant strains have normal patterns of aging and do not show the specific aging-related changes seen in SAMP strains. The fact that AKR mice have high levels of endogenous, ecotropic murine leukemia virus (MuLV) prompted an examination of the expression levels of MuLV in SAM strains. Analysis of brain, spleen and thymus samples revealed that seven of nine SAMP strains had high levels of MuLV and contained the Emv11 provirus (previously termed Akv1) that encodes the predominant MuLV found in AKR mice. In contrast, none of the SAMR strains had Emv11 or significant amounts of virus. The current findings represent an initial step in determining the role of MuLV in the accelerated senescence seen in SAMP strains. PMID:11850021

  11. Immune responses and protection against infection and abortion in cattle experimentally vaccinated with mutant strains of Brucella abortus.

    PubMed

    Cheville, N F; Stevens, M G; Jensen, A E; Tatum, F M; Halling, S M

    1993-10-01

    Twenty-four 10-month-old Polled Hereford heifers were inoculated SC with live cells of one of the following strains of Brucella abortus: S19 delta 31K (n = 4), S19 delta SOD (n = 4), RB51 (n = 4), and strain 19 (n = 6); controls (n = 6) were given saline solution. Heifers given the deletion mutants S19 delta 31K and S19 delta SOD, and those given strain 19 developed antibody responses to B abortus and cutaneous reactions to brucellin. Heifers given strain RB51 did not develop antibodies that reacted in the standard tube agglutination test, but sera reacted in tests, using an antibody dot-blot assay containing RB51 antigen. The S19 delta 31K and S19 delta SOD strains of B abortus isolated from lymph node tissue after vaccination did not differ genetically from the master stock strain. All heifers were bred naturally at 16 to 17 months of age, and were challenge-exposed intraconjunctivally with virulent B abortus strain 2308 during the fifth month of pregnancy. All vaccinated heifers were protected (ie, none aborted and none had B abortus isolated from their tissues after parturition). Calves born from vaccinated dams were free of B abortus. Antibody responses in heifers after challenge exposure were an indicator of immunity. All 5 control heifers (nonvaccinated) developed serum antibodies after challenge exposure; 3 aborted, and 1 delivered a small, weak calf at 8.5 months of gestation. Thus live mutant strains of B abortus can induce protective immunity when given at 10 months of age, and strain RB51 is a strong candidate for further testing.

  12. Newborn mouse lens proteome and its alteration by lysine 6 mutant ubiquitin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ubiquitin is a tag that often initiates degradation of proteins by the proteasome in the ubiquitin proteasome system. Targeted expression of K6W mutant ubiquitin (K6W-Ub) in the lens results in defects in lens development and cataract formation, suggesting critical functions for ubiquitin in lens. T...

  13. Comparative gene expression profiling in two congenic mouse strains following Bordetella pertussis infection

    PubMed Central

    Banus, Sander; Vandebriel, Rob J; Pennings, Jeroen LA; Gremmer, Eric R; Wester, Piet W; van Kranen, Henk J; Breit, Timo M; Demant, Peter; Mooi, Frits R; Hoebee, Barbara; Kimman, Tjeerd G

    2007-01-01

    Background Susceptibility to Bordetella pertussis infection varies widely. These differences can partly be explained by genetic host factors. HcB-28 mice are more resistant to B. pertussis infection than C3H mice, which could partially be ascribed to the B. pertussis susceptibility locus-1 (Bps1) on chromosome 12. The presence of C57BL/10 genome on this locus instead of C3H genome resulted in a decreased number of bacteria in the lung. To further elucidate the role of host genetic factors, in particular in the Bps1 locus, in B. pertussis infection, and to identify candidate genes within in this region, we compared expression profiles in the lungs of the C3H and HcB-28 mouse strains following B. pertussis inoculation. Twelve and a half percent of the genomes of these mice are from a different genetic background. Results Upon B. pertussis inoculation 2,353 genes were differentially expressed in the lungs of both mouse strains. Two hundred and six genes were differentially expressed between the two mouse strains, but, remarkably, none of these were up- or down-regulated upon B. pertussis infection. Of these 206 genes, 17 were located in the Bps1 region. Eight of these genes, which showed a strong difference in gene expression between the two mouse strains, map to the immunoglobulin heavy chain complex (Igh). Conclusion Gene expression changes upon B. pertussis infection are highly identical between the two mouse strains despite the differences in the course of B. pertussis infection. Because the genes that were differentially regulated between the mouse strains only showed differences in expression before infection, it appears likely that such intrinsic differences in gene regulation are involved in determining differences in susceptibility to B. pertussis infection. Alternatively, such genetic differences in susceptibility may be explained by genes that are not differentially regulated between these two mouse strains. Genes in the Igh complex, among which Igh-1a

  14. Mucosal-associated invariant T cell–rich congenic mouse strain allows functional evaluation

    PubMed Central

    Cui, Yue; Franciszkiewicz, Katarzyna; Mburu, Yvonne K.; Mondot, Stanislas; Le Bourhis, Lionel; Premel, Virginie; Martin, Emmanuel; Kachaner, Alexandra; Duban, Livine; Ingersoll, Molly A.; Rabot, Sylvie; Jaubert, Jean; De Villartay, Jean-Pierre; Soudais, Claire; Lantz, Olivier

    2015-01-01

    Mucosal-associated invariant T cells (MAITs) have potent antimicrobial activity and are abundant in humans (5%–10% in blood). Despite strong evolutionary conservation of the invariant TCR-α chain and restricting molecule MR1, this population is rare in laboratory mouse strains (≈0.1% in lymphoid organs), and lack of an appropriate mouse model has hampered the study of MAIT biology. Herein, we show that MAITs are 20 times more frequent in clean wild-derived inbred CAST/EiJ mice than in C57BL/6J mice. Increased MAIT frequency was linked to one CAST genetic trait that mapped to the TCR-α locus and led to higher usage of the distal Vα segments, including Vα19. We generated a MAIThi congenic strain that was then crossed to a transgenic Rorcgt-GFP reporter strain. Using this tool, we characterized polyclonal mouse MAITs as memory (CD44+) CD4–CD8lo/neg T cells with tissue-homing properties (CCR6+CCR7–). Similar to human MAITs, mouse MAITs expressed the cytokine receptors IL-7R, IL-18Rα, and IL-12Rβ and the transcription factors promyelocytic leukemia zinc finger (PLZF) and RAR-related orphan receptor γ (RORγt). Mouse MAITs produced Th1/2/17 cytokines upon TCR stimulation and recognized a bacterial compound in an MR1-dependent manner. During experimental urinary tract infection, MAITs migrated to the bladder and decreased bacterial load. Our study demonstrates that the MAIThi congenic strain allows phenotypic and functional characterization of naturally occurring mouse MAITs in health and disease. PMID:26524590

  15. Mucosal-associated invariant T cell-rich congenic mouse strain allows functional evaluation.

    PubMed

    Cui, Yue; Franciszkiewicz, Katarzyna; Mburu, Yvonne K; Mondot, Stanislas; Le Bourhis, Lionel; Premel, Virginie; Martin, Emmanuel; Kachaner, Alexandra; Duban, Livine; Ingersoll, Molly A; Rabot, Sylvie; Jaubert, Jean; De Villartay, Jean-Pierre; Soudais, Claire; Lantz, Olivier

    2015-11-01

    Mucosal-associated invariant T cells (MAITs) have potent antimicrobial activity and are abundant in humans (5%-10% in blood). Despite strong evolutionary conservation of the invariant TCR-α chain and restricting molecule MR1, this population is rare in laboratory mouse strains (≈0.1% in lymphoid organs), and lack of an appropriate mouse model has hampered the study of MAIT biology. Herein, we show that MAITs are 20 times more frequent in clean wild-derived inbred CAST/EiJ mice than in C57BL/6J mice. Increased MAIT frequency was linked to one CAST genetic trait that mapped to the TCR-α locus and led to higher usage of the distal Vα segments, including Vα19. We generated a MAIThi congenic strain that was then crossed to a transgenic Rorcgt-GFP reporter strain. Using this tool, we characterized polyclonal mouse MAITs as memory (CD44+) CD4-CD8lo/neg T cells with tissue-homing properties (CCR6+CCR7-). Similar to human MAITs, mouse MAITs expressed the cytokine receptors IL-7R, IL-18Rα, and IL-12Rβ and the transcription factors promyelocytic leukemia zinc finger (PLZF) and RAR-related orphan receptor γ (RORγt). Mouse MAITs produced Th1/2/17 cytokines upon TCR stimulation and recognized a bacterial compound in an MR1-dependent manner. During experimental urinary tract infection, MAITs migrated to the bladder and decreased bacterial load. Our study demonstrates that the MAIThi congenic strain allows phenotypic and functional characterization of naturally occurring mouse MAITs in health and disease.

  16. Anthraquinone dyes decolorization capacity of anamorphic Bjerkandera adusta CCBAS 930 strain and its HRP-like negative mutants.

    PubMed

    Korniłłowicz-Kowalska, Teresa; Rybczyńska, Kamila

    2014-06-01

    Cultures of the anamorphic fungus Bjerkandera adusta CCBAS 930 decolorizing, in stationary cultures, 0.01 % solutions of carminic acid and Poly R-478, were characterised by a strong increase in the activity of the horseradish peroxidase (HRP-like) and manganese-dependent peroxidase (MnP) at a low activity of lignin peroxidase. Genotypically modified mutants of B. adusta CCBAS 930: 930-5 and 930-14, with total or partial loss of decolorization capabilities relative to anthraquinonic dyes, showed inhibition of the activity of HRP-like peroxidase and MnP. Whereas, compared to the parental strain, in the mutant cultures there was an increase in the activity of lignin peroxidase and laccase. The paper presents a discussion of the role of the studied enzymatic activities in the process of decolorization of anthraquinonic dyes by the strain B. adusta CCBAS 930. PMID:24415463

  17. Transcriptional changes in the nuc-2A mutant strain of Neurospora crassa cultivated under conditions of phosphate shortage.

    PubMed

    Gras, Diana E; Silveira, Henrique C S; Peres, Nalu T A; Sanches, Pablo R; Martinez-Rossi, Nilce M; Rossi, Antonio

    2009-01-01

    The molecular mechanism that controls the response to phosphate shortage in Neurospora crassa involves four regulatory genes -nuc-2, preg, pgov, and nuc-1. Phosphate shortage is sensed by the nuc-2 gene, the product of which inhibits the functioning of the PREG-PGOV complex. This allows the translocation of the transcriptional factor NUC-1 into the nucleus, which activates the transcription of phosphate-repressible phosphatases. The nuc-2A mutant strain of N. crassa carries a loss-of-function mutation in the nuc-2 gene, which encodes an ankyrin-like repeat protein. In this study, we identified transcripts that are downregulated in the nuc-2A mutant strain. Functional grouping of these expressed sequence tags allowed the identification of genes that play essential roles in different cellular processes such as transport, transcriptional regulation, signal transduction, metabolism, protein synthesis, protein fate, and development. These results reveal novel aspects of the phosphorus-sensing network in N. crassa.

  18. [Study of the transcriptional and transpositional activities of the Tirant retrotransposon in Drosophila melanogaster strains mutant for the flamenco locus].

    PubMed

    Nefedova, L N; Urusov, F A; Romanova, N I; Shmel'kova, A O; Kim, A I

    2012-11-01

    Transpositions of the gypsy retrotransposon in the Drosophila melanogaster genome are controlled by the flamenco locus, which is represented as an accumulation of defective copies of transposable elements. In the present work, genetic control by the flamenco locus of the transcriptional and transpositional activities of the Tirant retrotransposon from the gypsy group was studied. Tissue-specific expression of Tirant was detected in the tissues of ovaries in a strain mutant for the flamenco locus. Tirant was found to be transpositionally active in isogenic D. melanogaster strains mutant for the flamenco locus. The sites of two new insertions have been localized by the method of subtractive hybridization. It has been concluded from the results obtained that the flamenco locus is involved in the genetic control of Tirant transpositions. PMID:23297482

  19. A new model for non-typeable Haemophilus influenzae middle ear infection in the Junbo mutant mouse.

    PubMed

    Hood, Derek; Moxon, Richard; Purnell, Tom; Richter, Caroline; Williams, Debbie; Azar, Ali; Crompton, Michael; Wells, Sara; Fray, Martin; Brown, Steve D M; Cheeseman, Michael T

    2016-01-01

    Acute otitis media, inflammation of the middle ear, is the most common bacterial infection in children and, as a consequence, is the most common reason for antimicrobial prescription to this age group. There is currently no effective vaccine for the principal pathogen involved, non-typeable Haemophilus influenzae (NTHi). The most frequently used and widely accepted experimental animal model of middle ear infection is in chinchillas, but mice and gerbils have also been used. We have established a robust model of middle ear infection by NTHi in the Junbo mouse, a mutant mouse line that spontaneously develops chronic middle ear inflammation in specific pathogen-free conditions. The heterozygote Junbo mouse (Jbo/+) bears a mutation in a gene (Evi1, also known as Mecom) that plays a role in host innate immune regulation; pre-existing middle ear inflammation promotes NTHi middle ear infection. A single intranasal inoculation with NTHi produces high rates (up to 90%) of middle ear infection and bacterial titres (10(4)-10(5) colony-forming units/µl) in bulla fluids. Bacteria are cleared from the majority of middle ears between day 21 and 35 post-inoculation but remain in approximately 20% of middle ears at least up to day 56 post-infection. The expression of Toll-like receptor-dependent response cytokine genes is elevated in the middle ear of the Jbo/+ mouse following NTHi infection. The translational potential of the Junbo model for studying antimicrobial intervention regimens was shown using a 3 day course of azithromycin to clear NTHi infection, and its potential use in vaccine development studies was shown by demonstrating protection in mice immunized with killed homologous, but not heterologous, NTHi bacteria. PMID:26611891

  20. A new model for non-typeable Haemophilus influenzae middle ear infection in the Junbo mutant mouse

    PubMed Central

    Hood, Derek; Moxon, Richard; Purnell, Tom; Richter, Caroline; Williams, Debbie; Azar, Ali; Crompton, Michael; Wells, Sara; Fray, Martin; Brown, Steve D. M.; Cheeseman, Michael T.

    2016-01-01

    ABSTRACT Acute otitis media, inflammation of the middle ear, is the most common bacterial infection in children and, as a consequence, is the most common reason for antimicrobial prescription to this age group. There is currently no effective vaccine for the principal pathogen involved, non-typeable Haemophilus influenzae (NTHi). The most frequently used and widely accepted experimental animal model of middle ear infection is in chinchillas, but mice and gerbils have also been used. We have established a robust model of middle ear infection by NTHi in the Junbo mouse, a mutant mouse line that spontaneously develops chronic middle ear inflammation in specific pathogen-free conditions. The heterozygote Junbo mouse (Jbo/+) bears a mutation in a gene (Evi1, also known as Mecom) that plays a role in host innate immune regulation; pre-existing middle ear inflammation promotes NTHi middle ear infection. A single intranasal inoculation with NTHi produces high rates (up to 90%) of middle ear infection and bacterial titres (104-105 colony-forming units/µl) in bulla fluids. Bacteria are cleared from the majority of middle ears between day 21 and 35 post-inoculation but remain in approximately 20% of middle ears at least up to day 56 post-infection. The expression of Toll-like receptor-dependent response cytokine genes is elevated in the middle ear of the Jbo/+ mouse following NTHi infection. The translational potential of the Junbo model for studying antimicrobial intervention regimens was shown using a 3 day course of azithromycin to clear NTHi infection, and its potential use in vaccine development studies was shown by demonstrating protection in mice immunized with killed homologous, but not heterologous, NTHi bacteria. PMID:26611891

  1. A mouse muscle-adapted enterovirus 71 strain with increased virulence in mice.

    PubMed

    Wang, Wei; Duo, Jianying; Liu, Jiangning; Ma, Chunmei; Zhang, Lianfeng; Wei, Qiang; Qin, Chuan

    2011-09-01

    Enterovirus 71 (EV71) infections can usually cause epidemic hand, foot, and mouth disease (HFMD), and occasionally lead to aseptic meningitis, encephalitis, and polio-like illness. Skeletal muscles have been thought to be crucial for the pathogenesis of EV71-related diseases. However, little is known about the virulence of mouse muscle-adapted EV71. The EV71 0805 were subjected to four passages in the mouse muscle to generate a mouse-adapted EV71 strain of 0805a. In comparison with the parental EV71 0805, the mouse muscle-adapted EV71 0805a displayed stronger cytotoxicity against Rhabdomyosarcoma (RD) cells and more efficient replication in RD cells. Furthermore, infection with the EV71 0805a significantly inhibited the gain of body weight, accompanied by increased muscle virus load and multiple tissue distribution in the infected mouse. Histological examinations indicated that infection with the EV71 0805 did not cause obvious pathogenic lesions in mice, while infection with the muscle-adapted 0805a resulted in severe necrotizing myositis in the skeletal and cardio muscles, and intestinitis in mice on day 5 post infection. Further analysis revealed many mutations in different regions of the genome of mouse muscle-adapted virus. Collectively, these data demonstrated the mouse muscle-adapted EV71 0805a with increased virulence in mice.

  2. Cyclic loading increases friction and changes cartilage surface integrity in lubricin-mutant mouse knees

    PubMed Central

    Drewniak, Elizabeth I; Jay, Gregory D; Fleming, Braden C; Zhang, Ling; Warman, Matthew L; Crisco, Joseph J

    2012-01-01

    Objective To investigate the effects of lubricin gene dosage and cyclic loading on whole joint coefficient of friction and articular cartilage surface integrity in mouse knee joints. Methods Joints from mice with 2 (Prg4+/+), 1 (Prg4+/−), or no (Prg4−/−) functioning lubricin alleles were subjected to 26 hours of cyclic loading using a custom-built pendulum. Coefficient of friction values were measured at multiple time points. Contralateral control joints were left unloaded. Following testing, joints were examined for histologic evidence of damage and cell viability. Results At baseline, the coefficient of friction values in Prg4−/− mice were significantly higher than those in Prg4+/+ and Prg4+/− mice (P < 0.001). Cyclic loading continuously increased the coefficient of friction in Prg4−/− mouse joints. In contrast, Prg4+/− and Prg4+/+ mouse joints had no coefficient of friction increases during the first 4 hours of loading. After 26 hours of loading, joints from all genotypes had increased coefficient of friction values compared to baseline and unloaded controls. Significantly greater increases occurred in Prg4−/− and Prg4+/− mouse joints compared to Prg4+/+ mouse joints. The coefficient of friction values were not significantly associated with histologic evidence of damage or loss of cell viability. Conclusion Our findings indicate that mice lacking lubricin have increased baseline coefficient of friction values and are not protected against further increases caused by loading. Prg4+/− mice are indistinguishable from Prg4+/+ mice at baseline, but have significantly greater coefficient of friction values following 26 hours of loading. Lubricin dosage affects joint properties during loading, and may have clinical implications in patients for whom injury or illness alters lubricin abundance. PMID:21905020

  3. Cox4i2, Ifit2, and Prdm11 Mutant Mice: Effective Selection of Genes Predisposing to an Altered Airway Inflammatory Response from a Large Compendium of Mutant Mouse Lines.

    PubMed

    Horsch, Marion; Aguilar-Pimentel, Juan Antonio; Bönisch, Clemens; Côme, Christophe; Kolster-Fog, Cathrine; Jensen, Klaus T; Lund, Anders H; Lee, Icksoo; Grossman, Lawrence I; Sinkler, Christopher; Hüttemann, Maik; Bohn, Erwin; Fuchs, Helmut; Ollert, Markus; Gailus-Durner, Valérie; de Angelis, Martin Hrabĕ; Beckers, Johannes

    2015-01-01

    We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype-envirotype interactions for other diseases.

  4. Cox4i2, Ifit2, and Prdm11 Mutant Mice: Effective Selection of Genes Predisposing to an Altered Airway Inflammatory Response from a Large Compendium of Mutant Mouse Lines

    PubMed Central

    Bönisch, Clemens; Côme, Christophe; Kolster-Fog, Cathrine; Jensen, Klaus T.; Lund, Anders H.; Lee, Icksoo; Grossman, Lawrence I.; Sinkler, Christopher; Hüttemann, Maik; Bohn, Erwin; Fuchs, Helmut; Ollert, Markus; Gailus-Durner, Valérie; Hrabĕ de Angelis, Martin; Beckers, Johannes

    2015-01-01

    We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype – envirotype interactions for other diseases. PMID:26263558

  5. Successful mouse cloning of an outbred strain by trichostatin A treatment after somatic nuclear transfer.

    PubMed

    Kishigami, Satoshi; Bui, Hong-Thuy; Wakayama, Sayaka; Tokunaga, Kenzo; Van Thuan, Nguyen; Hikichi, Takafusa; Mizutani, Eiji; Ohta, Hiroshi; Suetsugu, Rinako; Sata, Tetsutaro; Wakayama, Teruhiko

    2007-02-01

    Although the somatic cloning technique has been used for numerous applications and basic research of reprogramming in various species, extremely low success rates have plagued this technique for a decade. Further in mice, the "clonable" strains have been limited to mainly hybrid F1 strains such as B6D2F1. Recently, we established a new efficient cloning technique using trichostatin A (TSA) which leads to a 2-5 fold increase in success rates for mouse cloning of B6D2F1 cumulus cells. To further test the validity of this TSA cloning technique, we tried to clone the adult ICR mouse, an outbred strain, which has never been directly cloned before. Only when TSA was used did we obtain both male and female cloned mice from cumulus and fibroblast cells of adult ICR mice with 4-5% success rates, which is comparable to 5-7% of B6D2F1. Thus, the TSA treatment is the first cloning technique to allow us to successfully clone outbred mice, demonstrating that this technique not only improves the success rates of cloning from hybrid strains, but also enables mouse cloning from normally "unclonable" strains.

  6. Comparison of fracture healing among different inbred mouse strains.

    PubMed

    Manigrasso, Michaele B; O'Connor, J Patrick

    2008-06-01

    Quantitative trait locus analysis can be used to identify genes critically involved in biological processes. No such analysis has been applied to identifying genes that control bone fracture healing. To determine the feasibility of such an approach, healing of femur fractures was measured between C57BL/6, DBA/2, and C3H inbred strains of mice. Healing was assessed by radiography and histology and measured by histomorphometry and biomechanical testing. In all strains, radiographic bridging of the fracture was apparent after 3 weeks of healing. Histology showed that healing occurred through endochondral ossification in all strains. Histomorphometric measurements found more bone in the C57BL/6 fracture calluses 7 and 10 days after fracture. In contrast, more cartilage was present after 7 days in the C3H callus, which rapidly declined to levels less than those of C57BL/6 or DBA/2 mice by 14 days after fracture. An endochondral ossification index was calculated by multiplying the callus percent cartilage and bone areas as a measure of endochondral ossification. At 7 and 10 days after fracture, this value was higher in C57BL/6 mice. Using torsional mechanical testing, normalized structural and material properties of the C57BL/6 healing femurs were higher than values from the DBA/2 or C3H mice 4 weeks after fracture. The data indicate that fracture healing proceeds more rapidly in C57BL/6 mice and demonstrate that genetic variability significantly contributes to the process of bone regeneration. Large enough differences exist between C57BL/6 and DBA/2 or C3H mice to permit a quantitative trait locus analysis to identify genes controlling bone regeneration.

  7. Susceptibility to quantum dot induced lung inflammation differs widely among the Collaborative Cross founder mouse strains.

    PubMed

    Scoville, David K; White, Collin C; Botta, Dianne; McConnachie, Lisa A; Zadworny, Megan E; Schmuck, Stefanie C; Hu, Xiaoge; Gao, Xiaohu; Yu, Jianbo; Dills, Russell L; Sheppard, Lianne; Delaney, Martha A; Griffith, William C; Beyer, Richard P; Zangar, Richard C; Pounds, Joel G; Faustman, Elaine M; Kavanagh, Terrance J

    2015-12-01

    Quantum dots (QDs) are engineered semiconductor nanoparticles with unique physicochemical properties that make them potentially useful in clinical, research and industrial settings. However, a growing body of evidence indicates that like other engineered nanomaterials, QDs have the potential to be respiratory hazards, especially in the context of the manufacture of QDs and products containing them, as well as exposures to consumers using these products. The overall goal of this study was to investigate the role of mouse strain in determining susceptibility to QD-induced pulmonary inflammation and toxicity. Male mice from 8 genetically diverse inbred strains (the Collaborative Cross founder strains) were exposed to CdSe-ZnS core-shell QDs stabilized with an amphiphilic polymer. QD treatment resulted in significant increases in the percentage of neutrophils and levels of cytokines present in bronchoalveolar lavage fluid (BALF) obtained from NOD/ShiLtJ and NZO/HlLtJ mice relative to their saline (Sal) treated controls. Cadmium measurements in lung tissue indicated strain-dependent differences in disposition of QDs in the lung. Total glutathione levels in lung tissue were significantly correlated with percent neutrophils in BALF as well as with lung tissue Cd levels. Our findings indicate that QD-induced acute lung inflammation is mouse strain dependent, that it is heritable, and that the choice of mouse strain is an important consideration in planning QD toxicity studies. These data also suggest that formal genetic analyses using additional strains or recombinant inbred strains from these mice could be useful for discovering potential QD-induced inflammation susceptibility loci. PMID:26476918

  8. Functional Genomics Screening Utilizing Mutant Mouse Embryonic Stem Cells Identifies Novel Radiation-Response Genes

    PubMed Central

    Loesch, Kimberly; Galaviz, Stacy; Hamoui, Zaher; Clanton, Ryan; Akabani, Gamal; Deveau, Michael; DeJesus, Michael; Ioerger, Thomas; Sacchettini, James C.; Wallis, Deeann

    2015-01-01

    Elucidating the genetic determinants of radiation response is crucial to optimizing and individualizing radiotherapy for cancer patients. In order to identify genes that are involved in enhanced sensitivity or resistance to radiation, a library of stable mutant murine embryonic stem cells (ESCs), each with a defined mutation, was screened for cell viability and gene expression in response to radiation exposure. We focused on a cancer-relevant subset of over 500 mutant ESC lines. We identified 13 genes; 7 genes that have been previously implicated in radiation response and 6 other genes that have never been implicated in radiation response. After screening, proteomic analysis showed enrichment for genes involved in cellular component disassembly (e.g. Dstn and Pex14) and regulation of growth (e.g. Adnp2, Epc1, and Ing4). Overall, the best targets with the highest potential for sensitizing cancer cells to radiation were Dstn and Map2k6, and the best targets for enhancing resistance to radiation were Iqgap and Vcan. Hence, we provide compelling evidence that screening mutant ESCs is a powerful approach to identify genes that alter radiation response. Ultimately, this knowledge can be used to define genetic variants or therapeutic targets that will enhance clinical therapy. PMID:25853515

  9. Mutant PAX6 downregulates prohormone convertase 2 expression in mouse islets.

    PubMed

    Chen, Yuanyuan; Cao, Wenwan; Zhou, Shixin; Shen, Liang; Wen, Jinhua

    2013-11-01

    Transcriptional factor paired box 6 (PAX6) is very important for the development of the eyes, central nervous system, and pancreas. PAX6 mutations are associated with a diabetic phenotype and abnormal glucose metabolism. Our previous study showed that PAX6 directly bound to and activated the prohormone convertase 1/3 (Pc1/3) gene promoter and subsequently regulated proinsulin processing. Prohormone convertase 2 (PC2) is the essential enzyme for pancreatic proinsulin processing. To study the regulation of PAX6 in Pc2 expression, we did research on the pancreas of Pax6 R266Stop mutant mice, where truncated mutations happened in the C-terminal of the PAX6 protein. Our studies showed that the mutant PAX6 protein was stable and regulated the activity of Pc2 promoter as shown by luciferase activity assays. We found that the wild-type PAX6 protein imparts a transcriptional effect, and the mutant PAX6 can also regulate the downstream molecules. The results provide new insights into the mechanism of truncated PAX6 in regulating the functions of the pancreas and endocrine system. PMID:24047795

  10. Chromosomal changes in high- and low-invasive mouse lung adenocarcinoma cell strains derived from early passage mouse lung adenocarcinoma cell strains

    SciTech Connect

    Sargent, Linda M. Ensell, Mang X.; Ostvold, Anne-Carine; Baldwin, Kimberly T.; Kashon, Michael L.; Lowry, David T.; Senft, Jamie R.; Jefferson, Amy M.; Johnson, Robert C.; Li Zhi; Tyson, Frederick L.; Reynolds, Steven H.

    2008-11-15

    The incidence of adenocarcinoma of the lung is increasing in the United States, however, the difficulties in obtaining lung cancer families and representative samples of early to late stages of the disease have lead to the study of mouse models for lung cancer. We used Spectral Karyotyping (SKY), mapping with fluorescently labeled genomic clones (FISH), comparative genomic hybridization (CGH) arrays, gene expression arrays, Western immunoblot and real time polymerase chain reaction (PCR) to analyze nine pairs of high-invasive and low-invasive tumor cell strains derived from early passage mouse lung adenocarcinoma cells to detect molecular changes associated with tumor invasion. The duplication of chromosomes 1 and 15 and deletion of chromosome 8 were significantly associated with a high-invasive phenotype. The duplication of chromosome 1 at band C4 and E1/2-H1 were the most significant chromosomal changes in the high-invasive cell strains. Mapping with FISH and CGH array further narrowed the minimum region of duplication of chromosome 1 to 71-82 centimorgans (cM). Expression array analysis and confirmation by real time PCR demonstrated increased expression of COX-2, Translin (TB-RBP), DYRK3, NUCKS and Tubulin-{alpha}4 genes in the high-invasive cell strains. Elevated expression and copy number of these genes, which are involved in inflammation, cell movement, proliferation, inhibition of apoptosis and telomere elongation, were associated with an invasive phenotype. Similar linkage groups are altered in invasive human lung adenocarcinoma, implying that the mouse is a valid genetic model for the study of the progression of human lung adenocarcinoma.

  11. Autoantibodies against bromelainized mouse erythrocyte: strain distribution of serum idiotype expression and relative peritoneal cell activity.

    PubMed

    Kaushik, A; Poncet, P; Bussard, A

    1986-10-15

    Previously, we demonstrated that the naturally occurring mouse autoantibodies directed against bromelainized mouse red blood cells (BrMRBC) comprised a family of structurally related molecules bearing a common idiotypic determinant (CP) based on structural and idiotypic analysis of a series of anti-BrMRBC monoclonal autoantibodies derived from a fusion of peritoneal cells (PerC) with plasmacytomas. In the present studies, we have evaluated the quantitative expression of circulating CP idiotype related to autoantibodies against BrMRBC in relation to specific PerC anti-BrMRBC plaque-forming activity in an individual mouse of different strains. The data presented here show no direct relationship between serum CP idiotype expression and PerC anti-BrMRBC plaque-forming activity in an individual mouse of all strains tested. However, the circulating CP idiotype content is higher in strains, viz., CBA/J, NZB, C3H, BXSB, and Biozzi high responder (H) mice which exhibit a high perC autoantibody secretory activity against BrMRBC. The strains such as BALB/c, DBA2, SJL/J, CBA/N, and Biozzi low responder (L) express little or no circulating CP idiotype with a corresponding small or no PerC anti-BrMRBC activity. Furthermore, the PerC "auto"-immune phenomenon is markedly expressed in the normal CBA/J strain since these mice show a higher percentage ratio of CP idiotype over serum IgM (2.68%) as well as highest PerC anti-BrMRBC plaque-forming activity (11,319 +/- 18,029 plaques per million viable cells) compared to other normal and autoimmune strains tested. Nevertheless, the highest circulating serum CP idiotype (49.4 micrograms/ml) is observed in the autoimmune NZB mouse. The immunodeficient CBA/N mice fail to express detectable levels of CP idiotype in their serum. The experiments conducted in genetically selected outbred Biozzi (H and L) strain have revealed remarkable differences in serum CP idiotype expression as well as PerC anti-BrMRBC plaque-forming activity in these two

  12. Constitutive expression of the tzs gene from Agrobacterium tumefaciens virG mutant strains is responsible for improved transgenic plant regeneration in cotton meristem transformation.

    PubMed

    Ye, Xudong; Chen, Yurong; Wan, Yuechun; Hong, Yun-Jeong; Ruebelt, Martin C; Gilbertson, Larry A

    2016-03-01

    KEY MESSAGE : virG mutant strains of a nopaline type of Agrobacterium tumefaciens increase the transformation frequency in cotton meristem transformation. Constitutive cytokinin expression from the tzs gene in the virG mutant strains is responsible for the improvement. Strains of Agrobacterium tumefaciens were tested for their ability to improve cotton meristem transformation frequency. Two disarmed A. tumefaciens nopaline strains with either a virGN54D constitutively active mutation or virGI77V hypersensitive induction mutation significantly increased the transformation frequency in a cotton meristem transformation system. The virG mutant strains resulted in greener explants after three days of co-culture in the presence of light, which could be attributed to a cytokinin effect of the mutants. A tzs knockout strain of virGI77V mutant showed more elongated, less green explants and decreased cotton transformation frequency, as compared to a wild type parental strain, suggesting that expression of the tzs gene is required for transformation frequency improvement in cotton meristem transformation. In vitro cytokinin levels in culture media were tenfold higher in the virGN54D strain, and approximately 30-fold higher in the virGI77V strain, in the absence of acetosyringone induction, compared to the wild type strain. The cytokinin level in the virGN54D strain is further increased upon acetosyringone induction, while the cytokinin level in the virGI77V mutant is decreased by induction, suggesting that different tzs gene expression regulation mechanisms are present in the two virG mutant strains. Based on these data, we suggest that the increased cytokinin levels play a major role in increasing Agrobacterium attachment and stimulating localized division of the attached plant cells. PMID:26650837

  13. Comparative studies of lipopolysaccharide and exopolysaccharide from a virulent strain of Pseudomonas solanacearum and from three avirulent mutants.

    PubMed Central

    Drigues, P; Demery-Lafforgue, D; Trigalet, A; Dupin, P; Samain, D; Asselineau, J

    1985-01-01

    The composition of the Pseudomonas solanacearum lipolysaccharide (LPS) was found to be similar to that described for the LPS of enterobacteria. The lipid A contained fatty acids and glucosamine in a molar ratio of 5:2. The LPS fraction contained 2-keto-3-deoxyoctulosonic acid, L-glycero-D-mannoheptose, hexoses (glucose, rhamnose, and glucosamine), and a pentose (xylose). The LPSs from the wild-type strain (GMI1000), from the spontaneous rough mutant (GMI2000), and from their respective acridine orange-resistant (Acrr) mutants (GMI1178 and GMI2179) contained the same component sugars in their polysaccharide moieties, but the relative amounts of each sugar varied greatly. Spontaneous mutation to the rough type was characterized by a decrease in the ratio of rhamnose to glucose, whereas a reverse effect was seen for the acridine orange resistance mutation from the parent strains (GMI1000 and GMI2000) to the respective mutant strains (GMI1178 and GMI2179). The exopolysaccharide (EPS) from GMI1000 was found to be composed of two fractions: a heteropolysaccharide (galactosamine, glucose, and rhamnose) excluded from Sephadex G-50 and an additional glucan with a lower molecular weight. Strains GMI1000 and GMI1178 produced comparable amounts of EPS, GMI2179 synthesized less EPS, and GMI2000 produced no detectable EPS. High-pressure liquid chromatography and 13C nuclear magnetic resonance analyses revealed some differences between these EPSs. The glucan fraction seemed to be the major component of the EPS from GMI2179, whereas GMI1000 and GMI1178 EPSs contained both fractions and appeared to differ in the structures of their heteropolysaccharide fractions. Viscosity measurements confirmed differences between whole EPSs produced by the three strains. PMID:3988700

  14. The kinetic mechanism of wild-type and mutant mouse dihydrofolate reductases.

    PubMed

    Thillet, J; Adams, J A; Benkovic, S J

    1990-05-29

    A kinetic mechanism is presented for mouse dihydrofolate reductase that predicts all the steady-state parameters and full time-course kinetics. This mechanism was derived from association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance measurements. The major features of this kinetic mechanism are as follows: (1) the two native enzyme conformers, E1 and E2, bind ligands with varying affinities although only one conformer, E1, can support catalysis in the forward direction, (2) tetrahydrofolate dissociation is the rate-limiting step under steady-state turnover at low pH, and (3) the pH-independent rate of hydride transfer from NADPH to dihydrofolate is fast (khyd = 9000 s-1) and favorable (Keq = 100). The overall mechanism is similar in form to the Escherichia coli kinetic scheme (Fierke et al., 1987), although several differences are observed: (1) substrates and products predominantly bind the same form of the E. coli enzyme, and (2) the hydride transfer rate from NADPH to either folate or dihydrofolate is considerably faster for the mouse enzyme. The role of Glu-30 (Asp-27 in E. coli) in mouse DHFR has also been examined by using site-directed mutagenesis as a potential source of these differences. While aspartic acid is strictly conserved in all bacterial DHFRs, glutamic acid is conserved in all known eucaryotes. The two major effects of substituting Asp for Glu-30 in the mouse enzyme are (1) a decreased rate of folate reduction and (2) an increased rate of hydride transfer from NADPH to dihydrofolate.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Modeling Alzheimer’s Disease in Mouse without Mutant Protein Overexpression: Cooperative and Independent Effects of Aβ and Tau

    PubMed Central

    Guo, Qinxi; Li, Hongmei; Cole, Allysa L.; Hur, Ji-Yeun; Li, Yueming; Zheng, Hui

    2013-01-01

    Background Alzheimer’s disease (AD), the most common cause of dementia in the elderly, has two pathological hallmarks: Aβ plaques and aggregation of hyperphosphorylated tau (p-tau). Aβ is a cleavage product of Amyloid Precursor Protein (APP). Presenilin 1 (PS1) and presenilin 2 (PS2) are the catalytic subunit of γ-secretase, which cleaves APP and mediates Aβ production. Genetic mutations in APP, PSEN1 or PSEN2 can lead to early onset of familial AD (FAD). Although mutations in the tau encoding gene MAPT leads to a subtype of frontotemporal dementia and these mutations have been used to model AD tauopathy, no MAPT mutations have been found to be associated with AD. Results To model AD pathophysiology in mice without the gross overexpression of mutant transgenes, we created a humanized AD mouse model by crossing the APP and PSEN1 FAD knock-in mice with the htau mice which express wildtype human MAPT genomic DNA on mouse MAPT null background (APP/PS1/htau). The APP/PS1/htau mice displayed mild, age-dependent, Aβ plaques and tau hyperphosphorylation, thus successfully recapitulating the late-onset AD pathological hallmarks. Selected biochemical analyses, including p-tau western blot, γ-secretase activity assay, and Aβ ELISA, were performed to study the interaction between Aβ and p-tau. Subsequent behavioral studies revealed that the APP/PS1/htau mice showed reduced mobility in old ages and exaggerated fear response. Genetic analysis suggested that the fear phenotype is due to a synergic interaction between Aβ and p-tau, and it can be completely abolished by tau deletion. Conclusion The APP/PS1/htau model represents a valuable and disease-relevant late-onset pre-clinical AD animal model because it incorporates human AD genetics without mutant protein overexpression. Analysis of the mice revealed both cooperative and independent effects of Aβ and p-tau. PMID:24278307

  16. [Analysis of the structure and expression of the DIP1 gene in Drosophila melanogaster strains mutant for the flamenco gene].

    PubMed

    Nefedova, L N; Potanova, M V; Romanova, N I; Kim, A I

    2009-02-01

    DIP1 gene transcription was analyzed with the use of RT-PCR in three Drosophila melanogaster strains with the flamenco- phenotype (flam(SS), flam(MS), and flam(Ore)) and in one flamenco+ strain at the stages of embryos (0-24 h), third-instar larvae, and adult flies. The mutant strains flam(SS) and flam(Ore) lack an active copy of transposon gypsy. Theflam(MS) strain was obtained by introducing an active copy of gypsy in flies of theflam(SS) strain and is characterized by a high rate of gypsy transpositions. The experiments showed that at least five forms of DIP1 gene transcripts are produced. The form of cDNA corresponding to CDS DIP1-d was discovered only in embryos. It was found that DIP1 gene transcription depends on the age of flies: at the larval stage the level of transcription is significantly reduced. However, no reduction of gene transcription is observed in theflam(Ore) strain. These results suggest that the flamenco- phenotype may be associated with an alteration of DIP1 gene transcription, as in differentflamenco- strains the DIP1 gene expression is changed differently. PMID:19334614

  17. Enhanced desulfurization in a transposon-mutant strain of Rhodococcus erythropolis.

    PubMed

    Watanabe, Kimiko; Noda, Ken-ichi; Maruhashi, Kenji

    2003-08-01

    The dsz desulfurization gene cluster from Rhodococcus erythropolis strain KA2-5-1 was transferred into R. erythropolis strain MC1109, unable to desulfurize light gas oil (LGO), using a transposon-transposase complex. As a result, two recombinant strains, named MC0203 and MC0122, were isolated. Resting cells of strain MC0203 decreased the sulfur concentration of LGO from 120 mg l(-1) to 70 mg l(-1) in 2 h. The LGO-desulfurization activity of strain MC0203 was about twice that of strain MC0122 and KA2-5-1. The 10-methyl fatty acids of strain MC0203 were about 28%-41% that of strain MC1109. It is likely that strain MC0203 had a mutation involving alkylenation or methylation of delta9-unsaturated fatty acids caused by the transposon inserted in the chromosome, which increased the fluidity of cell membranes and enhanced the desulfurization activity.

  18. The morphogenesis of herpes simplex virus type 1 in infected parental mouse L fibroblasts and mutant gro29 cells.

    PubMed

    Jensen, Helle Lone; Norrild, Bodil

    2003-11-01

    Mutants of cell lines and viruses are important biological tools. The pathway of herpesvirus particle maturation and egress are contentious issues. The mutant gro29 line of mouse L cells is defective for egress of herpes simplex virus type 1 (HSV-1) virions, and a candidate for studies of virus-cell interactions. The properties of uninfected and HSV-1-infected L fibroblasts and gro29 cells investigated by protein assay, immunoblot, titration assay, immunofluorescence light microscopy and immunogold cryosection electron microscopy are reported. The ultrastructure of both HSV-1-infected L and gro29 cells confirmed primary envelopment of virions at the nuclear membranes followed by maturing multiple de-envelopments and re-envelopments in the endoplasmic reticulum and in the Golgi complex. The gro29 cells presented changed cytoskeleton, abolished egress of virions, and were defective in the trafficking of glycoproteins, giving rise to accumulation of viral particles and glycoproteins in the endoplasmic reticulum and the Golgi complex. The results suggest that gro29 cells harbour a causal underlying defect of the cytoskeleton in addition to the HSV-1-induced cytoskeletal changes.

  19. Characterization of a conditional interleukin-1 receptor 1 mouse mutant using the Cre/LoxP system.

    PubMed

    Abdulaal, Wesam H; Walker, Catherine R; Costello, Ryan; Redondo-Castro, Elena; Mufazalov, Ilgiz A; Papaemmanouil, Athina; Rothwell, Nancy J; Allan, Stuart M; Waisman, Ari; Pinteaux, Emmanuel; Müller, Werner

    2016-04-01

    IL-1 is a key cytokine known to drive chronic inflammation and to regulate many physiological, immunological, and neuroimmunological responses via actions on diverse cell types of the body. To determine the mechanisms of IL-1 actions as part of the inflammatory response in vivo, we generated a conditional IL-1 receptor 1 (IL-1R1) mouse mutant using the Cre/LoxP system (IL-1R1(fl/fl) ). In the mutant generated, exon 5, which encodes part of the extracellular-binding region of the receptor, is flanked by LoxP sites, thereby inactivating the two previously described functional IL-1R1 gene transcripts after Cre-mediated recombination. Using keratin 14-Cre driver mice, new IL-1R1 deficient (-/-) mice were subsequently generated, in which all signaling IL-1 receptor isoforms are deleted ubiquitously. Furthermore, using vav-iCre driver mice, we deleted IL-1 receptor isoforms in the hematopoietic system. In these mice, we show that both the IL-17 and IL-22 cytokine response is reduced, when mice are challenged by the helminth Trichuris muris. We are currently crossing IL-1R1(fl/fl) mice with different Cre-expressing mice in order to study mechanisms of acute and chronic inflammatory diseases.

  20. Initial locomotor sensitivity to cocaine varies widely among inbred mouse strains.

    PubMed

    Wiltshire, T; Ervin, R B; Duan, H; Bogue, M A; Zamboni, W C; Cook, S; Chung, W; Zou, F; Tarantino, L M

    2015-03-01

    Initial sensitivity to psychostimulants can predict subsequent use and abuse in humans. Acute locomotor activation in response to psychostimulants is commonly used as an animal model of initial drug sensitivity and has been shown to have a substantial genetic component. Identifying the specific genetic differences that lead to phenotypic differences in initial drug sensitivity can advance our understanding of the processes that lead to addiction. Phenotyping inbred mouse strain panels are frequently used as a first step for studying the genetic architecture of complex traits. We assessed locomotor activation following a single, acute 20 mg/kg dose of cocaine (COC) in males from 45 inbred mouse strains and observed significant phenotypic variation across strains indicating a substantial genetic component. We also measured levels of COC, the active metabolite, norcocaine and the major inactive metabolite, benzoylecgonine, in plasma and brain in the same set of inbred strains. Pharmacokinetic (PK) and behavioral data were significantly correlated, but at a level that indicates that PK alone does not account for the behavioral differences observed across strains. Phenotypic data from this reference population of inbred strains can be utilized in studies aimed at examining the role of psychostimulant-induced locomotor activation on drug reward and reinforcement and to test theories about addiction processes. Moreover, these data serve as a starting point for identifying genes that alter sensitivity to the locomotor stimulatory effects of COC.

  1. Initial locomotor sensitivity to cocaine varies widely among inbred mouse strains

    PubMed Central

    Wiltshire, T.; Ervin, R. B.; Duan, H.; Bogue, M. A.; Zamboni, W. C.; Cook, S.; Chung, W.; Zou, F.; Tarantino, L. M.

    2015-01-01

    Initial sensitivity to psychostimulants can predict subsequent use and abuse in humans. Acute locomotor activation in response to psychostimulants is commonly used as an animal model of initial drug sensitivity and has been shown to have a substantial genetic component. Identifying the specific genetic differences that lead to phenotypic differences in initial drug sensitivity can advance our understanding of the processes that lead to addiction. Phenotyping inbred mouse strain panels are frequently used as a first step for studying the genetic architecture of complex traits. We assessed locomotor activation following a single, acute 20 mg/kg dose of cocaine (COC) in males from 45 inbred mouse strains and observed significant phenotypic variation across strains indicating a substantial genetic component. We also measured levels of COC, the active metabolite, norcocaine and the major inactive metabolite, benzoylecgonine, in plasma and brain in the same set of inbred strains. Pharmacokinetic (PK) and behavioral data were significantly correlated, but at a level that indicates that PK alone does not account for the behavioral differences observed across strains. Phenotypic data from this reference population of inbred strains can be utilized in studies aimed at examining the role of psychostimulant-induced locomotor activation on drug reward and reinforcement and to test theories about addiction processes. Moreover, these data serve as a starting point for identifying genes that alter sensitivity to the locomotor stimulatory effects of COC. PMID:25727211

  2. Control of RFM strain endogenous retrovirus in RFM mouse cells.

    PubMed

    Tennant, R W; Otten, J A; Wang, T W; Liou, R S; Brown, A; Yang, W K

    1983-01-01

    RFM/Un mice express an endogenous type C retrovirus throughout their life span in many tissues; primary or established embryo fibroblast cell cultures do not express a virus but can be induced by exposure to 5-iodo-2'-deoxyuridine. All of our sources yielded a single ecotropic virus (RFV) which appeared to be related more closely to the endogenous N-tropic virus (WN1802N) of BALB/c mice than to Gross leukemia virus on the basis of two-dimensional gel electropherograms of virion proteins. No xenotropic or recombinant viruses were isolated by cocultivation techniques. RFV is N-tropic, and RFM/Un cells possess the Fv-1n allele, as indicated by restriction of B-tropic virus and susceptibility to Gross strain N-tropic virus. However, RFM cells are highly resistant to RFV and other endogenous N-tropic viruses. This resistance is expressed by two-hit titration kinetics and by inhibition of viral linear duplex DNA formation. This is similar to the effects of the Fv-1 locus, but preliminary work has shown no apparent genetic linkage between the two restrictions. The relative strength of the restriction, the presence of a single class of ecotropic virus, and the absence of recombinant viruses suggest that in RFM mice virus is expressed only in cells in which it is induced and not by cell-to-cell transmission.

  3. Ex vivo determination of bone tissue strains for an in vivo mouse tibial loading model

    PubMed Central

    Carriero, Alessandra; Abela, Lisa; Pitsillides, Andrew A.; Shefelbine, Sandra J.

    2014-01-01

    Previous studies introduced the digital image correlation (DIC) as a viable technique for measuring bone strain during loading. In this study, we investigated the sensitivity of a DIC system in determining surface strains in a mouse tibia while loaded in compression through the knee joint. Specifically, we examined the effect of speckle distribution, facet size and overlap, initial vertical alignment of the bone into the loading cups, rotation with respect to cameras, and ex vivo loading configurations on the strain contour maps measured with a DIC system. We loaded tibiae of C57BL/6 mice (12 and 18 weeks old male) up to 12 N at 8 N/min. Images of speckles on the bone surface were recorded at 1 N intervals and DIC was used to compute strains. Results showed that speckles must have the correct size and density with respect to the facet size of choice for the strain distribution to be computed and reproducible. Initial alignment of the bone within the loading cups does not influence the strain distribution measured during peak loading, but bones must be placed in front of the camera with the same orientation in order for strains to be comparable. Finally, the ex vivo loading configurations with the tibia attached to the entire mouse, or to the femur and foot, or only to the foot, showed different strain contour maps. This work provides a better understanding of parameters affecting full field strain measurements from DIC in ex vivo murine tibial loading tests. PMID:24835472

  4. Firefly Luciferase Mutants Allow Substrate-Selective Bioluminescence Imaging in the Mouse Brain.

    PubMed

    Adams, Spencer T; Mofford, David M; Reddy, G S Kiran Kumar; Miller, Stephen C

    2016-04-11

    Bioluminescence imaging is a powerful approach for visualizing specific events occurring inside live mice. Animals can be made to glow in response to the expression of a gene, the activity of an enzyme, or the growth of a tumor. But bioluminescence requires the interaction of a luciferase enzyme with a small-molecule luciferin, and its scope has been limited by the mere handful of natural combinations. Herein, we show that mutants of firefly luciferase can discriminate between natural and synthetic substrates in the brains of live mice. When using adeno-associated viral (AAV) vectors to express luciferases in the brain, we found that mutant luciferases that are inactive or weakly active with d-luciferin can light up brightly when treated with the aminoluciferins CycLuc1 and CycLuc2 or their respective FAAH-sensitive luciferin amides. Further development of selective luciferases promises to expand the power of bioluminescence and allow multiple events to be imaged in the same live animal.

  5. Firefly Luciferase Mutants Allow Substrate-Selective Bioluminescence Imaging in the Mouse Brain.

    PubMed

    Adams, Spencer T; Mofford, David M; Reddy, G S Kiran Kumar; Miller, Stephen C

    2016-04-11

    Bioluminescence imaging is a powerful approach for visualizing specific events occurring inside live mice. Animals can be made to glow in response to the expression of a gene, the activity of an enzyme, or the growth of a tumor. But bioluminescence requires the interaction of a luciferase enzyme with a small-molecule luciferin, and its scope has been limited by the mere handful of natural combinations. Herein, we show that mutants of firefly luciferase can discriminate between natural and synthetic substrates in the brains of live mice. When using adeno-associated viral (AAV) vectors to express luciferases in the brain, we found that mutant luciferases that are inactive or weakly active with d-luciferin can light up brightly when treated with the aminoluciferins CycLuc1 and CycLuc2 or their respective FAAH-sensitive luciferin amides. Further development of selective luciferases promises to expand the power of bioluminescence and allow multiple events to be imaged in the same live animal. PMID:26991209

  6. p62/SQSTM1 differentially removes the toxic mutant androgen receptor via autophagy and inclusion formation in a spinal and bulbar muscular atrophy mouse model.

    PubMed

    Doi, Hideki; Adachi, Hiroaki; Katsuno, Masahisa; Minamiyama, Makoto; Matsumoto, Shinjiro; Kondo, Naohide; Miyazaki, Yu; Iida, Madoka; Tohnai, Genki; Qiang, Qiang; Tanaka, Fumiaki; Yanagawa, Toru; Warabi, Eiji; Ishii, Tetsuro; Sobue, Gen

    2013-05-01

    Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders that are caused by the expansion of trinucleotide CAG repeats in the causative genes. Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease that is caused by the expansion of a polyQ tract within the androgen receptor (AR). p62 is a ubiquitin- and light-chain 3-binding protein that is known to regulate the degradation of targeted proteins via autophagy and inclusion formation. In this study, we examined the effects of p62 depletion and overexpression on cultured cells and in a transgenic mouse model that overexpressed the mutant AR. Here, we demonstrate that depletion of p62 significantly exacerbated motor phenotypes and the neuropathological outcome, whereas overexpression of p62 protected against mutant AR toxicity in SBMA mice. Depletion of p62 significantly increased the levels of monomeric mutant AR and mutant AR protein complexes in an SBMA mouse model via the impairment of autophagic degradation. In addition, p62 overexpression improved SBMA mouse phenotypes by inducing cytoprotective inclusion formation. Our results demonstrate that p62 provides two different therapeutic targets in SBMA pathogenesis: (1) autophagy-dependent degradation and (2) benevolent inclusion formation of the mutant AR.

  7. Increase in Ty1 cDNA Recombination in Yeast sir4 Mutant Strains at High Temperature

    PubMed Central

    Radford, Sarah J.; Boyle, Meredith L.; Sheely, Catherine J.; Graham, Joel; Haeusser, Daniel P.; Zimmerman, Leigh; Keeney, Jill B.

    2004-01-01

    Transposition of the Ty1 element of the yeast Saccharomyces cerevisiae is temperature sensitive. We have identified a null allele of the silent information regulator gene SIR4 as a host mutant that allows for transposition at high temperature. We show that the apparent increase in transposition activity in sir4 mutant strains at high temperature is dependent on the RAD52 gene and is thus likely resulting from an increase in Ty1 cDNA recombination, rather than in IN-mediated integration. General cellular recombination is not increased at high temperature, suggesting that the increase in recombination at high temperature in sir4 mutants is specific for Ty1 cDNA. Additionally, this high-temperature Ty1 recombination was found to be dependent on functional Sir2p and Sir3p. We speculate that the increase in recombination seen in sir4 mutants at high temperature may be due to changes in chromatin structure or Ty1 interactions with chromosomal structures resulting in higher recombination rates. PMID:15454529

  8. Tissue persistence and vaccine efficacy of tricarboxylic acid cycle and one-carbon metabolism mutant strains of Edwardsiella ictaluri.

    PubMed

    Dahal, Neeti; Abdelhamed, Hossam; Karsi, Attila; Lawrence, Mark L

    2014-06-30

    Edwardsiella ictaluri causes enteric septicemia in fish. Recently, we reported construction of E. ictaluri mutants with single and double gene deletions in tricarboxylic acid cycle (TCA) and one-carbon (C-1) metabolism. Here, we report the tissue persistence, virulence, and vaccine efficacy of TCA cycle (EiΔsdhC, EiΔfrdA, and EiΔmdh), C-1 metabolism (EiΔgcvP and EiΔglyA), and combination mutants (EiΔfrdAΔsdhC, EiΔgcvPΔsdhC, EiΔmdhΔsdhC, and EiΔgcvPΔglyA) in channel catfish. The tissue persistence study showed that EiΔsdhC, EiΔfrdA, EiΔfrdAΔsdhC, and EiΔgcvPΔsdhC were able to invade catfish and persist until 11 days post-infection. Vaccination of catfish fingerlings with all nine mutants provided significant (P<0.05) protection against subsequent challenge with the virulent parental strain. Vaccinated catfish fingerlings had 100% survival when subsequently challenged by immersion with wild-type E. ictaluri except for EiΔgcvPΔglyA and EiΔgcvP. Mutant EiΔgcvPΔsdhC was found to be very good at protecting catfish fry, as evidenced by 10-fold higher survival compared to non-vaccinated fish.

  9. Francisella tularensis live vaccine strain folate metabolism and pseudouridine synthase gene mutants modulate macrophage caspase-1 activation.

    PubMed

    Ulland, Tyler K; Janowski, Ann M; Buchan, Blake W; Faron, Matthew; Cassel, Suzanne L; Jones, Bradley D; Sutterwala, Fayyaz S

    2013-01-01

    Francisella tularensis is a Gram-negative bacterium and the causative agent of the disease tularemia. Escape of F. tularensis from the phagosome into the cytosol of the macrophage triggers the activation of the AIM2 inflammasome through a mechanism that is not well understood. Activation of the AIM2 inflammasome results in autocatalytic cleavage of caspase-1, resulting in the processing and secretion of interleukin-1β (IL-1β) and IL-18, which play a crucial role in innate immune responses to F. tularensis. We have identified the 5-formyltetrahydrofolate cycloligase gene (FTL_0724) as being important for F. tularensis live vaccine strain (LVS) virulence. Infection of mice in vivo with a F. tularensis LVS FTL_0724 mutant resulted in diminished mortality compared to infection of mice with wild-type LVS. The FTL_0724 mutant also induced increased inflammasome-dependent IL-1β and IL-18 secretion and cytotoxicity in macrophages in vitro. In contrast, infection of macrophages with a F. tularensis LVS rluD pseudouridine synthase (FTL_0699) mutant resulted in diminished IL-1β and IL-18 secretion from macrophages in vitro compared to infection of macrophages with wild-type LVS. In addition, the FTL_0699 mutant was not attenuated in vivo. These findings further illustrate that F. tularensis LVS possesses numerous genes that influence its ability to activate the inflammasome, which is a key host strategy to control infection with this pathogen in vivo.

  10. Francisella tularensis Live Vaccine Strain Folate Metabolism and Pseudouridine Synthase Gene Mutants Modulate Macrophage Caspase-1 Activation

    PubMed Central

    Ulland, Tyler K.; Janowski, Ann M.; Buchan, Blake W.; Faron, Matthew; Cassel, Suzanne L.; Jones, Bradley D.

    2013-01-01

    Francisella tularensis is a Gram-negative bacterium and the causative agent of the disease tularemia. Escape of F. tularensis from the phagosome into the cytosol of the macrophage triggers the activation of the AIM2 inflammasome through a mechanism that is not well understood. Activation of the AIM2 inflammasome results in autocatalytic cleavage of caspase-1, resulting in the processing and secretion of interleukin-1β (IL-1β) and IL-18, which play a crucial role in innate immune responses to F. tularensis. We have identified the 5-formyltetrahydrofolate cycloligase gene (FTL_0724) as being important for F. tularensis live vaccine strain (LVS) virulence. Infection of mice in vivo with a F. tularensis LVS FTL_0724 mutant resulted in diminished mortality compared to infection of mice with wild-type LVS. The FTL_0724 mutant also induced increased inflammasome-dependent IL-1β and IL-18 secretion and cytotoxicity in macrophages in vitro. In contrast, infection of macrophages with a F. tularensis LVS rluD pseudouridine synthase (FTL_0699) mutant resulted in diminished IL-1β and IL-18 secretion from macrophages in vitro compared to infection of macrophages with wild-type LVS. In addition, the FTL_0699 mutant was not attenuated in vivo. These findings further illustrate that F. tularensis LVS possesses numerous genes that influence its ability to activate the inflammasome, which is a key host strategy to control infection with this pathogen in vivo. PMID:23115038

  11. A search for radiosensitive mouse mutants by use of the micronucleus technique.

    PubMed

    van Buul, P P; Tuinenburg-Bolraap, A; Searle, A G; Natarajan, A T

    1987-01-01

    In order to identify radiosensitive mutations in mice, 26 genetically well defined mutations in 26 different combinations of homozygous, hemizygous or heterozygous conditions, together with normal mice and mutagen-sensitive MS/Ae mice were analysed for the induction of micronuclei by X-rays in bone-marrow cells. For each mutant two doses of 0.5 and 1.0 Gy, two sampling times of 18 and 27 h after irradiation and unirradiated controls were studied. Using our criteria, homozygous contrasted allele of steel (Slcon), scabby (scb), viable dominant spotting (Wv), quaking (qk), fidget (fi) and postaxial hemimelia (px), heterozygous lurcher (Lc), hemizygous gyro (Gy), the compounds Slcon/grizzle-belly (SlgbH) and Wv/rump-white (Rw) and MS/Ae mice, were regarded as radiosensitive, with Slcon/Slcon the highest in rank order. Homozygous wabbler-lethal (wl) and wasted (wst) showed hyposensitivity which for the latter may be connected with enhanced cell killing.

  12. Mouse chromosome 11 harbors genetic determinants of hippocampal strain-specific nicotinic receptor expression.

    PubMed

    Rogers, Scott W; Weis, Janis J; Ma, Ying; Teuscher, Cory; Gahring, Lorise C

    2008-01-01

    Differences between isogenic mouse strains in cellular expression of the neuronal nicotinic acetylcholine (ACh) receptor subunit alpha 4 (nAChR alpha 4) by the dorsal hippocampus are well known. To investigate further the genetic basis of these variations, expression of the nAChR alpha 4 subunit was measured in congenic mouse lines derived from two strains exhibiting notable divergence in the expression of this subunit: C3H and C57BL/6. Congenic lines carrying reciprocally introgressed regions (quantitative trait loci; QTL) from chromosomes 4, 5, and 12 each retained the phenotype most closely associated with the parental strain. However, in congenic lines harboring the reciprocal transfer of a chromosome 11 QTL, a characteristic difference in the ratio of interneurons versus astrocytes expressing nAChR alpha 4 in the CA1 region is reversed relative to the parental strain. These finding suggest that this chromosomal segment harbors genes that regulate strain distinct hippocampal morphology that is revealed by nAChR alpha 4 expression.

  13. BCL-2 and mutant NRAS interact physically and functionally in a mouse model of progressive myelodysplasia.

    PubMed

    Omidvar, Nader; Kogan, Scott; Beurlet, Stephanie; le Pogam, Carole; Janin, Anne; West, Robert; Noguera, Maria-Elena; Reboul, Murielle; Soulie, Annie; Leboeuf, Christophe; Setterblad, Niclas; Felsher, Dean; Lagasse, Eric; Mohamedali, Azim; Thomas, N Shaun B; Fenaux, Pierre; Fontenay, Michaela; Pla, Marika; Mufti, Ghulam J; Weissman, Irving; Chomienne, Christine; Padua, Rose Ann

    2007-12-15

    Myelodysplastic syndromes (MDS) are clonal stem cell hematologic disorders that evolve to acute myeloid leukemia (AML) and thus model multistep leukemogenesis. Activating RAS mutations and overexpression of BCL-2 are prognostic features of MDS/AML transformation. Using NRASD12 and BCL-2, we created two distinct models of MDS and AML, where human (h)BCL-2 is conditionally or constitutively expressed. Our novel transplantable in vivo models show that expression of hBCL-2 in a primitive compartment by mouse mammary tumor virus-long terminal repeat results in a disease resembling human MDS, whereas the myeloid MRP8 promoter induces a disease with characteristics of human AML. Expanded leukemic stem cell (Lin(-)/Sca-1(+)/c-Kit(+)) populations and hBCL-2 in the increased RAS-GTP complex within the expanded Sca-1(+) compartment are described in both MDS/AML-like diseases. Furthermore, the oncogenic compartmentalizations provide the proapoptotic versus antiapoptotic mechanisms, by activating extracellular signal-regulated kinase and AKT signaling, in determination of the neoplastic phenotype. When hBCL-2 is switched off with doxycycline in the MDS mice, partial reversal of the phenotype was observed with persistence of bone marrow blasts and tissue infiltration as RAS recruits endogenous mouse (m)BCL-2 to remain active, thus demonstrating the role of the complex in the disease. This represents the first in vivo progression model of MDS/AML dependent on the formation of a BCL-2:RAS-GTP complex. The colocalization of BCL-2 and RAS in the bone marrow of MDS/AML patients offers targeting either oncogene as a therapeutic strategy.

  14. Strain-Dependent Anterior Segment Dysgenesis and Progression to Glaucoma in Col4a1 Mutant Mice

    PubMed Central

    Mao, Mao; Smith, Richard S.; Alavi, Marcel V.; Marchant, Jeffrey K.; Cosma, Mihai; Libby, Richard T.; John, Simon W. M.; Gould, Douglas B.

    2015-01-01

    Purpose Mutations in the gene encoding collagen type IV alpha 1 (COL4A1) cause multisystem disorders including anterior segment dysgenesis (ASD) and optic nerve hypoplasia. The penetrance and severity of individual phenotypes depends on genetic context. Here, we tested the effects of a Col4a1 mutation in two different genetic backgrounds to compare how genetic context influences ocular dysgenesis, IOP, and progression to glaucoma. Methods Col4a1 mutant mice maintained on a C57BL/6J background were crossed to either 129S6/SvEvTac or CAST/EiJ and the F1 progeny were analyzed by slit-lamp biomicroscopy and optical coherence tomography. We also measured IOPs and compared tissue sections of eyes and optic nerves. Results. We found that the CAST/EiJ inbred strain has a relatively uniform and profound suppression on the effects of Col4a1 mutation and that mutant CASTB6F1 mice were generally only very mildly affected. In contrast, mutant 129B6F1 mice had more variable and severe ASD and IOP dysregulation that were associated with glaucomatous signs including lost or damaged retinal ganglion cell axons and excavation of the optic nerve head. Conclusions. Ocular defects in Col4a1 mutant mice model ASD and glaucoma that are observed in a subset of patients with COL4A1 mutations. We demonstrate that different inbred strains of mice give graded severities of ASD and we detected elevated IOP and glaucomatous damage in 129B6F1, but not CASTB6F1 mice that carried a Col4a1 mutation. These data demonstrate that genetic context differences are one factor that may contribute to the variable penetrance and severity of ASD and glaucoma in patients with COL4A1 mutations. PMID:26567795

  15. Differences in saccharin preference and genetic alterations of the Tas1r3 gene among senescence-accelerated mouse strains and their parental AKR/J strain.

    PubMed

    Niimi, Kimie; Takahashi, Eiki

    2014-05-10

    The senescence-accelerated mouse (SAM) is used as an animal model of senescence acceleration and age-associated disorders. SAM is derived from unexpected crosses between the AKR/J and unknown mouse strains. There are nine senescence-prone (SAMP) strains and three senescence-resistant (SAMR) strains. Although SAMP strains exhibit strain-specific and age-related pathological changes, the genes responsible for the pathologic changes in SAMP strains have not been comprehensively identified. In the present study, we evaluated sweet taste perception using the two-bottle test. We compared genotypes of the taste related gene, Tas1r3, using SAM strains and the parental AKR/J strain. The two-bottle test revealed that SAMR1 (R1), SAMP6 (P6), SAMP8 (P8), and SAMP10 (P10) mice were saccharin-preferring strains, whereas AKR/J did not prefer saccharin. All genotypes of the R1, P6, P8, and P10 strains at the polymorphic sites in Tas1r3, which is known to influence saccharin preference, were identical to those of C57BL6/J, a well-known saccharin-preferring strain, and were completely different from those of the parental AKR/J strain. These genetic alterations in SAM strains appear to arise from an unknown strain that is thought to have been crossed with AKR/J initially. PMID:24726396

  16. Differences in saccharin preference and genetic alterations of the Tas1r3 gene among senescence-accelerated mouse strains and their parental AKR/J strain.

    PubMed

    Niimi, Kimie; Takahashi, Eiki

    2014-05-10

    The senescence-accelerated mouse (SAM) is used as an animal model of senescence acceleration and age-associated disorders. SAM is derived from unexpected crosses between the AKR/J and unknown mouse strains. There are nine senescence-prone (SAMP) strains and three senescence-resistant (SAMR) strains. Although SAMP strains exhibit strain-specific and age-related pathological changes, the genes responsible for the pathologic changes in SAMP strains have not been comprehensively identified. In the present study, we evaluated sweet taste perception using the two-bottle test. We compared genotypes of the taste related gene, Tas1r3, using SAM strains and the parental AKR/J strain. The two-bottle test revealed that SAMR1 (R1), SAMP6 (P6), SAMP8 (P8), and SAMP10 (P10) mice were saccharin-preferring strains, whereas AKR/J did not prefer saccharin. All genotypes of the R1, P6, P8, and P10 strains at the polymorphic sites in Tas1r3, which is known to influence saccharin preference, were identical to those of C57BL6/J, a well-known saccharin-preferring strain, and were completely different from those of the parental AKR/J strain. These genetic alterations in SAM strains appear to arise from an unknown strain that is thought to have been crossed with AKR/J initially.

  17. In vitro and in vivo characterization of a Bordetella bronchiseptica mutant strain with a deep rough lipopolysaccharide structure.

    PubMed

    Sisti, Federico; Fernández, Julieta; Rodríguez, María Eugenia; Lagares, Antonio; Guiso, Nicole; Hozbor, Daniela Flavia

    2002-04-01

    Bordetella bronchiseptica is closely related to Bordetella pertussis, which produces respiratory disease primarily in mammals other than humans. However, its importance as a human pathogen is being increasingly recognized. Although a large amount of research on Bordetella has been generated regarding protein virulence factors, the participation of the surface lipopolysaccharide (LPS) during B. bronchiseptica infection is less understood. To get a better insight into this matter, we constructed and characterized the behavior of an LPS mutant with the deepest possible rough phenotype. We generated the defective mutant B. bronchiseptica LP39 on the waaC gene, which codes for a heptosyl transferase involved in the biosynthesis of the core region of the LPS molecule. Although in B. bronchiseptica LP39 the production of the principal virulence determinants adenylate cyclase-hemolysin, filamentous hemagglutinin, and pertactin persisted, the quantity of the two latter factors was diminished, with the levels of pertactin being the most greatly affected. Furthermore, the LPS of B. bronchiseptica LP39 did not react with sera obtained from mice that had been infected with the parental strain, indicating that this defective LPS is immunologically different from the wild-type LPS. In vivo experiments demonstrated that the ability to colonize the respiratory tract is reduced in the mutant, being effectively cleared from lungs within 5 days, whereas the parental strain survived at least for 30 days. In vitro experiments have demonstrated that, although B. bronchiseptica LP39 was impaired for adhesion to human epithelial cells, it is still able to survive within the host cells as efficiently as the parental strain. These results seem to indicate that the deep rough form of B. bronchiseptica LPS cannot represent a dominant phenotype at the first stage of colonization. Since isolates with deep rough LPS phenotype have already been obtained from human B. bronchiseptica chronic

  18. Recapitulation of the ovum mutant (Om) phenotype and loss of Om locus polarity in cloned mouse embryos.

    PubMed

    Gao, Shaorong; Wu, Guangming; Han, Zhiming; de la Casa-Esperón, Elena; Sapienza, Carmen; Latham, Keith E

    2005-02-01

    The ovum mutant (Om) locus in mice affects early interactions between sperm and egg that in turn affect viability of embryos beyond the morula stage. Crosses of DDK females to males of many other inbred strains are 95% lethal around the morula stage, whereas reciprocal crosses are fully viable. Available data indicate that the early lethality is the result of an interaction between a factor in the ooplasm and the paternal genome. In this study, we examined whether this lethal interaction would likewise occur in cloned embryos produced by somatic cell nuclear transfer. We find that the Om effect is recapitulated but that the parental origin effect at the Om locus is no longer evident in cloned embryos.

  19. [Separation of the biosynthesis products of a mutant strain of Actinomyces chrysomallus var. carotenoides and the identification of actinomycin antibiotics].

    PubMed

    Sverdlova, A N; Nefelova, M V; Silaev, A B

    1979-01-01

    An orange antibiotically active substance isolated from the mycelium of a mutant strain of Actinomyces chrysomallus var. carotenoides was identified as a mixture of actinomycins according to its light absorption spectra, circular dichroism spectra, IR spectra and chromatographic comparison with the standard samples. A scheme for successive extraction of the biologically active substances from the mycelium resulting in isolation of a fraction enriched with antibiotic substances and a fraction enriched with pigments is presented. A method for separation and purification of 3 groups of biologically active substances from the mycelium enriched extract was developed.

  20. Characterization of the endemic equilibrium and response to mutant injection in a multi-strain disease model.

    PubMed

    Aquino, Tomás; Bolster, Diogo; Nunes, Ana

    2015-03-01

    We explore a model of an antigenically diverse infection whose otherwise identical strains compete through cross-immunity. We assume that individuals may produce upon infection different numbers of antibody types, each of which matches the antigenic configuration of a particular epitope, and that one matching antibody type grants total immunity against a challenging strain. In order to reduce the number of equations involved in the analytic description of the dynamics, we follow the strategy proposed by Kryazhimskiy et al. (2007) and apply a low-order closure reminiscent of a pair approximation. Using this approximation, we go beyond the numerical studies of Kryazhimskiy et al. (2007) and explore the analytic properties of the ensuing model in the absence of mutation. We characterize its endemic equilibrium, comparing with the results of agent based simulations of the full model to assess the performance of the closure assumption. We show that a particular choice of immune response leads to a degenerate endemic equilibrium, where different strain prevalences may exist, breaking the symmetry of the model. Finally we study the behavior of the system under the injection of mutant strains. We find that the build up of diversity from a single founding strain is extremely unlikely for different choices of the population׳s immune response. PMID:25496729

  1. Impaired development of hippocampal mossy fibre synapses in mouse mutants for the presynaptic scaffold protein Bassoon.

    PubMed

    Lanore, Frederic; Blanchet, Christophe; Fejtova, Anna; Pinheiro, Paulo; Richter, Karin; Balschun, Detlef; Gundelfinger, Eckart; Mulle, Christophe

    2010-06-15

    Bassoon, a protein highly concentrated at the synaptic active zone, is thought to participate in the organization of the cytomatrix at the site of neurotransmitter release. Bassoon is amongst the first proteins to accumulate at newly formed synaptic junctions, raising the question of the functional role of this protein in the early stages of synaptic development. Here we show that the course of synaptic maturation of hippocampal mossy fibre (MF) synapses (glutamatergic synapses with multiple release sites) is markedly altered during the first 2 weeks of postnatal development in mutant mice lacking the central region of Bassoon (Bsn(-/-) mice). At postnatal day 7 (P7), Bsn(-/-) mice display large amplitude MF-EPSCs with decreased paired pulse ratios, an abnormality which may be linked to deficits in the organization of the presynaptic active zone. Surprisingly, 1 week later, decreased MF-EPSCs amplitude is observed in Bsn(-/-) mice, consistent with the inactivation of a subset of synaptic release sites. Finally, at more mature states a decreased posttetanic potentiation is observed at MF-synapses. These results support the notion that Bassoon is important for organizing the presynaptic active zone during the postnatal maturation of glutamatergic synapses.

  2. Jaw growth in the absence of teeth: the developmental morphology of edentulous mandibles using the p63 mouse mutant.

    PubMed

    Paradis, Mitchell R; Raj, Muhammad T; Boughner, Julia C

    2013-01-01

    Mammalian tooth and jaw development must be coordinated well enough that these systems continue to function together properly throughout growth, thus optimizing an animal's survival and fitness, as well as a species' success. The persistent question is how teeth and jaws remain developmentally and functionally viable despite sometimes monumental evolutionary changes to tooth and jaw shape and size. Here we used the p63 mouse mutant to test the effect of tooth development - or the lack thereof - on normal mandible developmental morphology. Using 3D geometric morphometrics, we compared for the first time mandible shape among mice with normal tooth and jaw development against p63 double knock-out mice, with failed tooth development but apparently normal jaw development. Mandible shape differed statistically between toothless (p63(-/-) ) and toothed (p63(+/-) , p63(+/+) ) mice as early as embryonic day (E) 18. As expected, most of the shape difference in the p63(-/-) mandibles was due to underdeveloped alveolar bone related to arrested odontogenesis in these E18-aged mice. Mandible shape did not differ statistically between p63(+/-) and p63(+/+) adult mice, which showed normal tooth development. Our results support the idea of a gene regulatory network that is exclusive to the mandible and independent of the dentition. This study also underscores the biomechanical impact of the teeth on the developing alveolar bone. Importantly, this work shows quantitatively that the p63 mutant is an apt model with which to study mandible morphogenesis in isolation of odontogenesis to clarify developmental relationships between the teeth and jaws.

  3. Regulation of TGF-β signalling by Fbxo11, the gene mutated in the Jeff otitis media mouse mutant

    PubMed Central

    Tateossian, Hilda; Hardisty-Hughes, Rachel E; Morse, Susan; Romero, Maria R; Hilton, Helen; Dean, Charlotte; Brown, Steve DM

    2009-01-01

    Background Jeff is a dominant mouse mutant displaying chronic otitis media. The gene underlying Jeff is Fbxo11, a member of the large F-box family, which are specificity factors for the SCF E3 ubiquitin ligase complex. Jeff homozygotes die shortly after birth displaying a number of developmental abnormalities including cleft palate and eyes open at birth. TGF-β signalling is involved in a number of epithelial developmental processes and we have investigated the impact of the Jeff mutation on the expression of this pathway. Results Phospho-Smad2 (pSmad2) is significantly upregulated in epithelia of Jeff homozygotes. Moreover, there was a significant increase in nuclear localization of pSmad2 in contrast to wild type. Mice heterozygous for both Jeff and Smad2 mutations recapitulate many of the features of the Jeff homozygous phenotype. However, tissue immunoprecipitations failed to detect any interaction between Fbxo11 and Smad2. Fbxo11 is known to neddylate p53, a co-factor of pSmad2, but we did not find any evidence of genetic interactions between Jeff and p53 mutants. Nevertheless, p53 levels are substantially reduced in Jeff mice suggesting that Fbxo11 plays a role in stabilizing p53. Conclusion Overall, our findings support a model whereby Fbxo11, possibly via stabilization of p53, is required to limit the accumulation of pSmad2 in the nucleus of epithelial cells of palatal shelves, eyelids and airways of the lungs. The finding that Fbxo11 impacts upon TGF-β signalling has important implications for our understanding of the underlying disease mechanisms of middle ear inflammatory disease. PMID:19580641

  4. The cytoplasmic NPM mutant induces myeloproliferation in a transgenic mouse model.

    PubMed

    Cheng, Ke; Sportoletti, Paolo; Ito, Keisuke; Clohessy, John G; Teruya-Feldstein, Julie; Kutok, Jeffery L; Pandolfi, Pier Paolo

    2010-04-22

    Although NPM1 gene mutations leading to aberrant cytoplasmic expression of nucleophosmin (NPMc(+)) are the most frequent genetic lesions in acute myeloid leukemia, there is yet no experimental model demonstrating their oncogenicity in vivo. We report the generation and characterization of a transgenic mouse model expressing the most frequent human NPMc(+) mutation driven by the myeloid-specific human MRP8 promoter (hMRP8-NPMc(+)). In parallel, we generated a similar wild-type NPM trans-genic model (hMRP8-NPM). Interestingly, hMRP8-NPMc(+) transgenic mice developed myeloproliferation in bone marrow and spleen, whereas nontransgenic littermates and hMRP8-NPM transgenic mice remained disease free. These findings provide the first in vivo evidence indicating that NPMc(+) confers a proliferative advantage in the myeloid lineage. No spontaneous acute myeloid leukemia was found in hMPR8-NPMc(+) or hMRP8-NPM mice. This model will also aid in the development of therapeutic regimens that specifically target NPMc(+).

  5. Copper distribution in fetus and placenta of the macular mutant mouse as a model of Menkes kinky hair disease.

    PubMed

    Xu, G Q; Yamano, T; Shimada, M

    1994-01-01

    Menkes kinky hair disease (MKHD) in humans is caused by a disturbance in copper homeostasis. A mutant mouse shows clinical and biochemical features very close to MKHD. In an attempt to elucidate the defect in copper transport, the copper distribution in various organs of 18-gestational-day-old macular mouse embryos, following administration by a single injection of saline (control) or 50 micrograms of CuCl2 on day 16 of gestation or by two injections on days 15 and 17 of gestation to the dams, was examined both biochemically and histochemically. The copper content in the hemizygous fetus (Ml/y) born to the homozygous mother, who had no copper injection during gestation, was lower in the brain and liver but higher in the placenta than in the respective organs of the normal fetus. When 50 micrograms of CuCl2 was injected into heterozygous dams (Ml/+) on day 16 of gestation, their hemizygous fetuses showed a slight increase in the copper content in the brain and liver, but the amount of copper in these organs was still less than that of the normal fetus. Conversely, the copper content in the placenta of the hemizygous fetus was far higher than that of the normal fetus. In the copper staining of the fetuses harvested from heterozygous dams, some fetuses showed copper deposition in the placenta, but not in the liver. The others showed no copper deposit in both the placenta and liver, thus indicating that the former were hemizygous for the mutation and the latter were normal littermates.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Brain immune cell composition and functional outcome after cerebral ischemia: comparison of two mouse strains

    PubMed Central

    Kim, Hyun Ah; Whittle, Stephanie C.; Lee, Seyoung; Chu, Hannah X.; Zhang, Shenpeng R.; Wei, Zihui; Arumugam, Thiruma V.; Vinh, Anthony; Drummond, Grant R.; Sobey, Christopher G.

    2014-01-01

    Inflammatory cells may contribute to secondary brain injury following cerebral ischemia. The C57Bl/6 mouse strain is known to exhibit a T helper 1-prone, pro-inflammatory type response to injury, whereas the FVB strain is relatively T helper 2-prone, or anti-inflammatory, in its immune response. We tested whether stroke outcome is more severe in C57Bl/6 than FVB mice. Male mice of each strain underwent sham surgery or 1 h occlusion of the middle cerebral artery followed by 23 h of reperfusion. Despite no difference in infarct size, C57Bl/6 mice displayed markedly greater functional deficits than FVB mice after stroke, as assessed by neurological scoring and hanging wire test. Total numbers of CD45+ leukocytes tended to be larger in the brains of C57Bl/6 than FVB mice after stroke, but there were marked differences in leukocyte composition between the two mouse strains. The inflammatory response in C57Bl/6 mice primarily involved T and B lymphocytes, whereas neutrophils, monocytes and macrophages were more prominent in FVB mice. Our data are consistent with the concept that functional outcome after stroke is dependent on the immune cell composition which develops following ischemic brain injury. PMID:25477780

  7. p53 suppression partially rescues the mutant phenotype in mouse models of DiGeorge syndrome

    PubMed Central

    Caprio, Cinzia; Baldini, Antonio

    2014-01-01

    T-box 1 (Tbx1), a gene encoding a T-box transcription factor, is required for embryonic development in humans and mice. Half dosage of this gene in humans causes most of the features of the DiGeorge or Velocardiofacial syndrome phenotypes, including aortic arch and cardiac outflow tract abnormalities. Here we found a strong genetic interaction between Tbx1 and transformation related protein 53 (Trp53). Indeed, genetic ablation of Trp53, or pharmacological inhibition of its protein product p53, rescues significantly the cardiovascular defects of Tbx1 heterozygous and hypomorphic mutants. We found that the Tbx1 and p53 proteins do not interact directly but both occupy a genetic element of Gbx2, which is required for aortic arch and cardiac outflow tract development, and is a known genetic interactor of Tbx1. We found that Gbx2 expression is down-regulated in Tbx1+/− embryos and is restored to normal levels in Tbx1+/−;Trp53+/− embryos. In addition, we found that the genetic element that binds both Tbx1 and p53 is highly enriched in H3K27 trimethylation, and upon p53 suppression H3K27me3 levels are reduced, along with Ezh2 enrichment. This finding suggests that the rescue of Gbx2 expression in Tbx1+/−;Trp53+/− embryos is due to reduction of repressive chromatin marks. Overall our data identify unexpected genetic interactions between Tbx1 and Trp53 and provide a proof of principle that developmental defects associated with reduced dosage of Tbx1 can be rescued pharmacologically. PMID:25197075

  8. Absence of linkage of apparently single gene mediated ADHD with the human syntenic region of the mouse mutant coloboma

    SciTech Connect

    Hess, E.J.; Rogan, P.K.; Domoto, M.

    1995-12-18

    Attention deficit disorder (ADHD) is a complex biobehavioral phenotype which affects up to 8% of the general population and often impairs social, academic, and job performance. Its origins are heterogeneous, but a significant genetic component is suggested by family and twin studies. The murine strain, coloboma, displays a spontaneously hyperactive phenotype that is responsive to dextroamphetamine and has been proposed as a genetic model for ADHD. Coloboma is a semi-dominant mutation that is caused by a hemizygous deletion of the SNAP-25 and other genes on mouse chromosome 2q. To test the possibility that the human homolog of the mouse coloboma gene(s) could be responsible for ADHD, we have carried out linkage studies with polymorphic markers in the region syntenic to coloboma (20p11-p12). Five families in which the pattern of inheritance of ADHD appears to be autosomal dominant were studied. Segregation analysis of the traits studied suggested that the best fitting model was a sex-influenced, single gene, Mendelian pattern. Several genetic models were evaluated based on estimates of penetrance, phenocopy rate, and allele frequency derived from our patient population and those of other investigators. No significant linkage was detected between the disease locus and markers spanning this chromosome 20 interval. 39 refs., 2 figs., 1 tab.

  9. Inactivation of cytochrome c oxidase by mutant SOD1s in mouse motoneuronal NSC-34 cells is independent from copper availability but is because of nitric oxide.

    PubMed

    Arciello, Mario; Capo, Concetta Rosa; Cozzolino, Mauro; Ferri, Alberto; Nencini, Monica; Carrì, Maria Teresa; Rossi, Luisa

    2010-01-01

    The copper-enzyme cytochrome c oxidase (Cytox) has been indicated as a primary molecular target of mutant copper, zinc superoxide dismutase (SOD1) in familial amyotrophic lateral sclerosis (fALS); however, the mechanism underlying its inactivation is still unclear. As the toxicity of mutant SOD1s could arise from their selective recruitment to mitochondria, it is conceivable that they might compete with Cytox for the mitochondrial copper pool causing Cytox inactivation. To investigate this issue, we used mouse motoneuronal neuroblastoma x spinal cord cell line-34, stably transfected for the inducible expression of low amounts of wild-type or mutant (G93A, H46R, and H80R) human SOD1s and compared the effects observed on Cytox with those obtained by copper depletion. We demonstrated that all mutants analyzed induced cell death and decreased the Cytox activity, but not the protein content of the Cytox subunit II, at difference with copper depletion that also affected subunit II protein. Copper supplementation did not counteract mutant hSOD1s toxicity. Otherwise, the treatment of neuroblastoma x spinal cord cell line-34 expressing G93A, H46R, or H80R hSOD1 mutants, and showing constitutive expression of iNOS and nNOS, with either a NO scavenger, or NOS inhibitors prevented the inhibition of Cytox activity and rescued cell viability. These results support the involvement of NO in mutant SOD1s-induced Cytox damage, and mitochondrial toxicity. PMID:19845829

  10. Regulators of pseudohyphal differentiation in Saccharomyces cerevisiae identified through multicopy suppressor analysis in ammonium permease mutant strains.

    PubMed Central

    Lorenz, M C; Heitman, J

    1998-01-01

    Nitrogen-starved diploid cells of the yeast Saccharomyces cerevisiae differentiate into a filamentous, pseudohyphal growth form. Recognition of nitrogen starvation is mediated, at least in part, by the ammonium permease Mep2p and the Galpha subunit Gpa2p. Genetic activation of the pheromone-responsive MAP kinase cascade, which is also required for filamentous growth, only weakly suppresses the filamentation defect of Deltamep2/Deltamep2 and Deltagpa2/Deltagpa2 strain. Surprisingly, deletion of Mep1p, an ammonium permease not previously thought to regulate differentiation, significantly enhances the potency of MAP kinase activation, such that the STE11-4 allele induces filamentation to near wild-type levels in Deltamep1/Deltamep1 Deltamep2/Deltamep2 and Deltamep1/Deltamep1 Deltagpa2/Deltagpa2 strains. To identify additional regulatory components, we isolated high-copy suppressors of the filamentation defect of the Deltamep1/Deltamep1 Deltamep2/Deltamep2 mutant. Multicopy expression of TEC1, PHD1, PHD2 (MSS10/MSN1/FUP4), MSN5, CDC6, MSS11, MGA1, SKN7, DOT6, HMS1, HMS2, or MEP2 each restored filamentation in a Deltamep1/Deltamep1 Deltamep2/Deltamep2 strain. Overexpression of SRK1 (SSD1), URE2, DAL80, MEP1, or MEP3 suppressed only the growth defect of the Deltamep1/Deltamep1 Deltamep2/Deltamep2 mutant strain. Characterization of these genes through deletion analysis and epistasis underscores the complexity of this developmental pathway and suggests that stress conditions other than nitrogen deprivation may also promote filamentous growth. PMID:9832522

  11. Industrial Robustness: Understanding the Mechanism of Tolerance for the Populus Hydrolysate-Tolerant Mutant Strain of Clostridium thermocellum

    PubMed Central

    Linville, Jessica L.; Rodriguez, Miguel; Land, Miriam; Syed, Mustafa H.; Engle, Nancy L.; Tschaplinski, Timothy J.; Mielenz, Jonathan R.; Cox, Chris D.

    2013-01-01

    Background An industrially robust microorganism that can efficiently degrade and convert lignocellulosic biomass into ethanol and next-generation fuels is required to economically produce future sustainable liquid transportation fuels. The anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum is a candidate microorganism for such conversions but it, like many bacteria, is sensitive to potential toxic inhibitors developed in the liquid hydrolysate produced during biomass processing. Microbial processes leading to tolerance of these inhibitory compounds found in the pretreated biomass hydrolysate are likely complex and involve multiple genes. Methodology/Principal Findings In this study, we developed a 17.5% v/v Populus hydrolysate tolerant mutant strain of C. thermocellum by directed evolution. The genome of the wild type strain, six intermediate population samples and seven single colony isolates were sequenced to elucidate the mechanism of tolerance. Analysis of the 224 putative mutations revealed 73 high confidence mutations. A longitudinal analysis of the intermediate population samples, a pan-genomic analysis of the isolates, and a hotspot analysis revealed 24 core genes common to all seven isolates and 8 hotspots. Genetic mutations were matched with the observed phenotype through comparison of RNA expression levels during fermentation by the wild type strain and mutant isolate 6 in various concentrations of Populus hydrolysate (0%, 10%, and 17.5% v/v). Conclusion/Significance The findings suggest that there are multiple mutations responsible for the Populus hydrolysate tolerant phenotype resulting in several simultaneous mechanisms of action, including increases in cellular repair, and altered energy metabolism. To date, this study provides the most comprehensive elucidation of the mechanism of tolerance to a pretreated biomass hydrolysate by C. thermocellum. These findings make important contributions to the development of industrially

  12. Elevated AIM2-mediated pyroptosis triggered by hypercytotoxic Francisella mutant strains is attributed to increased intracellular bacteriolysis

    PubMed Central

    Peng, Kaitian; Broz, Petr; Jones, Jonathan; Joubert, Lydia-Marie; Monack, Denise

    2011-01-01

    Intracellular bacterial pathogens Francisella novicida and the Live Vaccine Strain (LVS) are recognized in the macrophage cytosol by the AIM2 inflammasome, which leads to the activation of caspase-1 and the processing and secretion of active IL-1β, IL-18 and pyroptosis. Previous studies have reported that F. novicida and LVS mutants in specific genes (e.g., FTT0584, mviN and ripA) induce elevated inflammasome activation and hypercytotoxicity in host cells, leading to the proposal that F. novicida and LVS may have proteins that actively modulate inflammasome activation. However, there has been no direct evidence of such inflammasome evasion mechanisms. Here, we demonstrate for the first time that the above mutants, along with a wide range of F. novicida hypercytotoxic mutants that are deficient for membrane-associated proteins (ΔFTT0584, ΔmviN, ΔripA, ΔfopA and ΔFTN1217) or deficient for genes involved in O-antigen or LPS biosynthesis (ΔwbtA and ΔlpxH) lyse more intracellularly, thus activating increased levels of AIM2-dependent pyroptosis and other innate immune signaling pathways. This suggests that an inflammasome-specific evasion mechanism may not be present in F. novicida and LVS. Furthermore, future studies may need to consider increased bacterial lysis as a possible cause of elevated stimulation of multiple innate immune pathways when the protein composition or surface carbohydrates of the bacterial membrane is altered. PMID:21883803

  13. Attenuated mutant strain of Salmonella Typhimurium lacking the ZnuABC transporter contrasts tumor growth promoting anti-cancer immune response

    PubMed Central

    Chirullo, Barbara; Ammendola, Serena; Leonardi, Leonardo; Falcini, Roberto; Petrucci, Paola; Pistoia, Claudia; Vendetti, Silvia; Battistoni, Andrea; Pasquali, Paolo

    2015-01-01

    Salmonella Typhimurium has been shown to be highly effective as antitumor agent. The aim of this study was to investigate the tumor targeting efficacy and the mechanism of action of a specific attenuated mutant strain of Salmonella Typhimurium (STM) devoid of the whole operon coding for the high-affinity zinc transporter ZnuABC, which is required for bacterial growth in environments poor in zinc and for conferring full virulence to different Gram-negative pathogens. We showed that STM is able to penetrate and replicate into tumor cells in in vitro and in vivo models. The subcutaneous administration of STM in mammary adenocarcinoma mouse model led to both reduction of tumor growth and increase in life expectancy of STM treated mice. Moreover, investigating the potential mechanism behind the favorable clinical outcomes, we provide evidence that STM stimulates a potent inflammatory response and a specific immune pattern, recruiting a large number of innate and adaptive immune cells capable to contrast the immunosuppressive environment generated by tumors. PMID:26158862

  14. An Fgf8 Mouse Mutant Phenocopies Human 22q11 Deletion Syndrome

    PubMed Central

    Frank, Deborah U.; Fotheringham, Lori K.; Brewer, Judson A.; Muglia, Louis J.; Tristani-Firouzi, Martin; Capecchi, Mario R.; Moon, Anne M.

    2006-01-01

    SUMMARY Deletion of chromosome 22q11, the most common microdeletion detected in humans, is associated with a life-threatening array of birth defects. Although 90% of affected individuals share the same three megabase deletion, their phenotype is highly variable and includes craniofacial and cardiovascular anomalies, hypoplasia or aplasia of the thymus with associated deficiency of T cells, hypocalcemia with hypoplasia or aplasia of the parathyroids, and a variety of central nervous system abnormalities. Because ablation of neural crest in chicks produces many features of the deletion 22q11 syndrome, it has been proposed that haploinsufficiency in this region impacts neural crest function during cardiac and pharyngeal arch development. Few factors required for migration, survival, proliferation and subsequent differentiation of pharyngeal arch neural crest and mesoderm-derived mesenchyme into their respective cardiovascular, musculoskeletal, and glandular derivatives have been identified. However, the importance of epithelial-mesenchymal interactions and pharyngeal endoderm function is becoming increasingly clear. Fibroblast growth factor 8 is a signaling molecule expressed in the ectoderm and endoderm of the developing pharyngeal arches and known to play an important role in survival and patterning of first arch tissues. We demonstrate a dosage-sensitive requirement for FGF8 during development of pharyngeal arch, pharyngeal pouch and neural crest-derived tissues. We show that FGF8 deficient embryos have lethal malformations of the cardiac outflow tract, great vessels and heart due, at least in part, to failure to form the fourth pharyngeal arch arteries, altered expression of Fgf10 in the pharyngeal mesenchyme, and abnormal apoptosis in pharyngeal and cardiac neural crest. The Fgf8 mutants described herein display the complete array of cardiovascular, glandular and craniofacial phenotypes seen in human deletion 22q11 syndromes. This represents the first single gene

  15. Comparative evaluation of two vaccine candidates against experimental leishmaniasis due to Leishmania major infection in four inbred mouse strains.

    PubMed

    Benhnini, Fouad; Chenik, Mehdi; Laouini, Dhafer; Louzir, Hechmi; Cazenave, Pierre André; Dellagi, Koussay

    2009-11-01

    Experimental leishmaniasis in BALB/c and C57BL/6 mice are the most investigated murine models that were used for the preclinical evaluation of Leishmania vaccine candidates. We have previously described two new inbred mouse strains named PWK and MAI issued from feral founders that also support the development of experimental leishmaniasis due to L. major. In this study, we sought to determine whether different mouse inbred strains generate concordant or discordant results when used to evaluate the potential of Leishmania proteins to protect against experimental leishmaniasis. To this end, two Leishmania proteins, namely, LACK (for Leishmania homolog of receptor for activated C kinase) and LmPDI (for L. major protein disulfide isomerase) were compared for their capacity to protect against experimental leishmaniasis in PWK, MAI, BALB/c, and C57BL/6 inbred mouse strains. Our data show that the capacity of Leishmania proteins to confer protection depends on the mouse strain used, stressing the important role played by the genetic background in shaping the immune response against the pathogen. These results may have important implications for the preclinical evaluation of candidate Leishmania vaccines: rather than using a single mouse strain, a panel of different inbred strains of various genetic backgrounds should be tested in parallel. The antigen that confers protection in the larger range of inbred strains may have better chances to be also protective in outbred human populations and should be selected for clinical trials.

  16. Comparative Proteomic Analysis of Streptomyces lividans Wild-Type and ppk Mutant Strains Reveals the Importance of Storage Lipids for Antibiotic Biosynthesis

    PubMed Central

    Le Maréchal, Pierre; Decottignies, Paulette; Marchand, Christophe H.; Degrouard, Jeril; Jaillard, Danièle; Dulermo, Thierry; Froissard, Marine; Smirnov, Aleksey; Chapuis, Violaine

    2013-01-01

    Streptomyces lividans TK24 is a strain that naturally produces antibiotics at low levels, but dramatic overproduction of antibiotics occurs upon interruption of the ppk gene. However, the role of the Ppk enzyme in relation to the regulation of antibiotic biosynthesis remains poorly understood. In order to gain a better understanding of the phenotype of the ppk mutant, the proteomes of the wild-type (wt) and ppk mutant strains, grown for 96 h on R2YE medium limited in phosphate, were analyzed. Intracellular proteins were separated on two-dimensional (2D) gels, spots were quantified, and those showing a 3-fold variation or more were identified by mass spectrometry. The expression of 12 proteins increased and that of 29 decreased in the ppk mutant strain. Our results suggested that storage lipid degradation rather than hexose catabolism was taking place in the mutant. In order to validate this hypothesis, the triacylglycerol contents of the wt and ppk mutant strains of S. lividans as well as that of Streptomyces coelicolor M145, a strain that produces antibiotics at high levels and is closely related to S. lividans, were assessed using electron microscopy and thin-layer chromatography. These studies highlighted the large difference in triacylglycerol contents of the three strains and confirmed the hypothetical link between storage lipid metabolism and antibiotic biosynthesis in Streptomyces. PMID:23872561

  17. Comparative proteomic analysis of Streptomyces lividans Wild-Type and ppk mutant strains reveals the importance of storage lipids for antibiotic biosynthesis.

    PubMed

    Le Maréchal, Pierre; Decottignies, Paulette; Marchand, Christophe H; Degrouard, Jeril; Jaillard, Danièle; Dulermo, Thierry; Froissard, Marine; Smirnov, Aleksey; Chapuis, Violaine; Virolle, Marie-Joelle

    2013-10-01

    Streptomyces lividans TK24 is a strain that naturally produces antibiotics at low levels, but dramatic overproduction of antibiotics occurs upon interruption of the ppk gene. However, the role of the Ppk enzyme in relation to the regulation of antibiotic biosynthesis remains poorly understood. In order to gain a better understanding of the phenotype of the ppk mutant, the proteomes of the wild-type (wt) and ppk mutant strains, grown for 96 h on R2YE medium limited in phosphate, were analyzed. Intracellular proteins were separated on two-dimensional (2D) gels, spots were quantified, and those showing a 3-fold variation or more were identified by mass spectrometry. The expression of 12 proteins increased and that of 29 decreased in the ppk mutant strain. Our results suggested that storage lipid degradation rather than hexose catabolism was taking place in the mutant. In order to validate this hypothesis, the triacylglycerol contents of the wt and ppk mutant strains of S. lividans as well as that of Streptomyces coelicolor M145, a strain that produces antibiotics at high levels and is closely related to S. lividans, were assessed using electron microscopy and thin-layer chromatography. These studies highlighted the large difference in triacylglycerol contents of the three strains and confirmed the hypothetical link between storage lipid metabolism and antibiotic biosynthesis in Streptomyces.

  18. Francisella tularensis Schu S4 Lipopolysaccharide Core Sugar and O-Antigen Mutants Are Attenuated in a Mouse Model of Tularemia

    PubMed Central

    Rasmussen, Jed A.; Post, Deborah M. B.; Gibson, Bradford W.; Lindemann, Stephen R.; Apicella, Michael A.; Meyerholz, David K.

    2014-01-01

    The virulence factors mediating Francisella pathogenesis are being investigated, with an emphasis on understanding how the organism evades innate immunity mechanisms. Francisella tularensis produces a lipopolysaccharide (LPS) that is essentially inert and a polysaccharide capsule that helps the organism to evade detection by components of innate immunity. Using an F. tularensis Schu S4 mutant library, we identified strains that are disrupted for capsule and O-antigen production. These serum-sensitive strains lack both capsule production and O-antigen laddering. Analysis of the predicted protein sequences for the disrupted genes (FTT1236 and FTT1238c) revealed similarity to those for waa (rfa) biosynthetic genes in other bacteria. Mass spectrometry further revealed that these proteins are involved in LPS core sugar biosynthesis and the ligation of O antigen to the LPS core sugars. The 50% lethal dose (LD50) values of these strains are increased 100- to 1,000-fold for mice. Histopathology revealed that the immune response to the F. tularensis mutant strains was significantly different from that observed with wild-type-infected mice. The lung tissue from mutant-infected mice had widespread necrotic debris, but the spleens lacked necrosis and displayed neutrophilia. In contrast, the lungs of wild-type-infected mice had nominal necrosis, but the spleens had widespread necrosis. These data indicate that murine death caused by wild-type strains occurs by a mechanism different from that by which the mutant strains kill mice. Mice immunized with these mutant strains displayed >10-fold protective effects against virulent type A F. tularensis challenge. PMID:24452684

  19. Establishing a Markerless Genetic Exchange System for Methanosarcina mazei Strain Gö1 for Constructing Chromosomal Mutants of Small RNA Genes

    PubMed Central

    Ehlers, Claudia; Jäger, Dominik; Schmitz, Ruth A.

    2011-01-01

    A markerless genetic exchange system was successfully established in Methanosarcina mazei strain Gö1 using the hpt gene coding for hypoxanthine phosphoribosyltransferase. First, a chromosomal deletion mutant of the hpt gene was generated conferring resistance to the purine analog 8-aza-2,6-diaminopurine (8-ADP). The nonreplicating allelic exchange vector (pRS345) carrying the pac-resistance cassette for direct selection of chromosomal integration, and the hpt gene for counterselection was introduced into this strain. By a pop-in and ultimately pop-out event of the plasmid from the chromosome, allelic exchange is enabled. Using this system, we successfully generated a M. mazei deletion mutant of the gene encoding the regulatory non-coding RNA sRNA154. Characterizing M. mazeiΔsRNA154 under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of sRNA154 in regulation of nitrogen fixation by posttranscriptional regulation. PMID:21941461

  20. Deficits in Adult Neurogenesis, Contextual Fear Conditioning, and Spatial Learning in a Gfap Mutant Mouse Model of Alexander Disease

    PubMed Central

    Paylor, Richard; Messing, Albee

    2013-01-01

    Glial fibrillary acidic protein (GFAP) is the major intermediate filament of mature astrocytes in the mammalian CNS. Dominant gain of function mutations in GFAP lead to the fatal neurodegenerative disorder, Alexander disease (AxD), which is characterized by cytoplasmic protein aggregates known as Rosenthal fibers along with variable degrees of leukodystrophy and intellectual disability. The mechanisms by which mutant GFAP leads to these pleiotropic effects are unknown. In addition to astrocytes, GFAP is also expressed in other cell types, particularly neural stem cells that form the reservoir supporting adult neurogenesis in the hippocampal dentate gyrus and subventricular zone of the lateral ventricles. Here, we show that mouse models of AxD exhibit significant pathology in GFAP-positive radial glia-like cells in the dentate gyrus, and suffer from deficits in adult neurogenesis. In addition, they display impairments in contextual learning and spatial memory. This is the first demonstration of cognitive phenotypes in a model of primary astrocyte disease. PMID:24259590

  1. [Influence of mutant genes on crystallin synthesis in the forming mouse lens. II. Fidget and ocular retardation genes].

    PubMed

    Iakovlev, M I; Platonov, E S; Koniukhov, B V

    1977-01-01

    The beginning of synthesis and the localization of alpha- and gamma-crystallins in the developing lenses of the 10-13 and 15 days old mouse embryos of the genotypes fi/fi +/+, +/+ or/or, fi/fi or/or and +/+ +/+ were studied by means of indirect immunofluorescence. The synthesis of crystallins in the mutant embryos with the exception of the embryo +/+ or/or was shown to begin somewhat later than in the normal ones but to proceed in all defective lenses, irrespective of the degree of defect. Hence, the activation of the genes controlling the synthesis of alpha-crystallins begins at the early stages of lens development and the synthesis of these proteins proceeds even during the abnormal with the slowing down of the formation of primary lens fibers. In the cases of strong defects of morphogenesis in the fi/fi +/+ and, especially, fi/fi or/or, embryos gamma-crystallins were not detected. The synthesis of gamma-crystallins appears to begin at the final stages of lens fiber differentiation.

  2. SENCAR mouse skin tumorigenesis model versus other strains and stocks of mice

    SciTech Connect

    Slaga, T.J.

    1986-09-01

    The SENCAR mouse stock was selectively bred for eight generations for sensitivity to skin tumor induction by the two-stage tumorigenesis protocol using 7,12-dimethylbenz(a)anthracene (DMBA) as the initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as the promoter. The SENCAR mouse was derived by crossing Charles River CD-1 mice with skin-tumor-sensitive mice (STS). The SENCAR mice are much more sensitive to both DMBA tumor initiation and TPA tumor promotion than CD-1, BALB/c, and DBA/2 mice. An even greater difference in the sensitivity to two-stage skin tumorigenesis is apparent between SENCAR and C57BL/6 mice when using DMBA-TPA treatment. However, the SENCAR and C57BL/6 mice have a similar tumor response to DMBA-benzoyl peroxide treatment, suggesting that TPA is not an effective promoter in C57BL/6 mice. The DBA/2 mice respond in a similar manner to the SENCAR mice when using N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-TPA treatment. The SENCAR mouse model provides a good dose-response relationship for many carcinogens used as tumor initiators and for many compounds used as tumor promoter. When compared to other stocks and strains of mice, the SENCAR mouse has one of the largest data bases for carcinogens and promoters.

  3. Differential visual system organization and susceptibility to experimental models of optic neuropathies in three commonly used mouse strains.

    PubMed

    De Groef, Lies; Dekeyster, Eline; Geeraerts, Emiel; Lefevere, Evy; Stalmans, Ingeborg; Salinas-Navarro, Manuel; Moons, Lieve

    2016-04-01

    Mouse disease models have proven indispensable in glaucoma research, yet the complexity of the vast number of models and mouse strains has also led to confusing findings. In this study, we evaluated baseline intraocular pressure, retinal histology, and retinofugal projections in three mouse strains commonly used in glaucoma research, i.e. C57Bl/6, C57Bl/6-Tyr(c), and CD-1 mice. We found that the mouse strains under study do not only display moderate variations in their intraocular pressure, retinal architecture, and retinal ganglion cell density, also the retinofugal projections to the dorsal lateral geniculate nucleus and the superior colliculus revealed striking differences, potentially underlying diverging optokinetic tracking responses and visual acuity. Next, we reviewed the success rate of three models of (glaucomatous) optic neuropathies (intravitreal N-methyl-d-aspartic acid injection, optic nerve crush, and laser photocoagulation-induced ocular hypertension), looking for differences in disease susceptibility between these mouse strains. Different genetic backgrounds and albinism led to differential susceptibility to experimentally induced retinal ganglion cell death among these three mouse strains. Overall, CD-1 mice appeared to have the highest sensitivity to retinal ganglion cell damage, while the C57Bl/6 background was more resistant in the three models used. PMID:26791081

  4. Transcriptomics and Metabonomics Identify Essential Metabolic Signatures in Calorie Restriction (CR) Regulation across Multiple Mouse Strains.

    PubMed

    Collino, Sebastiano; Martin, François-Pierre J; Montoliu, Ivan; Barger, Jamie L; Da Silva, Laeticia; Prolla, Tomas A; Weindruch, Richard; Kochhar, Sunil

    2013-01-01

    Calorie restriction (CR) has long been used to study lifespan effects and oppose the development of a broad array of age-related biological and pathological changes (increase healthspan). Yet, a comprehensive comparison of the metabolic phenotype across different genetic backgrounds to identify common metabolic markers affected by CR is still lacking. Using a system biology approach comprising metabonomics and liver transcriptomics we revealed the effect of CR across multiple mouse strains (129S1/SvlmJ, C57BL6/J, C3H/HeJ, CBA/J, DBA/2J, JC3F1/J). Oligonucleotide microarrays identified 76 genes as differentially expressed in all six strains confirmed. These genes were subjected to quantitative RT-PCR analysis in the C57BL/6J mouse strain, and a CR-induced change expression was confirmed for 14 genes. To fully depict the metabolic pathways affected by CR and complement the changes observed through differential gene expression, the metabolome of C57BL6/J was further characterized in liver tissues, urine and plasma levels using a combination or targeted mass spectrometry and proton nuclear magnetic resonance spectroscopy. Overall, our integrated approach commonly confirms that energy metabolism, stress response, lipids regulators and the insulin/IGF-1 are key determinants factors involved in CR regulation. PMID:24958256

  5. Differential effects of sucrose and fructose on dietary obesity in four mouse strains

    PubMed Central

    Glendinnning, John I.; Breinager, Lindsey; Kyrillou, Emily; Lacuna, Kristine; Rocha, Rotsen; Sclafani, Anthony

    2010-01-01

    We examined sugar-induced obesity in mouse strains polymorphic for Tas1r3, a gene that codes for the T1R3 sugar taste receptor. The T1R3 receptor in the FVB and B6 strains has a higher affinity for sugars than that in the AKR and 129P3 strains. In Experiment 1, mice had 40 days of access to lab chow plus water, sucrose (10 or 34%), or fructose (10 or 34%) solutions. The strains consumed more of the sucrose than isocaloric fructose solutions. The pattern of strain differences in caloric intake from the 10% sugar solutions was FVB > 129P3 = B6 > AKR; and that from the 34% sugar solutions was FVB > 129P3 > B6 ≥ AKR. Despite consuming more sugar calories, the FVB mice resisted obesity altogether. The AKR and 129P3 mice became obese exclusively on the 34% sucrose diet, while the B6 mice did so on the 34% sucrose and 34% fructose diets. In Experiment 2, we compared total caloric intake from diets containing chow versus chow plus 34% sucrose. All strains consumed 15-29% more calories from the sucrose-supplemented diet. In Experiment 3, we compared the oral acceptability of the sucrose and fructose solutions, using lick tests. All strains licked more avidly for the 10% sucrose solutions. The results indicate that in mice (a) Tas1r3 genotype does not predict sugar-induced hyperphagia or obesity; (b) sucrose solutions stimulate higher daily intakes than isocaloric fructose solutions; and (c) susceptibility to sugar-induced obesity varies with strain, sugar concentration and sugar type. PMID:20600198

  6. Differential effects of sucrose and fructose on dietary obesity in four mouse strains.

    PubMed

    Glendinning, John I; Breinager, Lindsey; Kyrillou, Emily; Lacuna, Kristine; Rocha, Rotsen; Sclafani, Anthony

    2010-10-01

    We examined sugar-induced obesity in mouse strains polymorphic for Tas1r3, a gene that codes for the T1R3 sugar taste receptor. The T1R3 receptor in the FVB and B6 strains has a higher affinity for sugars than that in the AKR and 129P3 strains. In Experiment 1, mice had 40days of access to lab chow plus water, sucrose (10 or 34%), or fructose (10 or 34%) solutions. The strains consumed more of the sucrose than isocaloric fructose solutions. The pattern of strain differences in caloric intake from the 10% sugar solutions was FVB>129P3=B6>AKR; and that from the 34% sugar solutions was FVB>129P3>B6>/=AKR. Despite consuming more sugar calories, the FVB mice resisted obesity altogether. The AKR and 129P3 mice became obese exclusively on the 34% sucrose diet, while the B6 mice did so on the 34% sucrose and 34% fructose diets. In Experiment 2, we compared total caloric intake from diets containing chow versus chow plus 34% sucrose. All strains consumed between 11 and 25% more calories from the sucrose-supplemented diet. In Experiment 3, we compared the oral acceptability of the sucrose and fructose solutions, using lick tests. All strains licked more avidly for the 10% sucrose solutions. The results indicate that in mice (a) Tas1r3 genotype does not predict sugar-induced hyperphagia or obesity; (b) sucrose solutions stimulate higher daily intakes than isocaloric fructose solutions; and (c) susceptibility to sugar-induced obesity varies with strain, sugar concentration and sugar type.

  7. Comparative analysis of the behavioral and biomolecular parameters of four mouse strains.

    PubMed

    Nesher, Elimelech; Peskov, Vladimir; Rylova, Anna; Raz, Olga; Pinhasov, Albert

    2012-02-01

    The use of mice as experimental models in pharmacological and biochemical research began over 100 years ago, during which time different mice strains with specific features were developed. Numerous studies demonstrate that the pharmacological efficacy of various compounds significantly varies among different animal strains, a factor which must be considered when analyzing experimental data. The Sabra strain, developed more than 35 years ago, is widely used for research in Israel but has an unclear origin and is not characterized as well as other strains. Comparative analyses of the molecular characteristics of Sabra and other strains should help to understand their characteristics and to enhance the validity of their experimental use. Thus, four mouse strains-outbred ICR and Sabra as well as inbred C57Bl/6J and Balb/c were compared. Animals' weight, blood corticosterone and hippocampal BDNF mRNA levels were measured, and animals' behavior was compared using the EPM, open field, FST, and hot plate tests. We found that although Sabra mice are bigger and heavier than other tested lines, this is not reflected in behavior or in biomolecular features, wherein Sabra mice lay within the diapason of other tested animals. Thus, behavioral tests of anxiety-like behavior and locomotor activity revealed that Sabra mice scored close to the mean of all tested lines. Analysis of blood corticosterone levels did not show significant differences among tested strains. We also found a correlation between general and locomotor activity of the tested strains and their hippocampal BDNF mRNA expression. In summary, we may conclude that Sabra mice have traits similar to the better known lines, and therefore they are good subjects for neuroscience research.

  8. Genetic influences on exercise-induced adult hippocampal neurogenesis across 12 divergent mouse strains

    PubMed Central

    Clark, Peter J.; Kohman, Rachel A.; Miller, Daniel S.; Bhattacharya, Tushar K.; Brzezinska, Weronika J.; Rhodes, Justin S.

    2011-01-01

    New neurons are continuously born in the hippocampus of several mammalian species throughout adulthood. Adult neurogenesis represents a natural model for understanding how to grow and incorporate new nerve cells into pre-existing circuits in the brain. Finding molecules or biological pathways that increase neurogenesis has broad potential for regenerative medicine. One strategy is to identify mouse strains that display large versus small increases in neurogenesis in response to wheel running so the strains can be contrasted to find common genes or biological pathways associated with enhanced neuron formation. Therefore, mice from 12 different isogenic strains were housed with or without running wheels for 43 days to measure the genetic regulation of exercise-induced neurogenesis. The first 10 days mice received daily injections of BrdU to label dividing cells. Neurogenesis was measured as the total number of BrdU cells co-expressing NeuN mature neuronal marker in the hippocampal granule cell layer by immunohistochemistry. Exercise increased neurogenesis in all strains, but the magnitude significantly depended on genotype. Strain means for distance run on wheels, but not distance traveled in cages without wheels, were significantly correlated with strain mean level of neurogenesis. Further, certain strains displayed greater neurogenesis than others for a fixed level of running. Strain means for neurogenesis under sedentary conditions were not correlated with neurogenesis under runner conditions suggesting that different genes influence baseline versus exercise-induced neurogenesis. Genetic contributions to exercise-induced hippocampal neurogenesis suggest that it may be possible to identify genes and pathways associated with enhanced neuroplastic responses to exercise. PMID:21223504

  9. CYCLOPENTA-FUSED POLYCYCLIC AROMATIC HYDROCARBONS IN STRAIN A/J MOUSE LUNG: DNA ADDUCTS, ONCOGENE MUTATIONS, & TUMORIGENESIS

    EPA Science Inventory

    Cyclopenta-fused Polycyclic Aromatic Hydrocarbons in Strain AJJ Mouse Lung: DNA Adducts, Oncogene Mutations, and Tumorigenesis.

    We have examined the relationships between DNA adducts, Ki-ras oncogene mutations, DNA adducts, and adenoma induction in the lungs of strain A/J...

  10. Inversion of layer-specific cadherin expression profiles and maintenance of cytoarchitectonic areas in the allocortex of the reeler mutant mouse.

    PubMed

    Stoya, Gudrun; Redies, Christoph; Schmid-Hertel, Nicole

    2014-09-01

    Cadherins are calcium-depending cell adhesion proteins that play critical roles in brain morphogenesis and wiring. They provide an adhesive code for the development of cortical layers, due to their homophilic interactions and their restricted spatiotemporal expression patterns. In the adult organism, cadherins are involved in the maintenance and plasticity of neuronal circuits that play a role in learning. A well-known model for studying corticogenesis is the reeler mouse model. Numerous investigations of neocortical development suggest that, in the reeler mutant mouse, the lack of the protein Reelin results in cell-type and region-dependent changes of the neocortical layers. To investigate in detail how layer formation and regionalization is perturbed in the phylogenetically older archicortex of the adult reeler mutant mouse, we studied the expression of 11 different cadherins (Cdh4, Cdh7, Cdh8, Cdh11, Pcdh1, Pcdh7, Pcdh8, Pcdh9, Pcdh10, Pcdh17, and Pcdh19) and of the transcription factors ER81 and Cux2 by in situ hybridization in the (peri-)archicortex. All cadherins studied show a layer-specific expression in the (peri-)archicortex of the wildtype brain. In the archicortex of the reeler mutant, the cadherin-expressing cell layers are dispersed in the radial dimension, whereas in the periarchicortex the superficial and deep layers are inverted, both in the adult and during development. Possibly, this inversion relates to the histoarchitectural division of the reeler entorhinal cortex into an external and an internal zone. The regionalized, gradient-like expression of the cadherins is preserved in the reeler mutant mouse.

  11. Effect of crossing C57BL/6 and FVB mouse strains on basal cytokine expression.

    PubMed

    Szade, Agata; Nowak, Witold N; Szade, Krzysztof; Gese, Anna; Czypicki, Ryszard; Waś, Halina; Dulak, Józef; Józkowicz, Alicja

    2015-01-01

    C57BL/6 is the most often used laboratory mouse strain. However, sometimes it is beneficial to cross the transgenic mice on the C57BL/6 background to the other strain, such as FVB. Although this is a common strategy, the influence of crossing these different strains on homeostatic expression of cytokines is not known. Here we have investigated the differences in the expression of selected cytokines between C57BL/6J and C57BL/6JxFVB mice in serum and skeletal muscle. We have found that only few cytokines were altered by crossing of the strains. Concentrations of IL5, IL7, LIF, MIP-2, and IP-10 were higher in serum of C57BL/6J mice than in C57BL/6JxFVB mice, whereas concentration of G-CSF was lower in C57BL/6J. In the skeletal muscle only the concentration of VEGF was higher in C57BL/6J mice than in C57BL/6JxFVB mice. Concluding, the differences in cytokine expression upon crossing C57BL/6 and FVB strain in basal conditions are not profound.

  12. Effect of Crossing C57BL/6 and FVB Mouse Strains on Basal Cytokine Expression

    PubMed Central

    Szade, Agata; Nowak, Witold N.; Szade, Krzysztof; Gese, Anna; Czypicki, Ryszard; Waś, Halina; Dulak, Józef; Józkowicz, Alicja

    2015-01-01

    C57BL/6 is the most often used laboratory mouse strain. However, sometimes it is beneficial to cross the transgenic mice on the C57BL/6 background to the other strain, such as FVB. Although this is a common strategy, the influence of crossing these different strains on homeostatic expression of cytokines is not known. Here we have investigated the differences in the expression of selected cytokines between C57BL/6J and C57BL/6JxFVB mice in serum and skeletal muscle. We have found that only few cytokines were altered by crossing of the strains. Concentrations of IL5, IL7, LIF, MIP-2, and IP-10 were higher in serum of C57BL/6J mice than in C57BL/6JxFVB mice, whereas concentration of G-CSF was lower in C57BL/6J. In the skeletal muscle only the concentration of VEGF was higher in C57BL/6J mice than in C57BL/6JxFVB mice. Concluding, the differences in cytokine expression upon crossing C57BL/6 and FVB strain in basal conditions are not profound. PMID:25834307

  13. Effect of mouse strain as a background for Alzheimer’s disease models on the clearance of amyloid-β

    PubMed Central

    Qosa, Hisham; Kaddoumi, Amal

    2016-01-01

    Novel animal models of Alzheimer’s disease (AD) are relentlessly being developed and existing ones are being fine-tuned; however, these models face multiple challenges associated with the complexity of the disease where most of these models do not reproduce the full phenotypical disease spectrum. Moreover, different AD models express different phenotypes that could affect their validity to recapitulate disease pathogenesis and/or response to a drug. One of the most important and understudied differences between AD models is differences in the phenotypic characteristics of the background species. Here, we used the brain clearance index (BCI) method to investigate the effect of strain differences on the clearance of amyloid β (Aβ) from the brains of four mouse strains. These mouse strains, namely C57BL/6, FVB/N, BALB/c and SJL/J, are widely used as a background for the development of AD mouse models. Findings showed that while Aβ clearance across the blood-brain barrier (BBB) was comparable between the 4 strains, levels of LRP1, an Aβ clearance protein, was significantly lower in SJL/J mice compared to other mouse strains. Furthermore, these mouse strains showed a significantly different response to rifampicin treatment with regard to Aβ clearance and effect on brain level of its clearance-related proteins. Our results provide for the first time an evidence for strain differences that could affect ability of AD mouse models to recapitulate response to a drug, and opens a new research avenue that requires further investigation to successfully develop mouse models that could simulate clinically important phenotypic characteristics of AD. PMID:27478623

  14. Genetically diverse CC-founder mouse strains replicate the human influenza gene expression signature.

    PubMed

    Elbahesh, Husni; Schughart, Klaus

    2016-05-19

    Influenza A viruses (IAV) are zoonotic pathogens that pose a major threat to human and animal health. Influenza virus disease severity is influenced by viral virulence factors as well as individual differences in host response. We analyzed gene expression changes in the blood of infected mice using a previously defined set of signature genes that was derived from changes in the blood transcriptome of IAV-infected human volunteers. We found that the human signature was reproduced well in the founder strains of the Collaborative Cross (CC) mice, thus demonstrating the relevance and importance of mouse experimental model systems for studying human influenza disease.

  15. Pigmentation restored in mutant laboratory strain of the lady beetle Coleomegilla maculata through dietary supplementation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A laboratory colony of Coleomegilla maculata (DeGeer), ye, selected for a pigmentation deficiency, was restored to near wild type cuticle coloration by adding crushed heads and wings of the red colored parental strain to the diet. While the wings and other colored portions of the cuticle regained th...

  16. Primary human immunodeficiency virus type 1 viremia and central nervous system invasion in a novel hu-PBL-immunodeficient mouse strain.

    PubMed Central

    Koyanagi, Y; Tanaka, Y; Kira, J; Ito, M; Hioki, K; Misawa, N; Kawano, Y; Yamasaki, K; Tanaka, R; Suzuki, Y; Ueyama, Y; Terada, E; Tanaka, T; Miyasaka, M; Kobayashi, T; Kumazawa, Y; Yamamoto, N

    1997-01-01

    We established four new mouse strains with defective T and B cells as well as defects in innate immunological reactions using an NK cell depletion antibody and showed that all mutant mouse strains efficiently received human peripheral blood leukocyte (PBL) engraftment (hu-PBL-scid mice). Higher levels of human immunodeficiency virus type 1 (HIV-1) replication were observed in these new hu-PBL-scid mice than in conventional hu-PBL-C.B-17-scid mice. In one particular strain, hu-PBL-NOD-scid mice, high levels of HIV-1 viremia (more than 10(6) 50% infectious doses per ml) were detected after infection with HIV-1. The plasma viral load was about 100 to 1,000 times higher than that observed in other hu-PBL-scid mice infected with HIV-1. Although high-level viremia did not correlate with the total amount of HIV-1 RNA in cells from infected mice, high levels of free virions were detected only in hu-PBL-NOD-scid mice. HIV-1 viremia induced systemic HIV-1 infection involving the liver, lungs, and brain. PCR in situ hybridization confirmed that HIV-1-infected cells invaded the brain tissue of the hu-PBL-NOD-scid mice. Our results suggest that the genetic background, including innate immunity, is critical in the development of primary HIV-1 viremia and subsequent central nervous system invasion with HIV-1. The hu-PBL-NOD-scid mouse represents a useful model for the study of the pathogenesis of HIV-1 in vivo, especially brain involvement, and therapy of primary HIV-1 viremia. PMID:9032379

  17. Modelling Hepatitis B Virus Antiviral Therapy and Drug Resistant Mutant Strains

    NASA Astrophysics Data System (ADS)

    Bernal, Julie; Dix, Trevor; Allison, Lloyd; Bartholomeusz, Angeline; Yuen, Lilly

    Despite the existence of vaccines, the Hepatitis B virus (HBV) is still a serious global health concern. HBV targets liver cells. It has an unusual replication process involving an RNA pre-genome that the reverse transcriptase domain of the viral polymerase protein translates into viral DNA. The reverse transcription process is error prone and together with the high replication rates of the virus, allows the virus to exist as a heterogeneous population of mutants, known as a quasispecies, that can adapt and become resistant to antiviral therapy. This study presents an individual-based model of HBV inside an artificial liver, and associated blood serum, undergoing antiviral therapy. This model aims to provide insights into the evolution of the HBV quasispecies and the individual contribution of HBV mutations in the outcome of therapy.

  18. Using standard nomenclature to adequately name transgenes, knockout gene alleles and any mutation associated to a genetically modified mouse strain.

    PubMed

    Montoliu, Lluís; Whitelaw, C Bruce A

    2011-04-01

    Mice provide an unlimited source of animal models to study mammalian gene function and human diseases. The powerful genetic modification toolbox existing for the mouse genome enables the creation of, literally, thousands of genetically modified mouse strains, carrying spontaneous or induced mutations, transgenes or knock-out/knock-in alleles which, in addition, can exist in hundreds of different genetic backgrounds. Such an immense diversity of individuals needs to be adequately annotated, to ensure that the most relevant information is kept associated with the name of each mouse line, and hence, the scientific community can correctly interpret and benefit from the reported animal model. Therefore, rules and guidelines for correctly naming genes, alleles and mouse strains are required. The Mouse Genome Informatics Database is the authoritative source of official names for mouse genes, alleles, and strains. Nomenclature follows the rules and guidelines established by the International Committee on Standardized Genetic Nomenclature for Mice. Herewith, both from the International Society for Transgenic Technologies (ISTT) and from the scientific journal Transgenic Research, we would like to encourage all our colleagues to adhere and follow adequately the standard nomenclature rules when describing mouse models. The entire scientific community using genetically modified mice in experiments will benefit.

  19. Immobility responses between mouse strains correlate with distinct hippocampal serotonin transporter protein expression and function.

    PubMed

    Tang, Man; He, Tao; Meng, Qing-yan; Broussard, John Isaac; Yao, Lan; Diao, Yao; Sang, Xiu-bo; Liu, Qing-peng; Liao, Ying-jun; Li, Yuge; Zhao, Shulei

    2014-11-01

    Mouse strain differences in immobility and in sensitivity to antidepressants have been observed in the forced swimming test (FST) and the tail suspension test (TST). However, the neurotransmitter systems and neural substrates that contribute to these differences remain unknown. To investigate the role of the hippocampal serotonin transporter (5-HTT), we measured baseline immobility and the immobility responses to fluoxetine (FLX) in the FST and the TST in male CD-1, C57BL/6, DBA and BALB/c mice. We observed strain differences in baseline immobility time, with CD-1 mice showing the longest and DBA mice showing the shortest. In contrast, DBA and BALB/c mice showed the highest sensitivity to FLX, whereas CD-1 and C57BL/6 mice showed the lowest sensitivity. Also we found strain differences in both the total 5-HTT protein level and the membrane-bound 5-HTT level (estimated by V max) as follows: DBA>BALB/c>CD-1=C57BL/6. The uptake efficiency of the membrane-bound 5-HTT (estimated by 1/K m) was highest in DBA and BALB/c mice and lowest in CD-1 and C57BL/6 mice. A correlation analysis of subregions within the hippocampus revealed that immobility time was negatively correlated with V max and positively correlated with K m in the hippocampus. Therefore a higher uptake capacity of the membrane-bound 5-HTT in the hippocampus was associated with lower baseline immobility and greater sensitivity to FLX. These results suggest that alterations in hippocampal 5-HTT activity may contribute to mouse strain differences in the FST and the TST.

  20. A Myosin Va Mutant Mouse with Disruptions in Glutamate Synaptic Development and Mature Plasticity in Visual Cortex

    PubMed Central

    Yoshii, Akira; Zhao, Jianping; Pandian, Swarna; van Zundert, Brigitte; Constantine-Paton, Martha

    2013-01-01

    MyosinVa (MyoVa) mediates F-actin-based vesicular transport toward the plasma membrane and is found at neuronal postsynaptic densities (PSDs), but the role of MyoVa in synaptic development and function is largely unknown. Here, in studies using the dominant negative MyoVa neurological mutant mouse Flailer, we find that MyoVa plays an essential role in activity-dependent delivery of PSD-95 and other critical PSD molecules to synapses and in endocytosis of AMPA-type glutamate receptors (AMPAR) in the dendrites of CNS neurons. MyoVa is known to carry a complex containing the major scaffolding proteins of the mature PSD, PSD-95, SAPAP1/GKAP, Shank and Homer, to dendritic spine synapses. In Flailer, neurons show abnormal dendritic shaft localization of PSD-95, stargazin, dynamin3, AMPA glutamate receptors (AMPARs) and abnormal spine morphology. Flailer neurons also have abnormally high AMPAR miniature current frequencies and spontaneous AMPAR currents that are more frequent and larger than in WT while numbers of NMDAR containing synapses remain normal. The AMPAR abnormalities are consistent with a severely disrupted developmental regulation of long-term depression that we find in cortical Flailer neurons. Thus MyoVa plays a fundamentally important role both in localizing mature glutamate synapses to spines and in organizing the synapse for normal function. For this reason Flailer mice will be valuable in further dissecting the role of MyoVa in normal synaptic and circuit refinement and also in studies of neurological and neuropsychiatric diseases where disruptions of normal glutamate synapses are frequently observed. PMID:23658184

  1. Rett syndrome like phenotypes in the R255X Mecp2 mutant mouse are rescued by MECP2 transgene.

    PubMed

    Pitcher, Meagan R; Herrera, José A; Buffington, Shelly A; Kochukov, Mikhail Y; Merritt, Jonathan K; Fisher, Amanda R; Schanen, N Carolyn; Costa-Mattioli, Mauro; Neul, Jeffrey L

    2015-05-01

    Rett syndrome (RTT) is a severe neurodevelopmental disorder that is usually caused by mutations in Methyl-CpG-binding Protein 2 (MECP2). Four of the eight common disease causing mutations in MECP2 are nonsense mutations and are responsible for over 35% of all cases of RTT. A strategy to overcome disease-causing nonsense mutations is treatment with nonsense mutation suppressing drugs that allow expression of full-length proteins from mutated genes with premature in-frame stop codons. To determine if this strategy is useful in RTT, we characterized a new mouse model containing a knock-in nonsense mutation (p.R255X) in the Mecp2 locus (Mecp2(R255X)). To determine whether the truncated gene product acts as a dominant negative allele and if RTT-like phenotypes could be rescued by expression of wild-type protein, we genetically introduced an extra copy of MECP2 via an MECP2 transgene. The addition of MECP2 transgene to Mecp2(R255X) mice abolished the phenotypic abnormalities and resulted in near complete rescue. Expression of MECP2 transgene Mecp2(R255X) allele also rescued mTORC1 signaling abnormalities discovered in mice with loss of function and overexpression of Mecp2. Finally, we treated Mecp2(R255X) embryonic fibroblasts with the nonsense mutation suppressing drug gentamicin and we were able to induce expression of full-length MeCP2 from the mutant p.R255X allele. These data provide proof of concept that the p.R255X mutation of MECP2 is amenable to the nonsense suppression therapeutic strategy and provide guidelines for the extent of rescue that can be expected by re-expressing MeCP2 protein.

  2. Rett syndrome like phenotypes in the R255X Mecp2 mutant mouse are rescued by MECP2 transgene

    PubMed Central

    Pitcher, Meagan R.; Herrera, José A.; Buffington, Shelly A.; Kochukov, Mikhail Y.; Merritt, Jonathan K.; Fisher, Amanda R.; Schanen, N. Carolyn; Costa-Mattioli, Mauro; Neul, Jeffrey L.

    2015-01-01

    Rett syndrome (RTT) is a severe neurodevelopmental disorder that is usually caused by mutations in Methyl-CpG-binding Protein 2 (MECP2). Four of the eight common disease causing mutations in MECP2 are nonsense mutations and are responsible for over 35% of all cases of RTT. A strategy to overcome disease-causing nonsense mutations is treatment with nonsense mutation suppressing drugs that allow expression of full-length proteins from mutated genes with premature in-frame stop codons. To determine if this strategy is useful in RTT, we characterized a new mouse model containing a knock-in nonsense mutation (p.R255X) in the Mecp2 locus (Mecp2R255X). To determine whether the truncated gene product acts as a dominant negative allele and if RTT-like phenotypes could be rescued by expression of wild-type protein, we genetically introduced an extra copy of MECP2 via an MECP2 transgene. The addition of MECP2 transgene to Mecp2R255X mice abolished the phenotypic abnormalities and resulted in near complete rescue. Expression of MECP2 transgene Mecp2R255X allele also rescued mTORC1 signaling abnormalities discovered in mice with loss of function and overexpression of Mecp2. Finally, we treated Mecp2R255X embryonic fibroblasts with the nonsense mutation suppressing drug gentamicin and we were able to induce expression of full-length MeCP2 from the mutant p.R255X allele. These data provide proof of concept that the p.R255X mutation of MECP2 is amenable to the nonsense suppression therapeutic strategy and provide guidelines for the extent of rescue that can be expected by re-expressing MeCP2 protein. PMID:25634563

  3. Strain-dependent Damage in Mouse Lung After Carbon Ion Irradiation

    SciTech Connect

    Moritake, Takashi; Fujita, Hidetoshi; Yanagisawa, Mitsuru; Nakawatari, Miyako; Imadome, Kaori; Nakamura, Etsuko; Iwakawa, Mayumi; Imai, Takashi

    2012-09-01

    Purpose: To examine whether inherent factors produce differences in lung morbidity in response to carbon ion (C-ion) irradiation, and to identify the molecules that have a key role in strain-dependent adverse effects in the lung. Methods and Materials: Three strains of female mice (C3H/He Slc, C57BL/6J Jms Slc, and A/J Jms Slc) were locally irradiated in the thorax with either C-ion beams (290 MeV/n, in 6 cm spread-out Bragg peak) or with {sup 137}Cs {gamma}-rays as a reference beam. We performed survival assays and histologic examination of the lung with hematoxylin-eosin and Masson's trichrome staining. In addition, we performed immunohistochemical staining for hyaluronic acid (HA), CD44, and Mac3 and assayed for gene expression. Results: The survival data in mice showed a between-strain variance after C-ion irradiation with 10 Gy. The median survival time of C3H/He was significantly shortened after C-ion irradiation at the higher dose of 12.5 Gy. Histologic examination revealed early-phase hemorrhagic pneumonitis in C3H/He and late-phase focal fibrotic lesions in C57BL/6J after C-ion irradiation with 10 Gy. Pleural effusion was apparent in C57BL/6J and A/J mice, 168 days after C-ion irradiation with 10 Gy. Microarray analysis of irradiated lung tissue in the three mouse strains identified differential expression changes in growth differentiation factor 15 (Gdf15), which regulates macrophage function, and hyaluronan synthase 1 (Has1), which plays a role in HA metabolism. Immunohistochemistry showed that the number of CD44-positive cells, a surrogate marker for HA accumulation, and Mac3-positive cells, a marker for macrophage infiltration in irradiated lung, varied significantly among the three mouse strains during the early phase. Conclusions: This study demonstrated a strain-dependent differential response in mice to C-ion thoracic irradiation. Our findings identified candidate molecules that could be implicated in the between-strain variance to early

  4. Optimization of protocols for derivation of mouse embryonic stem cell lines from refractory strains, including the non obese diabetic mouse.

    PubMed

    Davies, Timothy J; Fairchild, Paul J

    2012-07-01

    The derivation of pluripotent embryonic stem cells (ESCs) from a variety of genetic backgrounds remains a desirable objective in the generation of mice functionally deficient in genes of interest and the modeling of human disease. Nevertheless, disparity in the ease with which different strains of mice yield ESC lines has long been acknowledged. Indeed, the generation of bona fide ESCs from the non obese diabetic (NOD) mouse, a well-characterized model of human type I diabetes, has historically proved especially difficult to achieve. Here, we report the development of protocols for the derivation of novel ESC lines from C57Bl/6 mice based on the combined use of high concentrations of leukemia inhibitory factor and serum-replacement, which is equally applicable to fresh and cryo-preserved embryos. Further, we demonstrate the success of this approach using Balb/K and CBA/Ca mice, widely considered to be refractory strains. CBA/Ca ESCs contributed to the somatic germ layers of chimeras and displayed a very high competence at germline transmission. Importantly, we were able to use the same protocol for the derivation of ESC lines from nonpermissive NOD mice. These ESCs displayed a normal karyotype that was robustly stable during long-term culture, were capable of forming teratomas in vivo and germline competent chimeras after injection into recipient blastocysts. Further, these novel ESC lines efficiently formed embryoid bodies in vitro and could be directed in their differentiation along the dendritic cell lineage, thus illustrating their potential application to the generation of cell types of relevance to the pathogenesis of type I diabetes. PMID:21933027

  5. Proliferation Potential of Müller Glia after Retinal Damage Varies between Mouse Strains

    PubMed Central

    Suga, Akiko; Sadamoto, Kazuyo; Fujii, Momo; Mandai, Michiko; Takahashi, Masayo

    2014-01-01

    Retinal Müller glia can serve as a source for regeneration of damaged retinal neurons in fish, birds and mammals. However, the proliferation rate of Müller glia has been reported to be low in the mammalian retina. To overcome this problem, growth factors and morphogens have been studied as potent promoters of Müller glial proliferation, but the molecular mechanisms that limit the proliferation of Müller glia in the mammalian retina remain unknown. In the present study, we found that the degree of damage-induced Müller glia proliferation varies across mouse strains. In mouse line 129×1/SvJ (129), there was a significantly larger proliferative response compared with that observed in C57BL/6 (B6) after photoreceptor cell death. Treatment with a Glycogen synthase kinase 3 (GSK3) inhibitor enhanced the proliferation of Müller glia in 129 but not in B6 mouse retinas. We therefore focused on the different gene expression patterns during retinal degeneration between B6 and 129. Expression levels of Cyclin D1 and Nestin correlated with the degree of Müller glial proliferation. A comparison of genome-wide gene expression between B6 and 129 showed that distinct sets of genes were upregulated in the retinas after damage, including immune response genes and chromatin remodeling factors. PMID:24747725

  6. Differential resistance/susceptibility patterns to pneumovirus infection among inbred mouse strains.

    PubMed

    Anh, Dao Bui Tran; Faisca, Pedro; Desmecht, Daniel J-M

    2006-09-01

    Respiratory syncytial virus (RSV) is a prominent cause of airway morbidity in children under 1 yr of age. It is assumed that host factors influence the severity of the disease presentation and thus the need for hospitalization. As a first step toward the identification of the underlying genes involved, this study was undertaken to establish whether inbred mouse strains differ in susceptibility to pneumonia virus of mice (PVM), the murine counterpart of RSV, which has been shown to accurately mimic the RSV disease of children. With this purpose in mind, double-chamber plethysmography and carbon monoxide uptake data were collected daily for 7 days after inoculation of PVM in six inbred strains of mice. In parallel, histological examinations and lung viral titration were carried out from day 5 to day 7 after inoculation. Pulmonary structure/function values reflected the success of viral replication in the lungs and revealed a pattern of continuous variation, with resistant, intermediate, and susceptible strains. The results suggest that SJL (resistant) and 129/Sv (susceptible) strains should be used in crossing experiments aimed at identifying genes controlling pneumovirus replication by the positional cloning approach. Similarly, crossing experiments using BALB/c or C57BL/6 (resistant) and DBA/2 or 129/Sv (susceptible) will allow the identification of the genes involved in the control of pulmonary inflammation during pneumovirus infection.

  7. General and social anxiety in the BTBR T+ tf/J mouse strain.

    PubMed

    Pobbe, Roger L H; Defensor, Erwin B; Pearson, Brandon L; Bolivar, Valerie J; Blanchard, D Caroline; Blanchard, Robert J

    2011-01-01

    BTBR T+ tf/J (BTBR) is an inbred mouse strain that shows behavioral traits with analogies to the three diagnostic symptoms of autism spectrum disorder (ASD); deficits in social interaction, impaired communication, and repetitive behaviors with restricted interests. Previous findings reveal that when compared to C57BL/6J (B6) and other inbred strains, BTBR exhibit normal to low anxiety-like traits in paradigms designed to assess anxiety-related behaviors. The current study assessed the generality of these anxiety findings. In experiment 1, B6 and BTBR mice were tested in the elevated plus maze (EPM), mouse defense test battery (MDTB) and elevated zero-maze. BTBR mice exhibited an anxiogenic profile in the EPM, with a reduction in open arm time and an increase in risk assessment behaviors, as compared to B6. In the MDTB, BTBR showed enhanced vocalization to the predator, and significantly less locomotor activity than B6 in the pre-threat situation, but significantly more locomotion than B6 following exposure to a predator threat, suggesting enhanced defensiveness to the predator. In the zero-maze, BTBR mice showed a significantly higher number of entries and time spent in the open segments of the apparatus, when compared to B6. In experiment 2, a three-chambered social preference test was used to evaluate effects of the systemic administration of an anxiolytic compound, diazepam, on B6 and BTBR social approach. Diazepam consistently increased time in the compartment containing the social stimulus, for both B6 and BTBR mice. However, in the vehicle treated groups, B6 mice spent significantly more time while BTBR mice spent significantly less time in the social stimulus compartment; after diazepam administration both B6 and BTBR strains significantly preferred the social stimulus chamber. These results suggest that while the anxiety responses of BTBR mice to novel situations (EPM and zero-maze) are inconsistent, BTBR mice appear to be more defensive to animate threat

  8. Experimenter effects on behavioral test scores of eight inbred mouse strains under the influence of ethanol.

    PubMed

    Bohlen, Martin; Hayes, Erika R; Bohlen, Benjamin; Bailoo, Jeremy D; Crabbe, John C; Wahlsten, Douglas

    2014-10-01

    Eight standard inbred mouse strains were evaluated for ethanol effects on a refined battery of behavioral tests in a study that was originally designed to assess the influence of rat odors in the colony on mouse behaviors. As part of the design of the study, two experimenters conducted the tests, and the study was carefully balanced so that equal numbers of mice in all groups and times of day were tested by each experimenter. A defect in airflow in the facility compromised the odor manipulation, and in fact the different odor exposure groups did not differ in their behaviors. The two experimenters, however, obtained markedly different results for three of the tests. Certain of the experimenter effects arose from the way they judged behaviors that were not automated and had to be rated by the experimenter, such as slips on the balance beam. Others were not evident prior to ethanol injection but had a major influence after the injection. For several measures, the experimenter effects were notably different for different inbred strains. Methods to evaluate and reduce the impact of experimenter effects in future research are discussed.

  9. Acquisition of nonspecific Bartonella strains by the northern grasshopper mouse (Onychomys leucogaster)

    USGS Publications Warehouse

    Bai, Y.; Kosoy, M.Y.; Cully, J.F.; Bala, T.; Ray, C.; Collinge, S.K.

    2007-01-01

    Rodent-associated Bartonella species are generally host-specific parasites in North America. Here evidence that Bartonella species can 'jump' between host species is presented. Northern grasshopper mice and other rodents were trapped in the western USA. A study of Bartonella infection in grasshopper mice demonstrated a high prevalence that varied from 25% to 90% by location. Bartonella infection was detected in other rodent species with a high prevalence as well. Sequence analyses of gltA identified 29 Bartonella variants in rodents, 10 of which were obtained from grasshopper mice. Among these 10, only six variants were specific to grasshopper mice, whereas four were identical to variants specific to deer mice or 13-lined ground squirrels. Fourteen of 90 sequenced isolates obtained from grasshopper mice were strains found more commonly in other rodent species and were apparently acquired from these animals. The ecological behavior of grasshopper mice may explain the occurrence of Bartonella strains in occasional hosts. The observed rate at which Bartonella jumps from a donor host species to the grasshopper mouse was directly proportional to a metric of donor host density and to the prevalence of Bartonella in the donor host, and inversely proportional to the same parameters for the grasshopper mouse. ?? 2007 Federation of European Microbiological Societies.

  10. Sperm morphology in two house mouse subspecies: do wild-derived strains and wild mice tell the same story?

    PubMed

    Albrechtová, Jana; Albrecht, Tomáš; Ďureje, Ludovít; Pallazola, Vincent A; Piálek, Jaroslav

    2014-01-01

    Being subject to intense post-copulatory selection, sperm size is a principal determining component of male fitness. Although previous studies have presented comparative sperm size data at higher taxonomic levels, information on the evolution of sperm size within species is generally lacking. Here, we studied two house mouse subspecies, Mus musculus musculus and Mus musculus domesticus, which undergo incipient speciation. We measured four sperm dimensions from cauda epididymis smears of 28 wild-caught mice of both subspecies. As inbred mouse strains are frequently used as proxies for exploring evolutionary processes, we further studied four wild-derived inbred strains from each subspecies. The subspecies differed significantly in terms of sperm head length and midpiece length, and these differences were consistent for wild mice and wild-derived strains pooled over genomes. When the inbred strains were analyzed individually, however, their strain-specific values were in some cases significantly shifted from subspecies-specific values derived from wild mice. We conclude that: (1) the size of sperm components differ in the two house mouse subspecies studied, and that (2) wild-derived strains reflect this natural polymorphism, serving as a potential tool to identify the genetic variation driving these evolutionary processes. Nevertheless, we suggest that more strains should be used in future experiments to account for natural variation and to avoid confounding results due to reduced variability and/or founder effect in the individual strains.

  11. Metabolic and behavioral effects of mutant huntingtin deletion in Sim1 neurons in the BACHD mouse model of Huntington’s disease

    PubMed Central

    Soylu-Kucharz, Rana; Baldo, Barbara; Petersén, Åsa

    2016-01-01

    Hypothalamic pathology, metabolic dysfunction and psychiatric symptoms are part of Huntington disease (HD), which is caused by an expanded CAG repeat in the huntingtin (HTT) gene. Inactivation of mutant HTT selectively in the hypothalamus prevents the development of metabolic dysfunction and depressive-like behavior in the BACHD mouse model. The hypothalamic paraventricular nucleus (PVN) is implicated in metabolic and emotional control, therefore we here tested whether inactivation of mutant HTT in the PVN affects metabolic and psychiatric manifestations of HD in BACHD mice. BACHD mice were crossed with mice expressing Cre-recombinase under the Sim1 promoter (Sim1-Cre) to inactivate mutant HTT in Sim1 expressing cells, i.e. the PVN of the hypothalamus. We found that inactivation of mutant HTT in Sim1 cells had a sex-specific effect on both the metabolic and the psychiatric phenotype, as these phenotypes were no longer different in male BACHD/Sim1-Cre mice compared to wild-type littermates. We also found a reduced number of GnRH neurons specifically in the anterior hypothalamus and an increased testes weight in male BACHD mice compared to wild-type littermates. Taken together, expression of mutant HTT in Sim1 cells may play a role for the development of metabolic dysfunction and depressive-like behavior in male BACHD mice. PMID:27334347

  12. Generation of stable mutants and targeted gene deletion strains in Cryptococcus neoformans through electroporation.

    PubMed

    Lin, Xiaorong; Chacko, Nadia; Wang, Linqi; Pavuluri, Yashwant

    2015-04-01

    Cryptococcus neoformans is the etiologic agent of cryptococcal meningitis that causes more than half a million deaths worldwide each year. This capsulated basidiomycetous yeast also serves as a model for micropathogenic studies. The ability to make stable mutants, either via ectopic integration or homologous recombination, has been accomplished using biolistic transformation. This technical advance has greatly facilitated the research on the basic biology and pathogenic mechanisms of this pathogen in the past two decades. However, biolistic transformation is costly, and its reproducibility varies widely. Here we found that stable ectopic integration or targeted gene deletion via homologous replacement could be accomplished through electroporative transformation. The stability of the transformants obtained through electroporation and the frequency of homologous replacement is highly dependent on the selective marker. A frequency of homologous recombination among the stable transformants obtained by electroporation is comparable to those obtained by biolistic transformation (∼10%) when dominant drug selection markers are used, which is much higher than what has been previously reported for electroporation when auxotrophic markers were used (0.001% to 0.1%). Furthermore, disruption of the KU80 gene or generation of gene deletion constructs using the split marker strategy, two approaches known to increase homologous replacement among transformants obtained through biolistic transformation, also increase the frequency of homologous replacement among transformants obtained through electroporation. Therefore, electroporation provides a low cost alternative for mutagenesis in Cryptococcus.

  13. Analysis of the mouse mutant Cloth-ears shows a role for the voltage-gated sodium channel Scn8a in peripheral neural hearing loss.

    PubMed

    Mackenzie, F E; Parker, A; Parkinson, N J; Oliver, P L; Brooker, D; Underhill, P; Lukashkina, V A; Lukashkin, A N; Holmes, C; Brown, S D M

    2009-10-01

    Deafness is the most common sensory disorder in humans and the aetiology of genetic deafness is complex. Mouse mutants have been crucial in identifying genes involved in hearing. However, many deafness genes remain unidentified. Using N-ethyl N-nitrosourea (ENU) mutagenesis to generate new mouse models of deafness, we identified a novel semi-dominant mouse mutant, Cloth-ears (Clth). Cloth-ears mice show reduced acoustic startle response and mild hearing loss from approximately 30 days old. Auditory-evoked brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) analyses indicate that the peripheral neural auditory pathway is impaired in Cloth-ears mice, but that cochlear function is normal. In addition, both Clth/Clth and Clth/+ mice display paroxysmal tremor episodes with behavioural arrest. Clth/Clth mice also show a milder continuous tremor during movement and rest. Longitudinal phenotypic analysis showed that Clth/+ and Clth/Clth mice also have complex defects in behaviour, growth, neurological and motor function. Positional cloning of Cloth-ears identified a point mutation in the neuronal voltage-gated sodium channel alpha-subunit gene, Scn8a, causing an aspartic acid to valine (D981V) change six amino acids downstream of the sixth transmembrane segment of the second domain (D2S6). Complementation testing with a known Scn8a mouse mutant confirmed that this mutation is responsible for the Cloth-ears phenotype. Our findings suggest a novel role for Scn8a in peripheral neural hearing loss and paroxysmal motor dysfunction. PMID:19737145

  14. Isolation of a novel mutant strain of Saccharomyces cerevisiae by an ethyl methane sulfonate-induced mutagenesis approach as a high producer of bioethanol.

    PubMed

    Mobini-Dehkordi, Mohsen; Nahvi, Iraj; Zarkesh-Esfahani, Hamid; Ghaedi, Kamran; Tavassoli, Manoochehr; Akada, Rinji

    2008-04-01

    In order to obtain mutant strains showing higher bioethanol production than wild-type strains, a commercial Saccharomyces cerevisiae type was subjected to mutagenesis using ethyl methane sulfonate (EMS). After adding EMS to a shaken yeast suspension, the viability of yeast cells was assessed by diluted sample inoculation to solid yeast-extract peptone glucose (YEPG) medium at 15-min intervals. At 45 min, the viability of yeast cells was estimated to be about 40%. Mutagenized cells were recovered from YEPG broth after incubation at 30 degrees C for 18 h. After this period, EMS-treated yeast cells were grown on solid aerobic low-peptone (ALP) medium containing 2-12% (v/v) ethanol. All plates were incubated at 30 degrees C for 2-6 d in order to form colonies. The mutant strains that tolerated high concentrations of ethanol were selected for bioethanol production in microfuge tubes containing fermentation medium. Formation of bioethanol in small tubes was detected by the distillation-colorimetric method. In addition, trehalose content and invertase activity were determined in each mutant strain. Among many isolated mutant strains, there were six isolated colonies that grew on ALP medium supplemented with 10% (v/v) ethanol and one of them produced bioethanol 17.3% more than the wild type.

  15. Pulmonary Responses to Stachybotrys chartarum and Its Toxins: Mouse Strain Affects Clearance and Macrophage Cytotoxicity

    PubMed Central

    Lichtenstein, Jamie H. Rosenblum; Molina, Ramon M.; Donaghey, Thomas C.; Amuzie, Chidozie J.; Pestka, James J.; Coull, Brent A.; Brain, Joseph D.

    2010-01-01

    We investigated differences in the pulmonary and systemic clearance of Stachybotrys chartarum spores in two strains of mice, BALB/c and C57BL/6J. To evaluate clearance, mice were intratracheally instilled with a suspension of radiolabeled S. chartarum spores or with unlabeled spores. The lungs of C57BL/6J mice showed more rapid spore clearance than the lungs of BALB/c mice, which correlated with increased levels of spore-associated radioactivity in the GI tracts of C57BL/6J as compared with BALB/c mice. To identify mechanisms responsible for mouse strain differences in spore clearance and previously described lung inflammatory responses, we exposed alveolar macrophages (AMs) lavaged from BALB/c and C57BL/6J mice to S. chartarum spores, S. chartarum spore toxin (SST), and satratoxin G (SG) in vitro. The S. chartarum spores were found to be highly toxic with most cells from either mouse strain being killed within 24 h when exposed to a spore:cell ratio of 1:75. The spores were more lethal to AMs from C57BL/6J than those from BALB/c mice. In mice, the SST elicited many of the same inflammatory responses as the spores in vivo, including AM recruitment, pulmonary hemorrhage, and cytokine production. Our data suggest that differences in pulmonary spore clearance may contribute to the differences in pulmonary responses to S. chartarum between BALB/c and C57BL/6J mice. Enhanced AM survival and subsequent macrophage-mediated inflammation may also contribute to the higher susceptibility of BALB/c mice to S. chartarum pulmonary effects. Analogous genetic differences among humans may contribute to reported variable sensitivity to S. chartarum. PMID:20385656

  16. Effects of Deletion of Mutant Huntingtin in Steroidogenic Factor 1 Neurons on the Psychiatric and Metabolic Phenotype in the BACHD Mouse Model of Huntington Disease

    PubMed Central

    Petersén, Åsa

    2014-01-01

    Psychiatric and metabolic features appear several years before motor disturbances in the neurodegenerative Huntington’s disease (HD), caused by an expanded CAG repeat in the huntingtin (HTT) gene. Although the mechanisms leading to these aspects are unknown, dysfunction in the hypothalamus, a brain region controlling emotion and metabolism, has been suggested. A direct link between the expression of the disease causing protein, huntingtin (HTT), in the hypothalamus and the development of metabolic and psychiatric-like features have been shown in the BACHD mouse model of HD. However, precisely which circuitry in the hypothalamus is critical for these features is not known. We hypothesized that expression of mutant HTT in the ventromedial hypothalamus, an area involved in the regulation of metabolism and emotion would be important for the development of these non-motor aspects. Therefore, we inactivated mutant HTT in a specific neuronal population of the ventromedial hypothalamus expressing the transcription factor steroidogenic factor 1 (SF1) in the BACHD mouse using cross-breeding based on a Cre-loxP system. Effects on anxiety-like behavior were assessed using the elevated plus maze and novelty-induced suppressed feeding test. Depressive-like behavior was assessed using the Porsolt forced swim test. Effects on the metabolic phenotype were analyzed using measurements of body weight and body fat, as well as serum insulin and leptin levels. Interestingly, the inactivation of mutant HTT in SF1-expressing neurons exerted a partial positive effect on the depressive-like behavior in female BACHD mice at 4 months of age. In this cohort of mice, no anxiety-like behavior was detected. The deletion of mutant HTT in SF1 neurons did not have any effect on the development of metabolic features in BACHD mice. Taken together, our results indicate that mutant HTT regulates metabolic networks by affecting hypothalamic circuitries that do not involve the SF1 neurons of the

  17. Transcriptomic analysis of Clostridium thermocellum Populus hydrolysate-tolerant mutant strain shows increased cellular efficiency in response to Populus hydrolysate compared to the wild type strain

    PubMed Central

    2014-01-01

    Background The thermophilic, anaerobic bacterium, Clostridium thermocellum is a model organism for consolidated processing due to its efficient fermentation of cellulose. Constituents of dilute acid pretreatment hydrolysate are known to inhibit C. thermocellum and other microorganisms. To evaluate the biological impact of this type of hydrolysate, a transcriptomic analysis of growth in hydrolysate-containing medium was conducted on 17.5% v/v Populus hydrolysate-tolerant mutant (PM) and wild type (WT) strains of C. thermocellum. Results In two levels of Populus hydrolysate medium (0% and 10% v/v), the PM showed both gene specific increases and decreases of gene expression compared to the wild-type strain. The PM had increased expression of genes in energy production and conversion, and amino acid transport and metabolism in both standard and 10% v/v Populus hydrolysate media. In particular, expression of the histidine metabolism increased up to 100 fold. In contrast, the PM decreased gene expression in cell division and sporulation (standard medium only), cell defense mechanisms, cell envelope, cell motility, and cellulosome in both media. The PM downregulated inorganic ion transport and metabolism in standard medium but upregulated it in the hydrolysate media when compared to the WT. The WT differentially expressed 1072 genes in response to the hydrolysate medium which included increased transcription of cell defense mechanisms, cell motility, and cellulosome, and decreased expression in cell envelope, amino acid transport and metabolism, inorganic ion transport and metabolism, and lipid metabolism, while the PM only differentially expressed 92 genes. The PM tolerates up to 17.5% v/v Populus hydrolysate and growth in it elicited 489 genes with differential expression, which included increased expression in energy production and conversion, cellulosome production, and inorganic ion transport and metabolism and decreased expression in transcription and cell

  18. Decreased coenzyme A levels in ridA mutant strains of Salmonella enterica result from inactivated serine hydroxymethyltransferase.

    PubMed

    Flynn, Jeffrey M; Christopherson, Melissa R; Downs, Diana M

    2013-08-01

    The RidA/Yer057/UK114 family of proteins is well represented across the domains of life and recent work has defined both an in vitro activity and an in vivo role for RidA. RidA proteins have enamine deaminase activity, and in their absence the reactive 2-aminoacrylate (2-AA) accumulates and inactivates at least some pyridoxal 5'-phosphate (PLP)-containing enzymes in Salmonella enterica. The conservation of RidA suggested that 2-AA was a ubiquitous cellular stressor that was generated in central metabolism. Phenotypically, strains of S. enterica that lack RidA accumulated significantly more pyruvate in the growth medium than wild-type strains. Here we dissected this ridA mutant phenotype and showed it was an indirect consequence of damage to serine hydroxymethyltransferase (GlyA; E.C. 2.1.2.1). The results here identified a fourth PLP enzyme as a target of enamine stress in Salmonella.

  19. Constitutive synthesis of enzymes of the protocatechuate pathway and of the beta-ketoadipate uptake system in mutant strains of Pseudomonas putida.

    PubMed Central

    Parke, D; Ornston, L N

    1976-01-01

    Mutant Pseudomonas putida strains that produce constitutive levels of the beta-ketoadipate uptake system are selected by the sequential transfer of cultures between mineral growth media supplemented with the noninducing growth substrate succinate and growth media containing beta-ketoadipate as the sole carbon and energy source. The mutant strains also produce constitutively three catabolic enzymes that give rise to beta-ketoadipate from the metabolic precursor beta-carboxy-cis, cis-muconate, and thus a single regulatory gene appears to govern the expression of the enzymes as well as the uptake system. The three enzymes that convert beta-carboxy-cis, cis-muconate to beta-ketoadipate are induced to higher levels when the orgainisms are grown with p-hydroxybenzoate (a compound that is catabolized via beta-ketoadipate); the beta-ketoadipate uptake system is partially repressed when the cells are grwon at the expense of p-hydroxybenzoate. The transferase that acts upon beta-ketoadipate remains inducible in the constitutive mutant strains. Thus a minimum of three biosynthetic controls must be exerted over the expression of the five genes. Since the regulatory mutation does not alter the expression of the gene for the transferase, the physiological target of the selection procedure appears to be mutant strains that produce the uptake system constitutively. Levels of the uptake system are higher in uninduced constitutive mutant cultures than in induced cultures of the wild type. Hence procedures analogous to the one we employed may be of general use in obtaining mutant strains that produce high levels of uptake systems. PMID:1262305

  20. Compressive viscoelasticity of freshly excised mouse skin is dependent on specimen thickness, strain level and rate.

    PubMed

    Wang, Yuxiang; Marshall, Kara L; Baba, Yoshichika; Lumpkin, Ellen A; Gerling, Gregory J

    2015-01-01

    Although the skin's mechanical properties are well characterized in tension, little work has been done in compression. Here, the viscoelastic properties of a population of mouse skin specimens (139 samples from 36 mice, aged 5 to 34 weeks) were characterized upon varying specimen thickness, as well as strain level and rate. Over the population, we observed the skin's viscoelasticity to be quite variable, yet found systematic correlation of residual stress ratio with skin thickness and strain, and of relaxation time constants with strain rates. In particular, as specimen thickness ranged from 211 to 671 μm, we observed significant variation in both quasi-linear viscoelasticity (QLV) parameters, the relaxation time constant (τ1 = 0.19 ± 0.10 s) and steady-state residual stress ratio (G∞ = 0.28 ± 0.13). Moreover, when τ1 was decoupled and fixed, we observed that G∞ positively correlated with skin thickness. Second, as steady-state stretch was increased (λ∞ from 0.22 to 0.81), we observed significant variation in both QLV parameters (τ1 = 0.26 ± 0.14 s, G∞ = 0.47 ± 0.17), and when τ1 was fixed, G∞ positively correlated with stretch level. Third, as strain rate was increased from 0.06 to 22.88 s-1, the median time constant τ1 varied from 1.90 to 0.31 s, and thereby negatively correlated with strain rate. These findings indicate that the natural range of specimen thickness, as well as experimental controls of compression level and rate, significantly influence measurements of skin viscoelasticity.

  1. Compressive Viscoelasticity of Freshly Excised Mouse Skin Is Dependent on Specimen Thickness, Strain Level and Rate

    PubMed Central

    Wang, Yuxiang; Marshall, Kara L.; Baba, Yoshichika; Lumpkin, Ellen A.; Gerling, Gregory J.

    2015-01-01

    Although the skin’s mechanical properties are well characterized in tension, little work has been done in compression. Here, the viscoelastic properties of a population of mouse skin specimens (139 samples from 36 mice, aged 5 to 34 weeks) were characterized upon varying specimen thickness, as well as strain level and rate. Over the population, we observed the skin’s viscoelasticity to be quite variable, yet found systematic correlation of residual stress ratio with skin thickness and strain, and of relaxation time constants with strain rates. In particular, as specimen thickness ranged from 211 to 671 μm, we observed significant variation in both quasi-linear viscoelasticity (QLV) parameters, the relaxation time constant (τ1 = 0.19 ± 0.10 s) and steady-state residual stress ratio (G∞ = 0.28 ± 0.13). Moreover, when τ1 was decoupled and fixed, we observed that G∞ positively correlated with skin thickness. Second, as steady-state stretch was increased (λ∞ from 0.22 to 0.81), we observed significant variation in both QLV parameters (τ1 = 0.26 ± 0.14 s, G∞ = 0.47 ± 0.17), and when τ1 was fixed, G∞ positively correlated with stretch level. Third, as strain rate was increased from 0.06 to 22.88 s−1, the median time constant τ1 varied from 1.90 to 0.31 s, and thereby negatively correlated with strain rate. These findings indicate that the natural range of specimen thickness, as well as experimental controls of compression level and rate, significantly influence measurements of skin viscoelasticity. PMID:25803703

  2. The Relationship between PROP and Ethanol Preferences: An Evaluation of 4 Inbred Mouse Strains

    PubMed Central

    White, Theresa L.; Dishaw, Laura V.; Sheehe, Paul R.; Youngentob, Steven L.

    2012-01-01

    Ethanol’s taste attributes undoubtedly contribute to the development of drug preference. Ethanol’s taste is both sweet and bitter. Taster status for bitter 6-n-propylthiouracil (PROP) has been proposed as a genetic marker for alcoholism; however, human results are conflicting. We collected preference scores for both tastants in 4 mouse strains selected on the basis of previously reported taste preference, with the generally accepted idea that inbred mice show minimal within-strain variation. Eighty-eight male mice (22 per strain) participated. The strains were as follows: C57BL/6J, ethanol preferring; BALB/cJ, ethanol avoiding; SWR/J, PROP avoiding; and C3HeB/FeJ, PROP neutral. Using a brief-access (1-min trials) 2-bottle preference test, we assessed the taste response of each strain to PROP and ethanol on separate days. Although PROP avoiding versus neutral mice could be segregated into significantly different populations, this was not the case for ethanol avoiding versus preferring mice, and all strains showed high variability. On average, only BALB/cJ, SWR/J, and C3HeB/FeJ mice conformed to their literature-reported preferences; nonetheless, there were a substantial number of discordant animals. C57BL/6J did not conform to previous results, indicating that they are ethanol preferring. Finally, we did not observe a significant relationship between PROP and ethanol preferences across strains. The high variability per strain and the number of animals in disagreement with their respective literature-reported preference raise concerns regarding their utility for investigations underlying mechanisms of taste-mediated ingestive responses. Absent postingestive consequences, the brief-access results suggest a possible degree of previously masked polymorphisms in taste preferences or a more recent drift in underlying genetic factors. The absence of a relationship between PROP and ethanol indicates that the bitter quality in ethanol may be more highly related to other

  3. Effects on hippocampus of lifelong absence of glucocorticoids in the pro-opiomelanocortin null mutant mouse reveal complex relationship between glucocorticoids and hippocampal structure and function.

    PubMed

    Ostwald, Dirk; Karpac, Jason; Hochgeschwender, Ute

    2006-01-01

    In humans changes in serum cortisol levels have been observed with aging, stress, and with affective disorders such as major depression and post-traumatic stress disorder. Corticosteroids are known to influence hippocampal structure and function; specifically, plasma corticosteroid levels have been inversely correlated with hippocampal cell proliferation, cell death, and impaired memory function. The relationship between corticosteroids and structure and function of the hippocampus has been studied in experimental systems in adult animals by increasing or decreasing corticosterone levels through pharmacological supplementation and through surgical removal of the adrenal gland. Here, we utilized the genetically engineered pro-opiomelanocortin (POMC) null mutant mouse, which because of the lack of all POMC peptides has no corticosterone from birth throughout life. The effect of this lifelong absence of corticosterone on the dentate gyrus of the hippocampus is a decrease in granule cell density, which correlated with a decrease in cell proliferation but not an increase in cell degeneration. Fine morphology of granule cells was unaltered. Analyses of gene expression revealed no changes in POMC null mutant vs wild-type hippocampus with respect to levels of expression of corticoid receptor genes or genes known to be regulated by corticosterone. Spatial learning as tested by the Morris water maze was not altered in the POMC null mutant mouse. Taken together with findings from other studies of the effects of altered levels of corticosteroids on the hippocampus, our results argue for a complex homeostasis in which disturbances of any one factor can offset the system in varying ways.

  4. Characterization of the Burkholderia mallei tonB Mutant and Its Potential as a Backbone Strain for Vaccine Development

    PubMed Central

    Mott, Tiffany M.; Vijayakumar, Sudhamathi; Sbrana, Elena; Endsley, Janice J.; Torres, Alfredo G.

    2015-01-01

    Background In this study, a Burkholderia mallei tonB mutant (TMM001) deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection models of murine glanders and melioidosis. Methodology/Principal Findings Compared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following B. mallei CSM001 challenge of mice previously receiving 1.5 x 104 CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of B. mallei-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with B. pseudomallei, TMM001-treated mice were significantly protected. While wild type was cleared in all B. mallei challenge studies, mice failed to clear TMM001. Conclusions/Significance Although further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis. PMID:26114445

  5. The relationship between nephron number, kidney size and body weight in two inbred mouse strains.

    PubMed

    Murawski, Inga J; Maina, Rita W; Gupta, Indra R

    2010-01-01

    While some reports in humans have shown that nephron number is positively correlated with height, body weight or kidney weight, other studies have not reproduced these findings. To understand the impact of genetic and environmental variation on these relationships, we examined whether nephron number correlates with body weight, kidney planar surface area, or kidney weight in two inbred mouse strains with contrasting kidney sizes but no overt renal pathology: C3H/HeJ and C57BL/6J. C3H/HeJ mice had smaller kidneys at birth and larger kidneys by adulthood, however there was no significant difference in nephron number between the two strains. We did observe a correlation between kidney size and body weight at birth and at adulthood for both strains. However, there was no relationship between nephron number and body weight or between nephron number and kidney size. From other studies, it appears that a greater than two-fold variation is required in each of these parameters in order to demonstrate these relationships, suggesting they are highly dependent on scale. Our results are therefore not surprising since there was a less than two-fold variation in each of the parameters examined. In summary, the relationship between nephron number and body or kidney size is most likely to be demonstrated when there is greater phenotypic variation either from genetic and/or environmental factors.

  6. Gliopathy of Demyelinating and Non-Demyelinating Strains of Mouse Hepatitis Virus

    PubMed Central

    Kenyon, Lawrence C.; Biswas, Kaushiki; Shindler, Kenneth S.; Nabar, Manasi; Stout, Marjorie; Hingley, Susan T.; Grinspan, Judith B.; Das Sarma, Jayasri

    2015-01-01

    Demyelination in the central nervous system induced by neurovirulent strains of Mouse Hepatitis Virus (MHV) is mediated by the viral spike glycoprotein, but it is not clear whether the mechanism of this disease pathology involves direct viral infection of oligodendrocytes. Detailed studies of glial cell tropism of MHV are presented, demonstrating that direct MHV infection of oligodendrocytes differs between demyelinating (RSA59) and non-demyelinating (RSMHV2) viral strains both in vitro and in vivo. Our results indicate that direct injury of mature oligodendrocytes is an important mechanism of virus-induced demyelination. In vivo, RSA59 infection was identified in spinal cord gray and white matter, but infected oligodendrocytes were restricted to white matter. In contrast, RSMHV2 infection was restricted to gray matter neurons and was not localized to oligodendrocytes. In vitro, RSA59 can infect both oligodendrocyte precursors and differentiated oligodendrocytes, whereas RSMHV2 can infect oligodendrocyte precursors but not differentiated oligodendrocytes. Viral spreading through axonal means to white matter and release of the demyelinating strain MHV at the nerve end is critical for oligodendrocytes infection and subsequent demyelination. Understanding the mechanisms by which known viruses effect demyelination in this animal model has important therapeutic implications in the treatment of human demyelinating disease. PMID:26733813

  7. Alterations in grooming activity and syntax in heterozygous SERT and BDNF knockout mice: the utility of behavior-recognition tools to characterize mutant mouse phenotypes.

    PubMed

    Kyzar, Evan J; Pham, Mimi; Roth, Andrew; Cachat, Jonathan; Green, Jeremy; Gaikwad, Siddharth; Kalueff, Allan V

    2012-12-01

    Serotonin transporter (SERT) and brain-derived neurotrophic factor (BDNF) are key modulators of molecular signaling, cognition and behavior. Although SERT and BDNF mutant mouse phenotypes have been extensively characterized, little is known about their self-grooming behavior. Grooming represents an important behavioral domain sensitive to environmental stimuli and is increasingly used as a model for repetitive behavioral syndromes, such as autism and attention deficit/hyperactivity disorder. The present study used heterozygous ((+/-)) SERT and BDNF male mutant mice on a C57BL/6J background and assessed their spontaneous self-grooming behavior applying both manual and automated techniques. Overall, SERT(+/-) mice displayed a general increase in grooming behavior, as indicated by more grooming bouts and more transitions between specific grooming stages. SERT(+/-) mice also aborted more grooming bouts, but showed generally unaltered activity levels in the observation chamber. In contrast, BDNF(+/-) mice displayed a global reduction in grooming activity, with fewer bouts and transitions between specific grooming stages, altered grooming syntax, as well as hypolocomotion and increased turning behavior. Finally, grooming data collected by manual and automated methods (HomeCageScan) significantly correlated in our experiments, confirming the utility of automated high-throughput quantification of grooming behaviors in various genetic mouse models with increased or decreased grooming phenotypes. Taken together, these findings indicate that mouse self-grooming behavior is a reliable behavioral biomarker of genetic deficits in SERT and BDNF pathways, and can be reliably measured using automated behavior-recognition technology.

  8. Discovery of Candidate Disease Genes in ENU–Induced Mouse Mutants by Large-Scale Sequencing, Including a Splice-Site Mutation in Nucleoredoxin

    PubMed Central

    Wilming, Laurens G.; Liu, Bin; Probst, Frank J.; Harrow, Jennifer; Grafham, Darren; Hentges, Kathryn E.; Woodward, Lanette P.; Maxwell, Andrea; Mitchell, Karen; Risley, Michael D.; Johnson, Randy; Hirschi, Karen; Lupski, James R.; Funato, Yosuke; Miki, Hiroaki; Marin-Garcia, Pablo; Matthews, Lucy; Coffey, Alison J.; Parker, Anne; Hubbard, Tim J.; Rogers, Jane; Bradley, Allan; Adams, David J.; Justice, Monica J.

    2009-01-01

    An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing. PMID:20011118

  9. Polymorphisms in the taste receptor gene (Tas1r3) region are associated with saccharin preference in 30 mouse strains.

    PubMed

    Reed, D R; Li, S; Li, X; Huang, L; Tordoff, M G; Starling-Roney, R; Taniguchi, K; West, D B; Ohmen, J D; Beauchamp, G K; Bachmanov, A A

    2004-01-28

    The results of recent studies suggest that the mouse Sac (saccharin preference) locus is identical to the Tas1r3 (taste receptor) gene. The goal of this study was to identify Tas1r3 sequence variants associated with saccharin preference in a large number of inbred mouse strains. Initially, we sequenced approximately 6.7 kb of the Tas1r3 gene and its flanking regions from six inbred mouse strains with high and low saccharin preference, including the strains in which the Sac alleles were described originally (C57BL/6J, Sac(b); DBA/2J, Sac(d)). Of the 89 sequence variants detected among these six strains, eight polymorphic sites were significantly associated with preferences for 1.6 mm saccharin. Next, each of these eight variant sites were genotyped in 24 additional mouse strains. Analysis of the genotype-phenotype associations in all 30 strains showed the strongest association with saccharin preference at three sites: nucleotide (nt) -791 (3 bp insertion/deletion), nt +135 (Ser45Ser), and nt +179 (Ile60Thr). We measured Tas1r3 gene expression, transcript size, and T1R3 immunoreactivity in the taste tissue of two inbred mouse strains with different Tas1r3 haplotypes and saccharin preferences. The results of these experiments suggest that the polymorphisms associated with saccharin preference do not act by blocking gene expression, changing alternative splicing, or interfering with protein translation in taste tissue. The amino acid substitution (Ile60Thr) may influence the ability of the protein to form dimers or bind sweeteners. Here, we present data for future studies directed to experimentally confirm the function of these polymorphisms and highlight some of the difficulties of identifying specific DNA sequence variants that underlie quantitative trait loci. PMID:14749438

  10. A Lipoprotein Receptor Cluster IV Mutant Preferentially Binds Amyloid-β and Regulates Its Clearance from the Mouse Brain*

    PubMed Central

    Sagare, Abhay P.; Bell, Robert D.; Srivastava, Alaka; Sengillo, Jesse D.; Singh, Itender; Nishida, Yoichiro; Chow, Nienwen; Zlokovic, Berislav V.

    2013-01-01

    Soluble low density lipoprotein receptor-related protein-1 (sLRP1) binds ∼70% of amyloid β-peptide (Aβ) in human plasma. In Alzheimer disease (AD) and individuals with mild cognitive impairment converting to AD, plasma sLRP1 levels are reduced and sLRP1 is oxidized, which results in diminished Aβ peripheral binding and higher levels of free Aβ in plasma. Experimental studies have shown that free circulating Aβ re-enters the brain and that sLRP1 and/or its recombinant wild type cluster IV (WT-LRPIV) prevent Aβ from entering the brain. Treatment of Alzheimer APPsw+/0 mice with WT-LRPIV has been shown to reduce brain Aβ pathology. In addition to Aβ, LRPIV binds multiple ligands. To enhance LRPIV binding for Aβ relative to other LRP1 ligands, we generated a library of LRPIV-derived fragments and full-length LRPIV variants with glycine replacing aspartic acid residues 3394, 3556, and 3674 in the calcium binding sites. Compared with WT-LRPIV, a lead LRPIV-D3674G mutant had 1.6- and 2.7-fold higher binding affinity for Aβ40 and Aβ42 in vitro, respectively, and a lower binding affinity for other LRP1 ligands (e.g. apolipoprotein E2, E3, and E4 (1.3–1.8-fold), tissue plasminogen activator (2.7-fold), matrix metalloproteinase-9 (4.1-fold), and Factor Xa (3.8-fold)). LRPIV-D3674G cleared mouse endogenous brain Aβ40 and Aβ42 25–27% better than WT-LRPIV. A 3-month subcutaneous treatment of APPsw+/0 mice with LRPIV-D3674G (40 μg/kg/day) reduced Aβ40 and Αβ42 levels in the hippocampus, cortex, and cerebrospinal fluid by 60–80% and improved cerebral blood flow responses and hippocampal function at 9 months of age. Thus, LRPIV-D3674G is an efficient new Aβ clearance therapy. PMID:23580652

  11. Attention to Background Strain Is Essential for Metabolic Research: C57BL/6 and the International Knockout Mouse Consortium.

    PubMed

    Fontaine, Danielle A; Davis, Dawn Belt

    2016-01-01

    The International Knockout Mouse Consortium (IKMC) introduces its targeted constructs into C57BL/6N embryonic stem cells. However, breeding with a Cre-recombinase and/or Flp-recombinase mouse is required for the generation of a null allele with the IKMC cassette. Many recombinase strains are in the C57BL/6J background, resulting in knockout animals on a mixed strain background. This can lead to variability in metabolic data and the use of improper control groups. While C57BL/6N and C57BL/6J are derived from the same parental C57BL/6 strain, there are key genotypic and phenotypic differences between these substrains. Many researchers may not even be aware of these differences, as the shorthand C57BL/6 is often used to describe both substrains. We found that 58% of articles involving genetically modified mouse models did not completely address background strain. This review will describe these two substrains and highlight the importance of separate consideration in mouse model development. Our aim is to increase awareness of this issue in the diabetes research community and to provide practical strategies to enable researchers to avoid mixed strain animals when using IKMC knockout mice. PMID:26696638

  12. Attention to Background Strain Is Essential for Metabolic Research: C57BL/6 and the International Knockout Mouse Consortium.

    PubMed

    Fontaine, Danielle A; Davis, Dawn Belt

    2016-01-01

    The International Knockout Mouse Consortium (IKMC) introduces its targeted constructs into C57BL/6N embryonic stem cells. However, breeding with a Cre-recombinase and/or Flp-recombinase mouse is required for the generation of a null allele with the IKMC cassette. Many recombinase strains are in the C57BL/6J background, resulting in knockout animals on a mixed strain background. This can lead to variability in metabolic data and the use of improper control groups. While C57BL/6N and C57BL/6J are derived from the same parental C57BL/6 strain, there are key genotypic and phenotypic differences between these substrains. Many researchers may not even be aware of these differences, as the shorthand C57BL/6 is often used to describe both substrains. We found that 58% of articles involving genetically modified mouse models did not completely address background strain. This review will describe these two substrains and highlight the importance of separate consideration in mouse model development. Our aim is to increase awareness of this issue in the diabetes research community and to provide practical strategies to enable researchers to avoid mixed strain animals when using IKMC knockout mice.

  13. Draft Genome Sequences of Two Heat-Resistant Mutant Strains (A52 and B41) of the Photosynthetic Hydrogen-Producing Bacterium Rhodobacter capsulatus.

    PubMed

    Gokce, Abdulmecit; Cakar, Zeynep Petek; Yucel, Meral; Ozcan, Orhan; Sencan, Sevde; Sertdemir, Ibrahim; Erguner, Bekir; Yuceturk, Betul; Sarac, Aydan; Yuksel, Bayram; Ozturk, Yavuz

    2016-01-01

    The draft genome sequences of two heat-resistant mutant strains, A52 and B41, derived from Rhodobacter capsulatus DSM 1710, and with different hydrogen production levels, are reported here. These sequences may help understand the molecular basis of heat resistance and hydrogen production in R. capsulatus.

  14. Draft Genome Sequences of Two Heat-Resistant Mutant Strains (A52 and B41) of the Photosynthetic Hydrogen-Producing Bacterium Rhodobacter capsulatus

    PubMed Central

    Gokce, Abdulmecit; Cakar, Zeynep Petek; Yucel, Meral; Ozcan, Orhan; Sencan, Sevde; Sertdemir, Ibrahim; Erguner, Bekir; Yuceturk, Betul; Sarac, Aydan; Yuksel, Bayram

    2016-01-01

    The draft genome sequences of two heat-resistant mutant strains, A52 and B41, derived from Rhodobacter capsulatus DSM 1710, and with different hydrogen production levels, are reported here. These sequences may help understand the molecular basis of heat resistance and hydrogen production in R. capsulatus. PMID:27284151

  15. Complete Closed Genome Sequences of a Mannheimia haemolytica Serotype A1 Leukotoxin Deletion Mutant and Its Wild-Type Parent Strain

    PubMed Central

    Harhay, Gregory P.; Smith, Timothy P. L.; Bono, James L.; Chitko-McKown, Carol G.

    2015-01-01

    Mannheimia haemolytica is a bacterial pathogen that secretes leukotoxin (LktA) which binds to leukocyte membranes via CD18, causing bacterial pneumonia in ruminants. We report the complete closed genome sequences of a leukotoxin mutant and its parent strain that are frequently used in respiratory disease studies. PMID:25953160

  16. luxS mutants of Serratia defective in autoinducer-2-dependent 'quorum sensing' show strain-dependent impacts on virulence and production of carbapenem and prodigiosin.

    PubMed

    Coulthurst, Sarah J; Kurz, C Léopold; Salmond, George P C

    2004-06-01

    The enzyme LuxS is responsible for the production of autoinducer-2 (AI-2), a molecule that has been implicated in quorum sensing in many bacterial species. This study investigated whether there is a luxS-dependent signalling system in the Gram-negative bacteria Serratia spp. Serratia marcescens is a broad-host-range pathogen and an important cause of nosocomial infections. Production of AI-2 activity was detected in S. marcescens ATCC 274 and Serratia ATCC 39006 and their luxS genes were sequenced. luxS mutants were constructed in these strains and were analysed to determine which phenotypes are regulated by luxS and therefore, potentially, by AI-2. The phenotypes of the luxS mutants included decreased carbapenem antibiotic production in Serratia ATCC 39006 and decreased prodigiosin and secreted haemolysin production in S. marcescens ATCC 274. The luxS mutant of S. marcescens ATCC 274 was also found to exhibit modestly reduced virulence in a Caenorhabditis elegans model. Finally, it was shown that the culture supernatant of a wild-type strain contains a signal, presumably AI-2, capable of complementing the prodigiosin defect of the luxS mutant of another strain, even when substantially diluted. It is concluded that luxS modulates virulence and antibiotic production in Serratia, in a strain-dependent manner, and that, for at least one phenotype, this regulation is via extracellular signalling.

  17. A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains

    PubMed Central

    2013-01-01

    Background The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms. Results We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems. Conclusions Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains. PMID:23902802

  18. The Brucella abortus phosphoglycerate kinase mutant is highly attenuated and induces protection superior to that of vaccine strain 19 in immunocompromised and immunocompetent mice.

    PubMed

    Trant, Cyntia G M C; Lacerda, Thais L S; Carvalho, Natalia B; Azevedo, Vasco; Rosinha, Gracia M S; Salcedo, Suzana P; Gorvel, Jean-Pierre; Oliveira, Sergio C

    2010-05-01

    Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that can function as virulence factors to better understand the host-pathogen interplay. Herein, we identified the gene encoding the phosphoglycerate kinase (PGK) of B. abortus strain 2308. To test the role of PGK in Brucella pathogenesis, a pgk deletion mutant was constructed. Replacement of the wild-type pgk by recombination was demonstrated by Southern and Western blot analyses. The B. abortus Delta pgk mutant strain exhibited extreme attenuation in bone marrow-derived macrophages and in vivo in BALB/c, C57BL/6, 129/Sv, and interferon regulatory factor-1 knockout (IRF-1 KO) mice. Additionally, at 24 h postinfection the Delta pgk mutant was not found within the same endoplasmic reticulum-derived compartment as the wild-type bacteria, but, instead, over 60% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP1. Furthermore, the B. abortus Delta pgk deletion mutant was used as a live vaccine. Challenge experiments revealed that the Delta pgk mutant strain induced protective immunity in 129/Sv or IRF-1 KO mice that was superior to the protection conferred by commercial strain 19 or RB51. Finally, the results shown here demonstrated that Brucella PGK is critical for full bacterial virulence and that a Delta pgk mutant may serve as a potential vaccine candidate in future studies. PMID:20194591

  19. The Effects of Modeled Microgravity on Growth Kinetics, Antibiotic Susceptibility, Cold Growth, and the Virulence Potential of a Yersinia pestis ymoA-Deficient Mutant and Its Isogenic Parental Strain

    PubMed Central

    Lawal, Abidat; Kirtley, Michelle L.; van Lier, Christina J.; Erova, Tatiana E.; Kozlova, Elena V.; Sha, Jian; Chopra, Ashok K.

    2013-01-01

    Abstract Previously, we reported that there was no enhancement in the virulence potential (as measured by cell culture infections) of the bacterial pathogen Yersinia pestis (YP) following modeled microgravity/clinorotation growth. We have now further characterized the effects of clinorotation (CR) on YP growth kinetics, antibiotic sensitivity, cold growth, and YP's virulence potential in a murine model of infection. Surprisingly, none of the aforementioned phenotypes were altered. To better understand why CR did not enhance YP's virulence potential as it did for other bacterial pathogens, a YP ΔymoA isogenic mutant in the KIM/D27 background strain that is unable to produce the histone-like YmoA protein and influences DNA topography was used in both cell culture and murine models of infection. YmoA represses type three secretion system (T3SS) virulence gene expression in the yersiniae. Similar to our CR-grown parental YP strain data, the CR-grown ΔymoA mutant induced reduced HeLa cell cytotoxicity with concomitantly decreased Yersinia outer protein E (YopE) and low calcium response V (LcrV) antigen production and secretion. Important, however, were our findings that, although no significant differences were observed in survival of mice infected intraperitoneally with either normal gravity (NG)- or CR-grown parental YP, the ΔymoA mutant induced significantly more mortality in infected mice than did the parental strain following CR growth. Taken together, our data demonstrate that CR did enhance the virulence potential of the YP ΔymoA mutant in a murine infection model (relative to the CR-grown parental strain), despite inducing less HeLa cell rounding in our cell culture infection assay due to reduced T3SS activity. Therefore, CR, which induces a unique type of bacterial stress, might be enhancing YP's virulence potential in vivo through a T3SS-independent mechanism when the histone-like YmoA protein is absent. Key Words: Type three secretion system (T3SS

  20. Improved generation of germline-competent embryonic stem cell lines from inbred mouse strains.

    PubMed

    Schoonjans, Luc; Kreemers, Veerle; Danloy, Sophie; Moreadith, Randall W; Laroche, Yves; Collen, Désiré

    2003-01-01

    Genetically altered mice may exhibit highly variable phenotypes due to the variation in genetic background, which can only be circumvented by generation of inbred, isogenic gene-targeted and control mice. Here we report that an embryonic stem (ES) cell culture medium conditioned by a rabbit fibroblast cell line transduced with genomic rabbit leukemia inhibitory factor allows efficient derivation and maintenance of ES cell lines from all of 10 inbred mouse strains tested, including some that were presumed to be nonpermissive for ES cell derivation (129/SvEv, 129/SvJ, C57BL/6N, C57BL/6JOla, CBA/CaOla, DBA/2N, DBA/1Ola, C3H/HeN, BALB/c, and FVB/N). Germline transmission was established by blastocyst injection of established ES cell lines after 10 or more passages from all of seven strains tested (129/SvJ, C57BL/6N, C57BL/6JOla, DBA/2N, DBA/1Ola, BALB/c, and FVB/N), by diploid aggregation of ES cell lines from all of four strains tested (129/SvEv, C57BL/6N, CBA/ CaOla, and FVB/N), or by tetraploid aggregation of ES cell lines from all of three strains tested (129/SvEv, C57BL/6N, and CBA/CaOla). Thus, these inbred ES cell lines may constitute useful tools to derive gene-targeted mice and isogenic controls in selected genetic backgrounds.

  1. Establishment of latent herpes simplex virus type 1 infection in resistant, sensitive, and immunodeficient mouse strains.

    PubMed

    Ellison, A R; Yang, L; Voytek, C; Margolis, T P

    2000-03-01

    Productive infection with herpes simplex virus (HSV) type 1 is limited by both innate and adaptive immune mechanisms. The purpose of the current study was to determine whether these mechanisms also play a role in the establishment of latent HSV infection. First we examined the trigeminal ganglia (TG) of severe combined immunodeficiency (SCID), interferon-gamma knockout (GKO), and beige (a strain deficient in natural killer cell activity) mice following ocular inoculation with HSV. Although infection of SCID mice was invariably lethal, we consistently found latently infected neurons in the TG of these animals at 2-4 days postinoculation. HSV infection of GKO and beige mice, while not lethal, was characterized by a greater number of productively infected TG neurons and/or a delay in the time to peak productive infection compared to C57BL/6 controls. However, as assayed by both in situ hybridization for LAT expression and quantitative PCR (Q-PCR) for viral DNA, we found that HSV established a latent infection in GKO and beige mice as efficiently as in C57BL/6 controls. We subsequently examined the TG of "HSV-sensitive" strains of mice (Swiss-Webster, CBA, and BALB/c) following ocular infection with HSV. At the peak of acute ganglionic infection the number of productively infected TG neurons in each of these mouse strains was about sevenfold greater than in the "HSV-resistant" strain C57BL/6, consistent with previously reported differences in susceptibility to lethal challenge with HSV. However, as assayed by both in situ hybridization for LAT and Q-PCR for viral DNA, we found that HSV established a latent infection in Swiss-Webster, CBA, and BALB/c mice as efficiently as in C57BL/6 controls. We conclude that HSV efficiently establishes latent infection in the TG of mice in the absence of innate and adaptive immune mechanisms that are essential for limiting productive viral infection.

  2. Marine Mammal Brucella Reference Strains Are Attenuated in a BALB/c Mouse Model.

    PubMed

    Nymo, Ingebjørg H; Arias, Maykel A; Pardo, Julián; Álvarez, María Pilar; Alcaraz, Ana; Godfroid, Jacques; Jiménez de Bagüés, María Pilar

    2016-01-01

    Brucellosis is a zoonosis of worldwide distribution with numerous animal host species. Since the novel isolation of Brucella spp. from marine mammals in 1994 the bacteria have been isolated from various marine mammal hosts. The marine mammal reference strains Brucella pinnipedialis 12890 (harbour seal, Phoca vitulina) and Brucella ceti 12891 (harbour porpoise, Phocoena phocoena) were included in genus Brucella in 2007, however, their pathogenicity in the mouse model is pending. Herein this is evaluated in BALB/c mice with Brucella suis 1330 as a control. Both marine mammal strains were attenuated, however, B. ceti was present at higher levels than B. pinnipedialis in blood, spleen and liver throughout the infection, in addition B. suis and B. ceti were isolated from brains and faeces at times with high levels of bacteraemia. In B. suis-infected mice serum cytokines peaked at day 7. In B. pinnipedialis-infected mice, levels were similar, but peaked predominantly at day 3 and an earlier peak in spleen weight likewise implied an earlier response. The inflammatory response induced pathology in the spleen and liver. In B. ceti-infected mice, most serum cytokine levels were comparable to those in uninfected mice, consistent with a limited inflammatory response, which also was indicated by restricted spleen and liver pathology. Specific immune responses against all three strains were detected in vitro after stimulation of splenocytes from infected mice with the homologous heat-killed brucellae. Antibody responses in vivo were also induced by the three brucellae. The immunological pattern of B. ceti in combination with persistence in organs and limited pathology has heretofore not been described for other brucellae. These two marine mammal wildtype strains show an attenuated pattern in BALB/c mice only previously described for Brucella neotomea. PMID:26959235

  3. Marine Mammal Brucella Reference Strains Are Attenuated in a BALB/c Mouse Model

    PubMed Central

    Nymo, Ingebjørg H.; Arias, Maykel A.; Pardo, Julián; Álvarez, María Pilar; Alcaraz, Ana; Godfroid, Jacques; Jiménez de Bagüés, María Pilar

    2016-01-01

    Brucellosis is a zoonosis of worldwide distribution with numerous animal host species. Since the novel isolation of Brucella spp. from marine mammals in 1994 the bacteria have been isolated from various marine mammal hosts. The marine mammal reference strains Brucella pinnipedialis 12890 (harbour seal, Phoca vitulina) and Brucella ceti 12891 (harbour porpoise, Phocoena phocoena) were included in genus Brucella in 2007, however, their pathogenicity in the mouse model is pending. Herein this is evaluated in BALB/c mice with Brucella suis 1330 as a control. Both marine mammal strains were attenuated, however, B. ceti was present at higher levels than B. pinnipedialis in blood, spleen and liver throughout the infection, in addition B. suis and B. ceti were isolated from brains and faeces at times with high levels of bacteraemia. In B. suis-infected mice serum cytokines peaked at day 7. In B. pinnipedialis-infected mice, levels were similar, but peaked predominantly at day 3 and an earlier peak in spleen weight likewise implied an earlier response. The inflammatory response induced pathology in the spleen and liver. In B. ceti-infected mice, most serum cytokine levels were comparable to those in uninfected mice, consistent with a limited inflammatory response, which also was indicated by restricted spleen and liver pathology. Specific immune responses against all three strains were detected in vitro after stimulation of splenocytes from infected mice with the homologous heat-killed brucellae. Antibody responses in vivo were also induced by the three brucellae. The immunological pattern of B. ceti in combination with persistence in organs and limited pathology has heretofore not been described for other brucellae. These two marine mammal wildtype strains show an attenuated pattern in BALB/c mice only previously described for Brucella neotomea. PMID:26959235

  4. Marine Mammal Brucella Reference Strains Are Attenuated in a BALB/c Mouse Model.

    PubMed

    Nymo, Ingebjørg H; Arias, Maykel A; Pardo, Julián; Álvarez, María Pilar; Alcaraz, Ana; Godfroid, Jacques; Jiménez de Bagüés, María Pilar

    2016-01-01

    Brucellosis is a zoonosis of worldwide distribution with numerous animal host species. Since the novel isolation of Brucella spp. from marine mammals in 1994 the bacteria have been isolated from various marine mammal hosts. The marine mammal reference strains Brucella pinnipedialis 12890 (harbour seal, Phoca vitulina) and Brucella ceti 12891 (harbour porpoise, Phocoena phocoena) were included in genus Brucella in 2007, however, their pathogenicity in the mouse model is pending. Herein this is evaluated in BALB/c mice with Brucella suis 1330 as a control. Both marine mammal strains were attenuated, however, B. ceti was present at higher levels than B. pinnipedialis in blood, spleen and liver throughout the infection, in addition B. suis and B. ceti were isolated from brains and faeces at times with high levels of bacteraemia. In B. suis-infected mice serum cytokines peaked at day 7. In B. pinnipedialis-infected mice, levels were similar, but peaked predominantly at day 3 and an earlier peak in spleen weight likewise implied an earlier response. The inflammatory response induced pathology in the spleen and liver. In B. ceti-infected mice, most serum cytokine levels were comparable to those in uninfected mice, consistent with a limited inflammatory response, which also was indicated by restricted spleen and liver pathology. Specific immune responses against all three strains were detected in vitro after stimulation of splenocytes from infected mice with the homologous heat-killed brucellae. Antibody responses in vivo were also induced by the three brucellae. The immunological pattern of B. ceti in combination with persistence in organs and limited pathology has heretofore not been described for other brucellae. These two marine mammal wildtype strains show an attenuated pattern in BALB/c mice only previously described for Brucella neotomea.

  5. Altered sensitivity to excitotoxic cell death and glutamate receptor expression between two commonly studied mouse strains

    PubMed Central

    Finn, Rozzy; Kovács, Attila D.; Pearce, David A.

    2011-01-01

    Alterations in glutamatergic synapse function have been implicated in the pathogenesis of many different neurological disorders including ischemia, epilepsy, Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease. While studying glutamate receptor function in juvenile Batten disease on the C57BL/6J and 129S6/SvEv mouse backgrounds, we noticed differences unlikely to be due to mutation difference alone. We report here that primary cerebellar granule cell cultures from C57BL/6J mice are more sensitive to NMDA-mediated cell death. Moreover, sensitivity to AMPA-mediated excitotoxicity is more variable and is dependent upon the treatment conditions and age of the cultures. Glutamate receptor surface expression levels examined in vitro by in situ ELISA and in vivo by Western blot in surface cross-linked cerebellar samples indicated that these differences in sensitivity are likely due to strain-dependent differences in cell surface receptor expression levels. We propose that differences in glutamate receptor expression and in excitotoxic vulnerability should be taken into consideration in the context of characterizing disease models on the C57BL/6J and 129S6/SvEv mouse backgrounds. PMID:20544821

  6. Differences in GABA-induced chloride ion influx in brain of inbred mouse strains

    SciTech Connect

    Yu, O.; Chiu, T.H.; Rosenberg, H.C.

    1986-03-01

    Audiogenic seizure-susceptible (AS) mice (DBA2J) are a widely used model of epilepsy. The precise pathophysiology of this mouse strain is not fully understood. One of the proposed mechanisms was a difference in GABA/BZ receptor affinity and population from that of audiogenic seizure resistant (ASR) mice. This study attempted to determine the difference in function of GABA/BZ receptor between DBA2J (AS) and C57BL6J (ASR) mice by directly measuring the GABA-induced chloride ion (/sup 36/Cl/sup -/) influx in twice washed crude brain homogenates. /sup 36/Cl/sup -/ influx was terminated by ice-cold buffer and collected by filtration. A concentration range of 2-1000 ..mu..M GABA and two age-matched groups (20-22 days and 40-42 days) were used. GABA-induced /sup 36/Cl/sup -/ influx was dose-dependent, and brain homogenates from DBA2J mice (20-22 days) were less sensitive to GABA-induced Cl/sup -/ ion influx than C57BL6J mice at both age groups. However, in older DBA2J mice (40-42 days), the sensitivity to GABA was intermediate between that of the younger AS mice and the control ASR mice. No significant difference in basal influx of Cl/sup -/ was observed between age groups and mouse strains, nor was there any significant difference between 20-22 days old and 40-42 days old C57BL6J mice. In conclusion, this study had demonstrated a malfunction may recover with age.

  7. INDUCTION OF DNA ADDUCTS, TUMORS, AND KI-RAS ONCOGENE MUTATIONS IN STRAIN A/J MOUSE LUNG BY IP. ADMINISTRATION OF DIBENZ[A,H]ANTHRACENE

    EPA Science Inventory

    Induction of DNA adducts, tumors, and Ki-ras oncogene mutations in strain AlJ mouse lung by ip. administration of dibenz[a,h]anthracene

    Previous studies of polycyclic aromatic hydrocarbon (P AH) induced lung tumors in the strain NJ mouse model system have demonstrated qua...

  8. Identification of a new Pmp22 mouse mutant and trafficking analysis of a Pmp22 allelic series suggesting that protein aggregates may be protective in Pmp22-associated peripheral neuropathy.

    PubMed

    Isaacs, Adrian M; Jeans, Alexander; Oliver, Peter L; Vizor, Lucie; Brown, Steve D M; Hunter, A Jackie; Davies, Kay E

    2002-09-01

    We have identified and characterized a new peripheral myelin protein 22 (Pmp22) mouse mutant. The mutation results in a serine to threonine amino acid substitution at residue 72, which is a hot spot for mutation in human PMP22, leading to the peripheral neuropathy Dejerine-Sottas syndrome. We have previously described two other Pmp22 mutants, providing an allelic series for gene function analysis. Pmp22 mutations generally lead to abnormal intracellular trafficking of Pmp22, and we show that each mutant protein in the allelic series has a unique pattern of intracellular localization in transfected cell lines. The mutant protein from the less severely affected mutants occurs in large aggregates, while the mutant protein from the most severely affected mutant occurs in a diffuse perinuclear pattern that largely colocalizes with wild-type protein. This suggests that large Pmp22 aggregates may be protective in this form of peripheral neuropathy. PMID:12359155

  9. Booster vaccination with safe, modified, live-attenuated mutants of Brucella abortus strain RB51 vaccine confers protective immunity against virulent strains of B. abortus and Brucella canis in BALB/c mice.

    PubMed

    Truong, Quang Lam; Cho, Youngjae; Kim, Kiju; Park, Bo-Kyoung; Hahn, Tae-Wook

    2015-11-01

    Brucella abortus attenuated strain RB51 vaccine (RB51) is widely used in prevention of bovine brucellosis. Although vaccination with this strain has been shown to be effective in conferring protection against bovine brucellosis, RB51 has several drawbacks, including residual virulence for animals and humans. Therefore, a safe and efficacious vaccine is needed to overcome these disadvantages. In this study, we constructed several gene deletion mutants (ΔcydC, ΔcydD and ΔpurD single mutants, and ΔcydCΔcydD and ΔcydCΔpurD double mutants) of RB51 with the aim of increasing the safety of the possible use of these mutants as vaccine candidates. The RB51ΔcydC, RB51ΔcydD, RB51ΔpurD, RB51ΔcydCΔcydD and RB51ΔcydCΔpurD mutants exhibited significant attenuation of virulence when assayed in murine macrophages in vitro or in BALB/c mice. A single intraperitoneal immunization with RB51ΔcydC, RB51ΔcydD, RB51ΔcydCΔcydD or RB51ΔcydCΔpurD mutants was rapidly cleared from mice within 3 weeks, whereas the RB51ΔpurD mutant and RB51 were detectable in spleens until 4 and 7 weeks, respectively. Vaccination with a single dose of RB51 mutants induced lower protective immunity in mice than did parental RB51. However, a booster dose of these mutants provided significant levels of protection in mice against challenge with either the virulent homologous B. abortus strain 2308 or the heterologous Brucella canis strain 26. In addition, these mutants were found to induce a mixed but T-helper-1-biased humoral and cellular immune response in immunized mice. These data suggest that immunization with a booster dose of attenuated RB51 mutants provides an attractive strategy to protect against either bovine or canine brucellosis.

  10. Construction of a Quadruple Auxotrophic Mutant of an Industrial Polyploid Saccharomyces cerevisiae Strain by Using RNA-Guided Cas9 Nuclease

    PubMed Central

    Zhang, Guo-Chang; Kong, In Iok; Kim, Heejin; Liu, Jing-Jing; Cate, Jamie H. D.

    2014-01-01

    Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries. PMID:25281382

  11. Complete Genome Sequence of Turicibacter sp. Strain H121, Isolated from the Feces of a Contaminated Germ-Free Mouse

    PubMed Central

    Auchtung, T. A.; Holder, M. E.; Gesell, J. R.; Ajami, N. J.; Duarte, R. T. D.; Itoh, K.; Caspi, R. R.; Petrosino, J. F.; Horai, R.

    2016-01-01

    Turicibacter bacteria are commonly detected in the gastrointestinal tracts and feces of humans and animals, but their phylogeny, ecological role, and pathogenic potential remain unclear. We present here the first complete genome sequence of Turicibacter sp. strain H121, which was isolated from the feces of a mouse line contaminated following germ-free derivation. PMID:27013036

  12. MK-801, a noncompetitive NMDA receptor antagonist, elicits circling behavior in the genetically inbred Balb/c mouse strain.

    PubMed

    Burket, Jessica A; Cannon, William R; Jacome, Luis F; Deutsch, Stephen I

    2010-11-20

    The Balb/c mouse is behaviorally hypersensitive to effects of MK-801 (dizocilpine), a noncompetitive NMDA receptor antagonist, and displays impaired sociability. In the current investigation, MK-801-elicited circling behavior in the genetically inbred Balb/c mouse strain that was either not or only minimally observed in similarly treated outbred Swiss-Webster mice. The ability of compounds to attenuate the intensity of MK-801-elicited circling behavior in the Balb/c mouse strain may serve as a preclinical screening paradigm for identifying effective NMDA receptor agonist interventions in the intact animal; ideally, these compounds would have therapeutic value in neuropsychiatric disorders associated with impaired sociability, such as schizophrenia and autism spectrum disorders (ASD).

  13. TRPV1 expression level in isolectin B₄-positive neurons contributes to mouse strain difference in cutaneous thermal nociceptive sensitivity.

    PubMed

    Ono, Kentaro; Ye, Yi; Viet, Chi T; Dang, Dongmin; Schmidt, Brian L

    2015-05-01

    Differential thermal nociception across inbred mouse strains has genetic determinants. Thermal nociception is largely attributed to the heat/capsaicin receptor transient receptor potential vanilloid 1 (TRPV1); however, the contribution of this channel to the genetics of thermal nociception has not been revealed. In this study we compared TRPV1 expression levels and electrophysiological properties in primary sensory neurons and thermal nociceptive behaviors between two (C57BL/6 and BALB/c) inbred mouse strains. Using immunofluorescence and patch-clamp physiology methods, we demonstrated that TRPV1 expression was significantly higher in isolectin B4 (IB4)-positive trigeminal sensory neurons of C57BL/6 relative to BALB/c; the expression in IB4-negative neurons was similar between the strains. Furthermore, using electrophysiological cell classification (current signature method), we showed differences between the two strains in capsaicin sensitivity in IB4-positive neuronal cell types 2 and 13, which were previously reported as skin nociceptors. Otherwise electrophysiological membrane properties of the classified cell types were similar in the two mouse strains. In publicly available nocifensive behavior data and our own behavior data from the using the two mouse strains, C57BL/6 exhibited higher sensitivity to heat stimulation than BALB/c, independent of sex and anatomical location of thermal testing (the tail, hind paw, and whisker pad). The TRPV1-selective antagonist JNJ-17203212 inhibited thermal nociception in both strains; however, removing IB4-positive trigeminal sensory neurons with IB4-conjugated saporin inhibited thermal nociception on the whisker pad in C57BL/6 but not in BALB/c. These results suggest that TRPV1 expression levels in IB4-positive type 2 and 13 neurons contributed to differential thermal nociception in skin of C57BL/6 compared with BALB/c.

  14. Calcium, potassium, iron, copper and zinc concentrations in the white and gray matter of the cerebellum and corpus callosum in brain of four genetic mouse strains

    NASA Astrophysics Data System (ADS)

    Sergeant, C.; Vesvres, M. H.; Devès, G.; Guillou, F.

    2005-04-01

    In the central nervous system, metallic cations are involved in oligodendrocyte maturation and myelinogenesis. Moreover, the metallic cations have been associated with pathogenesis, particularly multiple sclerosis and malignant gliomas. The brain is vulnerable to either a deficit or an excess of available trace elements. Relationship between trace metals and myelinogenesis is important in understanding a severe human pathology : the multiple sclerosis, which remains without efficient treatment. One approach to understand this disease has used mutant or transgenic mice presenting myelin deficiency or excess. But to date, the concentration of trace metals and mineral elements in white and gray matter areas in wild type brain is unknown. The aim of this study is to establish the reference concentrations of trace metals (iron, copper and zinc) and minerals (potassium and calcium) in the white and gray matter of the mouse cerebellum and corpus callosum. The brains of four different genetic mouse strains (C57Black6/SJL, C57Black6/D2, SJL and C3H) were analyzed. The freeze-dried samples were prepared to allow PIXE (Proton-induced X-ray emission) and RBS (Rutherford backscattering spectrometry) analyses with the nuclear microprobe in Bordeaux. The results obtained give the first reference values. Furthermore, one species out of the fours testes exhibited differences in calcium, iron and zinc concentrations in the white matter.

  15. Dataset of differentially regulated proteins in HUVECs challenged with wild type and UGM1 mutant Aspergillus fumigatus strains.

    PubMed

    Neves, Gabriela Westerlund Peixoto; Curty, Nathália; Kubitschek-Barreira, Paula Helena; Fontaine, Thierry; Souza, Gustavo Henrique Martins Ferreira; Cunha, Marcel Lyra; Goldman, Gustavo H; Beauvais, Anne; Latgé, Jean-Paul; Lopes-Bezerra, Leila M

    2016-12-01

    Invasive aspergillosis is the primary opportunistic invasive fungal infection described in neutropenic hematologic patients, caused by the angioinvasive pathogen Aspergillus fumigatus. The molecular mechanisms associated with A. fumigatus infection in the vascular endothelium are poorly understood. In this context, we used a high-throughput proteomic approach to unveil the proteins modulated in HUVECs after interaction with a wild type strain and the UGM1 mutant (Δugm1) of A. fumigatus. The proteomic analysis was also performed in HUVECs challenged with a galactosaminogalactan (GAG) purified from A. fumigatus cell wall. The dataset presented here correspond to all proteins identified that fit a 2-fold change criteria (log 2 ratio ≥ 1 or ≤ -1), disregarding the statistical validation cut off, in order to supplement the research article entitled "Modifications to the composition of the hyphal outer layer of Aspergillus fumigatus modulates the HUVEC proteins associated with inflammatory and stress responses" (G.W.P. Neves, N.A. Curty, P.H. Kubitschek-Barreira, T. Fontaine, G.H.M.F. Souza, M. Lyra Cunha, G.H. Goldman, A. Beauvais, J.P. Latgé, L.M. Lopes-Bezerra, 2016) [1]. The mass spectrometry proteomic data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002823. PMID:27622208

  16. Dataset of differentially regulated proteins in HUVECs challenged with wild type and UGM1 mutant Aspergillus fumigatus strains.

    PubMed

    Neves, Gabriela Westerlund Peixoto; Curty, Nathália; Kubitschek-Barreira, Paula Helena; Fontaine, Thierry; Souza, Gustavo Henrique Martins Ferreira; Cunha, Marcel Lyra; Goldman, Gustavo H; Beauvais, Anne; Latgé, Jean-Paul; Lopes-Bezerra, Leila M

    2016-12-01

    Invasive aspergillosis is the primary opportunistic invasive fungal infection described in neutropenic hematologic patients, caused by the angioinvasive pathogen Aspergillus fumigatus. The molecular mechanisms associated with A. fumigatus infection in the vascular endothelium are poorly understood. In this context, we used a high-throughput proteomic approach to unveil the proteins modulated in HUVECs after interaction with a wild type strain and the UGM1 mutant (Δugm1) of A. fumigatus. The proteomic analysis was also performed in HUVECs challenged with a galactosaminogalactan (GAG) purified from A. fumigatus cell wall. The dataset presented here correspond to all proteins identified that fit a 2-fold change criteria (log 2 ratio ≥ 1 or ≤ -1), disregarding the statistical validation cut off, in order to supplement the research article entitled "Modifications to the composition of the hyphal outer layer of Aspergillus fumigatus modulates the HUVEC proteins associated with inflammatory and stress responses" (G.W.P. Neves, N.A. Curty, P.H. Kubitschek-Barreira, T. Fontaine, G.H.M.F. Souza, M. Lyra Cunha, G.H. Goldman, A. Beauvais, J.P. Latgé, L.M. Lopes-Bezerra, 2016) [1]. The mass spectrometry proteomic data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002823.

  17. The cbs mutant strain of Rhodococcus erythropolis KA2-5-1 expresses high levels of Dsz enzymes in the presence of sulfate.

    PubMed

    Tanaka, Yasuhiro; Yoshikawa, Osamu; Maruhashi, Kenji; Kurane, Ryuichiro

    2002-11-01

    Two mutants of the dibenzothiophene-desulfurizing Rhodococcus erythropolis KA2-5-1, strains MS51 and MS316, which express a high level of desulfurizing activity in the presence of sulfate, were isolated using the transposome technique. The level of dibenzothiophene-desulfurization by cell-free extracts prepared from mutants MS51 and MS316 grown on sulfate was about five-fold higher than that by cell-free extracts of the wild-type. This result was consistent with results of Western-blot analysis using antisera specific for DszA, DszB and DszC, the enzymes involved in the desulfurization of dibenzothiophene. Gene analysis of the mutants revealed that the same gene was disrupted in mutants MS51 and MS316 and that the transposon-inserted gene in these strains was the gene for cystathionine beta-synthase, cbs. The cbs mutants also expressed high levels of Dsz enzymes when methionine was used as the sole source of sulfur.

  18. Molecular identification of an ABC transporter complex for manganese: analysis of a cyanobacterial mutant strain impaired in the photosynthetic oxygen evolution process.

    PubMed Central

    Bartsevich, V V; Pakrasi, H B

    1995-01-01

    During photosynthesis, the photosystem II (PSII) pigment-protein complex catalyzes oxygen evolution, a reaction in which a four-manganese ensemble plays a crucial role. Using a newly developed selection scheme, we have isolated BP13, a random photosynthesis-deficient mutant strain of the cyanobacterium, Synechocystis 6803. This mutant grew slowly under photoautotrophic conditions, and had a low oxygen evolution activity. Biochemical analysis revealed that the lesion in this mutant strain had specifically affected the Mn ensemble in PSII. Interestingly, incubation of BP13 cells with micromolar levels of added Mn induced rapid recovery of oxygen evolution activity. The mutant could be complemented with a fragment of wild-type chromosomal DNA containing three closely linked genes, mntA, mntB and mntC. These gene products showed significant sequence similarities with polypeptide components of bacterial permeases that are members of the 'ABC (ATP binding cassette) superfamily' of transporter proteins. We determined that in the BP13 strain, a single nucleotide change had resulted in the replacement of an alanine by an aspartic acid residue in MntA, a soluble protein containing ATP binding motifs. These results suggest that the mntCAB gene cluster encodes polypeptide components of a Mn transporter, the first such protein complex identified in any organism. PMID:7743991

  19. Opioid-dependent regulation of high and low fear responses in two inbred mouse strains.

    PubMed

    Szklarczyk, Klaudia; Korostynski, Michal; Cieslak, Przemyslaw Eligiusz; Wawrzczak-Bargiela, Agnieszka; Przewlocki, Ryszard

    2015-10-01

    The molecular mechanisms underlying the susceptibility or resilience to trauma-related disorders remain incompletely understood. Opioids modulate emotional learning, but the roles of specific receptors are unclear. Here, we aimed to analyze the contribution of the opioid system to fear responses in two inbred mouse strains exhibiting distinct behavioral phenotypes. SWR/J and C57BL/6J mice were subjected to five consecutive electric footshocks (1mA each), and the contextual freezing time was measured. Stress-induced alterations in gene expression were analyzed in the amygdala and the hippocampus. In both strains, the fear response was modulated using pharmacological tools. SWR/J mice did not develop conditioned fear but exhibited increased transcriptional expression of Pdyn and Penk in the amygdala region. Blocking opioid receptors prior to the footshocks using naltrexone (2 mg/kg) or naltrindole (5 mg/kg) increased the freezing responses in these animals. The C57BL/6J strain displayed high conditioned fear, although no alteration in the mRNA abundance of genes encoding opioid precursors was observed. Double-injection of morphine (20 mg/kg) following stress and upon context re-exposure prevented the enhancement of freezing. Moreover, selective delta and kappa agonists caused a reduction in conditioned fear responses. To summarize, the increased expression of the Pdyn and Penk genes corresponded to reduced intensity of fear responses. Blockade of the endogenous opioid system restored freezing behavior in stress-resistant animals. The pharmacological stimulation of the kappa and delta opioid receptors in stress-susceptible individuals may alleviate fear. Thus, subtype-selective opioid receptor agonists may protect against the development of trauma-related disorders.

  20. Rescue of cytochrome P450 oxidoreductase (Por) mouse mutants reveals functions in vasculogenesis, brain and limb patterning linked to retinoic acid homeostasis.

    PubMed

    Ribes, Vanessa; Otto, Diana M E; Dickmann, Leslie; Schmidt, Katy; Schuhbaur, Brigitte; Henderson, Colin; Blomhoff, Rune; Wolf, C Roland; Tickle, Cheryll; Dollé, Pascal

    2007-03-01

    Cytochrome P450 oxidoreductase (POR) acts as an electron donor for all cytochrome P450 enzymes. Knockout mouse Por(-/-) mutants, which are early embryonic (E9.5) lethal, have been found to have overall elevated retinoic acid (RA) levels, leading to the idea that POR early developmental function is mainly linked to the activity of the CYP26 RA-metabolizing enzymes (Otto et al., Mol. Cell. Biol. 23, 6103-6116). By crossing Por mutants with a RA-reporter lacZ transgene, we show that Por(-/-) embryos exhibit both elevated and ectopic RA signaling activity e.g. in cephalic and caudal tissues. Two strategies were used to functionally demonstrate that decreasing retinoid levels can reverse Por(-/-) phenotypic defects, (i) by culturing Por(-/-) embryos in defined serum-free medium, and (ii) by generating compound mutants defective in RA synthesis due to haploinsufficiency of the retinaldehyde dehydrogenase 2 (Raldh2) gene. Both approaches clearly improved the Por(-/-) early phenotype, the latter allowing mutants to be recovered up until E13.5. Abnormal brain patterning, with posteriorization of hindbrain cell fates and defective mid- and forebrain development and vascular defects were rescued in E9.5 Por(-/-) embryos. E13.5 Por(-/-); Raldh2(+/-) embryos exhibited abdominal/caudal and limb defects that strikingly phenocopy those of Cyp26a1(-/-) and Cyp26b1(-/-) mutants, respectively. Por(-/-); Raldh2(+/-) limb buds were truncated and proximalized and the anterior-posterior patterning system was not established. Thus, POR function is indispensable for the proper regulation of RA levels and tissue distribution not only during early embryonic development but also in later morphogenesis and molecular patterning of the brain, abdominal/caudal region and limbs. PMID:17126317

  1. Cyclic mechanical strain maintains Nanog expression through PI3K/Akt signaling in mouse embryonic stem cells

    SciTech Connect

    Horiuchi, Rie; Akimoto, Takayuki; Hong, Zhang; Ushida, Takashi

    2012-08-15

    Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17 Hz. The expression of Nanog was reduced during ES cell differentiation in response to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt. These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling. -- Highlights: Black-Right-Pointing-Pointer The expression of Nanog, which is an essential regulator of 'stemness' was reduced during embryonic stem (ES) cell differentiation. Black-Right-Pointing-Pointer Cyclic mechanical strain attenuated the reduction of Nanog expression. Black-Right-Pointing-Pointer Cyclic mechanical strain promoted PI3K-Akt signaling and mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor and an inhibitor of actin polymerization.

  2. Immunization with a Double-Mutant (R192G/L211A) of the Heat-Labile Enterotoxin of Escherichia coli Offers Partial Protection against Campylobacter jejuni in an Adult Mouse Intestinal Colonization Model.

    PubMed

    Albert, M John; Haridas, Shilpa; Ebenezer, Mathew; Raghupathy, Raj; Khan, Islam

    2015-01-01

    We have previously shown that antibodies to cholera toxin (CT) reacted with the major outer membrane proteins (MOMPs) from Campylobacter jejuni strains on Western blot. Further, oral immunization with CT significantly protected against challenge with C. jejuni in an adult mouse colonization model of infection. CT and the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli are structurally and functionally related. LT and its mutants including the double-mutant LT (R192G/L211A) (dmLT), are powerful mucosal adjuvants. Unlike LT which is reactogenic, dmLT has been shown to be safe for human use. In the current study, we determined whether rabbit anti-dmLT antibodies reacted with MOMPs from C. jejuni strains and whether immunization with dmLT would afford protection against C. jejuni. On Western blot, the MOMPs from C. jejuni 48 (Penner serotype O:19), C. jejuni 75 (O:3) and C. jejuni 111 (O:1,44) were probed with rabbit antibodies to dmLT or LT-E112K (a non-toxic LT mutant), which showed a lack of reaction. Adult BALB/c mice were orally immunized with dmLT and orally challenged with C. jejuni 48 or 111. Protection from colonization with the challenge bacteria was studied by enumerating Campylobacter colonies in feces daily for 9 days. Vaccination produced robust serum and stool antibody responses to dmLT and no antibody responses to C. jejuni MOMP. Vaccinated mice showed reduced colonization and excretion of both challenge strains compared to control mice. However, the differences were not statistically significant. The protective efficacy of the dmLT vaccine varied from 9.1% to 54.5%. The lack of cross-reaction between the MOMP and dmLT suggests that protection is not mediated by cross-reacting antibodies, but may be due to activation of innate immunity. As dmLT is safe for humans, it could be incorporated into a C. jejuni vaccine to enhance its efficacy. PMID:26540197

  3. Analysis of the presence of cell proliferation-related molecules in the Tgf-β3 null mutant mouse palate reveals misexpression of EGF and Msx-1.

    PubMed

    del Río, A; Barrio, M C; Murillo, J; Maldonado, E; López-Gordillo, Y; Martínez-Sanz, E; Martínez, M L; Martínez-Álvarez, C

    2011-01-01

    The Tgf-β(3) null mutant mouse palate presents several cellular anomalies that lead to the appearance of cleft palate. One of them concerns the cell proliferation of both the palatal medial edge epithelium and mesenchyme. In this work, our aim was to determine whether there was any variation in the presence/distribution of several cell proliferation-related molecules that could be responsible for the cell proliferation defects observed in these palates. Our results showed no difference in the presence of EGF-R, PDGF-A, TGF-β(2), Bmp-2, and Bmp-4, and differences were minimal for FGF-10 and Shh. However, the expression of EGF and Msx-1 changed substantially. The shift of the EGF protein expression was the one that most correlated with that of cell proliferation. This molecule is regulated by TGF-β(3), and experiments blocking its activity in culture suggest that EGF misexpression in the Tgf-β(3) null mutant mouse palate plays a role in the cell proliferation defect observed.

  4. Exome sequencing and arrayCGH detection of gene sequence and copy number variation between ILS and ISS mouse strains.

    PubMed

    Dumas, Laura; Dickens, C Michael; Anderson, Nathan; Davis, Jonathan; Bennett, Beth; Radcliffe, Richard A; Sikela, James M

    2014-06-01

    It has been well documented that genetic factors can influence predisposition to develop alcoholism. While the underlying genomic changes may be of several types, two of the most common and disease associated are copy number variations (CNVs) and sequence alterations of protein coding regions. The goal of this study was to identify CNVs and single-nucleotide polymorphisms that occur in gene coding regions that may play a role in influencing the risk of an individual developing alcoholism. Toward this end, two mouse strains were used that have been selectively bred based on their differential sensitivity to alcohol: the Inbred long sleep (ILS) and Inbred short sleep (ISS) mouse strains. Differences in initial response to alcohol have been linked to risk for alcoholism, and the ILS/ISS strains are used to investigate the genetics of initial sensitivity to alcohol. Array comparative genomic hybridization (arrayCGH) and exome sequencing were conducted to identify CNVs and gene coding sequence differences, respectively, between ILS and ISS mice. Mouse arrayCGH was performed using catalog Agilent 1 × 244 k mouse arrays. Subsequently, exome sequencing was carried out using an Illumina HiSeq 2000 instrument. ArrayCGH detected 74 CNVs that were strain-specific (38 ILS/36 ISS), including several ISS-specific deletions that contained genes implicated in brain function and neurotransmitter release. Among several interesting coding variations detected by exome sequencing was the gain of a premature stop codon in the alpha-amylase 2B (AMY2B) gene specifically in the ILS strain. In total, exome sequencing detected 2,597 and 1,768 strain-specific exonic gene variants in the ILS and ISS mice, respectively. This study represents the most comprehensive and detailed genomic comparison of ILS and ISS mouse strains to date. The two complementary genome-wide approaches identified strain-specific CNVs and gene coding sequence variations that should provide strong candidates to

  5. Regional changes of AQP0-dependent square array junction and gap junction associated with cortical cataract formation in the Emory mutant mouse.

    PubMed

    Biswas, Sondip K; Brako, Lawrence; Gu, Sumin; Jiang, Jean X; Lo, Woo-Kuen

    2014-10-01

    The Emory mutant mouse has been widely used as an animal model for human senile cataract since it develops late-onset hereditary cataract. Here, we focus on the regional changes of aquaporin-0 (AQP0) and connexins that are associated with the cortical cataract formation in the Emory mutant mice. Emory mutant and CFW wild-type mice at age 1-16 months were used in this study. By using an established photography system with dissecting microscopy, the opacities were first detected at the anterior or posterior lens center surface in Emory mice at age 7 months, and gradually extended toward the equator during the 16 months examined. Scanning EM verified that disorganized and fragmented fiber cells were associated with the areas of opacities within approximately 200 μm from the lens surface, indicating that Emory mouse cataracts belong to the cortical cataracts. Freeze-fracture TEM further confirmed that cortical cataracts exhibited extensive wavy square array junctions, small gap junctions and globules. Immunofluorescence analysis showed that in contrast to the high labeling intensity of AQP0-loop antibody, the labeling of AQP0 C-terminus antibody was decreased considerably in superficial fibers in Emory cataracts. Similarly, a significant decrease in the labeling of the antibody against Cx50 C-terminus, but not Cx46 C-terminus, occurred in superficial and outer cortical fibers in Emory cataracts. Western blotting further revealed that the C-termini of both AQP0 and Cx50 in Emory cataracts were decreased to over 50% to that of the wild-type. Thus, this systematic study concludes that the Emory mouse cataract belongs to the cortical cataract which is due to regional breakdown of superficial fibers associated with formation of AQP0-dependent wavy square array junctions, small gap junctions and globules. The marked decreases of the C-termini of both AQP0 and Cx50 in the superficial fibers may disturb the needed interaction between these two proteins during fiber cell

  6. Isolation and characterization of the ndhF gene of Synechococcus sp. strain PCC 7002 and initial characterization of an interposon mutant.

    PubMed Central

    Schluchter, W M; Zhao, J; Bryant, D A

    1993-01-01

    The ndhF gene of the unicellular marine cyanobacterium Synechococcus sp. strain PCC 7002 was cloned and characterized. NdhF is a subunit of the type 1, multisubunit NADH:plastoquinone oxidoreductase (NADH dehydrogenase). The nucleotide sequence of the gene predicts an extremely hydrophobic protein of 664 amino acids with a calculated mass of 72.9 kDa. The ndhF gene was shown to be single copy and transcribed into a monocistronic mRNA of 2,300 nucleotides. An ndhF null mutation was successfully constructed by interposon mutagenesis, demonstrating that NdhF is not required for cell viability under photoautotrophic growth conditions. The mutant strain exhibited a negligible rate of oxygen uptake in the dark, but its photosynthetic properties (oxygen evolution, chlorophyll/P700 ratio, and chlorophyll/P680 ratio) were generally similar to those of the wild type. Although the ndhF mutant strain grew as rapidly as the wild-type strain at high light intensity, the mutant grew more slowly than the wild type at lower light intensities and did not grow at all under photoheterotrophic conditions. The roles of the NADH:plastoquinone oxidoreductase in photosynthetic and respiratory electron transport are discussed. Images PMID:8501038

  7. Rapid, Sensitive, and Accurate Evaluation of Drug Resistant Mutant (NS5A-Y93H) Strain Frequency in Genotype 1b HCV by Invader Assay.

    PubMed

    Yoshimi, Satoshi; Ochi, Hidenori; Murakami, Eisuke; Uchida, Takuro; Kan, Hiromi; Akamatsu, Sakura; Hayes, C Nelson; Abe, Hiromi; Miki, Daiki; Hiraga, Nobuhiko; Imamura, Michio; Aikata, Hiroshi; Chayama, Kazuaki

    2015-01-01

    Daclatasvir and asunaprevir dual oral therapy is expected to achieve high sustained virological response (SVR) rates in patients with HCV genotype 1b infection. However, presence of the NS5A-Y93H substitution at baseline has been shown to be an independent predictor of treatment failure for this regimen. By using the Invader assay, we developed a system to rapidly and accurately detect the presence of mutant strains and evaluate the proportion of patients harboring a pre-treatment Y93H mutation. This assay system, consisting of nested PCR followed by Invader reaction with well-designed primers and probes, attained a high overall assay success rate of 98.9% among a total of 702 Japanese HCV genotype 1b patients. Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed. Our assay system showed a better lower detection limit of Y93H proportion than using direct sequencing, and Y93H frequencies obtained by this method correlated well with those of deep-sequencing analysis (r = 0.85, P <0.001). The proportion of the patients with the mutant strain estimated by this assay was 23.6% (164/694). Interestingly, patients with the Y93H mutant strain showed significantly lower ALT levels (p=8.8 x 10-4), higher serum HCV RNA levels (p=4.3 x 10-7), and lower HCC risk (p=6.9 x 10-3) than those with the wild type strain. Because the method is both sensitive and rapid, the NS5A-Y93H mutant strain detection system established in this study may provide important pre-treatment information valuable not only for treatment decisions but also for prediction of disease progression in HCV genotype 1b patients. PMID:26083687

  8. Mutant β-III spectrin causes mGluR1α mislocalization and functional deficits in a mouse model of spinocerebellar ataxia type 5.

    PubMed

    Armbrust, Karen R; Wang, Xinming; Hathorn, Tyisha J; Cramer, Samuel W; Chen, Gang; Zu, Tao; Kangas, Takashi; Zink, Anastasia N; Öz, Gülin; Ebner, Timothy J; Ranum, Laura P W

    2014-07-23

    Spinocerebellar ataxia type 5 (SCA5), a dominant neurodegenerative disease characterized by profound Purkinje cell loss, is caused by mutations in SPTBN2, a gene that encodes β-III spectrin. SCA5 is the first neurodegenerative disorder reported to be caused by mutations in a cytoskeletal spectrin gene. We have developed a mouse model to understand the mechanistic basis for this disease and show that expression of mutant but not wild-type β-III spectrin causes progressive motor deficits and cerebellar degeneration. We show that endogenous β-III spectrin interacts with the metabotropic glutamate receptor 1α (mGluR1α) and that mice expressing mutant β-III spectrin have cerebellar dysfunction with altered mGluR1α localization at Purkinje cell dendritic spines, decreased mGluR1-mediated responses, and deficient mGluR1-mediated long-term potentiation. These results indicate that mutant β-III spectrin causes mislocalization and dysfunction of mGluR1α at dendritic spines and connects SCA5 with other disorders involving glutamatergic dysfunction and synaptic plasticity abnormalities.

  9. A Sall4 Mutant Mouse Model Useful for Studying the Role of Sall4 in Early Embryonic Development and Organogenesis

    PubMed Central

    Warren, Madhuri; Wang, Wei; Spiden, Sarah; Chen-Murchie, Dongrong; Tannahill, David; Steel, Karen P.; Bradley, Allan

    2008-01-01

    Summary SALL4 is a homologue of the Drosophila homeotic gene spalt, a zinc finger transcription factor, required for inner cell mass proliferation in early embryonic development. It also interacts with other transcription factors to control the development of the anorectal region, kidney, heart, limbs, and brain. Truncating mutations in SALL4 cause Okihiro syndrome, manifest as Duane anomaly, radial ray defects and sensorineural and conductive deafness. We report the characterization of a novel murine Sall4 null allele created by bacterial recombineering in ES cells. Homozygous mutant mice exhibit early embryonic lethality. Heterozygous mutant mice recapitulate phenotypic features of Okihiro syndrome including deafness, lower anogenital tract abnormalities, renal hypoplasia, anencephaly, Hirschprung’s disease, and skeletal defects. This phenotype shows important differences in cardiac and ear manifestations to previously characterized Sall4 mutant alleles and should prove useful for the investigation of the influence of modifier alleles and protein interactions on the transcriptional regulatory function of Sall4. PMID:17216607

  10. Compound mouse mutants of bZIP transcription factors Mafg and Mafk reveal a regulatory network of non-crystallin genes associated with cataract.

    PubMed

    Agrawal, Smriti A; Anand, Deepti; Siddam, Archana D; Kakrana, Atul; Dash, Soma; Scheiblin, David A; Dang, Christine A; Terrell, Anne M; Waters, Stephanie M; Singh, Abhyudai; Motohashi, Hozumi; Yamamoto, Masayuki; Lachke, Salil A

    2015-07-01

    Although majority of the genes linked to early-onset cataract exhibit lens fiber cell-enriched expression, our understanding of gene regulation in these cells is limited to function of just eight transcription factors and largely in the context of crystallins. We report on small Maf transcription factors Mafg and Mafk as regulators of several non-crystallin human cataract-associated genes in fiber cells and establish their significance to this disease. We applied a bioinformatics tool for cataract gene discovery iSyTE to identify Mafg and its co-regulators in the lens, and generated various null-allelic combinations of Mafg:Mafk mouse mutants for phenotypic and molecular analysis. By age 4 months, Mafg-/-:Mafk+/- mutants exhibit lens defects that progressively develop into cataract. High-resolution phenotypic characterization of Mafg-/-:Mafk+/- mouse lens reveals severely disorganized fiber cells, while microarray-based expression profiling identifies 97 differentially regulated genes (DRGs). Integrative analysis of Mafg-/-:Mafk+/- lens-DRGs with (1) binding motifs and genomic targets of small Mafs and their regulatory partners, (2) iSyTE lens expression data, and (3) interactions between DRGs in the String database, unravel a detailed small Maf regulatory network in the lens, several nodes of which are linked to cataract. This approach identifies 36 high-priority candidates from the original 97 DRGs. Significantly, 8/36 (22%) DRGs are associated with cataracts in human (GSTO1, MGST1, SC4MOL, UCHL1) or mouse (Aldh3a1, Crygf, Hspb1, Pcbd1), suggesting a multifactorial etiology that includes oxidative stress and misregulation of sterol synthesis. These data identify Mafg and Mafk as new cataract-associated candidates and define their function in regulating largely non-crystallin genes linked to human cataract. PMID:25896808

  11. Gene expression in the mouse eye: an online resource for genetics using 103 strains of mice

    PubMed Central

    Lu, Lu; Freeman-Anderson, Natalie E.; Templeton, Justin P.; Nassr, Mohamed; Wang, Xusheng; Gu, Weikuan; Jiao, Yan; Williams, Robert W.

    2009-01-01

    Purpose Individual differences in patterns of gene expression account for much of the diversity of ocular phenotypes and variation in disease risk. We examined the causes of expression differences, and in their linkage to sequence variants, functional differences, and ocular pathophysiology. Methods mRNAs from young adult eyes were hybridized to oligomer microarrays (Affymetrix M430v2). Data were embedded in GeneNetwork with millions of single nucleotide polymorphisms, custom array annotation, and information on complementary cellular, functional, and behavioral traits. The data include male and female samples from 28 common strains, 68 BXD recombinant inbred lines, as well as several mutants and knockouts. Results We provide a fully integrated resource to map, graph, analyze, and test causes and correlations of differences in gene expression in the eye. Covariance in mRNA expression can be used to infer gene function, extract signatures for different cells or tissues, to define molecular networks, and to map quantitative trait loci that produce expression differences. These data can also be used to connect disease phenotypes with sequence variants. We demonstrate that variation in rhodopsin expression efficiently predicts candidate genes for eight uncloned retinal diseases, including WDR17 for the human RP29 locus. Conclusions The high level of strain variation in gene expression is a powerful tool that can be used to explore and test molecular networks underlying variation in structure, function, and disease susceptibility. The integration of these data into GeneNetwork provides users with a workbench to test linkages between sequence differences and eye structure and function. PMID:19727342

  12. Microarray analysis of gene expression in mouse (strain 129) embryonic stem cells after typical synthetic musk exposure.

    PubMed

    Shi, Jiachen; Li, Ming; Jiao, Zhihao; Zhang, Jing; Feng, Yixing; Shao, Bing

    2013-01-01

    Synthetic musks are widely used in personal-care products and can readily accumulate in the adipose tissue, breast milk, and blood of humans. In this study, the Affymetrix Mouse Genome GeneChip was used to identify alterations in gene expression of embryonic stem cells from the 129 strain of the laboratory mouse after treatment with the synthetic musk tonalide (AHTN). Among the 45,037 transcripts in the microarray, 2,879 genes were differentially expressed. According to the microarray analysis, the potential influence of AHTN on the development to embryo should be of concern, and the toxicological effects of it and related musk compounds should be studied further.

  13. Mouse adaptation of a sub-genogroup B5 strain of human enterovirus 71 is associated with a novel lysine to glutamic acid substitution at position 244 in protein VP1.

    PubMed

    Zaini, Zainun; Phuektes, Patchara; McMinn, Peter

    2012-07-01

    Most human enterovirus 71 (HEV71) strains infect only primates and are unable to cause clinically apparent infection in mice. Here we describe a mouse-adapted HEV71 strain that belongs to sub-genogroup B5 with increased virulence in newborn BALB/c mice. The mouse-virulent strain was initially selected by serial passage of a HEV71 clinical isolate (HEV71-B5) in Chinese hamster ovary (CHO) cells (CHO-B5), followed by serial passage in newborn mice. Virus from the fifth mouse passage was cultured twice on Vero cells and designated as MP-B5. MP-B5 induces severe disease of high mortality in newborn mice in a dose-dependent manner. Skeletal muscle is the primary site of virus replication and results in severe myositis. CHO-B5 harbours a single amino acid substitution (K(149) → I) in the VP2 capsid protein. Five additional nucleotide sequence changes were identified in MP-B5, two of which are located in the 5' UTR and the three within the open reading frame (ORF). Two of the ORF mutations resulted in deduced amino acid changes in the capsid protein VP1: S(241) → L and K(244) → E; the third ORF mutation was a synonymous C → T change at nucleotide position 6072 within the 3D polymerase gene. Infectious cDNA clone-derived mutant virus populations of HEV71 belonging to sub-genogroup B3 (CHO-26 M) that contain the VP1 mutations identified in MP-B5 were generated in order to determine the mutation(s) responsible for mouse virulence. Only viruses expressing the VP1 (K(244) → E) mutation were virulent in 5-day-old BALB/c mice, indicating that the VP1 (K(244) → E) change is the critical genetic determinant of mouse adaptation and virulence in this model.

  14. Oligonucleotide-directed mutagenesis of psbB, the gene encoding CP47, employing a deletion mutant strain of the cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Eaton-Rye, J J; Vermaas, W F

    1991-12-01

    A mutant strain of the cyanobacterium Synechocystis sp. PCC (Pasteur Culture Collection) 6803 has been developed in which psbB, the gene coding for the chlorophyl alpha-binding protein CP47 in Photosystem II (PSII), has been deleted. This deletion mutant can be used for the reintroduction of modified psbB into the cyanobacterium. To study the role of a large hydrophilic region in CP47, presumably located on the lumenal side of the thylakoid membrane between the fifth and sixth membrane-spanning regions, specific deletions have been introduced in psbB coding for regions within this domain. One psbB mutation leads to deletion of Gly-351 to Thr-365 in CP47, another psbB mutation was targeted towards deletion of Arg-384 to Val-392 in this protein. The deletion from Gly-351 to Thr-365 results in a loss of PSII activity and of photoautotrophic growth of the mutant, but the deletion between Arg-384 and Val-392 retains PSII activity and the ability to grow photoautotrophically. The mutant strain with the deletion from Gly-351 to Thr-365 does not assemble a stable PSII reaction center complex in its thylakoid membranes, and exhibits diminished levels of CP47 and of the reaction center proteins D1 and D2. In contrast to the Arg-384 to Val-392 portion of this domain, the region between Gly-351 and Thr-365 appears essential for the normal structure and function of photosystem II. PMID:1932693

  15. Genetic ablation of the mammillary bodies in the Foxb1 mutant mouse leads to selective deficit of spatial working memory.

    PubMed

    Radyushkin, Konstantin; Anokhin, Konstantin; Meyer, Barbara I; Jiang, Qiuhong; Alvarez-Bolado, Gonzalo; Gruss, Peter

    2005-01-01

    Mammillary bodies and the mammillothalamic tract are parts of a classic neural circuitry that has been implicated in severe memory disturbances accompanying Korsakoff's syndrome. However, the specific role of mammillary bodies in memory functions remains controversial, often being considered as just an extension of the hippocampal memory system. To study this issue we used mutant mice with a targeted mutation in the transcription factor gene Foxb1. These mice suffer perinatal degeneration of the medial and most of the lateral mammillary nuclei, as well as of the mammillothalamic bundle. Foxb1 mutant mice showed no deficits in such hippocampal-dependent tasks as contextual fear conditioning and social transmission of food preference. They were also not impaired in the spatial reference memory test in the radial arm maze. However, Foxb1 mutants showed deficits in the task for spatial navigation within the Barnes maze. Furthermore, they showed impairments in spatial working memory tasks such as the spontaneous alternation and the working memory test in the radial arm maze. Thus, our behavioural analysis of Foxb1 mutants suggests that the medial mammillary nuclei and mammillothalamic tract play a role in a specific subset of spatial tasks, which require combined use of both spatial and working memory functions. Therefore, the mammillary bodies and the mammillothalamic tract may form an important route through which the working memory circuitry receives spatial information from the hippocampus.

  16. Differential effects of acute morphine administrations on polymorphonuclear cell metabolism in various mouse strains.

    PubMed

    Di Francesco, P; Tavazzi, B; Gaziano, R; Lazzarino, G; Casalinuovo, I A; Di Pierro, D; Garaci, E

    1998-01-01

    This paper shows that an acute morphine treatment dose-dependently alters the energetic and oxidative metabolism of polymorphonuclear leukocytes obtained from BALB/c and DBA/2 mice, while phagocytic cells from C57BL/6 were not affected. In sensitive mouse strains, i.e. BALB/c and DBA/2, morphine decreased both ATP concentration and energy charge potential. At the same time, ATP catabolic products, i.e. nucleosides (inosine+adenosine) and oxypurines (hypoxanthine+xanthine+uric acid), significantly increased, indicating an imbalance between energy production and consumption. Morphine treatment also induced malondialdehyde and superoxide anions production in leukocyte cells from sensitive mice. The opiate antagonist naloxone blocked morphine-induced modifications by the lower morphine dose. The same parameters in cells from C57BL/6 mice were not affected. These findings confirm that: i) the phagocytic cells are an important target for the in vivo effects of morphine, and ii) the genotype-dependent variation influences the immunological responsiveness to opiates.

  17. MUTANT FREQUENCY AND MUTATIONAL SPECTRA IN THETK AND HPRT GENES OF N-ETHYL-N-NITROSOUREA TREATED MOUSE LYMPHOMA CELLS

    EPA Science Inventory

    Abstract

    The mouse lymphoma assay (MLA) utilizing the Tk locus is widely used to identify chemical mutagens. The autosomal location of the Tk locus allows for the detection of a wide range of mutational events, from point mutations to chromosome alterations. However, the ...

  18. Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model

    PubMed Central

    Chen, Song; Wang, Chenran; Yeo, Syn; Liang, Chun-Chi; Okamoto, Takako; Sun, Shaogang; Wen, Jian; Guan, Jun-Lin

    2016-01-01

    Autophagy is an evolutionarily conserved cellular process controlled through a set of essential autophagy genes (Atgs). However, there is increasing evidence that most, if not all, Atgs also possess functions independent of their requirement in canonical autophagy, making it difficult to distinguish the contributions of autophagy-dependent or -independent functions of a particular Atg to various biological processes. To distinguish these functions for FIP200 (FAK family-interacting protein of 200 kDa), an Atg in autophagy induction, we examined FIP200 interaction with its autophagy partner, Atg13. We found that residues 582–585 (LQFL) in FIP200 are required for interaction with Atg13, and mutation of these residues to AAAA (designated the FIP200-4A mutant) abolished its canonical autophagy function in vitro. Furthermore, we created a FIP200-4A mutant knock-in mouse model and found that specifically blocking FIP200 interaction with Atg13 abolishes autophagy in vivo, providing direct support for the essential role of the ULK1/Atg13/FIP200/Atg101 complex in the process beyond previous studies relying on the complete knockout of individual components. Analysis of the new mouse model showed that nonautophagic functions of FIP200 are sufficient to fully support embryogenesis by maintaining a protective role in TNFα-induced apoptosis. However, FIP200-mediated canonical autophagy is required to support neonatal survival and tumor cell growth. These studies provide the first genetic evidence linking an Atg's autophagy and nonautophagic functions to different biological processes in vivo. PMID:27013233

  19. Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model.

    PubMed

    Chen, Song; Wang, Chenran; Yeo, Syn; Liang, Chun-Chi; Okamoto, Takako; Sun, Shaogang; Wen, Jian; Guan, Jun-Lin

    2016-04-01

    Autophagy is an evolutionarily conserved cellular process controlled through a set of essential autophagy genes (Atgs). However, there is increasing evidence that most, if not all, Atgs also possess functions independent of their requirement in canonical autophagy, making it difficult to distinguish the contributions of autophagy-dependent or -independent functions of a particular Atg to various biological processes. To distinguish these functions for FIP200 (FAK family-interacting protein of 200 kDa), an Atg in autophagy induction, we examined FIP200 interaction with its autophagy partner, Atg13. We found that residues 582-585 (LQFL) in FIP200 are required for interaction with Atg13, and mutation of these residues to AAAA (designated the FIP200-4A mutant) abolished its canonical autophagy function in vitro. Furthermore, we created a FIP200-4A mutant knock-in mouse model and found that specifically blocking FIP200 interaction with Atg13 abolishes autophagy in vivo, providing direct support for the essential role of the ULK1/Atg13/FIP200/Atg101 complex in the process beyond previous studies relying on the complete knockout of individual components. Analysis of the new mouse model showed that nonautophagic functions of FIP200 are sufficient to fully support embryogenesis by maintaining a protective role in TNFα-induced apoptosis. However, FIP200-mediated canonical autophagy is required to support neonatal survival and tumor cell growth. These studies provide the first genetic evidence linking an Atg's autophagy and nonautophagic functions to different biological processes in vivo.

  20. Adhesion in vitro and in vivo associated with an adhesive antigen (F41) produced by a K99 mutant of the reference strain Escherichia coli B41.

    PubMed Central

    Morris, J A; Thorns, C; Scott, A C; Sojka, W J; Wells, G A

    1982-01-01

    A K99-negative mutant of the K99 reference strain Escherichia coli B41 (O101:K99) was isolated (strain B41M). It did not react with OK antiserum to a K12 K99+ recombinant or with OK antiserum to K99-positive organisms from the O8, O20, or O64 serogroups, but it did react with OK antiserum to K99-positive organisms from the O101 and O9 serogroups. The mutant hemagglutinated sheep erythrocytes and attached in vitro to calf enterocytes when cultured at 37 degrees C but not when grown at 18 degrees C. Attachment was mannose resistant but susceptible to heating and formaldehyde. These properties were associated with the presence of fimbriae. The isolated hemagglutinin migrated to the anode in immunoelectrophoresis experiments, competitively inhibited attachment of strain B41M to calf enterocytes, and could be demonstrated adhering to these cells in vitro by indirect immunofluorescent staining. The anionic hemagglutinin is referred to provisionally as F41. Germfree piglets infected with strain B41M developed diarrhea within 16 h. Scanning electron microscopy showed groups of bacteria adherent to the microvilli of villous enterocytes. Indirect immunofluorescent staining demonstrated the presence of F41 antigen in vivo. Images FIG. 1 FIG. 2 FIG. 4 PMID:6124503

  1. Regeneration of the ear after wounding in different mouse strains is dependent on the severity of wound trauma.

    PubMed

    Rajnoch, Charissa; Ferguson, Sharon; Metcalfe, Anthony D; Herrick, Sarah E; Willis, Hayley S; Ferguson, Mark W J

    2003-02-01

    The replacement and restoration of tissue mass after organ damage or injury in adult higher vertebrates is critical to the architecture and function of the organ. If replacement occurs with scar tissue, this often results in adverse effects on function and growth as well as an undesirable cosmetic appearance. However, certain mammals, such as the MRL/MpJ mouse, have shown a restricted capacity for regeneration, rather than scar tissue formation, after an excisional ear punch wound. To investigate the changes in tissue architecture leading to ear wound closure, initial ear wounding studies with a 2-mm clinical biopsy punch were performed on MRL/MpJ mice, by using C57BL/6 mice as a nonregenerative control strain. In contrast to previously reported studies on mouse ear regeneration, we observed that C57BL/6 mice in fact showed a limited regenerative capacity. One explanation for this difference could be attributed to the method of wounding used; both previous studies on mouse ear regeneration used a thumb punch, whereas our approach was to use a clinical biopsy punch. This approach led us to further investigate whether the severity of trauma applied influenced the rate of wound healing. We, therefore, compared the effects of the sharp clinical biopsy punch with that of a cruder thumb punch, and introduced a third strain of mouse, Balb/c, known to be a slow-healing strain. A new method to quantify ear punch hole closure was developed and a histologic investigation conducted up to 4 months after wounding. Image analysis data showed a reduction in original ear wound area of 85% in MRL/MpJ mice at 4 weeks and of 91.7% over 4 months by using a biopsy punch. In contrast, the crude thumb punch methodology resulted in an increase in wound area of up to 58% in Balb/c ears; thought to be due to increased necrosis of the wound site. All biopsy-punched wound areas plateaued in healing between days 28 and 112. Only 5 of 80 MRL/MpJ mouse ears showed no residual holes

  2. Structure, expression, and chromosome location of the gene for the beta subunit of brain-specific Ca2+/calmodulin-dependent protein kinase II identified by transgene integration in an embryonic lethal mouse mutant.

    PubMed Central

    Karls, U; Müller, U; Gilbert, D J; Copeland, N G; Jenkins, N A; Harbers, K

    1992-01-01

    The transgenic mouse strain CAT40 carries in its germ line one copy of a DNA construct consisting of the chloramphenicol acetyltransferase gene and the immunoglobulin heavy-chain enhancer. We show that transgene integration has resulted in a recessive lethal mutation that leads to death of homozygous CAT40 embryos shortly after implantation. The transgene has integrated adjacent to the 3' end of the gene coding for the beta subunit of the brain-specific Ca2+/calmodulin-dependent protein kinase II (Camk-2). The complete cDNA sequence of the Camk-2 gene and most of its exon/intron structure was determined. The deduced amino acid sequence is highly homologous to the previously described rat protein. The chromosomal location of the Camk-2 locus was mapped by interspecific backcross analysis to the proximal region of mouse chromosome 11. This region lacks previously identified recessive embryonic lethal mutations. During embryonic development, Camk-2-specific transcripts are first seen in the head section of 12.5-day-old embryos, and in adult mice the gene is expressed almost exclusively in the brain. Although transcription of the Camk-2 gene in heterozygous CAT40 mice is affected by transgene integration, it is unlikely that this gene is responsible for the mutant phenotype, since it is not expressed in blastocysts and the first transcripts during normal development are detected after the death of homozygous CAT40 embryos. Transgene integration is accompanied by a large deletion of cellular DNA; death is therefore most likely caused by the loss of a gene or genes that are important for early postimplantation development. Images PMID:1321343

  3. Genetic modulation of nephrocalcinosis in mouse models of ectopic mineralization: the Abcc6(tm1Jfk) and Enpp1(asj) mutant mice.

    PubMed

    Li, Qiaoli; Chou, David W; Price, Thea P; Sundberg, John P; Uitto, Jouni

    2014-06-01

    Ectopic mineralization of renal tissues in nephrocalcinosis is a complex, multifactorial process. The purpose of this study was to examine the role of genetic modulation and the role of diet in nephrocalcinosis using two established mouse models of ectopic mineralization, Abcc6(tm1Jfk) and Enpp1(asj) mice, which serve as models for pseudoxanthoma elasticum and generalized arterial calcification of infancy, two heritable disorders, respectively. These mutant mice, when on standard rodent diet, develop nephrocalcinosis only at a very late age. In contrast, when placed on an 'acceleration diet' composed of increased phosphate and reduced magnesium content, they showed extensive mineralization of the kidneys affecting primarily the medullary tubules as well as arcuate and renal arteries, as examined by histopathology and quantitated by chemical assay for calcium. Mineralization could also be detected noninvasively by micro computed tomography. Whereas the heterozygous mice did not develop nephrocalcinosis, compound heterozygous mice carrying both mutant alleles, Abcc6(tm1Jfk/+) and Enpp1(+/asj), developed ectopic mineralization similar to that noted in homozygous mice for either gene, indicating that deletion of one Abcc6 allele along with Enpp1 haploinsufficiency resulted in renal mineralization. Thus, synergistic genetic defects in the complex mineralization/antimineralization network can profoundly modulate the degree of ectopic mineralization in nephrocalcinosis.

  4. A reduction in Ptprq associated with specific features of the deafness phenotype of the miR-96 mutant mouse diminuendo

    PubMed Central

    Chen, Jing; Johnson, Stuart L; Lewis, Morag A; Hilton, Jennifer M; Huma, Andreea; Marcotti, Walter; Steel, Karen P

    2014-01-01

    miR-96 is a microRNA, a non-coding RNA gene which regulates a wide array of downstream genes. The miR-96 mouse mutant diminuendo exhibits deafness and arrested hair cell functional and morphological differentiation. We have previously shown that several genes are markedly downregulated in the diminuendo organ of Corti; one of these is Ptprq, a gene known to be important for maturation and maintenance of hair cells. In order to study the contribution that downregulation of Ptprq makes to the diminuendo phenotype, we carried out microarrays, scanning electron microscopy and single hair cell electrophysiology to compare diminuendo mutants (heterozygous and homozygous) with mice homozygous for a functional null allele of Ptprq. In terms of both morphology and electrophysiology, the auditory phenotype of mice lacking Ptprq resembles that of diminuendo heterozygotes, while diminuendo homozygotes are more severely affected. A comparison of transcriptomes indicates there is a broad similarity between diminuendo homozygotes and Ptprq-null mice. The reduction in Ptprq observed in diminuendo mice appears to be a major contributor to the morphological, transcriptional and electrophysiological phenotype, but does not account for the complete diminuendo phenotype. PMID:24446963

  5. Modelling MS: Chronic-Relapsing EAE in the NOD/Lt Mouse Strain.

    PubMed

    Dang, Phuc T; Bui, Quyen; D'Souza, Claretta S; Orian, Jacqueline M

    2015-01-01

    Modelling complex disorders presents considerable challenges, and multiple sclerosis (MS) is no exception to this rule. The aetiology of MS is unknown, and its pathophysiology is poorly understood. Moreover, the last two decades have witnessed a dramatic revision of the long-held view of MS as an inflammatory demyelinating white matter disease. Instead, it is now regarded as a global central nervous system (CNS) disorder with a neurodegenerative component. Currently, there is no animal model recapitulating MS immunopathogenesis. Available models are based on autoimmune-mediated demyelination, denoted experimental autoimmune encephalomyelitis (EAE) or virally or chemically induced demyelination. Of these, the EAE model has been the most commonly used. It has been extensively improved since its first description and now exists as a number of variants, including genetically modified and humanized versions. Nonetheless, EAE is a distinct disease, and each variant models only certain facets of MS. Whilst the search for more refined MS models must continue, it is important to further explore where mechanisms underlying EAE provide proof-of-principle for those driving MS pathogenesis. EAE variants generated with the myelin component myelin oligodendrocyte glycoprotein (MOG) have emerged as the preferred ones, because in this particular variant disease is associated with both T- and B-cell effector mechanisms, together with demyelination. MOG-induced EAE in the non-obese diabetic (NOD) mouse strain exhibits a chronic-relapsing EAE clinical profile and high disease incidence. We describe the generation of this variant, its contribution to the understanding of MS immune and pathogenetic mechanisms and potential for evaluation of candidate therapies.

  6. Comparative analysis of genetically engineered immunodeficient mouse strains as recipients for human myoblast transplantation.

    PubMed

    Silva-Barbosa, Suse D; Butler-Browne, Gillian S; Di Santo, James P; Mouly, Vincent

    2005-01-01

    The development of an optimized animal model for the in vivo analysis of human muscle cells remains an important goal in the search of therapy for muscular dystrophy. Here we examined the efficiency of human myoblast xenografts in three distinct immunodeficient mouse models. We found that different conditioning regimes used to provoke host muscle regeneration (i.e., cardiotoxin versus cryodamage) had a marked impact on xenograft success. Tibialis anterior muscle of Rag2-, Rag-/gammac-, and Rag-/gammac-/C5- mice was treated by cardiotoxin or cryodamage, submitted to enzymatic digestion, and analyzed by cytofluorometry to quantitate inflammatory cells. Human myoblasts were injected into pretreated muscles from immunodeficient recipients and the cell engraftment evaluated by immunocytochemistry, 4-8 weeks after transplantation. Donor cell differentiation and dispersion within the host muscles was also investigated. Host regeneration in cardiotoxin-treated mice was accompanied by a higher inflammatory cell infiltration when compared to that induced by cryodamage. Accordingly, when compared to the cardiotoxin group, more human myogenic cells were found after cryodamage. When the distinct immunodeficient mice were compared, we found that the alymphoid strain lacking the complement component C5 (Rag-/gammac-/C5- mice) was the most efficient host for human muscle xenografts, when compared with C5(+)Rag-/gammac- mice or Rag- mice. Our results demonstrate that cryolesion-conditioned muscles of Rag-/gammac-/C5- mice provide the best environment for long-term in vivo human myoblast differentiation, opening the way for a novel approach to study the pathophysiology of human muscle disorders. PMID:16285254

  7. Flocculus Purkinje cell signals in mouse Cacna1a calcium channel mutants of escalating severity: an investigation of the role of firing irregularity in ataxia

    PubMed Central

    Thumser, Zachary C.

    2014-01-01

    Mutation of the Cacna1a gene for the P/Q (CaV2.1) calcium channel invariably leads to cerebellar dysfunction. The dysfunction has been attributed to disrupted rhythmicity of cerebellar Purkinje cells, but the hypothesis remains unproven. If irregular firing rates cause cerebellar dysfunction, then the irregularity and behavioral deficits should covary in a series of mutant strains of escalating severity. We compared firing irregularity in floccular and anterior vermis Purkinje cells in the mildly affected rocker and moderately affected tottering Cacna1a mutants and normal C57BL/6 mice. We also measured the amplitude and timing of modulations of floccular Purkinje cell firing rate during the horizontal vestibuloocular reflex (VOR, 0.25–1 Hz) and the horizontal and vertical optokinetic reflex (OKR, 0.125–1 Hz). We recorded Purkinje cells selective for rotational stimulation about the vertical axis (VAPCs) and a horizontal axis (HAPCs). Irregularity scaled with behavioral deficit severity in the flocculus but failed to do so in the vermis, challenging the irregularity hypothesis. Mutant VAPCs exhibited unusually strong modulation during VOR and OKR, the response augmentation scaling with phenotypic severity. HAPCs exhibited increased OKR modulation but in tottering only. The data contradict prior claims that modulation amplitude is unaffected in tottering but support the idea that attenuated compensatory eye movements in Cacna1a mutants arise from defective transfer of Purkinje cell signals to downstream circuitry, rather than attenuated synaptic transmission within the cerebellar cortex. Shifts in the relative sizes of the VAPC and HAPC populations raise the possibility that Cacna1a mutations influence the development of floccular zone architecture. PMID:25143538

  8. Learning, memory and search strategies of inbred mouse strains with different visual abilities in the Barnes maze.

    PubMed

    O'Leary, Timothy P; Savoie, Vicki; Brown, Richard E

    2011-01-20

    Visuo-spatial learning and memory were assessed in male and female mice of 13 inbred strains on a small diameter mouse version of the Barnes maze surrounded by a wall and intra-maze visual cues. Mice completed acquisition and reversal training to assess learning, followed by a probe test to assess memory for the spatial location of the escape hole. The C57BL/6J and CAST/EiJ strains showed better learning performance than the other strains. A/J and 129/SvImJ strains showed poor learning performance, which may be due to their low rates of exploration. No differences in memory were found between strains in the probe test. Males showed better learning performance than females in the DBA/2J and C3H/HeJ strains, but there were no sex differences in the other strains. However, mice may not have used visuo-spatial cues to locate the escape hole in this maze, as (1) all strains primarily used the non-spatial serial/thigmotaxic search strategy, (2) no strains showed a reversal effect when the escape hole location was moved, and (3) learning and memory performance were not correlated with measures of visual ability. Multivariate and univariate analyses of variance indicated that mice with good visual ability performed better than mice with poor visual ability, but the effect sizes were small. The small diameter of the maze and the presence of a wall around the edge of the maze may promote thigmotaxis in mice, increasing the use of a non-visual search strategy, thereby reducing the influence of vision on performance and decreasing the sensitivity of this maze design to detect strain differences in visuo-spatial learning and memory. These results indicate that the design of the Barnes maze has a significant effect on learning and memory processes.

  9. A strategic study using mutant-strain entrapment in calcium alginate for the production of Saccharomyces cerevisiae cells with high invertase activity.

    PubMed

    Rossi-Alva, Juan Carlos; Rocha-Leão, Maria Helena Miguez

    2003-08-01

    Entrapped cells and entrapped cells grown inside of a calcium alginate matrix as well as free cells have been investigated using Saccharomyces cerevisiae mutant strains with regard to their pattern of growth and invertase activity. The repression of invertase by glucose and glucose-consumption ability were considered in the selection process of the mutants. Efficient sucrose bioconversion due to high invertase activity was obtained when entrapped mutant strain Q6R2 cells were grown within calcium alginate gel beads using sucrose plus glucose as the carbon source. Under these conditions, 1 mg (dry weight) of entrapped cells is able to produce 20 micromol of inverted sugar in 3 min (the maximum activity obtained was 20 units x mg(-1)). The experiments were carried out for 6 months without appreciable loss of either bead integrity or invertase activity. The biocatalyst was also stored at 4 degrees C for 6 months without appreciable loss of the invertase activity. This work shows that entrapped yeast cells with a weak ability to consume sugar may be used to produce inverted sugar.

  10. The mouse glucocorticoid receptor: mapping of functional domains by cloning, sequencing and expression of wild-type and mutant receptor proteins.

    PubMed Central

    Danielsen, M; Northrop, J P; Ringold, G M

    1986-01-01

    We have isolated mouse glucocorticoid receptor (GR) cDNAs which, when expressed in transfected mammalian cells, produce a fully functional GR protein. Sequence analysis reveals an open reading frame of 2349 bp which could encode a protein of approximately 86,000 daltons. We have also isolated two receptor cDNAs from mouse S49 nuclear transfer-deficient (nt-) cells which encode mutant forms of the receptor protein. One cDNA encodes a protein that is unable to bind hormone and represents the endogenous hormone binding deficient receptor recently discovered in S49 cells. The lesion in this receptor is due to a single amino acid substitution (Glu-546 to Gly). The second cDNA from nt- cells produces a receptor protein that is able to bind hormone but has reduced nuclear binding. This cDNA, therefore, encodes for the S49 nt- receptor which has been shown to have reduced affinity for DNA. The lesion maps to a single amino acid substitution (Arg-484 to His) located in a highly Cys, Lys, Arg-rich region of the protein previously implicated in DNA binding. Our studies provide unambiguous identification of receptor domains and specific amino acids critical for the hormone and DNA binding properties of this transcriptional regulatory protein. Contained within the first 106 amino acids of the mouse GR is a stretch of nine glutamines with two prolines which are related to the family of transcribed repetitive elements, opa, found in Drosophila melanogaster. A truncated receptor lacking these 106 amino acids is functionally indistinguishable from the wild-type receptor. Images Fig. 2. Fig. 5. Fig. 6. Fig. 7. PMID:3780669

  11. Rad51c- and Trp53-double-mutant mouse model reveals common features of homologous recombination-deficient breast cancers.

    PubMed

    Tumiati, M; Munne, P M; Edgren, H; Eldfors, S; Hemmes, A; Kuznetsov, S G

    2016-09-01

    Almost half of all hereditary breast cancers (BCs) are associated with germ-line mutations in homologous recombination (HR) genes. However, the tumor phenotypes associated with different HR genes vary, making it difficult to define the role of HR in BC predisposition. To distinguish between HR-dependent and -independent features of BCs, we generated a mouse model in which an essential HR gene, Rad51c, is knocked-out specifically in epidermal tissues. Rad51c is one of the key mediators of HR and a well-known BC predisposition gene. Here, we demonstrate that deletion of Rad51c invariably requires inactivation of the Trp53 tumor suppressor (TP53 in humans) to produce mammary carcinomas in 63% of female mice. Nonetheless, loss of Rad51c shortens the latency of Trp53-deficient mouse tumors from 11 to 6 months. Remarkably, the histopathological features of Rad51c-deficient mammary carcinomas, such as expression of hormone receptors and luminal epithelial markers, faithfully recapitulate the histopathology of human RAD51C-mutated BCs. Similar to other BC models, Rad51c/p53 double-mutant mouse mammary tumors also reveal a propensity for genomic instability, but lack the focal amplification of the Met locus or distinct mutational signatures reported for other HR genes. Using the human mammary epithelial cell line MCF10A, we show that deletion of TP53 can rescue RAD51C-deficient cells from radiation-induced cellular senescence, whereas it exacerbates their centrosome amplification and nuclear abnormalities. Altogether, our data indicate that a trend for genomic instability and inactivation of Trp53 are common features of HR-mediated BCs, whereas histopathology and somatic mutation patterns are specific for different HR genes. PMID:26820992

  12. MUTANT FREQUENCIES AND LOSS OF HETEROZYGOSITY INDUCED BY N-ETHYL-N-NITROSOUREA (ENU) IN THE THYMIDINE KINASE (TK) GENE OF L5178YTK+/-3.7.2C MOUSE LYMPHOMA CELLS

    EPA Science Inventory

    MUTANT FREQUENCIES AND LOSS HETEROZYGOSITY INDUCED BY N-ETHYK-N-NITROSOUREA (ENU) IN THE THYMIDINE KINASE (tk) GENE IF l5178Y/TK+/-3.7.2C MOUSE LYMPHOMA CELLS

    N-ethyl-N-nitrosourea (ENU) is a potent monofunctional-ethylating agent that has been found to be mutagenic in a w...

  13. The immune phenotype of AhR null mouse mutants: not a simple mirror of xenobiotic receptor over-activation.

    PubMed

    Esser, Charlotte

    2009-02-15

    Intrinsic and induced cell differentiation and the cellular response to endogenous and exogenous signals are hallmarks of the immune system. Specific and common signalling cascades ensure a highly flexible and adapted response. Increasing evidence suggests that gene modulation by the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, is an important part of these processes. For decades the AhR has been studied mainly for its toxic effects after artificial activation by man-made chemical pollutants such as dioxins. These studies gave important, albeit to some extent skewed, evidence for a mechanistic link between the AhR and the immune system. AhR null mutants and other mutants of the AhR signalling pathway have been generated and used to analyse the physiological function of the AhR, including for the developing and antigen-responding immune system. In this review I look at the natural immunological function(s) of the AhR.

  14. A targeted deletion/insertion in the mouse Pcsk1 locus is associated with homozygous embryo preimplantation lethality, mutant allele preferential transmission and heterozygous female susceptibility to dietary fat.

    PubMed

    Mbikay, Majambu; Croissandeau, Gilles; Sirois, Francine; Anini, Younes; Mayne, Janice; Seidah, Nabil G; Chrétien, Michel

    2007-06-15

    Proprotein convertase 1 (PC1) is a neuroendocrine proteinase involved in the proteolytic activation of precursors to hormones and neuropeptides. To determine the physiological importance of PC1, we produced a mutant mouse from embryonic stem cells in which its locus (Pcsk1) had been inactivated by homologous recombination. The inactivating mutation consisted of a 32.7-kb internal deletion and a 1.8 kb insertion of the bacterial neomycin resistance gene (neo) under the mouse phosphoglycerate kinase 1 protein (PGKneo). Intercross of Pcsk1(+/-) mice produced no Pcsk1(-/-) offspring or blastocysts; in addition, more than 80% of the offspring were Pcsk1(+/-). These observations suggested that the mutation caused preimplantation lethality of homozygous embryos and preferential transmission of the mutant allele. Interestingly, RT-PCR analysis on RNA from endocrine tissues from Pcsk1(+/-) mice revealed the presence of aberrant transcripts specifying the N-terminal half of the PC1 propeptide fused to neo gene product. Mass spectrometric profiles of proopiomelanocortin-derived peptides in the anterior pituitary were similar between Pcsk1(+/-) and Pcsk1(+/+) mice, but significantly different between male and female mice of the same genotype. Relative to their wild-type counterparts, female mutant mice exhibited stunted growth under a low fat diet, and catch-up growth under a high-fat diet. The complex phenotype exhibited by this Pcsk1 mutant mouse model may be due to PC1 deficiency aggravated by expression of aberrant gene products from the mutant allele. PMID:17490633

  15. Comparative Analysis of the Relationship between Trichloroethylene Metabolism and Tissue-Specific Toxicity among Inbred Mouse Strains: Kidney Effects

    PubMed Central

    Yoo, Hong Sik; Bradford, Blair U.; Kosyk, Oksana; Uehara, Takeki; Shymonyak, Svitlana; Collins, Leonard B.; Bodnar, Wanda M.; Ball, Louise M.; Gold, Avram; Rusyn, Ivan

    2014-01-01

    Trichloroethylene (TCE) is a well-known environmental and occupational toxicant that is classified as carcinogenic to humans based on the epidemiological evidence of an association with higher risk of renal cell carcinoma. A number of scientific issues critical for assessing human health risks from TCE remain unresolved, such as the amount of kidney-toxic glutathione conjugation metabolites formed, inter-species and -individual differences, and the mode of action for kidney carcinogenicity. We hypothesized that TCE metabolite levels in the kidney are associated with kidney-specific toxicity. Oral dosing with TCE was conducted in sub-acute (600 mg/kg/d; 5 days; 7 inbred mouse strains) and sub-chronic (100 or 400 mg/kg/d; 1, 2, or 4 weeks; 2 inbred mouse strains) designs. We evaluated the quantitative relationship between strain-, dose-, and time-dependent formation of TCE metabolites from cytochrome P450-mediated oxidation [trichloroacetic acid (TCA), dichloroacetic acid (DCA), and trichloroethanol] and glutathione conjugation [S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)glutathione], and various kidney toxicity phenotypes. In sub-acute study, we observed inter-strain differences in TCE metabolite levels in the kidney. In addition, we found that in several strains kidney-specific effects of TCE included induction of peroxisome proliferator-marker genes Cyp4a10 and Acox1, increased cell proliferation, and expression of KIM-1, a marker of tubular damage and regeneration. In sub-chronic study, peroxisome proliferator-marker gene induction and kidney toxicity diminished while cell proliferative response was elevated in a dose-dependent manner in NZW/LacJ, but not C57BL/6J mice. Overall, we show that TCE metabolite levels in the kidney are associated with kidney-specific toxicity and that these effects are strain-dependent. PMID:25424545

  16. A behavioural test battery to investigate tic-like symptoms, stereotypies, attentional capabilities, and spontaneous locomotion in different mouse strains.

    PubMed

    Proietti Onori, Martina; Ceci, Chiara; Laviola, Giovanni; Macrì, Simone

    2014-07-01

    The preclinical study of human disorders associated with comorbidities and for which the aetiology is still unclear may substantially benefit from multi-strain studies conducted in mice. The latter can help isolating experimental populations (strains) exhibiting distinct facets in the parameters isomorphic to the symptoms of a given disorder. Through a reverse-translation approach, multi-strain studies can inform both natural predisposing factors and environmental modulators. Thus, mouse strains selected for a particular trait may be leveraged to generate hypothesis-driven studies aimed at clarifying the potential role played by the environment in modulating the exhibition of the symptoms of interest. Tourette's syndrome (TS) constitutes a paradigmatic example whereby: it is characterized by a core symptom (tics) often associated with comorbidities (attention-deficit-hyperactivity and obsessive-compulsive symptoms); it has a clear genetic origin though specific genes are, as yet, unidentified; its course (exacerbations and remissions) is under the influence of environmental factors. Based on these considerations, we tested four mouse strains (ABH, C57, CD1, and SJL) - varying along a plethora of behavioural, neurochemical, and immunological parameters - on a test battery tailored to address the following domains: tics (through the i.p. administration of the selective 5-HT2 receptor agonist DOI, 5mg/kg); locomotion (spontaneous locomotion in the home-cage); perseverative responding in an attentional set shifting task; and behavioural stereotypies in response to a single amphetamine (10mg/kg, i.p.) injection. Present data demonstrate that while ABH and SJL mice respectively exhibit selective increments in amphetamine-induced sniffing behaviour and DOI-induced tic-like behaviours, C57 and CD1 mice show a distinct phenotype, compared to other strains, in several parameters. PMID:24675156

  17. Circadian clock control of connexin36 phosphorylation in retinal photoreceptors of the CBA/CaJ mouse strain.

    PubMed

    Zhang, Zhijing; Li, Hongyan; Liu, Xiaoqin; O'Brien, John; Ribelayga, Christophe P

    2015-01-01

    The gap-junction-forming protein connexin36 (Cx36) represents the anatomical substrate of photoreceptor electrical coupling in mammals. The strength of coupling is directly correlated to the phosphorylation of Cx36 at two regulatory sites: Ser110 and Ser293. Our previous work demonstrated that the extent of biotinylated tracer coupling between photoreceptor cells, which provides an index of the extent of electrical coupling, depends on the mouse strain. In the C57Bl/6J strain, light or dopamine reduces tracer coupling and Cx36 phosphorylation in photoreceptors. Conversely, darkness or a dopaminergic antagonist increases tracer coupling and Cx36 phosphorylation, regardless of the daytime. In the CBA/CaJ strain, photoreceptor tracer coupling is not only regulated by light and dopamine, but also by a circadian clock, a type of oscillator with a period close to 24 h and intrinsic to the retina, so that under prolonged dark-adapted conditions tracer coupling is broader at night compared to daytime. In the current study, we examined whether the modulation of photoreceptor coupling by a circadian clock in the CBA/CaJ mouse photoreceptors reflected a change in Cx36 protein expression and/or phosphorylation. We found no significant change in Cx36 expression or in the number of Cx36 gap junction among the conditions examined. However, we found that Cx36 phosphorylation is higher under dark-adapted conditions at night than in the daytime, and is the lowest under prolonged illumination at any time of the day/night cycle. Our observations are consistent with the view that the circadian clock regulation of photoreceptor electrical coupling is mouse strain-dependent and highlights the critical position of Cx36 phosphorylation in the control of photoreceptor coupling.

  18. Locating a modifier gene of Ovum mutant through crosses between DDK and C57BL/6J inbred strains in mice.

    PubMed

    Tan, Jing; Song, Gen Di; Song, Jia Sheng; Ren, Shi Hao; Li, Chun Li; Zheng, Zhen Yu; Zhao, Wei Dong

    2016-06-01

    A striking infertile phenotype has been discovered in the DDK strain of mouse. The DDK females are usually infertile when crossed with males of other inbred strains, whereas DDK males exhibit normal fertility in reciprocal crosses. This phenomenon is caused by mutation in the ovum (Om) locus on chromosome 11 and known as the DDK syndrome. Previously, some research groups reported that the embryonic mortality deviated from the semilethal rate in backcrosses between heterozygous (Om/+) females and males of other strains. This embryonic mortality exhibited an aggravated trend with increasing background genes of other strains. These results indicated that some modifier genes of Om were present in other strains. In the present study, a population of N₂2 (Om/+) females from the backcrosses between C57BL/6J (B6) and F₁ (B6♀ × DDK♂) was used to map potential modifier genes of Om. Quantitative trait locus showed that a major locus, namely Amom1 (aggravate modifier gene of Om 1), was located at the middle part of chromosome 9 in mice. The Amom1 could increase the expressivity of Om gene, thereby aggravating embryonic lethality when heterozygous (Om/+) females mated with males of B6 strain. Further, the 1.5 LOD-drop analysis indicated that the confidence interval was between 37.54 and 44.46 cM, ~6.92 cM. Amom1 is the first modifier gene of Om in the B6 background.

  19. Locating a modifier gene of Ovum mutant through crosses between DDK and C57BL/6J inbred strains in mice.

    PubMed

    Tan, Jing; Song, Gen Di; Song, Jia Sheng; Ren, Shi Hao; Li, Chun Li; Zheng, Zhen Yu; Zhao, Wei Dong

    2016-06-01

    A striking infertile phenotype has been discovered in the DDK strain of mouse. The DDK females are usually infertile when crossed with males of other inbred strains, whereas DDK males exhibit normal fertility in reciprocal crosses. This phenomenon is caused by mutation in the ovum (Om) locus on chromosome 11 and known as the DDK syndrome. Previously, some research groups reported that the embryonic mortality deviated from the semilethal rate in backcrosses between heterozygous (Om/+) females and males of other strains. This embryonic mortality exhibited an aggravated trend with increasing background genes of other strains. These results indicated that some modifier genes of Om were present in other strains. In the present study, a population of N₂2 (Om/+) females from the backcrosses between C57BL/6J (B6) and F₁ (B6♀ × DDK♂) was used to map potential modifier genes of Om. Quantitative trait locus showed that a major locus, namely Amom1 (aggravate modifier gene of Om 1), was located at the middle part of chromosome 9 in mice. The Amom1 could increase the expressivity of Om gene, thereby aggravating embryonic lethality when heterozygous (Om/+) females mated with males of B6 strain. Further, the 1.5 LOD-drop analysis indicated that the confidence interval was between 37.54 and 44.46 cM, ~6.92 cM. Amom1 is the first modifier gene of Om in the B6 background. PMID:27350672

  20. The genetic basis of strain-dependent differences in the early phase of radiation injury in mouse lung

    SciTech Connect

    Franko, A.J.; Sharplin, J.; Ward, W.F.; Hinz, J.M. )

    1991-06-01

    Substantial differences between mouse strains have been reported in the lesions present in the lung during the early phase of radiation injury. Some strains show only classical pneumonitis, while other strains develop substantial fibrosis and hyaline membranes which contribute appreciably to respiratory insufficiency, in addition to pneumonitis. Other strains are intermediate between these extremes. These differences correlate with intrinsic differences in activities of lung plasminogen activator and angiotensin converting enzyme. The genetic basis of these differences was assessed by examining histologically the early reaction in lungs of seven murine hybrids available commercially after whole-thorax irradiation. Crosses between fibrosing and nonfibrosing parents were uniformly nonfibrosing, and crosses between fibrosing and intermediate parents were uniformly intermediate. No evidence of sex linkage was seen. Thus the phenotype in which fibrosis is found is controlled by autosomal recessive determinants. Strains prone to radiation-induced pulmonary fibrosis and hyaline membranes exhibited intrinsically lower activities of lung plasminogen activator and angiotensin converting enzyme than either the nonfibrosing strains or the nonfibrosing hybrid crosses. The median time of death of the hybrids was genetically determined primarily by the longest-lived parent regardless of the types of lesions expressed.

  1. [Construction and function analysis of Bac operon mutants of bio-control strain Bacillus subtilis Bs-916].

    PubMed

    Luo, Chuping; Chen, Zhiyi; Liu, Yongfeng; Zhang, Jie; Liu, Youzhou; Wang, Xiaoyu; Nie, Yafeng

    2009-04-01

    Bacillus subtilis Bs-916 is an effective biocontrol agent in control rice sheath blight caused by Rhizoctonia solani. We identified and analyzed the operon Bac in Bs-916 responsible for synthesis of iturin-like lipopeptides. The research plays an important part in genetic engineered Bs-916 for further improving its bio-control activity. Taking advantage of homologous recombination method, the one mutant obtained by replacement of the Bac original promoter by a constitutive promoter P (repU) and designated BGG104; the other mutant was obtained by disruption of the Bac promoter by insertion and designated BGG105. The biological activities results showed the mutant BGG104 enhanced antagonistic activities against several pathogenic fungi and also clearly increased in hemolytic activities. However, the mutant BGG105 decreased clearly in both antagonistic activities and hemolytic activities. Crude lipopeptides were extracted with methanol from precipitates, which were obtained by adding 6 mol/L HCl to the cell-free culture broth. The results of reversed-phase high-performance liquid chromatography (HPLC) analysis of crude lipopeptides showed lipopeptides produced by Bs-916 have a different retention time compared to iturinA. The mutant BGG104 produced up to 3-fold more lipopeptides than Bs-916. However, the mutant BGG105 has not been detected countpart lipopeptides production. The molecular weights of the lipopeptides synthesized by Bac determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) were 1007.7 Da, 1021.7 Da and 1035.7 Da. They were presumed to belong to homologues differed by a structure of --CH2 and their molecular weights were not the same as the other iturin-like liopeptides', such as iturinA, mycosubtilin and bacillomycin, molecular weights. In conclusion, this paper showed the lipopeptides synthesized by Bac play crucial part in Bs-916 antifungal activities and the lipopeptides overproduction were able to enhance Bs

  2. Genetic analysis of developmental mechanisms in hydra. XVI. Effect of food on budding and developmental gradients in a mutant strain L4.

    PubMed

    Takano, J; Sugiyama, T

    1985-12-01

    Effect of food was examined on the budding rate and the developmental gradients of a mutant hydra strain L4. This mutant strain has very high levels of head-inhibition potential gradient along its body axis (Takano & Sugiyama, 1983). It also has a reduced budding capacity when it is cultured using brine shrimp nauplii as food, but its budding capacity is significantly improved when a small amount of tubifex worm tissue is added to its diet of brine shrimp (Takano, 1984). To test whether or not this change of budding rate is correlated with the change in the levels of the head-activation or head-inhibition potential gradients, L4 animals were cultured on the diet of brine shrimp with or without addition of tubifex worm tissue and the budding rates and the gradient levels were examined in these animals. The results showed that food affected the budding rate in L4 without affecting its gradient levels. This suggests that the gradient levels and the budding rate in L4 are are uncorrelated to each other, and that therefore the high levels of head-inhibition potential are not the cause for the low budding rate in this strain (cf., Takano & Sugiyama, 1983). PMID:3834025

  3. A sodium channel knockin mutant (NaV1.4-R669H) mouse model of hypokalemic periodic paralysis

    PubMed Central

    Wu, Fenfen; Mi, Wentao; Burns, Dennis K.; Fu, Yu; Gray, Hillery F.; Struyk, Arie F.; Cannon, Stephen C.

    2011-01-01

    Hypokalemic periodic paralysis (HypoPP) is an ion channelopathy of skeletal muscle characterized by attacks of muscle weakness associated with low serum K+. HypoPP results from a transient failure of muscle fiber excitability. Mutations in the genes encoding a calcium channel (CaV1.1) and a sodium channel (NaV1.4) have been identified in HypoPP families. Mutations of NaV1.4 give rise to a heterogeneous group of muscle disorders, with gain-of-function defects causing myotonia or hyperkalemic periodic paralysis. To address the question of specificity for the allele encoding the NaV1.4-R669H variant as a cause of HypoPP and to produce a model system in which to characterize functional defects of the mutant channel and susceptibility to paralysis, we generated knockin mice carrying the ortholog of the gene encoding the NaV1.4-R669H variant (referred to herein as R669H mice). Homozygous R669H mice had a robust HypoPP phenotype, with transient loss of muscle excitability and weakness in low-K+ challenge, insensitivity to high-K+ challenge, dominant inheritance, and absence of myotonia. Recovery was sensitive to the Na+/K+-ATPase pump inhibitor ouabain. Affected fibers had an anomalous inward current at hyperpolarized potentials, consistent with the proposal that a leaky gating pore in R669H channels triggers attacks, whereas a reduction in the amplitude of action potentials implies additional loss-of-function changes for the mutant NaV1.4 channels. PMID:21881211

  4. Principal component and cluster analysis of morphological variables reveals multiple discrete sub-phenotypes in weaver mouse mutants.

    PubMed

    Martí, Joaquín; Santa-Cruz, María C; Serra, Roger; Valero, Oliver; Molina, Vanessa; Hervás, José P; Villegas, Sandra

    2013-06-01

    The present study evaluates the usefulness of the principal component analysis-based cluster analysis in the categorization of several sub-phenotypes in the weaver mutant by using several morphological parameters from the cerebellar cortex of control, heterozygous (+/wv) and homozygous (wv/wv) weaver mice. The quantified parameters were length of the cerebellar cortex, area of the external granular layer, area of the molecular layer, number of the external granular layer cells (EGL), and number of Purkinje cells (PCs). The analysis indicated that at postnatal day 8, the genotype +/wv presented three sub-phenotypes tagged as +/wv (0), +/wv (1) and +/wv (2), whereas two sub-phenotypes designated as wv (0)/wv (1) and wv (0)/wv (2) were identified in the genotype wv/wv. The number of PCs for the genotype +/wv and the number of EGL cells for the genotype wv/wv were the variables that discriminated the best among sub-phenotypes. Each one of the sub-phenotypes showed specific abnormalities in the cytoarchitecture of the cerebellar cortex as well as in the foliar pattern. In particular, the wv (0)/wv (1) and wv (0)/wv (2) sub-phenotypes had the most altered cytoarchitectonics, followed by the +/wv (2) sub-phenotype and then by the +/wv (1) one. The sub-phenotype +/wv (0) was the less affected one. Apart from reporting for the first time the coexistence of several sub-phenotypes in the weaver mutant, our approach provides a new statistical tool that can be used to assess cerebellar morphology.

  5. Signal strains that can detect certain DNA replication and membrane mutants of Escherichia coli: Isolation of a new ssb allele, ssb-3

    SciTech Connect

    Schmellik-Sandage, C.S.; Tessman, E.S. )

    1990-08-01

    Mutations in several dna genes of Escherichia coli, when introduced into a strain with a lac fusion in the SOS gene sulA, resulted in formation of blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal). Unexpectedly, several lines of evidence indicated that the blue colony color was not primarily due to induction of the SOS system but rather was due to a membrane defect, along with the replication defect, making the cell X-Gal extrasensitive (phenotypically Xgx), possibly because of enhanced permeability to X-Gal or leakage of beta-galactosidase. (i) In most cases, beta-galactosidase specific activity increased only two- to threefold. (ii) Mutations conferring tolerance to colicin E1 resulted in blue colony color with no increase in beta-galactosidase specific activity. (iii) Mutations in either the dnaA, dnaB, dnaC, dnaE, dnaG, or ssb gene, when introduced into a strain containing a bioA::lac fusion, produced a blue colony color without an increase in beta-galactosidase synthesis. These lac fusion strains can serve as signal strains to detect dna mutations as well as membrane mutations. By localized mutagenesis of the 92-min region of the chromosome of the sulA::lac signal strain and picking blue colonies, we isolated a novel ssb allele that confers the same extreme UV sensitivity as a delta recA allele, which is a considerably greater sensitivity than that conferred by the two well-studied ssb alleles, ssb-1 and ssb-113. The technique also yielded dnaB mutants; fortuitously, uvrA mutants were also found.

  6. Signal strains that can detect certain DNA replication and membrane mutants of Escherichia coli: isolation of a new ssb allele, ssb-3.

    PubMed Central

    Schmellik-Sandage, C S; Tessman, E S

    1990-01-01

    Mutations in several dna genes of Escherichia coli, when introduced into a strain with a lac fusion in the SOS gene sulA, resulted in formation of blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal). Unexpectedly, several lines of evidence indicated that the blue colony color was not primarily due to induction of the SOS system but rather was due to a membrane defect, along with the replication defect, making the cell X-Gal extrasensitive (phenotypically Xgx), possibly because of enhanced permeability to X-Gal or leakage of beta-galactosidase. (i) In most cases, beta-galactosidase specific activity increased only two- to threefold. (ii) Mutations conferring tolerance to colicin E1 resulted in blue colony color with no increase in beta-galactosidase specific activity. (iii) Mutations in either the dnaA, dnaB, dnaC, dnaE, dnaG, or ssb gene, when introduced into a strain containing a bioA::lac fusion, produced a blue colony color without an increase in beta-galactosidase synthesis. These lac fusion strains can serve as signal strains to detect dna mutations as well as membrane mutations. By localized mutagenesis of the 92-min region of the chromosome of the sulA::lac signal strain and picking blue colonies, we isolated a novel ssb allele that confers the same extreme UV sensitivity as a delta recA allele, which is a considerably greater sensitivity than that conferred by the two well-studied ssb alleles, ssb-1 and ssb-113. The technique also yielded dnaB mutants; fortuitously, uvrA mutants were also found. PMID:2142938

  7. Sulphide Resistance in the Cyanobacterium Microcystis aeruginosa: a Comparative Study of Morphology and Photosynthetic Performance Between the Sulphide-Resistant Mutant and the Wild-Type Strain.

    PubMed

    Bañares-España, Elena; del Mar Fernández-Arjona, María; García-Sánchez, María Jesús; Hernández-López, Miguel; Reul, Andreas; Mariné, Mariona Hernández; Flores-Moya, Antonio

    2016-05-01

    The cyanobacterium Microcystis aeruginosa is a mesophilic freshwater organism, which cannot tolerate sulphide. However, it was possible to isolate a sulphide-resistant (S(r)) mutant strain that was able to survive in a normally lethal medium sulphide. In order to evaluate the cost of the mutation conferring sulphide resistance in the S(r) strain of M. aeruginosa, the morphology and the photosynthetic performance were compared to that found in the wild-type, sulphide-sensitive (S(s)) strain. An increase in size and a disrupted morphology was observed in S(r) cells in comparison to the S(s) counterpart. Phycoerythrin and phycocyanin levels were higher in the S(r) than in the S(s) cells, whereas a higher carotenoid content, per unit volume, was found in the S(s) strain. The irradiance-saturated photosynthetic oxygen-production rate (GPR max) and the photosynthetic efficiency (measured both by oxygen production and fluorescence, α(GPR) and α(ETR)) were lower in the S(r) strain than in the wild-type. These results appear to be the result of package effect. On the other hand, the S(r) strain showed higher quantum yield of non-photochemical quenching, especially those regulated mechanisms (estimated throughout qN and Y(NPQ)) and a significantly lower slope in the maximum quantum yield of light-adapted samples (Fv'/Fm') compared to the S(s) strain. These findings point to a change in the regulation of the quenching of the transition states (qT) in the S(r) strain which may be generated by a change in the distribution of thylakoidal membranes, which somehow could protect metalloenzymes of the electron transport chain from the lethal effect of sulphide. PMID:26677166

  8. Sulphide Resistance in the Cyanobacterium Microcystis aeruginosa: a Comparative Study of Morphology and Photosynthetic Performance Between the Sulphide-Resistant Mutant and the Wild-Type Strain.

    PubMed

    Bañares-España, Elena; del Mar Fernández-Arjona, María; García-Sánchez, María Jesús; Hernández-López, Miguel; Reul, Andreas; Mariné, Mariona Hernández; Flores-Moya, Antonio

    2016-05-01

    The cyanobacterium Microcystis aeruginosa is a mesophilic freshwater organism, which cannot tolerate sulphide. However, it was possible to isolate a sulphide-resistant (S(r)) mutant strain that was able to survive in a normally lethal medium sulphide. In order to evaluate the cost of the mutation conferring sulphide resistance in the S(r) strain of M. aeruginosa, the morphology and the photosynthetic performance were compared to that found in the wild-type, sulphide-sensitive (S(s)) strain. An increase in size and a disrupted morphology was observed in S(r) cells in comparison to the S(s) counterpart. Phycoerythrin and phycocyanin levels were higher in the S(r) than in the S(s) cells, whereas a higher carotenoid content, per unit volume, was found in the S(s) strain. The irradiance-saturated photosynthetic oxygen-production rate (GPR max) and the photosynthetic efficiency (measured both by oxygen production and fluorescence, α(GPR) and α(ETR)) were lower in the S(r) strain than in the wild-type. These results appear to be the result of package effect. On the other hand, the S(r) strain showed higher quantum yield of non-photochemical quenching, especially those regulated mechanisms (estimated throughout qN and Y(NPQ)) and a significantly lower slope in the maximum quantum yield of light-adapted samples (Fv'/Fm') compared to the S(s) strain. These findings point to a change in the regulation of the quenching of the transition states (qT) in the S(r) strain which may be generated by a change in the distribution of thylakoidal membranes, which somehow could protect metalloenzymes of the electron transport chain from the lethal effect of sulphide.

  9. NAD(P)+-Malic Enzyme Mutants of Sinorhizobium sp. Strain NGR234, but Not Azorhizobium caulinodans ORS571, Maintain Symbiotic N2 Fixation Capabilities

    PubMed Central

    Zhang, Ye; Aono, Toshihiro; Poole, Phillip

    2012-01-01

    C4-dicarboxylic acids appear to be metabolized via the tricarboxylic acid (TCA) cycle in N2-fixing bacteria (bacteroids) within legume nodules. In Sinorhizobium meliloti bacteroids from alfalfa, NAD+-malic enzyme (DME) is required for N2 fixation, and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont Rhizobium leguminosarum, pyruvate synthesis occurs via either DME or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK) and pyruvate kinase (PYK). Here we report that dme mutants of the broad-host-range Sinorhizobium sp. strain NGR234 formed nodules whose level of N2 fixation varied from 27 to 83% (plant dry weight) of the wild-type level, depending on the host plant inoculated. NGR234 bacteroids had significant PCK activity, and while single pckA and single dme mutants fixed N2 at reduced rates, a pckA dme double mutant had no N2-fixing activity (Fix−). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix− phenotype of S. meliloti dme mutants may be specific to the alfalfa-S. meliloti symbiosis. We therefore examined the ME-like genes azc3656 and azc0119 from Azorhizobium caulinodans, as azc3656 mutants were previously shown to form Fix− nodules on the tropical legume Sesbania rostrata. We found that purified AZC3656 protein is an NAD(P)+-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N2 fixation in A. caulinodans and S. meliloti, in other rhizobia this activity can be bypassed via another pathway(s). PMID:22307295

  10. Strain Differences in Behavioral Inhibition in a Go/No-go Task Demonstrated Using 15 Inbred Mouse Strains

    PubMed Central

    Gubner, Noah R.; Wilhelm, Clare J.; Phillips, Tamara J.; Mitchell, Suzanne H.

    2012-01-01

    Background High levels of impulsivity have been associated with a number of substance abuse disorders including alcohol abuse. Research has not yet revealed whether these high levels predate the development of alcohol abuse. Methods The current study examined impulsivity in 15 inbred strains of mice (A/HeJ, AKR/J, BALB/cJ, C3H/HeJ, C57BL/6J, C57L/J, C58/J, CBA/J, DBA/1J, DBA/2J, NZB/B1NJ, PL/J, SJL/J, SWR/J, and 129P3/J) using a Go/No-go task, which was designed to measure a subject’s ability to inhibit a behavior. Numerous aspects of response to ethanol and other drugs of abuse have been examined in these strains. Results There were significant strain differences in the number of responses made during the No-go signal (false alarms) and the extent to which strains responded differentially during the Go and No-go signals (d′). The rate of responding prior to the cue did not differ among strains, although there was a statistically significant correlation between false alarms and precue responding that was not related to basal activity level. Interstrain correlations suggested that false alarms and rate of responding were associated with strain differences in ethanol-related traits from the published literature. Conclusions The results of this study do support a link between innate level of impulsivity and response to ethanol and are consistent with a genetic basis for some measures of behavioral inhibition. PMID:20491731

  11. A Mouse Model for Candida glabrata Hematogenous Disseminated Infection Starting from the Gut: Evaluation of Strains with Different Adhesion Properties

    PubMed Central

    Atanasova, Ralitsa; Angoulvant, Adela; Tefit, Maurel; Gay, Frédérick; Guitard, Juliette; Mazier, Dominique; Fairhead, Cécile; Hennequin, Christophe

    2013-01-01

    Adhesion to digestive mucosa is considered a crucial first step in the pathogenicity of invasive Candida infections. Candida glabrata disseminated infections predominantly start from the gut. A mouse model of disseminated infection starting from the gut was set up. Hematogenous dissemination was obtained after a low-protein diet followed by a regimen of cyclophosphamide-methotrexate and an oral inoculation of the yeasts via the drinking water. The liver was the first organ infected (day 7 post-infection), and lethality was 100% at day 21 post-infection. This new mouse model was used to compare the mortality rate and fungal burden in deep organs induced by 5 strains exhibiting different levels of adhesion to enterocyte Caco-2 cells, as determined in a test on 36 C. glabrata strains. In this model, no statistical difference of lethality was demonstrated between the strains, and fungal burden varied in kidneys and lungs but without correlation with the level of adhesion to enterocytes. Further studies using the model developed here allow analysis of the crossing of the digestive mucosa by yeasts, and help relate this to yet-poorly understood adhesion phenotypes. PMID:23936069

  12. Characterization of cDNA encoding mouse DNA repair protein O sup 6 -methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant proteins in Escherichia coli

    SciTech Connect

    Shiota, Susumu; Tano, Keizo ); von Wronski, M.A.; Brent, T.P. ); Bigner, D.D. ); Mitra, S. )

    1992-02-25

    A mouse cDNA clone encoding O{sup 6}-methylguanine-DNA methyltransferase (MGMT), responsible for repair of mutagenic O{sup 6}-alkylguanine in DNA, was cloned from a {lambda}gt11 library. On the basis of an open reading frame in cDNA, the mouse protein contains 211 amino acids with a molecular mass of 22 kDa. The size and the predicted N-terminal sequence of the mouse protein were confirmed experimentally. The deduced amino acid sequence of the mouse MGMT is 70% homologous to that of the human MGMT. Cysteine-149 was shown to be the only alkyl acceptor residue in the mouse protein, in confirmation of the prediction based on conserved sequences of different MGMTs. Mouse MGMT protein is recognized by some monoclonal antibodies specific for human MGMT. Site-directed mutagenesis was utilized to reclone the mouse cDNA in a T7 promoter-based vector for overexpression of the native repair protein in Escherichia coli. The mouse protein has a tetrapeptide sequence, Pro-Glu-Gly-Val at positions 56-59, absent in the human protein. Neither deletion of this tetrapeptide nor substitution of valine-169 with alanine affected the activity of the mutant proteins.

  13. Progress in Using Mouse Inbred Strains, Consomics, and Mutants to Identify Genes Related to Stress, Anxiety, and Alcohol Phenotypes

    SciTech Connect

    Goldowitz, Daniel; Matthews, Douglas B.; Hamre, Kristin M.; Mittleman, Guy; Chesler, Elissa J; Becker, Howard C.; Lopez, Marcelo F.; Jones, Sara R.; Mathews, Tiffany A; Miles, Michael F.; Kerns, Robnet; Grant, Kathleen A.

    2006-01-01

    ALCOHOL ABUSE AND alcoholism result from the complex interplay of genetic and environmental factors. Stress is a factor that is widely thought to contribute to excessive drinking and alcoholism. One consequence of stressful experiences is anxiety, and there is a rich literature on the interactions between alcohol and anxiety. Less is known about brain mechanisms at the molecular, cellular, and system levels that mediate stress effects that contribute to excessive drinking and alcoholism. In addition, it is not clear whether and/or how genetic factors that contribute to excessive drinking interact with neural stress mechanisms.

  14. Establishment of c-myc-immortalized Kupffer cell line from a C57BL/6 mouse strain.

    PubMed

    Kitani, Hiroshi; Sakuma, Chisato; Takenouchi, Takato; Sato, Mitsuru; Yoshioka, Miyako; Yamanaka, Noriko

    2014-01-01

    We recently demonstrated in several mammalian species, a novel procedure to obtain liver-macrophages (Kupffer cells) in sufficient numbers and purity using a mixed primary culture of hepatocytes. In this study, we applied this method to the C57BL/6 mouse liver and established an immortalized Kupffer cell line from this mouse strain. The hepatocytes from the C57BL/6 adult mouse liver were isolated by a two-step collagenase perfusion method and cultured in T25 culture flasks. Similar to our previous studies, the mouse hepatocytes progressively changed their morphology into a fibroblastic appearance after a few days of culture. After 7-10 days of culture, Kupffer-like cells, which were contaminants in the hepatocyte fraction at the start of the culture, actively proliferated on the mixed fibroblastic cell sheet. At this stage, a retroviral vector containing the human c-myc oncogene and neomycin resistance gene was introduced into the mixed culture. Gentle shaking of the culture flask, followed by the transfer and brief incubation of the culture supernatant, resulted in a quick and selective adhesion of Kupffer cells to a plastic dish surface. After selection with G418 and cloning by limiting dilutions, a clonal cell line (KUP5) was established. KUP5 cells displayed typical macrophage morphology and were stably passaged at 4-5 days intervals for more than 5 months, with a population doubling time of 19 h. KUP5 cells are immunocytochemically positive for mouse macrophage markers, such as Mac-1, F4/80. KUP5 cells exhibited substantial phagocytosis of polystyrene microbeads and the release of inflammatory cytokines upon lipopolysaccharide stimulation. Taken together, KUP5 cells provide a useful means to study the function of Kupffer cells in vitro. PMID:25379377

  15. The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells.

    PubMed

    Parker, Andrew; Cross, Sally H; Jackson, Ian J; Hardisty-Hughes, Rachel; Morse, Susan; Nicholson, George; Coghill, Emma; Bowl, Michael R; Brown, Steve D M

    2015-12-01

    Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs) that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182) in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss.

  16. The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells

    PubMed Central

    Parker, Andrew; Cross, Sally H.; Jackson, Ian J.; Hardisty-Hughes, Rachel; Morse, Susan; Nicholson, George; Coghill, Emma; Bowl, Michael R.; Brown, Steve D. M.

    2015-01-01

    ABSTRACT Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs) that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182) in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss. PMID:26542706

  17. Efficacy of PARP inhibition in Pde6a mutant mouse models for retinitis pigmentosa depends on the quality and composition of individual human mutations

    PubMed Central

    Jiao, K; Sahaboglu, A; Zrenner, E; Ueffing, M; Ekström, P A R; Paquet-Durand, F

    2016-01-01

    Retinitis pigmentosa (RP), an inherited blinding disease, is caused by a variety of different mutations that affect retinal photoreceptor function and survival. So far there is neither effective treatment nor cure. We have previously shown that poly(ADP-ribose)polymerase (PARP) acts as a common and critical denominator of cell death in photoreceptors, qualifying it as a potential target for future therapeutic intervention. A significant fraction of RP-causing mutations affect the genes for the rod photoreceptor phosphodiesterase 6A (PDE6A) subunit, but it is not known whether they all engage the same death pathway. Analysing three homozygous point mutations (Pde6a R562W, D670G, and V685M) and one compound heterozygous Pde6aV685M/R562W mutation in mouse models that match human RP patients, we demonstrate excessive activation of PARP, which correlated in time with the progression of photoreceptor degeneration. The causal involvement of PARP activity in the neurodegenerative process was confirmed in organotypic retinal explant cultures treated with the PARP-selective inhibitor PJ34, using different treatment time-points and durations. Remarkably, the neuroprotective efficacy of PARP inhibition correlated inversely with the strength of the genetically induced insult, with the D670G mutant showing the best treatment effects. Our results highlight PARP as a target for neuroprotective interventions in RP caused by PDE6A mutations and are a first attempt towards personalized, genotype-matched therapy development for RP. In addition, for each of the different mutant situations, our work identifies windows of opportunity for an optimal treatment regimen for further in vivo experimentation and possibly clinical studies. PMID:27551530

  18. Mouse Genome Database: From sequence to phenotypes and disease models.

    PubMed

    Eppig, Janan T; Richardson, Joel E; Kadin, James A; Smith, Cynthia L; Blake, Judith A; Bult, Carol J

    2015-08-01

    The Mouse Genome Database (MGD, www.informatics.jax.org) is the international scientific database for genetic, genomic, and biological data on the laboratory mouse to support the research requirements of the biomedical community. To accomplish this goal, MGD provides broad data coverage, serves as the authoritative standard for mouse nomenclature for genes, mutants, and strains, and curates and integrates many types of data from literature and electronic sources. Among the key data sets MGD supports are: the complete catalog of mouse genes and genome features, comparative homology data for mouse and vertebrate genes, the authoritative set of Gene Ontology (GO) annotations for mouse gene functions, a comprehensive catalog of mouse mutations and their phenotypes, and a curated compendium of mouse models of human diseases. Here, we describe the data acquisition process, specifics about MGD's key data areas, methods to access and query MGD data, and outreach and user help facilities. PMID:26150326

  19. Low dose vaccination with attenuated Francisella tularensis strain SchuS4 mutants protects against tularemia independent of the route of vaccination.

    PubMed

    Rockx-Brouwer, Dedeke; Chong, Audrey; Wehrly, Tara D; Child, Robert; Crane, Deborah D; Celli, Jean; Bosio, Catharine M

    2012-01-01

    Tularemia, caused by the gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. <100 organisms, may address this concern. Herein we describe the ability of three defined, attenuated mutants of F. tularensis SchuS4, deleted for FTT0369c, FTT1676, or FTT0369c and FTT1676, respectively, to engender protective immunity against tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia.

  20. 1 + 1 = 3: Development and validation of a SNP-based algorithm to identify genetic contributions from three distinct inbred mouse strains.

    PubMed

    Gorham, James D; Ranson, Matthew S; Smith, Janebeth C; Gorham, Beverly J; Muirhead, Kristen-Ashley

    2012-12-01

    State-of-the-art, genome-wide assessment of mouse genetic background uses single nucleotide polymorphism (SNP) PCR. As SNP analysis can use multiplex testing, it is amenable to high-throughput analysis and is the preferred method for shared resource facilities that offer genetic background assessment of mouse genomes. However, a typical individual SNP query yields only two alleles (A vs. B), limiting the application of this methodology to distinguishing contributions from no more than two inbred mouse strains. By contrast, simple sequence length polymorphism (SSLP) analysis yields multiple alleles but is not amenable to high-throughput testing. We sought to devise a SNP-based technique to identify donor strain origins when three distinct mouse strains potentially contribute to the genetic makeup of an individual mouse. A computational approach was used to devise a three-strain analysis (3SA) algorithm that would permit identification of three genetic backgrounds while still using a binary-output SNP platform. A panel of 15 mosaic mice with contributions from BALB/c, C57Bl/6, and DBA/2 genetic backgrounds was bred and analyzed using a genome-wide SNP panel using 1449 markers. The 3SA algorithm was applied and then validated using SSLP. The 3SA algorithm assigned 85% of 1449 SNPs as informative for the C57Bl/6, BALB/c, or DBA/2 backgrounds, respectively. Testing the panel of 15 F2 mice, the 3SA algorithm predicted donor strain origins genome-wide. Donor strain origins predicted by the 3SA algorithm correlated perfectly with results from individual SSLP markers located on five different chromosomes (n=70 tests). We have established and validated an analysis algorithm based on binary SNP data that can successfully identify the donor strain origins of chromosomal regions in mice that are bred from three distinct inbred mouse strains. PMID:23204929

  1. The viable Mycobacterium tuberculosis H37Ra strain induces a stronger mouse macrophage response compared to the heat-inactivated H37Rv strain.

    PubMed

    He, Zong-Lin; Du, Fa-Wang; Du, Xian-Zhi

    2013-05-01

    Macrophages are the target cells for Mycobacterium tuberculosis (M. tuberculosis) as well as key effector cells for clearance of this pathogen. The aim of the present study was to measure and compare the responses of mouse peritoneal macrophages following exposure to the live M. tuberculosis H37Ra and heat-inactivated H37Rv strains. In vitro phagocytosis assays indicated that the macrophages had a higher capacity to engulf the live H37Ra strain compared to the inactivated H37Rv strain. Enzyme-linked immunosorbent assay (ELISA) demonstrated that H37Ra‑stimulated macrophages produced significantly increased concentrations of interleukin‑12p40 (IL‑12p40), tumor necrosis factor-α (TNF‑α) and interferon‑γ (IFN‑γ) compared to the untreated control cells. However, H37Rv exposure induced little to no increase in the levels of the cytokines examined. The results from ELISA were confirmed by reverse transcription-polymerase chain reaction (RT‑PCR) at the mRNA level. There was a dose-dependent increase in nitric oxide (NO) and hydrogen peroxide (H2O2) production from the H37Ra‑stimulated macrophages compared to the H37Rv‑stimulated ones. Confocal microscopy and flow cytometric analysis indicated that the IFN‑γ‑stimulated macrophages from viable H37Ra‑immunized mice had an enhanced surface expression of CD40 ligand (CD40L) compared to those from inactivated H37Rv‑immunized mice. Our data collectively indicate that exposure to the viable H37Ra strain induces a stronger macrophage response compared to exposure to the heat-inactivated H37Rv strain, which may be associated with the increased surface expression of CD40L in activated macrophages.

  2. The Cataract-linked Mutant Connexin50D47A Causes Endoplasmic Reticulum Stress in Mouse Lenses.

    PubMed

    Berthoud, Viviana M; Minogue, Peter J; Lambert, Paul A; Snabb, Joseph I; Beyer, Eric C

    2016-08-19

    Mice expressing connexin50D47A (Cx50D47A) exhibit nuclear cataracts and impaired differentiation. Cx50D47A does not traffic properly, and homozygous mutant lenses show increased levels of the stress-responsive αB-crystallins. Therefore, we assessed whether expression of Cx50D47A led to endoplasmic reticulum (ER) stress in the lens in vivo Although pharmacologic induction of ER stress can be transduced by three different pathways, we found no evidence for activation of the IRE1α or ATF6 pathways in Cx50D47A-expressing lenses. In contrast, heterozygous and homozygous Cx50D47A lenses showed an increase in phosphorylated PERK immunoreactivity and in the ratio of phosphorylated to total EIF2α (2.4- and 3.3-fold, respectively) compared with wild type. Levels of ATF4 were similar in wild type and heterozygous lenses but elevated in homozygotes (391%). In both heterozygotes and homozygotes, levels of calreticulin protein were increased (184 and 262%, respectively), as was Chop mRNA (1.9- and 12.4-fold, respectively). CHOP protein was increased in homozygotes (384%). TUNEL staining was increased in Cx50D47A lenses, especially in homozygous mice. Levels of two factors that may be pro-survival, Irs2 and Trib3, were greatly increased in homozygous lenses. These results suggest that expression of Cx50D47A induces ER stress, triggering activation of the PERK-ATF4 pathway, which potentially contributes to the lens pathology and leads to increased expression of anti-apoptotic factors, allowing cell survival.

  3. Open fermentative production of fuel ethanol from food waste by an acid-tolerant mutant strain of Zymomonas mobilis.

    PubMed

    Ma, Kedong; Ruan, Zhiyong; Shui, Zongxia; Wang, Yanwei; Hu, Guoquan; He, Mingxiong

    2016-03-01

    The aim of present study was to develop a process for open ethanol fermentation from food waste using an acid-tolerant mutant of Zymomonas mobilis (ZMA7-2). The mutant showed strong tolerance to acid condition of food waste hydrolysate and high ethanol production performance. By optimizing fermentation parameters, ethanol fermentation with initial glucose concentration of 200 g/L, pH value around 4.0, inoculum size of 10% and without nutrient addition was considered as best conditions. Moreover, the potential of bench scales fermentation and cell reusability was also examined. The fermentation in bench scales (44 h) was faster than flask scale (48 h), and the maximum ethanol concentration and ethanol yield (99.78 g/L, 0.50 g/g) higher than that of flask scale (98.31 g/L, 0.49 g/g). In addition, the stable cell growth and ethanol production profile in five cycles successive fermentation was observed, indicating the mutant was suitable for industrial ethanol production.

  4. The mouse genome informatics and the mouse genome database

    SciTech Connect

    Maltais, L.J.; Blackburn, R.E.; Bradt, D.W.

    1994-09-01

    The Mouse Genome Database (MGD) is a centralized, comprehensive database of the mouse genome that includes genetic mapping data, comparative mapping data, gene descriptions, mutant phenotype descriptions, strains and allelic polymorphism data, inbred strain characteristics, physical mapping data, and molecular probes and clones data. Data in MGD are obtained from the published literature and by electronic transfer from laboratories working on large backcross panels of mice. MGD provides tools that enable the user to search the database, retrieve data, generate reports, analyze data, annotate records, and build genetic maps. The Encyclopedia of the Mouse Genome provides a graphic user interface to mouse genome data. It consists of software tools including: LinkMap, a graphic display of genetic linkage maps with the ability to magnify regions of high locus density: CytoMap, a graphic display of cytogenetic maps showing banded chromosomes with cytogenetic locations of genes and chromosomal aberrations; CATS, a catalog searching tool for text retrieval of mouse locus descriptions. These software tools provide access to the following data sets: Chromosome Committee Reports, MIT Genome Center data, GBASE reports, Mouse Locus Catalog (MLC), and Mouse Cytogenetic Mapping Data. The MGD is available to the scientific community through the World Wide Web (WWW) and Gopher. In addition GBASE can be accessed via the Internet.

  5. Reproducibility of toxicity test data as a function of mouse strain, animal lot, and operator. [for bisphenol A polycarbonate

    NASA Technical Reports Server (NTRS)

    Hilado, C. J.; Furst, A.

    1978-01-01

    The toxicity screening test method developed at the University of San Francisco was evaluated for reproducibility. The variables addressed were strain of mouse, lot of animals, and operator. There was a significant difference in response between Swiss Webster mice and ICR mice, with the latter exhibiting greater resistance. These two strains of mice are not interchangeable in this procedure. Variation between individual animals was significant and unavoidable. In view of this variation, between-lot and between-operator variations appear to have no practical significance. The significant variation between individual animals stresses the need for average values based on at least four animals, and preferably values based on at least two experiments and eight animals. Efforts to compare materials should be based on the evaluation of relatively simple responses using substantial numbers of animals, rather than on elaborate evaluation of single animals

  6. Comparison of atypical Brachyspira spp. clinical isolates and classic strains in a mouse model of swine dysentery.

    PubMed

    Burrough, Eric; Strait, Erin; Kinyon, Joann; Bower, Leslie; Madson, Darin; Schwartz, Kent; Frana, Timothy; Songer, J Glenn

    2012-12-01

    Multiple Brachyspira spp. can colonize the porcine colon, and the presence of the strongly beta-hemolytic Brachyspira hyodysenteriae is typically associated with clinical swine dysentery. Recently, several Brachyspira spp. have been isolated from the feces of pigs with clinical disease suggestive of swine dysentery, yet these isolates were not identified as B. hyodysenteriae by genotypic or phenotypic methods. This study used a mouse model of swine dysentery to compare the pathogenic potential of seventeen different Brachyspira isolates including eight atypical clinical isolates, six typical clinical isolates, the standard strain of B. hyodysenteriae (B204), and reference strains of Brachyspira intermedia and Brachyspira innocens. Results revealed that strongly beta-hemolytic isolates induced significantly greater cecal inflammation than weakly beta-hemolytic isolates regardless of the genetic identification of the isolate, and that strongly beta-hemolytic isolates identified as 'Brachyspira sp. SASK30446' and B. intermedia by PCR produced lesions indistinguishable from those caused by B. hyodysenteriae in this model.

  7. An edited linkage map for the AXB and BXA recombinant inbred mouse strains.

    PubMed

    Sampson, S B; Higgins, D C; Elliot, R W; Taylor, B A; Lueders, K K; Koza, R A; Paigen, B

    1998-09-01

    We have updated the history of the AXB and BXA recombinant inbred (RI) strains, typed additional loci, and edited the AXB, BXA RI database. Thirteen of the original 51 AXB and BXA RI strains are either extinct or genetically contaminated, leaving 33 living strains available from The Jackson Laboratory. However, we found a high degree of similarity among three sets of strains, indicating that these strains are not independent, which leaves 27 independent RI strains in the set. Accordingly, we modified the database by combining the AXB and BXA RI sets and eliminating strains that were genetically contaminated or extinct with no available DNA. We added 92 newly typed loci, retyped some questionable genotypings, and removed loci with excessive double crossovers or an insufficient number of typed strains. The edited strain distribution pattern (SDP) is available on the World Wide Web (WWW) (http://www. informatics.jax.org/riset.html) and now includes over 700 loci. Each locus is linked to adjacent loci with a LOD score of at least 3.0 with a few described exceptions. We also carried out a second editing designed for the analysis of quantitative trait loci by deleting extinct strains and loci with identical SDPs; this edited database is also available on the WWW.

  8. A conditional mouse mutant in the tumor suppressor SdhD gene unveils a link between p21(WAF1/Cip1) induction and mitochondrial dysfunction.

    PubMed

    Millán-Uclés, Africa; Díaz-Castro, Blanca; García-Flores, Paula; Báez, Alicia; Pérez-Simón, José Antonio; López-Barneo, José; Piruat, José I

    2014-01-01

    Mutations in mitochondrial complex II (MCII; succinate dehydrogenase, Sdh) genes cause familiar pheochromocytoma/paraganglioma tumors. Several mechanisms have been proposed to account for Sdh-mutation-induced tumorigenesis, the most accepted of which is based on the constitutive expression of the hypoxia-inducible factor 1α (Hif1α) at normal oxygen tension, a theory referred to as "pseudo-hypoxic drive". Other molecular processes, such as oxidative stress, apoptosis, or chromatin remodeling have been also proposed to play a causative role. Nevertheless, the actual contribution of each of these mechanisms has not been definitively established. Moreover, the biological factors that determine the tissue-specificity of these tumors have not been identified. In this work, we made use of the inducible SDHD-ESR mouse, a conditional mutant in the SdhD gene, which encodes the small subunit of MCII, and that acts as a tumor suppressor gene in humans. The analysis of the Hif1α pathway in SDHD-ESR tissues and in two newly derived cell lines after complete SdhD loss -a requirement for hereditary paraganglioma type-1 tumor formation in humans- partially recapitulated the "pseudo-hypoxic" response and rendered inconsistent results. Therefore, we performed microarray analysis of adrenal medulla and kidney in order to identify other early gene expression changes elicited by SdhD deletion. Our results revealed that each mutant tissue displayed different variations in their gene expression profiles affecting to different biological processes. However, we found that the Cdkn1a gene was up-regulated in both tissues. This gene encodes the cyclin-dependent kinase inhibitor p21(WAF1/Cip1), a factor implicated in cell cycle, senescence, and cancer. The two SDHD-ESR cell lines also showed accumulation of this protein. This new and unprecedented evidence for a link between SdhD dysfunction and p21(WAF1/Cip1) will open new avenues for the study of the mechanisms that cause tumors in Sdh

  9. Pacemaker-neuron-dependent disturbance of the molecular clockwork by a Drosophila CLOCK mutant homologous to the mouse Clock mutation.

    PubMed

    Lee, Euna; Cho, Eunjoo; Kang, Doo Hyun; Jeong, Eun Hee; Chen, Zheng; Yoo, Seung-Hee; Kim, Eun Young

    2016-08-16

    Circadian clocks are composed of transcriptional/translational feedback loops (TTFLs) at the cellular level. In Drosophila TTFLs, the transcription factor dCLOCK (dCLK)/CYCLE (CYC) activates clock target gene expression, which is repressed by the physical interaction with PERIOD (PER). Here, we show that amino acids (AA) 657-707 of dCLK, a region that is homologous to the mouse Clock exon 19-encoded region, is crucial for PER binding and E-box-dependent transactivation in S2 cells. Consistently, in transgenic flies expressing dCLK with an AA657-707 deletion in the Clock (Clk(out)) genetic background (p{dClk-Δ};Clk(out)), oscillation of core clock genes' mRNAs displayed diminished amplitude compared with control flies, and the highly abundant dCLKΔ657-707 showed significantly decreased binding to PER. Behaviorally, the p{dClk-Δ};Clk(out) flies exhibited arrhythmic locomotor behavior in the photic entrainment condition but showed anticipatory activities of temperature transition and improved free-running rhythms in the temperature entrainment condition. Surprisingly, p{dClk-Δ};Clk(out) flies showed pacemaker-neuron-dependent alterations in molecular rhythms; the abundance of dCLK target clock proteins was reduced in ventral lateral neurons (LNvs) but not in dorsal neurons (DNs) in both entrainment conditions. In p{dClk-Δ};Clk(out) flies, however, strong but delayed molecular oscillations in temperature cycle-sensitive pacemaker neurons, such as DN1s and DN2s, were correlated with delayed anticipatory activities of temperature transition. Taken together, our study reveals that the LNv molecular clockwork is more sensitive than the clockwork of DNs to dysregulation of dCLK by AA657-707 deletion. Therefore, we propose that the dCLK/CYC-controlled TTFL operates differently in subsets of pacemaker neurons, which may contribute to their specific functions. PMID:27489346

  10. Development of a Nuclear Transformation System for Oleaginous Green Alga Lobosphaera (Parietochloris) incisa and Genetic Complementation of a Mutant Strain, Deficient in Arachidonic Acid Biosynthesis

    PubMed Central

    Khozin-Goldberg, Inna; Leu, Stefan; Shapira, Michal; Kaye, Yuval; Tourasse, Nicolas; Vallon, Olivier; Boussiba, Sammy

    2014-01-01

    Microalgae are considered a promising source for various high value products, such as carotenoids, ω-3 and ω-6 polyunsaturated fatty acids (PUFA). The unicellular green alga Lobosphaera (Parietochloris) incisa is an outstanding candidate for the efficient phototrophic production of arachidonic acid (AA), an essential ω-6 PUFA for infant brain development and a widely used ingredient in the baby formula industry. Although phototrophic production of such algal products has not yet been established, estimated costs are considered to be 2–5 times higher than competing heterotrophic production costs. This alga accumulates unprecedented amounts of AA within triacylglycerols and the molecular pathway of AA biosynthesis in L. incisa has been previously elucidated. Thus, progress in transformation and metabolic engineering of this high value alga could be exploited for increasing the efficient production of AA at competitive prices. We describe here the first successful transformation of L. incisa using the ble gene as a selection marker, under the control of the endogenous RBCS promoter. Furthermore, we have succeeded in the functional complementation of the L. incisa mutant strain P127, containing a mutated, inactive version of the delta-5 (Δ5) fatty acid desaturase gene. A copy of the functional Δ5 desaturase gene, linked to the ble selection marker, was transformed into the P127 mutant. The resulting transformants selected for zeocine resistant, had AA biosynthesis partially restored, indicating the functional complementation of the mutant strain with the wild-type gene. The results of this study present a platform for the successful genetic engineering of L. incisa and its long-chain PUFA metabolism. PMID:25133787

  11. Metabolic Engineering of Light and Dark Biochemical Pathways in Wild-Type and Mutant Strains of Synechocystis PCC 6803 for Maximal, 24-Hour Production of Hydrogen Gas

    SciTech Connect

    Ely, Roger L.; Chaplen, Frank W.R.

    2014-03-11

    enhanced H2 production profiles using selected culture conditions and inhibitors of specific pathways in WT cells and an NDH-1 mutant; 3. Create Synechocystis PCC 6803 mutant strains with modified hydrogenases exhibiting increased O2 tolerance and greater H2 production; and 4. Integrate enhanced hydrogenase mutants and culture and metabolic factor studies to maximize 24-hour H2 production.

  12. A mouse model for Betacoronavirus subgroup 2c using a bat coronavirus strain HKU5 variant.

    PubMed

    Agnihothram, Sudhakar; Yount, Boyd L; Donaldson, Eric F; Huynh, Jeremy; Menachery, Vineet D; Gralinski, Lisa E; Graham, Rachel L; Becker, Michelle M; Tomar, Sakshi; Scobey, Trevor D; Osswald, Heather L; Whitmore, Alan; Gopal, Robin; Ghosh, Arun K; Mesecar, Andrew; Zambon, Maria; Heise, Mark; Denison, Mark R; Baric, Ralph S

    2014-03-25

    Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks. Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (β-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5) and the nucleocapsid (N) protein, which are significant drug and vaccine targets. BtCoV HKU5 has been described in silico but has not been shown to replicate in culture, thus hampering drug and vaccine studies against subgroup 2c β-CoVs. We report the synthetic reconstruction and testing of BtCoV HKU5 containing the severe acute respiratory syndrome (SARS)-CoV spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus replicates efficiently in cell culture and in young and aged mice, where the virus targets airway and alveolar epithelial cells. Unlike some subgroup 2b SARS-CoV vaccines that elicit a strong eosinophilia following challenge, we demonstrate that BtCoV HKU5 and MERS-CoV N-expressing Venezuelan equine encephalitis virus replicon particle (VRP) vaccines do not cause extensive eosinophilia following BtCoV HKU5-SE challenge. Passage of BtCoV HKU5-SE in young mice resulted in enhanced virulence, causing 20% weight loss, diffuse alveolar damage, and hyaline membrane formation in aged mice. Passaged virus was characterized by mutations in the nsp13, nsp14, open reading frame 5 (ORF5) and M genes. Finally, we identified an inhibitor active against the nsp5 proteases of subgroup 2c β-CoVs. Synthetic-genome platforms capable of reconstituting emerging zoonotic viral pathogens or their phylogenetic relatives provide new strategies for identifying broad-based therapeutics, evaluating vaccine outcomes, and studying viral pathogenesis. IMPORTANCE The 2012 outbreak of MERS-CoV raises the specter

  13. Characterization of a novel NKG2D and NKp46 double-mutant mouse reveals subtle variations in the NK cell repertoire

    PubMed Central

    Sheppard, Sam; Triulzi, Chiara; Ardolino, Michele; Serna, Daniel; Zhang, Lily; Raulet, David H.

    2013-01-01

    The immunoreceptors NKG2D and NKp46 are known for their capacity to activate natural killer (NK) cell cytotoxicity and secretory responses in the contexts of tumors and infections, yet their roles in NK cell education remain unclear. Here, we provide the first characterization of mice deficient for both NKG2D and NKp46 receptors to address the relevance of their concomitant absence during NK cell development and function. Our findings reveal that NK cells develop normally in double-mutant (DKO) mice. Mice lacking NKG2D but not NKp46 showed subtle differences in the percentages of NK cells expressing inhibitory Ly49 receptors and the adhesion molecule DNAM-1. A slightly increased percentage of terminally differentiated NK cells and functional response to in vitro stimuli was observed in some experiments. These alterations were modest and did not affect NK cell function in vivo in response to mouse cytomegalovirus infection. NKp46 deficiency alone, or in combination with NKG2D deficiency, had no effect on frequency or function of NK cells. PMID:23649470

  14. 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.

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

    PubMed Central

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

    2016-01-01

    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

  16. Complement gene expression in hepatic and extrahepatic tissues of NZB and NZB x W (F1) mouse strains.

    PubMed Central

    Passwell, J H; Schreiner, G F; Wetsel, R A; Colten, H R

    1990-01-01

    To study the role of local production of complement proteins during the evolution of a naturally occurring immune complex disease, C3, C4, C2 and Factor B mRNA expression was assessed in several tissues of the inbred mouse strains NZB and (NZB x W) F1 hybrid. In the NZB/W F1 hybrid strain, coincident with the development of glomerulonephritis a marked increase in kidney C3 and C4 mRNA was observed; Factor B mRNA, which is expressed as a doublet in kidney and intestine, showed an increase in expression of the smaller transcript. This alteration of kidney C3, C4 and Factor B mRNA is identical to that noted in association with lupus nephritis in the MRL lpr/lpr strain and following in vivo administration of endotoxin to the BALB/c strain. The development of systemic lupus erythematosis (SLE) in the NZB/W F1 was not associated with a marked change in hepatic complement gene expression. These findings support the hypothesis that local production of complement may play a role in the pathogenesis of glomerulonephritis and other tissue injury in SLE. Images Figure 1 Figure 2 PMID:2228028

  17. Fentanyl effects on breath generation in C57BL/6J and A/J mouse strains.

    PubMed

    Fechtner, Linnea; El Ali, Mazen; Sattar, Abdus; Moore, Michael; Strohl, Kingman P

    2015-08-15

    We examined the effect of fentanyl on chemoresponsiveness in mouse strains divergent in the expression of spontaneous and post-hypoxic pauses. Frequency and tidal volume were recorded with plethysmography in A/J and C57BL/6J (B6) male mice. Mice selected at random received an intraperitoneal (IP) injection of either saline, low dose fentanyl (LDF = 0.04 mg/kg), or high dose fentanyl (HDF = 0.4 mg/kg) under hypoxia (8% O2) or hyperoxia (100%O2). LDF produced a decrease in frequency during hypoxia in B6, but not A/J, mice. HDF significantly decreased frequency and tidal volume in both strains under hypoxia and hyperoxia (p<0.01); naloxone, an opioid antagonist, reversed this response. The acute administration of fentanyl at any dose did not promote apneas in strains of mice exhibiting regular or irregular respiratory patterns. However, higher doses depressed respiratory frequency in both strains. The B6 mice responded with a depressive response to hypoxia that did not recover with reoxygenation, but did recover with time or naloxone.

  18. Different rankings of inbred mouse strains on the Morris maze and a refined 4-arm water escape task.

    PubMed

    Wahlsten, Douglas; Cooper, Sean F; Crabbe, John C

    2005-11-30

    The submerged platform or Morris water escape task is widely used to study genetic variation in spatial learning and memory, but interpretation is sometimes difficult because of wall hugging, jumping off the platform, floating or non-spatial swim strategies. We modified the task by introducing four wide arms into the circular tank and adding features that reduced, eliminated, or compensated for several competing behaviors. Three versions of the 4-arm task were evaluated in detail, and the third version yielded good results for six of eight inbred strains. Furthermore, the 4-arm task could be scored adequately without computerized video tracking. Although performance on the 4-arm task was generally superior to the Morris maze, the extent of the improvement was strain dependent. Two strains with retinal degeneration (C3H/HeJ, FVB/NJ) performed poorly on both the Morris and 4-arm mazes, whereas C57BL/6J and DBA/2J did well on both mazes. A/J performed poorly on the Morris task but became very proficient on the 4-arm maze, despite its strong tendency to hug the walls of the tank. The BALB/cByJ strain, on the other hand, exhibited the best probe trial performance on the Morris maze but was very slow in acquiring the 4-arm task. We conclude that no single task can reveal the full richness of spatially guided behavior in a wide range of mouse genotypes. PMID:16191444

  19. Brucella pinnipedialis hooded seal (Cystophora cristata) strain in the mouse model with concurrent exposure to PCB 153.

    PubMed

    Nymo, Ingebjørg H; das Neves, Carlos G; Tryland, Morten; Bårdsen, Bård-Jørgen; Santos, Renato Lima; Turchetti, Andreia Pereira; Janczak, Andrew M; Djønne, Berit; Lie, Elisabeth; Berg, Vidar; Godfroid, Jacques

    2014-05-01

    Brucellosis, a worldwide zoonosis, is linked to reproductive problems in primary hosts. A high proportion of Brucella-positive hooded seals (Cystophora cristata) have been detected in the declined Northeast Atlantic stock. High concentrations of polychlorinated biphenyls (PCBs) have also been discovered in top predators in the Arctic, including the hooded seal, PCB 153 being most abundant. The aim of this study was to assess the pathogenicity of Brucella pinnipedialis hooded seal strain in the mouse model and to evaluate the outcome of Brucella spp. infection after exposure of mice to PCB 153. BALB/c mice were infected with B. pinnipedialis hooded seal strain or Brucella suis 1330, and half from each group was exposed to PCB 153 through the diet. B. pinnipedialis showed a reduced pathogenicity in the mouse model as compared to B. suis 1330. Exposure to PCB 153 affected neither the immunological parameters, nor the outcome of the infection. Altogether this indicates that it is unlikely that B. pinnipedialis contribute to the decline of hooded seals in the Northeast Atlantic.

  20. Brucella pinnipedialis hooded seal (Cystophora cristata) strain in the mouse model with concurrent exposure to PCB 153.

    PubMed

    Nymo, Ingebjørg H; das Neves, Carlos G; Tryland, Morten; Bårdsen, Bård-Jørgen; Santos, Renato Lima; Turchetti, Andreia Pereira; Janczak, Andrew M; Djønne, Berit; Lie, Elisabeth; Berg, Vidar; Godfroid, Jacques

    2014-05-01

    Brucellosis, a worldwide zoonosis, is linked to reproductive problems in primary hosts. A high proportion of Brucella-positive hooded seals (Cystophora cristata) have been detected in the declined Northeast Atlantic stock. High concentrations of polychlorinated biphenyls (PCBs) have also been discovered in top predators in the Arctic, including the hooded seal, PCB 153 being most abundant. The aim of this study was to assess the pathogenicity of Brucella pinnipedialis hooded seal strain in the mouse model and to evaluate the outcome of Brucella spp. infection after exposure of mice to PCB 153. BALB/c mice were infected with B. pinnipedialis hooded seal strain or Brucella suis 1330, and half from each group was exposed to PCB 153 through the diet. B. pinnipedialis showed a reduced pathogenicity in the mouse model as compared to B. suis 1330. Exposure to PCB 153 affected neither the immunological parameters, nor the outcome of the infection. Altogether this indicates that it is unlikely that B. pinnipedialis contribute to the decline of hooded seals in the Northeast Atlantic. PMID:24534631

  1. In vivo-selected mutations in methyl-directed mismatch repair suppress the virulence attenuation of Salmonella dam mutant strains following intraperitoneal, but not oral, infection of naïve mice.

    PubMed

    Heithoff, Douglas M; Badie, Golnaz; Julio, Steven M; Enioutina, Elena Y; Daynes, Raymond A; Sinsheimer, Robert L; Mahan, Michael J

    2007-07-01

    Salmonella enterica serovar Typhimurium that lacks the DNA adenine methylase (Dam) ectopically expresses multiple genes that are preferentially expressed during infection, is attenuated for virulence, and confers heightened immunity in vaccinated hosts. The safety of dam mutant Salmonella vaccines was evaluated by screening within infected mice for isolates that have an increased capacity to cause disease relative to the attenuated parental strain. Since dam mutant strains are sensitive to the DNA base analog 2-aminopurine (2-AP), we screened for 2-AP-resistant (2-AP(r)) isolates in systemic tissues of mice infected with dam mutant Salmonella. Such 2-AP(r) derivatives were isolated following intraperitoneal but not oral administration and were shown to be competent for infectivity via intraperitoneal but not oral infection of naïve mice. These 2-AP(r) derivatives were deficient in methyl-directed mismatch repair and were resistant to nitric oxide, yet they retained the bile-sensitive phenotype of the parental dam mutant strain. Additionally, introduction of a mutH null mutation into dam mutant cells suppressed the inherent defects in intraperitoneal infectivity and nitric oxide resistance, as well as overexpression of SpvB, an actin cytotoxin required for Salmonella systemic survival. These data suggest that restoration of intraperitoneal virulence of dam mutant strains is associated with deficiencies in methyl-directed mismatch repair that correlate with the production of systemically related virulence functions.

  2. SKHIN/Sprd, a new genetically defined inbred hairless mouse strain for UV-induced skin carcinogenesis studies.

    PubMed

    Perez, Carlos; Parker-Thornburg, Jan; Mikulec, Carol; Kusewitt, Donna F; Fischer, Susan M; Digiovanni, John; Conti, Claudio J; Benavides, Fernando

    2012-03-01

    Strains of mice vary in their susceptibility to ultra-violet (UV) radiation-induced skin tumors. Some strains of hairless mice (homozygous for the spontaneous Hr(hr) mutation) are particularly susceptible to these tumors. The skin tumors that develop in hairless mice resemble, both at the morphologic and molecular levels, UV-induced squamous cell carcinomas (SCC) and their precursors in human. The most commonly employed hairless mice belong to the SKH1 stock. However, these mice are outbred and their genetic background is not characterized, which makes them a poor model for genetic studies. We have developed a new inbred strain from outbred SKH1 mice that we named SKHIN/Sprd (now at generation F31). In order to characterize the genetic background of this new strain, we genotyped a cohort of mice at F30 with 92 microsatellites and 140 single nucleotide polymorphisms (SNP) evenly distributed throughout the mouse genome. We also exposed SKHIN/Sprd mice to chronic UV irradiation and showed that they are as susceptible to UV-induced skin carcinogenesis as outbred SKH1 mice. In addition, we proved that, albeit with low efficiency, inbred SKHIN/Sprd mice are suitable for transgenic production by classical pronuclear microinjection. This new inbred strain will be useful for the development of transgenic and congenic strains on a hairless inbred background as well as the establishment of syngeneic tumor cell lines. These new tools can potentially help elucidate a number of features of the cutaneous response to UV irradiation in humans, including the effect of genetic background and modifier genes.

  3. Protective effects of recombinant staphylococcal enterotoxin type C mutant vaccine against experimental bovine infection by a strain of Staphylococcus aureus isolated from subclinical mastitis in dairy cattle.

    PubMed

    Chang, Byoung Sun; Moon, Jin San; Kang, Hyun-Mi; Kim, Young-In; Lee, Hong-Kyun; Kim, Jong-Duk; Lee, Byung-Saeng; Koo, Hye Cheong; Park, Yong Ho

    2008-04-16

    Staphylococcus aureus is one of the main etiological agents of bovine mastitis; however, antibiotics that are effective against bovine strains of S. aureus are not currently available. Staphylococcal enterotoxin type C (SEC), a superantigen, is the enterotoxin most frequently expressed by bovine strains of S. aureus and one of immunogenic determinants. The purpose of this study was to evaluate the protective effectiveness of recombinant SEC mutant vaccine (MastaVactrade mark) against experimentally induced bovine infection. Three representative SEC secreting strains were selected from 9 candidate isolates that showed various intensities of pathogenicity on mice and inoculated into 5 lactating dairy cattle at a concentration of 50-5.0x10(8) CFU per quarter. The optimal experimental bovine subclinical mastitis model was produced by inoculation with 50 CFU of S. aureus 409 per quarter, a level which was not lethal to mice. After the experimental model was determined, other 3 cattle were intramuscularly administered three doses of vaccine at day 0, at 2 wks and at 6 wks. Nine quarters of 3 vaccinated cattle and 8 quarters of 3 control cattle were then challenged with S. aureus 409. An SEC-specific ELISA test conducted at 4 wks post-immunization confirmed the presence of a high antibody titer against SEC in all vaccinated cattle. The somatic cell counts from the vaccinated group remained relatively low, whereas those of control group increased significantly after challenge with S. aureus. After challenge, S. aureus was not isolated from any cattle in the vaccinated group, whereas it was isolated from 75% of the cattle in the control group. These results indicate that recombinant SEC mutant vaccine had a protective effect against S. aureus intramammary infection in lactating cattle.

  4. Infection with street strain rabies virus induces modulation of the microRNA profile of the mouse brain

    PubMed Central

    2012-01-01

    Background Rabies virus (RABV) causes a fatal infection of the central nervous systems (CNS) of warm-blooded animals. Once the clinical symptoms develop, rabies is almost invariably fatal. The mechanism of RABV pathogenesis remains poorly understood. Recent studies have shown that microRNA (miRNA) plays an important role in the pathogenesis of viral infections. Our recent findings have revealed that infection with laboratory-fixed rabies virus strain can induce modulation of the microRNA profile of mouse brains. However, no previous report has evaluated the miRNA expression profile of mouse brains infected with RABV street strain. Results The results of microarray analysis show that miRNA expression becomes modulated in the brains of mice infected with street RABV. Quantitative real-time PCR assay of the differentially expressed miRNAs confirmed the results of microarray assay. Functional analysis showed the differentially expressed miRNAs to be involved in many immune-related signaling pathways, such as the Jak-STAT signaling pathway, the MAPK signaling pathway, cytokine-cytokine receptor interactions, and Fc gamma R-mediated phagocytosis. The predicted expression levels of the target genes of these modulated miRNAs were found to be correlated with gene expression as measured by DNA microarray and qRT-PCR. Conclusion RABV causes significant changes in the miRNA expression profiles of infected mouse brains. Predicted target genes of the differentially expression miRNAs are associated with host immune response, which may provide important information for investigation of RABV pathogenesis and therapeutic method. PMID:22882874

  5. Draft Genome Sequences of Five Novel Polyketide Synthetase-Containing Mouse Escherichia coli Strains

    PubMed Central

    Mannion, Anthony; Shen, Zeli; Feng, Yan; Garcia, Alexis

    2016-01-01

    We report herein the draft genomes of five novel Escherichia coli strains isolated from surveillance and experimental mice housed at MIT and the Whitehead Institute and describe their genomic characteristics in context with the polyketide synthetase (PKS)-containing pathogenic E. coli strains NC101, IHE3034, and A192PP.

  6. Body Composition QTLs Identified in Intercross Populations Are Reproducible in Consomic Mouse Strains

    PubMed Central

    Lin, Cailu; Fesi, Brad D.; Marquis, Michael; Bosak, Natalia P.; Theodorides, Maria L.; Avigdor, Mauricio; McDaniel, Amanda H.; Duke, Fujiko F.; Lysenko, Anna; Khoshnevisan, Amin; Gantick, Brian R.; Arayata, Charles J.; Nelson, Theodore M.; Bachmanov, Alexander A.; Reed, Danielle R.

    2015-01-01

    Genetic variation contributes to individual differences in obesity, but defining the exact relationships between naturally occurring genotypes and their effects on fatness remains elusive. As a step toward positional cloning of previously identified body composition quantitative trait loci (QTLs) from F2 crosses of mice from the C57BL/6ByJ and 129P3/J inbred strains, we sought to recapture them on a homogenous genetic background of consomic (chromosome substitution) strains. Male and female mice from reciprocal consomic strains originating from the C57BL/6ByJ and 129P3/J strains were bred and measured for body weight, length, and adiposity. Chromosomes 2, 7, and 9 were selected for substitution because previous F2 intercross studies revealed body composition QTLs on these chromosomes. We considered a QTL confirmed if one or both sexes of one or both reciprocal consomic strains differed significantly from the host strain in the expected direction after correction for multiple testing. Using these criteria, we confirmed two of two QTLs for body weight (Bwq5-6), three of three QTLs for body length (Bdln3-5), and three of three QTLs for adiposity (Adip20, Adip26 and Adip27). Overall, this study shows that despite the biological complexity of body size and composition, most QTLs for these traits are preserved when transferred to consomic strains; in addition, studying reciprocal consomic strains of both sexes is useful in assessing the robustness of a particular QTL. PMID:26551037

  7. Hidden in plain sight: spike-wave discharges in mouse inbred strains.

    PubMed

    Letts, V A; Beyer, B J; Frankel, W N

    2014-07-01

    Twenty-seven inbred strains of mice were tested for spike-wave discharge (SWD) activity by video-electroencephalographic recordings over a 24-h recording period. Eight strains had reproducible, frequent SWDs, including five strains (C57BLKS/J, CBA/J, DBA/1J, NOR/LtJ, SM/J) previously undiagnosed for this distinctive phenotype. Eighteen other strains exhibited no such activity. Spike-wave discharges usually occurred while the subject was motionless, and in a significant number of annotated instances coincided with an arrest of the subject's relatively unrestrained locomotor activity, which resumed immediately after the discharge ended. In all five new strains, SWDs were suppressed by ethosuximide administration. From the genealogy of inbred strains, we suggest that two ancestors, A and DBA, transmitted genotypes required for SWD in all positive strains. Together these strains with SWDs provide new opportunities to understand the genetic core susceptibility of this distinctive electroencephalographic activity and to explore its relationship to absence epilepsy, a human disorder for which few genes are known. PMID:24861780

  8. The effect of mouse strain on herpes simplex virus type 1 (HSV-1) infection of the central nervous system (CNS)

    PubMed Central

    2012-01-01

    Background Mice infected with HSV-1 can develop lethal encephalitis or virus induced CNS demyelination. Multiple factors affect outcome including route of infection, virus and mouse strain. When infected with a sub-lethal dose of HSV-1 strain 2 via the oral mucosa, susceptible SJL/J, A/J, and PL/J mice develop demyelinating lesions throughout the brain. In contrast, lesions are restricted to the brainstem (BST) in moderately resistant BALB/c mice and are absent in resistant BL/6 mice. The reasons for the strain differences are unknown. Methods In this study, we combine histology, immunohistochemistry, and in-situ hybridization to investigate the relationship between virus and the development of lesions during the early stage (< 24 days PI) of demyelination in different strains of mice. Results Initially, viral DNA and antigen positive cells appear sequentially in non-contiguous areas throughout the brains of BALB/c, SJL/J, A/J, and PL/J mice but are restricted to an area of the BST of BL/6 mice. In SJL/J, A/J, and PL/J mice, this is followed by the development of 'focal' areas of virus infected neuronal and non-neuronal cells throughout the brain. The 'focal' areas follow a hierarchical order and co-localize with developing demyelinating lesions. When antigen is cleared, viral DNA positive cells can remain in areas of demyelination; consistent with a latent infection. In contrast, 'focal' areas are restricted to the BST of BALB/c mice and do not occur in BL/6 mice. Conclusions The results of this study indicate that susceptible mouse strains, infected with HSV-1 via the oral mucosa, develop CNS demyelination during the first 24 days PI in several stages. These include: the initial spread of virus and infection of cells in non-contiguous areas throughout the brain, the development of 'focal' areas of virus infected neuronal and non-neuronal cells, the co-localization of 'focal' areas with developing demyelinating lesions, and latent infection in a number of the

  9. The mouse resources at the RIKEN BioResource center.

    PubMed

    Yoshiki, Atsushi; Ike, Fumio; Mekada, Kazuyuki; Kitaura, Yasuyuki; Nakata, Hatsumi; Hiraiwa, Noriko; Mochida, Keiji; Ijuin, Maiko; Kadota, Masayo; Murakami, Ayumi; Ogura, Atsuo; Abe, Kuniya; Moriwaki, Kazuo; Obata, Yuichi

    2009-04-01

    Mice are one of the most important model organisms for studying biological phenomena and diseases processes in life sciences. The biomedical research community has succeeded in launching large scale strategic knockout mouse projects around the world. RIKEN BRC, a comprehensive government funded biological resource center was established in 2001. RIKEN BRC has been acting as the core facility for the mouse resources of the National BioResource Project (NBRP) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan since 2002. RIKEN BRC is a founding member of the Federation of International Mouse Resources (FIMRe) together with the Jackson Laboratory, the European Mouse Mutant Archive, and other centers, and has participated in the International Mouse Strain Resource (IMSR) to distribute mouse strains worldwide. With the support of the scientific community, RIKEN BRC has collected over 3,800 strains including inbred, transgenic, knockout, wild-derived, and ENU-induced mutant strains. Excellent mouse models for human diseases and gene functions from academic organizations and private companies are distributed through RIKEN BRC. To meet research and social needs, our mice will be rederived to a specific pathogen-free state, strictly monitored for their health, and accurately tested for their genetic modifications and backgrounds. Users can easily access our mouse resources through the internet and obtain the mouse strains for a minimal fee. Cryopreservation of embryos and sperm is used for efficient preservation of the increasing number of mouse resources. RIKEN BRC collaborates with FIMRe members to support Japanese scientists in the use of valuable mouse resources from around the world.

  10. Mouse hepatitis virus type 4 (JHM strains). induced fatal central nervous system disease. I. genetic control and murine neuron as the susceptible site of disease

    PubMed Central

    1981-01-01

    Mouse hepatitis virus (JHM strain) type 4 induces acute encephalitis followed by death in many strains of laboratory mice. Immunohistochemical study in vivo and analysis of mouse neuronal cells in vitro both indicate that the target cells in this infection is the neuron. Further, examination of several inbred mouse strains and neuronal cells from them shows that disease expression is controlled by a single autosomal gene action at the level of the neuronal cell. Susceptibility is dominant but not H-2 linked. However, cultured neuronal cells and macrophages from SJL/J mice, which are resistant to this infection, fail to make significant amounts of infectious virus after an appropriate viral inoculation. Apparently the defect is not at the level of the virus-cell receptor, because these cells, in part, express viral antigens. PMID:6265583

  11. Food intake, water intake, and drinking spout side preference of 28 mouse strains.

    PubMed

    Bachmanov, Alexander A; Reed, Danielle R; Beauchamp, Gary K; Tordoff, Michael G

    2002-11-01

    Male mice from 28 inbred strains (129P3/J, A/J, AKR/J, BALB/cByJ, BUB/BnJ, C3H/HeJ, C57BL/6J, C57L/J, CAST/Ei, CBA/J, CE/J, DBA/2J, FVB/NJ, I/LnJ, KK/H1J, LP/J, NOD/LtJ, NZB/B1NJ, P/J, PL/J, RBF/DnJ, RF/J, RIIIS/J, SEA/GnJ, SJL/J, SM/J, SPRET/Ei, and SWR/J) were fed chow and had access to two water bottles. Body weight, food intake, water intake, and drinking spout side preference were measured. There were large strain differences in all the measures collected, with at least a two-fold difference between strains with the lowest and the highest trait values. Estimates of heritability ranged from 0.36 (spout side preference) to 0.87 (body weight). Body weight, food intake, and water intake were interrelated among the strains, although substantial strain variation in food and water intakes independent from body weight was present. The strain differences described here provide useful information for designing mutagenesis screens and choosing strains for genetic mapping studies.

  12. Thermoregulatory responses of two mouse Mus musculus strains selectively bred for high and low food intake.

    PubMed

    Selman, C; Korhonen, T K; Bünger, L; Hill, W G; Speakman, J R

    2001-11-01

    We examined the thermoregulatory responses of male and female mice Mus musculus that had been divergently selected on voluntary food intake, corrected for body mass, to produce a high-intake and a low-intake strain. Resting metabolic rate was determined by indirect calorimetry (at 30 degrees C, 25 degrees C, 15 degrees C and 5 degrees C). Body temperature responses were measured in a separate group of mice in a parallel protocol. High-intake mice had significantly elevated body masses compared to low-intake mice in both sexes. Lower critical temperature in both strains appeared to be around 28 degrees C. At 30 degrees C there was a significant strain effect on resting metabolic rate, with high strain mice having greater metabolism than low strain mice. Sex and body mass were not significant main effects on resting metabolic rate and there were no significant interactions. Body temperature measured at 30 degrees C, 25 degrees C, 15 degrees C and 5 degrees C differed significantly between sexes (females higher) and there was a significant sexxbody mass interaction effect, but there was no difference between strains. Thermal conductance was significantly related to strain and sex, mice from the high strain and males having greater thermal conductances than mice from the low strain and females. Artificial selection has resulted in high-intake mice having greater body masses and greater thermal conductances, which together account for up to 45% of the elevated daily energy demands that underpin the increase in food intake. The greater levels of food intake were also associated with higher resting metabolic rates at 30 degrees C.

  13. 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

  14. Live attenuated mutants of Francisella tularensis protect rabbits against aerosol challenge with a virulent type A strain.

    PubMed

    Reed, Douglas S; Smith, Le'kneitah P; Cole, Kelly Stefano; Santiago, Araceli E; Mann, Barbara J; Barry, Eileen M

    2014-05-01

    Francisella tularensis, a Gram-negative bacterium, is the causative agent of tularemia. No licensed vaccine is currently available for protection against tularemia, although an attenuated strain, dubbed the live vaccine strain (LVS), is given to at-risk laboratory personnel as an investigational new drug (IND). In an effort to develop a vaccine that offers better protection, recombinant attenuated derivatives of a virulent type A strain, SCHU S4, were evaluated in New Zealand White (NZW) rabbits. Rabbits vaccinated via scarification with the three attenuated derivatives (SCHU S4 ΔguaBA, ΔaroD, and ΔfipB strains) or with LVS developed a mild fever, but no weight loss was detected. Twenty-one days after vaccination, all vaccinated rabbits were seropositive for IgG to F. tularensis lipopolysaccharide (LPS). Thirty days after vaccination, all rabbits were challenged with aerosolized SCHU S4 at doses ranging from 50 to 500 50% lethal doses (LD50). All rabbits developed fevers and weight loss after challenge, but the severity was greater for mock-vaccinated rabbits. The ΔguaBA and ΔaroD SCHU S4 derivatives provided partial protection against death (27 to 36%) and a prolonged time to death compared to results for the mock-vaccinated group. In contrast, LVS and the ΔfipB strain both prolonged the time to death, but there were no survivors from the challenge. This is the first demonstration of vaccine efficacy against aerosol challenge with virulent type A F. tularensis in a species other than a rodent since the original work with LVS in the 1960s. The ΔguaBA and ΔaroD SCHU S4 derivatives warrant further evaluation and consideration as potential vaccines for tularemia and for identification of immunological correlates of protection.

  15. Unusual ∆7,12,19 C35:3 Alkenone Produced by the Mutant Emiliania huxleyi strain CCMP2758 in Culture

    NASA Astrophysics Data System (ADS)

    Zheng, Y.; Huang, Y.; Zhang, Y.; Dillon, J. T.

    2015-12-01

    Alkenones with chain length ranging from C37 to C40 are highly specific biomarkers for certain haptophyte algae in ocean and lake sediments and have been widely used for paleoclimate studies. Short chain alkenones (e.g., C35 and C36) have been found in environmental and culture samples but the origin and structures of these compounds are not fully understood. The benchmark marine alkenone producer, Emiliania huxleyi CCMP2758 strain (the mutant of strain CCMP1742, NEPCC55a) was reported to make 35:2 alkenone when cultured at 15 °C (Prahl et al., 2006). Here we show, when this strain is cultured at lower temperatures (e.g., 4°C), CCMP2758 produces large amount of 35:3 alkenone with unusual double bond positions of ∆7,12,19. We determined the double bond positions of the C35:3 methyl ketonee based on GC-MS analysis of cyclobutylimine derivatives and dimethyl disulfide derivatives respectively, and provide the first temperature calibrations based on the unsaturation ratios of C35 alkenones. Previous studies have found 35:2 alkenone with three methylene interruption in the Black Sea sediment, but it is the first time that an alkenone with a mixed three and five methylene interruption is found. The discovery of short chain alkenones with unusual double bond positions may shed new light to alkenone biosynthesis.

  16. Aeromonas Caviae Strain Induces Th1 Cytokine Response in Mouse Intestinal Tract

    EPA Science Inventory

    Aeromonas caviae has been associated with human gastrointestinal disease. Strains of this species typically lack virulence factors (VFs) such as enterotoxins and hemolysins that are produced by other human pathogens of the Aeromonas genus. Microarray profiling of murine small i...

  17. COMPARISON OF SYSTEMIC AND MUCOSAL ROUTES OF SENSITIZATION TO OVALBUMIN ANTIGEN IN THREE MOUSE STRAINS

    EPA Science Inventory

    Several studies have shown strain differences in allergic lung responses following ovalbumin (OVA) antigen sensitization and challenge. The purpose of this study was to determine whether these differences were maintained between systemic and mucosal sensitization routes, and to ...

  18. The mode of cauliflower mosaic virus propagation in the plant allows rapid amplification of viable mutant strains.

    PubMed

    Riederer, M A; Grimsley, N H; Hohn, B; Jiricny, J

    1992-06-01

    We inoculated the leaves of turnip plants (Brassica campestris spp. rapa cv. Just Right) with two cauliflower mosaic viruses (CaMVs) with different small mutations in a dispensable region of the viral genome, and followed the spread of the virus infection through the plant. Surprisingly, analysis of viral DNA in single primary chlorotic lesions revealed the presence of both mutants. In contrast, the secondary chlorotic lesions and systemically infected leaves contained virus molecules of either one or the other type only. Infection of plants with different ratios of the two reporter viruses showed that this ratio is not conserved during systemic virus spread. Infection with CaMV DNA in the form of heteroduplexes containing a single mismatched base pair, in which each strand carried a distinct diagnostic marker, provided us with evidence that the mismatch was subjected to a repair process in the host plant.

  19. 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.

  20. Resistance of novel mouse strains different in MHC class I and the NKC domain to the development of experimental tumors.

    PubMed

    Fišerová, Anna; Richter, Jan; Čapková, Katarína; Bieblová, Jana; Mikyšková, Romana; Reiniš, Milan; Indrová, Marie

    2016-08-01

    To elucidate the immunological mechanisms critical for tumor progression, we bred novel mouse strains, different in the NKC and H-2D domains. We used inbreeding to generate hybrids of Balb/c and C57BL/6 of stable H-2Db+d-NK1.1neg and H-2Db-d+NK1.1high phenotypes. We analyzed the growth of three established MHC class I-deficient tumor cell lines: TC-1/A9 tumor (HPV-associated) and B16F10 melanoma, both syngeneic to C57BL/6, and the MCB8 (3-methycholanthrene-induced tumor) syngeneic to Balb/c. Furthermore, we induced colorectal carcinoma by azoxymethane-DSS treatment to test the susceptibility to chemically-induced primary cancer. We found that the novel strains spontaneously regressed the tumor transplants syngeneic to both Balb/c (MCB8) and C57BL/6 (B16F10 and TC-1/A9) mice. The H2-Db+d-NK1.1neg, but not the H2-Db-d+NK1.1high strain was also highly resistant to chemically-induced colorectal cancer in comparison to the parental mice. The immune changes during TC-1/A9 cancer development involved an increase of the NK cell distribution in the peripheral blood and spleen along with higher expression of NKG2D activation antigen; this was in correlation with the time-dependent rise of cytotoxic activity in comparison to C57BL/6 mice. The TC-1/A9 cancer regression was accompanied by higher proportion of B cells in the spleen and B220+/CD86+ activated antigen-presenting B cells distributed in the lymphoid organs, as well as in the periphery. The changes in the T-cell population were represented mainly by the prevalence of T helper cells reflected by grown CD4/CD8 ratio, most prominent in the b+d-NK1.1neg strain. The results of the present study imply usefulness of the two novel mouse strains as an experimental model for further studies of tumor resistance mechanisms. PMID:27279019

  1. RIII S/J (H-2r). An inbred mouse strain with a massive deletion of T cell receptor V beta genes

    PubMed Central

    1989-01-01

    We have identified an inbred strain of mouse, RIII S/J (H-2r), that has the largest known deletion of the TCR V beta genes by screening with mAb and TCR V beta specific probes. Upon screening of PBL with mAb F23.1, which is specific for V beta 8 TCR, RIII S/J was found to be negative. On further screening with mAb KJ 23a, which is specific for V beta 17a TCR, RIII S/J was completely negative. We next tested RIII S/J with mAb 44-22-1, which is specific for V beta 6 TCR, and found it also to be negative. The (B10 X RIII)F1 mice showed a 50% expression of V beta 6 gene, indicating a genomic rather than a clonal deletion. mAb KJ25, detecting V beta 3, was positive in RIII S/J, denoting the downstream boundary for the deletion. Southern blot analysis of liver DNA using TCR V beta-specific probes confirmed the deletion of V beta 8 gene subfamily and V beta 5 gene subfamily, along with V beta 9, V beta 11, V beta 12, and V beta 13 genes similar to the known TCR V beta deletion mutants (SWR, SJL, C57L, and C57Br). In addition, RIII S/J is missing V beta 6, V beta 15, and V beta 17 genes. Our mapping of the deletion indicates that RIII S/J has lost approximately 130 kb of V beta chromosome and with it 13 V beta genes out of the known 21 V beta genes of the TCR. The deletion is marked by the presence of V beta 10 gene upstream and V beta 3 gene downstream. PMID:2525171

  2. Chromosomes 6 and 13 harbor genes that regulate pubertal timing in mouse chromosome substitution strains.

    PubMed

    Krewson, Thomas D; Supelak, Pamela J; Hill, Annie E; Singer, Jonathan B; Lander, Eric S; Nadeau, Joseph H; Palmert, Mark R

    2004-10-01

    Variation in the onset of puberty among inbred strains of mice suggests that quantitative trait loci (QTLs) affect neurological and hormonal aspects of sexual maturation. Taking a novel approach toward identifying factors that regulate the hypothalamic-pituitary-gonadal (HPG) axis, we evaluated pubertal timing [as assessed by vaginal opening (VO)] in two inbred strains of mice, A/J and C57BL/6J (B6), and in a panel of chromosome substitution strains (CSSs) generated from A/J and B6 mice. In each CSS, a single chromosome from A/J has been substituted in a homozygous fashion for the corresponding chromosome in B6, partitioning the A/J genome into 22 strains with a common host (B6) background. VO occurred significantly earlier in A/J compared with B6 mice. Although the majority of the CSSs assessed had a timing of VO that was similar to the progenitor B6 strain, CSSs for chromosomes 6 and 13 each displayed significantly earlier time of VO than B6 mice. F1 (B6 x CSS) mice for chromosomes 6 and 13 displayed phenotypes that were intermediate between the CSS and B6 strains, suggesting that the trait was inherited in a codominant manner. These findings demonstrate that chromosomes 6 and 13 harbor QTLs that control the timing of VO. Identification of the responsible genes may reveal factors that regulate the maturation of the HPG axis and determine the timing of puberty.

  3. Genetic control of mammalian meiotic recombination. I. Variation in exchange frequencies among males from inbred mouse strains.

    PubMed

    Koehler, Kara E; Cherry, Jonathan P; Lynn, Audrey; Hunt, Patricia A; Hassold, Terry J

    2002-09-01

    Genetic background effects on the frequency of meiotic recombination have long been suspected in mice but never demonstrated in a systematic manner, especially in inbred strains. We used a recently described immunostaining technique to assess meiotic exchange patterns in male mice. We found that among four different inbred strains--CAST/Ei, A/J, C57BL/6, and SPRET/Ei--the mean number of meiotic exchanges per cell and, thus, the recombination rates in these genetic backgrounds were significantly different. These frequencies ranged from a low of 21.5 exchanges in CAST/Ei to a high of 24.9 in SPRET/Ei. We also found that, as expected, these crossover events were nonrandomly distributed and displayed positive interference. However, we found no evidence for significant differences in the patterns of crossover positioning between strains with different exchange frequencies. From our observations of >10,000 autosomal synaptonemal complexes, we conclude that achiasmate bivalents arise in the male mouse at a frequency of 0.1%. Thus, special mechanisms that segregate achiasmate chromosomes are unlikely to be an important component of mammalian male meiosis.

  4. Interaction between the endocannabinoid and serotonergic system in the exhibition of head twitch response in four mouse strains.

    PubMed

    Ceci, Chiara; Proietti Onori, Martina; Macrì, Simone; Laviola, Giovanni

    2015-04-01

    More than 10 % of children during school years suffer from a transient tic disorder, and 1 % has a particular type of tic disorder known as Tourette syndrome. At present, there is no available treatment that can improve tics without considerable side effects. Recent evidence indicates that tetrahydrocannabinol (THC), the principal psychoactive component of cannabis, reduced in mice the head twitch responses, a tic pharmacologically induced by the selective serotonin 5-HT2 receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI). THC has some considerable side effects that render its use problematic. In this view, cyclohexyl-carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB597), an indirect cannabinoid agonist that enhances endogenous anandamide levels, can constitute a valid alternative to the use of direct CB1 receptor agonists. We investigated whether URB597 may reduce the exhibition of DOI-induced head twitch responses in mice. Moreover, to address whether the effects of URB597 on DOI-induced behavioral response constitute a general phenomenon, we evaluated four (ABH, C57BL/6N, SJL/J, CD-1) mouse strains. These strains have been selected in order to represent an ample spectrum of genetic background and phenotypic variation. Predictably, DOI induced consistent tic-like behaviors in all mice. While URB597 exerted slight sedation in C57BN/6L mice, this cannabinoid agonist remarkably mitigated the exhibition of DOI-induced head twitch in all strains. Present data may disclose novel avenues for the pharmacological treatment of tic disorders.

  5. Novel mutant human immunodeficiency virus type 1 strains with high degree of resistance to cynomolgus macaque TRIMCyp generated by random mutagenesis.

    PubMed

    Sultana, Tahmina; Nakayama, Emi E; Tobita, Satoshi; Yokoyama, Masaru; Seki, Yohei; Saito, Akatsuki; Nomaguchi, Masako; Adachi, Akio; Akari, Hirofumi; Sato, Hironori; Shioda, Tatsuo

    2016-04-01

    Old World monkey TRIM5α strongly suppresses human immunodeficiency virus type 1 (HIV-1) replication. A fusion protein comprising cynomolgus macaque (CM) TRIM5 and cyclophilin A (CM TRIMCyp) also potently suppresses HIV-1 replication. However, CM TRIMCyp fails to suppress a mutant HIV-1 that encodes a mutant capsid protein containing a SIVmac239-derived loop between α-helices 4 and 5 (L4/5). There are seven amino acid differences between L4/5 of HIV-1 and SIVmac239. Here, we investigated the minimum numbers of amino acid substitutions that would allow HIV-1 to evade CM TRIMCyp-mediated suppression. We performed random PCR mutagenesis to construct a library of HIV-1 variants containing mutations in L4/5, and then we recovered replication-competent viruses from CD4+ MT4 cells that expressed high levels of CM TRIMCyp. CM TRIMCyp-resistant viruses were obtained after three rounds of selection in MT4 cells expressing CM TRIMCyp and these were found to contain four amino acid substitutions (H87R, A88G, P90D and P93A) in L4/5. We then confirmed that these substitutions were sufficient to confer CM TRIMCyp resistance to HIV-1. In a separate experiment using a similar method, we obtained novel CM TRIM5α-resistant HIV-1 strains after six rounds of selection and rescue. Analysis of these mutants revealed that V86A and G116E mutations in the capsid region conferred partial resistance to CM TRIM5α without substantial fitness cost when propagated in MT4 cells expressing CM TRIM5α. These results confirmed and further extended the previous notion that CM TRIMCyp and CM TRIM5α recognize the HIV-1 capsid in different manners. PMID:26795727

  6. Tubulointerstitial Nephritis Due to a Mutant Polyomavirus BK Virus Strain, BKV(Cin), Causing End-Stage Renal Disease

    PubMed Central

    Smith, R. D.; Galla, J. H.; Skahan, K.; Anderson, P.; Linnemann, C. C.; Ault, G. S.; Ryschkewitsch, C. F.; Stoner, G. L.

    1998-01-01

    A renal biopsy from a 36-year-old man with AIDS showed a severe tubulointerstitial nephritis with intranuclear inclusions in epithelial cells. Electron microscopy revealed the characteristic findings of a polyomavirus (PyV) infection, and immunofluorescenc