Nuclear Expression of a Mitochondrial DNA Gene: Mitochondrial Targeting of Allotopically Expressed Mutant ATP6 in Transgenic Mice

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David A. Dunn

2012-01-01

Full Text Available Nuclear encoding of mitochondrial DNA transgenes followed by mitochondrial targeting of the expressed proteins (allotopic expression; AE represents a potentially powerful strategy for creating animal models of mtDNA disease. Mice were created that allotopically express either a mutant (A6M or wildtype (A6W mt-Atp6 transgene. Compared to non-transgenic controls, A6M mice displayed neuromuscular and motor deficiencies (wire hang, pole, and balance beam analyses; P0.05. This study illustrates a mouse model capable of circumventing in vivo mitochondrial mutations. Moreover, it provides evidence supporting AE as a tool for mtDNA disease research with implications in development of DNA-based therapeutics.

Epididymis response partly compensates for spermatozoa oxidative defects in snGPx4 and GPx5 double mutant mice.

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Anaïs Noblanc

Full Text Available We report here that spermatozoa of mice lacking both the sperm nucleus glutathione peroxidase 4 (snGPx4 and the epididymal glutathione peroxidase 5 (GPx5 activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H(2O(2-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice.

Anti-H-Y responses of H-2b mutant mice.

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Simpson, E; Gordon, R D; Chandler, P R; Bailey, D

1978-10-01

Two strains of H-2b mutant mice, H-2ba and H-2bf, in which the mutational event took place at H-2K, make anti-H-Y cytotoxic T cell responses which are H-2-restricted, Db-associated and indistinguishable in target cell specificity from those of H-2b mice. Thus, alteration of the H-2K molecule affects neither the Ir gene controlling the response, nor the associative antigen. On the other hand, one H-2Db mutant strain, H-2bo, although it makes a good anti-H-Y cytotoxic response, shows target cell specificity restricted to its own Dbo antigen(s), and neither H-2b, H-2ba or H-2bf anti-H-Y cytotoxic cells kill H-2bo male target cells. Thus, the alteration of the H-2Db molecule does not affect the Ir gene of H-2b mice, but it does alter the H-2Db-associative antigen.

Sensorimotor learning in Dab1(scm) (scrambler) mutant mice.

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Lalonde, R; Strazielle, C

2011-04-15

Homozygous Dab1(scm) mouse mutants with cell ectopias in cerebellar cortex and neocortex were compared with non-ataxic controls on two tests of motor coordination: rotorod and grid climbing. Even at the minimal speed of 4 rpm and unlike controls, none of the Dab1(scm) mutants reached criterion on the constant speed rotorod. In contrast, Dab1(scm) mutants improved their performances on the vertical grid over the course of the same number of trials. Thus, despite massive cerebellar degeneration, sensorimotor learning for equilibrium is still possible, indicating the potential usefulness of the grid-climbing test in determining residual functions in mice with massive cerebellar damage. Copyright © 2010. Published by Elsevier B.V.

Immunohistochemical studies of lens crystallins in the dysgenetic lens (dyl) mutant mice

NARCIS (Netherlands)

Brahma, S.K.; Sanyal, S.

1984-01-01

The lens in the dyl mutant mice shows a persistent lens-ectodermal connection as well as degeneration and extrusion of lens materials after the initial differentiation of the fibres. Immunohistochemical investigation of the ontogeny of the lens crystallins in this developing mutant lens has been

GH and IGF1: roles in energy metabolism of long-living GH mutant mice.

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Brown-Borg, Holly M; Bartke, Andrzej

2012-06-01

Of the multiple theories to explain exceptional longevity, the most robust of these has centered on the reduction of three anabolic protein hormones, growth hormone (GH), insulin-like growth factor, and insulin. GH mutant mice live 50% longer and exhibit significant differences in several aspects of energy metabolism as compared with wild-type mice. Mitochondrial metabolism is upregulated in the absence of GH, whereas in GH transgenic mice and dwarf mice treated with GH, multiple aspects of these pathways are suppressed. Core body temperature is markedly lower in dwarf mice, yet whole-body metabolism, as measured by indirect calorimetry, is surprisingly higher in Ames dwarf and Ghr-/- mice compared with normal controls. Elevated adiponectin, a key antiinflammatory cytokine, is also very likely to contribute to longevity in these mice. Thus, several important components related to energy metabolism are altered in GH mutant mice, and these differences are likely critical in aging processes and life-span extension.

Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis.

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Marty, Caroline; Pecquet, Christian; Nivarthi, Harini; El-Khoury, Mira; Chachoua, Ilyas; Tulliez, Micheline; Villeval, Jean-Luc; Raslova, Hana; Kralovics, Robert; Constantinescu, Stefan N; Plo, Isabelle; Vainchenker, William

2016-03-10

Frameshift mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and myelofibrosis patients. To address the contribution of the CALR mutants to the pathogenesis of myeloproliferative neoplasms, we engrafted lethally irradiated recipient mice with bone marrow cells transduced with retroviruses expressing these mutants. In contrast to wild-type CALR, CALRdel52 (type I) and, to a lesser extent, CALRins5 (type II) induced thrombocytosis due to a megakaryocyte (MK) hyperplasia. Disease was transplantable into secondary recipients. After 6 months, CALRdel52-, in contrast to rare CALRins5-, transduced mice developed a myelofibrosis associated with a splenomegaly and a marked osteosclerosis. Monitoring of virus-transduced populations indicated that CALRdel52 leads to expansion at earlier stages of hematopoiesis than CALRins5. However, both mutants still specifically amplified the MK lineage and platelet production. Moreover, a mutant deleted of the entire exon 9 (CALRdelex9) did not induce a disease, suggesting that the oncogenic property of CALR mutants was related to the new C-terminus peptide. To understand how the CALR mutants target the MK lineage, we used a cell-line model and demonstrated that the CALR mutants, but not CALRdelex9, specifically activate the thrombopoietin (TPO) receptor (MPL) to induce constitutive activation of Janus kinase 2 and signal transducer and activator of transcription 5/3/1. We confirmed in c-mpl- and tpo-deficient mice that expression of Mpl, but not of Tpo, was essential for the CALR mutants to induce thrombocytosis in vivo, although Tpo contributes to disease penetrance. Thus, CALR mutants are sufficient to induce thrombocytosis through MPL activation. © 2016 by The American Society of Hematology.

mPeriod2 Brdm1 and other single Period mutant mice have normal food anticipatory activity.

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Pendergast, Julie S; Wendroth, Robert H; Stenner, Rio C; Keil, Charles D; Yamazaki, Shin

2017-11-14

Animals anticipate the timing of food availability via the food-entrainable oscillator (FEO). The anatomical location and timekeeping mechanism of the FEO are unknown. Several studies showed the circadian gene, Period 2, is critical for FEO timekeeping. However, other studies concluded that canonical circadian genes are not essential for FEO timekeeping. In this study, we re-examined the effects of the Per2 Brdm1 mutation on food entrainment using methods that have revealed robust food anticipatory activity in other mutant lines. We examined food anticipatory activity, which is the output of the FEO, in single Period mutant mice. Single Per1, Per2, and Per3 mutant mice had robust food anticipatory activity during restricted feeding. In addition, we found that two different lines of Per2 mutant mice (ldc and Brdm1) anticipated restricted food availability. To determine if FEO timekeeping persisted in the absence of the food cue, we assessed activity during fasting. Food anticipatory (wheel-running) activity in all Period mutant mice was also robust during food deprivation. Together, our studies demonstrate that the Period genes are not necessary for the expression of food anticipatory activity.

Primary coenzyme Q deficiency in Pdss2 mutant mice causes isolated renal disease.

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Min Peng

2008-04-01

Full Text Available Coenzyme Q (CoQ is an essential electron carrier in the respiratory chain whose deficiency has been implicated in a wide variety of human mitochondrial disease manifestations. Its multi-step biosynthesis involves production of polyisoprenoid diphosphate in a reaction that requires the enzymes be encoded by PDSS1 and PDSS2. Homozygous mutations in either of these genes, in humans, lead to severe neuromuscular disease, with nephrotic syndrome seen in PDSS2 deficiency. We now show that a presumed autoimmune kidney disease in mice with the missense Pdss2(kd/kd genotype can be attributed to a mitochondrial CoQ biosynthetic defect. Levels of CoQ9 and CoQ10 in kidney homogenates from B6.Pdss2(kd/kd mutants were significantly lower than those in B6 control mice. Disease manifestations originate specifically in glomerular podocytes, as renal disease is seen in Podocin/cre,Pdss2(loxP/loxP knockout mice but not in conditional knockouts targeted to renal tubular epithelium, monocytes, or hepatocytes. Liver-conditional B6.Alb/cre,Pdss2(loxP/loxP knockout mice have no overt disease despite demonstration that their livers have undetectable CoQ9 levels, impaired respiratory capacity, and significantly altered intermediary metabolism as evidenced by transcriptional profiling and amino acid quantitation. These data suggest that disease manifestations of CoQ deficiency relate to tissue-specific respiratory capacity thresholds, with glomerular podocytes displaying the greatest sensitivity to Pdss2 impairment.

Mutation-related differences in exploratory, spatial and depressive-like behavior in pcd and Lurcher cerebellar mutant mice

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Jan eTuma

2015-05-01

Full Text Available The cerebellum is not only essential for motor coordination but is also involved in cognitive and affective processes. These functions of the cerebellum and mechanisms of their disorders in cerebellar injury are not completely understood. There is a wide spectrum of cerebellar mutant mice which are used as models of hereditary cerebellar degenerations. Nevertheless, they differ in pathogenesis of manifestation of the particular mutation and also in the strain background. The aim of this work was to compare spatial navigation, learning and memory in pcd and Lurcher mice, two of the most frequently used cerebellar mutants. The mice were tested in the open field for exploration behavior, in the Morris water maze with visible as well as reversal hidden platform tasks and in the forced swimming test for motivation assessment. Lurcher mice showed different space exploration activity in the open field and a lower tendency to depressive-like behavior in the forced swimming test compared with pcd mice. Severe deficit of spatial navigation was shown in both cerebellar mutants. However, the overall performance of Lurcher mice was better than that of pcd mutants. Lurcher mice showed the ability of visual guidance despite difficulties with the direct swim towards a goal. In the probe trial test, Lurcher mice preferred the visible platform rather than the more recent localization of the hidden goal.

Alterations in grooming activity and syntax in heterozygous SERT and BDNF knockout mice: the utility of behavior-recognition tools to characterize mutant mouse phenotypes.

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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. Copyright © 2012 Elsevier Inc. All rights reserved.

AAV-Mediated Administration of Myostatin Pro-Peptide Mutant in Adult Ldlr Null Mice Reduces Diet-Induced Hepatosteatosis and Arteriosclerosis

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Guo, Wen; Wong, Siu; Bhasin, Shalender

2013-01-01

Genetic disruption of myostatin or its related signaling is known to cause strong protection against diet-induced metabolic disorders. The translational value of these prior findings, however, is dependent on whether such metabolically favorable phenotype can be reproduced when myostatin blockade begins at an adult age. Here, we reported that AAV-mediated delivery of a myostatin pro-peptide D76A mutant in adult mice attenuates the development of hepatic steatosis and arteriosclerosis, two common diet-induced metabolic diseases. A single dose of AAV-D76A in adult Ldlr null mice resulted in sustained expression of myostatin pro-peptide in the liver. Compared to vehicle-treated mice, D76A-treated mice gained similar amount of lean and fat mass when fed a high fat diet. However, D76A-treated mice displayed significantly reduced aortic lesions and liver fat, in association with a reduction in hepatic expression of lipogenic genes and improvement in liver insulin sensitivity. This suggests that muscle and fat may not be the primary targets of treatment under our experimental condition. In support to this argument, we show that myostatin directly up-regulated lipogenic genes and increased fat accumulation in cultured liver cells. We also show that both myostatin and its receptor were abundantly expressed in mouse aorta. Cultured aortic endothelial cells responded to myostatin with a reduction in eNOS phosphorylation and an increase in ICAM-1 and VCAM-1 expression. Conclusions: AAV-mediated expression of myostatin pro-peptide D76A mutant in adult Ldlr null mice sustained metabolic protection without remarkable impacts on body lean and fat mass. Further investigations are needed to determine whether direct impact of myostatin on liver and aortic endothelium may contribute to the related metabolic phenotypes. PMID:23936482

Cp/Heph mutant mice have iron-induced neurodegeneration diminished by deferiprone

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Zhao, Liangliang; Hadziahmetovic, Majda; Wang, Chenguang; Xu, Xueying; Song, Ying; Jinnah, H.A.; Wodzinska, Jolanta; Iacovelli, Jared; Wolkow, Natalie; Krajacic, Predrag; Weissberger, Alyssa Cwanger; Connelly, John; Spino, Michael; Lee, Michael K.; Connor, James; Giasson, Benoit; Harris, Z. Leah; Dunaief, Joshua L.

2016-01-01

Brain iron accumulates in several neurodegenerative diseases and can cause oxidative damage, but mechanisms of brain iron homeostasis are incompletely understood. Patients with mutations in the cellular iron-exporting ferroxidase ceruloplasmin (Cp) have brain iron accumulation causing neurodegeneration. Here, we assessed the brains of mice with combined mutation of Cp and its homolog hephaestin. Compared to single mutants, brain iron accumulation was accelerated in double mutants in the cerebellum, substantia nigra, and hippocampus. Iron accumulated within glia, while neurons were iron deficient. There was loss of both neurons and glia. Mice developed ataxia and tremor, and most died by 9 months. Treatment with the oral iron chelator deferiprone diminished brain iron levels, protected against neuron loss, and extended lifespan. Ferroxidases play important, partially overlapping roles in brain iron homeostasis by facilitating iron export from glia, making iron available to neurons. PMID:26303407

Impaired insulin secretion and glucose intolerance in synaptotagmin-7 null mutant mice

DEFF Research Database (Denmark)

Gustavsson, Natalia; Lao, Ye; Maximov, Anton

2008-01-01

and insulin release. Here, we show that synaptotagmin-7 is required for the maintenance of systemic glucose tolerance and glucose-stimulated insulin secretion. Mutant mice have normal insulin sensitivity, insulin production, islet architecture and ultrastructural organization, and metabolic and calcium...... secretion in pancreatic beta-cells. Of these other synaptotagmins, synaptotagmin-7 is one of the most abundant and is present in pancreatic beta-cells. To determine whether synaptotagmin-7 regulates Ca(2+)-dependent insulin secretion, we analyzed synaptotagmin-7 null mutant mice for glucose tolerance...... responses but exhibit impaired glucose-induced insulin secretion, indicating a calcium-sensing defect during insulin-containing secretory granule exocytosis. Taken together, our findings show that synaptotagmin-7 functions as a positive regulator of insulin secretion and may serve as a calcium sensor...

Absence-like and tonic seizures in aspartoacylase/attractin double-mutant mice.

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Gohma, Hiroshi; Kuramoto, Takashi; Matalon, Reuben; Surendran, Sankar; Tyring, Stephen; Kitada, Kazuhiro; Sasa, Masashi; Serikawa, Tadao

2007-04-01

The Spontaneously Epileptic Rat (SER), a double-mutant for tremor and zitter mutations, shows spontaneous occurrences of absence-like and tonic seizures. Several lines of evidence suggest that the combined effect of Aspa and Atrn mutations is the most likely cause of the epileptic phenotype of the SER. To address this issue, we produced a new double-mutant mouse line carrying both homozygous Aspa-knockout and Atrn(mg-3J) mutant alleles. The Aspa/Atrn double-mutant mice exhibited absence-like and tonic seizures that were characterized by the appearance of 5-7 Hz spike-wave-like complexes and low voltage fast waves on EEGs. These results demonstrate directly that the simultaneous loss of the Aspa and Atrn gene functions causes epileptic seizures in the mouse and suggest that both Aspa and Atrn deficiencies might be responsible for epileptic seizures in the SER.

Transthyretin knockout mice display decreased susceptibility to AMPA-induced neurodegeneration

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Nunes, Ana Filipa; Montero, Maria; Franquinho, Filipa

2009-01-01

Transthyretin (TTR) has been regarded as a neuroprotective protein given that TTR knockout (KO) mice display increased susceptibility for amyloid beta deposition and memory deficits during aging. In parallel, TTR KO mice have increased levels of neuropeptide Y (NPY), which promotes neuroprotectio...

Probing ADAMTS13 substrate specificity using phage display.

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Karl C Desch

Full Text Available Von Willebrand factor (VWF is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2' and P11', for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13-VWF exosite interactions outside of VWF73.

GH and IGF1: Roles in Energy Metabolism of Long-Living GH Mutant Mice

OpenAIRE

Brown-Borg, Holly M.; Bartke, Andrzej

2012-01-01

Of the multiple theories to explain exceptional longevity, the most robust of these has centered on the reduction of three anabolic protein hormones, growth hormone (GH), insulin-like growth factor, and insulin. GH mutant mice live 50% longer and exhibit significant differences in several aspects of energy metabolism as compared with wild-type mice. Mitochondrial metabolism is upregulated in the absence of GH, whereas in GH transgenic mice and dwarf mice treated with GH, multiple aspects of t...

[Accumulation of the bvg- Bordetella pertussis a virulent mutants in the process of experimental whooping cough in mice].

Science.gov (United States)

Medkova, A Iu; Siniashina, L N; Rumiantseva, Iu P; Voronina, O L; Kunda, M S; Karataev, G I

2013-01-01

The duration of the persistence and dynamics of accumulation of insertion bvg- Bordetella pertussis mutants were studied in lungs of laboratory mice after intranasal and intravenous challenge by virulent bacteria of the causative agent of whooping cough. The capability of the virulent B. pertussis bacteria to long-term persistence in the body of mice was tested. Using the real-time PCR approximately hundred genome equivalents of the B. pertussis DNA were detected in lungs of mice in two months after infection regardless of the way of challenge. Using the bacterial test bacteria were identified during only four weeks after challenge. Bvg- B. pertussis avirulent mutants were accumulated for the infection time. The percentage of the avirulent bacteria in the B. pertussis population reached 50% in 7-9 weeks after challenge. The obtained results show that the laboratory mice can be used for study of the B. pertussis insertion mutant formation dynamics in vivo and confirm the hypothesis about insertional bvg- B. pertussis virulent mutants accumulation during development of pertussis infection in human.

Mapping pathological phenotypes in Reelin mutant mice

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Caterina eMichetti

2014-09-01

Full Text Available Autism Spectrum Disorders (ASD are neurodevelopmental disorders with multifactorial origin characterized by social communication and behavioural perseveration deficits. Several studies showed an association between the reelin gene mutation and increased risk of ASD and a reduced reelin expression in some brain regions of ASD subjects, suggesting a role for reelin deficiency in ASD etiology. Reelin is a large extracellular matrix glycoprotein playing important roles during development of the central nervous system. To deeply investigate the role of reelin dysfunction as vulnerability factor in ASD, we investigated the behavioural, neurochemical and brain morphological features of reeler male mice. We recently reported a genotype-dependent deviation in ultrasonic vocal repertoire and a general delay in motor development in reeler pups. We now report that adult male heterozygous reeler mice did not show social behaviour and communication deficits during male-female social interactions. Wildtype and heterozygous mice also showed a typical light/dark locomotor activity profile, with a peak during the central interval of the dark phase. However, when faced with a mild stressful stimulus (a saline injection only heterozygous mice showed an over response to stress. At the end of the behavioural studies, we conducted high performance liquid chromatography and magnetic resonance imaging and spectroscopy to investigate whether reelin mutation influences brain monoamine and metabolites levels in regions involved in ASD. Low levels of dopamine in cortex and high levels of glutamate and taurine in hippocampus were detected in heterozygous mice, in line with clinical data collected on ASD children. Altogether, our data detected subtle but relevant neurochemical abnormalities in reeler mice supporting this mutant line, particularly male subjects, as a valid experimental model to estimate the contribution played by reelin deficiency in the global ASD

Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models

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Sara eAmorim-Vaz

2015-05-01

Full Text Available The aim of the present study was to identify C. albicans transcription factors (TF involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens quantified in kidneys. Mutants of unannotated genes which generated a kidney fungal burden significantly different from that of wild-type were selected and individually examined in G. mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25 % of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects, a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching fungal burden phenotypes were observed in 50 % of the cases, highlighting the bias due to host effects. In contrast, 33.4 % concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the pool effect. After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adaptation.

Altered metabolism of growth hormone receptor mutant mice: a combined NMR metabonomics and microarray study.

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Horst Joachim Schirra

Full Text Available BACKGROUND: Growth hormone is an important regulator of post-natal growth and metabolism. We have investigated the metabolic consequences of altered growth hormone signalling in mutant mice that have truncations at position 569 and 391 of the intracellular domain of the growth hormone receptor, and thus exhibit either low (around 30% maximum or no growth hormone-dependent STAT5 signalling respectively. These mutations result in altered liver metabolism, obesity and insulin resistance. METHODOLOGY/PRINCIPAL FINDINGS: The analysis of metabolic changes was performed using microarray analysis of liver tissue and NMR metabonomics of urine and liver tissue. Data were analyzed using multivariate statistics and Gene Ontology tools. The metabolic profiles characteristic for each of the two mutant groups and wild-type mice were identified with NMR metabonomics. We found decreased urinary levels of taurine, citrate and 2-oxoglutarate, and increased levels of trimethylamine, creatine and creatinine when compared to wild-type mice. These results indicate significant changes in lipid and choline metabolism, and were coupled with increased fat deposition, leading to obesity. The microarray analysis identified changes in expression of metabolic enzymes correlating with alterations in metabolite concentration both in urine and liver. Similarity of mutant 569 to the wild-type was seen in young mice, but the pattern of metabolites shifted to that of the 391 mutant as the 569 mice became obese after six months age. CONCLUSIONS/SIGNIFICANCE: The metabonomic observations were consistent with the parallel analysis of gene expression and pathway mapping using microarray data, identifying metabolites and gene transcripts involved in hepatic metabolism, especially for taurine, choline and creatinine metabolism. The systems biology approach applied in this study provides a coherent picture of metabolic changes resulting from impaired STAT5 signalling by the growth hormone

Probing ADAMTS13 Substrate Specificity using Phage Display

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Desch, Karl C.; Kretz, Colin; Yee, Andrew; Gildersleeve, Robert; Metzger, Kristin; Agrawal, Nidhi; Cheng, Jane; Ginsburg, David

2015-01-01

Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2’ and P11’, for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13–VWF exosite interactions outside of VWF73. PMID:25849793

Ectopic Mineralization and Conductive Hearing Loss in Enpp1asj Mutant Mice, a New Model for Otitis Media and Tympanosclerosis.

Science.gov (United States)

Tian, Cong; Harris, Belinda S; Johnson, Kenneth R

2016-01-01

Otitis media (OM), inflammation of the middle ear, is a common cause of hearing loss in children and in patients with many different syndromic diseases. Studies of the human population and mouse models have revealed that OM is a multifactorial disease with many environmental and genetic contributing factors. Here, we report on otitis media-related hearing loss in asj (ages with stiffened joints) mutant mice, which bear a point mutation in the Enpp1 gene. Auditory-evoked brainstem response (ABR) measurements revealed that around 90% of the mutant mice (Enpp1asj/asj) tested had moderate to severe hearing impairment in at least one ear. The ABR thresholds were variable and generally elevated with age. We found otitis media with effusion (OME) in all of the hearing-impaired Enpp1asj/asj mice by anatomic and histological examinations. The volume and inflammatory cell content of the effusion varied among the asj mutant mice, but all mutants exhibited a thickened middle ear epithelium with fibrous polyps and more mucin-secreting goblet cells than controls. Other abnormalities observed in the Enpp1 mutant mice include over-ossification at the round window ridge, thickened and over-calcified stapedial artery, fusion of malleus and incus, and white patches on the inside of tympanic membrane, some of which are typical symptoms of tympanosclerosis. An excessive yellow discharge was detected in the outer ear canal of older asj mutant mice, with 100% penetrance by 5 months of age, and contributes to the progressive nature of the hearing loss. This is the first report of hearing loss and ear pathology associated with an Enpp1 mutation in mice. The Enpp1asj mutant mouse provides a new animal model for studying tympanosclerotic otitis and otitis media with effusion, and also provides a specific model for the hearing loss recently reported to be associated with human ENPP1 mutations causing generalized arterial calcification of infancy and hypophosphatemic rickets.

Vapb/Amyotrophic lateral sclerosis 8 knock-in mice display slowly progressive motor behavior defects accompanying ER stress and autophagic response.

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Larroquette, Frédérique; Seto, Lesley; Gaub, Perrine L; Kamal, Brishna; Wallis, Deeann; Larivière, Roxanne; Vallée, Joanne; Robitaille, Richard; Tsuda, Hiroshi

2015-11-15

Missense mutations (P56S) in Vapb are associated with autosomal dominant motor neuron diseases: amyotrophic lateral sclerosis and lower motor neuron disease. Although transgenic mice overexpressing the mutant vesicle-associated membrane protein-associated protein B (VAPB) protein with neuron-specific promoters have provided some insight into the toxic properties of the mutant proteins, their role in pathogenesis remains unclear. To identify pathological defects in animals expressing the P56S mutant VAPB protein at physiological levels in the appropriate tissues, we have generated Vapb knock-in mice replacing wild-type Vapb gene with P56S mutant Vapb gene and analyzed the resulting pathological phenotypes. Heterozygous P56S Vapb knock-in mice show mild age-dependent defects in motor behaviors as characteristic features of the disease. The homozygous P56S Vapb knock-in mice show more severe defects compared with heterozygous mice reflecting the dominant and dose-dependent effects of P56S mutation. Significantly, the knock-in mice demonstrate accumulation of P56S VAPB protein and ubiquitinated proteins in cytoplasmic inclusions, selectively in motor neurons. The mutant mice demonstrate induction of ER stress and autophagic response in motor neurons before obvious onset of behavioral defects, suggesting that these cellular biological defects might contribute to the initiation of the disease. The P56S Vapb knock-in mice could be a valuable tool to gain a better understanding of the mechanisms by which the disease arises. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Toll-like receptor 4 mutant and null mice retain morphine-induced tolerance, hyperalgesia, and physical dependence.

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Theresa Alexandra Mattioli

Full Text Available The innate immune system modulates opioid-induced effects within the central nervous system and one target that has received considerable attention is the toll-like receptor 4 (TLR4. Here, we examined the contribution of TLR4 in the development of morphine tolerance, hyperalgesia, and physical dependence in two inbred mouse strains: C3H/HeJ mice which have a dominant negative point mutation in the Tlr4 gene rendering the receptor non-functional, and B10ScNJ mice which are TLR4 null mutants. We found that neither acute antinociceptive response to a single dose of morphine, nor the development of analgesic tolerance to repeated morphine treatment, was affected by TLR4 genotype. Likewise, opioid induced hyperalgesia and opioid physical dependence (assessed by naloxone precipitated withdrawal were not altered in TLR4 mutant or null mice. We also examined the behavioural consequence of two stereoisomers of naloxone: (- naloxone, an opioid receptor antagonist, and (+ naloxone, a purported antagonist of TLR4. Both stereoisomers of naloxone suppressed opioid induced hyperalgesia in wild-type control, TLR4 mutant, and TLR4 null mice. Collectively, our data suggest that TLR4 is not required for opioid-induced analgesic tolerance, hyperalgesia, or physical dependence.

Analysis on expression of gene for flower shape in Dendrobium sonia mutants using differential display technique

International Nuclear Information System (INIS)

Affrida Abu Hassan; Ahmad Syazni Kamarudin; Nurul Nadia Aminuddin; Mohd Nazir Basiran

2004-01-01

In vitro mutagenesis on Dendrobium Sonia in MINT has produced mutants with wide range of flower form and colour variations. Among the mutants are plants with different flower size and shape. These changes could be caused by alterations to the expression level of the genes responsible for the characteristics. In this studies, Differential Display technique was used to identify and analyse altered gene expression at the mRNA level. Total RNA of the control and mutants were reversed transcribed using three anchored oligo-d T primers. Subsequently, these cDNAs were Pcr amplified in combination with 16 arbitrary primers. The amplified products were electrophoresed side by side on agarose gel. Differentially expressed bands are isolated for further analysis. (Author)

Mutant Mice Lacking the p53 C-Terminal Domain Model Telomere Syndromes

NARCIS (Netherlands)

Simeonova, I.; Jaber, S.; Draskovic, I.; Bardot, B.; Fang, M.; Bouarich-Bourimi, R.; Lejour, V.; Charbonnier, L.; Soudais, C.; Bourdon, J.C.; Huerre, M.; Londono-Vallejo, A.; Toledo, F.

2013-01-01

Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53(Delta31), a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis,

Deficient plasticity in the primary visual cortex of alpha-calcium/calmodulin-dependent protein kinase II mutant mice.

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Gordon, J A; Cioffi, D; Silva, A J; Stryker, M P

1996-09-01

The recent characterization of plasticity in the mouse visual cortex permits the use of mutant mice to investigate the cellular mechanisms underlying activity-dependent development. As calcium-dependent signaling pathways have been implicated in neuronal plasticity, we examined visual cortical plasticity in mice lacking the alpha-isoform of calcium/calmodulin-dependent protein kinase II (alpha CaMKII). In wild-type mice, brief occlusion of vision in one eye during a critical period reduces responses in the visual cortex. In half of the alpha CaMKII-deficient mice, visual cortical responses developed normally, but visual cortical plasticity was greatly diminished. After intensive training, spatial learning in the Morris water maze was severely impaired in a similar fraction of mutant animals. These data indicate that loss of alpha CaMKII results in a severe but variable defect in neuronal plasticity.

Progression of Hepatic Adenoma to Carcinoma in Ogg1 Mutant Mice Induced by Phenobarbital

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Anna Kakehashi

2017-01-01

Full Text Available The carcinogenic potential of phenobarbital (PB was assessed in a mouse line carrying a mutant Mmh allele of the Mmh/Ogg1 gene encoding the enzyme oxoguanine DNA glycosylase (Ogg1 responsible for the repair of 8-hydroxy-2′-deoxyguanosine (8-OHdG. Mmh homozygous mutant (Ogg1−/− and wild-type (Ogg1+/+ male and female, 10-week-old, mice were treated with 500 ppm PB in diet for 78 weeks. Hepatocellular carcinomas (HCCs were found in PB-treated Ogg1−/− mice, while Ogg1+/+ animals developed only hepatocellular adenomas (HCAs at the same rate. This was coordinated with PB-induced significant elevation of 8-OHdG formation in DNA and cell proliferation in adjacent liver of Ogg1−/− mice. Proteome analysis predicted activation of transcriptional factor Nrf2 in the livers and HCAs of PB-administered Ogg1+/+ mice; however, its activation was insufficient or absent in the livers and HCCs of Ogg1−/− mice, respectively. Significant elevation of phase I and II metabolizing enzymes was demonstrated in both Ogg1−/− and Ogg1+/+ animals. Treatment of Ogg1−/− mice with PB resulted in significant elevation of cell proliferation in the liver. These results indicate that PB induced progression from HCA to HCC in Ogg1−/− mice, due to persistent accumulation of DNA oxidative base modifications and suppression of Nrf2-mediated oxidative stress response, resulting in significant elevation of cell proliferation.

Alzheimer’s Disease Mutant Mice Exhibit Reduced Brain Tissue Stiffness Compared to Wild-type Mice in both Normoxia and following Intermittent Hypoxia Mimicking Sleep Apnea

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Maria José Menal

2018-01-01

Full Text Available BackgroundEvidence from patients and animal models suggests that obstructive sleep apnea (OSA may increase the risk of Alzheimer’s disease (AD and that AD is associated with reduced brain tissue stiffness.AimTo investigate whether intermittent hypoxia (IH alters brain cortex tissue stiffness in AD mutant mice exposed to IH mimicking OSA.MethodsSix-eight month old (B6C3-Tg(APPswe,PSEN1dE985Dbo/J AD mutant mice and wild-type (WT littermates were subjected to IH (21% O2 40 s to 5% O2 20 s; 6 h/day or normoxia for 8 weeks. After euthanasia, the stiffness (E of 200-μm brain cortex slices was measured by atomic force microscopy.ResultsTwo-way ANOVA indicated significant cortical softening and weight increase in AD mice compared to WT littermates, but no significant effects of IH on cortical stiffness and weight were detected. In addition, reduced myelin was apparent in AD (vs. WT, but no significant differences emerged in the cortex extracellular matrix components laminin and glycosaminoglycans when comparing baseline AD and WT mice.ConclusionAD mutant mice exhibit reduced brain tissue stiffness following both normoxia and IH mimicking sleep apnea, and such differences are commensurate with increased edema and demyelination in AD.

Mutant Huntingtin Gene-Dose Impacts on Aggregate Deposition, DARPP32 Expression and Neuroinflammation in HdhQ150 Mice

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Young, Douglas; Mayer, Franziska; Vidotto, Nella; Schweizer, Tatjana; Berth, Ramon; Abramowski, Dorothee; Shimshek, Derya R.; van der Putten, P. Herman; Schmid, Peter

2013-01-01

Huntington's disease (HD) is an autosomal dominant, progressive and fatal neurological disorder caused by an expansion of CAG repeats in exon-1 of the huntingtin gene. The encoded poly-glutamine stretch renders mutant huntingtin prone to aggregation. HdhQ150 mice genocopy a pathogenic repeat (∼150 CAGs) in the endogenous mouse huntingtin gene and model predominantly pre-manifest HD. Treating early is likely important to prevent or delay HD, and HdhQ150 mice may be useful to assess therapeutic strategies targeting pre-manifest HD. This requires appropriate markers and here we demonstrate, that pre-symptomatic HdhQ150 mice show several dramatic mutant huntingtin gene-dose dependent pathological changes including: (i) an increase of neuronal intra-nuclear inclusions (NIIs) in brain, (ii) an increase of extra-nuclear aggregates in dentate gyrus, (iii) a decrease of DARPP32 protein and (iv) an increase in glial markers of neuroinflammation, which curiously did not correlate with local neuronal mutant huntingtin inclusion-burden. HdhQ150 mice developed NIIs also in all retinal neuron cell-types, demonstrating that retinal NIIs are not specific to human exon-1 R6 HD mouse models. Taken together, the striking and robust mutant huntingtin gene-dose related changes in aggregate-load, DARPP32 levels and glial activation markers should greatly facilitate future testing of therapeutic strategies in the HdhQ150 HD mouse model. PMID:24086450

Indy mutants: live long and prosper

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Stewart eFrankel

2012-02-01

Full Text Available Indy encodes the fly homologue of a mammalian transporter of di and tricarboxylatecomponents of the Krebs cycle. Reduced expression of fly Indy or two of the C. elegansIndy homologs leads to an increase in life span. Fly and worm tissues that play key roles inintermediary metabolism are also the places where Indy genes are expressed. One of themouse homologs of Indy (mIndy is mainly expressed in the liver. It has been hypothesizedthat decreased INDY activity creates a state similar to caloric restriction (CR. Thishypothesis is supported by the physiological similarities between Indy mutant flies on highcalorie food and control flies on CR, such as increased physical activity and decreases inweight, egg production, triglyceride levels, starvation resistance, and insulin signaling. Inaddition, Indy mutant flies undergo changes in mitochondrial biogenesis also observed inCR animals. Recent findings with mIndy knockout mice support and extend the findingsfrom flies. mIndy-/- mice display an increase in hepatic mitochondrial biogenesis, lipidoxidation and decreased hepatic lipogenesis. When mIndy-/- mice are fed high calorie foodthey are protected from adiposity and insulin resistance. These findings point to INDY as apotential drug target for the treatment of metabolic syndrome, type 2 diabetes and obesity.

Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice.

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Carlos A Santiviago

2009-07-01

Full Text Available Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to the method as Array-based analysis of cistrons under selection (ABACUS.

Interferon β induces clearance of mutant ataxin 7 and improves locomotion in SCA7 knock-in mice.

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Chort, Alice; Alves, Sandro; Marinello, Martina; Dufresnois, Béatrice; Dornbierer, Jean-Gabriel; Tesson, Christelle; Latouche, Morwena; Baker, Darren P; Barkats, Martine; El Hachimi, Khalid H; Ruberg, Merle; Janer, Alexandre; Stevanin, Giovanni; Brice, Alexis; Sittler, Annie

2013-06-01

We showed previously, in a cell model of spinocerebellar ataxia 7, that interferon beta induces the expression of PML protein and the formation of PML protein nuclear bodies that degrade mutant ataxin 7, suggesting that the cytokine, used to treat multiple sclerosis, might have therapeutic value in spinocerebellar ataxia 7. We now show that interferon beta also induces PML-dependent clearance of ataxin 7 in a preclinical model, SCA7(266Q/5Q) knock-in mice, and improves motor function. Interestingly, the presence of mutant ataxin 7 in the mice induces itself the expression of endogenous interferon beta and its receptor. Immunohistological studies in brains from two patients with spinocerebellar ataxia 7 confirmed that these modifications are also caused by the disease in humans. Interferon beta, administered intraperitoneally three times a week in the knock-in mice, was internalized with its receptor in Purkinje and other cells and translocated to the nucleus. The treatment induced PML protein expression and the formation of PML protein nuclear bodies and decreased mutant ataxin 7 in neuronal intranuclear inclusions, the hallmark of the disease. No reactive gliosis or other signs of toxicity were observed in the brain or internal organs. The performance of the SCA7(266Q/5Q) knock-in mice was significantly improved on two behavioural tests sensitive to cerebellar function: the Locotronic® Test of locomotor function and the Beam Walking Test of balance, motor coordination and fine movements, which are affected in patients with spinocerebellar ataxia 7. In addition to motor dysfunction, SCA7(266Q/5Q) mice present abnormalities in the retina as in patients: ataxin 7-positive neuronal intranuclear inclusions that were reduced by interferon beta treatment. Finally, since neuronal death does not occur in the cerebellum of SCA7(266Q/5Q) mice, we showed in primary cell cultures expressing mutant ataxin 7 that interferon beta treatment improves Purkinje cell survival.

Neurologic function during developmental and adult stages in Dab1(scm) (scrambler) mutant mice.

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Jacquelin, C; Strazielle, C; Lalonde, R

2012-01-01

Homozygous Dab1(scm) mouse mutants with cell ectopias in cerebellar cortex, hippocampus, and neocortex were compared to non-ataxic controls on the SHIRPA primary screening battery on postnatal days 8, 15, and 22, as well as in the adult period. Dab1(scm) mutants were distinguished from non-ataxic controls as early as postnatal day 8 based on body tremor, gait anomalies, and body weight. On postnatal day 15, motor coordination deficits were evident on horizontal bar and inclined or vertical grid tests in association with a weaker grip strength. Likewise, mutants were distinguished from controls on drop righting and hindpaw clasping tests. Further differences were detected on postnatal day 22 in the form of fewer visual placing, touch escape, trunk curl, freezing, and vocalization responses, as well as squares traversed in the open-field. Evaluation at the adult age demonstrated similar impairments, indicative of permanent motor alterations. Neuronal metabolic activity was estimated by cytochrome oxidase histochemistry on cerebellar sections. Cerebellar cortical layers and efferent deep nuclei of Dab1(scm) mice appeared hypometabolic relative to non-ataxic mice despite normal metabolism in both regular and ectopic Purkinje cells. Copyright © 2011 Elsevier B.V. All rights reserved.

Diet-induced obesity increases the frequency of Pig-a mutant erythrocytes in male C57BL/6J mice.

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Wickliffe, Jeffrey K; Dertinger, Stephen D; Torous, Dorothea K; Avlasevich, Svetlana L; Simon-Friedt, Bridget R; Wilson, Mark J

2016-12-01

Obesity increases the risk of a number of chronic diseases in humans including several cancers. Biological mechanisms responsible for such increased risks are not well understood at present. Increases in systemic inflammation and oxidative stress, endogenous production of mutagenic metabolites, altered signaling in proliferative pathways, and increased sensitivity to exogenous mutagens and carcinogens are some of the potential contributing factors. We hypothesize that obesity creates an endogenously mutagenic environment in addition to increasing the sensitivity to environmental mutagens. To test this hypothesis, we examined two in vivo genotoxicity endpoints. Pig-a mutant frequencies and micronucleus frequencies were determined in blood cells in two independent experiments in 30-week old male mice reared on either a high-fat diet (60% calories from fat) that exhibit an obese phenotype or a normal-fat diet (10% calories from fat) that do not exhibit an obese phenotype. Mice were assayed again at 52 weeks of age in one of the experiments. N-ethyl-N-nitrosourea (ENU) was used as a positive mutation control in one experiment. ENU induced a robust Pig-a mutant and micronucleus response in both phenotypes. Obese, otherwise untreated mice, did not differ from non-obese mice with respect to Pig-a mutant frequencies in reticulocytes or micronucleus frequencies. However, such mice, had significantly higher and sustained Pig-a mutant frequencies (increased 2.5-3.7-fold, p obese mice (based on measurements collected at 30 weeks or 30 and 52 weeks of age). This suggests that obesity, in the absence of exposure to an exogenous mutagen, is itself mutagenic. Environ. Mol. Mutagen. 57:668-677, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Alternating hemiplegia of childhood-related neural and behavioural phenotypes in Na+,K+-ATPase α3 missense mutant mice.

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Greer S Kirshenbaum

Full Text Available Missense mutations in ATP1A3 encoding Na(+,K(+-ATPase α3 have been identified as the primary cause of alternating hemiplegia of childhood (AHC, a motor disorder with onset typically before the age of 6 months. Affected children tend to be of short stature and can also have epilepsy, ataxia and learning disability. The Na(+,K(+-ATPase has a well-known role in maintaining electrochemical gradients across cell membranes, but our understanding of how the mutations cause AHC is limited. Myshkin mutant mice carry an amino acid change (I810N that affects the same position in Na(+,K(+-ATPase α3 as I810S found in AHC. Using molecular modelling, we show that the Myshkin and AHC mutations display similarly severe structural impacts on Na(+,K(+-ATPase α3, including upon the K(+ pore and predicted K(+ binding sites. Behavioural analysis of Myshkin mice revealed phenotypic abnormalities similar to symptoms of AHC, including motor dysfunction and cognitive impairment. 2-DG imaging of Myshkin mice identified compromised thalamocortical functioning that includes a deficit in frontal cortex functioning (hypofrontality, directly mirroring that reported in AHC, along with reduced thalamocortical functional connectivity. Our results thus provide validation for missense mutations in Na(+,K(+-ATPase α3 as a cause of AHC, and highlight Myshkin mice as a starting point for the exploration of disease mechanisms and novel treatments in AHC.

Differential gene expression in ADAM10 and mutant ADAM10 transgenic mice

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Postina Rolf

2009-02-01

Full Text Available Abstract Background In a transgenic mouse model of Alzheimer disease (AD, cleavage of the amyloid precursor protein (APP by the α-secretase ADAM10 prevented amyloid plaque formation, and alleviated cognitive deficits. Furthermore, ADAM10 overexpression increased the cortical synaptogenesis. These results suggest that upregulation of ADAM10 in the brain has beneficial effects on AD pathology. Results To assess the influence of ADAM10 on the gene expression profile in the brain, we performed a microarray analysis using RNA isolated from brains of five months old mice overexpressing either the α-secretase ADAM10, or a dominant-negative mutant (dn of this enzyme. As compared to non-transgenic wild-type mice, in ADAM10 transgenic mice 355 genes, and in dnADAM10 mice 143 genes were found to be differentially expressed. A higher number of genes was differentially regulated in double-transgenic mouse strains additionally expressing the human APP[V717I] mutant. Overexpression of proteolytically active ADAM10 affected several physiological pathways, such as cell communication, nervous system development, neuron projection as well as synaptic transmission. Although ADAM10 has been implicated in Notch and β-catenin signaling, no significant changes in the respective target genes were observed in adult ADAM10 transgenic mice. Real-time RT-PCR confirmed a downregulation of genes coding for the inflammation-associated proteins S100a8 and S100a9 induced by moderate ADAM10 overexpression. Overexpression of the dominant-negative form dnADAM10 led to a significant increase in the expression of the fatty acid-binding protein Fabp7, which also has been found in higher amounts in brains of Down syndrome patients. Conclusion In general, there was only a moderate alteration of gene expression in ADAM10 overexpressing mice. Genes coding for pro-inflammatory or pro-apoptotic proteins were not over-represented among differentially regulated genes. Even a decrease of

Tau passive immunotherapy in mutant P301L mice: antibody affinity versus specificity.

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Cristina d'Abramo

Full Text Available The use of antibodies to treat neurodegenerative diseases has undergone rapid development in the past decade. To date, immunotherapeutic approaches to Alzheimer's disease have mostly targeted amyloid beta as it is a secreted protein that can be found in plasma and CSF and is consequently accessible to circulating antibodies. Few recent publications have suggested the utility of treatment of tau pathology with monoclonal antibodies to tau. Our laboratory has begun a systematic study of different classes of tau monoclonal antibodies using mutant P301L mice. Three or seven months old mutant tau mice were inoculated weekly with tau monoclonal antibodies at a dose of 10 mg/Kg, until seven or ten months of age were reached respectively. Our data strongly support the notion that in P301L animals treated with MC1, a conformational monoclonal antibody specific for PHF-tau, the rate of development of tau pathology is effectively reduced, while injecting DA31, a high affinity tau sequence antibody, does not exert such benefit. MC1 appears superior to DA31 in overall effects, suggesting that specificity is more important than affinity in therapeutic applications. Unfortunately the survival rate of the P301L treated mice was not improved when immunizing either with MC1 or PHF1, a high affinity phospho-tau antibody previously reported to be efficacious in reducing pathological tau. These data demonstrate that passive immunotherapy in mutant tau models may be efficacious in reducing the development of tau pathology, but a great deal of work remains to be done to carefully select the tau epitopes to target.

Dearth and Delayed Maturation of Testicular Germ Cells in Fanconi Anemia E Mutant Male Mice.

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Chun Fu

Full Text Available After using a self-inactivating lentivirus for non-targeted insertional mutagenesis in mice, we identified a transgenic family with a recessive mutation that resulted in reduced fertility in homozygous transgenic mice. The lentiviral integration site was amplified by inverse PCR. Sequencing revealed that integration had occurred in intron 8 of the mouse Fance gene, which encodes the Fanconi anemia E (Fance protein. Fanconi anemia (FA proteins play pivotal roles in cellular responses to DNA damage and Fance acts as a molecular bridge between the FA core complex and Fancd2. To investigate the reduced fertility in the mutant males, we analyzed postnatal development of testicular germ cells. At one week after birth, most tubules in the mutant testes contained few or no germ cells. Over the next 2-3 weeks, germ cells accumulated in a limited number of tubules, so that some tubules contained germ cells around the full periphery of the tubule. Once sufficient numbers of germ cells had accumulated, they began to undergo the later stages of spermatogenesis. Immunoassays revealed that the Fancd2 protein accumulated around the periphery of the nucleus in normal developing spermatocytes, but we did not detect a similar localization of Fancd2 in the Fance mutant testes. Our assays indicate that although Fance mutant males are germ cell deficient at birth, the extant germ cells can proliferate and, if they reach a threshold density, can differentiate into mature sperm. Analogous to previous studies of FA genes in mice, our results show that the Fance protein plays an important, but not absolutely essential, role in the initial developmental expansion of the male germ line.

Searching for biomarkers of CDKL5 disorder: early-onset visual impairment in CDKL5 mutant mice.

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Mazziotti, Raffaele; Lupori, Leonardo; Sagona, Giulia; Gennaro, Mariangela; Della Sala, Grazia; Putignano, Elena; Pizzorusso, Tommaso

2017-06-15

CDKL5 disorder is a neurodevelopmental disorder still without a cure. Murine models of CDKL5 disorder have been recently generated raising the possibility of preclinical testing of treatments. However, unbiased, quantitative biomarkers of high translational value to monitor brain function are still missing. Moreover, the analysis of treatment is hindered by the challenge of repeatedly and non-invasively testing neuronal function. We analyzed the development of visual responses in a mouse model of CDKL5 disorder to introduce visually evoked responses as a quantitative method to assess cortical circuit function. Cortical visual responses were assessed in CDKL5 null male mice, heterozygous females, and their respective control wild-type littermates by repeated transcranial optical imaging from P27 until P32. No difference between wild-type and mutant mice was present at P25-P26 whereas defective responses appeared from P27-P28 both in heterozygous and homozygous CDKL5 mutant mice. These results were confirmed by visually evoked potentials (VEPs) recorded from the visual cortex of a different cohort. The previously imaged mice were also analyzed at P60-80 using VEPs, revealing a persistent reduction of response amplitude, reduced visual acuity and defective contrast function. The level of adult impairment was significantly correlated with the reduction in visual responses observed during development. Support vector machine showed that multi-dimensional visual assessment can be used to automatically classify mutant and wt mice with high reliability. Thus, monitoring visual responses represents a promising biomarker for preclinical and clinical studies on CDKL5 disorder. © The Author 2017. Published by Oxford University Press.

Trace elements monitored with neutron activation analysis durig neurodegeneration in brains of mutant mice

Czech Academy of Sciences Publication Activity Database

Kranda, Karel; Kučera, Jan; Bäurle, J.

2006-01-01

Roč. 269, č. 3 (2006), s. 555-559 ISSN 0236-5731 Institutional research plan: CEZ:AV0Z10480505 Keywords : trace elements * neutron activation analysis * brain neurodegeneration * mutant mice Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 0.509, year: 2006

A cerebellar learning model of vestibulo-ocular reflex adaptation in wild-type and mutant mice.

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Clopath, Claudia; Badura, Aleksandra; De Zeeuw, Chris I; Brunel, Nicolas

2014-05-21

Mechanisms of cerebellar motor learning are still poorly understood. The standard Marr-Albus-Ito theory posits that learning involves plasticity at the parallel fiber to Purkinje cell synapses under control of the climbing fiber input, which provides an error signal as in classical supervised learning paradigms. However, a growing body of evidence challenges this theory, in that additional sites of plasticity appear to contribute to motor adaptation. Here, we consider phase-reversal training of the vestibulo-ocular reflex (VOR), a simple form of motor learning for which a large body of experimental data is available in wild-type and mutant mice, in which the excitability of granule cells or inhibition of Purkinje cells was affected in a cell-specific fashion. We present novel electrophysiological recordings of Purkinje cell activity measured in naive wild-type mice subjected to this VOR adaptation task. We then introduce a minimal model that consists of learning at the parallel fibers to Purkinje cells with the help of the climbing fibers. Although the minimal model reproduces the behavior of the wild-type animals and is analytically tractable, it fails at reproducing the behavior of mutant mice and the electrophysiology data. Therefore, we build a detailed model involving plasticity at the parallel fibers to Purkinje cells' synapse guided by climbing fibers, feedforward inhibition of Purkinje cells, and plasticity at the mossy fiber to vestibular nuclei neuron synapse. The detailed model reproduces both the behavioral and electrophysiological data of both the wild-type and mutant mice and allows for experimentally testable predictions. Copyright © 2014 the authors 0270-6474/14/347203-13$15.00/0.

Sequential super-stereotypy of an instinctive fixed action pattern in hyper-dopaminergic mutant mice: a model of obsessive compulsive disorder and Tourette's

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Houchard Kimberly R

2005-02-01

Full Text Available Abstract Background Excessive sequential stereotypy of behavioral patterns (sequential super-stereotypy in Tourette's syndrome and obsessive compulsive disorder (OCD is thought to involve dysfunction in nigrostriatal dopamine systems. In sequential super-stereotypy, patients become trapped in overly rigid sequential patterns of action, language, or thought. Some instinctive behavioral patterns of animals, such as the syntactic grooming chain pattern of rodents, have sufficiently complex and stereotyped serial structure to detect potential production of overly-rigid sequential patterns. A syntactic grooming chain is a fixed action pattern that serially links up to 25 grooming movements into 4 predictable phases that follow 1 syntactic rule. New mutant mouse models allow gene-based manipulation of brain function relevant to sequential patterns, but no current animal model of spontaneous OCD-like behaviors has so far been reported to exhibit sequential super-stereotypy in the sense of a whole complex serial pattern that becomes stronger and excessively rigid. Here we used a hyper-dopaminergic mutant mouse to examine whether an OCD-like behavioral sequence in animals shows sequential super-stereotypy. Knockdown mutation of the dopamine transporter gene (DAT causes extracellular dopamine levels in the neostriatum of these adult mutant mice to rise to 170% of wild-type control levels. Results We found that the serial pattern of this instinctive behavioral sequence becomes strengthened as an entire entity in hyper-dopaminergic mutants, and more resistant to interruption. Hyper-dopaminergic mutant mice have stronger and more rigid syntactic grooming chain patterns than wild-type control mice. Mutants showed sequential super-stereotypy in the sense of having more stereotyped and predictable syntactic grooming sequences, and were also more likely to resist disruption of the pattern en route, by returning after a disruption to complete the pattern from the

Conserved role of unc-79 in ethanol responses in lightweight mutant mice.

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David J Speca

2010-08-01

Full Text Available The mechanisms by which ethanol and inhaled anesthetics influence the nervous system are poorly understood. Here we describe the positional cloning and characterization of a new mouse mutation isolated in an N-ethyl-N-nitrosourea (ENU forward mutagenesis screen for animals with enhanced locomotor activity. This allele, Lightweight (Lwt, disrupts the homolog of the Caenorhabditis elegans (C. elegans unc-79 gene. While Lwt/Lwt homozygotes are perinatal lethal, Lightweight heterozygotes are dramatically hypersensitive to acute ethanol exposure. Experiments in C. elegans demonstrate a conserved hypersensitivity to ethanol in unc-79 mutants and extend this observation to the related unc-80 mutant and nca-1;nca-2 double mutants. Lightweight heterozygotes also exhibit an altered response to the anesthetic isoflurane, reminiscent of unc-79 invertebrate mutant phenotypes. Consistent with our initial mapping results, Lightweight heterozygotes are mildly hyperactive when exposed to a novel environment and are smaller than wild-type animals. In addition, Lightweight heterozygotes exhibit increased food consumption yet have a leaner body composition. Interestingly, Lightweight heterozygotes voluntarily consume more ethanol than wild-type littermates. The acute hypersensitivity to and increased voluntary consumption of ethanol observed in Lightweight heterozygous mice in combination with the observed hypersensitivity to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants suggests a novel conserved pathway that might influence alcohol-related behaviors in humans.

Growth Hormone-Releaser Diet Attenuates Cognitive Dysfunction in Klotho Mutant Mice via Insulin-Like Growth Factor-1 Receptor Activation in a Genetic Aging Model

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Seok Joo Park

2014-09-01

Full Text Available BackgroundIt has been recognized that a defect in klotho gene expression accelerates the degeneration of multiple age-sensitive traits. Accumulating evidence indicates that aging is associated with declines in cognitive function and the activity of growth hormone (GH/insulin-like growth factor-1 (IGF-1.MethodsIn this study, we examined whether a GH-releaser diet could be effective in protecting against cognitive impairment in klotho mutant mice.ResultsThe GH-releaser diet significantly induced the expression of IGF-1 and IGF-1 receptors in the hippocampus of klotho mutant mice. Klotho mutant mice showed significant memory impairments as compared with wild-type mice. In addition, the klotho mutation significantly decreased the expression of cell survival/antiapoptotic factors, including phospho-Akt (p-Akt/phospho-glycogen synthase kinase3β (p-GSK3β, phospho-extracellular signal-related kinase (p-ERK, and Bcl-2, but significantly increased those of cell death/proapoptotic factors, such as phospho-c-jun N-terminal kinase (p-JNK, Bax, and cleaved caspase-3 in the hippocampus. Treatment with GH-releaser diet significantly attenuated both decreases in the expression of cell survival/antiapoptotic factors and increases in the expression of cell death/proapoptotic factors in the hippocampus of klotho mutant mice. In addition, klotho mutation-induced oxidative stress was significantly attenuated by the GH-releaser diet. Consequently, a GH-releaser diet significantly improved memory function in the klotho mutant mice. GH-releaser diet-mediated actions were significantly reversed by JB-1, an IGF-1 receptor antagonist.ConclusionThe results suggest that a GH-releaser diet attenuates oxidative stress, proapoptotic changes and consequent dysfunction in klotho mutant mice by promoting IGF-1 expression and IGF-1 receptor activation.

Mice Expressing a "Hyper-Sensitive" Form of the Cannabinoid Receptor 1 (CB1 Are Neither Obese Nor Diabetic.

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David J Marcus

Full Text Available Multiple lines of evidence implicate the endocannabinoid signaling system in the modulation of metabolic disease. Genetic or pharmacological inactivation of CB1 in rodents leads to reduced body weight, resistance to diet-induced obesity, decreased intake of highly palatable food, and increased energy expenditure. Cannabinoid agonists stimulate feeding in rodents and increased levels of endocannabinoids can disrupt lipid metabolism. Therefore, the hypothesis that sustained endocannabinoid signaling can lead to obesity and diabetes was examined in this study using S426A/S430A mutant mice expressing a desensitization-resistant CB1 receptor. These mice display exaggerated and prolonged responses to acute administration of phytocannabinoids, synthetic cannabinoids, and endocannabinoids. As a consequence these mice represent a novel model for determining the effect of enhanced endocannabinoid signaling on metabolic disease. S426A/S430A mutants consumed equivalent amounts of both high fat (45% and low fat (10% chow control diet compared to wild-type littermate controls. S426A/S430A mutants and wild-type mice fed either high or low fat control diet displayed similar fasting blood glucose levels and normal glucose clearance following a 2 g/kg glucose challenge. Furthermore, S426A/S430A mutants and wild-type mice consumed similar amounts of chow following an overnight fast. While both THC and JZL195 significantly increased food intake two hours after injection, this increase was similar between the S426A/S430A mutant and wildtype control mice Our results indicate that S426A/S430A mutant mice expressing the desensitization-resistant form of CB1 do not exhibit differences in body weight, food intake, glucose homeostasis, or re-feeding following a fast.

BAX and tumor suppressor TRP53 are important in regulating mutagenesis in spermatogenic cells in mice.

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Xu, Guogang; Vogel, Kristine S; McMahan, C Alex; Herbert, Damon C; Walter, Christi A

2010-12-01

During the first wave of spermatogenesis, and in response to ionizing radiation, elevated mutant frequencies are reduced to a low level by unidentified mechanisms. Apoptosis is occurring in the same time frame that the mutant frequency declines. We examined the role of apoptosis in regulating mutant frequency during spermatogenesis. Apoptosis and mutant frequencies were determined in spermatogenic cells obtained from Bax-null or Trp53-null mice. The results showed that spermatogenic lineage apoptosis was markedly decreased in Bax-null mice and was accompanied by a significantly increased spontaneous mutant frequency in seminiferous tubule cells compared to that of wild-type mice. Apoptosis profiles in the seminiferous tubules for Trp53-null were similar to control mice. Spontaneous mutant frequencies in pachytene spermatocytes and in round spermatids from Trp53-null mice were not significantly different from those of wild-type mice. However, epididymal spermatozoa from Trp53-null mice displayed a greater spontaneous mutant frequency compared to that from wild-type mice. A greater proportion of spontaneous transversions and a greater proportion of insertions/deletions 15 days after ionizing radiation were observed in Trp53-null mice compared to wild-type mice. Base excision repair activity in mixed germ cell nuclear extracts prepared from Trp53-null mice was significantly lower than that for wild-type controls. These data indicate that BAX-mediated apoptosis plays a significant role in regulating spontaneous mutagenesis in seminiferous tubule cells obtained from neonatal mice, whereas tumor suppressor TRP53 plays a significant role in regulating spontaneous mutagenesis between postmeiotic round spermatid and epididymal spermatozoon stages of spermiogenesis.

Conditioned taste aversion memory and c-Fos induction are disrupted in RIIbeta-protein kinase A mutant mice.

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Koh, Ming Teng; Clarke, Sharon N D A; Spray, Kristina J; Thiele, Todd E; Bernstein, Ilene L

2003-07-14

The cAMP-dependent protein kinase (PKA) signaling pathway has been implicated in many forms of learning. The present studies examined conditioned taste aversion (CTA) learning, an amygdala-dependent task, in mice with a targeted disruption of a gene for a specific regulatory subunit of PKA (RIIbeta), which is selectively expressed in amygdala. Null mutant (RIIbeta(-/-)) mice and littermate controls (RIIbeta(+/+)) were tested for protein synthesis-independent short-term memory (STM) and protein synthesis-dependent long-term memory (LTM) for CTAs. The ability of the unconditioned stimulus (US) drug, LiCl, to induce c-Fos in regions thought to be important in this learning was also determined. RIIbeta(-/-) mice showed significant impairment in CTA memory when tested 24h after training (LTM). In contrast, STM was normal. With regard to the c-Fos response to LiCl, the US drug, significant elevations were evident in brainstem (nucleus of the solitary tract) and pontine (parabrachial nucleus) regions, in mutants as well as wild-type controls. However, in amygdala, elevations were seen in controls but were absent in the mutants. These findings suggest that disruption of PKA signaling interferes with LTM consolidation of CTA and that a possible mediator of this effect is interference with c-Fos expression in amygdala which may be necessary for CTA memory.

Characteristics of gait ataxia in δ2 glutamate receptor mutant mice, ho15J.

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Eri Takeuchi

Full Text Available The cerebellum plays a fundamental, but as yet poorly understood, role in the control of locomotion. Recently, mice with gene mutations or knockouts have been used to investigate various aspects of cerebellar function with regard to locomotion. Although many of the mutant mice exhibit severe gait ataxia, kinematic analyses of limb movements have been performed in only a few cases. Here, we investigated locomotion in ho15J mice that have a mutation of the δ2 glutamate receptor. The cerebellum of ho15J mice shows a severe reduction in the number of parallel fiber-Purkinje synapses compared with wild-type mice. Analysis of hindlimb kinematics during treadmill locomotion showed abnormal hindlimb movements characterized by excessive toe elevation during the swing phase, and by severe hyperflexion of the ankles in ho15J mice. The great trochanter heights in ho15J mice were lower than in wild-type mice throughout the step cycle. However, there were no significant differences in various temporal parameters between ho15J and wild-type mice. We suggest that dysfunction of the cerebellar neuronal circuits underlies the observed characteristic kinematic abnormality of hindlimb movements during locomotion of ho15J mice.

Female preproenkephalin-knockout mice display altered emotional responses

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Ragnauth, A.; Schuller, A.; Morgan, M.; Chan, J.; Ogawa, S.; Pintar, J.; Bodnar, R. J.; Pfaff, D. W.

2001-01-01

The endogenous opioid system has been implicated in sexual behavior, palatable intake, fear, and anxiety. The present study examined whether ovariectomized female transgenic preproenkephalin-knockout (PPEKO) mice and their wild-type and heterozygous controls displayed alterations in fear and anxiety paradigms, sucrose intake, and lordotic behavior. To examine stability of responding, three squads of the genotypes were tested across seasons over a 20-month period. In a fear-conditioning paradigm, PPEKO mice significantly increased freezing to both fear and fear + shock stimuli relative to controls. In the open field, PPEKO mice spent significantly less time and traversed significantly less distance in the center of an open field than wild-type controls. Further, PPEKO mice spent significantly less time and tended to be less active on the light side of a dark–light chamber than controls, indicating that deletion of the enkephalin gene resulted in exaggerated responses to fear or anxiety-provoking environments. These selective deficits were observed consistently across testing squads spanning 20 months and different seasons. In contrast, PPEKO mice failed to differ from corresponding controls in sucrose, chow, or water intake across a range (0.0001–20%) of sucrose concentrations and failed to differ in either lordotic or female approach to male behaviors when primed with estradiol and progesterone, thereby arguing strongly for the selectivity of a fear and anxiety deficit which was not caused by generalized and nonspecific debilitation. These transgenic data strongly suggest that opioids, and particularly enkephalin gene products, are acting naturally to inhibit fear and anxiety. PMID:11172058

Enhanced Adsorption and Recovery of Uranyl Ions by NikR Mutant-Displaying Yeast

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Kouichi Kuroda

2014-04-01

Full Text Available Uranium is one of the most important metal resources, and the technology for the recovery of uranyl ions (UO22+ from aqueous solutions is required to ensure a semi-permanent supply of uranium. The NikR protein is a Ni2+-dependent transcriptional repressor of the nickel-ion uptake system in Escherichia coli, but its mutant protein (NikRm is able to selectively bind uranyl ions in the interface of the two monomers. In this study, NikRm protein with ability to adsorb uranyl ions was displayed on the cell surface of Saccharomyces cerevisiae. To perform the binding of metal ions in the interface of the two monomers, two metal-binding domains (MBDs of NikRm were tandemly fused via linker peptides and displayed on the yeast cell surface by fusion with the cell wall-anchoring domain of yeast α-agglutinin. The NikRm-MBD-displaying yeast cells with particular linker lengths showed the enhanced adsorption of uranyl ions in comparison to the control strain. By treating cells with citrate buffer (pH 4.3, the uranyl ions adsorbed on the cell surface were recovered. Our results indicate that the adsorption system by yeast cells displaying tandemly fused MBDs of NikRm is effective for simple and concentrated recovery of uranyl ions, as well as adsorption of uranyl ions.

Serum cholinesterases are differentially regulated in normal and dystrophin-deficient mutant mice

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Andrea R. Durrant

2012-06-01

Full Text Available The cholinesterases, acetylcholinesterase and butyrylcholinesterase (pseudocholinesterase, are abundant in the nervous system and in other tissues. The role of acetylcholinesterase in terminating transmitter action in the peripheral and central nervous system is well understood. However, both knowledge of the function(s of the cholinesterases in serum, and of their metabolic and endocrine regulation under normal and pathological conditions, is limited. This study investigates acetylcholinesterase and butyrylcholinesterase in sera of dystrophin-deficient mdx mutant mice, an animal model for the human Duchenne muscular dystrophy and in control healthy mice. The data show systematic and differential variations in the concentrations of both enzymes in the sera, and specific changes dictated by alteration of hormonal balance in both healthy and dystrophic mice. While acetylcholinesterase in mdx-sera is elevated, butyrylcholinesterase is markedly diminished, resulting in an overall cholinesterase decrease compared to sera of healthy controls. The androgen testosterone (T is a negative modulator of butyrylcholinesterase, but not of acetylcholinesterase, in male mouse sera. T-removal elevated both butyrylcholinesterase activity and the butyrylcholinesterase/acetylcholinesterase ratio in mdx male sera to values resembling those in healthy control male mice. Mechanisms of regulation of the circulating cholinesterases and their impairment in the dystrophic mice are suggested, and clinical implications for diagnosis and treatment are considered.

Radio-sensitivity of the cells from amyotrophic lateral sclerosis model mice transfected with human mutant SOD1

International Nuclear Information System (INIS)

Wate, Reika; Ito, Hidefumi; Kusaka, Hirofumi; Takahashi, Sentaro; Kubota, Yoshihisa; Suetomi, Katsutoshi; Sato, Hiroshi; Okayasu, Ryuichi

2005-01-01

In order to clarify the possible involvement of oxidative damage induced by ionizing radiation in the onset and/or progression of familial amyotrophic lateral sclerosis (ALS), we studied radio-sensitivity in primary cells derived from ALS model mice expressing human mutant Cu/Zn superoxide dismutase (SOD1). The primary mouse cells expressed both mouse and the mutant human SOD1. The cell survival of the transgenic mice (with mutant SOD1), determined by counting cell numbers at a scheduled time after X-irradiation, is very similar to that of cells from wild type animals. The induction and repair of DNA damage in the transgenic cells, measured by single cell gel electrophoresis and pulsed field gel electrophoresis, are also similar to those of wild type cells. These results indicate that the human mutant SOD1 gene does not seem to contribute to the alteration of radio-sensitivity, at least in the fibroblastic cells used here. Although it is necessary to consider the difference in cell types between fibroblastic and neuronal cells, the present results may suggest that ionizing radiation is not primarily responsible for the onset of familial ALS with the SOD1 mutation, and that the excess risks are probably not a concern for radiation diagnosis and therapy in familial ALS patients. (author)

Hypolipidemic effects of starch and γ-oryzanol from wx/ae double-mutant rice on BALB/c.KOR-Apoe(shl) mice.

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Nakaya, Makoto; Shojo, Aiko; Hirai, Hiroaki; Matsumoto, Kenji; Kitamura, Shinichi

2013-01-01

waxy/amylose-extender (wx/ae) double-mutant japonica rice (Oryza sativa L.) produces resistant starch (RS) and a large amount of γ-oryzanol. Our previous study has shown the hypolipidemic effect of wx/ae brown rice on mice. To identify the functional constituents of the hypolipidemic activity in wx/ae rice, we prepared pure wx/ae starch and γ-oryzanol from wx/ae rice and investigated their effect on the lipid metabolism in BALB/c.KOR/Stm Slc-Apoe(shl) mice. The mice were fed for 3 weeks a diet containing non-mutant rice starch, non-mutant rice starch plus γ-oryzanol, wx/ae starch, or wx/ae starch plus γ-oryzanol. γ-Oryzanol by itself had no effect on the lipid metabolism, and wx/ae starch prevented an accumulation of triacylglycerol (TAG) in the liver. Interestingly, the combination of wx/ae starch plus γ-oryzanol not only prevented a TAG accumulation in the liver, but also partially suppressed the rise in plasma TAG concentration, indicating that wx/ae starch and γ-oryzanol could have a synergistic effect on the lipid metabolism.

Ectopic norrin induces growth of ocular capillaries and restores normal retinal angiogenesis in Norrie disease mutant mice.

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Ohlmann, Andreas; Scholz, Michael; Goldwich, Andreas; Chauhan, Bharesh K; Hudl, Kristiane; Ohlmann, Anne V; Zrenner, Eberhart; Berger, Wolfgang; Cvekl, Ales; Seeliger, Mathias W; Tamm, Ernst R

2005-02-16

Norrie disease is an X-linked retinal dysplasia that presents with congenital blindness, sensorineural deafness, and mental retardation. Norrin, the protein product of the Norrie disease gene (NDP), is a secreted protein of unknown biochemical function. Norrie disease (Ndp(y/-)) mutant mice that are deficient in norrin develop blindness, show a distinct failure in retinal angiogenesis, and completely lack the deep capillary layers of the retina. We show here that the transgenic expression of ectopic norrin under control of a lens-specific promoter restores the formation of a normal retinal vascular network in Ndp(y/-) mutant mice. The improvement in structure correlates with restoration of neuronal function in the retina. In addition, lenses of transgenic mice with ectopic expression of norrin show significantly more capillaries in the hyaloid vasculature that surrounds the lens during development. In vitro, lenses of transgenic mice in coculture with microvascular endothelial cells induce proliferation of the cells. Transgenic mice with ectopic expression of norrin show more bromodeoxyuridine-labeled retinal progenitor cells at embryonic day 14.5 and thicker retinas at postnatal life than wild-type littermates, indicating a putative direct neurotrophic effect of norrin. These data provide direct evidence that norrin induces growth of ocular capillaries and that pharmacologic modulation of norrin might be used for treatment of the vascular abnormalities associated with Norrie disease or other vascular disorders of the retina.

Social approach behaviors are similar on conventional versus reverse lighting cycles, and in replications across cohorts, in BTBR T+ tf/J, C57BL/6J, and vasopressin receptor 1B mutant mice

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Mu Yang

2007-11-01

Full Text Available Mice are a nocturnal species, whose social behaviors occur primarily during the dark phase of the circadian cycle. However, laboratory rodents are frequently tested during their light phase, for practical reasons. We investigated the question of whether light phase testing presents a methodological pitfall for investigating mouse social approach behaviors. Three lines of mice were systematically compared. One cohort of each line was raised in a conventional lighting schedule and tested during the light phase, under white light illumination; another cohort was raised in a reverse lighting schedule and tested during their dark phase, under dim red light. Male C57BL/6J (B6 displayed high levels of sociability in our three-chambered automated social approach task when tested in either phase. BTBR T+ tf/J (BTBR displayed low levels of sociability in either phase. Five cohorts of vasopressin receptor subtype 1b (Avpr1b null mutants, heterozygotes, and wildtype littermate controls were tested in the same social approach paradigm: three in the dark phase and two in the light phase. All three genotypes displayed normal sociability in four out of the five replications. In the juvenile play test, testing phase had no effect on play soliciting behaviors in Avpr1b mice, but had modest effects on nose sniff and huddling. Taken together, these findings indicate that testing phase is not a crucial factor for studying some forms of social approach in juvenile and adult mice.

Mice deficient in phosphodiesterase-4A display anxiogenic-like behavior.

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Hansen, Rolf T; Conti, Marco; Zhang, Han-Ting

2014-08-01

Phosphodiesterases (PDEs) are a super family of enzymes responsible for the halting of intracellular cyclic nucleotide signaling and may represent novel therapeutic targets for treatment of cognitive disorders. PDE4 is of considerable interest to cognitive research because it is highly expressed in the brain, particularly in the cognition-related brain regions. Recently, the functional role of PDE4B and PDE4D, two of the four PDE4 subtypes (PDE4A, B, C, and D), in behavior has begun to be identified; however, the role of PDE4A in the regulation of behavior is still unknown. The purpose of this study was to characterize the functional role of PDE4A in behavior. The role of PDE4A in behavior was evaluated through a battery of behavioral tests using PDE4A knockout (KO) mice; urine corticosterone levels were also measured. PDE4A KO mice exhibited improved memory in the step-through-passive-avoidance test. They also displayed anxiogenic-like behavior in elevated-plus maze, holeboard, light-dark transition, and novelty suppressed feeding tests. Consistent with the anxiety profile, PDE4A KO mice had elevated corticosterone levels compared with wild-type controls post-stress. Interestingly, PDE4A KO mice displayed no change in object recognition, Morris water maze, forced swim, tail suspension, and duration of anesthesia induced by co-administration of xylazine and ketamine (suggesting that PDE4A KO may not be emetic). These results suggest that PDE4A may be important in the regulation of emotional memory and anxiety-like behavior, but not emesis. PDE4A could possibly represent a novel therapeutic target in the future for anxiety or disorders affecting memory.

Dopaminergic neuronal loss, reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2.

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David Ramonet

2011-04-01

Full Text Available Mutations in the leucine-rich repeat kinase 2 (LRRK2 gene cause late-onset, autosomal dominant familial Parkinson's disease (PD and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s through which familial mutations precipitate neuronal degeneration and PD.

Inactivation of JAK2/STAT3 Signaling Axis and Downregulation of M1 mAChR Cause Cognitive Impairment in klotho Mutant Mice, a Genetic Model of Aging

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Park, Seok-Joo; Shin, Eun-Joo; Min, Sun Seek; An, Jihua; Li, Zhengyi; Hee Chung, Yoon; Hoon Jeong, Ji; Bach, Jae-Hyung; Nah, Seung-Yeol; Kim, Won-Ki; Jang, Choon-Gon; Kim, Yong-Sun; Nabeshima, Yo-ichi; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2013-01-01

We previously reported cognitive dysfunction in klotho mutant mice. In the present study, we further examined novel mechanisms involved in cognitive impairment in these mice. Significantly decreased janus kinase 2 (JAK2) and signal transducer and activator of transcription3 (STAT3) phosphorylation were observed in the hippocampus of klotho mutant mice. A selective decrease in protein expression and binding density of the M1 muscarinic cholinergic receptor (M1 mAChR) was observed in these mice. Cholinergic parameters (ie, acetylcholine (ACh), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE)) and NMDAR-dependent long-term potentiation (LTP) were significantly impaired in klotho mutant mice. McN-A-343 (McN), an M1 mAChR agonist, significantly attenuated these impairments. AG490 (AG), a JAK2 inhibitor, counteracted the attenuating effects of McN, although AG did not significantly alter the McN-induced effect on AChE. Furthermore, AG significantly inhibited the attenuating effects of McN on decreased NMDAR-dependent LTP, protein kinase C βII, p-ERK, p-CREB, BDNF, and p-JAK2/p-STAT3-expression in klotho mutant mice. In addition, k252a, a BDNF receptor tyrosine kinase B (TrkB) inhibitor, significantly counteracted McN effects on decreased ChAT, ACh, and M1 mAChR and p-JAK2/p-STAT3 expression. McN-induced effects on cognitive impairment in klotho mutant mice were consistently counteracted by either AG or k252a. Our results suggest that inactivation of the JAK2/STAT3 signaling axis and M1 mAChR downregulation play a critical role in cognitive impairment observed in klotho mutant mice. PMID:23389690

Lef1 haploinsufficient mice display a low turnover and low bone mass phenotype in a gender- and age-specific manner.

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Tommy Noh

Full Text Available We investigated the role of Lef1, one of the four transcription factors that transmit Wnt signaling to the genome, in the regulation of bone mass. Microcomputed tomographic analysis of 13- and 17-week-old mice revealed significantly reduced trabecular bone mass in Lef1(+/- females compared to littermate wild-type females. This was attributable to decreased osteoblast activity and bone formation as indicated by histomorphometric analysis of bone remodeling. In contrast to females, bone mass was unaffected by Lef1 haploinsufficiency in males. Similarly, females were substantially more responsive than males to haploinsufficiency in Gsk3beta, a negative regulator of the Wnt pathway, displaying in this case a high bone mass phenotype. Lef1 haploinsufficiency also led to low bone mass in males lacking functional androgen receptor (AR (tfm mutants. The protective skeletal effect of AR against Wnt-related low bone mass is not necessarily a result of direct interaction between the AR and Wnt signaling pathways, because Lef1(+/- female mice had normal bone mass at the age of 34 weeks. Thus, our results indicate an age- and gender-dependent role for Lef1 in regulating bone formation and bone mass in vivo. The resistance to Lef1 haploinsufficiency in males with active AR and in old females could be due to the reduced bone turnover in these mice.

A mutation in the dynein heavy chain gene compensates for energy deficit of mutant SOD1 mice and increases potentially neuroprotective IGF-1

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Larmet Yves

2011-04-01

Full Text Available Abstract Background Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons. ALS patients, as well as animal models such as mice overexpressing mutant SOD1s, are characterized by increased energy expenditure. In mice, this hypermetabolism leads to energy deficit and precipitates motor neuron degeneration. Recent studies have shown that mutations in the gene encoding the dynein heavy chain protein are able to extend lifespan of mutant SOD1 mice. It remains unknown whether the protection offered by these dynein mutations relies on a compensation of energy metabolism defects. Results SOD1(G93A mice were crossbred with mice harboring the dynein mutant Cramping allele (Cra/+ mice. Dynein mutation increased adipose stores in compound transgenic mice through increasing carbohydrate oxidation and sparing lipids. Metabolic changes that occurred in double transgenic mice were accompanied by the normalization of the expression of key mRNAs in the white adipose tissue and liver. Furthermore, Dynein Cra mutation rescued decreased post-prandial plasma triglycerides and decreased non esterified fatty acids upon fasting. In SOD1(G93A mice, the dynein Cra mutation led to increased expression of IGF-1 in the liver, increased systemic IGF-1 and, most importantly, to increased spinal IGF-1 levels that are potentially neuroprotective. Conclusions These findings suggest that the protection against SOD1(G93A offered by the Cramping mutation in the dynein gene is, at least partially, mediated by a reversal in energy deficit and increased IGF-1 availability to motor neurons.

Ultrasonic vocalizations of adult male Foxp2-mutant mice: behavioral contexts of arousal and emotion.

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Gaub, S; Fisher, S E; Ehret, G

2016-02-01

Adult mouse ultrasonic vocalizations (USVs) occur in multiple behavioral and stimulus contexts associated with various levels of arousal, emotion and social interaction. Here, in three experiments of increasing stimulus intensity (water; female urine; male interacting with adult female), we tested the hypothesis that USVs of adult males express the strength of arousal and emotion via different USV parameters (18 parameters analyzed). Furthermore, we analyzed two mouse lines with heterozygous Foxp2 mutations (R552H missense, S321X nonsense), known to produce severe speech and language disorders in humans. These experiments allowed us to test whether intact Foxp2 function is necessary for developing full adult USV repertoires, and whether mutations of this gene influence instinctive vocal expressions based on arousal and emotion. The results suggest that USV calling rate characterizes the arousal level, while sound pressure and spectrotemporal call complexity (overtones/harmonics, type of frequency jumps) may provide indices of levels of positive emotion. The presence of Foxp2 mutations did not qualitatively affect the USVs; all USV types that were found in wild-type animals also occurred in heterozygous mutants. However, mice with Foxp2 mutations displayed quantitative differences in USVs as compared to wild-types, and these changes were context dependent. Compared to wild-type animals, heterozygous mutants emitted mainly longer and louder USVs at higher minimum frequencies with a higher occurrence rate of overtones/harmonics and complex frequency jump types. We discuss possible hypotheses about Foxp2 influence on emotional vocal expressions, which can be investigated in future experiments using selective knockdown of Foxp2 in specific brain circuits. © 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Transient elevation of element contents as a result of neuronal death in mutant-mice cerebellum studied by neutron activation analysis

International Nuclear Information System (INIS)

Kranda, K.; Kucera, J.; Baeurle, J.

2006-01-01

Accumulation of some metals, in particular iron or manganese, has long been considered to trigger or accentuate neurodegenerative processes in humans. The two most frequently cited examples are Parkinson's and Alzheimer diseases, where excitotoxic processes lead to neuronal death. However, these neuropathies are somewhat unsuitable for investigating the time course of the metal accumulation because the applied analytical methods such as neutron activation analysis (NAA) are invasive. Hence, only one measurement can be made after the patient's death. Animal models of Parkinson's type neurodegeneration, such as mice mutants, are more suitable as a larger number of animals can be investigated at various postnatal ages. In this study we used one type of mice mutants weaver, where primary neurodegeneration is principally confined to the cerebellum and centred in time around the postnatal age of six days. Elemental composition of brain segments with dry mass as low as 0.5 mg, which were isolated from weaver and wild type (normal) mice were investigated using a combination of INAA and RNAA. Elevated concentration of the following elements Fe, Zn, Cu, K, Na, Rb, and Br that were observed in the weaver's cerebella closely followed the time course of neurodegeneration documented for this type of mutant. The transient elevation of these elements never preceded the onset of neurodegeneration but closely mirrored its time course reaching its peak on the sixth day. The concentration of these elements in the weaver's cerebella declined afterwards to converge on the elemental time course observed in the wild type mice. In conclusion, metal and other elemental elevation observed in the cerebella of these mutants are an expression of neurodegenerative processes rather than its precondition. (author)

Hyperactivity and learning deficits in transgenic mice bearing a human mutant thyroid hormone beta1 receptor gene.

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McDonald, M P; Wong, R; Goldstein, G; Weintraub, B; Cheng, S Y; Crawley, J N

1998-01-01

Resistance to thyroid hormone (RTH) is a human syndrome mapped to the thyroid receptor beta (TRbeta) gene on chromosome 3, representing a mutation of the ligand-binding domain of the TRbeta gene. The syndrome is characterized by reduced tissue responsiveness to thyroid hormone and elevated serum levels of thyroid hormones. A common behavioral phenotype associated with RTH is attention deficit hyperactivity disorder (ADHD). To test the hypothesis that RTH produces attention deficits and/or hyperactivity, transgenic mice expressing a mutant TRbeta gene were generated. The present experiment tested RTH transgenic mice from the PV kindred on behavioral tasks relevant to the primary features of ADHD: hyperactivity, sustained attention (vigilance), learning, and impulsivity. Male transgenic mice showed elevated locomotor activity in an open field compared to male wild-type littermate controls. Both male and female transgenic mice exhibited impaired learning of an autoshaping task, compared to wild-type controls. On a vigilance task in an operant chamber, there were no differences between transgenics and controls on the proportion of hits, response latency, or duration of stimulus tolerated. On an operant go/no-go task measuring sustained attention and impulsivity, there were no differences between controls and transgenics. These results indicate that transgenic mice bearing a mutant human TRbeta gene demonstrate several behavioral characteristics of ADHD and may serve a valuable heuristic role in elucidating possible candidate genes in converging pathways for other causes of ADHD.

Hyperactivity and Learning Deficits in Transgenic Mice Bearing a Human Mutant Thyroid Hormone β1 Receptor Gene

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McDonald, Michael P.; Wong, Rosemary; Goldstein, Gregory; Weintraub, Bruce; Cheng, Sheue-yann; Crawley, Jacqueline N.

1998-01-01

Resistance to thyroid hormone (RTH) is a human syndrome mapped to the thyroid receptor β (TRβ) gene on chromosome 3, representing a mutation of the ligandbinding domain of the TRβ gene. The syndrome is characterized by reduced tissue responsiveness to thyroid hormone and elevated serum levels of thyroid hormones. A common behavioral phenotype associated with RTH is attention deficit hyperactivity disorder (ADHD). To test the hypothesis that RTH produces attention deficits and/or hyperactivity, transgenic mice expressing a mutant TRβ gene were generated. The present experiment tested RTH transgenic mice from the PV kindred on behavioral tasks relevant to the primary features of ADHD: hyperactivity, sustained attention (vigilance), learning, and impulsivity. Male transgenic mice showed elevated locomotor activity in an open field compared to male wild-type littermate controls. Both male and female transgenic mice exhibited impaired learning of an autoshaping task, compared to wild-type controls. On a vigilance task in an operant chamber, there were no differences between transgenics and controls on the proportion of hits, response latency, or duration of stimulus tolerated. On an operant go/no-go task measuring sustained attention and impulsivity, there were no differences between controls and transgenics. These results indicate that transgenic mice bearing a mutant human TRβ gene demonstrate several behavioral characteristics of ADHD and may serve a valuable heuristic role in elucidating possible candidate genes in converging pathways for other causes of ADHD. PMID:10454355

Three Herpes Simplex Virus Type 1 Latency-Associated Transcript Mutants with Distinct and Asymmetric Effects on Virulence in Mice Compared with Rabbits

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Perng, Guey-Chuen; Esmaili, Daniel; Slanina, Susan M.; Yukht, Ada; Ghiasi, Homayon; Osorio, Nelson; Mott, Kevin R.; Maguen, Barak; Jin, Ling; Nesburn, Anthony B.; Wechsler, Steven L.

2001-01-01

Herpes simplex virus type 1 latency-associated transcript (LAT)-null mutants have decreased reactivation but normal virulence in rabbits and mice. We report here on dLAT1.5, a mutant with LAT nucleotides 76 to 1667 deleted. Following ocular infection of rabbits, dLAT1.5 reactivated at a lower rate than its wild-type parent McKrae (6.1 versus 11.8%; P = 0.0025 [chi-square test]). Reactivation was restored in the marker-rescued virus dLAT1.5R (12.6%; P = 0.53 versus wild type), confirming the importance of the deleted region in spontaneous reactivation. Compared with wild-type or marker-rescued virus, dLAT1.5 had similar or slightly reduced virulence in rabbits (based on survival following ocular infection). In contrast, in mice, dLAT1.5 had increased virulence (P Wechsler, J. Virol. 73:920–929, 1999), had decreased virulence in mice (P = 0.03). In addition, we also found that dLAT371, a LAT mutant that we previously reported to have wild-type virulence in rabbits (G. C. Perng, S. M. Slanina, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 70:2014–2018, 1996), had decreased virulence in mice (P < 0.05). Thus, these three mutants, each of which encodes a different LAT RNA, have different virulence phenotypes. dLAT1.5 had wild-type virulence in rabbits but increased virulence in mice. In contrast, LAT2.9A had increased virulence in rabbits but decreased virulence in mice, and dLAT371 had wild-type virulence in rabbits but decreased virulence in mice. Taken together, these results suggest that (i) the 5′ end of LAT and/or a gene that overlaps part of this region is involved in viral virulence, (ii) this virulence appears to have species-specific effects, and (iii) regulation of this virulence may be complex. PMID:11533165

Overexpression of survival motor neuron improves neuromuscular function and motor neuron survival in mutant SOD1 mice.

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Turner, Bradley J; Alfazema, Neza; Sheean, Rebecca K; Sleigh, James N; Davies, Kay E; Horne, Malcolm K; Talbot, Kevin

2014-04-01

Spinal muscular atrophy results from diminished levels of survival motor neuron (SMN) protein in spinal motor neurons. Low levels of SMN also occur in models of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1) and genetic reduction of SMN levels exacerbates the phenotype of transgenic SOD1(G93A) mice. Here, we demonstrate that SMN protein is significantly reduced in the spinal cords of patients with sporadic ALS. To test the potential of SMN as a modifier of ALS, we overexpressed SMN in 2 different strains of SOD1(G93A) mice. Neuronal overexpression of SMN significantly preserved locomotor function, rescued motor neurons, and attenuated astrogliosis in spinal cords of SOD1(G93A) mice. Despite this, survival was not prolonged, most likely resulting from SMN mislocalization and depletion of gems in motor neurons of symptomatic mice. Our results reveal that SMN upregulation slows locomotor deficit onset and motor neuron loss in this mouse model of ALS. However, disruption of SMN nuclear complexes by high levels of mutant SOD1, even in the presence of SMN overexpression, might limit its survival promoting effects in this specific mouse model. Studies in emerging mouse models of ALS are therefore warranted to further explore the potential of SMN as a modifier of ALS. Copyright © 2014 Elsevier Inc. All rights reserved.

Bacteriophage-resistant mutants in Yersinia pestis: identification of phage receptors and attenuation for mice.

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Andrey A Filippov

Full Text Available BACKGROUND: Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. METHODOLOGY/PRINCIPAL FINDINGS: The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD₅₀ and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. CONCLUSIONS/SIGNIFICANCE: We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.

Insulin signaling displayed a differential tissue-specific response to low-dose dihydrotestosterone in female mice.

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Andrisse, Stanley; Billings, Katelyn; Xue, Ping; Wu, Sheng

2018-04-01

Hyperandrogenemia and hyperinsulinemia are believed to play prominent roles in polycystic ovarian syndrome (PCOS). We explored the effects of low-dose dihydrotestosterone (DHT), a model of PCOS, on insulin signaling in metabolic and reproductive tissues in a female mouse model. Insulin resistance in the energy storage tissues is associated with type 2 diabetes. Insulin signaling in the ovaries and pituitary either directly or indirectly stimulates androgen production. Energy storage and reproductive tissues were isolated and molecular assays were performed. Livers and white adipose tissue (WAT) from DHT mice displayed lower mRNA and protein expression of insulin signaling intermediates. However, ovaries and pituitaries of DHT mice exhibited higher expression levels of insulin signaling genes/proteins. Insulin-stimulated p-AKT levels were blunted in the livers and WAT of the DHT mice but increased or remained the same in the ovaries and pituitaries compared with controls. Glucose uptake decreased in liver and WAT but was unchanged in pituitary and ovary of DHT mice. Plasma membrane GLUTs were decreased in liver and WAT but increased in ovary and pituitary of DHT mice. Skeletal muscle insulin-signaling genes were not lowered in DHT mice compared with control. DHT mice did not display skeletal muscle insulin resistance. Insulin-stimulated glucose transport increased in skeletal muscles of DHT mice compared with controls. DHT mice were hyperinsulinemic. However, the differential mRNA and protein expression pattern was independent of hyperinsulinemia in cultured hepatocytes and pituitary cells. These findings demonstrate a differential effect of DHT on the insulin-signaling pathway in energy storage vs. reproductive tissues independent of hyperinsulinemia.

Zebrafish mutants in the von Hippel-Lindau tumor suppressor display a hypoxic response and recapitulate key aspects of Chuvash polycythemia

NARCIS (Netherlands)

van Rooijen, E.; Voest, E.E.; Logister, I.; Korving, J.; Schwerte, T.; Schulte-Merker, S.; Giles, R.H.; van Eeden, F.J.

2009-01-01

We have generated 2 zebrafish lines carrying inactivating germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene ortholog vhl. Mutant embryos display a general systemic hypoxic response, including the up-regulation of hypoxia-induced genes by 1 day after fertilization and a severe

Mice genetically depleted of brain serotonin display social impairments, communication deficits and repetitive behaviors: possible relevance to autism.

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Michael J Kane

Full Text Available Autism is a complex neurodevelopmental disorder characterized by impaired reciprocal social interaction, communication deficits and repetitive behaviors. A very large number of genes have been linked to autism, many of which encode proteins involved in the development and function of synaptic circuitry. However, the manner in which these mutated genes might participate, either individually or together, to cause autism is not understood. One factor known to exert extremely broad influence on brain development and network formation, and which has been linked to autism, is the neurotransmitter serotonin. Unfortunately, very little is known about how alterations in serotonin neuronal function might contribute to autism. To test the hypothesis that serotonin dysfunction can contribute to the core symptoms of autism, we analyzed mice lacking brain serotonin (via a null mutation in the gene for tryptophan hydroxylase 2 (TPH2 for behaviors that are relevant to this disorder. Mice lacking brain serotonin (TPH2-/- showed substantial deficits in numerous validated tests of social interaction and communication. These mice also display highly repetitive and compulsive behaviors. Newborn TPH2-/- mutant mice show delays in the expression of key developmental milestones and their diminished preference for maternal scents over the scent of an unrelated female is a forerunner of more severe socialization deficits that emerge in weanlings and persist into adulthood. Taken together, these results indicate that a hypo-serotonin condition can lead to behavioral traits that are highly characteristic of autism. Our findings should stimulate new studies that focus on determining how brain hyposerotonemia during critical neurodevelopmental periods can alter the maturation of synaptic circuits known to be mis-wired in autism and how prevention of such deficits might prevent this disorder.

Preserved otolith organ function in caspase-3-deficient mice with impaired horizontal semicircular canal function.

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Armstrong, Patrick A; Wood, Scott J; Shimizu, Naoki; Kuster, Kael; Perachio, Adrian; Makishima, Tomoko

2015-06-01

Genetically engineered mice are valuable models for elucidation of auditory and vestibular pathology. Our goal was to establish a comprehensive vestibular function testing system in mice using: (1) horizontal angular vestibulo-ocular reflex (hVOR) to evaluate semicircular canal function and (2) otolith-ocular reflex (OOR) to evaluate otolith organ function and to validate the system by characterizing mice with vestibular dysfunction. We used pseudo off-vertical axis rotation to induce an otolith-only stimulus using a custom-made centrifuge. For the OOR, horizontal slow-phase eye velocity and vertical eye position were evaluated as a function of acceleration. Using this system, we characterized hVOR and OOR in the caspase-3 (Casp3) mutant mice. Casp3 (-/-) mice had severely impaired hVOR gain, while Casp3 (+/-) mice had an intermediate response compared to WT mice. Evaluation of OOR revealed that at low-to-mid frequencies and stimulus intensity, Casp3 mutants and WT mice had similar responses. At higher frequencies and stimulus intensity, the Casp3 mutants displayed mildly reduced otolith organ-related responses. These findings suggest that the Casp3 gene is important for the proper function of the semicircular canals but less important for the otolith organ function.

Preserved otolith organ function in caspase-3 deficient mice with impaired horizontal semicircular canal function

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Armstrong, Patrick A; Wood, Scott J; Shimizu, Naoki; Kuster, Kael; Perachio, Adrian; Makishima, Tomoko

2015-01-01

Genetically engineered mice are valuable models for elucidation of auditory and vestibular pathology. Our goal was to establish a comprehensive vestibular function testing system in mice using: 1) horizontal angular vestibular-ocular reflex (hVOR) to evaluate semicircular canal function, and 2) otolith-ocular reflex (OOR) to evaluate otolith organ function, and to validate the system by characterizing mice with vestibular dysfunction. We used pseudo-off vertical axis rotation (pOVAR) to induce an otolith-only stimulus using a custom-made centrifuge. For the OOR, horizontal slow phase eye velocity (HEV) and vertical eye position (VEP) was evaluated as a function of acceleration. Using this system, we characterized hVOR and OOR in the caspase-3 (Casp3) mutant mice. Casp3 −/− mice had severely impaired hVOR gain, while Casp3 +/− mice had an intermediate response compared to WT mice. Evaluation of OOR revealed that at low to mid frequencies and stimulus intensity, Casp3 mutants and WT mice had similar responses. At higher frequencies and stimulus intensity, the Casp3 mutants displayed mildly reduced otolith organ related responses. These findings suggest that the Casp3 gene is important for the proper function of the semicircular canals but less important for the otolith organ function. PMID:25827332

Mutant Mice Lacking the p53 C-Terminal Domain Model Telomere Syndromes

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Iva Simeonova

2013-06-01

Full Text Available Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53Δ31, a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis, hallmarks of syndromes caused by short telomeres. Indeed, p53Δ31/Δ31 mice had short telomeres and other phenotypic traits associated with the telomere disease dyskeratosis congenita and its severe variant the Hoyeraal-Hreidarsson syndrome. Heterozygous p53+/Δ31 mice were only mildly affected, but decreased levels of Mdm4, a negative regulator of p53, led to a dramatic aggravation of their symptoms. Importantly, several genes involved in telomere metabolism were downregulated in p53Δ31/Δ31 cells, including Dyskerin, Rtel1, and Tinf2, which are mutated in dyskeratosis congenita, and Terf1, which is implicated in aplastic anemia. Together, these data reveal that a truncating mutation can activate p53 and that p53 plays a major role in the regulation of telomere metabolism.

Elevated Fibroblast Growth Factor Signaling Is Critical for the Pathogenesis of the Dwarfism in Evc2/Limbin Mutant Mice.

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Zhang, Honghao; Kamiya, Nobuhiro; Tsuji, Takehito; Takeda, Haruko; Scott, Greg; Rajderkar, Sudha; Ray, Manas K; Mochida, Yoshiyuki; Allen, Benjamin; Lefebvre, Veronique; Hung, Irene H; Ornitz, David M; Kunieda, Tetsuo; Mishina, Yuji

2016-12-01

Ellis-van Creveld (EvC) syndrome is a skeletal dysplasia, characterized by short limbs, postaxial polydactyly, and dental abnormalities. EvC syndrome is also categorized as a ciliopathy because of ciliary localization of proteins encoded by the two causative genes, EVC and EVC2 (aka LIMBIN). While recent studies demonstrated important roles for EVC/EVC2 in Hedgehog signaling, there is still little known about the pathophysiological mechanisms underlying the skeletal dysplasia features of EvC patients, and in particular why limb development is affected, but not other aspects of organogenesis that also require Hedgehog signaling. In this report, we comprehensively analyze limb skeletogenesis in Evc2 mutant mice and in cell and tissue cultures derived from these mice. Both in vivo and in vitro data demonstrate elevated Fibroblast Growth Factor (FGF) signaling in Evc2 mutant growth plates, in addition to compromised but not abrogated Hedgehog-PTHrP feedback loop. Elevation of FGF signaling, mainly due to increased Fgf18 expression upon inactivation of Evc2 in the perichondrium, critically contributes to the pathogenesis of limb dwarfism. The limb dwarfism phenotype is partially rescued by inactivation of one allele of Fgf18 in the Evc2 mutant mice. Taken together, our data uncover a novel pathogenic mechanism to understand limb dwarfism in patients with Ellis-van Creveld syndrome.

Elevated Fibroblast Growth Factor Signaling Is Critical for the Pathogenesis of the Dwarfism in Evc2/Limbin Mutant Mice.

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Honghao Zhang

2016-12-01

Full Text Available Ellis-van Creveld (EvC syndrome is a skeletal dysplasia, characterized by short limbs, postaxial polydactyly, and dental abnormalities. EvC syndrome is also categorized as a ciliopathy because of ciliary localization of proteins encoded by the two causative genes, EVC and EVC2 (aka LIMBIN. While recent studies demonstrated important roles for EVC/EVC2 in Hedgehog signaling, there is still little known about the pathophysiological mechanisms underlying the skeletal dysplasia features of EvC patients, and in particular why limb development is affected, but not other aspects of organogenesis that also require Hedgehog signaling. In this report, we comprehensively analyze limb skeletogenesis in Evc2 mutant mice and in cell and tissue cultures derived from these mice. Both in vivo and in vitro data demonstrate elevated Fibroblast Growth Factor (FGF signaling in Evc2 mutant growth plates, in addition to compromised but not abrogated Hedgehog-PTHrP feedback loop. Elevation of FGF signaling, mainly due to increased Fgf18 expression upon inactivation of Evc2 in the perichondrium, critically contributes to the pathogenesis of limb dwarfism. The limb dwarfism phenotype is partially rescued by inactivation of one allele of Fgf18 in the Evc2 mutant mice. Taken together, our data uncover a novel pathogenic mechanism to understand limb dwarfism in patients with Ellis-van Creveld syndrome.

Elevated Fibroblast Growth Factor Signaling Is Critical for the Pathogenesis of the Dwarfism in Evc2/Limbin Mutant Mice

Science.gov (United States)

Zhang, Honghao; Kamiya, Nobuhiro; Tsuji, Takehito; Takeda, Haruko; Scott, Greg; Ray, Manas K.; Mochida, Yoshiyuki; Lefebvre, Veronique; Hung, Irene H.; Kunieda, Tetsuo; Mishina, Yuji

2016-01-01

Ellis-van Creveld (EvC) syndrome is a skeletal dysplasia, characterized by short limbs, postaxial polydactyly, and dental abnormalities. EvC syndrome is also categorized as a ciliopathy because of ciliary localization of proteins encoded by the two causative genes, EVC and EVC2 (aka LIMBIN). While recent studies demonstrated important roles for EVC/EVC2 in Hedgehog signaling, there is still little known about the pathophysiological mechanisms underlying the skeletal dysplasia features of EvC patients, and in particular why limb development is affected, but not other aspects of organogenesis that also require Hedgehog signaling. In this report, we comprehensively analyze limb skeletogenesis in Evc2 mutant mice and in cell and tissue cultures derived from these mice. Both in vivo and in vitro data demonstrate elevated Fibroblast Growth Factor (FGF) signaling in Evc2 mutant growth plates, in addition to compromised but not abrogated Hedgehog-PTHrP feedback loop. Elevation of FGF signaling, mainly due to increased Fgf18 expression upon inactivation of Evc2 in the perichondrium, critically contributes to the pathogenesis of limb dwarfism. The limb dwarfism phenotype is partially rescued by inactivation of one allele of Fgf18 in the Evc2 mutant mice. Taken together, our data uncover a novel pathogenic mechanism to understand limb dwarfism in patients with Ellis-van Creveld syndrome. PMID:28027321

Mice expressing a "hyper-sensitive" form of the CB1 cannabinoid receptor (CB1 show modestly enhanced alcohol preference and consumption.

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David J Marcus

Full Text Available We recently characterized S426A/S430A mutant mice expressing a desensitization-resistant form of the CB1 receptor. These mice display an enhanced response to endocannabinoids and ∆9-THC. In this study, S426A/S430A mutants were used as a novel model to test whether ethanol consumption, morphine dependence, and reward for these drugs are potentiated in mice with a "hyper-sensitive" form of CB1. Using an unlimited-access, two-bottle choice, voluntary drinking paradigm, S426A/S430A mutants exhibit modestly increased intake and preference for low (6% but not higher concentrations of ethanol. S426A/S430A mutants and wild-type mice show similar taste preference for sucrose and quinine, exhibit normal sensitivity to the hypothermic and ataxic effects of ethanol, and have normal blood ethanol concentrations following administration of ethanol. S426A/S430A mutants develop robust conditioned place preference for ethanol (2 g/kg, morphine (10 mg/kg, and cocaine (10 mg/kg, demonstrating that drug reward is not changed in S426A/S430A mutants. Precipitated morphine withdrawal is also unchanged in opioid-dependent S426A/S430A mutant mice. Although ethanol consumption is modestly changed by enhanced CB1 signaling, reward, tolerance, and acute sensitivity to ethanol and morphine are normal in this model.

Mutations in the Schmallenberg Virus Gc Glycoprotein Facilitate Cellular Protein Synthesis Shutoff and Restore Pathogenicity of NSs Deletion Mutants in Mice.

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Varela, Mariana; Pinto, Rute Maria; Caporale, Marco; Piras, Ilaria M; Taggart, Aislynn; Seehusen, Frauke; Hahn, Kerstin; Janowicz, Anna; de Souza, William Marciel; Baumgärtner, Wolfgang; Shi, Xiaohong; Palmarini, Massimo

2016-06-01

Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild-type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach high titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild-type SBV, as it has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate for both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an interferon (IFN) antagonist. Therefore, SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, for the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins, while the ability to control the production of IFN maps to the NSs protein. The identification of viral determinants of pathogenesis is key to the development of prophylactic and intervention measures. In this study, we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that mutations in the Gc

Expanding the body mass range: associations between BMR and tissue morphology in wild type and mutant dwarf mice (David mice).

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Meyer, Carola W; Neubronner, Juliane; Rozman, Jan; Stumm, Gabi; Osanger, Andreas; Stoeger, Claudia; Augustin, Martin; Grosse, Johannes; Klingenspor, Martin; Heldmaier, Gerhard

2007-02-01

We sought to identify associations of basal metabolic rate (BMR) with morphological traits in laboratory mice. In order to expand the body mass (BM) range at the intra-strain level, and to minimize relevant genetic variation, we used male and female wild type mice (C3HeB/FeJ) and previously unpublished ENU-induced dwarf mutant littermates (David mice), covering a body mass range from 13.5 g through 32.3 g. BMR was measured at 30 degrees C, mice were killed by means of CO(2 )overdose, and body composition (fat mass and lean mass) was subsequently analyzed by dual X-ray absorptiometry (DEXA), after which mice were dissected into 12 (males) and 10 (females) components, respectively. Across the 44 individuals, 43% of the variation in the basal rates of metabolism was associated with BM. The latter explained 47% to 98% of the variability in morphology of the different tissues. Our results demonstrate that sex is a major determinant of body composition and BMR in mice: when adjusted for BM, females contained many larger organs, more fat mass, and less lean mass compared to males. This could be associated with a higher mass adjusted BMR in females. Once the dominant effects of sex and BM on BMR and tissue mass were removed, and after accounting for multiple comparisons, no further significant association between individual variation in BMR and tissue mass emerged.

Mice expressing a “hyper-sensitive” form of the CB1 cannabinoid receptor (CB1) show modestly enhanced alcohol preference and consumption

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Gonek, Maciej; Zee, Michael L.; Farnsworth, Jill C.; Amin, Randa A.; Andrews, Mary-Jeanette; Davis, Brian J.; Mackie, Ken; Morgan, Daniel J.

2017-01-01

We recently characterized S426A/S430A mutant mice expressing a desensitization-resistant form of the CB1 receptor. These mice display an enhanced response to endocannabinoids and ∆9-THC. In this study, S426A/S430A mutants were used as a novel model to test whether ethanol consumption, morphine dependence, and reward for these drugs are potentiated in mice with a “hyper-sensitive” form of CB1. Using an unlimited-access, two-bottle choice, voluntary drinking paradigm, S426A/S430A mutants exhibit modestly increased intake and preference for low (6%) but not higher concentrations of ethanol. S426A/S430A mutants and wild-type mice show similar taste preference for sucrose and quinine, exhibit normal sensitivity to the hypothermic and ataxic effects of ethanol, and have normal blood ethanol concentrations following administration of ethanol. S426A/S430A mutants develop robust conditioned place preference for ethanol (2 g/kg), morphine (10 mg/kg), and cocaine (10 mg/kg), demonstrating that drug reward is not changed in S426A/S430A mutants. Precipitated morphine withdrawal is also unchanged in opioid-dependent S426A/S430A mutant mice. Although ethanol consumption is modestly changed by enhanced CB1 signaling, reward, tolerance, and acute sensitivity to ethanol and morphine are normal in this model. PMID:28426670

ENU-mutagenesis mice with a non-synonymous mutation in Grin1 exhibit abnormal anxiety-like behaviors, impaired fear memory, and decreased acoustic startle response

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2013-01-01

Background The Grin1 (glutamate receptor, ionotropic, NMDA1) gene expresses a subunit of N-methyl-D-aspartate (NMDA) receptors that is considered to play an important role in excitatory neurotransmission, synaptic plasticity, and brain development. Grin1 is a candidate susceptibility gene for neuropsychiatric disorders, including schizophrenia, bipolar disorder, and attention deficit/hyperactivity disorder (ADHD). In our previous study, we examined an N-ethyl-N-nitrosourea (ENU)-generated mutant mouse strain (Grin1Rgsc174/Grin1+) that has a non-synonymous mutation in Grin1. These mutant mice showed hyperactivity, increased novelty-seeking to objects, and abnormal social interactions. Therefore, Grin1Rgsc174/Grin1+ mice may serve as a potential animal model of neuropsychiatric disorders. However, other behavioral characteristics related to these disorders, such as working memory function and sensorimotor gating, have not been fully explored in these mutant mice. In this study, to further investigate the behavioral phenotypes of Grin1Rgsc174/Grin1+ mice, we subjected them to a comprehensive battery of behavioral tests. Results There was no significant difference in nociception between Grin1Rgsc174/Grin1+ and wild-type mice. The mutants did not display any abnormalities in the Porsolt forced swim and tail suspension tests. We confirmed the previous observations that the locomotor activity of these mutant mice increased in the open field and home cage activity tests. They displayed abnormal anxiety-like behaviors in the light/dark transition and the elevated plus maze tests. Both contextual and cued fear memory were severely deficient in the fear conditioning test. The mutant mice exhibited slightly impaired working memory in the eight-arm radial maze test. The startle amplitude was markedly decreased in Grin1Rgsc174/Grin1+ mice, whereas no significant differences between genotypes were detected in the prepulse inhibition (PPI) test. The mutant mice showed no obvious

Serotonin hyperinnervation and upregulated 5-HT2A receptor expression and motor-stimulating function in nigrostriatal dopamine-deficient Pitx3 mutant mice.

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Li, Li; Qiu, Guozhen; Ding, Shengyuan; Zhou, Fu-Ming

2013-01-23

The striatum receives serotonin (5-hydroxytryptamine, 5-HT) innervation and expresses 5-HT2A receptors (5-HT2ARs) and other 5-HT receptors, raising the possibility that the striatal 5-HT system may undergo adaptive changes after chronic severe dopamine (DA) loss and contribute to the function and dysfunction of the striatum. Here we show that in transcription factor Pitx3 gene mutant mice with a selective, severe DA loss in the dorsal striatum mimicking the DA denervation in late Parkinson's disease (PD), both the 5-HT innervation and the 5-HT2AR mRNA expression were increased in the dorsal striatum. Functionally, while having no detectable motor effect in wild type mice, the 5-HT2R agonist 2,5-dimethoxy-4-iodoamphetamine increased both the baseline and l-dopa-induced normal ambulatory and dyskinetic movements in Pitx3 mutant mice, whereas the selective 5-HT2AR blocker volinanserin had the opposite effects. These results demonstrate that Pitx3 mutant mice are a convenient and valid mouse model to study the compensatory 5-HT upregulation following the loss of the nigrostriatal DA projection and that the upregulated 5-HT2AR function in the DA deficient dorsal striatum may enhance both normal and dyskinetic movements. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

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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.

Adenovirus Particles that Display the Plasmodium falciparum Circumsporozoite Protein NANP Repeat Induce Sporozoite-Neutralizing Antibodies in Mice

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Palma, Christopher; Overstreet, Michael G.; Guedon, Jean-Marc; Hoiczyk, Egbert; Ward, Cameron; Karen, Kasey A.; Zavala, Fidel; Ketner, Gary

2011-01-01

Adenovirus particles can be engineered to display exogenous peptides on their surfaces by modification of viral capsid proteins, and particles that display pathogen-derived peptides can induce protective immunity. We constructed viable recombinant adenoviruses that display B-cell epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) in the major adenovirus capsid protein, hexon. Recombinants induced high-titer antibodies against CSP when injected intraperitoneally into mice. Serum obtained from immunized mice recognized both recombinant PfCSP protein and P. falciparum sporozoites, and neutralized P. falciparum sporozoites in vitro. Replicating adenovirus vaccines have provided economical protection against adenovirus disease for over three decades. The recombinants described here may provide a path to an affordable malaria vaccine in the developing world. PMID:21199707

Neuropathology in mice expressing mouse alpha-synuclein.

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Claus Rieker

Full Text Available α-Synuclein (αSN in human is tightly linked both neuropathologically and genetically to Parkinson's disease (PD and related disorders. Disease-causing properties in vivo of the wildtype mouse ortholog (mαSN, which carries a threonine at position 53 like the A53T human mutant version that is genetically linked to PD, were never reported. To this end we generated mouse lines that express mαSN in central neurons at levels reaching up to six-fold compared to endogenous mαSN. Unlike transgenic mice expressing human wildtype or mutant forms of αSN, these mαSN transgenic mice showed pronounced ubiquitin immunopathology in spinal cord and brainstem. Isoelectric separation of mαSN species revealed multiple isoforms including two Ser129-phosphorylated species in the most severely affected brain regions. Neuronal Ser129-phosphorylated αSN occurred in granular and small fibrillar aggregates and pathological staining patterns in neurites occasionally revealed a striking ladder of small alternating segments staining either for Ser129-phosphorylated αSN or ubiquitin but not both. Axonal degeneration in long white matter tracts of the spinal cord, with breakdown of myelin sheaths and degeneration of neuromuscular junctions with loss of integrity of the presynaptic neurofilament network in mαSN transgenic mice, was similar to what we have reported for mice expressing human αSN wildtype or mutant forms. In hippocampal neurons, the mαSN protein accumulated and was phosphorylated but these neurons showed no ubiquitin immunopathology. In contrast to the early-onset motor abnormalities and muscle weakness observed in mice expressing human αSN, mαSN transgenic mice displayed only end-stage phenotypic alterations that manifested alongside with neuropathology. Altogether these findings show that increased levels of wildtype mαSN does not induce early-onset behavior changes, but drives end-stage pathophysiological changes in murine neurons that are

Adenovirus Particles that Display the Plasmodium falciparum Circumsporozoite Protein NANP Repeat Induce Sporozoite-Neutralizing Antibodies in Mice

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Palma, Christopher; Overstreet, Michael G.; Guedon, Jean-Marc; Hoiczyk, Egbert; Ward, Cameron; Karen, Kasey A.; Zavala, Fidel; Ketner, Gary

2011-01-01

Adenovirus particles can be engineered to display exogenous peptides on their surfaces by modification of viral capsid proteins, and particles that display pathogen-derived peptides can induce protective immunity. We constructed viable recombinant adenoviruses that display B-cell epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) in the major adenovirus capsid protein, hexon. Recombinants induced high-titer antibodies against CSP when injected intraperitoneally into mice...

K-RasV14I recapitulates Noonan syndrome in mice

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Hernández-Porras, Isabel; Fabbiano, Salvatore; Schuhmacher, Alberto J.; Aicher, Alexandra; Cañamero, Marta; Cámara, Juan Antonio; Cussó, Lorena; Desco, Manuel; Heeschen, Christopher; Mulero, Francisca; Bustelo, Xosé R.; Guerra, Carmen; Barbacid, Mariano

2014-01-01

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. NS also is associated with a risk for developing myeloproliferative disorders (MPD), including juvenile myelomonocytic leukemia (JMML). Mutations responsible for NS occur in at least 11 different loci including KRAS. Here we describe a mouse model for NS induced by K-RasV14I, a recurrent KRAS mutation in NS patients. K-RasV14I–mutant mice displayed multiple NS-associated developmental defects such as growth delay, craniofacial dysmorphia, cardiac defects, and hematologic abnormalities including a severe form of MPD that resembles human JMML. Homozygous animals had perinatal lethality whose penetrance varied with genetic background. Exposure of pregnant mothers to a MEK inhibitor rescued perinatal lethality and prevented craniofacial dysmorphia and cardiac defects. However, Mek inhibition was not sufficient to correct these defects when mice were treated after weaning. Interestingly, Mek inhibition did not correct the neoplastic MPD characteristic of these mutant mice, regardless of the timing at which the mice were treated, thus suggesting that MPD is driven by additional signaling pathways. These genetically engineered K-RasV14I–mutant mice offer an experimental tool for studying the molecular mechanisms underlying the clinical manifestations of NS. Perhaps more importantly, they should be useful as a preclinical model to test new therapies aimed at preventing or ameliorating those deficits associated with this syndrome. PMID:25359213

Mutant Brucella abortus membrane fusogenic protein induces protection against challenge infection in mice.

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de Souza Filho, Job Alves; de Paulo Martins, Vicente; Campos, Priscila Carneiro; Alves-Silva, Juliana; Santos, Nathalia V; de Oliveira, Fernanda Souza; Menezes, Gustavo B; Azevedo, Vasco; Cravero, Silvio Lorenzo; Oliveira, Sergio Costa

2015-04-01

Brucella species can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of the Brucella membrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed that B. abortus Δmfp::kan and Δomp19::kan deletion mutant strains have reduced persistence in vivo in C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kan or Δomp19::kan strain expressing green fluorescent protein (GFP) approximately 80% or 65% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments. B. abortus Δomp19::kan was attenuated in vivo but had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kan strain had a lower virulence in these same mouse models. Furthermore, Δmfp::kan and Δomp19::kan strains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kan strain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kan strain induced protection similar to that of RB51. Thus, these results demonstrate that Brucella Mfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kan mutant may serve as a potential vaccine candidate in future studies. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Fanconi anemia group A and C double-mutant mice: functional evidence for a multi-protein Fanconi anemia complex.

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Noll, Meenakshi; Battaile, Kevin P; Bateman, Raynard; Lax, Timothy P; Rathbun, Keany; Reifsteck, Carol; Bagby, Grover; Finegold, Milton; Olson, Susan; Grompe, Markus

2002-07-01

Fanconi anemia (FA) is a genetically heterogeneous disorder associated with defects in at least eight genes. The biochemical function(s) of the FA proteins are unknown, but together they define the FA pathway, which is involved in cellular responses to DNA damage and in other cellular processes. It is currently unknown whether all FA proteins are involved in controlling a single function or whether some of the FA proteins have additional roles. The aim of this study was 1) to determine whether the FA group A and group C genes have identical or partially distinct functions, and 2) to have a better model for human FA. We generated mice with a targeted mutation in fanca and crossed them with fancc disrupted animals. Several phenotypes including sensitivity to DNA cross linkers and ionizing radiation, hematopoietic colony growth, and germ cell loss were analyzed in fanca-/-, fancc-/-, fanca/fancc double -/-, and controls. Fibroblast cells and hematopoietic precursors from fanca/fancc double-mutant mice were not more sensitive to MMC than those of either single mutant. fanca/fancc double mutants had no evidence for an additive phenotype at the cellular or organismal level. These results support a model where both FANCA and FANCC are part of a multi-protein nuclear FA complex with identical function in cellular responses to DNA damage and germ cell survival.

A knock-in/knock-out mouse model of HSPB8-associated distal hereditary motor neuropathy and myopathy reveals toxic gain-of-function of mutant Hspb8.

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Bouhy, Delphine; Juneja, Manisha; Katona, Istvan; Holmgren, Anne; Asselbergh, Bob; De Winter, Vicky; Hochepied, Tino; Goossens, Steven; Haigh, Jody J; Libert, Claude; Ceuterick-de Groote, Chantal; Irobi, Joy; Weis, Joachim; Timmerman, Vincent

2018-01-01

Mutations in the small heat shock protein B8 gene (HSPB8/HSP22) have been associated with distal hereditary motor neuropathy, Charcot-Marie-Tooth disease, and recently distal myopathy. It is so far not clear how mutant HSPB8 induces the neuronal and muscular phenotypes and if a common pathogenesis lies behind these diseases. Growing evidence points towards a role of HSPB8 in chaperone-associated autophagy, which has been shown to be a determinant for the clearance of poly-glutamine aggregates in neurodegenerative diseases but also for the maintenance of skeletal muscle myofibrils. To test this hypothesis and better dissect the pathomechanism of mutant HSPB8, we generated a new transgenic mouse model leading to the expression of the mutant protein (knock-in lines) or the loss-of-function (functional knock-out lines) of the endogenous protein Hspb8. While the homozygous knock-in mice developed motor deficits associated with degeneration of peripheral nerves and severe muscle atrophy corroborating patient data, homozygous knock-out mice had locomotor performances equivalent to those of wild-type animals. The distal skeletal muscles of the post-symptomatic homozygous knock-in displayed Z-disk disorganisation, granulofilamentous material accumulation along with Hspb8, αB-crystallin (HSPB5/CRYAB), and desmin aggregates. The presence of the aggregates correlated with reduced markers of effective autophagy. The sciatic nerve of the homozygous knock-in mice was characterized by low autophagy potential in pre-symptomatic and Hspb8 aggregates in post-symptomatic animals. On the other hand, the sciatic nerve of the homozygous knock-out mice presented a normal morphology and their distal muscle displayed accumulation of abnormal mitochondria but intact myofiber and Z-line organisation. Our data, therefore, suggest that toxic gain-of-function of mutant Hspb8 aggregates is a major contributor to the peripheral neuropathy and the myopathy. In addition, mutant Hspb8 induces

Leiomodin-3-deficient mice display nemaline myopathy with fast-myofiber atrophy

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Lei Tian

2015-06-01

Full Text Available Nemaline myopathy (NM is one of the most common forms of congenital myopathy, and affects either fast myofibers, slow myofibers, or both. However, an animal model for congenital myopathy with fast-myofiber-specific atrophy is not available. Furthermore, mutations in the leiomodin-3 (LMOD3 gene have recently been identified in a group of individuals with NM. However, it is not clear how loss of LMOD3 leads to NM. Here, we report a mouse mutant in which the piggyBac (PB transposon is inserted into the Lmod3 gene and disrupts its expression. Lmod3PB/PB mice show severe muscle weakness and postnatal growth retardation. Electron microscopy and immunofluorescence studies of the mutant skeletal muscles revealed the presence of nemaline bodies, a hallmark of NM, and disorganized sarcomeric structures. Interestingly, Lmod3 deficiency caused muscle atrophy specific to the fast fibers. Together, our results show that Lmod3 is required in the fast fibers for sarcomere integrity, and this study offers the first NM mouse model with muscle atrophy that is specific to fast fibers. This model could be a valuable resource for interrogating myopathy pathogenesis and developing therapeutics for NM as well as other pathophysiological conditions with preferential atrophy of fast fibers, including cancer cachexia and sarcopenia.

Over-Expression of Porcine Myostatin Missense Mutant Leads to A Gender Difference in Skeletal Muscle Growth between Transgenic Male and Female Mice.

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Ma, Dezun; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Jiang, Shengwang; Xiao, Gaojun; Cui, Wentao

2015-08-24

Myostatin, a transforming growth factor-β family member, is a negative regulator of skeletal muscle development and growth. Piedmontese cattle breeds have a missense mutation, which results in a cysteine to tyrosine substitution in the mature myostatin protein (C313Y). This loss-of-function mutation in myostatin results in a double-muscled phenotype in cattle. Myostatin propeptide is an inhibitor of myostatin activity and is considered a potential agent to stimulate muscle growth in livestock. In this study, we generated transgenic mice overexpressing porcine myostatin missense mutant (pmMS), C313Y, and wild-type porcine myostatin propeptide (ppMS), respectively, to examine their effects on muscle growth in mice. Enhanced muscle growth was observed in both pmMS and ppMS transgenic female mice and also in ppMS transgenic male mice. However, there was no enhanced muscle growth observed in pmMS transgenic male mice. To explore why there is such a big difference in muscle growth between pmMS and ppMS transgenic male mice, the expression level of androgen receptor (AR) mutant AR45 was measured by Western blot. Results indicated that AR45 expression significantly increased in pmMS transgenic male mice while it decreased dramatically in ppMS transgenic male mice. Our data demonstrate that both pmMS and ppMS act as myostatin inhibitors in the regulation of muscle growth, but the effect of pmMS in male mice is reversed by an increased AR45 expression. These results provide useful insight and basic theory to future studies on improving pork quality by genetically manipulating myostatin expression or by regulating myostatin activity.

Transgenic mice expressing constitutive active MAPKAPK5 display gender-dependent differences in exploration and activity

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Moens Ugo

2007-11-01

Full Text Available Abstract Background The mitogen-activated protein kinases, MAPKs for short, constitute cascades of signalling pathways involved in the regulation of several cellular processes that include cell proliferation, differentiation and motility. They also intervene in neurological processes like fear conditioning and memory. Since little remains known about the MAPK-Activated Protein Kinase, MAPKAPK5, we constructed the first MAPKAPK knockin mouse model, using a constitutive active variant of MAPKAPK5 and analyzed the resulting mice for changes in anxiety-related behaviour. Methods We performed primary SHIRPA observations during background breeding into the C57BL/6 background and assessed the behaviour of the background-bred animals on the elevated plus maze and in the light-dark test. Our results were analyzed using Chi-square tests and homo- and heteroscedatic T-tests. Results Female transgenic mice displayed increased amounts of head dips and open arm time on the maze, compared to littermate controls. In addition, they also explored further into the open arm on the elevated plus maze and were less active in the closed arm compared to littermate controls. Male transgenic mice displayed no differences in anxiety, but their locomotor activity increased compared to non-transgenic littermates. Conclusion Our results revealed anxiety-related traits and locomotor differences between transgenic mice expressing constitutive active MAPKAPK5 and control littermates.

Intramuscular Immunization of Mice with a Live-Attenuated Triple Mutant of Yersinia pestis CO92 Induces Robust Humoral and Cell-Mediated Immunity To Completely Protect Animals against Pneumonic Plague.

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Tiner, Bethany L; Sha, Jian; Ponnusamy, Duraisamy; Baze, Wallace B; Fitts, Eric C; Popov, Vsevolod L; van Lier, Christina J; Erova, Tatiana E; Chopra, Ashok K

2015-12-01

Earlier, we showed that the Δlpp ΔmsbB Δail triple mutant of Yersinia pestis CO92 with deleted genes encoding Braun lipoprotein (Lpp), an acyltransferase (MsbB), and the attachment invasion locus (Ail), respectively, was avirulent in a mouse model of pneumonic plague. In this study, we further evaluated the immunogenic potential of the Δlpp ΔmsbB Δail triple mutant and its derivative by different routes of vaccination. Mice were immunized via the subcutaneous (s.c.) or the intramuscular (i.m.) route with two doses (2 × 10(6) CFU/dose) of the above-mentioned triple mutant with 100% survivability of the animals. Upon subsequent pneumonic challenge with 70 to 92 50% lethal doses (LD(50)) of wild-type (WT) strain CO92, all of the mice survived when immunization occurred by the i.m. route. Since Ail has virulence and immunogenic potential, a mutated version of Ail devoid of its virulence properties was created, and the genetically modified ail replaced the native ail gene on the chromosome of the Δlpp ΔmsbB double mutant, creating a Δlpp ΔmsbB::ailL2 vaccine strain. This newly generated mutant was attenuated similarly to the Δlpp ΔmsbB Δail triple mutant when administered by the i.m. route and provided 100% protection to animals against subsequent pneumonic challenge. Not only were the two above-mentioned mutants cleared rapidly from the initial i.m. site of injection in animals with no histopathological lesions, the immunized mice did not exhibit any disease symptoms during immunization or after subsequent exposure to WT CO92. These two mutants triggered balanced Th1- and Th2-based antibody responses and cell-mediated immunity. A substantial increase in interleukin-17 (IL-17) from the T cells of vaccinated mice, a cytokine of the Th17 cells, further augmented their vaccine potential. Thus, the Δlpp ΔmsbB Δail and Δlpp ΔmsbB::ailL2 mutants represent excellent vaccine candidates for plague, with the latter mutant still retaining Ail immunogenicity but

Laminin alpha2 deficiency and muscular dystrophy; genotype-phenotype correlation in mutant mice

DEFF Research Database (Denmark)

Guo, L T; Zhang, X U; Kuang, W

2003-01-01

2, lacking domain VI. Interestingly, all mutants lack laminin alpha2 in peripheral nerve. We have demonstrated previously, that overexpression of the human laminin alpha2 in skeletal muscle in dy(2J)/dy(2J) and dy(W)/dy(W) mice under the control of a striated muscle-specific creatine kinase promoter......Deficiency of laminin alpha2 is the cause of one of the most severe muscular dystrophies in humans and other species. It is not yet clear how particular mutations in the laminin alpha2 chain gene affect protein expression, and how abnormal levels or structure of the protein affect disease. Animal...

Skeletal development of mice lacking bone sialoprotein (BSP--impairment of long bone growth and progressive establishment of high trabecular bone mass.

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Wafa Bouleftour

Full Text Available Adult Ibsp-knockout mice (BSP-/- display shorter stature, lower bone turnover and higher trabecular bone mass than wild type, the latter resulting from impaired bone resorption. Unexpectedly, BSP knockout also affects reproductive behavior, as female mice do not construct a proper "nest" for their offsprings. Multiple crossing experiments nonetheless indicated that the shorter stature and lower weight of BSP-/- mice, since birth and throughout life, as well as their shorter femur and tibia bones are independent of the genotype of the mothers, and thus reflect genetic inheritance. In BSP-/- newborns, µCT analysis revealed a delay in membranous primary ossification, with wider cranial sutures, as well as thinner femoral cortical bone and lower tissue mineral density, reflected in lower expression of bone formation markers. However, trabecular bone volume and osteoclast parameters of long bones do not differ between genotypes. Three weeks after birth, osteoclast number and surface drop in the mutants, concomitant with trabecular bone accumulation. The growth plates present a thinner hypertrophic zone in newborns with lower whole bone expression of IGF-1 and higher IHH in 6 days old BSP-/- mice. At 3 weeks the proliferating zone is thinner and the hypertrophic zone thicker in BSP-/- than in BSP+/+ mice of either sex, maybe reflecting a combination of lower chondrocyte proliferation and impaired cartilage resorption. Six days old BSP-/- mice display lower osteoblast marker expression but higher MEPE and higher osteopontin(Opn/Runx2 ratio. Serum Opn is higher in mutants at day 6 and in adults. Thus, lack of BSP alters long bone growth and membranous/cortical primary bone formation and mineralization. Endochondral development is however normal in mutant mice and the accumulation of trabecular bone observed in adults develops progressively in the weeks following birth. Compensatory high Opn may allow normal endochondral development in BSP-/- mice

Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice.

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Iglesias, Ainhoa; Murga, Matilde; Laresgoiti, Usua; Skoudy, Anouchka; Bernales, Irantzu; Fullaondo, Asier; Moreno, Bernardino; Lloreta, José; Field, Seth J; Real, Francisco X; Zubiaga, Ana M

2004-05-01

E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.

Functional verification of a porcine myostatin propeptide mutant.

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Ma, Dezun; Jiang, Shengwang; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Xiao, Gaojun; Yang, Jinzeng; Cui, Wentao

2015-10-01

Myostatin is a member of TGF-β superfamily that acts as a key negative regulator in development and growth of embryonic and postnatal muscles. In this study, the inhibitory activities of recombinant porcine myostatin propeptide and its mutated form (at the cleavage site of metalloproteinases of BMP-1/TLD family) against murine myostatin was evaluated in vivo by intraperitoneal injection into mice. Results showed that both wild type and mutated form of porcine propeptide significantly inhibited myostatin activity in vivo. The average body weight of mice receiving wild type propeptide or its mutated form increased by 12.5 % and 24.14%, respectively, compared to mice injected with PBS, implying that the in vivo efficacy of porcine propeptide mutant is greater than its wild type propeptide. Transgenic mice expressing porcine myostatin propeptide mutant were generated to further verify the results obtained from mice injected with recombinant porcine propeptide mutant. Compared with wild type (non-transgenic) mice, relative weight of gastrocnemius, rectusfemoris, and tibialis anterior increased by 22.14 %, 34.13 %, 25.37%, respectively, in transgenic male mice, and by 19.90 %, 42.47 %, 45.61%, respectively, in transgenic female mice. Our data also demonstrated that the mechanism by which muscle growth enhancement is achieved by these propeptides is due to an increase in fiber sizes, not by an increase in number of fiber cells.

Functional motor recovery from motoneuron axotomy is compromised in mice with defective corticospinal projections.

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Yuetong Ding

Full Text Available Brachial plexus injury (BPI and experimental spinal root avulsion result in loss of motor function in the affected segments. After root avulsion, significant motoneuron function is restored by re-implantation of the avulsed root. How much this functional recovery depends on corticospinal inputs is not known. Here, we studied that question using Celsr3|Emx1 mice, in which the corticospinal tract (CST is genetically absent. In adult mice, we tore off right C5-C7 motor and sensory roots and re-implanted the right C6 roots. Behavioral studies showed impaired recovery of elbow flexion in Celsr3|Emx1 mice compared to controls. Five months after surgery, a reduced number of small axons, and higher G-ratio of inner to outer diameter of myelin sheaths were observed in mutant versus control mice. At early stages post-surgery, mutant mice displayed lower expression of GAP-43 in spinal cord and of myelin basic protein (MBP in peripheral nerves than control animals. After five months, mutant animals had atrophy of the right biceps brachii, with less newly formed neuromuscular junctions (NMJs and reduced peak-to-peak amplitudes in electromyogram (EMG, than controls. However, quite unexpectedly, a higher motoneuron survival rate was found in mutant than in control mice. Thus, following root avulsion/re-implantation, the absence of the CST is probably an important reason to hamper axonal regeneration and remyelination, as well as target re-innervation and formation of new NMJ, resulting in lower functional recovery, while fostering motoneuron survival. These results indicate that manipulation of corticospinal transmission may help improve functional recovery following BPI.

Brevican-deficient mice display impaired hippocampal CA1 long-term potentiation but show no obvious deficits in learning and memory

DEFF Research Database (Denmark)

Brakebusch, Cord; Seidenbecher, Constanze I; Asztely, Fredrik

2002-01-01

to be less prominent in mutant than in wild-type mice. Brevican-deficient mice showed significant deficits in the maintenance of hippocampal long-term potentiation (LTP). However, no obvious impairment of excitatory and inhibitory synaptic transmission was found, suggesting a complex cause for the LTP defect....... Detailed behavioral analysis revealed no statistically significant deficits in learning and memory. These data indicate that brevican is not crucial for brain development but has restricted structural and functional roles....

Baculovirus virions displaying Plasmodium berghei circumsporozoite protein protect mice against malaria sporozoite infection

International Nuclear Information System (INIS)

Yoshida, Shigeto; Kondoh, Daisuke; Arai, Eriko; Matsuoka, Hiroyuki; Seki, Chisato; Tanaka, Takao; Okada, Masaji; Ishii, Akira

2003-01-01

The display of foreign proteins on the surface of baculovirus virions has provided a tool for the analysis of protein-protein interactions and for cell-specific targeting in gene transfer applications. To evaluate the baculovirus display system as a vaccine vehicle, we have generated a recombinant baculovirus (AcNPV-CSPsurf) that displays rodent malaria Plasmodium berghei circumsporozoite protein (PbCSP) on the virion surface as a fusion protein with the major baculovirus envelope glycoprotein gp64. The PbCSP-gp64 fusion protein was incorporated and oligomerized on the virion surface and led to a 12-fold increase in the binding activity of AcNPV-CSPsurf virions to HepG2 cells. Immunization with adjuvant-free AcNPV-CSPsurf virions induced high levels of antibodies and gamma interferon-secreting cells against PbCSP and protected 60% of mice against sporozoite challenge. These data demonstrate that AcNPV-CSPsurf displays sporozoite-like PbCSP on the virion surface and possesses dual potentials as a malaria vaccine candidate and a liver-directed gene delivery vehicle

Brucella abortus 2308ΔNodVΔNodW double-mutant is highly attenuated and confers protection against wild-type challenge in BALB/c mice.

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Li, Zhiqiang; Wang, Shuli; Zhang, Jinliang; Yang, Guangli; Yuan, Baodong; Huang, Jie; Han, Jincheng; Xi, Li; Xiao, Yanren; Chen, Chuangfu; Zhang, Hui

2017-05-01

Brucellosis is an important zoonotic disease of worldwide distribution, which causes animal and human disease. However, the current Brucella abortus (B. abortus) vaccines (S19 and RB51) have several drawbacks, including residual virulence for animals and humans. Moreover, S19 cannot allow serological differentiation between infected and vaccinated animals. We constructed double deletion (ΔNodVΔNodW) mutant from virulent B. abortus 2308 (S2308) by deleting the genes encoding two-component regulatory system (TCS) in chromosome II in S2308.2308ΔNodVΔNodW was significantly reduced survival in murine macrophages (RAW 264.7) and BALB/c mice. Moreover, the inoculated mice showed no splenomegaly. The mutant induced high protective immunity in BALB/c mice against challenge with S2308, and elicited an anti-Brucella-specific immunoglobulin G (IgG) response and induced the secretion of gamma interferon (IFN-γ) and interleukin-4 (IL-4). Moreover, NODV and NODW antigens would allow the serological differentiation between infected and vaccinated animals. These results suggest that 2308ΔNodVΔNodW mutant is a potential live attenuated vaccine candidate and can be used effectively against bovine brucellosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phenotypic characterization of skeletal abnormalities of osteopotentia mutant mice by micro-CT: a descriptive approach with emphasis on reconstruction techniques

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Roemer, Frank W. [Department of Radiology, Klinikum Augsburg, Augsburg (Germany); Boston University School of Medicine, Quantitative Imaging Center, Boston, MA (United States); University of California, San Francisco, Osteoporosis and Arthritis Research Group, San Francisco, CA (United States); Boston University Medical Center, Department of Radiology, Boston, MA (United States); Mohr, Andreas [University of California, San Francisco, Osteoporosis and Arthritis Research Group, San Francisco, CA (United States); Sligo General Hospital, Department of Radiology, Sligo (Ireland); Guermazi, Ali [Boston University School of Medicine, Quantitative Imaging Center, Boston, MA (United States); University of California, San Francisco, Osteoporosis and Arthritis Research Group, San Francisco, CA (United States); Jiang, Yebin [University of California, San Francisco, Osteoporosis and Arthritis Research Group, San Francisco, CA (United States); University of Michigan Medical School, Osteoporosis and Arthritis Laboratory, Musculoskeletal Division, Department of Radiology, Ann Arbor, MI (United States); Schlechtweg, Philipp [University of Erlangen, Department of Radiology, Erlangen (Germany); Genant, Harry K. [University of California, San Francisco, Osteoporosis and Arthritis Research Group, San Francisco, CA (United States); CCBR-SYNARC, Inc., San Francisco, CA (United States); Sohaskey, Michael L. [University of California, Berkeley, Department of Molecular and Cell Biology and Center for Integrative Genomics, Berkeley, CA (United States)

2011-08-15

The novel protein osteopotentia (Opt) has recently been described as an essential regulator of postnatal osteoblast maturation and might possibly be responsible for some of the rarer types of osteogenesis imperfecta. Our aim was the evaluation of micro CT for the qualitative morphological assessment of skeletal abnormalities of Osteopotentia-mutant mice in comparison to radiography and histology. Four homozygous mice with insertional mutations in the Opt gene and three wild-type controls were examined ex vivo using radiography and micro-CT. Two of the homozygous animals were evaluated histologically (trichrome reagent). For the micro-CT evaluation three-dimensional (3D) surface reconstructions and two-dimensional (2D) multiplanar reformations (MPRs) were applied. The Opt-homozygous mice exhibited severe growth. The radiographic examinations showed osteopenia and fractures with hypertrophic callus formation and pseudarthroses of the forelimbs and ribs. Micro-CT confirmed these findings and was able to demonstrate additional fractures especially at smaller bones such as the metacarpals and phalanges. Additional characterization and superior delineation of cortices and fracture fragments was achieved by 2D MPRs. Histological correlation verified several of these imaging findings. Micro-CT is able to screen Opt-mutant mice for osseous pathologies and furthermore characterize these anomalies. The modality seems superior to conventional radiography, but is not able to demonstrate cellular pathology. However, histology is destructive and more time- and material-consuming than micro-CT. Additional information may be gathered by 2D MPRs. (orig.)

Phenotypic characterization of skeletal abnormalities of osteopotentia mutant mice by micro-CT: a descriptive approach with emphasis on reconstruction techniques

International Nuclear Information System (INIS)

Roemer, Frank W.; Mohr, Andreas; Guermazi, Ali; Jiang, Yebin; Schlechtweg, Philipp; Genant, Harry K.; Sohaskey, Michael L.

2011-01-01

The novel protein osteopotentia (Opt) has recently been described as an essential regulator of postnatal osteoblast maturation and might possibly be responsible for some of the rarer types of osteogenesis imperfecta. Our aim was the evaluation of micro CT for the qualitative morphological assessment of skeletal abnormalities of Osteopotentia-mutant mice in comparison to radiography and histology. Four homozygous mice with insertional mutations in the Opt gene and three wild-type controls were examined ex vivo using radiography and micro-CT. Two of the homozygous animals were evaluated histologically (trichrome reagent). For the micro-CT evaluation three-dimensional (3D) surface reconstructions and two-dimensional (2D) multiplanar reformations (MPRs) were applied. The Opt-homozygous mice exhibited severe growth. The radiographic examinations showed osteopenia and fractures with hypertrophic callus formation and pseudarthroses of the forelimbs and ribs. Micro-CT confirmed these findings and was able to demonstrate additional fractures especially at smaller bones such as the metacarpals and phalanges. Additional characterization and superior delineation of cortices and fracture fragments was achieved by 2D MPRs. Histological correlation verified several of these imaging findings. Micro-CT is able to screen Opt-mutant mice for osseous pathologies and furthermore characterize these anomalies. The modality seems superior to conventional radiography, but is not able to demonstrate cellular pathology. However, histology is destructive and more time- and material-consuming than micro-CT. Additional information may be gathered by 2D MPRs. (orig.)

A novel intergenic ETnII-β insertion mutation causes multiple malformations in polypodia mice.

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Jessica A Lehoczky

Full Text Available Mouse early transposon insertions are responsible for ~10% of spontaneous mutant phenotypes. We previously reported the phenotypes and genetic mapping of Polypodia, (Ppd, a spontaneous, X-linked dominant mutation with profound effects on body plan morphogenesis. Our new data shows that mutant mice are not born in expected Mendelian ratios secondary to loss after E9.5. In addition, we refined the Ppd genetic interval and discovered a novel ETnII-β early transposon insertion between the genes for Dusp9 and Pnck. The ETn inserted 1.6 kb downstream and antisense to Dusp9 and does not disrupt polyadenylation or splicing of either gene. Knock-in mice engineered to carry the ETn display Ppd characteristic ectopic caudal limb phenotypes, showing that the ETn insertion is the Ppd molecular lesion. Early transposons are actively expressed in the early blastocyst. To explore the consequences of the ETn on the genomic landscape at an early stage of development, we compared interval gene expression between wild-type and mutant ES cells. Mutant ES cell expression analysis revealed marked upregulation of Dusp9 mRNA and protein expression. Evaluation of the 5' LTR CpG methylation state in adult mice revealed no correlation with the occurrence or severity of Ppd phenotypes at birth. Thus, the broad range of phenotypes observed in this mutant is secondary to a novel intergenic ETn insertion whose effects include dysregulation of nearby interval gene expression at early stages of development.

Kharon1 null mutants of Leishmania mexicana are avirulent in mice and exhibit a cytokinesis defect within macrophages.

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Khoa D Tran

Full Text Available In a variety of eukaryotes, flagella play important roles both in motility and as sensory organelles that monitor the extracellular environment. In the parasitic protozoan Leishmania mexicana, one glucose transporter isoform, LmxGT1, is targeted selectively to the flagellar membrane where it appears to play a role in glucose sensing. Trafficking of LmxGT1 to the flagellar membrane is dependent upon interaction with the KHARON1 protein that is located at the base of the flagellar axoneme. Remarkably, while Δkharon1 null mutants are viable as insect stage promastigotes, they are unable to survive as amastigotes inside host macrophages. Although Δkharon1 promastigotes enter macrophages and transform into amastigotes, these intracellular parasites are unable to execute cytokinesis and form multinucleate cells before dying. Notably, extracellular axenic amastigotes of Δkharon1 mutants replicate and divide normally, indicating a defect in the mutants that is only exhibited in the intra-macrophage environment. Although the flagella of Δkharon1 amastigotes adhere to the phagolysomal membrane of host macrophages, the morphology of the mutant flagella is often distorted. Additionally, these null mutants are completely avirulent following injection into BALB/c mice, underscoring the critical role of the KHARON1 protein for viability of intracellular amastigotes and disease in the animal model of leishmaniasis.

Impact of Non-Invasively Induced Motor Deficits on Tibial Cortical Properties in Mutant Lurcher Mice.

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Alena Jindrová

Full Text Available It has been shown that Lurcher mutant mice have significantly altered motor abilities, regarding their motor coordination and muscular strength because of olivorecebellar degeneration. We assessed the response of the cross-sectional geometry and lacuno-canalicular network properties of the tibial mid-diaphyseal cortical bone to motor differences between Lurcher and wild-type (WT male mice from the B6CBA strain. The first data set used in the cross-sectional geometry analysis consists of 16 mice of 4 months of age and 32 mice of 9 months of age. The second data set used in the lacunar-canalicular network analysis consists of 10 mice of 4 months of age. We compared two cross-sectional geometry and four lacunar-canalicular properties by I-region using the maximum and minimum second moment of area and anatomical orientation as well as H-regions using histological differences within a cross section. We identified inconsistent differences in the studied cross-sectional geometry properties between Lurcher and WT mice. The biggest significant difference between Lurcher and WT mice is found in the number of canaliculi, whereas in the other studied properties are only limited. Lurcher mice exhibit an increased number of canaliculi (p < 0.01 in all studied regions compared with the WT controls. The number of canaliculi is also negatively correlated with the distance from the centroid in the Lurcher and positively correlated in the WT mice. When the Lurcher and WT sample is pooled, the number of canaliculi and lacunar volume is increased in the posterior Imax region, and in addition, midcortical H-region exhibit lower number of canaliculi, lacuna to lacuna distance and increased lacunar volume. Our results indicate, that the importance of precise sample selection within cross sections in future studies is highlighted because of the histological heterogeneity of lacunar-canalicular network properties within the I-region and H-region in the mouse cortical

Glycine receptor mutants of the mouse: what are possible routes of inhibitory compensation?

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Natascha eSchaefer

2012-10-01

Full Text Available Defects in glycinergic inhibition result in a complex neuromotor disorder in humans known as hyperekplexia (OMIM 149400 with similar phenotypes in rodents characterized by an exaggerated startle reflex and hypertonia. Analogous to genetic defects in humans, single point mutations, microdeletions, or insertions in the Glra1 gene but also in the Glrb gene underlie the pathology in mice. The mutations either localized in the α (spasmodic, oscillator, cincinnati, Nmf11 or the β (spastic subunit of the GlyR are much less tolerated in mice than in humans, leaving the question for the existence of different regulatory elements of the pathomechanisms in humans and rodents. In addition to the spontaneous mutations, new insights into understanding of the regulatory pathways in hyperekplexia or glycine encephalopathy arose from the constantly increasing number of knock-out as well as knock-in mutants of GlyRs. Over the last five years, various efforts using in vivo whole cell recordings provided a detailed analysis of the kinetic parameters underlying glycinergic dysfunction. Presynaptic compensation as well as postsynaptic compensatory mechanisms in these mice by other GlyR subunits or GABAA receptors, and the role of extra-synaptic GlyRs is still a matter of debate. A recent study on the mouse mutant oscillator, displayed a novel aspect for compensation of functionality by complementation of receptor domains that fold independently. This review focuses on defects in glycinergic neurotransmission in mice discussed with the background of human hyperekplexia en route to strategies of compensation.

miR-155 Controls Lymphoproliferation in LAT Mutant Mice by Restraining T-Cell Apoptosis via SHIP-1/mTOR and PAK1/FOXO3/BIM Pathways.

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Alexandre K Rouquette-Jazdanian

Full Text Available Linker for Activation of T cells (LAT is an adapter protein that is essential for T cell function. Knock-in mice with a LAT mutation impairing calcium flux develop a fatal CD4+ lymphoproliferative disease. miR-155 is a microRNA that is correlated with hyperproliferation in a number of cancers including lymphomas and leukemias and is overexpressed in mutant LAT T cells. To test whether miR-155 was merely indicative of T cell activation or whether it contributes to lymphoproliferative disease in mutant LAT mice, we interbred LAT mutant and miR-155-deficient mice. miR-155 deficiency markedly inhibited lymphoproliferative disease by stimulating BIM-dependent CD4+ T cell apoptosis, even though ERK activation and T cell proliferation were increased in double mutant CD4+ T cells. Bim/Bcl2l11 expression is activated by the forkhead transcription factor FOXO3. Using miR-155-deficient, LAT mutant T cells as a discovery tool, we found two connected pathways that impact the nuclear translocation and activation of FOXO3 in T cells. One pathway is mediated by the inositide phosphatase SHIP-1 and the serine/threonine kinases AKT and PDK1. The other pathway involves PAK1 and JNK kinase activation. We define crosstalk between the two pathways via the kinase mTOR, which stabilizes PAK1. This study establishes a role for PAK1 in T cell apoptosis, which contrasts to its previously identified role in T cell proliferation. Furthermore, miR-155 regulates the delicate balance between PAK1-mediated proliferation and apoptosis in T cells impacting lymphoid organ size and function.

Mice deficient in transmembrane prostatic acid phosphatase display increased GABAergic transmission and neurological alterations.

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Heidi O Nousiainen

Full Text Available Prostatic acid phosphatase (PAP, the first diagnostic marker and present therapeutic target for prostate cancer, modulates nociception at the dorsal root ganglia (DRG, but its function in the central nervous system has remained unknown. We studied expression and function of TMPAP (the transmembrane isoform of PAP in the brain by utilizing mice deficient in TMPAP (PAP-/- mice. Here we report that TMPAP is expressed in a subpopulation of cerebral GABAergic neurons, and mice deficient in TMPAP show multiple behavioral and neurochemical features linked to hyperdopaminergic dysregulation and altered GABAergic transmission. In addition to increased anxiety, disturbed prepulse inhibition, increased synthesis of striatal dopamine, and augmented response to amphetamine, PAP-deficient mice have enlarged lateral ventricles, reduced diazepam-induced loss of righting reflex, and increased GABAergic tone in the hippocampus. TMPAP in the mouse brain is localized presynaptically, and colocalized with SNARE-associated protein snapin, a protein involved in synaptic vesicle docking and fusion, and PAP-deficient mice display altered subcellular distribution of snapin. We have previously shown TMPAP to reside in prostatic exosomes and we propose that TMPAP is involved in the control of GABAergic tone in the brain also through exocytosis, and that PAP deficiency produces a distinct neurological phenotype.

MASTR: A Technique for Mosaic Mutant Analysis with Spatial and Temporal Control of Recombination Using Conditional Floxed Alleles in Mice

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Zhimin Lao

2012-08-01

Full Text Available Mosaic mutant analysis, the study of cellular defects in scattered mutant cells in a wild-type environment, is a powerful approach for identifying critical functions of genes and has been applied extensively to invertebrate model organisms. A highly versatile technique has been developed in mouse: MASTR (mosaic mutant analysis with spatial and temporal control of recombination, which utilizes the increasing number of floxed alleles and simultaneously combines conditional gene mutagenesis and cell marking for fate analysis. A targeted allele (R26MASTR was engineered; the allele expresses a GFPcre fusion protein following FLP-mediated recombination, which serves the dual function of deleting floxed alleles and marking mutant cells with GFP. Within 24 hr of tamoxifen administration to R26MASTR mice carrying an inducible FlpoER transgene and a floxed allele, nearly all GFP-expressing cells have a mutant allele. The fate of single cells lacking FGF8 or SHH signaling in the developing hindbrain was analyzed using MASTR, and it was revealed that there is only a short time window when neural progenitors require FGFR1 for viability and that granule cell precursors differentiate rapidly when SMO is lost. MASTR is a powerful tool that provides cell-type-specific (spatial and temporal marking of mosaic mutant cells and is broadly applicable to developmental, cancer, and adult stem cell studies.

Attenuated bioluminescent Brucella melitensis mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) confer protection in mice.

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Rajashekara, Gireesh; Glover, David A; Banai, Menachem; O'Callaghan, David; Splitter, Gary A

2006-05-01

In vivo bioluminescence imaging is a persuasive approach to investigate a number of issues in microbial pathogenesis. Previously, we have applied bioluminescence imaging to gain greater insight into Brucella melitensis pathogenesis. Endowing Brucella with bioluminescence allowed direct visualization of bacterial dissemination, pattern of tissue localization, and the contribution of Brucella genes to virulence. In this report, we describe the pathogenicity of three attenuated bioluminescent B. melitensis mutants, GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091), and the dynamics of bioluminescent virulent bacterial infection following vaccination with these mutants. The virB4, galE, and BMEI1090-BMEI1091 mutants were attenuated in interferon regulatory factor 1-deficient (IRF-1(-/-)) mice; however, only the GR019 (virB4) mutant was attenuated in cultured macrophages. Therefore, in vivo imaging provides a comprehensive approach to identify virulence genes that are relevant to in vivo pathogenesis. Our results provide greater insights into the role of galE in virulence and also suggest that BMEI1090 and downstream genes constitute a novel set of genes involved in Brucella virulence. Survival of the vaccine strain in the host for a critical period is important for effective Brucella vaccines. The galE mutant induced no changes in liver and spleen but localized chronically in the tail and protected IRF-1(-/-) and wild-type mice from virulent challenge, implying that this mutant may serve as a potential vaccine candidate in future studies and that the direct visualization of Brucella may provide insight into selection of improved vaccine candidates.

RNAi-mediated Gene Silencing of Mutant Myotilin Improves Myopathy in LGMD1A Mice

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Jian Liu

2014-01-01

Full Text Available Recent progress suggests gene therapy may one day be an option for treating some forms of limb girdle muscular dystrophy (LGMD. Nevertheless, approaches targeting LGMD have so far focused on gene replacement strategies for recessive forms of the disease. In contrast, no attempts have been made to develop molecular therapies for any of the eight dominantly inherited forms of LGMD. Importantly, the emergence of RNA interference (RNAi therapeutics in the last decade provided new tools to combat dominantly inherited LGMDs with molecular therapy. In this study, we describe the first RNAi-based, preclinical gene therapy approach for silencing a gene associated with dominant LGMD. To do this, we developed adeno-associated viral vectors (AAV6 carrying designed therapeutic microRNAs targeting mutant myotilin (MYOT, which is the underlying cause of LGMD type 1A (LGMD1A. Our best MYOT-targeted microRNA vector (called miMYOT significantly reduced mutant myotilin mRNA and soluble protein expression in muscles of LGMD1A mice (the TgT57I model both 3 and 9 months after delivery, demonstrating short- and long-term silencing effects. This MYOT gene silencing subsequently decreased deposition of MYOT-seeded intramuscular protein aggregates, which is the hallmark feature of LGMD1A. Histological improvements were accompanied by significant functional correction, as miMYOT-treated animals showed increased muscle weight and improved specific force in the gastrocnemius, which is one of the most severely affected muscles in TgT57I mice and patients with dominant myotilin mutations. These promising results in a preclinical model of LGMD1A support the further development of RNAi-based molecular therapy as a prospective treatment for LGMD1A. Furthermore, this study sets a foundation that may be refined and adapted to treat other dominant LGMD and related disorders.

Dynamics of competitive population abundance of Lactobacillus plantarum ivi gene mutants in faecel samples after passage through the gastrointestinal tract of mice

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Bron, P.A.; Meijer, M.; Bongers, R.S.; Vos, de W.M.; Kleerebezem, M.

2007-01-01

This study aims to evaluate the impact of mutation of previously identified in vivo-induced (ivi) genes on the persistence and survival of Lactobacillus plantarum WCFS1 in the gastrointestinal (GI) tract of mice. Methods and Results:¿ Nine Lact. plantarum ivi gene replacement mutants were

A Mycobacterium leprae Hsp65 mutant as a candidate for mitigating lupus aggravation in mice.

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Eliana B Marengo

Full Text Available Hsp60 is an abundant and highly conserved family of intracellular molecules. Increased levels of this family of proteins have been observed in the extracellular compartment in chronic inflammation. Administration of M. leprae Hsp65 [WT] in [NZBxNZW]F(1 mice accelerates the Systemic Lupus Erythematosus [SLE] progression whereas the point mutated K(409A Hsp65 protein delays the disease. Here, the biological effects of M. leprae Hsp65 Leader pep and K(409A pep synthetic peptides, which cover residues 352-371, are presented. Peptides had immunomodulatory effects similar to that observed with their respective proteins on survival and the combined administration of K(409A+Leader pep or K(409A pep+WT showed that the mutant forms were able to inhibit the deleterious effect of WT on mortality, indicating the neutralizing potential of the mutant molecules in SLE progression. Molecular modeling showed that replacing Lysine by Alanine affects the electrostatic potential of the 352-371 region. The number of interactions observed for WT is much higher than for Hsp65 K(409A and mouse Hsp60. The immunomodulatory effects of the point-mutated protein and peptide occurred regardless of the catalytic activity. These findings may be related to the lack of effect on survival when F(1 mice were inoculated with Hsp60 or K(409A pep. Our findings indicate the use of point-mutated Hsp65 molecules, such as the K(409A protein and its corresponding peptide, that may minimize or delay the onset of SLE, representing a new approach to the treatment of autoimmune diseases.

High amplitude phase resetting in rev-erbalpha/per1 double mutant mice.

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Corinne Jud

Full Text Available Over time, organisms developed various strategies to adapt to their environment. Circadian clocks are thought to have evolved to adjust to the predictable rhythms of the light-dark cycle caused by the rotation of the Earth around its own axis. The rhythms these clocks generate persist even in the absence of environmental cues with a period of about 24 hours. To tick in time, they continuously synchronize themselves to the prevailing photoperiod by appropriate phase shifts. In this study, we disrupted two molecular components of the mammalian circadian oscillator, Rev-Erbalpha and Period1 (Per1. We found that mice lacking these genes displayed robust circadian rhythms with significantly shorter periods under constant darkness conditions. Strikingly, they showed high amplitude resetting in response to a brief light pulse at the end of their subjective night phase, which is rare in mammals. Surprisingly, Cry1, a clock component not inducible by light in mammals, became slightly inducible in these mice. Taken together, Rev-Erbalpha and Per1 may be part of a mechanism preventing drastic phase shifts in mammals.

Reanalysis of parabiosis of obesity mutants in the age of leptin.

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Zeng, Wenwen; Lu, Yi-Hsueh; Lee, Jonah; Friedman, Jeffrey M

2015-07-21

In this study we set out to explain the differing effects of parabiosis with genetically diabetic (db) mice versus administration of recombinant leptin. Parabiosis of db mutant, which overexpress leptin, to wildtype (WT) or genetically obese (ob) mice has been reported to cause death by starvation, whereas leptin infusions do not produce lethality at any dose or mode of delivery tested. Leptin is not posttranslationally modified other than a single disulphide bond, raising the possibility that it might require additional factor(s) to exert the maximal appetite-suppressing effect. We reconfirmed the lethal effect of parabiosis of db mutant on WT mice and further showed that this lethality could not be rescued by administration of ghrelin or growth hormone. We then initiated a biochemical fractionation of a high-molecular-weight leptin complex from human plasma and identified clusterin as a major component of this leptin-containing complex. However, in contrast to previous reports, we failed to observe a leptin-potentiating effect of either exogenous or endogenous clusterin, and parabiosis of db clusterin(-/-) double-mutant to WT mice still caused lethality. Intriguingly, in parabiotic pairs of two WT mice, leptin infusion into one of the mice led to an enhanced starvation response during calorie restriction as evidenced by increased plasma ghrelin and growth-hormone levels. Moreover, leptin treatment resulted in death of the parabiotic pairs. These data suggest that the appetite suppression in WT mice after parabiosis to db mutants is the result of induced hyperleptinemia combined with the stress or other aspect(s) of the parabiosis procedure.

Phenotype of transgenic mice carrying a very low copy number of the mutant human G93A superoxide dismutase-1 gene associated with amyotrophic lateral sclerosis.

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Jeffrey S Deitch

Full Text Available Amyotrophic lateral sclerosis (ALS is a progressive neurodegenerative disease of the motor neuron. While most cases of ALS are sporadic, 10% are familial (FALS with 20% of FALS caused by a mutation in the gene that codes for the enzyme Cu/Zn superoxide dismutase (SOD1. There is variability in sporadic ALS as well as FALS where even within the same family some siblings with the same mutation do not manifest disease. A transgenic (Tg mouse model of FALS containing 25 copies of the mutant human SOD1 gene demonstrates motor neuron pathology and progressive weakness similar to ALS patients, leading to death at approximately 130 days. The onset of symptoms and survival of these transgenic mice are directly related to the number of copies of the mutant gene. We report the phenotype of a very low expressing (VLE G93A SOD1 Tg carrying only 4 copies of the mutant G93ASOD1 gene. While weakness can start at 9 months, only 74% of mice 18 months or older demonstrate disease. The VLE mice show decreased motor neurons compared to wild-type mice as well as increased cytoplasmic translocation of TDP-43. In contrast to the standard G93A SOD1 Tg mouse which always develops motor weakness leading to death, not all VLE animals manifested clinical disease or shortened life span. In fact, approximately 20% of mice older than 24 months had no motor symptoms and only 18% of VLE mice older than 22 months reached end stage. Given the variable penetrance of clinical phenotype, prolonged survival, and protracted loss of motor neurons the VLE mouse provides a new tool that closely mimics human ALS. This tool will allow the study of pathologic events over time as well as the study of genetic and environmental modifiers that may not be causative, but can exacerbate or accelerate motor neuron disease.

Abnormal iron metabolism and oxidative stress in mice expressing a mutant form of the ferritin light polypeptide gene

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Barbeito, Ana G.; Garringer, Holly J.; Baraibar, Martin A.; Gao, Xiaoying; Arredondo, Miguel; Núñez, Marco T.; Smith, Mark A.; Ghetti, Bernardino; Vidal, Ruben

2009-01-01

Insertional mutations in exon 4 of the ferritin light chain (FTL) gene are associated with hereditary ferritinopathy (HF) or neuroferritinopathy, an autosomal dominant neurodegenerative disease characterized by progressive impairment of motor and cognitive functions. To determine the pathogenic mechanisms by which mutations in FTL lead to neurodegeneration, we investigated iron metabolism and markers of oxidative stress in the brain of transgenic (Tg) mice that express the mutant human FTL498-499InsTC cDNA. Compared with wild-type mice, brain extracts from Tg (FTL-Tg) mice showed an increase in the cytoplasmic levels of both FTL and ferritin heavy chain polypeptides, a decrease in the protein and mRNA levels of transferrin receptor-1, and a significant increase in iron levels. Transgenic mice also showed the presence of markers for lipid peroxidation, protein carbonyls, and nitrone–protein adducts in the brain. However, gene expression analysis of iron management proteins in the liver of Tg mice indicates that the FTL-Tg mouse liver is iron deficient. Our data suggest that disruption of iron metabolism in the brain has a primary role in the process of neurodegeneration in HF and that the pathogenesis of HF is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates in the brain. PMID:19519778

Altered depression-related behavior and neurochemical changes in serotonergic neurons in mutant R406W human tau transgenic mice.

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Egashira, Nobuaki; Iwasaki, Katsunori; Takashima, Akihiko; Watanabe, Takuya; Kawabe, Hideyuki; Matsuda, Tomomi; Mishima, Kenichi; Chidori, Shozo; Nishimura, Ryoji; Fujiwara, Michihiro

2005-10-12

Mutant R406W human tau was originally identified in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and causes a hereditary tauopathy that clinically resembles Alzheimer's disease (AD). In the current study, we examined the performance of R406W transgenic (Tg) mice in the forced swimming test, a test with high predictivity of antidepressant efficacy in human depression, and found an enhancement of the immobility time. In contrast, the motor function and anxiety-related emotional response of R406W Tg mice were normal. Furthermore, a selective serotonin reuptake inhibitor (SSRI), fluvoxamine (100 mg/kg, p.o.), significantly reduced this enhancement of the immobility time, whereas a noradrenaline reuptake inhibitor, desipramine, had no effect. In an in vivo microdialysis study, R406W Tg mice exhibited a significantly decreased extracellular 5-hydroxyindoleacetic acid (5-HIAA) level in the frontal cortex and also exhibited a tendency toward a decreased extracellular 5-hydroxytryptamine (5-HT) level. Moreover, fluvoxamine, which reduced the enhancement of the immobility time, significantly increased the extracellular 5-HT level in R406W Tg mice. These results suggest that R406W Tg mice exhibit changes in depression-related behavior involving serotonergic neurons and provide an animal model for investigating AD with depression.

The Endocannabinoid System across Postnatal Development in Transmembrane Domain Neuregulin 1 Mutant Mice

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Rose Chesworth

2018-02-01

Full Text Available The use of cannabis is a well-established component risk factor for schizophrenia, particularly in adolescent individuals with genetic predisposition for the disorder. Alterations to the endocannabinoid system have been found in the prefrontal cortex of patients with schizophrenia. Thus, we assessed whether molecular alterations exist in the endocannabinoid signalling pathway during brain development in a mouse model for the schizophrenia risk gene neuregulin 1 (Nrg1. We analysed transcripts encoding key molecules of the endocannabinoid system in heterozygous transmembrane domain Nrg1 mutant mice (Nrg1 TM HET, which is known to have increased sensitivity to cannabis exposure. Tissue from the prelimbic cortex and hippocampus of male and female Nrg1 TM HET mice and wild type-like littermates was collected at postnatal days (PNDs 7, 10, 14, 21, 28, 35, 49, and 161. Quantitative polymerase chain reaction was conducted to assess mRNA levels of cannabinoid receptor 1 (CB1R and enzymes for the synthesis and breakdown of the endocannabinoid 2-arachidonoylglycerol [i.e., diacylglycerol lipase alpha (DAGLα, monoglyceride lipase (MGLL, and α/β-hydrolase domain-containing 6 (ABHD6]. No sex differences were found for any transcripts in either brain region; thus, male and female data were pooled. Hippocampal and cortical mRNA expression of DAGLα, MGLL, and ABHD6 increased until PND 21–35 and then decreased and stabilised for the rest of postnatal development. Hippocampal CB1R mRNA expression increased until PND 21 and decreased after this age. Expression levels of these endocannabinoid markers did not differ in Nrg1 TM HET compared to control mice at any time point. Here, we demonstrate dynamic changes in the developmental trajectory of several key endocannabinoid system transcripts in the mouse brain, which may correspond with periods of endocannabinoid system maturation. Nrg1 TM HET mutation did not alter the developmental trajectory of the

Intact interval timing in circadian CLOCK mutants.

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Cordes, Sara; Gallistel, C R

2008-08-28

While progress has been made in determining the molecular basis for the circadian clock, the mechanism by which mammalian brains time intervals measured in seconds to minutes remains a mystery. An obvious question is whether the interval-timing mechanism shares molecular machinery with the circadian timing mechanism. In the current study, we trained circadian CLOCK +/- and -/- mutant male mice in a peak-interval procedure with 10 and 20-s criteria. The mutant mice were more active than their wild-type littermates, but there were no reliable deficits in the accuracy or precision of their timing as compared with wild-type littermates. This suggests that expression of the CLOCK protein is not necessary for normal interval timing.

Mice deficient for striatal Vesicular Acetylcholine Transporter (VAChT) display impaired short-term but normal long-term object recognition memory.

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Palmer, Daniel; Creighton, Samantha; Prado, Vania F; Prado, Marco A M; Choleris, Elena; Winters, Boyer D

2016-09-15

Substantial evidence implicates Acetylcholine (ACh) in the acquisition of object memories. While most research has focused on the role of the cholinergic basal forebrain and its cortical targets, there are additional cholinergic networks that may contribute to object recognition. The striatum contains an independent cholinergic network comprised of interneurons. In the current study, we investigated the role of this cholinergic signalling in object recognition using mice deficient for Vesicular Acetylcholine Transporter (VAChT) within interneurons of the striatum. We tested whether these striatal VAChT(D2-Cre-flox/flox) mice would display normal short-term (5 or 15min retention delay) and long-term (3h retention delay) object recognition memory. In a home cage object recognition task, male and female VAChT(D2-Cre-flox/flox) mice were impaired selectively with a 15min retention delay. When tested on an object location task, VAChT(D2-Cre-flox/flox) mice displayed intact spatial memory. Finally, when object recognition was tested in a Y-shaped apparatus, designed to minimize the influence of spatial and contextual cues, only females displayed impaired recognition with a 5min retention delay, but when males were challenged with a 15min retention delay, they were also impaired; neither males nor females were impaired with the 3h delay. The pattern of results suggests that striatal cholinergic transmission plays a role in the short-term memory for object features, but not spatial location. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of Brucella abortus mutant strain Δ22915, a potential vaccine candidate.

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Bao, Yanqing; Tian, Mingxing; Li, Peng; Liu, Jiameng; Ding, Chan; Yu, Shengqing

2017-04-04

Brucellosis, caused by Brucella spp., is an important zoonosis worldwide. Vaccination is an effective strategy for protection against Brucella infection in livestock in developing countries and in wildlife in developed countries. However, current vaccine strains including S19 and RB51 are pathogenic to humans and pregnant animals, limiting their use. In this study, we constructed the Brucella abortus (B. abortus) S2308 mutant strain Δ22915, in which the putative lytic transglycosylase gene BAB_RS22915 was deleted. The biological properties of mutant strain Δ22915 were characterized and protection of mice against virulent S2308 challenge was evaluated. The mutant strain Δ22915 showed reduced survival within RAW264.7 cells and survival in vivo in mice. In addition, the mutant strain Δ22915 failed to escape fusion with lysosomes within host cells, and caused no observable pathological damage. RNA-seq analysis indicated that four genes associated with amino acid/nucleotide transport and metabolism were significantly upregulated in mutant strain Δ22915. Furthermore, inoculation of ∆22915 at 10 5 colony forming units induced effective host immune responses and long-term protection of BALB/c mice. Therefore, mutant strain ∆22915 could be used as a novel vaccine candidate in the future to protect animals against B. abortus infection.

Msh2 acts in medium-spiny striatal neurons as an enhancer of CAG instability and mutant huntingtin phenotypes in Huntington's disease knock-in mice.

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Marina Kovalenko

Full Text Available The CAG trinucleotide repeat mutation in the Huntington's disease gene (HTT exhibits age-dependent tissue-specific expansion that correlates with disease onset in patients, implicating somatic expansion as a disease modifier and potential therapeutic target. Somatic HTT CAG expansion is critically dependent on proteins in the mismatch repair (MMR pathway. To gain further insight into mechanisms of somatic expansion and the relationship of somatic expansion to the disease process in selectively vulnerable MSNs we have crossed HTT CAG knock-in mice (HdhQ111 with mice carrying a conditional (floxed Msh2 allele and D9-Cre transgenic mice, in which Cre recombinase is expressed specifically in MSNs within the striatum. Deletion of Msh2 in MSNs eliminated Msh2 protein in those neurons. We demonstrate that MSN-specific deletion of Msh2 was sufficient to eliminate the vast majority of striatal HTT CAG expansions in HdhQ111 mice. Furthermore, MSN-specific deletion of Msh2 modified two mutant huntingtin phenotypes: the early nuclear localization of diffusely immunostaining mutant huntingtin was slowed; and the later development of intranuclear huntingtin inclusions was dramatically inhibited. Therefore, Msh2 acts within MSNs as a genetic enhancer both of somatic HTT CAG expansions and of HTT CAG-dependent phenotypes in mice. These data suggest that the selective vulnerability of MSNs may be at least in part contributed by the propensity for somatic expansion in these neurons, and imply that intervening in the expansion process is likely to have therapeutic benefit.

Cardiac dysfunction in Pkd1-deficient mice with phenotype rescue by galectin-3 knockout

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Balbo, Bruno E.; Amaral, Andressa G.; Fonseca, Jonathan M.; de Castro, Isac; Salemi, Vera M.; Souza, Leandro E.; dos Santos, Fernando; Irigoyen, Maria C.; Qian, Feng; Chammas, Roger; Onuchic, Luiz F.

2016-01-01

Alterations in myocardial wall texture stand out among ADPKD cardiovascular manifestations, in hypertensive and normotensive patients. To elucidate their pathogenesis, we analyzed the cardiac phenotype in Pkd1cond/cond:Nestincre (CYG+) cystic mice exposed to increased blood pressure, at 5–6 and 20–24 weeks of age, and Pkd1+/− (HTG+) noncystic mice at 5–6 and 10–13 weeks. Echocardiographic analyses revealed decreased myocardial deformation and systolic function in CYG+ and HTG+ mice, as well as diastolic dysfunction in older CYG+ mice, compared to their Pkd1cond/cond and Pkd1+/+ controls. Hearts from CYG+ and HTG+ mice presented reduced polycystin-1 expression, increased apoptosis and mild fibrosis. Since galectin-3 has been associated with heart dysfunction, we studied it as a potential modifier of the ADPKD cardiac phenotype. Double-mutant Pkd1cond/cond:Nestincre;Lgals3−/− (CYG−) and Pkd1+/−;Lgals3−/− (HTG−) mice displayed improved cardiac deformability and systolic parameters compared to single-mutants, not differing from their controls. CYG− and HTG− showed decreased apoptosis and fibrosis. Analysis of a severe cystic model (Pkd1V/V; VVG+) showed that Pkd1V/V;Lgals3−/− (VVG−) mice have longer survival, decreased cardiac apoptosis and improved heart function compared to VVG+. CYG− and VVG− animals showed no difference in renal cystic burden compared to CYG+ and VVG+ mice. Thus, myocardial dysfunction occurs in different Pkd1-deficient models and suppression of galectin-3 expression rescues this phenotype. PMID:27475230

Rational design of a live attenuated dengue vaccine: 2'-o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques.

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Roland Züst

Full Text Available Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

Are Sema5a mutant mice a good model of autism? A behavioral analysis of sensory systems, emotionality and cognition

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Gunn, Rhian K.; Huentelman, Matthew J.; Brown, Richard E.

2011-01-01

Semaphorin 5A (Sema5A) expression is reduced in the brain of individuals with autism, thus mice with reduced Sema5A levels may serve as a model of this neurodevelopmental disorder. We tested male and female Sema5a knockout mice (B6.129P2SEMA5A/J) and C57BL/6J controls for emotionality, visual ability, prepulse inhibition, motor learning and cognition. Overall, there were only two genotype differences in emotionality: Sema5a mutant mice had more stretch-attend postures in the elevated plus-maze and more defecations in the open field. All mice could see, but Sema5a mice had better visual ability than C57BL/6J mice. There were no genotype differences in sensory-motor gating. Sema5a mice showed higher levels of activity in the elevated plus-maze and light/dark transition box, and there were sex by genotype differences in the Rotarod, suggesting a sex difference in balance and coordination differentially affected by Sema5a. There were no genotype effects on cognition: Sema5a mice did not differ from C57BL/6J in the Morris water maze, set-shifting or cued and contextual fear conditioning. In the social recognition test, all mice preferred social stimuli, but there was no preference for social novelty, thus the Sema5A mice do not have a deficit in social behavior. Overall, there were a number of sex differences, with females showing greater activity and males performing better in tests of spatial learning and memory, but no deficits in the behavior of Sema5A mice. We conclude that the Sema5a mice do not meet the behavioral criteria for a mouse model of autism. PMID:21777623

The subcutaneous inoculation of pH 6 antigen mutants of Yersinia pestis does not affect virulence and immune response in mice.

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Anisimov, Andrey P; Bakhteeva, Irina V; Panfertsev, Evgeniy A; Svetoch, Tat'yana E; Kravchenko, Tat'yana B; Platonov, Mikhail E; Titareva, Galina M; Kombarova, Tat'yana I; Ivanov, Sergey A; Rakin, Alexander V; Amoako, Kingsley K; Dentovskaya, Svetlana V

2009-01-01

Two isogenic sets of Yersinia pestis strains were generated, composed of wild-type strains 231 and I-1996, their non-polar pH 6(-) mutants with deletions in the psaA gene that codes for its structural subunit or the whole operon, as well as strains with restored ability for temperature- and pH-dependent synthesis of adhesion pili or constitutive production of pH 6 antigen. The mutants were generated by site-directed mutagenesis of the psa operon and subsequent complementation in trans. It was shown that the loss of synthesis or constitutive production of pH 6 antigen did not influence Y. pestis virulence or the average survival time of subcutaneously inoculated BALB/c naïve mice or animals immunized with this antigen.

Paternal Aging Affects Behavior in Pax6 Mutant Mice: A Gene/Environment Interaction in Understanding Neurodevelopmental Disorders.

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Yoshizaki, Kaichi; Furuse, Tamio; Kimura, Ryuichi; Tucci, Valter; Kaneda, Hideki; Wakana, Shigeharu; Osumi, Noriko

2016-01-01

Neurodevelopmental disorders such as autism spectrum disorder (ASD) and attention deficit and hyperactivity disorder (ADHD) have increased over the last few decades. These neurodevelopmental disorders are characterized by a complex etiology, which involves multiple genes and gene-environmental interactions. Various genes that control specific properties of neural development exert pivotal roles in the occurrence and severity of phenotypes associated with neurodevelopmental disorders. Moreover, paternal aging has been reported as one of the factors that contribute to the risk of ASD and ADHD. Here we report, for the first time, that paternal aging has profound effects on the onset of behavioral abnormalities in mice carrying a mutation of Pax6, a gene with neurodevelopmental regulatory functions. We adopted an in vitro fertilization approach to restrict the influence of additional factors. Comprehensive behavioral analyses were performed in Sey/+ mice (i.e., Pax6 mutant heterozygotes) born from in vitro fertilization of sperm taken from young or aged Sey/+ fathers. No body weight changes were found in the four groups, i.e., Sey/+ and wild type (WT) mice born to young or aged father. However, we found important differences in maternal separation-induced ultrasonic vocalizations of Sey/+ mice born from young father and in the level of hyperactivity of Sey/+ mice born from aged fathers in the open-field test, respectively, compared to WT littermates. Phenotypes of anxiety were observed in both genotypes born from aged fathers compared with those born from young fathers. No significant difference was found in social behavior and sensorimotor gating among the four groups. These results indicate that mice with a single genetic risk factor can develop different phenotypes depending on the paternal age. Our study advocates for serious considerations on the role of paternal aging in breeding strategies for animal studies.

Paternal Aging Affects Behavior in Pax6 Mutant Mice: A Gene/Environment Interaction in Understanding Neurodevelopmental Disorders.

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Kaichi Yoshizaki

Full Text Available Neurodevelopmental disorders such as autism spectrum disorder (ASD and attention deficit and hyperactivity disorder (ADHD have increased over the last few decades. These neurodevelopmental disorders are characterized by a complex etiology, which involves multiple genes and gene-environmental interactions. Various genes that control specific properties of neural development exert pivotal roles in the occurrence and severity of phenotypes associated with neurodevelopmental disorders. Moreover, paternal aging has been reported as one of the factors that contribute to the risk of ASD and ADHD. Here we report, for the first time, that paternal aging has profound effects on the onset of behavioral abnormalities in mice carrying a mutation of Pax6, a gene with neurodevelopmental regulatory functions. We adopted an in vitro fertilization approach to restrict the influence of additional factors. Comprehensive behavioral analyses were performed in Sey/+ mice (i.e., Pax6 mutant heterozygotes born from in vitro fertilization of sperm taken from young or aged Sey/+ fathers. No body weight changes were found in the four groups, i.e., Sey/+ and wild type (WT mice born to young or aged father. However, we found important differences in maternal separation-induced ultrasonic vocalizations of Sey/+ mice born from young father and in the level of hyperactivity of Sey/+ mice born from aged fathers in the open-field test, respectively, compared to WT littermates. Phenotypes of anxiety were observed in both genotypes born from aged fathers compared with those born from young fathers. No significant difference was found in social behavior and sensorimotor gating among the four groups. These results indicate that mice with a single genetic risk factor can develop different phenotypes depending on the paternal age. Our study advocates for serious considerations on the role of paternal aging in breeding strategies for animal studies.

A novel p53 mutational hotspot in skin tumors from UV-irradiated Xpc mutant mice alters transactivation functions.

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Inga, Alberto; Nahari, Dorit; Velasco-Miguel, Susana; Friedberg, Errol C; Resnick, Michael A

2002-08-22

A mutation in codon 122 of the mouse p53 gene resulting in a T to L amino acid substitution (T122-->L) is frequently associated with skin cancer in UV-irradiated mice that are both homozygous mutant for the nucleotide excision repair (NER) gene Xpc (Xpc(-/-)) and hemizygous mutant for the p53 gene. We investigated the functional consequences of the mouse T122-->L mutation when expressed either in mammalian cells or in the yeast Saccharomyces cerevisiae. Similar to a non-functional allele, high expression of the T122-->L allele in p53(-/-) mouse embryo fibroblasts and human Saos-2 cells failed to suppress growth. However, the T122-->L mutant p53 showed wild-type transactivation levels with Bax and MDM2 promoters when expressed in either cell type and retained transactivation of the p21 and the c-Fos promoters in one cell line. Using a recently developed rheostatable p53 induction system in yeast we assessed the T122-->L transactivation capacity at low levels of protein expression using 12 different p53 response elements (REs). Compared to wild-type p53 the T122-->L protein manifested an unusual transactivation pattern comprising reduced and enhanced activity with specific REs. The high incidence of the T122-->L mutant allele in the Xpc(-/-) background suggests that both genetic and epigenetic conditions may facilitate the emergence of particular functional p53 mutations. Furthermore, the approach that we have taken also provides for the dissection of functions that may be retained in many p53 tumor alleles.

Reduced alcohol consumption in mice lacking preprodynorphin.

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Blednov, Yuri A; Walker, Danielle; Martinez, Marni; Harris, R Adron

2006-10-01

Many studies suggest a role for endogenous opioid peptides and their receptors in regulation of ethanol intake. It is commonly accepted that the kappa-opioid receptors and their endogenous ligands, dynorphins, produce a dysphoric state and therefore may be responsible for avoidance of alcohol. We used mutant mice lacking preprodynorphin in a variety of behavioral tests of alcohol actions. Null mutant female, but not male, mice showed significantly lower preference for alcohol and consumed lower amounts of alcohol in a two-bottle choice test as compared with wild-type littermates. In the same test, knockout mice of both sexes showed a strong reduction of preference for saccharin compared to control mice. In contrast, under conditions of limited (4 h) access (light phase of the light/dark cycle), null mutant mice did not show any differences in consumption of saccharin, but they showed significantly reduced intake of sucrose. To determine the possible cause for reduction of ethanol preference and intake, we studied other ethanol-related behaviors in mice lacking the preprodynorphin gene. There were no differences between null mutant and wild-type mice in ethanol-induced loss of righting reflex, acute ethanol withdrawal, ethanol-induced conditioned place preference, or conditioned taste aversion to ethanol. These results indicate that deletion of preprodynorphin leads to substantial reduction of alcohol intake in female mice, and suggest that this is caused by decreased orosensory reward of alcohol (sweet taste and/or palatability).

Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae.

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Matsuoka, Hiroyuki; Hashimoto, Kazuya; Saijo, Aki; Takada, Yuki; Kondo, Akihiko; Ueda, Mitsuyoshi; Ooshima, Hiroshi; Tachibana, Taro; Azuma, Masayuki

2014-02-01

A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β-Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9-fold higher than that of the wild-type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild-type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild-type strain. The relative value (mnn2 deletion mutant/wild-type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β-glucosidase activity using p-nitrophenyl-β-glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high-molecular-weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.

Resistance to organophosphorus agent toxicity in transgenic mice expressing the G117H mutant of human butyrylcholinesterase

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Wang Yuxia; Ticu Boeck, Andreea; Duysen, Ellen G.; Van Keuren, Margaret; Saunders, Thomas L.; Lockridge, Oksana

2004-01-01

Organophosphorus toxicants (OP) include chemical nerve agents and pesticides. The goal of this work was to find out whether an animal could be made resistant to OP toxicity by genetic engineering. The human butyrylcholinesterase (BChE) mutant G117H was chosen for study because it has the unusual ability to hydrolyze OP as well as acetylcholine, and it is resistant to inhibition by OP. Human G117H BChE, under the control of the ROSA26 promoter, was expressed in all tissues of transgenic mice. A stable transgenic mouse line expressed 0.5 μg/ml of human G117H BChE in plasma as well as 2 μg/ml of wild-type mouse BChE. Intestine, kidneys, stomach, lungs, heart, spleen, liver, brain, and muscle expressed 0.6-0.15 μg/g of G117H BChE. Transgenic mice were normal in behavior and fertility. The LD50 dose of echothiophate for wild-type mice was 0.1 mg/kg sc. This dose caused severe cholinergic signs of toxicity and lethality in wild-type mice, but caused no deaths and only mild toxicity in transgenic animals. The mechanism of protection was investigated by measuring acetylcholinesterase (AChE) and BChE activity. It was found that AChE and endogenous BChE were inhibited to the same extent in echothiophate-treated wild type and transgenic mice. This led to the hypothesis that protection against echothiophate toxicity was not explained by hydrolysis of echothiophate. In conclusion, the transgenic G117H BChE mouse demonstrates the factors required to achieve protection from OP toxicity in a vertebrate animal

Proteostasis and ageing: insights from long-lived mutant mice.

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Sands, William A; Page, Melissa M; Selman, Colin

2017-10-15

The global increase in life expectancy is creating significant medical, social and economic challenges to current and future generations. Consequently, there is a need to identify the fundamental mechanisms underlying the ageing process. This knowledge should help develop realistic interventions capable of combatting age-related disease, and thus improving late-life health and vitality. While several mechanisms have been proposed as conserved lifespan determinants, the loss of proteostasis - where proteostasis is defined here as the maintenance of the proteome - appears highly relevant to both ageing and disease. Several studies have shown that multiple proteostatic mechanisms, including the endoplasmic reticulum (ER)-induced unfolded protein response (UPR), the ubiquitin-proteasome system (UPS) and autophagy, appear indispensable for longevity in many long-lived invertebrate mutants. Similarly, interspecific comparisons suggest that proteostasis may be an important lifespan determinant in vertebrates. Over the last 20 years a number of long-lived mouse mutants have been described, many of which carry single-gene mutations within the growth-hormone, insulin/IGF-1 or mTOR signalling pathways. However, we still do not know how these mutations act mechanistically to increase lifespan and healthspan, and accordingly whether mechanistic commonality occurs between different mutants. Recent evidence supports the premise that the successful maintenance of the proteome during ageing may be linked to the increased lifespan and healthspan of long-lived mouse mutants. © 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Selective Transgenic Expression of Mutant Ubiquitin in Purkinje Cell Stripes in the Cerebellum.

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Verheijen, Bert M; Gentier, Romina J G; Hermes, Denise J H P; van Leeuwen, Fred W; Hopkins, David A

2017-06-01

The ubiquitin-proteasome system (UPS) is one of the major mechanisms for protein breakdown in cells, targeting proteins for degradation by enzymatically conjugating them to ubiquitin molecules. Intracellular accumulation of ubiquitin-B +1 (UBB +1 ), a frameshift mutant of ubiquitin-B, is indicative of a dysfunctional UPS and has been implicated in several disorders, including neurodegenerative disease. UBB +1 -expressing transgenic mice display widespread labeling for UBB +1 in brain and exhibit behavioral deficits. Here, we show that UBB +1 is specifically expressed in a subset of parasagittal stripes of Purkinje cells in the cerebellar cortex of a UBB +1 -expressing mouse model. This expression pattern is reminiscent of that of the constitutively expressed Purkinje cell antigen HSP25, a small heat shock protein with neuroprotective properties.

Myg1-deficient mice display alterations in stress-induced responses and reduction of sex-dependent behavioural differences.

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Philips, Mari-Anne; Abramov, Urho; Lilleväli, Kersti; Luuk, Hendrik; Kurrikoff, Kaido; Raud, Sirli; Plaas, Mario; Innos, Jürgen; Puussaar, Triinu; Kõks, Sulev; Vasar, Eero

2010-02-11

Myg1 (Melanocyte proliferating gene 1) is a highly conserved and ubiquitously expressed gene, which encodes a protein with mitochondrial and nuclear localization. In the current study we demonstrate a gradual decline of Myg1 expression during the postnatal development of the mouse brain that suggests relevance for Myg1 in developmental processes. To study the effects of Myg1 loss-of-function, we created Myg1-deficient (-/-) mice by displacing the entire coding sequence of the gene. Initial phenotyping, covering a multitude of behavioural, cognitive, neurological, physiological and stress-related responses, revealed that homozygous Myg1 (-/-) mice are vital, fertile and display no gross abnormalities. Myg1 (-/-) mice showed an inconsistent pattern of altered anxiety-like behaviour in different tests. The plus-maze and social interaction tests revealed that male Myg1 (-/-) mice were significantly less anxious than their wild-type littermates; female (-/-) mice showed increased anxiety in the locomotor activity arena. Restraint-stress significantly reduced the expression of the Myg1 gene in the prefrontal cortex of female wild-type mice and restrained female (-/-) mice showed a blunted corticosterone response, suggesting involvement of Myg1 in stress-induced responses. The main finding of the present study was that Myg1 invalidation decreases several behavioural differences between male and female animals that were obvious in wild-type mice, indicating that Myg1 contributes to the expression of sex-dependent behavioural differences in mice. Taken together, we provide evidence for the involvement of Myg1 in anxiety- and stress-related responses and suggest that Myg1 contributes to the expression of sex-dependent behavioural differences.

Brucella abortus mutants lacking ATP-binding cassette transporter proteins are highly attenuated in virulence and confer protective immunity against virulent B. abortus challenge in BALB/c mice.

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Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Park, Bo-Kyoung; Hahn, Tae-Wook

2016-06-01

Brucella abortus RB51 is an attenuated vaccine strain that has been most frequently used for bovine brucellosis. Although it is known to provide good protection in cattle, it still has some drawbacks including resistance to rifampicin, residual virulence and pathogenicity in humans. Thus, there has been a continuous interest on new safe and effective bovine vaccine candidates. In the present study, we have constructed unmarked mutants by deleting singly cydD and cydC genes, which encode ATP-binding cassette transporter proteins, from the chromosome of the virulent Brucella abortus isolate from Korean cow (referred to as IVK15). Both IVK15ΔcydD and ΔcydC mutants showed increased sensitivity to metal ions, hydrogen peroxide and acidic pH, which are mimic to intracellular environment during host infection. Additionally, the mutants exhibited a significant growth defect in RAW264.7 cells and greatly attenuated in mice. Vaccination of mice with either IVK15ΔcydC or IVK15ΔcydD mutant could elicit an anti-Brucella specific immunoglobulin G (IgG) and IgG subclass responses as well as enhance the secretion of interferon-gamma, and provided better protection against challenge with B. abortus strain 2308 than with the commercial B. abortus strain RB51 vaccine. Collectively, these results suggest that both IVK15ΔcydC and IVK15ΔcydD mutants could be an attenuated vaccine candidate against B. abortus. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mice with Sort1 deficiency display normal cognition but elevated anxiety-like behavior.

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Ruan, Chun-Sheng; Yang, Chun-Rui; Li, Jia-Yi; Luo, Hai-Yun; Bobrovskaya, Larisa; Zhou, Xin-Fu

2016-07-01

Exposure to stressful life events plays a central role in the development of mood disorders in vulnerable individuals. However, the mechanisms that link mood disorders to stress are poorly understood. Brain-derived neurotrophic factor (BDNF) has long been implicated in positive regulation of depression and anxiety, while its precursor (proBDNF) recently showed an opposing effect on such mental illnesses. P75(NTR) and sortilin are co-receptors of proBDNF, however, the role of these receptors in mood regulation is not established. Here, we aimed to investigate the role of sortilin in regulating mood-related behaviors and its role in the proBDNF-mediated mood abnormality in mice. We found that sortilin was up-regulated in neocortex (by 78.3%) and hippocampus (by 111%) of chronically stressed mice as assessed by western blot analysis. These changes were associated with decreased mobility in the open field test and increased depression-like behavior in the forced swimming test. We also found that sortilin deficiency in mice resulted in hyperlocomotion in the open field test and increased anxiety-like behavior in both the open field and elevated plus maze tests. No depression-like behavior in the forced swimming test and no deficit in spatial cognition in the Morris water maze test were found in the Sort1-deficient mice. Moreover, the intracellular and extracellular levels of mature BDNF and proBDNF were not changed when sortilin was absent in vivo and in vitro. Finally, we found that both WT and Sort1-deficient mice injected with proBDNF in lateral ventricle displayed increased depression-like behavior in the forced swimming test but not anxiety-like behaviors in the open field and elevated plus maze tests. The present study suggests that sortilin functions as a negative regulator of mood performance and can be a therapeutic target for the treatment of mental illness. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

Homeostatic imbalance between apoptosis and cell renewal in the liver of premature aging Xpd mice.

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Jung Yoon Park

2008-06-01

Full Text Available Unrepaired or misrepaired DNA damage has been implicated as a causal factor in cancer and aging. Xpd(TTD mice, harboring defects in nucleotide excision repair and transcription due to a mutation in the Xpd gene (R722W, display severe symptoms of premature aging but have a reduced incidence of cancer. To gain further insight into the molecular basis of the mutant-specific manifestation of age-related phenotypes, we used comparative microarray analysis of young and old female livers to discover gene expression signatures distinguishing Xpd(TTD mice from their age-matched wild type controls. We found a transcription signature of increased apoptosis in the Xpd(TTD mice, which was confirmed by in situ immunohistochemical analysis and found to be accompanied by increased proliferation. However, apoptosis rate exceeded the rate of proliferation, resulting in homeostatic imbalance. Interestingly, a metabolic response signature was observed involving decreased energy metabolism and reduced IGF-1 signaling, a major modulator of life span. We conclude that while the increased apoptotic response to endogenous DNA damage contributes to the accelerated aging phenotypes and the reduced cancer incidence observed in the Xpd(TTD mice, the signature of reduced energy metabolism is likely to reflect a compensatory adjustment to limit the increased genotoxic stress in these mutants. These results support a general model for premature aging in DNA repair deficient mice based on cellular responses to DNA damage that impair normal tissue homeostasis.

The central cannabinoid CB1 receptor is required for diet-induced obesity and rimonabant's antiobesity effects in mice.

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Pang, Zhen; Wu, Nancy N; Zhao, Weiguang; Chain, David C; Schaffer, Erica; Zhang, Xin; Yamdagni, Preeti; Palejwala, Vaseem A; Fan, Chunpeng; Favara, Sarah G; Dressler, Holly M; Economides, Kyriakos D; Weinstock, Daniel; Cavallo, Jean S; Naimi, Souad; Galzin, Anne-Marie; Guillot, Etienne; Pruniaux, Marie-Pierre; Tocci, Michael J; Polites, H Greg

2011-10-01

Cannabinoid receptor CB1 is expressed abundantly in the brain and presumably in the peripheral tissues responsible for energy metabolism. It is unclear if the antiobesity effects of rimonabant, a CB1 antagonist, are mediated through the central or the peripheral CB1 receptors. To address this question, we generated transgenic mice with central nervous system (CNS)-specific knockdown (KD) of CB1, by expressing an artificial microRNA (AMIR) under the control of the neuronal Thy1.2 promoter. In the mutant mice, CB1 expression was reduced in the brain and spinal cord, whereas no change was observed in the superior cervical ganglia (SCG), sympathetic trunk, enteric nervous system, and pancreatic ganglia. In contrast to the neuronal tissues, CB1 was undetectable in the brown adipose tissue (BAT) or the liver. Consistent with the selective loss of central CB1, agonist-induced hypothermia was attenuated in the mutant mice, but the agonist-induced delay of gastrointestinal transit (GIT), a primarily peripheral nervous system-mediated effect, was not. Compared to wild-type (WT) littermates, the mutant mice displayed reduced body weight (BW), adiposity, and feeding efficiency, and when fed a high-fat diet (HFD), showed decreased plasma insulin, leptin, cholesterol, and triglyceride levels, and elevated adiponectin levels. Furthermore, the therapeutic effects of rimonabant on food intake (FI), BW, and serum parameters were markedly reduced and correlated with the degree of CB1 KD. Thus, KD of CB1 in the CNS recapitulates the metabolic phenotype of CB1 knockout (KO) mice and diminishes rimonabant's efficacy, indicating that blockade of central CB1 is required for rimonabant's antiobesity actions.

Characterization and protective property of Brucella abortus cydC and looP mutants.

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Truong, Quang Lam; Cho, Youngjae; Barate, Abhijit Kashinath; Kim, Suk; Hahn, Tae-Wook

2014-11-01

Brucella abortus readily multiplies in professional or nonprofessional phagocytes in vitro and is highly virulent in mice. Isogenic mutants of B. abortus biovar 1 strain IVKB9007 lacking the ATP/GDP-binding protein motif A (P-loop) (named looP; designated here the IVKB9007 looP::Tn5 mutant) and the ATP-binding/permease protein (cydC; designated here the IVKB9007 cydC::Tn5 mutant) were identified and characterized by transposon mutagenesis using the mini-Tn5Km2 transposon. Both mutants were found to be virtually incapable of intracellular replication in both murine macrophages (RAW264.7) and the HeLa cell line, and their virulence was significantly impaired in BALB/c mice. Respective complementation of the IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants restored their ability to survive in vitro and in vivo to a level comparable with that of the wild type. These findings indicate that the cydC and looP genes play important roles in the virulence of B. abortus. In addition, intraperitoneal immunization of mice with a dose of the live IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants provided a high degree of protection against challenge with pathogenic B. abortus strain 544. Both mutants should be evaluated further as a live attenuated vaccine against bovine brucellosis for their ability to stimulate a protective immune response. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

ALS mutant SOD1 interacts with G3BP1 and affects stress granule dynamics.

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Gal, Jozsef; Kuang, Lisha; Barnett, Kelly R; Zhu, Brian Z; Shissler, Susannah C; Korotkov, Konstantin V; Hayward, Lawrence J; Kasarskis, Edward J; Zhu, Haining

2016-10-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Mutations in Cu/Zn superoxide dismutase (SOD1) are responsible for approximately 20 % of the familial ALS cases. ALS-causing SOD1 mutants display a gain-of-toxicity phenotype, but the nature of this toxicity is still not fully understood. The Ras GTPase-activating protein-binding protein G3BP1 plays a critical role in stress granule dynamics. Alterations in the dynamics of stress granules have been reported in several other forms of ALS unrelated to SOD1. To our surprise, the mutant G93A SOD1 transgenic mice exhibited pathological cytoplasmic inclusions that co-localized with G3BP1-positive granules in spinal cord motor neurons. The co-localization was also observed in fibroblast cells derived from familial ALS patient carrying SOD1 mutation L144F. Mutant SOD1, unlike wild-type SOD1, interacted with G3BP1 in an RNA-independent manner. Moreover, the interaction is specific for G3BP1 since mutant SOD1 showed little interaction with four other RNA-binding proteins implicated in ALS. The RNA-binding RRM domain of G3BP1 and two particular phenylalanine residues (F380 and F382) are critical for this interaction. Mutant SOD1 delayed the formation of G3BP1- and TIA1-positive stress granules in response to hyperosmolar shock and arsenite treatment in N2A cells. In summary, the aberrant mutant SOD1-G3BP1 interaction affects stress granule dynamics, suggesting a potential link between pathogenic SOD1 mutations and RNA metabolism alterations in ALS.

PP4 is essential for germinal center formation and class switch recombination in mice.

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Ming-Yu Chen

Full Text Available PP4 is a serine/threonine phosphatase required for immunoglobulin (Ig VDJ recombination and pro-B/pre-B cell development in mice. To elucidate the role of PP4 in mature B cells, we ablated the catalytic subunit of murine PP4 in vivo utilizing the CD23 promoter and cre-loxP recombination and generated CD23(crePP4(F/F mice. The development of follicular and marginal zone B cells was unaffected in these mutants, but the proliferation of mature PP4-deficient B cells stimulated by in vitro treatment with either anti-IgM antibody (Ab or LPS was partially impaired. Interestingly, the induction of CD80 and CD86 expression on these stimulated B cells was normal. Basal levels of serum Igs of all isotypes were strongly reduced in CD23(crePP4(F/F mice, and their B cells showed a reduced efficiency of class switch recombination (CSR in vitro upon stimulation by LPS or LPS plus IL-4. When CD23(crePP4(F/F mice were challenged with either the T cell-dependent antigen TNP-KLH or the T cell-independent antigen TNP-Ficoll, or by H1N1 virus infection, the mutant animals failed to form germinal centers (GCs in the spleen and the draining mediastinal lymph nodes, and did not efficiently mount antigen-specific humoral responses. In the resting state, PP4-deficient B cells exhibited pre-existing DNA fragmentation. Upon stimulation by DNA-damaging drug etoposide in vitro, mutant B cells showed increased cleavage of caspase 3. In addition, the mutant B cells displayed impaired CD40-mediated MAPK activation, abnormal IgM-mediated NF-κB activation, and reduced S phase entry upon IgM/CD40-stimulation. Taken together, our results establish a novel role for PP4 in CSR, and reveal crucial functions for PP4 in the maintenance of genomic stability, GC formation, and B cell-mediated immune responses.

Pharmacologic Treatment Assigned for Niemann Pick Type C1 Disease Partly Changes Behavioral Traits in Wild-Type Mice.

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Schlegel, Victoria; Thieme, Markus; Holzmann, Carsten; Witt, Martin; Grittner, Ulrike; Rolfs, Arndt; Wree, Andreas

2016-11-09

Niemann-Pick Type C1 (NPC1) is an autosomal recessive inherited disorder characterized by accumulation of cholesterol and glycosphingolipids. Previously, we demonstrated that BALB/c-npc1 nih Npc1 -/- mice treated with miglustat, cyclodextrin and allopregnanolone generally performed better than untreated Npc1 -/- animals. Unexpectedly, they also seemed to accomplish motor tests better than their sham-treated wild-type littermates. However, combination-treated mutant mice displayed worse cognition performance compared to sham-treated ones. To evaluate effects of these drugs in healthy BALB/c mice, we here analyzed pharmacologic effects on motor and cognitive behavior of wild-type mice. For combination treatment mice were injected with allopregnanolone/cyclodextrin weekly, starting at P7. Miglustat injections were performed daily from P10 till P23. Starting at P23, miglustat was embedded in the chow. Other mice were treated with miglustat only, or sham-treated. The battery of behavioral tests consisted of accelerod, Morris water maze, elevated plus maze, open field and hot-plate tests. Motor capabilities and spontaneous motor behavior were unaltered in both drug-treated groups. Miglustat-treated wild-type mice displayed impaired spatial learning compared to sham- and combination-treated mice. Both combination- and miglustat-treated mice showed enhanced anxiety in the elevated plus maze compared to sham-treated mice. Additionally, combination treatment as well as miglustat alone significantly reduced brain weight, whereas only combination treatment reduced body weight significantly. Our results suggest that allopregnanolone/cyclodextrin ameliorate most side effects of miglustat in wild-type mice.

Pharmacologic Treatment Assigned for Niemann Pick Type C1 Disease Partly Changes Behavioral Traits in Wild-Type Mice

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Victoria Schlegel

2016-11-01

Full Text Available Niemann-Pick Type C1 (NPC1 is an autosomal recessive inherited disorder characterized by accumulation of cholesterol and glycosphingolipids. Previously, we demonstrated that BALB/c-npc1nihNpc1−/− mice treated with miglustat, cyclodextrin and allopregnanolone generally performed better than untreated Npc1−/− animals. Unexpectedly, they also seemed to accomplish motor tests better than their sham-treated wild-type littermates. However, combination-treated mutant mice displayed worse cognition performance compared to sham-treated ones. To evaluate effects of these drugs in healthy BALB/c mice, we here analyzed pharmacologic effects on motor and cognitive behavior of wild-type mice. For combination treatment mice were injected with allopregnanolone/cyclodextrin weekly, starting at P7. Miglustat injections were performed daily from P10 till P23. Starting at P23, miglustat was embedded in the chow. Other mice were treated with miglustat only, or sham-treated. The battery of behavioral tests consisted of accelerod, Morris water maze, elevated plus maze, open field and hot-plate tests. Motor capabilities and spontaneous motor behavior were unaltered in both drug-treated groups. Miglustat-treated wild-type mice displayed impaired spatial learning compared to sham- and combination-treated mice. Both combination- and miglustat-treated mice showed enhanced anxiety in the elevated plus maze compared to sham-treated mice. Additionally, combination treatment as well as miglustat alone significantly reduced brain weight, whereas only combination treatment reduced body weight significantly. Our results suggest that allopregnanolone/cyclodextrin ameliorate most side effects of miglustat in wild-type mice.

Mice lacking the synaptic adhesion molecule Neph2/Kirrel3 display moderate hyperactivity and defective novel object preference

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Su Yeon eChoi

2015-07-01

Full Text Available Synaptic adhesion molecules regulate diverse aspects of neuronal synapse development, including synapse specificity, formation, and maturation. Neph2, also known as Kirrel3, is an immunoglobulin superfamily adhesion molecule implicated in intellectual disability, neurocognitive delay associated with Jacobsen syndrome, and autism spectrum disorders. We here report mice lacking Neph2 (Neph2–/– mice display moderate hyperactivity in a familiar but not novel environment and novel object recognition deficit with normal performances in Morris water maze spatial learning and memory, contextual fear conditioning and extinction, and pattern separation tests. These mice show normal levels of anxiety-like behaviors, social interaction, and repetitive behaviors. At the synapse level, Neph2–/– dentate gyrus granule cells exhibit unaltered dendritic spine density and spontaneous excitatory synaptic transmission. These results suggest that Neph2 is important for normal locomotor activity and object recognition memory.

Modulating Wnt Signaling Rescues Palate Morphogenesis in Pax9 Mutant Mice.

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Li, C; Lan, Y; Krumlauf, R; Jiang, R

2017-10-01

Cleft palate is a common birth defect caused by disruption of palatogenesis during embryonic development. Although mutations disrupting components of the Wnt signaling pathway have been associated with cleft lip and palate in humans and mice, the mechanisms involving canonical Wnt signaling and its regulation in secondary palate development are not well understood. Here, we report that canonical Wnt signaling plays an important role in Pax9-mediated regulation of secondary palate development. We found that cleft palate pathogenesis in Pax9-deficient embryos is accompanied by significantly reduced expression of Axin2, an endogenous target of canonical Wnt signaling, in the developing palatal mesenchyme, particularly in the posterior regions of the palatal shelves. We found that expression of Dkk2, encoding a secreted Wnt antagonist, is significantly increased whereas the levels of active β-catenin protein, the essential transcriptional coactivator of canonical Wnt signaling, is significantly decreased in the posterior regions of the palatal shelves in embryonic day 13.5 Pax9-deficent embryos in comparison with control littermates. We show that small molecule-mediated inhibition of Dickkopf (DKK) activity in utero during palatal shelf morphogenesis partly rescued secondary palate development in Pax9-deficient embryos. Moreover, we found that genetic inactivation of Wise, which is expressed in the developing palatal shelves and encodes another secreted antagonist of canonical Wnt signaling, also rescued palate morphogenesis in Pax9-deficient mice. Furthermore, whereas Pax9 del/del embryos exhibit defects in palatal shelf elevation/reorientation and significant reduction in accumulation of hyaluronic acid-a high molecular extracellular matrix glycosaminoglycan implicated in playing an important role in palatal shelf elevation-80% of Pax9 del/del ;Wise -/- double-mutant mouse embryos exhibit rescued palatal shelf elevation/reorientation, accompanied by restored

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

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Man Hei Cheng

Full Text Available 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

Ex-vivo assessment and non-invasive in vivo imaging of internal hemorrhages in Aga2/+ mutant mice

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Ermolayev, Vladimir [Institute for Biological and Medical Imaging, Helmholtz Zentrum München, Building 56, Ingolstädter Landstraße 1, D-85764 Neuherberg (Germany); Cohrs, Christian M. [Institute for Experimental Genetics, Helmholtz Zentrum München, Ingolstädter Landstraße 1, D-85764 Neuherberg (Germany); Mohajerani, Pouyan; Ale, Angelique [Institute for Biological and Medical Imaging, Helmholtz Zentrum München, Building 56, Ingolstädter Landstraße 1, D-85764 Neuherberg (Germany); Hrabé de Angelis, Martin [Institute for Experimental Genetics, Helmholtz Zentrum München, Ingolstädter Landstraße 1, D-85764 Neuherberg (Germany); Ntziachristos, Vasilis, E-mail: v.ntziachristos@tum.de [Institute for Biological and Medical Imaging, Helmholtz Zentrum München, Building 56, Ingolstädter Landstraße 1, D-85764 Neuherberg (Germany)

2013-03-08

Highlights: ► Aga2/+ mice, model for Osteogenesis imperfecta, have type I collagen mutation. ► Aga2/+ mice display both moderate and severe phenotypes lethal 6–11th postnatal. ► Internal hemorrhages studied in Aga2/+ vs. control mice at 6 and 9 days postnatal. ► Anatomical and functional findings in-vivo contrasted to the ex-vivo appearance. -- Abstract: Mutations in type I collagen genes (COL1A1/2) typically lead to Osteogenesis imperfecta, the most common heritable cause of skeletal fractures and bone deformation in humans. Heterozygous Col1a1{sup Aga2/+}, animals with a dominant mutation in the terminal C-propeptide domain of type I collagen develop typical skeletal hallmarks and internal hemorrhages starting from 6 day after birth. The disease progression for Aga2/+ mice, however, is not uniform differing between severe phenotype lethal at the 6–11th day of life, and moderate-to-severe one with survival to adulthood. Herein we investigated whether a new modality that combines X-ray computer tomography with fluorescence tomography in one hybrid system can be employed to study internal bleedings in relation to bone fractures and obtain insights into disease progression. The disease phenotype was characterized on Aga2/+ vs. wild type mice between 6 and 9 days postnatal. Anatomical and functional findings obtained in-vivo were contrasted to the ex-vivo appearance of the same tissues under cryo-slicing.

Ex-vivo assessment and non-invasive in vivo imaging of internal hemorrhages in Aga2/+ mutant mice

International Nuclear Information System (INIS)

Ermolayev, Vladimir; Cohrs, Christian M.; Mohajerani, Pouyan; Ale, Angelique; Hrabé de Angelis, Martin; Ntziachristos, Vasilis

2013-01-01

Highlights: ► Aga2/+ mice, model for Osteogenesis imperfecta, have type I collagen mutation. ► Aga2/+ mice display both moderate and severe phenotypes lethal 6–11th postnatal. ► Internal hemorrhages studied in Aga2/+ vs. control mice at 6 and 9 days postnatal. ► Anatomical and functional findings in-vivo contrasted to the ex-vivo appearance. -- Abstract: Mutations in type I collagen genes (COL1A1/2) typically lead to Osteogenesis imperfecta, the most common heritable cause of skeletal fractures and bone deformation in humans. Heterozygous Col1a1 Aga2/+ , animals with a dominant mutation in the terminal C-propeptide domain of type I collagen develop typical skeletal hallmarks and internal hemorrhages starting from 6 day after birth. The disease progression for Aga2/+ mice, however, is not uniform differing between severe phenotype lethal at the 6–11th day of life, and moderate-to-severe one with survival to adulthood. Herein we investigated whether a new modality that combines X-ray computer tomography with fluorescence tomography in one hybrid system can be employed to study internal bleedings in relation to bone fractures and obtain insights into disease progression. The disease phenotype was characterized on Aga2/+ vs. wild type mice between 6 and 9 days postnatal. Anatomical and functional findings obtained in-vivo were contrasted to the ex-vivo appearance of the same tissues under cryo-slicing

Liver tumor formation by a mutant retinoblastoma protein in the transgenic mice is caused by an upregulation of c-Myc target genes

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Wang, Bo; Hikosaka, Keisuke; Sultana, Nishat; Sharkar, Mohammad Tofael Kabir [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Noritake, Hidenao [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Department of Internal Medicine, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Kimura, Wataru; Wu, Yi-Xin [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Kobayashi, Yoshimasa [Department of Internal Medicine, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Uezato, Tadayoshi [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Miura, Naoyuki, E-mail: nmiura@hama-med.ac.jp [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan)

2012-01-06

Highlights: Black-Right-Pointing-Pointer Fifty percent of the mutant Rb transgenic mice produced liver tumors. Black-Right-Pointing-Pointer In the tumor, Foxm1, Skp2, Bmi1 and AP-1 mRNAs were up-regulated. Black-Right-Pointing-Pointer No increase in expression of the Myc-target genes was observed in the non-tumorous liver. Black-Right-Pointing-Pointer Tumor formation depends on up-regulation of the Myc-target genes. -- Abstract: The retinoblastoma (Rb) tumor suppressor encodes a nuclear phosphoprotein that regulates cellular proliferation, apoptosis and differentiation. In order to adapt itself to these biological functions, Rb is subjected to modification cycle, phosphorylation and dephosphorylation. To directly determine the effect of phosphorylation-resistant Rb on liver development and function, we generated transgenic mice expressing phosphorylation-resistant human mutant Rb (mt-Rb) under the control of the rat hepatocyte nuclear factor-1 gene promoter/enhancer. Expression of mt-Rb in the liver resulted in macroscopic neoplastic nodules (adenomas) with {approx}50% incidence within 15 months old. Interestingly, quantitative reverse transcriptase-PCR analysis showed that c-Myc was up-regulated in the liver of mt-Rb transgenic mice irrespective of having tumor tissues or no tumor. In tumor tissues, several c-Myc target genes, Foxm1, c-Jun, c-Fos, Bmi1 and Skp2, were also up-regulated dramatically. We determined whether mt-Rb activated the Myc promoter in the HTP9 cells and demonstrated that mt-Rb acted as an inhibitor of wild-type Rb-induced repression on the Myc promoter. Our results suggest that continued upregulation of c-Myc target genes promotes the liver tumor formation after about 1 year of age.

Comparative Hair Restorer Efficacy of Medicinal Herb on Nude (Foxn1nu Mice

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Shahnaz Begum

2014-01-01

Full Text Available Eclipta alba (L. Hassk, Asiasarum sieboldii (Miq. F. Maek (Asiasari radix, and Panax ginseng C. A. Mey (red ginseng are traditionally acclaimed for therapeutic properties of various human ailments. Synergistic effect of each standardized plant extract was investigated for hair growth potential on nude mice, as these mutant mice genetically lack hair due to abnormal keratinization. Dried plant samples were ground and extracted by methanol. Topical application was performed on the back of nude mice daily up to completion of two hair growth generations. The hair density and length of Eclipta alba treated mice were increased significantly P>0.001 than control mice. Hair growth area was also distinctly visible in Eclipta alba treated mice. On the other hand, Asiasari radix and Panax ginseng treated mice developing hair loss were recognized from the abortive boundaries of hair coverage. Histomorphometric observation of nude mice skin samples revealed an increase in number of hair follicles (HFs. The presence of follicular keratinocytes was confirmed by BrdU labeling, S-phase cells in HFs. Therefore, Eclipta alba extract and/or phytochemicals strongly displayed incomparability of hair growth promotion activity than others. Thus, the standardized Eclipta alba extract can be used as an effective, alternative, and complementary treatment against hair loss.

Role of protein kinase C family in the cerebellum-dependent adaptive learning of horizontal optokinetic response eye movements in mice.

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Shutoh, Fumihiro; Katoh, Akira; Ohki, Masafumi; Itohara, Shigeyoshi; Tonegawa, Susumu; Nagao, Soichi

2003-07-01

Among the subtypes of the Ca2+-dependent protein kinase C (PKC), which play a crucial role in long-term depression (LTD), both alpha and gamma are expressed in the cerebellar floccular Purkinje cells. To reveal the functional differences of PKC subtypes, we examined the adaptability of ocular reflexes of PKCgamma mutant mice, which show mild ataxia and normal LTD. In mutant mice, gains of the horizontal optokinetic eye response (HOKR) were reduced. Adaptation of the HOKR was not affected but its retinal slip dependency was altered in mutant mice. Sustained 1-h sinusoidal screen oscillation, which induced a relatively large amount of retinal slips in both mutant and wild-type mice, increased the HOKR gain in wild-type mice but not in mutant mice. In contrast, exposure to 1 h of sustained slower screen oscillations, which induced relatively small retinal slips in mutant and wild-type mice, increased the HOKR gain in both mutant and wild-type mice. Adaptation of the HOKR of the mutant mice to slow screen oscillation and those of wild-type mice to fast and slow screen oscillations were all abolished by local applications of a PKC inhibitor (chelerythrine) within the flocculi. Electrophysiological and anatomical studies showed no appreciable changes in the sources and magnitudes of climbing fibre inputs, which mediate retinal slip signals to the flocculus in the mutant mice. These results suggest that PKCgamma has a modulatory role in determining retinal slip dependency, and other PKC subtypes, e.g. PKCalpha, may play a crucial role in the adaptation of the HOKR.

Non-motor and motor features in LRRK2 transgenic mice.

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Zoë Bichler

Full Text Available Non-motor symptoms are increasingly recognized as important features of Parkinson's disease (PD. LRRK2 mutations are common causes of familial and sporadic PD. Non-motor features have not been yet comprehensively evaluated in LRRK2 transgenic mouse models.Using a transgenic mouse model overexpressing the R1441G mutation of the human LRRK2 gene, we have investigated the longitudinal correlation between motor and non-motor symptoms and determined if specific non-motor phenotypes precede motor symptoms.We investigated the onset of motor and non-motor phenotypes on the LRRK2(R1441G BAC transgenic mice and their littermate controls from 4 to 21 month-old using a battery of behavioral tests. The transgenic mutant mice displayed mild hypokinesia in the open field from 16 months old, with gastrointestinal dysfunctions beginning at 6 months old. Non-motor features such as depression and anxiety-like behaviors, sensorial functions (pain sensitivity and olfaction, and learning and memory abilities in the passive avoidance test were similar in the transgenic animals compared to littermate controls.LRRK2(R1441G BAC transgenic mice displayed gastrointestinal dysfunction at an early stage but did not have abnormalities in fine behaviors, olfaction, pain sensitivity, mood disorders and learning and memory compared to non-transgenic littermate controls. The observations on olfaction and gastrointestinal dysfunction in this model validate findings in human carriers. These mice did recapitulate mild Parkinsonian motor features at late stages but compensatory mechanisms modulating the progression of PD in these models should be further evaluated.

LRRK2 knockout mice have an intact dopaminergic system but display alterations in exploratory and motor co-ordination behaviors

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2012-01-01

Mutations in the LRRK2 gene are the most common cause of genetic Parkinson’s disease. Although the mechanisms behind the pathogenic effects of LRRK2 mutations are still not clear, data emerging from in vitro and in vivo models suggests roles in regulating neuronal polarity, neurotransmission, membrane and cytoskeletal dynamics and protein degradation. We created mice lacking exon 41 that encodes the activation hinge of the kinase domain of LRRK2. We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis. Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice. However, LRRK2 knockout mice displayed abnormal exploratory activity in the open-field test. Moreover, LRRK2 knockout mice stayed longer than their wild type littermates on the accelerated rod during rotarod testing. Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes. PMID:22647713

Chronic exposure of mutant DISC1 mice to lead produces sex-dependent abnormalities consistent with schizophrenia and related mental disorders: a gene-environment interaction study.

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Abazyan, Bagrat; Dziedzic, Jenifer; Hua, Kegang; Abazyan, Sofya; Yang, Chunxia; Mori, Susumu; Pletnikov, Mikhail V; Guilarte, Tomas R

2014-05-01

The glutamatergic hypothesis of schizophrenia suggests that hypoactivity of the N-methyl-D-aspartate receptor (NMDAR) is an important factor in the pathophysiology of schizophrenia and related mental disorders. The environmental neurotoxicant, lead (Pb(2+)), is a potent and selective antagonist of the NMDAR. Recent human studies have suggested an association between prenatal Pb(2+) exposure and the increased likelihood of schizophrenia later in life, possibly via interacting with genetic risk factors. In order to test this hypothesis, we examined the neurobehavioral consequences of interaction between Pb(2+) exposure and mutant disrupted in schizophrenia 1 (mDISC1), a risk factor for major psychiatric disorders. Mutant DISC1 and control mice born by the same dams were raised and maintained on a regular diet or a diet containing moderate levels of Pb(2+). Chronic, lifelong exposure of mDISC1 mice to Pb(2+) was not associated with gross developmental abnormalities but produced sex-dependent hyperactivity, exaggerated responses to the NMDAR antagonist, MK-801, mildly impaired prepulse inhibition of the acoustic startle, and enlarged lateral ventricles. Together, these findings support the hypothesis that environmental toxins could contribute to the pathogenesis of mental disease in susceptible individuals.

Replicable in vivo physiological and behavioral phenotypes of the Shank3B null mutant mouse model of autism.

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Dhamne, Sameer C; Silverman, Jill L; Super, Chloe E; Lammers, Stephen H T; Hameed, Mustafa Q; Modi, Meera E; Copping, Nycole A; Pride, Michael C; Smith, Daniel G; Rotenberg, Alexander; Crawley, Jacqueline N; Sahin, Mustafa

2017-01-01

behavioral domain, including social, repetitive, cognitive, anxiety-related, sensory, and motor categories of assays. Relative to WT mice, Shank3B KO mice displayed a dramatic resistance to PTZ seizure induction and an enhancement of gamma band oscillatory EEG activity indicative of enhanced inhibitory tone. These findings replicated in two separate cohorts. Behaviorally, Shank3B KO mice exhibited repetitive grooming, deficits in aspects of reciprocal social interactions and vocalizations, and reduced open field activity, as well as variable deficits in sensory responses, anxiety-related behaviors, learning and memory. Robust animal models and quantitative, replicable biomarkers of neural dysfunction are needed to decrease risk and enable successful drug discovery and development for ASD and other neurodevelopmental disorders. Complementary to the replicated behavioral phenotypes of the Shank3B mutant mouse is the new identification of a robust, translational in vivo neurophysiological phenotype. Our findings provide strong evidence for robustness and replicability of key translational phenotypes in Shank3B mutant mice and support the usefulness of this mouse model of ASD for therapeutic discovery.

ERK Regulates Renal Cell Proliferation and Renal Cyst Expansion in inv Mutant Mice

International Nuclear Information System (INIS)

Okumura, Yasuko; Sugiyama, Noriyuki; Tanimura, Susumu; Nishida, Masashi; Hamaoka, Kenji; Kohno, Michiaki; Yokoyama, Takahiko

2009-01-01

Nephronophthisis (NPHP) is the most frequent genetic cause of end-stage kidney disease in children and young adults. Inv mice are a model for human nephronophthisis type 2 (NPHP2) and characterized by multiple renal cysts and situs inversus. Renal epithelial cells in inv cystic kidneys show increased cell proliferation. We studied the ERK pathway to understand the mechanisms that induce cell proliferation and renal cyst progression in inv kidneys. We studied the effects of ERK suppression by administering PD184352, an oral mitogen-activated protein kinase kinase (MEK) inhibitor on renal cyst expansion, extracellular signal-regulated protein kinase (ERK) activity, bromo-deoxyuridine (BrdU) incorporation and expression of cell-cycle regulators in invΔC kidneys. Phosphorylated ERK (p-ERK) level increased along with renal cyst enlargement. Cell-cycle regulators showed a high level of expression in invΔC kidneys. PD184352 successfully decreased p-ERK level and inhibited renal cyst enlargement. The inhibitor also decreased expression of cell-cycle regulators and BrdU incorporation in renal epithelial cells. The present results showed that ERK regulated renal cell proliferation and cyst expansion in inv mutants

Knockin mouse with mutant Gα11 mimics human inherited hypocalcemia and is rescued by pharmacologic inhibitors

DEFF Research Database (Denmark)

Roszko, Kelly L; Bi, Ruiye; Gorvin, Caroline M

2017-01-01

in patients with autosomal-dominant hypocalcemia type 2 (ADH2), an inherited disorder of hypocalcemia, low parathyroid hormone (PTH), and hyperphosphatemia. We have generated knockin mice harboring the point mutation GNA11 c.C178T (p.Arg60Cys) identified in ADH2 patients. The mutant mice faithfully replicated...... human ADH2. They also exhibited low bone mineral density and increased skin pigmentation. Treatment with NPS 2143, a negative allosteric modulator of the calcium-sensing receptor (CASR), increased PTH and calcium concentrations in WT and mutant mice, suggesting that the gain-of-function effect of GNA11...

Plasmodium yoelii: induction of attenuated mutants by irradiation

International Nuclear Information System (INIS)

Waki, S.; Yonome, I.; Suzuki, M.

1986-01-01

When erythrocytic forms of Plasmodium yoelii nigeriensis, which is invariably fatal in mice, were exposed to X rays, the dose to reduce surviving parasites to one millionth was 100 gray (10 Krad). A suspension of 5 X 10(6) per ml of parasitized erythrocyte was irradiated at 100 gray, and 0.2 ml aliquots were inoculated into 22 mice. Eleven mice showed patent parasitemia, and in these the growth curves were less steep than that found in nonirradiated parasites. The infections of 8 mice of the 11 were self-resolving, and the attenuated feature of the parasites maintained following a limited number of blood passages. The parasites were slowly growing even in nude mice and cause self-resolving infections in intact mice. BALB/c mice immunized with the attenuated parasites were protected against subsequent challenge infections with the original virulent erythrocytic and sporogonic forms. These findings indicate that attenuated mutants of malaria parasites can be readily induced by this method

Radiation carcinogenesis in radiosensitive mutant Scid mice

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Ogiu, Toshiaki; Ishii-Ohba, Hiroko; Kobayashi, Shigeru; Nishimura, Mayumi; Shimada, Yoshiya; Tsuji, Hideo; Watanabe, Fumiaki; Suzuki, Fumio; Sado, Toshihiko

2000-01-01

The Scid mice were established as a severe combined immunodeficient mouse strain lacking both T- and B-cell functions. Scid (homozygote), its parent strain C.B-17 (wild-type) and their hybrid F1 (heterozygote) were used for analysis of the relationship between sensitivity to acute effects of ionizing radiation and radiation-tumor development. Acute effects were studied using γ-rays and LD 50(30) was found to be 4.05 Gy in Scid, 6.5 Gy in F1 and 7.2 Gy in C.B-17. When bone marrow cells were irradiated with X-rays in vitro, survival curves of C.B-17 and F1 cells showed a region of shoulder with D 0 =0.68 and 0.67 Gy, respectively, while those of Scid were of no shoulder with D 0 =0.46 Gy. Scid mice died due to tumors (most were thymic lymphoma, T/L) 20-40 weeks after irradiation with 1-3 Gy γ-rays but C.B-17 and F1 survived longer. Bone marrow transplantation was found effective to prevent the radiation T/L. FACS analysis for surface antigens of those T/L cells suggested the change of Ras oncogenes. The change of Notch 1 gene was suggested by Southern hybridization and thus a possible role of defective DNA-PK in mice alone (not in rats and humans) was suggested as well. (K.H.)

Differential interaction of Apolipoprotein-E isoforms with insulin receptors modulates brain insulin signaling in mutant human amyloid precursor protein transgenic mice.

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Chan, Elizabeth S; Chen, Christopher; Cole, Gregory M; Wong, Boon-Seng

2015-09-08

It is unclear how human apolipoprotein E4 (ApoE4) increases the risk for Alzheimer's disease (AD). Although Aβ levels can lead to insulin signaling impairment, these experiments were done in the absence of human ApoE. To examine ApoE role, we crossed the human ApoE-targeted replacement mice with mutant human amyloid precursor protein (APP) mice. In 26 week old mice with lower Aβ levels, the expression and phosphorylation of insulin signaling proteins remained comparable among APP, ApoE3xAPP and ApoE4xAPP mouse brains. When the mice aged to 78 weeks, these proteins were markedly reduced in APP and ApoE4xAPP mouse brains. While Aβ can bind to insulin receptor, how ApoE isoforms modulate this interaction remains unknown. Here, we showed that ApoE3 had greater association with insulin receptor as compared to ApoE4, regardless of Aβ42 concentration. In contrast, ApoE4 bound more Aβ42 with increasing peptide levels. Using primary hippocampal neurons, we showed that ApoE3 and ApoE4 neurons are equally sensitive to physiological levels of insulin. However, in the presence of Aβ42, insulin failed to elicit a downstream response only in ApoE4 hippocampal neurons. Taken together, our data show that ApoE genotypes can modulate this Aβ-mediated insulin signaling impairment.

Reduced Gut Acidity Induces an Obese-Like Phenotype in Drosophila melanogaster and in Mice.

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Wei-Sheng Lin

Full Text Available In order to identify genes involved in stress and metabolic regulation, we carried out a Drosophila P-element-mediated mutagenesis screen for starvation resistance. We isolated a mutant, m2, that showed a 23% increase in survival time under starvation conditions. The P-element insertion was mapped to the region upstream of the vha16-1 gene, which encodes the c subunit of the vacuolar-type H+-ATPase. We found that vha16-1 is highly expressed in the fly midgut, and that m2 mutant flies are hypomorphic for vha16-1 and also exhibit reduced midgut acidity. This deficit is likely to induce altered metabolism and contribute to accelerated aging, since vha16-1 mutant flies are short-lived and display increases in body weight and lipid accumulation. Similar phenotypes were also induced by pharmacological treatment, through feeding normal flies and mice with a carbonic anhydrase inhibitor (acetazolamide or proton pump inhibitor (PPI, lansoprazole to suppress gut acid production. Our study may thus provide a useful model for investigating chronic acid suppression in patients.

Immunization with mutant HPV16 E7 protein inhibits the growth of TC-1 cells in tumor-bearing mice.

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Li, Yan-Li; Ma, Zhong-Liang; Zhao, Yue; Zhang, Jing

2015-04-01

Two human papillomavirus (HPV) 16 oncogenic proteins, E6 and E7, are co-expressed in the majority of HPV16-induced cervical cancer cells. Thus, the E6 and E7 proteins are good targets for developing therapeutic vaccines for cervical cancer. In the present study, immunization with the mutant non-transforming HPV16 E7 (mE7) protein was demonstrated to inhibit the growth of TC-1 cells in the TC-1 mouse model. The HPV16 mE7 gene was amplified by splicing overlap extension polymerase chain reaction using pET-28a(+)-E7 as a template, and the gene was cloned into pET-28a(+) to form pET-28a(+)-mE7. Compared with the E7 protein, mE7 lacks amino acid residues 94-98, and at residue 24, there is a Cys to Gly substitution. pET-28a(+)-mE7 was then introduced into Escherichia coli Rosetta. The expression of mE7 was induced by isopropyl β-D-1-thiogalactopyranoside. The mE7 protein was purified using Ni-NTA agarose and detected by SDS-PAGE and western blot analysis. In the tumor prevention model, no tumor was detected in the mice vaccinated with the mE7 protein. After 40 days, the tumor-free mice and control mice were challenged with 2×10 5 TC-1 cells. All control mice developed tumors six days later, but mE7 immunized mice were tumor free until 90 days. In the tumor therapy model, the TC-1 cells were initially injected subcutaneously, and the mice were subsequently vaccinated. Vaccination against the mE7 protein may significantly inhibit TC-1 cell growth compared to the control. These results demonstrated that immunization with the HPV16 mE7 protein elicited a long-term protective immunity against TC-1 tumor growth and generated a significant inhibition of TC-1 growth in a TC-1 mouse model.

Mutant TDP-43 within motor neurons drives disease onset but not progression in amyotrophic lateral sclerosis.

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Ditsworth, Dara; Maldonado, Marcus; McAlonis-Downes, Melissa; Sun, Shuying; Seelman, Amanda; Drenner, Kevin; Arnold, Eveline; Ling, Shuo-Chien; Pizzo, Donald; Ravits, John; Cleveland, Don W; Da Cruz, Sandrine

2017-06-01

Mutations in TDP-43 cause amyotrophic lateral sclerosis (ALS), a fatal paralytic disease characterized by degeneration and premature death of motor neurons. The contribution of mutant TDP-43-mediated damage within motor neurons was evaluated using mice expressing a conditional allele of an ALS-causing TDP-43 mutant (Q331K) whose broad expression throughout the central nervous system mimics endogenous TDP-43. TDP-43 Q331K mice develop age- and mutant-dependent motor deficits from degeneration and death of motor neurons. Cre-recombinase-mediated excision of the TDP-43 Q331K gene from motor neurons is shown to delay onset of motor symptoms and appearance of TDP-43-mediated aberrant nuclear morphology, and abrogate subsequent death of motor neurons. However, reduction of mutant TDP-43 selectively in motor neurons did not prevent age-dependent degeneration of axons and neuromuscular junction loss, nor did it attenuate astrogliosis or microgliosis. Thus, disease mechanism is non-cell autonomous with mutant TDP-43 expressed in motor neurons determining disease onset but progression defined by mutant acting within other cell types.

Effect of CCS on the accumulation of FALS SOD1 mutant-containing aggregates and on mitochondrial translocation of SOD1 mutants: implication of a free radical hypothesis.

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Kim, Ha Kun; Chung, Youn Wook; Chock, P Boon; Yim, Moon B

2011-05-15

Missense mutations of SOD1 are linked to familial amyotrophic lateral sclerosis (FALS) through a yet-to-be identified toxic-gain-of-function. One of the proposed mechanisms involves enhanced aggregate formation. However, a recent study showed that dual transgenic mice overexpressing both G93A and CCS copper chaperone (G93A/CCS) exhibit no SOD1-positive aggregates yet show accelerated FALS symptoms with enhanced mitochondrial pathology compared to G93A mice. Using a dicistronic mRNA to simultaneously generate hSOD1 mutants, G93A, A4V and G85R, and hCCS in AAV293 cells, we revealed: (i) CCS is degraded primarily via a macroautophagy pathway. It forms a stable heterodimer with inactive G85R, and via its novel copper chaperone-independent molecular chaperone activity facilitates G85R degradation via a macroautophagy-mediated pathway. For active G93A and A4V, CCS catalyzes their maturation to form active and soluble homodimers. (ii) CCS reduces, under non-oxidative conditions, yet facilitates in the presence of H(2)O(2), mitochondrial translocation of inactive SOD1 mutants. These results, together with previous reports showing FALS SOD1 mutants enhanced free radical-generating activity, provide a mechanistic explanation for the observations with G93A/CCS dual transgenic mice and suggest that free radical generation by FALS SOD1, enhanced by CCS, may, in part, be responsible for the FALS SOD1 mutant-linked aggregation, mitochondrial translocation, and degradation. Published by Elsevier Inc.

Biodistribution patterns of native and mutant 99mTc-labelled annexin V in mice

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Mariani, G.; Erba, P.; Pellegrino, D.; Volterrani, D.; Lazzeri, E.; Freer, G.; Bevilacqua, G.; Blankenberg, F.G.; Tait, J.F.; Strauss, H.W.

2003-01-01

Full text: Annexin is a 36 kD protein with high binding affinity to phosphatidylserine (PS), a phospholipid exposed on the membrane surface of cells upon activation of the enzyme caspase, the first step of apoptosis. Radiolabeled annexin V could thus be used for imaging apoptosis in-vivo. When the 319 amino acid protein is made by recombinant techniques and expressed as the human material, it can be radiolabeled with 99mTc after derivatization with a bifunctional agent such as HYNIC. Alternatively, the amino acid structure of the protein can be modified by producing annexin V mutants with an endogenous chelation site for 99mTc, the NH2 residue Ala-Gly-Gly-Cys-Gly-His-Met. Mutant annexin has similar affinity for membrane-bound PS as unmodified annexin. This study was performed to compare the biodistribution of 99mTc-labeled HYNIC annexin (HyA) to mutant annexin (MuA). 99mTc-labeling efficiency of the two annexin preparations was >99% by gel chromatography on Sephadex G10 columns. Groups of adult male mice (n 10, body weight 18-25 grams) were injected iv with either HyA or MuA (1-3 MBq, 3-9 μg/animal). Animals were sacrificed one hour later and dissected for organ biodistribution. Similar biodistribution was performed after pretreatment with cyclophosphamide (150 mg/kg ip 6-15 hr prior to the study). The results of the biodistribution study showed significantly reduced (p<0.05 to p<0.01) uptake of MuA versus HyA in the kidneys (Δ- 81.4%), spleen (Δ- 58.2%), liver (Δ- 56.2%), and bone marrow (Δ- 33.7%), while it was increased in lymph nodes (Δ+ 131%, p<0.001). Pretreatment with the pro-apoptotic agent cyclophosphamide induced significantly increased uptake of MuA (p<0.05) versus baseline in the heart (Δ+ 34.7%), spleen (Δ+ 30.1%) and bowel (Δ+ 44.5%), while uptake of HyA was increased only in the spleen (Δ+ 44.1%). The marked reduction in the renal, splenic, liver, and bone marrow localization of MuA compared to HyA in control animals outlines a pattern of

Mutations in Dnaaf1 and Lrrc48 Cause Hydrocephalus, Laterality Defects, and Sinusitis in Mice

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Seungshin Ha

2016-08-01

Full Text Available We have previously described a forward genetic screen in mice for abnormalities of brain development. Characterization of two hydrocephalus mutants by whole-exome sequencing after whole-genome SNP mapping revealed novel recessive mutations in Dnaaf1 and Lrrc48. Mouse mutants of these two genes have not been previously reported. The Dnaaf1 mutant carries a mutation at the splice donor site of exon 4, which results in abnormal transcripts. The Lrrc48 mutation is a missense mutation at a highly conserved leucine residue, which is also associated with a decrease in Lrrc48 transcription. Both Dnaaf1 and Lrrc48 belong to a leucine-rich repeat-containing protein family and are components of the ciliary axoneme. Their Chlamydomonas orthologs are known to be required for normal ciliary beat frequency or flagellar waveform, respectively. Some Dnaaf1 or Lrrc48 homozygote mutants displayed laterality defects, suggesting a motile cilia defect in the embryonic node. Mucus accumulation and neutrophil infiltration in the maxillary sinuses suggested sinusitis. Dnaaf1 mutants showed postnatal lethality, and none survived to weaning age. Lrrc48 mutants survive to adulthood, but had male infertility. ARL13B immunostaining showed the presence of motile cilia in the mutants, and the distal distribution of DNAH9 in the axoneme of upper airway motile cilia appeared normal. The phenotypic abnormalities suggest that mutations in Dnaaf1 and Lrrc48 cause defects in motile cilia function.

Cerebrovascular defects in Foxc1 mutants correlate with aberrant WNT and VEGF-A pathways downstream of retinoic acid from the meninges.

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Mishra, Swati; Choe, Youngshik; Pleasure, Samuel J; Siegenthaler, Julie A

2016-12-01

Growth and maturation of the cerebrovasculature is a vital event in neocortical development however mechanisms that control cerebrovascular development remain poorly understood. Mutations in or deletions that include the FOXC1 gene are associated with congenital cerebrovascular anomalies and increased stroke risk in patients. Foxc1 mutant mice display severe cerebrovascular hemorrhage at late gestational ages. While these data demonstrate Foxc1 is required for cerebrovascular development, its broad expression in the brain vasculature combined with Foxc1 mutant's complex developmental defects have made it difficult to pinpoint its function(s). Using global and conditional Foxc1 mutants, we find 1) significant cerebrovascular growth defects precede cerebral hemorrhage and 2) expression of Foxc1 in neural crest-derived meninges and brain pericytes, though not endothelial cells, is required for normal cerebrovascular development. We provide evidence that reduced levels of meninges-derived retinoic acid (RA), caused by defects in meninges formation in Foxc1 mutants, is a major contributing factor to the cerebrovascular growth defects in Foxc1 mutants. We provide data that suggests that meninges-derived RA ensures adequate growth of the neocortical vasculature via regulating expression of WNT pathway proteins and neural progenitor derived-VEGF-A. Our findings offer the first evidence for a role of the meninges in brain vascular development and provide new insight into potential causes of cerebrovascular defects in patients with FOXC1 mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

A novel triple repeat mutant tau transgenic model that mimics aspects of pick's disease and fronto-temporal tauopathies.

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Edward Rockenstein

Full Text Available Tauopathies are a group of disorders leading to cognitive and behavioral impairment in the aging population. While four-repeat (4R Tau is more abundant in corticobasal degeneration, progressive supranuclear palsy, and Alzheimer's disease, three-repeat (3R Tau is the most abundant splice, in Pick's disease. A number of transgenic models expressing wild-type and mutant forms of the 4R Tau have been developed. However, few models of three-repeat Tau are available. A transgenic mouse model expressing three-repeat Tau was developed bearing the mutations associated with familial forms of Pick's disease (L266V and G272V mutations. Two lines expressing high (Line 13 and low (Line 2 levels of the three-repeat mutant Tau were analyzed. By Western blot, using antibodies specific to three-repeat Tau, Line 13 expressed 5-times more Tau than Line 2. The Tau expressed by these mice was most abundant in the frontal-temporal cortex and limbic system and was phosphorylated at residues detected by the PHF-1, AT8, CP9 and CP13 antibodies. The higher-expressing mice displayed hyperactivity, memory deficits in the water maze and alterations in the round beam. The behavioral deficits started at 6-8 months of age and were associated with a progressive increase in the accumulation of 3R Tau. By immunocytochemistry, mice from Line 13 displayed extensive accumulation of 3R Tau in neuronal cells bodies in the pyramidal neurons of the neocortex, CA1-3 regions, and dentate gyrus of the hippocampus. Aggregates in the granular cells had a globus appearance and mimic Pick's-like inclusions. There were abundant dystrophic neurites, astrogliosis and synapto-dendritic damage in the neocortex and hippocampus of the higher expresser line. The hippocampal lesions were moderately argyrophilic and Thioflavin-S negative. By electron microscopy, discrete straight filament aggregates were detected in some neurons in the hippocampus. This model holds promise for better understanding the

Hearts of dystonia musculorum mice display normal morphological and histological features but show signs of cardiac stress.

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Justin G Boyer

2010-03-01

Full Text Available Dystonin is a giant cytoskeletal protein belonging to the plakin protein family and is believed to crosslink the major filament systems in contractile cells. Previous work has demonstrated skeletal muscle defects in dystonin-deficient dystonia musculorum (dt mice. In this study, we show that the dystonin muscle isoform is localized at the Z-disc, the H zone, the sarcolemma and intercalated discs in cardiac tissue. Based on this localization pattern, we tested whether dystonin-deficiency leads to structural defects in cardiac muscle. Desmin intermediate filament, microfilament, and microtubule subcellular organization appeared normal in dt hearts. Nevertheless, increased transcript levels of atrial natriuretic factor (ANF, 66% beta-myosin heavy chain (beta-MHC, 95% and decreased levels of sarcoplasmic reticulum calcium pump isoform 2A (SERCA2a, 26%, all signs of cardiac muscle stress, were noted in dt hearts. Hearts from two-week old dt mice were assessed for the presence of morphological and histological alterations. Heart to body weight ratios as well as left ventricular wall thickness and left chamber volume measurements were similar between dt and wild-type control mice. Hearts from dt mice also displayed no signs of fibrosis or calcification. Taken together, our data provide new insights into the intricate structure of the sarcomere by situating dystonin in cardiac muscle fibers and suggest that dystonin does not significantly influence the structural organization of cardiac muscle fibers during early postnatal development.

Mast cell-deficient Kit(W-sh) "Sash" mutant mice display aberrant myelopoiesis leading to the accumulation of splenocytes that act as myeloid-derived suppressor cells.

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Michel, Anastasija; Schüler, Andrea; Friedrich, Pamela; Döner, Fatma; Bopp, Tobias; Radsak, Markus; Hoffmann, Markus; Relle, Manfred; Distler, Ute; Kuharev, Jörg; Tenzer, Stefan; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Schild, Hansjörg; Schmitt, Edgar; Becker, Marc; Stassen, Michael

2013-06-01

Mast cell-deficient Kit(W-sh) "sash" mice are widely used to investigate mast cell functions. However, mutations of c-Kit also affect additional cells of hematopoietic and nonimmune origin. In this study, we demonstrate that Kit(W-sh) causes aberrant extramedullary myelopoiesis characterized by the expansion of immature lineage-negative cells, common myeloid progenitors, and granulocyte/macrophage progenitors in the spleen. A consistent feature shared by these cell types is the reduced expression of c-Kit. Populations expressing intermediate and high levels of Ly6G, a component of the myeloid differentiation Ag Gr-1, are also highly expanded in the spleen of sash mice. These cells are able to suppress T cell responses in vitro and phenotypically and functionally resemble myeloid-derived suppressor cells (MDSC). MDSC typically accumulate in tumor-bearing hosts and are able to dampen immune responses. Consequently, transfer of MDSC from naive sash mice into line 1 alveolar cell carcinoma tumor-bearing wild-type littermates leads to enhanced tumor progression. However, although it can also be observed in sash mice, accelerated growth of transplanted line 1 alveolar cell carcinoma tumors is a mast cell-independent phenomenon. Thus, the Kit(W-sh) mutation broadly affects key steps in myelopoiesis that may have an impact on mast cell research.

Herpes Simplex Virus 1 Mutant with Point Mutations in UL39 Is Impaired for Acute Viral Replication in Mice, Establishment of Latency, and Explant-Induced Reactivation.

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Mostafa, Heba H; Thompson, Thornton W; Konen, Adam J; Haenchen, Steve D; Hilliard, Joshua G; Macdonald, Stuart J; Morrison, Lynda A; Davido, David J

2018-04-01

In the process of generating herpes simplex virus 1 (HSV-1) mutations in the viral regulatory gene encoding infected cell protein 0 (ICP0), we isolated a viral mutant, termed KOS-NA, that was severely impaired for acute replication in the eyes and trigeminal ganglia (TG) of mice, defective in establishing a latent infection, and reactivated poorly from explanted TG. To identify the secondary mutation(s) responsible for the impaired phenotypes of this mutant, we sequenced the KOS-NA genome and noted that it contained two nonsynonymous mutations in UL39 , which encodes the large subunit of ribonucleotide reductase, ICP6. These mutations resulted in lysine-to-proline (residue 393) and arginine-to-histidine (residue 950) substitutions in ICP6. To determine whether alteration of these amino acids was responsible for the KOS-NA phenotypes in vivo , we recombined the wild-type UL39 gene into the KOS-NA genome and rescued its acute replication phenotypes in mice. To further establish the role of UL39 in KOS-NA's decreased pathogenicity, the UL39 mutations were recombined into HSV-1 (generating UL39 mut ), and this mutant virus showed reduced ocular and TG replication in mice comparable to that of KOS-NA. Interestingly, ICP6 protein levels were reduced in KOS-NA-infected cells relative to the wild-type protein. Moreover, we observed that KOS-NA does not counteract caspase 8-induced apoptosis, unlike wild-type strain KOS. Based on alignment studies with other HSV-1 ICP6 homologs, our data suggest that amino acid 950 of ICP6 likely plays an important role in ICP6 accumulation and inhibition of apoptosis, consequently impairing HSV-1 pathogenesis in a mouse model of HSV-1 infection. IMPORTANCE HSV-1 is a major human pathogen that infects ∼80% of the human population and can be life threatening to infected neonates or immunocompromised individuals. Effective therapies for treatment of recurrent HSV-1 infections are limited, which emphasizes a critical need to understand in

Subthreshold membrane potential oscillations in inferior olive neurons are dynamically regulated by P/Q- and T-type calcium channels: a study in mutant mice.

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Choi, Soonwook; Yu, Eunah; Kim, Daesoo; Urbano, Francisco J; Makarenko, Vladimir; Shin, Hee-Sup; Llinás, Rodolfo R

2010-08-15

The role of P/Q- and T-type calcium channels in the rhythmic oscillatory behaviour of inferior olive (IO) neurons was investigated in mutant mice. Mice lacking either the CaV2.1 gene of the pore-forming alpha1A subunit for P/Q-type calcium channel, or the CaV3.1 gene of the pore-forming alpha1G subunit for T-type calcium channel were used. In vitro intracellular recording from IO neurons reveals that the amplitude and frequency of sinusoidal subthreshold oscillations (SSTOs) were reduced in the CaV2.1-/- mice. In the CaV3.1-/- mice, IO neurons also showed altered patterns of SSTOs and the probability of SSTO generation was significantly lower (15%, 5 of 34 neurons) than that of wild-type (78%, 31 of 40 neurons) or CaV2.1-/- mice (73%, 22 of 30 neurons). In addition, the low-threshold calcium spike and the sustained endogenous oscillation following rebound potentials were absent in IO neurons from CaV3.1-/- mice. Moreover, the phase-reset dynamics of oscillatory properties of single neurons and neuronal clusters in IO were remarkably altered in both CaV2.1-/- and CaV3.1-/- mice. These results suggest that both alpha1A P/Q- and alpha1G T-type calcium channels are required for the dynamic control of neuronal oscillations in the IO. These findings were supported by results from a mathematical IO neuronal model that incorporated T and P/Q channel kinetics.

Effects of metallothionein on zinc metabolism in lethal-milk mutant mice

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Grider, A. Jr.

1986-01-01

The lethal-milk mice (C57BL/6J-Im) exhibit various pleiotropic effects, including a congenital otolith defect, production of zinc-deficient milk, and clinical signs of a systemic Zn deficiency by one year of age. The clinical signs include alopecia, dermatitis, and skin lesions. The systemic zinc deficiency may be due to increased levels of metallothionein (MT) in the intestine and/or liver of Im mice. The untreated Im mice contain twice as much intestinal MT as do C57BL/6J-(+/sup im//+ /sup Im/) (B6) controls. This was determined by a sulfhydryl assay, by the 109 Cd-saturation/hemolysate method, and by the 65 Zn-binding assay. Various concentrations of Cd or Zn were added to the drinking water three days before assaying for MT. Compared to B6 mice, the Im mice exhibited more MT in their liver by the 65 Zn-MT binding assay (3-fold) and by the 109 Cd-saturation/hemolysate method (18-fold). The effects of the two zinc treatments did not differ significantly between Im and B6 mice. The retention and excretion of 65 Zn (administered intraperitoneally) were determined over a 14-day period, but the results did not different between the Im and B6 mice. The increased concentrations of MT within the Im mice was not significantly different for the intestine and liver. Based on these data and other studies, the Im mice may exhibit alterations in zinc homeostasis due to some deregulation of MT metabolism, including the inner ear of the fetus, the lactating mammary gland, and the intestine and liver of adults by one year of age

Effects of metallothionein on zinc metabolism in lethal-milk mutant mice

Energy Technology Data Exchange (ETDEWEB)

Grider, A. Jr.

1986-01-01

The lethal-milk mice (C57BL/6J-Im) exhibit various pleiotropic effects, including a congenital otolith defect, production of zinc-deficient milk, and clinical signs of a systemic Zn deficiency by one year of age. The clinical signs include alopecia, dermatitis, and skin lesions. The systemic zinc deficiency may be due to increased levels of metallothionein (MT) in the intestine and/or liver of Im mice. The untreated Im mice contain twice as much intestinal MT as do C57BL/6J-(+/sup im//+ /sup Im/) (B6) controls. This was determined by a sulfhydryl assay, by the /sup 109/Cd-saturation/hemolysate method, and by the /sup 65/Zn-binding assay. Various concentrations of Cd or Zn were added to the drinking water three days before assaying for MT. Compared to B6 mice, the Im mice exhibited more MT in their liver by the /sup 65/Zn-MT binding assay (3-fold) and by the /sup 109/Cd-saturation/hemolysate method (18-fold). The effects of the two zinc treatments did not differ significantly between Im and B6 mice. The retention and excretion of /sup 65/Zn (administered intraperitoneally) were determined over a 14-day period, but the results did not different between the Im and B6 mice. The increased concentrations of MT within the Im mice was not significantly different for the intestine and liver. Based on these data and other studies, the Im mice may exhibit alterations in zinc homeostasis due to some deregulation of MT metabolism, including the inner ear of the fetus, the lactating mammary gland, and the intestine and liver of adults by one year of age.

Erythroblast differentiation at spleen in Q137E mutant ribosomal protein S19 gene knock-in C57BL/6J mice.

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Yamanegi, Koji; Yamada, Naoko; Nakasho, Keiji; Nishiura, Hiroshi

2018-01-01

We recently found that erythroblast-like cells derived from human leukaemia K562 cells express C5a receptor (C5aR) and produce its antagonistic and agonistic ligand ribosomal protein S19 (RP S19) polymer, which is cross-linked between K122 and Q137 by tissue transglutaminases. RP S19 polymer binds to the reciprocal C5aRs on erythroblast-like cells and macrophage-like cells derived from human monocytic THP-1 cells and promotes differentiation into reticulocyte-like cells through enucleation in vitro. To examine the roles of RP S19 polymer in mouse erythropoiesis, we prepared Q137E mutant RP S19 gene knock-in C57BL/6J mice. In contrast to wild-type mice, erythroblast numbers at the preliminary stage (CD71 high /TER119 low ) in spleen based on transferrin receptor (CD71) and glycophorin A (TER119) values and erythrocyte numbers in orbital artery bloods were not largely changed in knock-in mice. Conversely, erythroblast numbers at the early stage (CD71 high /TER119 high ) were significantly decreased in spleen by knock-in mice. The reduction of early erythroblast numbers in spleen was enhanced by the phenylhydrazine-induced pernicious anemia model knock-in mice and was rescued by a functional analogue of RP S19 dimer S-tagged C5a/RP S19. These data indicated that RP S19 polymer plays the roles in the early erythroblast differentiation of C57BL/6J mouse spleen. Copyright © 2017 Elsevier GmbH. All rights reserved.

Deletion of vanilloid receptor (TRPV1) in mice alters behavioral effects of ethanol

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Blednov, Y.A.; Harris, R.A.

2009-01-01

The vanilloid receptor TRPV1 is activated by ethanol and this may be important for some of the central and peripheral actions of ethanol. To determine if this receptor has a role in ethanol-mediated behaviors, we studied null mutant mice in which the Trpv1 gene was deleted. Mice lacking this gene showed significantly higher preference for ethanol and consumed more ethanol in a two-bottle choice test as compared with wild type littermates. Null mutant mice showed shorter duration of loss of righting reflex induced by low doses of ethanol (3.2 and 3.4 g/kg) and faster recovery from motor incoordination induced by ethanol (2 g/kg). However, there were no differences between null mutant and wild type mice in severity of ethanol-induced acute withdrawal (4 g/kg) or conditioned taste aversion to ethanol (2.5 g/kg). Two behavioral phenotypes (decreased sensitivity to ethanol-induced sedation and faster recovery from ethanol-induced motor incoordination) seen in null mutant mice were reproduced in wild type mice by injection of a TRPV1 antagonist, capsazepine (10 mg/kg). These two ethanol behaviors were changed in the opposite direction after injection of capsaicin, a selective TRPV1 agonist, in wild type mice. The studies provide the first evidence that TRPV1 is important for specific behavioral actions of ethanol. PMID:19705551

Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/Wv mutant mouse colon.

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Tamada, Hiromi; Kiyama, Hiroshi

2015-01-01

Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/Wv mice carrying W and Wv mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/Wv mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/Wv mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/Wv mutant colon.The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers,but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/Wv mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/Wv mutant mice.

Behavioral and other phenotypes in a cytoplasmic Dynein light intermediate chain 1 mutant mouse.

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Banks, Gareth T; Haas, Matilda A; Line, Samantha; Shepherd, Hazel L; Alqatari, Mona; Stewart, Sammy; Rishal, Ida; Philpott, Amelia; Kalmar, Bernadett; Kuta, Anna; Groves, Michael; Parkinson, Nicholas; Acevedo-Arozena, Abraham; Brandner, Sebastian; Bannerman, David; Greensmith, Linda; Hafezparast, Majid; Koltzenburg, Martin; Deacon, Robert; Fainzilber, Mike; Fisher, Elizabeth M C

2011-04-06

The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system.

Mutant PrP Suppresses Glutamatergic Neurotransmission in Cerebellar Granule Neurons by Impairing Membrane Delivery of VGCC α2δ-1 Subunit

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Senatore, Assunta; Colleoni, Simona; Verderio, Claudia; Restelli, Elena; Morini, Raffaella; Condliffe, Steven B.; Bertani, Ilaria; Mantovani, Susanna; Canovi, Mara; Micotti, Edoardo; Forloni, Gianluigi; Dolphin, Annette C.; Matteoli, Michela; Gobbi, Marco; Chiesa, Roberto

2012-01-01

Summary How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α2δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α2δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies. PMID:22542184

RhoE deficiency produces postnatal lethality, profound motor deficits and neurodevelopmental delay in mice.

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Enric Mocholí

Full Text Available Rnd proteins are a subfamily of Rho GTPases involved in the control of actin cytoskeleton dynamics and other cell functions such as motility, proliferation and survival. Unlike other members of the Rho family, Rnd proteins lack GTPase activity and therefore remain constitutively active. We have recently described that RhoE/Rnd3 is expressed in the Central Nervous System and that it has a role in promoting neurite formation. Despite their possible relevance during development, the role of Rnd proteins in vivo is not known. To get insight into the in vivo function of RhoE we have generated mice lacking RhoE expression by an exon trapping cassette. RhoE null mice (RhoE gt/gt are smaller at birth, display growth retardation and early postnatal death since only half of RhoE gt/gt mice survive beyond postnatal day (PD 15 and 100% are dead by PD 29. RhoE gt/gt mice show an abnormal body position with profound motor impairment and impaired performance in most neurobehavioral tests. Null mutant mice are hypoactive, show an immature locomotor pattern and display a significant delay in the appearance of the hindlimb mature responses. Moreover, they perform worse than the control littermates in the wire suspension, vertical climbing and clinging, righting reflex and negative geotaxis tests. Also, RhoE ablation results in a delay of neuromuscular maturation and in a reduction in the number of spinal motor neurons. Finally, RhoE gt/gt mice lack the common peroneal nerve and, consequently, show a complete atrophy of the target muscles. This is the first model to study the in vivo functions of a member of the Rnd subfamily of proteins, revealing the important role of Rnd3/RhoE in the normal development and suggesting the possible involvement of this protein in neurological disorders.

Unexpected Cartilage Phenotype in CD4-Cre-Conditional SOS-Deficient Mice.

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Guittard, Geoffrey; Gallardo, Devorah L; Li, Wenmei; Melis, Nicolas; Lui, Julian C; Kortum, Robert L; Shakarishvili, Nicholas G; Huh, Sunmee; Baron, Jeffrey; Weigert, Roberto; Kramer, Joshua A; Samelson, Lawrence E; Sommers, Connie L

2017-01-01

RAS signaling is central to many cellular processes and SOS proteins promote RAS activation. To investigate the role of SOS proteins in T cell biology, we crossed Sos1 f/f Sos2 -/- mice to CD4-Cre transgenic mice. We previously reported an effect of these mutations on T cell signaling and T cell migration. Unexpectedly, we observed nodules on the joints of greater than 90% of these mutant mice at 5 months of age, especially on the carpal joints. As the mice aged further, some also displayed joint stiffness, hind limb paralysis, and lameness. Histological analysis indicated that the abnormal growth in joints originated from dysplastic chondrocytes. Second harmonic generation imaging of the carpal nodules revealed that nodules were encased by rich collagen fibrous networks. Nodules formed in mice also deficient in RAG2, indicating that conventional T cells, which undergo rearrangement of the T cell antigen receptor, are not required for this phenotype. CD4-Cre expression in a subset of cells, either immune lineage cells (e.g., non-conventional T cells) or non-immune lineage cells (e.g., chondrocytes) likely mediates the dramatic phenotype observed in this study. Disruptions of genes in the RAS signaling pathway are especially likely to cause this phenotype. These results also serve as a cautionary tale to those intending to use CD4-Cre transgenic mice to specifically delete genes in conventional T cells.

PAC1- and VPAC2 receptors in light regulated behavior and physiology: Studies in single and double mutant mice.

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Jens Hannibal

Full Text Available The two sister peptides, pituitary adenylate cyclase activating polypeptide (PACAP and vasoactive intestinal polypeptide (VIP and their receptors, the PAC1 -and the VPAC2 receptors, are involved in regulation of the circadian timing system. PACAP as a neurotransmitter in the retinohypothalamic tract (RHT and VIP as a neurotransmitter, involved in synchronization of SCN neurons. Behavior and physiology in VPAC2 deficient mice are strongly regulated by light most likely as a result of masking. Consequently, we used VPAC2 and PAC1/VPAC2 double mutant mice in comparison with PAC1 receptor deficient mice to further elucidate the role of PACAP in the light mediated regulation of behavior and physiology of the circadian system. We compared circadian rhythms in mice equipped with running wheels or implanted radio-transmitter measuring core body temperature kept in a full photoperiod ((FPP(12:12 h light dark-cycles (LD and skeleton photo periods (SPP at high and low light intensity. Furthermore, we examined the expression of PAC1- and VPAC2 receptors in the SCN of the different genotypes in combination with visualization of PACAP and VIP and determined whether compensatory changes in peptide and/or receptor expression in the reciprocal knockouts (KO (PAC1 and VPAC2 had occurred. Our data demonstrate that in although being closely related at both ligand and receptor structure/sequence, PACAP/PAC1 receptor signaling are independent of VIP/VPAC2 receptor signaling and vice versa. Furthermore, lack of either of the receptors does not result in compensatory changes at neither the physiological or anatomical level. PACAP/PAC1 signaling is important for light regulated behavior, VIP/VPAC2signaling for stable clock function and both signaling pathways may play a role in shaping diurnality versus nocturnality.

PERCEPTION OF SWEET TASTE IS IMPORTANT FOR VOLUNTARY ALCOHOL CONSUMPTION IN MICE

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Blednov, Y.A.; Walker, D.; Martinez, M.; Levine, M.; Damak, S.; Margolskee, R.F.

2007-01-01

To directly evaluate the association between taste perception and alcohol intake, we used three different mutant mice, each lacking a gene expressed in taste buds and critical to taste transduction: α-gustducin (Gnat3), Tas1r3 or Trpm5. Null mutant mice lacking any of these three genes showed lower preference score for alcohol and consumed less alcohol in a two-bottle choice test, as compared with wild-type littermates. These null mice also showed lower preference score for saccharin solution...

Zebrafish bandoneon mutants display behavioral defects due to a mutation in the glycine receptor β-subunit

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Hirata, Hiromi; Saint-Amant, Louis; Downes, Gerald B.; Cui, Wilson W.; Zhou, Weibin; Granato, Michael; Kuwada, John Y.

2005-01-01

Bilateral alternation of muscle contractions requires reciprocal inhibition between the two sides of the hindbrain and spinal cord, and disruption of this inhibition should lead to simultaneous activation of bilateral muscles. At 1 day after fertilization, wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. Similar simultaneous contractions are observed in wild-type embryos treated with strychnine, a blocker of the inhibitory glycine receptor (GlyR). This result suggests that glycinergic synaptic transmission is defective in beo mutants. Muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the CNS. Recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. Furthermore, immunostaining with an antibody against GlyR showed clusters in wild-type neurons but not in beo neurons. These data suggest that the failure of GlyRs to aggregate at synaptic sites causes impairment of glycinergic transmission and abnormal behavior in beo mutants. Indeed, mutations in the GlyR β-subunit, which are thought to be required for proper localization of GlyRs, were identified as the basis for the beo mutation. These data demonstrate that GlyRβ is essential for physiologically relevant clustering of GlyRs in vivo. Because GlyR mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, the zebrafish beo mutant should be a useful animal model for this condition. PMID:15928085

Variations of L- and D-amino acid levels in the brain of wild-type and mutant mice lacking D-amino acid oxidase activity.

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Du, Siqi; Wang, Yadi; Weatherly, Choyce A; Holden, Kylie; Armstrong, Daniel W

2018-05-01

D-amino acids are now recognized to be widely present in organisms and play essential roles in biological processes. Some D-amino acids are metabolized by D-amino acid oxidase (DAO), while D-Asp and D-Glu are metabolized by D-aspartate oxidase (DDO). In this study, levels of 22 amino acids and the enantiomeric compositions of the 19 chiral proteogenic entities have been determined in the whole brain of wild-type ddY mice (ddY/DAO +/+ ), mutant mice lacking DAO activity (ddY/DAO -/- ), and the heterozygous mice (ddY/DAO +/- ) using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). No significant differences were observed for L-amino acid levels among the three strains except for L-Trp which was markedly elevated in the DAO +/- and DAO -/- mice. The question arises as to whether this is an unknown effect of DAO inactivity. The three highest levels of L-amino acids were L-Glu, L-Asp, and L-Gln in all the three strains. The lowest L-amino acid level was L-Cys in ddY/DAO +/- and ddY/DAO -/- mice, while L-Trp showed the lowest level in ddY/DAO +/+ mice. The highest concentration of D-amino acid was found to be D-Ser, which also had the highest % D value (~ 25%). D-Glu had the lowest % D value (~ 0.01%) in all the three strains. Significant differences of D-Leu, D-Ala, D-Ser, D-Arg, and D-Ile were observed in ddY/DAO +/- and ddY/DAO -/- mice compared to ddY/DAO +/+ mice. This work provides the most complete baseline analysis of L- and D-amino acids in the brains of ddY/DAO +/+ , ddY/DAO +/- , and ddY/DAO -/- mice yet reported. It also provides the most effective and efficient analytical approach for measuring these analytes in biological samples. This study provides fundamental information on the role of DAO in the brain and may be relevant for future development involving novel drugs for DAO regulation.

Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation

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Fresno Manuel

2011-07-01

Full Text Available Abstract Background Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. Results In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II, to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus, infected with mutant parasites did not develop any sign of pathology. Conclusions The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis.

Juvenile spermatogonial depletion (jsd): a genetic defect of germ cell proliferation of male mice.

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Beamer, W G; Cunliffe-Beamer, T L; Shultz, K L; Langley, S H; Roderick, T H

1988-05-01

Adult C57BL/6J male mice homozygous for the mutant gene, juvenile spermatogonial depletion (jsd/jsd), show azoosper4ia and testes reduced to one-third normal size, but are otherwise phenotypically normal. In contrast, adult jsd/jsd females are fully fertile. This feature facilitated mapping the jsd gene to the centromeric end of chromosome 1; the gene order is jsd-Isocitrate dehydrogenase-1 (Idh-1)-Peptidase-3 (Pep-3). Analysis of testicular histology from jsd/jsd mice aged 3-10 wk revealed that these mutant mice experience one wave of spermatogenesis, but fail to continue mitotic proliferation of type A spermatogonial cells at the basement membrane. As a consequence, histological sections of testes from mutant mice aged 8-52 wk showed tubules populated by modest numbers of Sertoli cells, with only an occasional spermatogonial cell. Some sperm with normal morphology and motility were observed in epididymides of 6.5- but not in 8-wk or older mutants. Treatment with retinol failed to alter the loss of spermatogenesis in jsd/jsd mice. Analyses of serum hormones of jsd/jsd males showed that testosterone levels were normal at all ages--a finding corroborated by normal seminal vesicle and vas deferens weights, whereas serum follicle-stimulating hormone levels were significantly elevated in mutant mice from 4 to 20 wk of age. We hypothesize the jsd/jsd male may be deficient in proliferative signals from Sertoli cells that are needed for spermatogenesis.

Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/W(v) mutant mouse colon.

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Tamada, Hiromi; Kiyama, Hiroshi

2015-01-01

Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/W(v) mice carrying W and W(v) mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/W(v) mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/W(v) mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/W(v) mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/W(v) mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/W(v) mutant mice.

Comparison of effects of p53 null and gain-of-function mutations on salivary tumors in MMTV-Hras transgenic mice.

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Dadi Jiang

Full Text Available p53 is an important tumor suppressor gene which is mutated in ~50% of all human cancers. Some of these mutants appear to have acquired novel functions beyond merely losing wild-type functions. To investigate these gain-of-function effects in vivo, we generated mice of three different genotypes: MMTV-Hras/p53(+/+, MMTV-Hras/p53(-/-, and MMTV-Hras/p53R172H/R172H. Salivary tumors from these mice were characterized with regard to age of tumor onset, tumor growth rates, cell cycle distribution, apoptotic levels, tumor histopathology, as well as response to doxorubicin treatment. Microarray analysis was also performed to profile gene expression. The MMTV-Hras/p53(-/- and MMTV-Hras/p53R172H/R172H mice displayed similar properties with regard to age of tumor onset, tumor growth rates, tumor histopathology, and response to doxorubicin, while both groups were clearly distinct from the MMTV-Hras/p53(+/+ mice by these measurements. In addition, the gene expression profiles of the MMTV-Hras/p53(-/- and MMTV-Hras/p53(R172H/R172H tumors were tightly clustered, and clearly distinct from the profiles of the MMTV-Hras/p53(+/+ tumors. Only a small group of genes showing differential expression between the MMTV-Hras/p53(-/- and MMTV-Hras/p53(R172H/R172H tumors, that did not appear to be regulated by wild-type p53, were identified. Taken together, these results indicate that in this MMTV-Hras-driven salivary tumor model, the major effect of the p53 R172H mutant is due to the loss of wild-type p53 function, with little or no gain-of-function effect on tumorigenesis, which may be explained by the tissue- and tumor type-specific properties of this gain-of-function mutant of p53.

Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia

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Chao Liu

2015-08-01

Full Text Available Apolipoprotein C-II (APOC2 is an obligatory activator of lipoprotein lipase. Human patients with APOC2 deficiency display severe hypertriglyceridemia while consuming a normal diet, often manifesting xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia is also an important risk factor for development of cardiovascular disease. Animal models to study hypertriglyceridemia are limited, with no Apoc2-knockout mouse reported. To develop a genetic model of hypertriglyceridemia, we generated an apoc2 mutant zebrafish characterized by the loss of Apoc2 function. apoc2 mutants show decreased plasma lipase activity and display chylomicronemia and severe hypertriglyceridemia, which closely resemble the phenotype observed in human patients with APOC2 deficiency. The hypertriglyceridemia in apoc2 mutants is rescued by injection of plasma from wild-type zebrafish or by injection of a human APOC2 mimetic peptide. Consistent with a previous report of a transient apoc2 knockdown, apoc2 mutant larvae have a minor delay in yolk consumption and angiogenesis. Furthermore, apoc2 mutants fed a normal diet accumulate lipid and lipid-laden macrophages in the vasculature, which resemble early events in the development of human atherosclerotic lesions. In addition, apoc2 mutant embryos show ectopic overgrowth of pancreas. Taken together, our data suggest that the apoc2 mutant zebrafish is a robust and versatile animal model to study hypertriglyceridemia and the mechanisms involved in the pathogenesis of associated human diseases.

Epiphyseal abnormalities, trabecular bone loss and articular chondrocyte hypertrophy develop in the long bones of postnatal Ext1-deficient mice.

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Sgariglia, Federica; Candela, Maria Elena; Huegel, Julianne; Jacenko, Olena; Koyama, Eiki; Yamaguchi, Yu; Pacifici, Maurizio; Enomoto-Iwamoto, Motomi

2013-11-01

Long bones are integral components of the limb skeleton. Recent studies have indicated that embryonic long bone development is altered by mutations in Ext genes and consequent heparan sulfate (HS) deficiency, possibly due to changes in activity and distribution of HS-binding/growth plate-associated signaling proteins. Here we asked whether Ext function is continuously required after birth to sustain growth plate function and long bone growth and organization. Compound transgenic Ext1(f/f);Col2CreERT mice were injected with tamoxifen at postnatal day 5 (P5) to ablate Ext1 in cartilage and monitored over time. The Ext1-deficient mice exhibited growth retardation already by 2weeks post-injection, as did their long bones. Mutant growth plates displayed a severe disorganization of chondrocyte columnar organization, a shortened hypertrophic zone with low expression of collagen X and MMP-13, and reduced primary spongiosa accompanied, however, by increased numbers of TRAP-positive osteoclasts at the chondro-osseous border. The mutant epiphyses were abnormal as well. Formation of a secondary ossification center was significantly delayed but interestingly, hypertrophic-like chondrocytes emerged within articular cartilage, similar to those often seen in osteoarthritic joints. Indeed, the cells displayed a large size and round shape, expressed collagen X and MMP-13 and were surrounded by an abundant Perlecan-rich pericellular matrix not seen in control articular chondrocytes. In addition, ectopic cartilaginous outgrowths developed on the lateral side of mutant growth plates over time that resembled exostotic characteristic of children with Hereditary Multiple Exostoses, a syndrome caused by Ext mutations and HS deficiency. In sum, the data do show that Ext1 is continuously required for postnatal growth and organization of long bones as well as their adjacent joints. Ext1 deficiency elicits defects that can occur in human skeletal conditions including trabecular bone loss

Voronoi-based spatial analysis reveals selective interneuron changes in the cortex of FALS mice.

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Minciacchi, Diego; Kassa, Roman M; Del Tongo, Claudia; Mariotti, Raffaella; Bentivoglio, Marina

2009-01-01

The neurodegenerative disease amyotrophic lateral sclerosis affects lower motoneurons and corticospinal cells. Mice expressing human mutant superoxide dismutase (SOD)1 provide widely investigated models of the familial form of disease, but information on cortical changes in these mice is still limited. We here analyzed the spatial organization of interneurons characterized by parvalbumin immunoreactivity in the motor, somatosensory, and visual cortical areas of SOD1(G93A) mice. Cell number and sociological spatial behavior were assessed by digital charts of cell location in cortical samples, cell counts, and generation of two-dimensional Voronoi diagrams. In end-stage SOD1-mutant mice, an increase of parvalbumin-containing cortical interneurons was found in the motor and somatosensory areas (about 35% and 20%, respectively) with respect to wild-type littermates. Changes in cell spatial distribution, as documented by Voronoi-derived coefficients of variation, indicated increased tendency of parvalbumin cells to aggregate into clusters in the same areas of the SOD1-mutant cortex. Counts and coefficients of variation of parvalbumin cells in the visual cortex gave instead similar results in SOD1-mutant and wild-type mice. Analyses of motor and somatosensory areas in presymptomatic SOD1-mutant mice provided findings very similar to those obtained at end-stage, indicating early changes of interneurons in these cortical areas during the pathology. Altogether the data reveal in the SOD1-mutant mouse cortex an altered architectonic pattern of interneurons, which selectively affects areas involved in motor control. The findings, which can be interpreted as pathogenic factors or early disease-related adaptations, point to changes in the cortical regulation and modulation of the motor circuit during motoneuron disease.

Differential effects of lesion mimic mutants in barley on disease development by facultative pathogens

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McGrann, Graham R. D.; Steed, , Andrew; Burt, Christopher; Nicholson, Paul; Brown, James K. M.

2015-01-01

Lesion mimic mutants display spontaneous necrotic spots and chlorotic leaves as a result of mis-regulated cell death programmes. Typically these mutants have increased resistance to biotrophic pathogens but their response to facultative fungi that cause necrotrophic diseases is less well studied. The effect of altered cell death regulation on the development of disease caused by Ramularia collo-cygni, Fusarium culmorum and Oculimacula yallundae was explored using a collection of barley necrotic (nec) lesion mimic mutants. nec8 mutants displayed lower levels of all three diseases compared to nec9 mutants, which had increased R. collo-cygni but decreased F. culmorum disease symptoms. nec1 mutants reduced disease development caused by both R. collo-cygni and F. culmorum. The severity of the nec1-induced lesion mimic phenotype and F. culmorum symptom development was reduced by mutation of the negative cell death regulator MLO. The significant reduction in R. collo-cygni symptoms caused by nec1 was completely abolished in the presence of the mlo-5 allele and both symptoms and fungal biomass were greater than in the wild-type. These results indicate that physiological pathways involved in regulation of cell death interact with one another in their effects on different fungal pathogens. PMID:25873675

Glutaminase-Deficient Mice Display Hippocampal Hypoactivity, Insensitivity to Pro-Psychotic Drugs and Potentiated Latent Inhibition: Relevance to Schizophrenia

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Gaisler-Salomon, Inna; Miller, Gretchen M; Chuhma, Nao; Lee, Sooyeon; Zhang, Hong; Ghoddoussi, Farhad; Lewandowski, Nicole; Fairhurst, Stephen; Wang, Yvonne; Conjard-Duplany, Agnès; Masson, Justine; Balsam, Peter; Hen, René; Arancio, Ottavio; Galloway, Matthew P; Moore, Holly M; Small, Scott A; Rayport, Stephen

2009-01-01

Dysregulated glutamatergic neurotransmission has been strongly implicated in the pathophysiology of schizophrenia (SCZ). Recently, presynaptic modulation of glutamate transmission has been shown to have therapeutic promise. We asked whether genetic knockdown of glutaminase (gene GLS1) to reduce glutamatergic transmission presynaptically by slowing the recycling of glutamine to glutamate, would produce a phenotype relevant to SCZ and its treatment. GLS1 heterozygous (GLS1 het) mice showed about a 50% global reduction in glutaminase activity, and a modest reduction in glutamate levels in brain regions relevant to SCZ pathophysiology, but displayed neither general behavioral abnormalities nor SCZ-associated phenotypes. Functional imaging, measuring regional cerebral blood volume, showed hippocampal hypometabolism mainly in the CA1 subregion and subiculum, the inverse of recent clinical imaging findings in prodromal and SCZ patients. GLS1 het mice were less sensitive to the behavioral stimulating effects of amphetamine, showed a reduction in amphetamine-induced striatal dopamine release and in ketamine-induced frontal cortical activation, suggesting that GLS1 het mice are resistant to the effects of these pro-psychotic challenges. Moreover, GLS1 het mice showed clozapine-like potentiation of latent inhibition, suggesting that reduction in glutaminase has antipsychotic-like properties. These observations provide further support for the pivotal role of altered glutamatergic synaptic transmission in the pathophysiology of SCZ, and suggest that presynaptic modulation of the glutamine–glutamate pathway through glutaminase inhibition may provide a new direction for the pharmacotherapy of SCZ. PMID:19516252

Effector and naturally occurring regulatory T cells display no abnormalities in activation induced cell death in NOD mice.

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Ayelet Kaminitz

Full Text Available BACKGROUND: Disturbed peripheral negative regulation might contribute to evolution of autoimmune insulitis in type 1 diabetes. This study evaluates the sensitivity of naïve/effector (Teff and regulatory T cells (Treg to activation-induced cell death mediated by Fas cross-linking in NOD and wild-type mice. PRINCIPAL FINDINGS: Both effector (CD25(-, FoxP3(- and suppressor (CD25(+, FoxP3(+ CD4(+ T cells are negatively regulated by Fas cross-linking in mixed splenocyte populations of NOD, wild type mice and FoxP3-GFP trangeneess. Proliferation rates and sensitivity to Fas cross-linking are dissociated in Treg cells: fast cycling induced by IL-2 and CD3/CD28 stimulation improve Treg resistance to Fas-ligand (FasL in both strains. The effector and suppressor CD4(+ subsets display balanced sensitivity to negative regulation under baseline conditions, IL-2 and CD3/CD28 stimulation, indicating that stimulation does not perturb immune homeostasis in NOD mice. Effective autocrine apoptosis of diabetogenic cells was evident from delayed onset and reduced incidence of adoptive disease transfer into NOD.SCID by CD4(+CD25(- T cells decorated with FasL protein. Treg resistant to Fas-mediated apoptosis retain suppressive activity in vitro. The only detectable differential response was reduced Teff proliferation and upregulation of CD25 following CD3-activation in NOD mice. CONCLUSION: These data document negative regulation of effector and suppressor cells by Fas cross-linking and dissociation between sensitivity to apoptosis and proliferation in stimulated Treg. There is no evidence that perturbed AICD in NOD mice initiates or promotes autoimmune insulitis.

Genetic background of Prop1(df) mutants provides remarkable protection against hypothyroidism-induced hearing impairment.

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Fang, Qing; Giordimaina, Alicia M; Dolan, David F; Camper, Sally A; Mustapha, Mirna

2012-04-01

Hypothyroidism is a cause of genetic and environmentally induced deafness. The sensitivity of cochlear development and function to thyroid hormone (TH) mandates understanding TH action in this sensory organ. Prop1(df) and Pou1f1(dw) mutant mice carry mutations in different pituitary transcription factors, each resulting in pituitary thyrotropin deficiency. Despite the same lack of detectable serum TH, these mutants have very different hearing abilities: Prop1(df) mutants are mildly affected, while Pou1f1(dw) mutants are completely deaf. Genetic studies show that this difference is attributable to the genetic backgrounds. Using embryo transfer, we discovered that factors intrinsic to the fetus are the major contributor to this difference, not maternal effects. We analyzed Prop1(df) mutants to identify processes in cochlear development that are disrupted in other hypothyroid animal models but protected in Prop1(df) mutants by the genetic background. The development of outer hair cell (OHC) function is delayed, but Prestin and KCNQ4 immunostaining appear normal in mature Prop1(df) mutants. The endocochlear potential and KCNJ10 immunostaining in the stria vascularis are indistinguishable from wild type, and no differences in neurofilament or synaptophysin staining are evident in Prop1(df) mutants. The synaptic vesicle protein otoferlin normally shifts expression from OHC to IHC as temporary afferent fibers beneath the OHC regress postnatally. Prop1(df) mutants exhibit persistent, abnormal expression of otoferlin in apical OHC, suggesting delayed maturation of synaptic function. Thus, the genetic background of Prop1(df) mutants is remarkably protective for most functions affected in other hypothyroid mice. The Prop1(df) mutant is an attractive model for identifying the genes that protect against deafness.

Calcilytic Ameliorates Abnormalities of Mutant Calcium-Sensing Receptor (CaSR) Knock-In Mice Mimicking Autosomal Dominant Hypocalcemia (ADH).

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Dong, Bingzi; Endo, Itsuro; Ohnishi, Yukiyo; Kondo, Takeshi; Hasegawa, Tomoka; Amizuka, Norio; Kiyonari, Hiroshi; Shioi, Go; Abe, Masahiro; Fukumoto, Seiji; Matsumoto, Toshio

2015-11-01

Activating mutations of calcium-sensing receptor (CaSR) cause autosomal dominant hypocalcemia (ADH). ADH patients develop hypocalcemia, hyperphosphatemia, and hypercalciuria, similar to the clinical features of hypoparathyroidism. The current treatment of ADH is similar to the other forms of hypoparathyroidism, using active vitamin D3 or parathyroid hormone (PTH). However, these treatments aggravate hypercalciuria and renal calcification. Thus, new therapeutic strategies for ADH are needed. Calcilytics are allosteric antagonists of CaSR, and may be effective for the treatment of ADH caused by activating mutations of CaSR. In order to examine the effect of calcilytic JTT-305/MK-5442 on CaSR harboring activating mutations in the extracellular and transmembrane domains in vitro, we first transfected a mutated CaSR gene into HEK cells. JTT-305/MK-5442 suppressed the hypersensitivity to extracellular Ca(2+) of HEK cells transfected with the CaSR gene with activating mutations in the extracellular and transmembrane domains. We then selected two activating mutations locating in the extracellular (C129S) and transmembrane (A843E) domains, and generated two strains of CaSR knock-in mice to build an ADH mouse model. Both mutant mice mimicked almost all the clinical features of human ADH. JTT-305/MK-5442 treatment in vivo increased urinary cAMP excretion, improved serum and urinary calcium and phosphate levels by stimulating endogenous PTH secretion, and prevented renal calcification. In contrast, PTH(1-34) treatment normalized serum calcium and phosphate but could not reduce hypercalciuria or renal calcification. CaSR knock-in mice exhibited low bone turnover due to the deficiency of PTH, and JTT-305/MK-5442 as well as PTH(1-34) increased bone turnover and bone mineral density (BMD) in these mice. These results demonstrate that calcilytics can reverse almost all the phenotypes of ADH including hypercalciuria and renal calcification, and suggest that calcilytics can become a

Gender-specific effects of endogenous testosterone: female alpha-estrogen receptor-deficient C57Bl/6J mice develop glomerulosclerosis.

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Elliot, S J; Berho, M; Korach, K; Doublier, S; Lupia, E; Striker, G E; Karl, M

2007-08-01

Young female mice on a C57Bl/6J (B6) background are considered glomerulosclerosis (GS)-resistant but aging B6 mice develop mild GS. Estrogen deficiency accelerates while estrogen replacement retards GS in young sclerosis-prone oligosyndactyly mutant mice on an ROP background. To explore the effects of sex hormones on glomerular structure and function in the context of gender and genetic background, we studied mice in which the estrogen-receptor (ER) genes alpha- or -beta were deleted (alpha- or betaER knockout (KO)) and crossed into the B6 background. We also studied ovariectomized (Ovx) B6 mice given testosterone. Male and female betaERKO and male alphaERKO mice had no glomerular dysfunction at 9 months of age; however, alphaERKO female mice displayed albuminuria and GS. Ovx prevented glomerular dysfunction in alphaERKO female mice by eliminating endogenous testosterone production while exogenous testosterone induced GS in Ovx B6 mice. Androgen receptor (AR) expression and function was found in microdissected glomeruli and cultured mesangial cells. Testosterone compared to placebo increased both AR expression and TGF-beta1 mRNA levels in glomeruli isolated from female B6 mice. Estrogen deficiency had no deleterious effects on the glomeruli in B6 mice. Our study shows that genetic traits strongly influence the GS-promoting effects of estrogen deficiency while testosterone induces GS in a gender-specific manner.

Loss of circadian clock accelerates aging in neurodegeneration-prone mutants.

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Krishnan, Natraj; Rakshit, Kuntol; Chow, Eileen S; Wentzell, Jill S; Kretzschmar, Doris; Giebultowicz, Jadwiga M

2012-03-01

Circadian clocks generate rhythms in molecular, cellular, physiological, and behavioral processes. Recent studies suggest that disruption of the clock mechanism accelerates organismal senescence and age-related pathologies in mammals. Impaired circadian rhythms are observed in many neurological diseases; however, it is not clear whether loss of rhythms is the cause or result of neurodegeneration, or both. To address this important question, we examined the effects of circadian disruption in Drosophila melanogaster mutants that display clock-unrelated neurodegenerative phenotypes. We combined a null mutation in the clock gene period (per(01)) that abolishes circadian rhythms, with a hypomorphic mutation in the carbonyl reductase gene sniffer (sni(1)), which displays oxidative stress induced neurodegeneration. We report that disruption of circadian rhythms in sni(1) mutants significantly reduces their lifespan compared to single mutants. Shortened lifespan in double mutants was coupled with accelerated neuronal degeneration evidenced by vacuolization in the adult brain. In addition, per(01)sni(1) flies showed drastically impaired vertical mobility and increased accumulation of carbonylated proteins compared to age-matched single mutant flies. Loss of per function does not affect sni mRNA expression, suggesting that these genes act via independent pathways producing additive effects. Finally, we show that per(01) mutation accelerates the onset of brain pathologies when combined with neurodegeneration-prone mutation in another gene, swiss cheese (sws(1)), which does not operate through the oxidative stress pathway. Taken together, our data suggest that the period gene may be causally involved in neuroprotective pathways in aging Drosophila. Copyright © 2011 Elsevier Inc. All rights reserved.

ATM loss leads to synthetic lethality in BRCA1 BRCT mutant mice associated with exacerbated defects in homology-directed repair.

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Chen, Chun-Chin; Kass, Elizabeth M; Yen, Wei-Feng; Ludwig, Thomas; Moynahan, Mary Ellen; Chaudhuri, Jayanta; Jasin, Maria

2017-07-18

BRCA1 is essential for homology-directed repair (HDR) of DNA double-strand breaks in part through antagonism of the nonhomologous end-joining factor 53BP1. The ATM kinase is involved in various aspects of DNA damage signaling and repair, but how ATM participates in HDR and genetically interacts with BRCA1 in this process is unclear. To investigate this question, we used the Brca1 S1598F mouse model carrying a mutation in the BRCA1 C-terminal domain of BRCA1. Whereas ATM loss leads to a mild HDR defect in adult somatic cells, we find that ATM inhibition leads to severely reduced HDR in Brca1 S1598F cells. Consistent with a critical role for ATM in HDR in this background, loss of ATM leads to synthetic lethality of Brca1 S1598F mice. Whereas both ATM and BRCA1 promote end resection, which can be regulated by 53BP1, 53bp1 deletion does not rescue the HDR defects of Atm mutant cells, in contrast to Brca1 mutant cells. These results demonstrate that ATM has a role in HDR independent of the BRCA1-53BP1 antagonism and that its HDR function can become critical in certain contexts.

ER stress inhibitor attenuates hearing loss and hair cell death in Cdh23erl/erl mutant mice.

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Hu, Juan; Li, Bo; Apisa, Luke; Yu, Heping; Entenman, Shami; Xu, Min; Stepanyan, Ruben; Guan, Bo-Jhih; Müller, Ulrich; Hatzoglou, Maria; Zheng, Qing Yin

2016-11-24

Hearing loss is one of the most common sensory impairments in humans. Mouse mutant models helped us to better understand the mechanisms of hearing loss. Recently, we have discovered that the erlong (erl) mutation of the cadherin23 (Cdh23) gene leads to hearing loss due to hair cell apoptosis. In this study, we aimed to reveal the molecular pathways upstream to apoptosis in hair cells to exploit more effective therapeutics than an anti-apoptosis strategy. Our results suggest that endoplasmic reticulum (ER) stress is the earliest molecular event leading to the apoptosis of hair cells and hearing loss in erl mice. We also report that the ER stress inhibitor, Salubrinal (Sal), could delay the progression of hearing loss and preserve hair cells. Our results provide evidence that therapies targeting signaling pathways in ER stress development prevent hair cell apoptosis at an early stage and lead to better outcomes than those targeting downstream factors, such as tip-link degeneration and apoptosis.

Iron metabolism mutant hbd mice have a deletion in Sec15l1, which has homology to a yeast gene for vesicle docking.

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White, Robert A; Boydston, Leigh A; Brookshier, Terri R; McNulty, Steven G; Nsumu, Ndona N; Brewer, Brandon P; Blackmore, Krista

2005-12-01

Defects in iron absorption and utilization lead to iron deficiency and anemia. While iron transport by transferrin receptor-mediated endocytosis is well understood, it is not completely clear how iron is transported from the endosome to the mitochondria where heme is synthesized. We undertook a positional cloning project to identify the causative mutation for the hemoglobin-deficit (hbd) mouse mutant, which suffers from a microcytic, hypochromic anemia apparently due to defective iron transport in the endocytosis cycle. As shown by previous studies, reticulocyte iron accumulation in homozygous hbd/hbd mice is deficient despite normal binding of transferrin to its receptor and normal transferrin uptake in the cell. We have identified a strong candidate gene for hbd, Sec15l1, a homologue to yeast SEC15, which encodes a key protein in vesicle docking. The hbd mice have an exon deletion in Sec15l1, which is the first known mutation of a SEC gene homologue in mammals.

Tyrosine 842 in the activation loop is required for full transformation by the oncogenic mutant FLT3-ITD.

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Kazi, Julhash U; Chougule, Rohit A; Li, Tianfeng; Su, Xianwei; Moharram, Sausan A; Rupar, Kaja; Marhäll, Alissa; Gazi, Mohiuddin; Sun, Jianmin; Zhao, Hui; Rönnstrand, Lars

2017-07-01

The type III receptor tyrosine kinase FLT3 is frequently mutated in acute myeloid leukemia. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. Among these tyrosine residues, Y842 is located in the so-called activation loop. The position of this tyrosine residue is well conserved in all receptor tyrosine kinases. It has been reported that phosphorylation of the activation loop tyrosine is critical for catalytic activity for some but not all receptor tyrosine kinases. The role of Y842 residue in FLT3 signaling has not yet been studied. In this report, we show that Y842 is not important for FLT3 activation or ubiquitination but plays a critical role in regulating signaling downstream of the receptor as well as controlling receptor stability. We found that mutation of Y842 in the FLT3-ITD oncogenic mutant background reduced cell viability and increased apoptosis. Furthermore, the introduction of the Y842 mutation in the FLT3-ITD background led to a dramatic reduction in in vitro colony forming capacity. Additionally, mice injected with cells expressing FLT3-ITD/Y842F displayed a significant delay in tumor formation, compared to FLT3-ITD expressing cells. Microarray analysis comparing gene expression regulated by FLT3-ITD versus FLT3-ITD/Y842F demonstrated that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we showed that cells expressing FLT3-ITD/Y842F display impaired activity of the RAS/ERK pathway due to reduced interaction between FLT3 and SHP2 leading to reduced SHP2 activation. Thus, we suggest that Y842 is critical for FLT3-mediated RAS/ERK signaling and cellular transformation.

Lgl1 Is Required for Olfaction and Development of Olfactory Bulb in Mice

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Li, Zhenzu; Zhang, Tingting; Lin, Zhuchun; Hou, Congzhe; Zhang, Jian; Men, Yuqin; Li, Huashun

2016-01-01

Lethal giant larvae 1 (Lgl1) was initially identified as a tumor suppressor in Drosophila and functioned as a key regulator of epithelial polarity and asymmetric cell division. In this study, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in olfactory bulb (OB). Next, we examined the effects of Lgl1 loss in the OB. First, we determined the expression patterns of Lgl1 in the neurogenic regions of the embryonic dorsal region of the LGE (dLGE) and postnatal OB. Furthermore, the Lgl1 conditional mutants exhibited abnormal morphological characteristics of the OB. Our behavioral analysis exhibited greatly impaired olfaction in Lgl1 mutant mice. To elucidate the possible mechanisms of impaired olfaction in Lgl1 mutant mice, we investigated the development of the OB. Interestingly, reduced thickness of the MCL and decreased density of mitral cells (MCs) were observed in Lgl1 mutant mice. Additionally, we observed a dramatic loss in SP8+ interneurons (e.g. calretinin and GABAergic/non-dopaminergic interneurons) in the GL of the OB. Our results demonstrate that Lgl1 is required for the development of the OB and the deletion of Lgl1 results in impaired olfaction in mice. PMID:27603780

Noonan syndrome-causing SHP2 mutants impair ERK-dependent chondrocyte differentiation during endochondral bone growth.

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Tajan, Mylène; Pernin-Grandjean, Julie; Beton, Nicolas; Gennero, Isabelle; Capilla, Florence; Neel, Benjamin G; Araki, Toshiyuki; Valet, Philippe; Tauber, Maithé; Salles, Jean-Pierre; Yart, Armelle; Edouard, Thomas

2018-04-12

Growth retardation is a constant feature of Noonan syndrome (NS) but its physiopathology remains poorly understood. We previously reported that hyperactive NS-causing SHP2 mutants impair the systemic production of insulin-like growth factor 1 (IGF1) through hyperactivation of the RAS/extracellular signal-regulated kinases (ERK) signalling pathway. Besides endocrine defects, a direct effect of these mutants on growth plate has not been explored, although recent studies have revealed an important physiological role for SHP2 in endochondral bone growth. We demonstrated that growth plate length was reduced in NS mice, mostly due to a shortening of the hypertrophic zone and to a lesser extent of the proliferating zone. These histological features were correlated with decreased expression of early chondrocyte differentiation markers, and with reduced alkaline phosphatase staining and activity, in NS murine primary chondrocytes. Although IGF1 treatment improved growth of NS mice, it did not fully reverse growth plate abnormalities, notably the decreased hypertrophic zone. In contrast, we documented a role of RAS/ERK hyperactivation at the growth plate level since 1) NS-causing SHP2 mutants enhance RAS/ERK activation in chondrocytes in vivo (NS mice) and in vitro (ATDC5 cells) and 2) inhibition of RAS/ERK hyperactivation by U0126 treatment alleviated growth plate abnormalities and enhanced chondrocyte differentiation. Similar effects were obtained by chronic treatment of NS mice with statins.In conclusion, we demonstrated that hyperactive NS-causing SHP2 mutants impair chondrocyte differentiation during endochondral bone growth through a local hyperactivation of the RAS/ERK signalling pathway, and that statin treatment may be a possible therapeutic approach in NS.

Comprehensive identification of Salmonella enterica serovar typhimurium genes required for infection of BALB/c mice.

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Roy R Chaudhuri

2009-07-01

Full Text Available Genes required for infection of mice by Salmonella Typhimurium can be identified by the interrogation of random transposon mutant libraries for mutants that cannot survive in vivo. Inactivation of such genes produces attenuated S. Typhimurium strains that have potential for use as live attenuated vaccines. A quantitative screen, Transposon Mediated Differential Hybridisation (TMDH, has been developed that identifies those members of a large library of transposon mutants that are attenuated. TMDH employs custom transposons with outward-facing T7 and SP6 promoters. Fluorescently-labelled transcripts from the promoters are hybridised to whole-genome tiling microarrays, to allow the position of the transposon insertions to be determined. Comparison of microarray data from the mutant library grown in vitro (input with equivalent data produced after passage of the library through mice (output enables an attenuation score to be determined for each transposon mutant. These scores are significantly correlated with bacterial counts obtained during infection of mice using mutants with individual defined deletions of the same genes. Defined deletion mutants of several novel targets identified in the TMDH screen are effective live vaccines.

Biochemical Analysis of Two Single Mutants that Give Rise to a Polymorphic G6PD A-Double Mutant

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Edson Jiovany Ramírez-Nava

2017-10-01

Full Text Available Glucose-6-phosphate dehydrogenase (G6PD is a key regulatory enzyme that plays a crucial role in the regulation of cellular energy and redox balance. Mutations in the gene encoding G6PD cause the most common enzymopathy that drives hereditary nonspherocytic hemolytic anemia. To gain insights into the effects of mutations in G6PD enzyme efficiency, we have investigated the biochemical, kinetic, and structural changes of three clinical G6PD variants, the single mutations G6PD A+ (Asn126AspD and G6PD Nefza (Leu323Pro, and the double mutant G6PD A− (Asn126Asp + Leu323Pro. The mutants showed lower residual activity (≤50% of WT G6PD and displayed important kinetic changes. Although all Class III mutants were located in different regions of the three-dimensional structure of the enzyme and were not close to the active site, these mutants had a deleterious effect over catalytic activity and structural stability. The results indicated that the G6PD Nefza mutation was mainly responsible for the functional and structural alterations observed in the double mutant G6PD A−. Moreover, our study suggests that the G6PD Nefza and G6PD A− mutations affect enzyme functions in a similar fashion to those reported for Class I mutations.

Simulated space radiation-induced mutants in the mouse kidney display widespread genomic change.

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Mitchell S Turker

Full Text Available Exposure to a small number of high-energy heavy charged particles (HZE ions, as found in the deep space environment, could significantly affect astronaut health following prolonged periods of space travel if these ions induce mutations and related cancers. In this study, we used an in vivo mutagenesis assay to define the mutagenic effects of accelerated 56Fe ions (1 GeV/amu, 151 keV/μm in the mouse kidney epithelium exposed to doses ranging from 0.25 to 2.0 Gy. These doses represent fluences ranging from 1 to 8 particle traversals per cell nucleus. The Aprt locus, located on chromosome 8, was used to select induced and spontaneous mutants. To fully define the mutagenic effects, we used multiple endpoints including mutant frequencies, mutation spectrum for chromosome 8, translocations involving chromosome 8, and mutations affecting non-selected chromosomes. The results demonstrate mutagenic effects that often affect multiple chromosomes for all Fe ion doses tested. For comparison with the most abundant sparsely ionizing particle found in space, we also examined the mutagenic effects of high-energy protons (1 GeV, 0.24 keV/μm at 0.5 and 1.0 Gy. Similar doses of protons were not as mutagenic as Fe ions for many assays, though genomic effects were detected in Aprt mutants at these doses. Considered as a whole, the data demonstrate that Fe ions are highly mutagenic at the low doses and fluences of relevance to human spaceflight, and that cells with considerable genomic mutations are readily induced by these exposures and persist in the kidney epithelium. The level of genomic change produced by low fluence exposure to heavy ions is reminiscent of the extensive rearrangements seen in tumor genomes suggesting a potential initiation step in radiation carcinogenesis.

Simulated space radiation-induced mutants in the mouse kidney display widespread genomic change.

Science.gov (United States)

Turker, Mitchell S; Grygoryev, Dmytro; Lasarev, Michael; Ohlrich, Anna; Rwatambuga, Furaha A; Johnson, Sorrel; Dan, Cristian; Eckelmann, Bradley; Hryciw, Gwen; Mao, Jian-Hua; Snijders, Antoine M; Gauny, Stacey; Kronenberg, Amy

2017-01-01

Exposure to a small number of high-energy heavy charged particles (HZE ions), as found in the deep space environment, could significantly affect astronaut health following prolonged periods of space travel if these ions induce mutations and related cancers. In this study, we used an in vivo mutagenesis assay to define the mutagenic effects of accelerated 56Fe ions (1 GeV/amu, 151 keV/μm) in the mouse kidney epithelium exposed to doses ranging from 0.25 to 2.0 Gy. These doses represent fluences ranging from 1 to 8 particle traversals per cell nucleus. The Aprt locus, located on chromosome 8, was used to select induced and spontaneous mutants. To fully define the mutagenic effects, we used multiple endpoints including mutant frequencies, mutation spectrum for chromosome 8, translocations involving chromosome 8, and mutations affecting non-selected chromosomes. The results demonstrate mutagenic effects that often affect multiple chromosomes for all Fe ion doses tested. For comparison with the most abundant sparsely ionizing particle found in space, we also examined the mutagenic effects of high-energy protons (1 GeV, 0.24 keV/μm) at 0.5 and 1.0 Gy. Similar doses of protons were not as mutagenic as Fe ions for many assays, though genomic effects were detected in Aprt mutants at these doses. Considered as a whole, the data demonstrate that Fe ions are highly mutagenic at the low doses and fluences of relevance to human spaceflight, and that cells with considerable genomic mutations are readily induced by these exposures and persist in the kidney epithelium. The level of genomic change produced by low fluence exposure to heavy ions is reminiscent of the extensive rearrangements seen in tumor genomes suggesting a potential initiation step in radiation carcinogenesis.

Cyclic GMP-AMP Displays Mucosal Adjuvant Activity in Mice

OpenAIRE

Škrnjug, Ivana; Guzmán, Carlos Alberto; Ruecker, Christine

2014-01-01

The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP) after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice....

Ames dwarf (Prop1(df)/Prop1(df)) mice display increased sensitivity of the major GH-signaling pathways in liver and skeletal muscle.

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Miquet, Johanna G; Muñoz, Marina C; Giani, Jorge F; González, Lorena; Dominici, Fernando P; Bartke, Andrzej; Turyn, Daniel; Sotelo, Ana I

2010-04-01

Growth hormone (GH) is an anabolic hormone that regulates growth and metabolism. Ames dwarf mice are natural mutants for Prop1, with impaired development of anterior pituitary and undetectable levels of circulating GH, prolactin and TSH. They constitute an endocrine model of life-long GH-deficiency. The main signaling cascades activated by GH binding to its receptor are the JAK2/STATs, PI-3K/Akt and the MAPK Erk1/2 pathways. We have previously reported that GH-induced STAT5 activation was higher in Ames dwarf mice liver compared to non-dwarf controls. The aim of this study was to evaluate the principal components of the main GH-signaling pathways under GH-deficiency in liver and skeletal muscle, another GH-target tissue. Ames dwarf mice and their non-dwarf siblings were assessed. Animals were injected i.p. with GH or saline 15min before tissue removal. Protein content and phosphorylation of signaling mediators were determined by immunoblotting of tissue solubilizates. GH was able to induce STAT5 and STAT3 tyrosine phosphorylation in both liver and muscle, but the response was higher for Ames dwarf mice than for non-dwarf controls. When Erk1/2 activation was assessed in liver, only dwarf mice showed GH-induced phosphorylation, while in muscle no response to the hormone was found in either genotype. GH-induced Akt phosphorylation at Ser473 in liver was only detected in dwarf mice. In skeletal muscle, both normal and dwarf mice responded to a GH stimulus, although dwarf mice presented higher GH activation levels. The phosphorylation of GSK-3, a substrate of Akt, increased upon hormone stimulation only in dwarf mice in both tissues. In contrast, no differences in the phosphorylation of mTOR, another substrate of Akt, were observed after GH stimulus, either in normal or dwarf mice in liver, while we were unable to determine mTOR in muscle. Protein content of GH-receptor and of the signaling mediators studied did not vary between normal and dwarf animals in the assessed

Obese Mice Fed a Diet Supplemented with Enzyme-Treated Wheat Bran Display Marked Shifts in the Liver Metabolome Concurrent with Altered Gut Bacteria

DEFF Research Database (Denmark)

Kieffer, Dorothy A.; Piccolo, Brian D.; Marco, Maria L.

2016-01-01

) associated with specific microbes may be involved. Objective: The objective of this study was to characterize ETWB-driven shifts in the cecal microbiome and to identify correlates between microbial changes and diet-related differences in liver metabolism in diet-induced obese mice that typically display......Background: Enzyme-treated wheat bran (ETWB) contains a fermentable dietary fiber previously shown to decrease liver triglycerides (TGs) and modify the gut microbiome in mice. It is not clear which mechanisms explain how ETWB feeding affects hepatic metabolism, but factors (i.e., xenometabolites...... steatosis. Methods: Five-week-old male C57BL/6J mice fed a 45%-lard based fat diet supplemented with ETWB (20% wt:wt) or rapidly digestible starch (control) (n = 15/group) for 10 wk were characterized by using a multi-omics approach. Multivariate statistical analysis was used to identify variables that were...

Time-place learning and memory persist in mice lacking functional Per1 and Per2 clock genes.

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Mulder, C; Van Der Zee, E A; Hut, R A; Gerkema, M P

2013-12-01

With time-place learning, animals link a stimulus with the location and the time of day. This ability may optimize resource localization and predator avoidance in daily changing environments. Time-place learning is a suitable task to study the interaction of the circadian system and memory. Previously, we showed that time-place learning in mice depends on the circadian system and Cry1 and/or Cry2 clock genes. We questioned whether time-place learning is Cry specific or also depends on other core molecular clock genes. Here, we show that Per1/Per2 double mutant mice, despite their arrhythmic phenotype, acquire time-place learning similar to wild-type mice. As well as an established role in circadian rhythms, Per genes have also been implicated in the formation and storage of memory. We found no deficiencies in short-term spatial working memory in Per mutant mice compared to wild-type mice. Moreover, both Per mutant and wild-type mice showed similar long-term memory for contextual features of a paradigm (a mild foot shock), measured in trained mice after a 2-month nontesting interval. In contrast, time-place associations were lost in both wild-type and mutant mice after these 2 months, suggesting a lack of maintained long-term memory storage for this type of information. Taken together, Cry-dependent time-place learning does not require Per genes, and Per mutant mice showed no PER-specific short-term or long-term memory deficiencies. These results limit the functional role of Per clock genes in the circadian regulation of time-place learning and memory.

Autophagy and UPR in alpha-crystallin mutant knock-in mouse models of hereditary cataracts.

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Andley, Usha P; Goldman, Joshua W

2016-01-01

Knock-in mice provide useful models of congenital and age-related cataracts caused by α-crystallin mutations. R49C αA-crystallin and R120G αB-crystallin mutations are linked with hereditary cataracts. Knock-in αA-R49C+/- heterozygotes develop cataracts by 1-2months, whereas homozygote mice have cataracts at birth. The R49C mutation drastically reduces lens protein water solubility and causes cell death in knock-in mouse lenses. Mutant crystallin cannot function as a chaperone, which leads to protein aggregation and lens opacity. Protein aggregation disrupts the lens fiber cell structure and normal development and causes cell death in epithelial and fiber cells. We determined what aspects of the wild-type phenotype are age-dependently altered in the mutant lens. Wild-type, heterozygote (αA-R49C+/-), and homozygote (αA-R49C+/+) mouse lenses were assessed pre- and postnatally for lens morphology (electron microscopy, immunohistochemistry), and autophagy or unfolded protein response markers (immunoblotting). Morphology was altered by embryonic day 17 in R49C+/+ lenses; R49C+/- lens morphology was unaffected at this stage. Active autophagy in the lens epithelium of mutant lenses was indicated by the presence of autophagosomes using electron microscopy. Protein p62 levels, which are degraded specifically by autophagy, increased in αA-R49C mutant versus wild-type lenses, suggesting autophagy inhibition in the mutant lenses. The unfolded protein response marker XBP-1 was upregulated in adult lenses of αB-R120G+/+ mice, suggesting its role in lens opacification. Mutated crystallins alter lens morphology, autophagy, and stress responses. Therapeutic modulation of autophagic pathways may improve protein degradation in cataractous lenses and reduce lens opacity. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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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. Copyright © 2014 Elsevier B.V. All rights reserved.

Cardiomyocyte specific deletion of Crif1 causes mitochondrial cardiomyopathy in mice.

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Juhee Shin

Full Text Available Mitochondria are key organelles dedicated to energy production. Crif1, which interacts with the large subunit of the mitochondrial ribosome, is indispensable for the mitochondrial translation and membrane insertion of respiratory subunits. To explore the physiological function of Crif1 in the heart, Crif1(f/f mice were crossed with Myh6-cre/Esr1 transgenic mice, which harbor cardiomyocyte-specific Cre activity in a tamoxifen-dependent manner. The tamoxifen injections were given at six weeks postnatal, and the mutant mice survived only five months due to hypertrophic heart failure. In the mutant cardiac muscles, mitochondrial mass dramatically increased, while the inner structure was altered with lack of cristae. Mutant cardiac muscles showed decreased rates of oxygen consumption and ATP production, suggesting that Crif1 plays a critical role in the maintenance of both mitochondrial structure and respiration in cardiac muscles.

Brucella abortusΔcydCΔcydD and ΔcydCΔpurD double-mutants are highly attenuated and confer long-term protective immunity against virulent Brucella abortus.

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Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Kim, Kiju; Hahn, Tae-Wook

2016-01-04

We constructed double deletion (ΔcydCΔcydD and ΔcydCΔpurD) mutants from virulent Brucella abortus biovar 1 field isolate (BA15) by deleting the genes encoding an ATP-binding cassette-type transporter (cydC and cydD genes) and a phosphoribosylamine-glycine ligase (purD). Both BA15ΔcydCΔcydD and BA15ΔcydCΔpurD double-mutants exhibited significant attenuation of virulence when assayed in murine macrophages or in BALB/c mice. Both double-mutants were readily cleared from spleens by 4 weeks post-inoculation even when inoculated at the dose of 10(8) CFU per mouse. Moreover, the inoculated mice showed no splenomegaly, which indicates that the mutants are highly attenuated. Importantly, the attenuation of in vitro and in vivo growth did not impair the ability of these mutants to confer long-term protective immunity in mice against challenge with B. abortus strain 2308. Vaccination of mice with either mutant induced humoral and cell-mediated immune responses, and provided significantly better protection than commercial B. abortus strain RB51 vaccine. These results suggest that highly attenuated BA15ΔcydCΔcydD and BA15ΔcydCΔpurD mutants can be used effectively as potential live vaccine candidates against bovine brucellosis. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Mice deficient of glutamatergic signaling from intrinsically photosensitive retinal ganglion cells exhibit abnormal circadian photoentrainment.

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Nicole Purrier

Full Text Available Several aspects of behavior and physiology, such as sleep and wakefulness, blood pressure, body temperature, and hormone secretion exhibit daily oscillations known as circadian rhythms. These circadian rhythms are orchestrated by an intrinsic biological clock in the suprachiasmatic nuclei (SCN of the hypothalamus which is adjusted to the daily environmental cycles of day and night by the process of photoentrainment. In mammals, the neuronal signal for photoentrainment arises from a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs that send a direct projection to the SCN. ipRGCs also mediate other non-image-forming (NIF visual responses such as negative masking of locomotor activity by light, and the pupillary light reflex (PLR via co-release of neurotransmitters glutamate and pituitary adenylate cyclase-activating peptide (PACAP from their synaptic terminals. The relative contribution of each neurotransmitter system for the circadian photoentrainment and other NIF visual responses is still unresolved. We investigated the role of glutamatergic neurotransmission for circadian photoentrainment and NIF behaviors by selective ablation of ipRGC glutamatergic synaptic transmission in mice. Mutant mice displayed delayed re-entrainment to a 6 h phase shift (advance or delay in the light cycle and incomplete photoentrainment in a symmetrical skeleton photoperiod regimen (1 h light pulses between 11 h dark periods. Circadian rhythmicity in constant darkness also was reduced in some mutant mice. Other NIF responses such as the PLR and negative masking responses to light were also partially attenuated. Overall, these results suggest that glutamate from ipRGCs drives circadian photoentrainment and negative masking responses to light.

Effect of abscisic acid on stomatal opening in isolated epidermal strips of abi mutants of Arabidopsis thaliana

NARCIS (Netherlands)

Roelfsema, MRG; Prins, HBA

Abscisic acid-insensitive mutants of Arabidopsis thaliana L. var. Landsberg erecta were selected for their decreased sensitivity to ABA during germination. Two of these mutants, abi-1 and abi-2, display a wilty phenotype as adult plants, indicating disturbed water relations. Experiments were

DNMT3AR882H mutant and Tet2 inactivation cooperate in the deregulation of DNA methylation control to induce lymphoid malignancies in mice

DEFF Research Database (Denmark)

Scourzic, L; Couronné, L; Pedersen, Marianne Terndrup

2016-01-01

malignancies with one mouse developing an AITL-like disease, two mice presenting acute myeloid leukemia (AML)-like and two others T-cell acute lymphoblastic leukemia (T-ALL)-like diseases within 6 months following transplantation. Serial transplantations of DNMT3A(R882H) Tet2(-/-) progenitors led...... to a differentiation bias toward the T-cell compartment, eventually leading to AITL-like disease in 9/12 serially transplanted recipients. Expression profiling suggested that DNMT3A(R882H) Tet2(-/-) T-ALLs resemble those of NOTCH1 mutant. Methylation analysis of DNMT3A(R882H) Tet2(-/-) T-ALLs showed a global increase...... in DNA methylation affecting tumor suppressor genes and local hypomethylation affecting genes involved in the Notch pathway. Our data confirm the transformation potential of DNMT3A(R882H) Tet2(-/-) progenitors and represent the first cooperative model in mice involving Tet2 inactivation driving lymphoid...

5-HT2C Receptor Desensitization Moderates Anxiety in 5-HTT Deficient Mice: From Behavioral to Cellular Evidence

Science.gov (United States)

Martin, Cédric BP; Martin, Vincent S.; Trigo, José M.; Chevarin, Caroline; Maldonado, Rafael; Fink, Latham H.; Cunningham, Kathryn A.; Hamon, Michel; Lanfumey, Laurence

2015-01-01

Background: Desensitization and blockade of 5-HT2C receptors (5-HT2CR) have long been thought to be central in the therapeutic action of antidepressant drugs. However, besides behavioral pharmacology studies, there is little in vivo data documenting antidepressant-induced 5-HT2CR desensitization in specific brain areas. Methods: Mice lacking the 5-HT reuptake carrier (5-HTT-/-) were used to model the consequences of chronic 5-HT reuptake inhibition with antidepressant drugs. The effect of this mutation on 5-HT2CR was evaluated at the behavioral (social interaction, novelty-suppressed feeding, and 5-HT2CR–induced hypolocomotion tests), the neurochemical, and the cellular (RT-qPCR, mRNA editing, and c-fos–induced expression) levels. Results: Although 5-HTT-/- mice had an anxiogenic profile in the novelty-suppressed feeding test, they displayed less 5-HT2CR–mediated anxiety in response to the agonist m-chlorophenylpiperazine in the social interaction test. In addition, 5-HT2CR–mediated inhibition of a stress-induced increase in 5-HT turnover, measured in various brain areas, was markedly reduced in 5-HTT-/- mutants. These indices of tolerance to 5-HT2CR stimulation were associated neither with altered levels of 5-HT2CR protein and mRNA nor with changes in pre-mRNA editing in the frontal cortex. However, basal c-fos mRNA production in cells expressing 5-HT2CR was higher in 5-HTT-/- mutants, suggesting an altered basal activity of these cells following sustained 5-HT reuptake carrier inactivation. Furthermore, the increased c-fos mRNA expression in 5-HT2CR–like immune-positive cortical cells observed in wild-type mice treated acutely with the 5-HT2CR agonist RO-60,0175 was absent in 5-HTT-/- mutants. Conclusions: Such blunted responsiveness of the 5-HT2CR system, observed at the cell signaling level, probably contributes to the moderation of the anxiety phenotype in 5-HTT-/- mice. PMID:25522398

Lack of tau proteins rescues neuronal cell death and decreases amyloidogenic processing of APP in APP/PS1 mice.

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Leroy, Karelle; Ando, Kunie; Laporte, Vincent; Dedecker, Robert; Suain, Valérie; Authelet, Michèle; Héraud, Céline; Pierrot, Nathalie; Yilmaz, Zehra; Octave, Jean-Noël; Brion, Jean-Pierre

2012-12-01

Lack of tau expression has been reported to protect against excitotoxicity and to prevent memory deficits in mice expressing mutant amyloid precursor protein (APP) identified in familial Alzheimer disease. In APP mice, mutant presenilin 1 (PS1) enhances generation of Aβ42 and inhibits cell survival pathways. It is unknown whether the deficient phenotype induced by concomitant expression of mutant PS1 is rescued by absence of tau. In this study, we have analyzed the effect of tau deletion in mice expressing mutant APP and PS1. Although APP/PS1/tau(+/+) mice had a reduced survival, developed spatial memory deficits at 6 months and motor impairments at 12 months, these deficits were rescued in APP/PS1/tau(-/-) mice. Neuronal loss and synaptic loss in APP/PS1/tau(+/+) mice were rescued in the APP/PS1/tau(-/-) mice. The amyloid plaque burden was decreased by roughly 50% in the cortex and the spinal cord of the APP/PS1/tau(-/-) mice. The levels of soluble and insoluble Aβ40 and Aβ42, and the Aβ42/Aβ40 ratio were reduced in APP/PS1/tau(-/-) mice. Levels of phosphorylated APP, of β-C-terminal fragments (CTFs), and of β-secretase 1 (BACE1) were also reduced, suggesting that β-secretase cleavage of APP was reduced in APP/PS1/tau(-/-) mice. Our results indicate that tau deletion had a protective effect against amyloid induced toxicity even in the presence of mutant PS1 and reduced the production of Aβ. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse)

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Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R.; Marzban, Hassan

2014-01-01

The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18–19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22–23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation. PMID:24722417

Comparison between medium-chain acyl-CoA dehydrogenase mutant proteins overexpressed in bacterial and mammalian cells

DEFF Research Database (Denmark)

Jensen, T G; Bross, P; Andresen, B S

1995-01-01

." Upon expression in E. coli, these mutant proteins produce activity levels in the range of the wild-type enzyme only if the chaperonins GroESL are co-overproduced. When overexpressed in COS cells, the pure folding mutants display enzyme activities comparable to the wild-type enzyme. The results suggest...

Forebrain-Specific Loss of BMPRII in Mice Reduces Anxiety and Increases Object Exploration.

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McBrayer, Zofeyah L; Dimova, Jiva; Pisansky, Marc T; Sun, Mu; Beppu, Hideyuki; Gewirtz, Jonathan C; O'Connor, Michael B

2015-01-01

To investigate the role of Bone Morphogenic Protein Receptor Type II (BMPRII) in learning, memory, and exploratory behavior in mice, a tissue-specific knockout of BMPRII in the post-natal hippocampus and forebrain was generated. We found that BMPRII mutant mice had normal spatial learning and memory in the Morris water maze, but showed significantly reduced swimming speeds with increased floating behavior. Further analysis using the Porsolt Swim Test to investigate behavioral despair did not reveal any differences in immobility between mutants and controls. In the Elevated Plus Maze, BMPRII mutants and Smad4 mutants showed reduced anxiety, while in exploratory tests, BMPRII mutants showed more interest in object exploration. These results suggest that loss of BMPRII in the mouse hippocampus and forebrain does not disrupt spatial learning and memory encoding, but instead impacts exploratory and anxiety-related behaviors.

Forebrain-Specific Loss of BMPRII in Mice Reduces Anxiety and Increases Object Exploration.

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Zofeyah L McBrayer

Full Text Available To investigate the role of Bone Morphogenic Protein Receptor Type II (BMPRII in learning, memory, and exploratory behavior in mice, a tissue-specific knockout of BMPRII in the post-natal hippocampus and forebrain was generated. We found that BMPRII mutant mice had normal spatial learning and memory in the Morris water maze, but showed significantly reduced swimming speeds with increased floating behavior. Further analysis using the Porsolt Swim Test to investigate behavioral despair did not reveal any differences in immobility between mutants and controls. In the Elevated Plus Maze, BMPRII mutants and Smad4 mutants showed reduced anxiety, while in exploratory tests, BMPRII mutants showed more interest in object exploration. These results suggest that loss of BMPRII in the mouse hippocampus and forebrain does not disrupt spatial learning and memory encoding, but instead impacts exploratory and anxiety-related behaviors.

A broad phenotypic screen identifies novel phenotypes driven by a single mutant allele in Huntington's disease CAG knock-in mice.

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Sabine M Hölter

Full Text Available Huntington's disease (HD is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the HTT gene encoding huntingtin. The disease has an insidious course, typically progressing over 10-15 years until death. Currently there is no effective disease-modifying therapy. To better understand the HD pathogenic process we have developed genetic HTT CAG knock-in mouse models that accurately recapitulate the HD mutation in man. Here, we describe results of a broad, standardized phenotypic screen in 10-46 week old heterozygous HdhQ111 knock-in mice, probing a wide range of physiological systems. The results of this screen revealed a number of behavioral abnormalities in HdhQ111/+ mice that include hypoactivity, decreased anxiety, motor learning and coordination deficits, and impaired olfactory discrimination. The screen also provided evidence supporting subtle cardiovascular, lung, and plasma metabolite alterations. Importantly, our results reveal that a single mutant HTT allele in the mouse is sufficient to elicit multiple phenotypic abnormalities, consistent with a dominant disease process in patients. These data provide a starting point for further investigation of several organ systems in HD, for the dissection of underlying pathogenic mechanisms and for the identification of reliable phenotypic endpoints for therapeutic testing.

Characterization of amyloid beta peptides from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein.

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Pype, Stefan; Moechars, Dieder; Dillen, Lieve; Mercken, Marc

2003-02-01

Alzheimer's disease (AD) is marked by the presence of neurofibrillary tangles and amyloid plaques in the brain of patients. To study plaque formation, we report on further quantitative and qualitative analysis of human and mouse amyloid beta peptides (Abeta) from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP). Using enzyme-linked immunosorbant assays (ELISAs) specific for either human or rodent Abeta, we found that the peptides from both species aggregated to form plaques. The ratios of deposited Abeta1-42/1-40 were in the order of 2-3 for human and 8-9 for mouse peptides, indicating preferential deposition of Abeta42. We also determined the identity and relative levels of other Abeta variants present in protein extracts from soluble and insoluble brain fractions. This was done by combined immunoprecipitation and mass spectrometry (IP/MS). The most prominent peptides truncated either at the carboxyl- or the amino-terminus were Abeta1-38 and Abeta11-42, respectively, and the latter was strongly enriched in the extracts of deposited peptides. Taken together, our data indicate that plaques of APP-London transgenic mice consist of aggregates of multiple human and mouse Abeta variants, and the human variants that we identified were previously detected in brain extracts of AD patients.

Cystogenesis in ARPKD results from increased apoptosis in collecting duct epithelial cells of Pkhd1 mutant kidneys

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Hu, Bo; He, Xiusheng; Li, Ao; Qiu, Qingchao; Li, Cunxi; Liang, Dan; Zhao, Ping; Ma, Jie; Coffey, Robert J.; Zhan, Qimin; Wu, Guanqing

2011-01-01

Mutations in the PKHD1 gene result in autosomal recessive polycystic kidney disease (ARPKD) in humans. To determine the molecular mechanism of the cystogenesis in ARPKD, we recently generated a mouse model for ARPKD that carries a targeted mutation in the mouse orthologue of human PKHD1. The homozygous mutant mice display hepatorenal cysts whose phenotypes are similar to those of human ARPKD patients. By littermates of this mouse, we developed two immortalized renal collecting duct cell lines with Pkhd1 and two without. Under nonpermissive culture conditions, the Pkhd1 -/- renal cells displayed aberrant cell-cell contacts and tubulomorphogenesis. The Pkhd1 -/- cells also showed significantly reduced cell proliferation and elevated apoptosis. To validate this finding in vivo, we examined proliferation and apoptosis in the kidneys of Pkhd1 -/- mice and their wildtype littermates. Using proliferation (PCNA and Histone-3) and apoptosis (TUNEL and caspase-3) markers, similar results were obtained in the Pkhd1 -/- kidney tissues as in the cells. To identify the molecular basis of these findings, we analyzed the effect of Pkhd1 loss on multiple putative signaling regulators. We demonstrated that the loss of Pkhd1 disrupts multiple major phosphorylations of focal adhesion kinase (FAK), and these disruptions either inhibit the Ras/C-Raf pathways to suppress MEK/ERK activity and ultimately reduce cell proliferation, or suppress PDK1/AKT to upregulate Bax/caspase-9/caspase-3 and promote apoptosis. Our findings indicate that apoptosis may be a major player in the cyst formation in ARPKD, which may lead to new therapeutic strategies for human ARPKD.

The Essential Role of Mbd5 in the Regulation of Somatic Growth and Glucose Homeostasis in Mice

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Du, Yarui; Liu, Bo; Guo, Fan; Xu, Guifang; Ding, Yuqiang; Liu, Yong; Sun, Xin; Xu, Guoliang

2012-01-01

Methyl-CpG binding domain protein 5 (MBD5) belongs to the MBD family proteins, which play central roles in transcriptional regulation and development. The significance of MBD5 function is highlighted by recent studies implicating it as a candidate gene involved in human 2q23.1 microdeletion syndrome. To investigate the physiological role of Mbd5, we generated knockout mice. The Mbd5-deficient mice showed growth retardation, wasting and pre-weaning lethality. The observed growth retardation was associated with the impairment of GH/IGF-1 axis in Mbd5-null pups. Conditional knockout of Mbd5 in the brain resulted in the similar phenotypes as whole body deletion, indicating that Mbd5 functions in the nervous system to regulate postnatal growth. Moreover, the mutant mice also displayed enhanced glucose tolerance and elevated insulin sensitivity as a result of increased insulin signaling, ultimately resulting in disturbed glucose homeostasis and hypoglycemia. These results indicate Mbd5 as an essential factor for mouse postnatal growth and maintenance of glucose homeostasis. PMID:23077600

Increased BRAF Heterodimerization Is the Common Pathogenic Mechanism for Noonan Syndrome-Associated RAF1 Mutants

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Wu, Xue; Yin, Jiani; Simpson, Jeremy; Kim, Kyoung-Han; Gu, Shengqing; Hong, Jenny H.; Bayliss, Peter; Backx, Peter H.

2012-01-01

Noonan syndrome (NS) is a relatively common autosomal dominant disorder characterized by congenital heart defects, short stature, and facial dysmorphia. NS is caused by germ line mutations in several components of the RAS–RAF–MEK–extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway, including both kinase-activating and kinase-impaired alleles of RAF1 (∼3 to 5%), which encodes a serine-threonine kinase for MEK1/2. To investigate how kinase-impaired RAF1 mutants cause NS, we generated knock-in mice expressing Raf1D486N. Raf1D486N/+ (here D486N/+) female mice exhibited a mild growth defect. Male and female D486N/D486N mice developed concentric cardiac hypertrophy and incompletely penetrant, but severe, growth defects. Remarkably, Mek/Erk activation was enhanced in Raf1D486N-expressing cells compared with controls. RAF1D486N, as well as other kinase-impaired RAF1 mutants, showed increased heterodimerization with BRAF, which was necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also required heterodimerization to enhance MEK/ERK activation. Our results suggest that an increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations. PMID:22826437

Analysis of Msx1; Msx2 double mutants reveals multiple roles for Msx genes in limb development.

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Lallemand, Yvan; Nicola, Marie-Anne; Ramos, Casto; Bach, Antoine; Cloment, Cécile Saint; Robert, Benoît

2005-07-01

The homeobox-containing genes Msx1 and Msx2 are highly expressed in the limb field from the earliest stages of limb formation and, subsequently, in both the apical ectodermal ridge and underlying mesenchyme. However, mice homozygous for a null mutation in either Msx1 or Msx2 do not display abnormalities in limb development. By contrast, Msx1; Msx2 double mutants exhibit a severe limb phenotype. Our analysis indicates that these genes play a role in crucial processes during limb morphogenesis along all three axes. Double mutant limbs are shorter and lack anterior skeletal elements (radius/tibia, thumb/hallux). Gene expression analysis confirms that there is no formation of regions with anterior identity. This correlates with the absence of dorsoventral boundary specification in the anterior ectoderm, which precludes apical ectodermal ridge formation anteriorly. As a result, anterior mesenchyme is not maintained, leading to oligodactyly. Paradoxically, polydactyly is also frequent and appears to be associated with extended Fgf activity in the apical ectodermal ridge, which is maintained up to 14.5 dpc. This results in a major outgrowth of the mesenchyme anteriorly, which nevertheless maintains a posterior identity, and leads to formation of extra digits. These defects are interpreted in the context of an impairment of Bmp signalling.

Overactivation of Hedgehog Signaling Alters Development of the Ovarian Vasculature in Mice1

Science.gov (United States)

Ren, Yi; Cowan, Robert G.; Migone, Fernando F.; Quirk, Susan M.

2012-01-01

ABSTRACT The hedgehog (HH) signaling pathway is critical for ovarian function in Drosophila, but its role in the mammalian ovary has not been defined. Previously, expression of a dominant active allele of the HH signal transducer protein smoothened (SMO) in Amhr2cre/+SmoM2 mice caused anovulation in association with a lack of smooth muscle in the theca of developing follicles. The current study examined events during the first 2 wk of life in Amhr2cre/+SmoM2 mice to gain insight into the cause of anovulation. Expression of transcriptional targets of HH signaling, Gli1, Ptch1, and Hhip, which are used as measures of pathway activity, were elevated during the first several days of life in Amhr2cre/+SmoM2 mice compared to controls but were similar to controls in older mice. Microarray analysis showed that genes with increased expression in 2-day-old mutants compared to controls were enriched for the processes of vascular and tube development and steroidogenesis. The density of platelet endothelial cell adhesion molecule (PECAM)-labeled endothelial tubes was increased in the cortex of newborn ovaries of mutant mice. Costaining of preovulatory follicles for PECAM and smooth muscle actin showed that muscle-type vascular support cells are deficient in theca of mutant mice. Expression of genes for steroidogenic enzymes that are normally expressed in the fetal adrenal gland were elevated in newborn ovaries of mutant mice. In summary, overactivation of HH signaling during early life alters gene expression and vascular development and this is associated with the lifelong development of anovulatory follicles in which the thecal vasculature fails to mature appropriately. PMID:22402963

A chilling sensitive mutant of Arabidopsis with altered steryl-ester metabolism

International Nuclear Information System (INIS)

Hugly, S.; McCourt, P.; Somerville, C.; Browse, J.; Patterson, G.W.

1990-01-01

A chilling-sensitive mutant of Arabidopsis thaliana was isolated and subjected to genetic, physiological, and biochemical analysis. The chilling-sensitive nature of the mutant line is due to a single recessive nuclear mutation at a locus designated chs1. In contrast to wild-type plants, which are not adversely affected by low temperatures, the chs1 mutant is killed by several days of exposure to temperatures below 18 degree C. Following exposure to chilling temperatures, the mutant displays two common symptoms of chilling injury - leaf chlorosis and electrolyte leakage. In these respects, the physiological response of the mutant to low temperatures mimics the response observed in some naturally occurring chilling sensitive species. The biochemical basis of chilling sensitivity was explored by examining the pattern of incorporation of 14 CO 2 into soluble metabolites and lipids in wild-type and mutant plants. The only difference observed between the mutant and wild type was that following low temperature treatment, the mutant accumulated 10-fold more radioactivity in a specific class of neutral lipids which were identified by a variety of criteria to be steryl-esters. The accumulation of radioactivity in the steryl-ester fraction occurs 24 hours before there is any visible evidence of chilling injury

Human cathepsin L rescues the neurodegeneration and lethality incathepsin B/L double deficient mice

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Sevenich, Lisa; Pennacchio, Len A.; Peters, Christoph; Reinheckel, Thomas

2006-01-09

Cathepsin B (CTSB) and cathepsin L (CTSL) are two widelyexpressed cysteine proteases thought to predominantly reside withinlysosomes. Functional analysis of CTSL in humans is complicated by theexistence of two CTSL-like homologues (CTSL and CTSL2), in contrast tomice which contain only one CTSL enzyme. Thus transgenic expression ofhuman CTSL in CTSL deficient mice provides an opportunity to study the invivo functions of this human protease without interference by its highlyrelated homologue. While mice with single gene deficiencies for murineCTSB or CTSL survive without apparent neuromuscular impairment, murineCTSB/CTSL double deficient mice display degeneration of cerebellarPurkinje cells and neurons of the cerebral cortex, resulting in severehypotrophy, motility defects, and lethality during their third to fourthweek of life. Here we show that expression of human CTSL through agenomic transgene results in widespread expression of human CTSL in themouse which is capable of rescuing the lethality found in CTSB/CTSLdouble-deficient animals. Human CTSL is expressed in the brain of thesecompound mutants predominantly in neurons of the cerebral cortex and inPurkinje cells of the cerebellum, where it appears to prevent neuronalcell death.

Ethanol-related behaviors in mice lacking the sigma-1 receptor.

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Valenza, Marta; DiLeo, Alyssa; Steardo, Luca; Cottone, Pietro; Sabino, Valentina

2016-01-15

The Sigma-1 receptor (Sig-1R) is a chaperone protein that has been implicated in drug abuse and addiction. Multiple studies have characterized the role the Sig-1R plays in psychostimulant addiction; however, fewer studies have specifically investigated its role in alcohol addiction. We have previously shown that antagonism of the Sig-1R reduces excessive drinking and motivation to drink, whereas agonism induces binge-like drinking in rodents. The objectives of these studies were to investigate the impact of Sig-1R gene deletion in C57Bl/6J mice on ethanol drinking and other ethanol-related behaviors. We used an extensive panel of behavioral tests to examine ethanol actions in male, adult mice lacking Oprs1, the gene encoding the Sig-1R. To compare ethanol drinking behavior, Sig-1 knockout (KO) and wild type (WT) mice were subject to a two-bottle choice, continuous access paradigm with different concentrations of ethanol (3-20% v/v) vs. water. Consumption of sweet and bitter solutions was also assessed in Sig-1R KO and WT mice. Finally, motor stimulant sensitivity, taste aversion and ataxic effects of ethanol were assessed. Sig-1R KO mice displayed higher ethanol intake compared to WT mice; the two genotypes did not differ in their sweet or bitter taste perception. Sig-1R KO mice showed lower sensitivity to ethanol stimulant effects, but greater sensitivity to its taste aversive effects. Ethanol-induced sedation was instead unaltered in the mutants. Our results prove that the deletion of the Sig-1R increases ethanol consumption, likely by decreasing its rewarding effects, and therefore indicating that the Sig-1R is involved in modulation of the reinforcing effects of alcohol. Copyright © 2015 Elsevier B.V. All rights reserved.

Graft-versus-host reaction and immune function. III. Functional pre-T cells in the bone marrow of graft-versus-host-reactive mice displaying T cell immunodeficiency

International Nuclear Information System (INIS)

Seddik, M.; Seemayer, T.A.; Lapp, W.S.

1986-01-01

Studies were performed to determine whether pre-T cells develop normally in the bone marrow of mice displaying thymic dysplasia and T cell immunodeficiency as a consequence of a graft-versus-host (GVH) reaction. GVH reactions were induced in CBAxAF1 mice by the injection of A strain lymphoid cells. To test for the presence of pre-T cells in GVH-reactive mice, bone marrow from GVH-reactive mice (GVHBM) was injected into irradiated syngeneic F1 mice and 30-40 days later thymic morphology and function were studied. Morphology studies showed nearly normal thymic architectural restoration; moreover, such glands contained normal numbers of Thy-1-positive cells. Functional pre-T cells were evaluated by transferring thymocytes from the irradiated GVHBM-reconstituted mice into T-cell-deprived mice. These thymocytes reconstituted allograft reactivity, T helper cell function and Con A and PHA mitogen responses of T-cell-deprived mice. These results suggest that the pre-T cell population in the bone marrow is not affected by the GVH reaction. Therefore, the T cell immunodeficiency associated with the GVH reaction is not due to a deficiency of pre-T cells in the bone marrow but is more likely associated with GVH-induced thymic dysplasia

The secreted L-arabinose isomerase displays anti-hyperglycemic effects in mice.

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Rhimi, Moez; Bermudez-Humaran, Luis G; Huang, Yuan; Boudebbouze, Samira; Gaci, Nadia; Garnier, Alexandrine; Gratadoux, Jean-Jacques; Mkaouar, Héla; Langella, Philippe; Maguin, Emmanuelle

2015-12-21

The L-arabinose isomerase is an intracellular enzyme which converts L-arabinose into L-ribulose in living systems and D-galactose into D-tagatose in industrial processes and at industrial scales. D-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The D-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive L-arabinose isomerase should be thermoactive and acidotolerant with high catalytic efficiency. While many reports focused on the set out of a low cost process for the industrial production of D-tagatose, these procedures remain costly. When compared to intracellular enzymes, the production of extracellular ones constitutes an interesting strategy to increase the suitability of the biocatalysts. The L-arabinose isomerase (L-AI) from Lactobacillus sakei was expressed in Lactococcus lactis in fusion with the signal peptide of usp45 (SP(Usp45)). The L-AI protein and activity were detected only in the supernatant of the induced cultures of the recombinant L. lactis demonstrating the secretion in the medium of the intracellular L. sakei L-AI in an active form. Moreover, we showed an improvement in the enzyme secretion using either (1) L. lactis strains deficient for their two major proteases, ClpP and HtrA, or (2) an enhancer of protein secretion in L. lactis fused to the recombinant L-AI with the SP(Usp45). Th L-AI enzyme secreted by the recombinant L. lactis strains or produced intracellularly in E. coli, showed the same functional properties than the native enzyme. Furthermore, when mice are fed with the L. lactis strain secreting the L-AI and galactose, tagatose was produced in vivo and reduced the glycemia index. We report for the first time the secretion of the intracellular L-arabinose isomerase in the supernatant of food grade L. lactis cultures with hardly display other secreted proteins. The secreted L-AI originated from the food

Increased ethanol consumption despite taste aversion in mice with a human tryptophan hydroxylase 2 loss of function mutation.

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Lemay, Francis; Doré, François Y; Beaulieu, Jean-Martin

2015-11-16

Polymorphisms in the gene encoding the brain serotonin synthesis enzyme Tph2 have been identified in mental illnesses, with co-morbidity of substance use disorder. However, little is known about the impact of Tph2 gene variants on addiction. Mice expressing a human Tph2 loss of function variant were used to investigate consequences of aversive conditions on ethanol intake. Mice were familiarized either with ethanol or a solution containing both ethanol and the bittering agent quinine. Effect of familiarization to ethanol or an ethanol-quinine solution was then evaluated using a two-bottles preference test in Tph2-KI and control littermates. Mice from both genotypes displayed similar levels of ethanol consumption and quinine avoidance when habituated to ethanol alone. In contrast, addition of quinine to ethanol during the familiarization period resulted in a reduction of avoidance for the quinine-ethanol solution only in mutant mice. These results indicate that loss of function mutation in Tph2 results in greater motivation for ethanol consumption under aversive conditions and may confer enhanced sensitivity to alcohol use disorder. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Tauopathic changes in the striatum of A53T α-synuclein mutant mouse model of Parkinson's disease.

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Jonathan Wills

2011-03-01

Full Text Available Tauopathic pathways lead to degenerative changes in Alzheimer's disease and there is evidence that they are also involved in the neurodegenerative pathology of Parkinson's disease [PD]. We have examined tauopathic changes in striatum of the α-synuclein (α-Syn A53T mutant mouse. Elevated levels of α-Syn were observed in striatum of the adult A53T α-Syn mice. This was accompanied by increases in hyperphosphorylated Tau [p-Tau], phosphorylated at Ser202, Ser262 and Ser396/404, which are the same toxic sites also seen in Alzheimer's disease. There was an increase in active p-GSK-3β, hyperphosphorylated at Tyr216, a major and primary kinase known to phosphorylate Tau at multiple sites. The sites of hyperphosphorylation of Tau in the A53T mutant mice were similar to those seen in post-mortem striata from PD patients, attesting to their pathophysiological relevance. Increases in p-Tau were not due to alterations on protein phosphatases in either A53T mice or in human PD, suggesting lack of involvement of these proteins in tauopathy. Extraction of striata with Triton X-100 showed large increases in oligomeric forms of α-Syn suggesting that α-Syn had formed aggregates the mutant mice. In addition, increased levels of p-GSK-3β and pSer396/404 were also found associated with aggregated α-Syn. Differential solubilization to measure protein binding to cytoskeletal proteins demonstrated that p-Tau in the A53T mutant mouse were unbound to cytoskeletal proteins, consistent with dissociation of p-Tau from the microtubules upon hyperphosphorylation. Interestingly, α-Syn remained tightly bound to the cytoskeleton, while p-GSK-3β was seen in the cytoskeleton-free fractions. Immunohistochemical studies showed that α-Syn, pSer396/404 Tau and p-GSK-3β co-localized with one another and was aggregated and accumulated into large inclusion bodies, leading to cell death of Substantia nigral neurons. Together, these data demonstrate an elevated state of

Primary Cilia in the Murine Cerebellum and in Mutant Models of Medulloblastoma.

Science.gov (United States)

Di Pietro, Chiara; Marazziti, Daniela; La Sala, Gina; Abbaszadeh, Zeinab; Golini, Elisabetta; Matteoni, Rafaele; Tocchini-Valentini, Glauco P

2017-01-01

Cellular primary cilia crucially sense and transduce extracellular physicochemical stimuli. Cilium-mediated developmental signaling is tissue and cell type specific. Primary cilia are required for cerebellar differentiation and sonic hedgehog (Shh)-dependent proliferation of neuronal granule precursors. The mammalian G-protein-coupled receptor 37-like 1 is specifically expressed in cerebellar Bergmann glia astrocytes and participates in regulating postnatal cerebellar granule neuron proliferation/differentiation and Bergmann glia and Purkinje neuron maturation. The mouse receptor protein interacts with the patched 1 component of the cilium-associated Shh receptor complex. Mice heterozygous for patched homolog 1 mutations, like heterozygous patched 1 humans, have a higher incidence of Shh subgroup medulloblastoma (MB) and other tumors. Cerebellar cells bearing primary cilia were identified during postnatal development and in adulthood in two mouse strains with altered Shh signaling: a G-protein-coupled receptor 37-like 1 null mutant and an MB-susceptible, heterozygous patched homolog 1 mutant. In addition to granule and Purkinje neurons, primary cilia were also expressed by Bergmann glia astrocytes in both wild-type and mutant animals, from birth to adulthood. Variations in ciliary number and length were related to the different levels of neuronal and glial cell proliferation and maturation, during postnatal cerebellar development. Primary cilia were also detected in pre-neoplastic MB lesions in heterozygous patched homolog 1 mutant mice and they could represent specific markers for the development and analysis of novel cerebellar oncogenic models.

Aged PROP1 deficient dwarf mice maintain ACTH production.

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Igor O Nasonkin

Full Text Available Humans with PROP1 mutations have multiple pituitary hormone deficiencies (MPHD that typically advance from growth insufficiency diagnosed in infancy to include more severe growth hormone (GH deficiency and progressive reduction in other anterior pituitary hormones, eventually including adrenocorticotropic hormone (ACTH deficiency and hypocortisolism. Congenital deficiencies of GH, prolactin, and thyroid stimulating hormone have been reported in the Prop1(null (Prop1(-/- and the Ames dwarf (Prop1(df/df mouse models, but corticotroph and pituitary adrenal axis function have not been thoroughly investigated. Here we report that the C57BL6 background sensitizes mutants to a wasting phenotype that causes approximately one third to die precipitously between weaning and adulthood, while remaining homozygotes live with no signs of illness. The wasting phenotype is associated with severe hypoglycemia. Circulating ACTH and corticosterone levels are elevated in juvenile and aged Prop1 mutants, indicating activation of the pituitary-adrenal axis. Despite this, young adult Prop1 deficient mice are capable of responding to restraint stress with further elevation of ACTH and corticosterone. Low blood glucose, an expected side effect of GH deficiency, is likely responsible for the elevated corticosterone level. These studies suggest that the mouse model differs from the human patients who display progressive hormone loss and hypocortisolism.

Impaired genome maintenance suppresses the growth hormone--insulin-like growth factor 1 axis in mice with Cockayne syndrome.

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Ingrid van der Pluijm

2007-01-01

Full Text Available Cockayne syndrome (CS is a photosensitive, DNA repair disorder associated with progeria that is caused by a defect in the transcription-coupled repair subpathway of nucleotide excision repair (NER. Here, complete inactivation of NER in Csb(m/m/Xpa(-/- mutants causes a phenotype that reliably mimics the human progeroid CS syndrome. Newborn Csb(m/m/Xpa(-/- mice display attenuated growth, progressive neurological dysfunction, retinal degeneration, cachexia, kyphosis, and die before weaning. Mouse liver transcriptome analysis and several physiological endpoints revealed systemic suppression of the growth hormone/insulin-like growth factor 1 (GH/IGF1 somatotroph axis and oxidative metabolism, increased antioxidant responses, and hypoglycemia together with hepatic glycogen and fat accumulation. Broad genome-wide parallels between Csb(m/m/Xpa(-/- and naturally aged mouse liver transcriptomes suggested that these changes are intrinsic to natural ageing and the DNA repair-deficient mice. Importantly, wild-type mice exposed to a low dose of chronic genotoxic stress recapitulated this response, thereby pointing to a novel link between genome instability and the age-related decline of the somatotroph axis.

Differential requirement for utrophin in the induced pluripotent stem cell correction of muscle versus fat in muscular dystrophy mice.

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Amanda J Beck

Full Text Available Duchenne muscular dystrophy (DMD is an incurable degenerative muscle disorder. We injected WT mouse induced pluripotent stem cells (iPSCs into mdx and mdx∶utrophin mutant blastocysts, which are predisposed to develop DMD with an increasing degree of severity (mdx <<< mdx∶utrophin. In mdx chimeras, iPSC-dystrophin was supplied to the muscle sarcolemma to effect corrections at morphological and functional levels. Dystrobrevin was observed in dystrophin-positive and, at a lesser extent, utrophin-positive areas. In the mdx∶utrophin mutant chimeras, although iPSC-dystrophin was also supplied to the muscle sarcolemma, mice still displayed poor skeletal muscle histopathology, and negligible levels of dystrobrevin in dystrophin- and utrophin-negative areas. Not only dystrophin-expressing tissues are affected by iPSCs. Mdx and mdx∶utrophin mice have reduced fat/body weight ratio, but iPSC injection normalized this parameter in both mdx and mdx∶utrophin chimeras, despite the fact that utrophin was compromised in the mdx∶utrophin chimeric fat. The results suggest that the presence of utrophin is required for the iPSC-corrections in skeletal muscle. Furthermore, the results highlight a potential (utrophin-independent non-cell autonomous role for iPSC-dystrophin in the corrections of non-muscle tissue like fat, which is intimately related to the muscle.

Applications of Protein Thermodynamic Database for Understanding Protein Mutant Stability and Designing Stable Mutants.

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Gromiha, M Michael; Anoosha, P; Huang, Liang-Tsung

2016-01-01

Protein stability is the free energy difference between unfolded and folded states of a protein, which lies in the range of 5-25 kcal/mol. Experimentally, protein stability is measured with circular dichroism, differential scanning calorimetry, and fluorescence spectroscopy using thermal and denaturant denaturation methods. These experimental data have been accumulated in the form of a database, ProTherm, thermodynamic database for proteins and mutants. It also contains sequence and structure information of a protein, experimental methods and conditions, and literature information. Different features such as search, display, and sorting options and visualization tools have been incorporated in the database. ProTherm is a valuable resource for understanding/predicting the stability of proteins and it can be accessed at http://www.abren.net/protherm/ . ProTherm has been effectively used to examine the relationship among thermodynamics, structure, and function of proteins. We describe the recent progress on the development of methods for understanding/predicting protein stability, such as (1) general trends on mutational effects on stability, (2) relationship between the stability of protein mutants and amino acid properties, (3) applications of protein three-dimensional structures for predicting their stability upon point mutations, (4) prediction of protein stability upon single mutations from amino acid sequence, and (5) prediction methods for addressing double mutants. A list of online resources for predicting has also been provided.

The ovary is an alternative site of origin for high-grade serous ovarian cancer in mice.

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Kim, Jaeyeon; Coffey, Donna M; Ma, Lang; Matzuk, Martin M

2015-06-01

Although named "ovarian cancer," it has been unclear whether the cancer actually arises from the ovary, especially for high-grade serous carcinoma (HGSC), also known as high-grade serous ovarian cancer, the most common and deadliest ovarian cancer. In addition, the tumor suppressor p53 is the most frequently mutated gene in HGSC. However, whether mutated p53 can cause HGSC remains unknown. In this study, we bred a p53 mutation, p53(R172H), into conditional Dicer-Pten double-knockout (DKO) mice, a mouse model duplicating human HGSC, to generate triple-mutant (TKO) mice. Like DKO mice, these TKO mice develop metastatic HGSCs originating from the fallopian tube. Unlike DKO mice, however, even after fallopian tubes are removed in TKO mice, ovaries alone can develop metastatic HGSCs, indicating that a p53 mutation can drive HGSC arising from the ovary. To confirm this, we generated p53(R172H)-Pten double-mutant mice, one of the genetic control lines for TKO mice. As anticipated, these double-mutant mice also develop metastatic HGSCs from the ovary, verifying the HGSC-forming ability of ovaries with a p53 mutation. Our study therefore shows that ovaries harboring a p53 mutation, as well as fallopian tubes, can be a distinct tissue source of high-grade serous ovarian cancer in mice.

Dopamine D3 receptor knockout mice exhibit abnormal nociception in a sex-different manner.

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Liu, Peng; Xing, Bo; Chu, Zheng; Liu, Fei; Lei, Gang; Zhu, Li; Gao, Ya; Chen, Teng; Dang, Yong-Hui

2017-07-01

Pain is a complex and subjective experience. Previous studies have shown that mice lacking the dopamine D3 receptor (D3RKO) exhibit hypoalgesia, indicating a role of the D3 receptor in modulation of nociception. Given that there are sex differences in pain perception, there may be differences in responses to nociceptive stimuli between male and female D3RKO mice. In the current study, we examined the role of the D3 receptor in modulating nociception in male and female D3RKO mice. Acute thermal pain was modeled by hot-plate test. This test was performed at different temperatures including 52°C, 55°C, and 58°C. The von Frey hair test was applied to evaluate mechanical pain. And persistent pain produced by peripheral tissue injury and inflammation was modeled by formalin test. In the hot-plate test, compared with wild-type (WT) mice, D3RKO mice generally exhibited longer latencies at each of the three temperatures. Specially, male D3RKO mice showed hypoalgesia compared with male WT mice when the temperature was 55°C, while for the female mice, there was a statistical difference between genotypes when the test condition was 52°C. In the von Frey hair test, both male and female D3RKO mice exhibited hypoalgesia. In the formalin test, the male D3RKO mice displayed a similar nociceptive behavior as their sex-matched WT littermates, whereas significantly depressed late-phase formalin-induced nociceptive behaviors were observed in the female mutants. These findings indicated that the D3 receptor affects nociceptive behaviors in a sex-specific manner and that its absence induces more analgesic behavior in the female knockout mice. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Cox4i2, Ifit2, and Prdm11 Mutant Mice

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Horsch, Marion; Aguilar-Pimentel, Juan Antonio; Bönisch, Clemens

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 imm...

A spontaneous and novel Pax3 mutant mouse that models Waardenburg syndrome and neural tube defects.

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Ohnishi, Tetsuo; Miura, Ikuo; Ohba, Hisako; Shimamoto, Chie; Iwayama, Yoshimi; Wakana, Shigeharu; Yoshikawa, Takeo

2017-04-05

Genes responsible for reduced pigmentation phenotypes in rodents are associated with human developmental defects, such as Waardenburg syndrome, where patients display congenital deafness along with various abnormalities mostly related to neural crest development deficiency. In this study, we identified a spontaneous mutant mouse line Rwa, which displays variable white spots on mouse bellies and white digits and tail, on a C57BL/6N genetic background. Curly tail and spina bifida were also observed, although at a lower penetrance. These phenotypes were dominantly inherited by offspring. We searched for the genetic mechanism of the observed phenotypes. We harnessed a rapid mouse gene mapping system newly developed in our laboratories to identify a responsible gene. We detected a region within chromosome 1 as a probable locus for the causal mutation. Dense mapping using interval markers narrowed the locus down to a 670-kbp region, containing four genes including Pax3, a gene known to be implicated in the types I and III Waardenburg syndrome. Extensive mutation screening of Pax3 detected an 841-bp deletion, spanning the promoter region and intron 1 of the gene. The defective allele of Pax3, named Pax3 Rwa , lacked the first coding exon and co-segregated perfectly with the phenotypes, confirming its causal nature. The genetic background of Rwa mice is almost identical to that of inbred C57BL/6N. These results highlight Pax3 Rwa mice as a beneficial tool for analyzing biological processes involving Pax3, in particular the development and migration of neural crest cells and melanocytes. Copyright © 2017 Elsevier B.V. All rights reserved.

Sex-dependent Differences in Intestinal Tumorigenesis Induced in Apc1638N/+ Mice by Exposure to {gamma} Rays

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Trani, Daniela [Department of Biochemistry and Molecular and Cellular Biology, Georgetown University, Washington, District of Columbia (United States); Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia (United States); Maastricht Radiation Oncology (MaastRO) Lab, GROW-School for Oncology and Developmental Biology, University of Maastricht (Netherlands); Moon, Bo-Hyun [Department of Biochemistry and Molecular and Cellular Biology, Georgetown University, Washington, District of Columbia (United States); Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia (United States); Kallakury, Bhaskar; Hartmann, Dan P. [Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia (United States); Datta, Kamal [Department of Biochemistry and Molecular and Cellular Biology, Georgetown University, Washington, District of Columbia (United States); Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia (United States); Fornace, Albert J., E-mail: af294@georgetown.edu [Department of Biochemistry and Molecular and Cellular Biology, Georgetown University, Washington, District of Columbia (United States); Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia (United States); Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah (Saudi Arabia)

2013-01-01

Purpose: The purpose of the present study was to assess the effect of 1 and 5 Gy radiation doses and to investigate the interplay of gender and radiation with regard to intestinal tumorigenesis in an adenomatous polyposis coli (APC) mutant mouse model. Methods and Materials: Apc1638N/+ female and male mice were exposed whole body to either 1 Gy or 5 Gy of {gamma} rays and euthanized when most of the treated mice became moribund. Small and large intestines were processed to determine tumor burden, distribution, and grade. Expression of proliferation marker Ki-67 and estrogen receptor (ER)-{alpha} were also assessed by immunohistochemistry. Results: We observed that, with both 1 Gy and 5 Gy of {gamma} rays, females displayed reduced susceptibility to radiation-induced intestinal tumorigenesis compared with males. As for radiation effect on small intestinal tumor progression, although no substantial differences were found in the relative frequency and degree of dysplasia of adenomas in irradiated animals compared with controls, invasive carcinomas were found in 1-Gy- and 5-Gy-irradiated animals. Radiation exposure was also shown to induce an increase in protein levels of proliferation marker Ki-67 and sex-hormone receptor ER-{alpha} in both non tumor mucosa and intestinal tumors from irradiated male mice. Conclusions: We observed important sex-dependent differences in susceptibility to radiation-induced intestinal tumorigenesis in Apc1638N/+ mutants. Furthermore, our data provide evidence that exposure to radiation doses as low as 1 Gy can induce a significant increase in intestinal tumor multiplicity as well as enhance tumor progression in vivo.

Weaver mutant mouse cerebellar granule cells respond normally to chronic depolarization

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Bjerregaard, Annette; Mogensen, Helle Smidt; Hack, N

1997-01-01

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 prote...

Cdx and Hox genes differentially regulate posterior axial growth in mammalian embryos

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Young, Teddy; Rowland, Jennifer Elizabeth; van de Ven, Cesca; Bialecka, Monika; Novoa, Ana; Carapuco, Marta; van Nes, Johan; de Graaff, Wim; Duluc, Isabelle; Freund, Jean-Noël; Beck, Felix; Mallo, Moises; Deschamps, Jacqueline

2009-01-01

Hox and Cdx transcription factors regulate embryonic positional identities. Cdx mutant mice display posterior body truncations of the axial skeleton, neuraxis, and caudal urorectal structures. We show that trunk Hox genes stimulate axial extension, as they can largely rescue these Cdx mutant

Immunobiology of congenitally athymic-asplenic mice

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Gershwin, M.E.; Ahmed, A.; Ikeda, R.M.; Shifrine, M.; Wilson, F.

1978-01-01

A study has been made of congenitally athymic-asplenic mice obtained by the mating of nude by hereditarily asplenic (Dh/+) mice. The mice survived for up to 9 months, under specific pathogen-free conditions, with no evidence for increased risk of spontaneous neoplasia. Although lymphocyte surface markers and sera immunoglobulin levels of athymic-asplenic mice were similar to those of their nude and asplenic littermates, there were a number of major immunologic differences. The athymic-asplenic mice appeared more immunologically compromised than nude mice. There was an elevated rate of growth and a lower inoculated cell threshold needed for successful transplantation of a human malignant melanoma. There was no evidence for auto-antibody production in mice up to 9 months of age. Congenitally athymic-asplenic mice can be used for a variety of studies in which other immunologically deprived mouse mutants are desired. (author)

Expression of truncated PITX3 in the developing lens leads to microphthalmia and aphakia in mice.

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Kenta Wada

Full Text Available Microphthalmia is a severe ocular disorder, and this condition is typically caused by mutations in transcription factors that are involved in eye development. Mice carrying mutations in these transcription factors would be useful tools for defining the mechanisms underlying developmental eye disorders. We discovered a new spontaneous recessive microphthalmos mouse mutant in the Japanese wild-derived inbred strain KOR1/Stm. The homozygous mutant mice were histologically characterized as microphthalmic by the absence of crystallin in the lens, a condition referred to as aphakia. By positional cloning, we identified the nonsense mutation c.444C>A outside the genomic region that encodes the homeodomain of the paired-like homeodomain transcription factor 3 gene (Pitx3 as the mutation responsible for the microphthalmia and aphakia. We examined Pitx3 mRNA expression of mutant mice during embryonic stages using RT-PCR and found that the expression levels are higher than in wild-type mice. Pitx3 over-expression in the lens during developmental stages was also confirmed at the protein level in the microphthalmos mutants via immunohistochemical analyses. Although lens fiber differentiation was not observed in the mutants, strong PITX3 protein signals were observed in the lens vesicles of the mutant lens. Thus, we speculated that abnormal PITX3, which lacks the C-terminus (including the OAR domain as a result of the nonsense mutation, is expressed in mutant lenses. We showed that the expression of the downstream genes Foxe3, Prox1, and Mip was altered because of the Pitx3 mutation, with large reductions in the lens vesicles in the mutants. Similar profiles were observed by immunohistochemical analysis of these proteins. The expression profiles of crystallins were also altered in the mutants. Therefore, we speculated that the microphthalmos/aphakia in this mutant is caused by the expression of truncated PITX3, resulting in the abnormal expression of

Simultaneous analysis of multiple Mycobacterium tuberculosis knockdown mutants in vitro and in vivo.

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Antje Blumenthal

2010-12-01

Full Text Available Mycobacterium tuberculosis (Mtb represents one of the most persistent bacterial threats to human health and new drugs are needed to limit its impact. Conditional knockdown mutants can help validate new drug targets, but the analysis of individual mutants is laborious and time consuming. Here, we describe quantitative DNA tags (qTags and their use to simultaneously analyze conditional Mtb knockdown mutants that allowed silencing the glyoxylate and methylcitrate cycles (via depletion of isocitrate lyase, ICL, the serine protease Rv3671c, and the core subunits of the mycobacterial proteasome, PrcB and PrcA. The impact of gene silencing in multi-strain cultures was determined by measuring the relative abundance of mutant-specific qTags with real-time PCR. This achieved accurate quantification over a broad range of qTag abundances and depletion of ICL, Rv3671c, or PrcBA resulted in the expected impairment of growth of Mtb with butyrate as the primary carbon source, survival during oxidative stress, acid stress and starvation. The impact of depleting ICL, Rv3671c, or PrcBA in multi-strain mouse infections was analyzed with two approaches. We first measured the relative abundance of mutant-specific qTags in total chromosomal DNA isolated from bacteria that were recovered from infected lungs on agar plates. We then developed a two-step amplification procedure, which allowed us to measure the abundances of individual mutants directly in infected lung tissue. Both strategies confirmed that inactivation of Rv3671c and PrcBA severely reduced persistence of Mtb in mice. The multi-strain infections furthermore suggested that silencing ICL not only prevented growth of Mtb during acute infections but also prevented survival of Mtb during chronic infections. Analyses of the ICL knockdown mutant in single-strain infections confirmed this and demonstrated that silencing of ICL during chronic infections impaired persistence of Mtb to the extent that the pathogen

Drosophila PINK1 and parkin loss-of-function mutants display a range of non-motor Parkinson's disease phenotypes.

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Julienne, Hannah; Buhl, Edgar; Leslie, David S; Hodge, James J L

2017-08-01

Parkinson's disease (PD) is more commonly associated with its motor symptoms and the related degeneration of dopamine (DA) neurons. However, it is becoming increasingly clear that PD patients also display a wide range of non-motor symptoms, including memory deficits and disruptions of their sleep-wake cycles. These have a large impact on their quality of life, and often precede the onset of motor symptoms, but their etiology is poorly understood. The fruit fly Drosophila has already been successfully used to model PD, and has been used extensively to study relevant non-motor behaviours in other contexts, but little attention has yet been paid to modelling non-motor symptoms of PD in this genetically tractable organism. We examined memory performance and circadian rhythms in flies with loss-of-function mutations in two PD genes: PINK1 and parkin. We found learning and memory abnormalities in both mutant genotypes, as well as a weakening of circadian rhythms that is underpinned by electrophysiological changes in clock neurons. Our study paves the way for further work that may help us understand the mechanisms underlying these neglected aspects of PD, thus identifying new targets for treatments to address these non-motor problems specifically and perhaps even to halt disease progression in its prodromal phase. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

A small molecule inhibitor of mutant IDH2 rescues cardiomyopathy in a D-2-hydroxyglutaric aciduria type II mouse model.

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Wang, Fang; Travins, Jeremy; Lin, Zhizhong; Si, Yaguang; Chen, Yue; Powe, Josh; Murray, Stuart; Zhu, Dongwei; Artin, Erin; Gross, Stefan; Santiago, Stephanie; Steadman, Mya; Kernytsky, Andrew; Straley, Kimberly; Lu, Chenming; Pop, Ana; Struys, Eduard A; Jansen, Erwin E W; Salomons, Gajja S; David, Muriel D; Quivoron, Cyril; Penard-Lacronique, Virginie; Regan, Karen S; Liu, Wei; Dang, Lenny; Yang, Hua; Silverman, Lee; Agresta, Samuel; Dorsch, Marion; Biller, Scott; Yen, Katharine; Cang, Yong; Su, Shin-San Michael; Jin, Shengfang

2016-11-01

D-2-hydroxyglutaric aciduria (D2HGA) type II is a rare neurometabolic disorder caused by germline gain-of-function mutations in isocitrate dehydrogenase 2 (IDH2), resulting in accumulation of D-2-hydroxyglutarate (D2HG). Patients exhibit a wide spectrum of symptoms including cardiomyopathy, epilepsy, developmental delay and limited life span. Currently, there are no effective therapeutic interventions. We generated a D2HGA type II mouse model by introducing the Idh2R140Q mutation at the native chromosomal locus. Idh2R140Q mice displayed significantly elevated 2HG levels and recapitulated multiple defects seen in patients. AGI-026, a potent, selective inhibitor of the human IDH2R140Q-mutant enzyme, suppressed 2HG production, rescued cardiomyopathy, and provided a survival benefit in Idh2R140Q mice; treatment withdrawal resulted in deterioration of cardiac function. We observed differential expression of multiple genes and metabolites that are associated with cardiomyopathy, which were largely reversed by AGI-026. These findings demonstrate the potential therapeutic benefit of an IDH2R140Q inhibitor in patients with D2HGA type II.

Lithium improves hippocampal neurogenesis, neuropathology and cognitive functions in APP mutant mice.

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Anna Fiorentini

Full Text Available BACKGROUND: Alzheimer's disease (AD is a neurodegenerative disorder characterized by progressive deterioration of cognitive functions, extracellular β-amyloid (Aβ plaques and intracellular neurofibrillary tangles within neocortex and hippocampus. Adult hippocampal neurogenesis plays an important role in learning and memory processes and its abnormal regulation might account for cognitive impairments associated with AD. METHODOLOGY/PRINCIPAL FINDINGS: The double transgenic (Tg CRND8 mice (overexpressing the Swedish and Indiana mutations in the human amyloid precursor protein, aged 2 and 6 months, were used to examine in vivo the effects of 5 weeks lithium treatment. BrdU labelling showed a decreased neurogenesis in the subgranular zone of Tg mice compared to non-Tg mice. The decrease of hippocampal neurogenesis was accompanied by behavioural deficits and worsened with age and pathology severity. The differentiation into neurons and maturation of the proliferating cells were also markedly impaired in the Tg mice. Lithium treatment to 2-month-old Tg mice significantly stimulated the proliferation and neuron fate specification of newborn cells and fully counteracted the transgene-induced impairments of cognitive functions. The drug, by the inhibition of GSK-3β and subsequent activation of Wnt/ß-catenin signalling promoted hippocampal neurogenesis. Finally, the data show that the lithium's ability to stimulate neurogenesis and cognitive functions was lost in the aged Tg mice, thus indicating that the lithium-induced facilitation of neurogenesis and cognitive functions declines as brain Aβ deposition and pathology increases. CONCLUSIONS: Lithium, when given on time, stimulates neurogenesis and counteracts AD-like pathology.

Altered fast- and slow-twitch muscle fibre characteristics in female mice with a (S248F) knock-in mutation of the brain neuronal nicotinic acetylcholine receptor.

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Cannata, David J; Finkelstein, David I; Gantois, Ilse; Teper, Yaroslav; Drago, John; West, Jan M

2009-01-01

We generated a mouse line with a missense mutation (S248F) in the gene (CHRNA4) encoding the alpha4 subunit of neuronal nicotinic acetylcholine receptor (nAChR). Mutant mice demonstrate brief nicotine induced dystonia that resembles the clinical events seen in patients with the same mutation. Drug-induced dystonia is more pronounced in female mice, thus our aim was to determine if the S248F mutation changed the properties of fast- and slow-twitch muscle fibres from female mutant mice. Reverse transcriptase-PCR confirmed CHRNA4 gene expression in the brain but not skeletal muscles in normal and mutant mice. Ca(2+) and Sr(2+) force activation curves were obtained using skinned muscle fibres prepared from slow-twitch (soleus) and fast-twitch (EDL) muscles. Two significant results were found: (1) the (pCa(50) - pSr(50)) value from EDL fibres was smaller in mutant mice than in wild type (1.01 vs. 1.30), (2) the percentage force produced at pSr 5.5 was larger in mutants than in wild type (5.76 vs. 0.24%). Both results indicate a shift to slow-twitch characteristics in the mutant. This conclusion is supported by the identification of the myosin heavy chain (MHC) isoforms. Mutant EDL fibres expressed MHC I (usually only found in slow-twitch fibres) as well as MHC IIa. Despite the lack of spontaneous dystonic events, our findings suggest that mutant mice may be having subclinical events or the mutation results in a chronic alteration to muscle neural input.

Blocking antibodies induced by immunization with a hypoallergenic parvalbumin mutant reduce allergic symptoms in a mouse model of fish allergy.

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Freidl, Raphaela; Gstoettner, Antonia; Baranyi, Ulrike; Swoboda, Ines; Stolz, Frank; Focke-Tejkl, Margarete; Wekerle, Thomas; van Ree, Ronald; Valenta, Rudolf; Linhart, Birgit

2017-06-01

Fish is a frequent elicitor of severe IgE-mediated allergic reactions. Beside avoidance, there is currently no allergen-specific therapy available. Hypoallergenic variants of the major fish allergen, parvalbumin, for specific immunotherapy based on mutation of the 2 calcium-binding sites have been developed. This study sought to establish a mouse model of fish allergy resembling human disease and to investigate whether mouse and rabbit IgG antibodies induced by immunization with a hypoallergenic mutant of the major carp allergen protect against allergic symptoms in sensitized mice. C3H/HeJ mice were sensitized with recombinant wildtype Cyp c 1 or carp extract by intragastric gavage. Antibody, cellular immune responses, and epitope specificity in sensitized mice were investigated by ELISA, rat basophil leukemia assay, T-cell proliferation experiments using recombinant wildtype Cyp c 1, and overlapping peptides spanning the Cyp c 1 sequence. Anti-hypoallergenic Cyp c 1 mutant mouse and rabbit sera were tested for their ability to inhibit IgE recognition of Cyp c 1, Cyp c 1-specific basophil degranulation, and Cyp c 1-induced allergic symptoms in the mouse model. A mouse model of fish allergy mimicking human disease regarding IgE epitope recognition and symptoms as close as possible was established. Administration of antisera generated in mice and rabbits by immunization with a hypoallergenic Cyp c 1 mutant inhibited IgE binding to Cyp c 1, Cyp c 1-induced basophil degranulation, and allergic symptoms caused by allergen challenge in sensitized mice. Antibodies induced by immunization with a hypoallergenic Cyp c 1 mutant protect against allergic reactions in a murine model of fish allergy. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Fitness and virulence of a coxsackievirus mutant that can circumnavigate the need for phosphatidylinositol 4-kinase class III beta

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Thibaut, Hendrik Jan; van der Schaar, Hilde M; Lanke, Kjerstin H W; Verbeken, Erik; Andrews, Martin; Leyssen, Pieter; Neyts, Johan; van Kuppeveld, Frank J M

2014-01-01

Coxsackieviruses require phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) for replication but can bypass this need by an H57Y mutation in protein 3A (3A-H57Y). We show that mutant coxsackievirus is not outcompeted by wild-type virus during 10 passages in vitro. In mice, the mutant virus proved as

Additional sex combs-like 1 belongs to the enhancer of trithorax and Polycomb Group and genetically interacts with Cbx2 in mice

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Fisher, C.L.; Lee, I.; Bloyer, S.; Bozza, S.; Chevalier, J.; Dahl, A; Bodner, C.; Helgason, C. D.; Hess, J.L.; Humphries, R.K.; Brock, H.W.

2009-01-01

The Additional sex combs (Asx) gene of Drosophila behaves genetically as an enhancer of trithorax and Polycomb (ETP) in displaying bidirectional homeotic phenotypes, suggesting that is required for maintenance of both activation and silencing of Hox genes. There are 3 murine homologs of Asx called Additional sex combs-like1, 2, and-3. Asxl1 is required for normal adult hematopoiesis; however its embryonic function is unknown. We used a targeted mouse mutant line Asxl1tm1Bc to determine if Asxl1 is required to silence and activate Hox genes in mice during axial patterning. The mutant embryos exhibit simultaneous anterior and posterior transformations of the axial skeleton, consistent with a role for Asxl1 in activation and silencing of Hox genes. Transformations of the axial skeleton are enhanced in compound mutant embryos for the Polycomb group gene M33/Cbx2. Hox a4, a7, and c8 are derepressed in Asxl1tm1Bc mutants in the antero-posterior axis, but Hox c8 expression is reduced in the brain of mutants, consistent with Asxl1 being required both for activation and repression of Hox genes. We discuss the genetic and molecular definition of ETPs, and suggest that the function of Asxl1 depends on its cellular context. PMID:19833123

Impact of Campylobacter jejuni cj0268c knockout mutation on intestinal colonization, translocation, and induction of immunopathology in gnotobiotic IL-10 deficient mice.

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Markus M Heimesaat

Full Text Available BACKGROUND: Although Campylobacter jejuni infections have a high prevalence worldwide and represent a significant socioeconomic burden, the underlying molecular mechanisms of induced intestinal immunopathology are still not well understood. We have recently generated a C. jejuni mutant strain NCTC11168::cj0268c, which has been shown to be involved in cellular adhesion and invasion. The immunopathological impact of this gene, however, has not been investigated in vivo so far. METHODOLOGY/PRINCIPAL FINDINGS: Gnotobiotic IL-10 deficient mice were generated by quintuple antibiotic treatment and perorally infected with C. jejuni mutant strain NCTC11168::cj0268c, its complemented version (NCTC11168::cj0268c-comp-cj0268c, or the parental strain NCTC11168. Kinetic analyses of fecal pathogen loads until day 6 post infection (p.i. revealed that knockout of cj0268c did not compromise intestinal C. jejuni colonization capacities. Whereas animals irrespective of the analysed C. jejuni strain developed similar clinical symptoms of campylobacteriosis (i.e. enteritis, mice infected with the NCTC11168::cj0268c mutant strain displayed significant longer small as well as large intestinal lengths indicative for less distinct C. jejuni induced pathology when compared to infected control groups at day 6 p.i. This was further supported by significantly lower apoptotic and T cell numbers in the colonic mucosa and lamina propria, which were paralleled by lower intestinal IFN-γ and IL-6 concentrations at day 6 following knockout mutant NCTC11168::cj0268c as compared to parental strain infection. Remarkably, less intestinal immunopathology was accompanied by lower IFN-γ secretion in ex vivo biopsies taken from mesenteric lymphnodes of NCTC11168::cj0268c infected mice versus controls. CONCLUSION/SIGNIFICANCE: We here for the first time show that the cj0268c gene is involved in mediating C. jejuni induced immunopathogenesis in vivo. Future studies will provide further

Human liver cell trafficking mutants: characterization and whole exome sequencing.

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Fei Yuan

Full Text Available The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α''. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.

Abnormal trafficking of endogenously expressed BMPR2 mutant allelic products in patients with heritable pulmonary arterial hypertension.

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Andrea L Frump

Full Text Available More than 200 heterozygous mutations in the type 2 BMP receptor gene, BMPR2, have been identified in patients with Heritable Pulmonary Arterial Hypertension (HPAH. More severe clinical outcomes occur in patients with BMPR2 mutations by-passing nonsense-mediated mRNA decay (NMD negative mutations. These comprise 40% of HPAH mutations and are predicted to express BMPR2 mutant products. However expression of endogenous NMD negative BMPR2 mutant products and their effect on protein trafficking and signaling function have never been described. Here, we characterize the expression and trafficking of an HPAH-associated NMD negative BMPR2 mutation that results in an in-frame deletion of BMPR2 EXON2 (BMPR2ΔEx2 in HPAH patient-derived lymphocytes and in pulmonary endothelial cells (PECs from mice carrying the same in-frame deletion of Exon 2 (Bmpr2 (ΔEx2/+ mice. The endogenous BMPR2ΔEx2 mutant product does not reach the cell surface and is retained in the endoplasmic reticulum. Moreover, chemical chaperones 4-PBA and TUDCA partially restore cell surface expression of Bmpr2ΔEx2 in PECs, suggesting that the mutant product is mis-folded. We also show that PECs from Bmpr2 (ΔEx2/+ mice have defects in the BMP-induced Smad1/5/8 and Id1 signaling axis, and that addition of chemical chaperones restores expression of the Smad1/5/8 target Id1. These data indicate that the endogenous NMD negative BMPRΔEx2 mutant product is expressed but has a folding defect resulting in ER retention. Partial correction of this folding defect and restoration of defective BMP signaling using chemical chaperones suggests that protein-folding agents could be used therapeutically in patients with these NMD negative BMPR2 mutations.

TLR2−/− Mice Display Decreased Severity of Giardiasis via Enhanced Proinflammatory Cytokines Production Dependent on AKT Signal Pathway

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Xin Li

2017-09-01

Full Text Available Giardia infection is one of the most common causes of waterborne diarrheal disease in a wide array of mammalian hosts, including humans globally. Although numerous studies have indicated that adaptive immune responses are important for Giardia defense, however, whether the host innate immune system such as TLRs recognizes Giardia remains poorly understood. TLR2 plays a crucial role in pathogen recognition, innate immunity activation, and the eventual pathogen elimination. In this study, we investigated the role of TLR2 as a non-protective inflammatory response on controlling the severity of giardiasis. RT-PCR analysis suggested that TLR2 expression was increased in vitro. We demonstrated that Giardia lamblia-induced cytokines expression by the activation of p38 and ERK pathways via TLR2. Interestingly, the expression of IL-12 p40, TNF-α, and IL-6, but not IFN-γ, was enhanced in TLR2-blocked and TLR2−/− mouse macrophages exposed to G. lamblia trophozoites compared with wild-type (WT mouse macrophages. Further analysis demonstrated that G. lamblia trophozoites reduced cytokines secretion by activating AKT pathway in WT mouse macrophages. Immunohistochemical staining in G. lamblia cysts infected TLR2−/− and WT mice showed that TLR2 was highly expressed in duodenum in infected WT mice. Also, infected TLR2−/− and AKT-blocked mice showed an increased production of IL-12 p40 and IFN-γ compared with infected WT mice at the early stage during infection. Interestingly, infected TLR2−/− and AKT-blocked mice displayed a decreased parasite burden, an increased weight gain rate, and short parasite persistence. Histological morphometry showed shortened villus length, hyperplastic crypt and decreased ratio of villus height/crypt depth in infected WT mice compared with in infected TLR2−/− and AKT-blocked mice. Together, our results suggested that TLR2 deficiency leads to alleviation of giardiasis and reduction of parasite burden through

CISH has no non-redundant functions in glucose homeostasis or beta cell proliferation during pregnancy in mice.

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Jiao, Yang; Rieck, Sebastian; Le Lay, John; Kaestner, Klaus H

2013-11-01

Increased beta cell proliferation during pregnancy is mediated by the Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5) signalling pathway in response to increased lactogen levels. Activation of the pathway leads to transcriptional upregulation of Cish (encoding cytokine-inducible SH2 domain-containing protein), a member of the suppressor of cytokine signalling (SOCS) family of genes, forming a negative-feedback loop. Here, we examined whether conditional gene ablation of Cish in the pancreas improves beta cell proliferation and beta cell function during pregnancy in mice. We derived mice with a novel, conditional loxP allele for Cish. Pancreas-specific ablation of Cish was achieved by crossing Cish (loxP/loxP) mice with Pdx1-Cre (Early) mice. Beta cell proliferation was quantified by BrdU labelling. Glucose homeostasis was examined with glucose tolerance tests and determination of plasma insulin levels. The expression of other Socs genes and target genes of p-STAT5 related to beta cell function and beta cell proliferation was determined by quantitative PCR. There was no difference in beta cell proliferation or glucose homeostasis between the Cish mutant group and the control group. The p-STAT5 protein level was the same in Cish mutant and control mice. Socs2 gene expression was higher in Cish mutant than control mice at pregnancy day 9.5. The expression of other Socs genes was the same between control and mutant mice. Our results show that CISH has no non-redundant functions in beta cell proliferation or glucose homeostasis during pregnancy in mice. Socs2 might compensate for the loss of Cish during pregnancy.

Chemical rescue of cleft palate and midline defects in conditional GSK-3beta mice.

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Liu, Karen J; Arron, Joseph R; Stankunas, Kryn; Crabtree, Gerald R; Longaker, Michael T

2007-03-01

Glycogen synthase kinase-3beta (GSK-3beta) has integral roles in a variety of biological processes, including development, diabetes, and the progression of Alzheimer's disease. As such, a thorough understanding of GSK-3beta function will have a broad impact on human biology and therapeutics. Because GSK-3beta interacts with many different pathways, its specific developmental roles remain unclear. We have discovered a genetic requirement for GSK-3beta in midline development. Homozygous null mice display cleft palate, incomplete fusion of the ribs at the midline and bifid sternum as well as delayed sternal ossification. Using a chemically regulated allele of GSK-3beta (ref. 6), we have defined requirements for GSK-3beta activity during discrete temporal windows in palatogenesis and skeletogenesis. The rapamycin-dependent allele of GSK-3beta produces GSK-3beta fused to a tag, FRB* (FKBP/rapamycin binding), resulting in a rapidly destabilized chimaeric protein. In the absence of drug, GSK-3beta(FRB)*(/FRB)* mutants appear phenotypically identical to GSK-3beta-/- mutants. In the presence of drug, GSK-3betaFRB* is rapidly stabilized, restoring protein levels and activity. Using this system, mutant phenotypes were rescued by restoring endogenous GSK-3beta activity during two distinct periods in gestation. This technology provides a powerful tool for defining windows of protein function during development.

Adaptive and Behavioral Changes in Kynurenine 3-Monooxygenase Knockout Mice: Relevance to Psychotic Disorders.

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Erhardt, Sophie; Pocivavsek, Ana; Repici, Mariaelena; Liu, Xi-Cong; Imbeault, Sophie; Maddison, Daniel C; Thomas, Marian A R; Smalley, Joshua L; Larsson, Markus K; Muchowski, Paul J; Giorgini, Flaviano; Schwarcz, Robert

2017-11-15

Kynurenine 3-monooxygenase converts kynurenine to 3-hydroxykynurenine, and its inhibition shunts the kynurenine pathway-which is implicated as dysfunctional in various psychiatric disorders-toward enhanced synthesis of kynurenic acid, an antagonist of both α7 nicotinic acetylcholine and N-methyl-D-aspartate receptors. Possibly as a result of reduced kynurenine 3-monooxygenase activity, elevated central nervous system levels of kynurenic acid have been found in patients with psychotic disorders, including schizophrenia. In the present study, we investigated adaptive-and possibly regulatory-changes in mice with a targeted deletion of Kmo (Kmo -/- ) and characterized the kynurenine 3-monooxygenase-deficient mice using six behavioral assays relevant for the study of schizophrenia. Genome-wide differential gene expression analyses in the cerebral cortex and cerebellum of these mice identified a network of schizophrenia- and psychosis-related genes, with more pronounced alterations in cerebellar tissue. Kynurenic acid levels were also increased in these brain regions in Kmo -/- mice, with significantly higher levels in the cerebellum than in the cerebrum. Kmo -/- mice exhibited impairments in contextual memory and spent less time than did controls interacting with an unfamiliar mouse in a social interaction paradigm. The mutant animals displayed increased anxiety-like behavior in the elevated plus maze and in a light/dark box. After a D-amphetamine challenge (5 mg/kg, intraperitoneal), Kmo -/- mice showed potentiated horizontal activity in the open field paradigm. Taken together, these results demonstrate that the elimination of Kmo in mice is associated with multiple gene and functional alterations that appear to duplicate aspects of the psychopathology of several neuropsychiatric disorders. Copyright © 2016. Published by Elsevier Inc.

Sex-dependent novelty response in neurexin-1α mutant mice.

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Marijke C Laarakker

Full Text Available Neurexin-1 alpha (NRXN1α belongs to the family of cell adhesion molecules (CAMs, which are involved in the formation of neuronal networks and synapses. NRXN1α gene mutations have been identified in neuropsychiatric diseases including Schizophrenia (SCZ and Autism Spectrum Disorder (ASD. In order to get a better understanding of the pleiotropic behavioral manifestations caused by NRXN1α gene mutations, we performed a behavioral study of Nrxn1α heterozygous knock-out (+/- mice and observed increased responsiveness to novelty and accelerated habituation to novel environments compared to wild type (+/+ litter-mates. However, this effect was mainly observed in male mice, strongly suggesting that gender-specific mechanisms play an important role in Nrxn1α-induced phenotypes.

Thymidine Kinase-Negative Herpes Simplex Virus 1 Can Efficiently Establish Persistent Infection in Neural Tissues of Nude Mice.

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Huang, Chih-Yu; Yao, Hui-Wen; Wang, Li-Chiu; Shen, Fang-Hsiu; Hsu, Sheng-Min; Chen, Shun-Hua

2017-02-15

Herpes simplex virus 1 (HSV-1) establishes latency in neural tissues of immunocompetent mice but persists in both peripheral and neural tissues of lymphocyte-deficient mice. Thymidine kinase (TK) is believed to be essential for HSV-1 to persist in neural tissues of immunocompromised mice, because infectious virus of a mutant with defects in both TK and UL24 is detected only in peripheral tissues, but not in neural tissues, of severe combined immunodeficiency mice (T. Valyi-Nagy, R. M. Gesser, B. Raengsakulrach, S. L. Deshmane, B. P. Randazzo, A. J. Dillner, and N. W. Fraser, Virology 199:484-490, 1994, https://doi.org/10.1006/viro.1994.1150). Here we find infiltration of CD4 and CD8 T cells in peripheral and neural tissues of mice infected with a TK-negative mutant. We therefore investigated the significance of viral TK and host T cells for HSV-1 to persist in neural tissues using three genetically engineered mutants with defects in only TK or in both TK and UL24 and two strains of nude mice. Surprisingly, all three mutants establish persistent infection in up to 100% of brain stems and 93% of trigeminal ganglia of adult nude mice at 28 days postinfection, as measured by the recovery of infectious virus. Thus, in mouse neural tissues, host T cells block persistent HSV-1 infection, and viral TK is dispensable for the virus to establish persistent infection. Furthermore, we found 30- to 200-fold more virus in neural tissues than in the eye and detected glycoprotein C, a true late viral antigen, in brainstem neurons of nude mice persistently infected with the TK-negative mutant, suggesting that adult mouse neurons can support the replication of TK-negative HSV-1. Acyclovir is used to treat herpes simplex virus 1 (HSV-1)-infected immunocompromised patients, but treatment is hindered by the emergence of drug-resistant viruses, mostly those with mutations in viral thymidine kinase (TK), which activates acyclovir. TK mutants are detected in brains of immunocompromised

Chir99021 and Valproic acid reduce the proliferative advantage of Apc mutant cells.

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Langlands, Alistair J; Carroll, Thomas D; Chen, Yu; Näthke, Inke

2018-02-15

More than 90% of colorectal cancers carry mutations in Apc that drive tumourigenesis. A 'just-right' signalling model proposes that Apc mutations stimulate optimal, but not excessive Wnt signalling, resulting in a growth advantage of Apc mutant over wild-type cells. Reversal of this growth advantage constitutes a potential therapeutic approach. We utilised intestinal organoids to compare the growth of Apc mutant and wild-type cells. Organoids derived from Apc Min/+ mice recapitulate stages of intestinal polyposis in culture. They eventually form spherical cysts that reflect the competitive growth advantage of cells that have undergone loss of heterozygosity (LOH). We discovered that this emergence of cysts was inhibited by Chiron99021 and Valproic acid, which potentiates Wnt signalling. Chiron99021 and Valproic acid restrict the growth advantage of Apc mutant cells while stimulating that of wild-type cells, suggesting that excessive Wnt signalling reduces the relative fitness of Apc mutant cells. As a proof of concept, we demonstrated that Chiron99021-treated Apc mutant organoids were rendered susceptible to TSA-induced apoptosis, while wild-type cells were protected.

Sharing mutants and experimental information prepublication using FgMutantDb (https://scabusa.org/FgMutantDb).

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Baldwin, Thomas T; Basenko, Evelina; Harb, Omar; Brown, Neil A; Urban, Martin; Hammond-Kosack, Kim E; Bregitzer, Phil P

2018-06-01

There is no comprehensive storage for generated mutants of Fusarium graminearum or data associated with these mutants. Instead, researchers relied on several independent and non-integrated databases. FgMutantDb was designed as a simple spreadsheet that is accessible globally on the web that will function as a centralized source of information on F. graminearum mutants. FgMutantDb aids in the maintenance and sharing of mutants within a research community. It will serve also as a platform for disseminating prepublication results as well as negative results that often go unreported. Additionally, the highly curated information on mutants in FgMutantDb will be shared with other databases (FungiDB, Ensembl, PhytoPath, and PHI-base) through updating reports. Here we describe the creation and potential usefulness of FgMutantDb to the F. graminearum research community, and provide a tutorial on its use. This type of database could be easily emulated for other fungal species. Published by Elsevier Inc.

Compensation for PKMζ in long-term potentiation and spatial long-term memory in mutant mice.

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Tsokas, Panayiotis; Hsieh, Changchi; Yao, Yudong; Lesburguères, Edith; Wallace, Emma Jane Claire; Tcherepanov, Andrew; Jothianandan, Desingarao; Hartley, Benjamin Rush; Pan, Ling; Rivard, Bruno; Farese, Robert V; Sajan, Mini P; Bergold, Peter John; Hernández, Alejandro Iván; Cottrell, James E; Shouval, Harel Z; Fenton, André Antonio; Sacktor, Todd Charlton

2016-05-17

PKMζ is a persistently active PKC isoform proposed to maintain late-LTP and long-term memory. But late-LTP and memory are maintained without PKMζ in PKMζ-null mice. Two hypotheses can account for these findings. First, PKMζ is unimportant for LTP or memory. Second, PKMζ is essential for late-LTP and long-term memory in wild-type mice, and PKMζ-null mice recruit compensatory mechanisms. We find that whereas PKMζ persistently increases in LTP maintenance in wild-type mice, PKCι/λ, a gene-product closely related to PKMζ, persistently increases in LTP maintenance in PKMζ-null mice. Using a pharmacogenetic approach, we find PKMζ-antisense in hippocampus blocks late-LTP and spatial long-term memory in wild-type mice, but not in PKMζ-null mice without the target mRNA. Conversely, a PKCι/λ-antagonist disrupts late-LTP and spatial memory in PKMζ-null mice but not in wild-type mice. Thus, whereas PKMζ is essential for wild-type LTP and long-term memory, persistent PKCι/λ activation compensates for PKMζ loss in PKMζ-null mice.

Evaluation of Mobile Phones for Large Display Interaction

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Bauer, Jens; Thelen, Sebastian; Ebert, Achim

2012-01-01

Large displays have become more and more common in the last few years. While interaction with these displays can be conducted using standard methods such as computer mouse and keyboard, this approach causes issues in multi-user environments, where the various conditions for providing multiple keyboards and mice, together with the facilities to employ them, cannot be met. To solve this problem, interaction using mobile phones was proposed by several authors. Previous solutions were specialized...

Slitrk1-deficient mice display elevated anxiety-like behavior and noradrenergic abnormalities.

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Katayama, K; Yamada, K; Ornthanalai, V G; Inoue, T; Ota, M; Murphy, N P; Aruga, J

2010-02-01

Mutations in SLITRK1 are found in patients with Tourette's syndrome and trichotillomania. SLITRK1 encodes a transmembrane protein containing leucine-rich repeats that is produced predominantly in the nervous system. However, the role of this protein is largely unknown, except that it can modulate neurite outgrowth in vitro. To clarify the role of Slitrk1 in vivo, we developed Slitrk1-knockout mice and analyzed their behavioral and neurochemical phenotypes. Slitrk1-deficient mice exhibited elevated anxiety-like behavior in the elevated plus-maze test as well as increased immobility time in forced swimming and tail suspension tests. Neurochemical analysis revealed that Slitrk1-knockout mice had increased levels of norepinephrine and its metabolite 3-methoxy-4-hydroxyphenylglycol. Administration of clonidine, an alpha2-adrenergic agonist that is frequently used to treat patients with Tourette's syndrome, attenuated the anxiety-like behavior of Slitrk1-deficient mice in the elevated plus-maze test. These results lead us to conclude that noradrenergic mechanisms are involved in the behavioral abnormalities of Slitrk1-deficient mice. Elevated anxiety due to Slitrk1 dysfunction may contribute to the pathogenesis of neuropsychiatric diseases such as Tourette's syndrome and trichotillomania.

Aberrant Muscle Antigen Exposure in Mice Is Sufficient to Cause Myositis in a Treg Cell–Deficient Milieu

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Young, Nicholas A; Sharma, Rahul; Friedman, Alexandra K; Kaffenberger, Benjamin H; Bolon, Brad; Jarjour, Wael N

2013-01-01

Objective Myositis is associated with muscle-targeted inflammation and is observed in some Treg cell–deficient mouse models. Because an autoimmune pathogenesis has been strongly implicated, the aim of this study was to investigate the hypothesis that abnormal exposure to muscle antigens, as observed in muscle injury, can induce autoimmune-mediated myositis in susceptible hosts. Methods FoxP3 mutant (scurfy) mice were mated to synaptotagmin VII (Syt VII) mutant mice, which resulted in a new mouse strain that combines impaired membrane resealing with Treg cell deficiency. Lymphocyte preparations from double-mutant mice were adoptively transferred intraperitoneally, with or without purified Treg cells, into recombination-activating gene 1 (RAG-1)–null recipients. Lymph node cells from mice with the FoxP3 mutation were transferred into RAG-1–null mice either 1) intraperitoneally in conjunction with muscle homogenate or purified myosin protein or 2) intramuscularly with or without cotransfer of purified Treg cells. Results FoxP3-deficient mouse lymph node cells transferred in conjunction with myosin protein or muscle homogenate induced robust skeletal muscle inflammation. The infiltrates consisted predominantly of CD4+ and CD8+ T cells, a limited number of macrophages, and no B cells. Significant inflammation was also seen in similar experiments using lymph node cells from FoxP3/Syt VII double-mutant mice but was absent in experiments using adoptive transfer of FoxP3 mutant mouse cells alone. The cotransfer of Treg cells completely suppressed myositis. Conclusion These data, derived from a new, reproducible model, demonstrate the critical roles of Treg cell deficiency and aberrant muscle antigen exposure in the priming of autoreactive cells to induce myositis. This mouse system has multifaceted potential for examining the interplay in vivo between tissue injury and autoimmunity. PMID:24022275

Wild-Type, but Not Mutant N296H, Human Tau Restores Aβ-Mediated Inhibition of LTP in Tau−/− mice

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Mariana Vargas-Caballero

2017-04-01

Full Text Available Microtubule associated protein tau (MAPT is involved in the pathogenesis of Alzheimer's disease and many forms of frontotemporal dementia (FTD. We recently reported that Aβ-mediated inhibition of hippocampal long-term potentiation (LTP in mice requires tau. Here, we asked whether expression of human MAPT can restore Aβ-mediated inhibition on a mouse Tau−/− background and whether human tau with an FTD-causing mutation (N296H can interfere with Aβ-mediated inhibition of LTP. We used transgenic mouse lines each expressing the full human MAPT locus using bacterial artificial chromosome technology. These lines expressed all six human tau protein isoforms on a Tau−/− background. We found that the human wild-type MAPT H1 locus was able to restore Aβ42-mediated impairment of LTP. In contrast, Aβ42 did not reduce LTP in slices in two independently generated transgenic lines expressing tau protein with the mutation N296H associated with frontotemporal dementia (FTD. Basal phosphorylation of tau measured as the ratio of AT8/Tau5 immunoreactivity was significantly reduced in N296H mutant hippocampal slices. Our data show that human MAPT is able to restore Aβ42-mediated inhibition of LTP in Tau−/− mice. These results provide further evidence that tau protein is central to Aβ-induced LTP impairment and provide a valuable tool for further analysis of the links between Aβ, human tau and impairment of synaptic function.

Rubisco mutants of Chlamydomonas reinhardtii enhance photosynthetic hydrogen production.

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Pinto, T S; Malcata, F X; Arrabaça, J D; Silva, J M; Spreitzer, R J; Esquível, M G

2013-06-01

Molecular hydrogen (H2) is an ideal fuel characterized by high enthalpy change and lack of greenhouse effects. This biofuel can be released by microalgae via reduction of protons to molecular hydrogen catalyzed by hydrogenases. The main competitor for the reducing power required by the hydrogenases is the Calvin cycle, and rubisco plays a key role therein. Engineered Chlamydomonas with reduced rubisco levels, activity and stability was used as the basis of this research effort aimed at increasing hydrogen production. Biochemical monitoring in such metabolically engineered mutant cells proceeded in Tris/acetate/phosphate culture medium with S-depletion or repletion, both under hypoxia. Photosynthetic activity, maximum photochemical efficiency, chlorophyll and protein levels were all measured. In addition, expression of rubisco, hydrogenase, D1 and Lhcb were investigated, and H2 was quantified. At the beginning of the experiments, rubisco increased followed by intense degradation. Lhcb proteins exhibited monomeric isoforms during the first 24 to 48 h, and D1 displayed sensitivity under S-depletion. Rubisco mutants exhibited a significant decrease in O2 evolution compared with the control. Although the S-depleted medium was much more suitable than its complete counterpart for H2 production, hydrogen release was observed also in sealed S-repleted cultures of rubisco mutated cells under low-moderate light conditions. In particular, the rubisco mutant Y67A accounted for 10-15-fold higher hydrogen production than the wild type under the same conditions and also displayed divergent metabolic parameters. These results indicate that rubisco is a promising target for improving hydrogen production rates in engineered microalgae.

Oral administration of human papillomavirus type 16 E7 displayed on Lactobacillus casei induces E7-specific antitumor effects in C57/BL6 mice.

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Poo, Haryoung; Pyo, Hyun-Mi; Lee, Tae-Young; Yoon, Sun-Woo; Lee, Jong-Soo; Kim, Chul-Joong; Sung, Moon-Hee; Lee, Seung-Hoon

2006-10-01

The mounting of a specific immune response against the human papillomavirus type 16 E7 protein (HPV16 E7) is important for eradication of HPV16 E7-expressing cancer cells from the cervical mucosa. To induce a mucosal immune response by oral delivery of the E7 antigen, we expressed the HPV16 E7 antigen on the surface of Lactobacillus casei by employing a novel display system in which the poly-gamma-glutamic acid (gamma-PGA) synthetase complex A (PgsA) from Bacillus subtilis (chungkookjang) was used as an anchoring motif. After surface expression of the HPV16 E7 protein was confirmed by Western blot, flow cytometry and immunofluorescence microscopy, mice were orally inoculated with L. casei-PgsA-E7. E7-specific serum IgG and mucosal IgA productions were enhanced after oral administration and significantly enhanced after boosting. Systemic and local cellular immunities were significantly increased after boosting, as shown by increased counts of lymphocytes (SI = 9.7 +/- 1.8) and IFN-gamma secreting cells [510 +/- 86 spot-forming cells/10(6)cells] among splenocytes and increased IFN-gamma in supernatants of vaginal lymphocytes. Furthermore, in an E7-based mouse tumor model, animals receiving orally administered L. casei-PgsA-E7 showed reduced tumor size and increased survival rate versus mice receiving control (L. casei-PgsA) immunization. These results collectively indicate that the oral administration of E7 displayed on lactobacillus induces cellular immunity and antitumor effects in mice. Copyright 2006 Wiley-Liss, Inc.

ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus.

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Frankel, Matthew B; Wojcik, Brandon M; DeDent, Andrea C; Missiakas, Dominique M; Schneewind, Olaf

2010-10-01

The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. © 2010 Blackwell Publishing Ltd.

Deficiency of Lipoprotein Lipase in Neurons Decreases AMPA Receptor Phosphorylation and Leads to Neurobehavioral Abnormalities in Mice.

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Tian Yu

Full Text Available Alterations in lipid metabolism have been found in several neurodegenerative disorders, including Alzheimer's disease. Lipoprotein lipase (LPL hydrolyzes triacylglycerides in lipoproteins and regulates lipid metabolism in multiple organs and tissues, including the central nervous system (CNS. Though many brain regions express LPL, the functions of this lipase in the CNS remain largely unknown. We developed mice with neuron-specific LPL deficiency that became obese on chow by 16 wks in homozygous mutant mice (NEXLPL-/- and 10 mo in heterozygous mice (NEXLPL+/-. In the present study, we show that 21 mo NEXLPL+/- mice display substantial cognitive function decline including poorer learning and memory, and increased anxiety with no difference in general motor activities and exploratory behavior. These neurobehavioral abnormalities are associated with a reduction in the 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl propanoic acid (AMPA receptor subunit GluA1 and its phosphorylation, without any alterations in amyloid β accumulation. Importantly, a marked deficit in omega-3 and omega-6 polyunsaturated fatty acids (PUFA in the hippocampus precedes the development of the neurobehavioral phenotype of NEXLPL+/- mice. And, a diet supplemented with n-3 PUFA can improve the learning and memory of NEXLPL+/- mice at both 10 mo and 21 mo of age. We interpret these findings to indicate that LPL regulates the availability of PUFA in the CNS and, this in turn, impacts the strength of synaptic plasticity in the brain of aging mice through the modification of AMPA receptor and its phosphorylation.

Targeted Deletion of Kynurenine 3-Monooxygenase in Mice

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Giorgini, Flaviano; Huang, Shao-Yi; Sathyasaikumar, Korrapati V.; Notarangelo, Francesca M.; Thomas, Marian A. R.; Tararina, Margarita; Wu, Hui-Qiu; Schwarcz, Robert; Muchowski, Paul J.

2013-01-01

Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway (KP) of tryptophan degradation, has been suggested to play a major role in physiological and pathological events involving bioactive KP metabolites. To explore this role in greater detail, we generated mice with a targeted genetic disruption of Kmo and present here the first biochemical and neurochemical characterization of these mutant animals. Kmo−/− mice lacked KMO activity but showed no obvious abnormalities in the activity of four additional KP enzymes tested. As expected, Kmo−/− mice showed substantial reductions in the levels of its enzymatic product, 3-hydroxykynurenine, in liver, brain, and plasma. Compared with wild-type animals, the levels of the downstream metabolite quinolinic acid were also greatly decreased in liver and plasma of the mutant mice but surprisingly were only slightly reduced (by ∼20%) in the brain. The levels of three other KP metabolites: kynurenine, kynurenic acid, and anthranilic acid, were substantially, but differentially, elevated in the liver, brain, and plasma of Kmo−/− mice, whereas the liver and brain content of the major end product of the enzymatic cascade, NAD+, did not differ between Kmo−/− and wild-type animals. When assessed by in vivo microdialysis, extracellular kynurenic acid levels were found to be significantly elevated in the brains of Kmo−/− mice. Taken together, these results provide further evidence that KMO plays a key regulatory role in the KP and indicate that Kmo−/− mice will be useful for studying tissue-specific functions of individual KP metabolites in health and disease. PMID:24189070

Escherichia coli ArgR mutants defective in cer/Xer recombination, but not in DNA binding.

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Sénéchal, Hélène; Delesques, Jérémy; Szatmari, George

2010-04-01

The Escherichia coli arginine repressor (ArgR) is an L-arginine-dependent DNA-binding protein that controls the expression of the arginine biosynthetic genes and is required as an accessory factor for Xer site-specific recombination at cer and related recombination sites in plasmids. We used the technique of pentapeptide scanning mutagenesis to isolate a series of ArgR mutants that were considerably reduced in cer recombination, but were still able to repress an argA::lacZ fusion. DNA sequence analysis showed that all of the mutants mapped to the same nucleotide, resulting in a five amino acid insertion between residues 149 and 150 of ArgR, corresponding to the end of the alpha6 helix. A truncated ArgR containing a stop codon at residue 150 displayed the same phenotype as the protein with the five amino acid insertion, and both mutants displayed sequence-specific DNA-binding activity that was L-arginine dependent. These results show that the C-terminus of ArgR is more important in cer/Xer site-specific recombination than in DNA binding.

Cyclic GMP-AMP displays mucosal adjuvant activity in mice.

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Ivana Škrnjug

Full Text Available The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice. A characteristic of the cGAMP-induced immune response is the slightly reduced induction of interleukin-17 as a hallmark of Th17 activity--a distinct feature that is not observed with other cyclic di-nucleotide adjuvants. We further characterize the innate immune stimulation activity in vitro on murine bone marrow-derived dendritic cells and human dendritic cells. The observed results suggest the consideration of cGAMP as a candidate mucosal adjuvant for human vaccines.

Cyclic GMP-AMP displays mucosal adjuvant activity in mice.

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Škrnjug, Ivana; Guzmán, Carlos Alberto; Rueckert, Christine; Ruecker, Christine

2014-01-01

The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP) after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice. A characteristic of the cGAMP-induced immune response is the slightly reduced induction of interleukin-17 as a hallmark of Th17 activity--a distinct feature that is not observed with other cyclic di-nucleotide adjuvants. We further characterize the innate immune stimulation activity in vitro on murine bone marrow-derived dendritic cells and human dendritic cells. The observed results suggest the consideration of cGAMP as a candidate mucosal adjuvant for human vaccines.

Partial rescue of postnatal growth plate abnormalities in Ihh mutants by expression of a constitutively active PTH/PTHrP receptor.

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Maeda, Yukiko; Schipani, Ernestina; Densmore, Michael J; Lanske, Beate

2010-02-01

Indian hedgehog (Ihh) is essential for chondrocyte proliferation/differentiation and osteoblast differentiation during prenatal endochondral bone formation. Ihh expression in postnatal chondrocytes has a non-redundant role in maintaining a growth plate and sustaining trabecular bone after birth. Loss of Ihh in postnatal chondrocytes results in fusion of the growth plate and a decrease in trabecular bone. In order to normalize this abnormal chondrocyte phenotype and to investigate whether a putative rescue of the growth plate anomalies is sufficient to correct the severe alterations in the bone, we expressed a constitutively active PTH/PTHrP receptor (an Ihh downstream target) in the chondrocytes of Col2 alpha 1-Cre ER; Ihh(dld) mice by mating Col2 alpha 1-Cre ER; Ihh(fl/fl) mice with Col2 alpha 1-constitutively active PTH/PTHrP receptor transgenic mice (Jansen, J). Col2 alpha 1-Cre ER; Ihh(f/f); J mice were then injected with tamoxifen at P0 to generate Col2 alpha 1-Cre ER; Ihh(d/d); J mice. In contrast with the previously reported growth plate phenotype of Col2 alpha 1-Cre ER; Ihh(d/d) mice that displayed ectopic chondrocyte hypertrophy at P7, growth plates of Col2 alpha 1-Cre ER; Ihh(d/d); J double mutants were well organized, and exhibited a gene expression pattern similar to the one of control mice. However, expression of osteoblast markers and Dkk1, a Wnt signaling target, remains decreased in the bone collar of Col2 alpha 1-Cre ER; Ihh(d/d); J mice when compared to control mice despite the rescue of abnormal chondrocyte differentiation. Moreover, proliferation of chondrocytes was still significantly impaired in Col2 alpha 1-Cre ER; Ihh(d/d); J mice, and this eventually led to the fusion of the growth plate at P14. In summary, we have demonstrated that expression of a Jansen receptor in chondrocytes was able to rescue abnormal chondrocyte differentiation but not impaired chondrocyte proliferation and the bone anomalies in mice lacking the Ihh gene in

Alopecia in a viable phospholipase C delta 1 and phospholipase C delta 3 double mutant.

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Fabian Runkel

Full Text Available BACKGROUND: Inositol 1,4,5trisphosphate (IP(3 and diacylglycerol (DAG are important intracellular signalling molecules in various tissues. They are generated by the phospholipase C family of enzymes, of which phospholipase C delta (PLCD forms one class. Studies with functional inactivation of Plcd isozyme encoding genes in mice have revealed that loss of both Plcd1 and Plcd3 causes early embryonic death. Inactivation of Plcd1 alone causes loss of hair (alopecia, whereas inactivation of Plcd3 alone has no apparent phenotypic effect. To investigate a possible synergy of Plcd1 and Plcd3 in postnatal mice, novel mutations of these genes compatible with life after birth need to be found. METHODOLOGY/PRINCIPAL FINDINGS: We characterise a novel mouse mutant with a spontaneously arisen mutation in Plcd3 (Plcd3(mNab that resulted from the insertion of an intracisternal A particle (IAP into intron 2 of the Plcd3 gene. This mutation leads to the predominant expression of a truncated PLCD3 protein lacking the N-terminal PH domain. C3H mice that carry one or two mutant Plcd3(mNab alleles are phenotypically normal. However, the presence of one Plcd3(mNab allele exacerbates the alopecia caused by the loss of functional Plcd1 in Del(9olt1Pas mutant mice with respect to the number of hair follicles affected and the body region involved. Mice double homozygous for both the Del(9olt1Pas and the Plcd3(mNab mutations survive for several weeks and exhibit total alopecia associated with fragile hair shafts showing altered expression of some structural genes and shortened phases of proliferation in hair follicle matrix cells. CONCLUSIONS/SIGNIFICANCE: The Plcd3(mNab mutation is a novel hypomorphic mutation of Plcd3. Our investigations suggest that Plcd1 and Plcd3 have synergistic effects on the murine hair follicle in specific regions of the body surface.

Mice lacking Ras-GRF1 show contextual fear conditioning but not spatial memory impairments: convergent evidence from two independently generated mouse mutant lines

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Raffaele ed'Isa

2011-12-01

Full Text Available Ras-GRF1 is a neuronal specific guanine exchange factor that, once activated by both ionotropic and metabotropic neurotransmitter receptors, can stimulate Ras proteins, leading to long-term phosphorylation of downstream signaling. The two available reports on the behavior of two independently generated Ras-GRF1 deficient mouse lines provide contrasting evidence on the role of Ras-GRF1 in spatial memory and contextual fear conditioning. These discrepancies may be due to the distinct alterations introduced in the mouse genome by gene targeting in the two lines that could differentially affect expression of nearby genes located in the imprinted region containing the Ras-grf1 locus. In order to determine the real contribution of Ras-GRF1 to spatial memory we compared in Morris Water Maze learning the Brambilla’s mice with a third mouse line (GENA53 in which a nonsense mutation was introduced in the Ras-GRF1 coding region without additional changes in the genome and we found that memory in this task is normal. Also, we measured both contextual and cued fear conditioning, which were previously reported to be affected in the Brambilla’s mice, and we confirmed that contextual learning but not cued conditioning is impaired in both mouse lines. In addition, we also tested both lines for the first time in conditioned place aversion in the Intellicage, an ecological and remotely controlled behavioral test, and we observed normal learning. Finally, based on previous reports of other mutant lines suggesting that Ras-GRF1 may control body weight, we also measured this non-cognitive phenotype and we confirmed that both Ras-GRF1 deficient mutants are smaller than their control littermates. In conclusion, we demonstrate that Ras-GRF1 has no unique role in spatial memory while its function in contextual fear conditioning is likely to be due not only to its involvement in amygdalar functions but possibly to some distinct hippocampal connections specific to

Mast cells have no impact on cutaneous leishmaniasis severity and related Th2 differentiation in resistant and susceptible mice.

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Paul, Christoph; Wolff, Svenja; Zapf, Thea; Raifer, Hartmann; Feyerabend, Thorsten B; Bollig, Nadine; Camara, Bärbel; Trier, Claudia; Schleicher, Ulrike; Rodewald, Hans-Reimer; Lohoff, Michael

2016-01-01

The genus leishmania comprises different protozoan parasites which are causative agents of muco-cutaneous and systemic, potentially lethal diseases. After infection with the species Leishmania major, resistant mice expand Th1 cells which stimulate macrophages for Leishmania destruction. In contrast, susceptible mice generate Th2 cells which deactivate macrophages, leading to systemic spread of the pathogens. Th-cell differentiation is determined within the first days, and Th2 cell differentiation requires IL-4, whereby the initial IL-4 source is often unknown. Mast cells are potential sources of IL-4, and hence their role in murine leishmaniasis has previously been studied in mast cell-deficient Kit mutant mice, although these mice display immunological phenotypes beyond mast cell deficiency. We therefore readdressed this question by infecting Kit-independent mast cell-deficient mice that are Th1 (C57BL/6 Cpa(Cre) ) or Th2 (BALB/c Cpa(Cre) ) prone with L. major. Using different parasite doses and intra- or subcutaneous infection routes, the results demonstrate no role of mast cells on lesion size development, parasite load, immune cell phenotypes expanding in draining lymph nodes, and cytokine production during murine cutaneous leishmaniasis. Thus, other cell types such as ILCs or T cells have to be considered as primary source of Th2-driving IL-4. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nootropic nefiracetam inhibits proconvulsant action of peripheral-type benzodiazepines in epileptic mutant EL mice.

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Nakamoto, Yurie; Shiotani, Tadashi; Watabe, Shigeo; Nabeshima, Toshitaka; Yoshii, Mitsunobu

2004-10-01

Piracetam and structurally related nootropics are known to potentiate the anticonvulsant effects of antiepileptic drugs. It remains to be seen, however, whether these nootropics inhibit proconvulsant actions of many toxic agents including Ro 5-4864, a specific agonist for peripheral-type benzodiazepine receptors (PBR). The present study was designed to address this issue using EL mice, an animal model of epilepsy. In behavioral pharmacological experiments, EL mice were highly susceptible to convulsions induced by Ro 5-4864 (i.p.) in comparison with nonepileptic DDY mice. Nefiracetam administered orally to EL mice inhibited spontaneous seizures. In DDY mice, convulsions induced by Ro 5-4864 were prevented by nefiracetam when administered by i.v. injection. Aniracetam (i.v.) was partially effective, but piracetam and oxiracetam were ineffective as anticonvulsants. Binding assay for brain tissues revealed a higher density of mitochondrial PBR in EL mice compared with DDY mice. Binding of the PBR ligands Ro 5-4864 to either EL or DDY mouse brain was inhibited by micromolar concentrations of these nootropic agents in the sequence of nefiracetam > aniracetam > oxiracetam, piracetam. This rank order is identical to potency as anticonvulsants. These data suggest that nefiracetam may prevent toxic effects of PBR agonists through interacting with PBR.

Grain-filling duration and grain yield relationships in wheat mutants

International Nuclear Information System (INIS)

Larik, A.S.

1987-01-01

Nine stable mutants of bread wheat along with their mother cultivars were investigated for grain-filling characteristics in relation to grain yield. Significant differences among mutants for grain-filling duration and grain-filling index were observed. Inspite of the consistent differences in grain-filling duration there was no significant association between grain-filling duration and grain yield in C-591 and Nayab mutants. Failure to detect an yield advantage due to differences in grain-filling duration in these genotypes suggests that any advantage derived from alteration of grain-filling period may have been outweighed by the coincident changes in length of the vegetative period. Other factors such as synchrony of anthesis may have limited out ability to find an association between grainfilling duration and grain yield. On the contrary, significant association between grain-filling duration and grain yield displayed by indus-66 indus-66 mutants derived from gamma rays, shows the ability of gamma rays to induce functional alternations in the pattern of gene arrangements controlling these traits. Thus, the vaability observed in these physiological traits suggests that selection for these traits could be useful in improving grain yield. (author)

A thermo-sensitive purple leaf rice mutant--PLM12 and its genetical study

International Nuclear Information System (INIS)

Wu Guanting; Wang Xianyu; Jin Wei

2001-01-01

PLM12 was a thermo-sensitive purple leaf mutant selected from Indica rice variety Luqingzao 1 treated with pingyangmycin in combination with γ-rays, and for display of its mutant character, a relatively high temperature was required. Compared with its original parent, many major agronomic traits of PLM12 changed to varied extents. Based on spikelet cutting experiment, it was believed that significant decreases in number of filled grains per panicle, fertility, and 1000-grain weight in PLM12 resulted mainly from a great decline in photosynthetic capacity and serious lack of photosynthate in purple leaves. It was indicated by genetic analysis that expression of the mutant phenotype in PLM12 was conditioned by a single recessive major nuclear gene and modified by several minor genes

Induction of endoplasmic reticulum stress by deletion of Grp78 depletes Apc mutant intestinal epithelial stem cells.

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van Lidth de Jeude, J F; Meijer, B J; Wielenga, M C B; Spaan, C N; Baan, B; Rosekrans, S L; Meisner, S; Shen, Y H; Lee, A S; Paton, J C; Paton, A W; Muncan, V; van den Brink, G R; Heijmans, J

2017-06-15

Intestinal epithelial stem cells are highly sensitive to differentiation induced by endoplasmic reticulum (ER) stress. Colorectal cancer develops from mutated intestinal epithelial stem cells. The most frequent initiating mutation occurs in Apc, which results in hyperactivated Wnt signalling. This causes hyperproliferation and reduced sensitivity to chemotherapy, but whether these mutated stem cells are sensitive to ER stress induced differentiation remains unknown. Here we examined this by generating mice in which both Apc and ER stress repressor chaperone Grp78 can be conditionally deleted from the intestinal epithelium. For molecular studies, we used intestinal organoids derived from these mice. Homozygous loss of Apc alone resulted in crypt elongation, activation of the Wnt signature and accumulation of intestinal epithelial stem cells, as expected. This phenotype was however completely rescued on activation of ER stress by additional deletion of Grp78. In these Apc-Grp78 double mutant animals, stem cells were rapidly lost and repopulation occurred by non-mutant cells that had escaped recombination, suggesting that Apc-Grp78 double mutant stem cells had lost self-renewal capacity. Although in Apc-Grp78 double mutant mice the Wnt signature was lost, these intestines exhibited ubiquitous epithelial presence of nuclear β-catenin. This suggests that ER stress interferes with Wnt signalling downstream of nuclear β-catenin. In conclusion, our findings indicate that ER stress signalling results in loss of Apc mutated intestinal epithelial stem cells by interference with the Wnt signature. In contrast to many known inhibitors of Wnt signalling, ER stress acts downstream of β-catenin. Therefore, ER stress poses a promising target in colorectal cancers, which develop as a result of Wnt activating mutations.

Brucella abortus nicotinamidase (PncA) contributes to its intracellular replication and infectivity in mice.

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Kim, Suk; Kurokawa, Daisuke; Watanabe, Kenta; Makino, Sou-Ichi; Shirahata, Toshikazu; Watarai, Masahisa

2004-05-15

Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and non-professional phagocytes, and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. Nicotinamidase/pyrazinamidase mutant (pncA mutant) of Brucella abortus failed to replicate in HeLa cells, and showed a lower rate of intracellular replication than that of wild-type strain in macrophages. Addition of nicotinic acid, but not nicotinamide, into medium supported intracellular replication of pncA mutant in HeLa cells and macrophages. The pncA mutant was not co-localizing with either late endosomes or lysosomes. The B. abortus virB4 mutant was completely cleared from the spleens of mice after 4 weeks, while the pncA mutant showed a 1.5-log reduction of the number of bacteria isolated from spleens after 10 weeks. Although pncA mutant showed reduced virulence in mice and defective intracellular replication, its ability to confer protection against the virulent B. abortus strain 544 was fully retained. These results suggest that PncA does not contribute to intracellular trafficking of B. abortus, but contributes to utilization of nutrients required for intracellular growth. Our results indicate that detailed characterizations of the pncA mutant may help the improvement of currently available live vaccines. Copyright 2004 Federation of European Microbiological Societies

Chronic Proliferative Dermatitis in Mice: NFκB Activation Autoinflammatory Disease

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Yanhua Liang

2011-01-01

Full Text Available Autoinflammatory diseases are a heterogeneous group of congenital diseases characterized by the presence of recurrent inflammation, in the absence of infectious agents, detectable autoantibodies or antigen-specific autoreactive T-cells. SHARPIN deficient mice presents multiorgan chronic inflammation without known autoantibodies or autoreactive T-cells, designated Sharpincpdm. Histological studies demonstrated epidermal hyperproliferation, Th-2 inflammation, and keratinocyte apoptosis in this mutant. The mutant mice have decreased behavioral mobility, slower growth, and loss of body weight. Epidermal thickness and mitotic epidermal cells increase along with disease development. K5/K14 expression is distributed through all layers of epidermis, along with K6 expression in interfollicular epidermis, suggesting epidermal hyperproliferation. K1/K10 is only detectable in outer layers of spinosum epidermis, reflecting accelerated keratinocyte migration. Alpha smooth muscle actin is overexpressed in skin blood vessels, which may release the elevated white blood cells to dermis. CD3+CD45+ cells and granulocytes, especially eosinophils and mast cells, aggregate in the mutant skin. TUNEL assay, together with Annexin-V/propidium iodide FACS analysis, confirmed the increase of apoptotic keratinocytes in skin. These data validate and provide new lines of evidence of the proliferation-inflammation-apoptosis triad in Sharpincpdm mice, an NFκB activation autoinflammatory disease.

Inhibition of PTP1B Restores IRS1-Mediated Hepatic Insulin Signaling in IRS2-Deficient Mice

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González-Rodríguez, Águeda; Gutierrez, Jose A. Mas; Sanz-González, Silvia; Ros, Manuel; Burks, Deborah J.; Valverde, Ángela M.

2010-01-01

OBJECTIVE Mice with complete deletion of insulin receptor substrate 2 (IRS2) develop hyperglycemia, impaired hepatic insulin signaling, and elevated gluconeogenesis, whereas mice deficient for protein tyrosine phosphatase (PTP)1B display an opposing hepatic phenotype characterized by increased sensitivity to insulin. To define the relationship between these two signaling pathways in the regulation of liver metabolism, we used genetic and pharmacological approaches to study the effects of inhibiting PTP1B on hepatic insulin signaling and expression of gluconeogenic enzymes in IRS2−/− mice. RESEARCH DESIGN AND METHODS We analyzed glucose homeostasis and insulin signaling in liver and isolated hepatocytes from IRS2−/− and IRS2−/−/PTP1B−/− mice. Additionally, hepatic insulin signaling was assessed in control and IRS2−/− mice treated with resveratrol, an antioxidant present in red wine. RESULTS In livers of hyperglycemic IRS2−/− mice, the expression levels of PTP1B and its association with the insulin receptor (IR) were increased. The absence of PTP1B in the double-mutant mice restored hepatic IRS1-mediated phosphatidylinositol (PI) 3-kinase/Akt/Foxo1 signaling. Moreover, resveratrol treatment of hyperglycemic IRS2−/− mice decreased hepatic PTP1B mRNA and inhibited PTP1B activity, thereby restoring IRS1-mediated PI 3-kinase/Akt/Foxo1 signaling and peripheral insulin sensitivity. CONCLUSIONS By regulating the phosphorylation state of IR, PTB1B determines sensitivity to insulin in liver and exerts a unique role in the interplay between IRS1 and IRS2 in the modulation of hepatic insulin action. PMID:20028942

Mutants induced in winter rye (Secale cereale L.): Short straw-mutant No. 2714 and late-senescence mutant

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Muszynski, S; Darlewska, M [Department of Plant Breeding and Seed Science, Warsaw Agricultural University, Warsaw (Poland)

1990-01-01

Full text: Mutants were induced by treating dormant seeds with ionizing radiation (fast neutrons) or chemicals (N-nitroso-N-ethyl urea or sodium azide). Among several mutants obtained, of special value is the short-straw mutant No. 2714 and a late senescent mutant. (author)

A novel ENU-mutation in ankyrin-1 disrupts malaria parasite maturation in red blood cells of mice.

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Andreas Greth

Full Text Available The blood stage of the plasmodium parasite life cycle is responsible for the clinical symptoms of malaria. Epidemiological studies have identified coincidental malarial endemicity and multiple red blood cell (RBC disorders. Many RBC disorders result from mutations in genes encoding cytoskeletal proteins and these are associated with increased protection against malarial infections. However the mechanisms underpinning these genetic, host responses remain obscure. We have performed an N-ethyl-N-nitrosourea (ENU mutagenesis screen and have identified a novel dominant (haploinsufficient mutation in the Ank-1 gene (Ank1(MRI23420 of mice displaying hereditary spherocytosis (HS. Female mice, heterozygous for the Ank-1 mutation showed increased survival to infection by Plasmodium chabaudi adami DS with a concomitant 30% decrease in parasitemia compared to wild-type, isogenic mice (wt. A comparative in vivo red cell invasion and parasite growth assay showed a RBC-autonomous effect characterised by decreased proportion of infected heterozygous RBCs. Within approximately 6-8 hours post-invasion, TUNEL staining of intraerythrocytic parasites, showed a significant increase in dead parasites in heterozygotes. This was especially notable at the ring and trophozoite stages in the blood of infected heterozygous mutant mice compared to wt (p<0.05. We conclude that increased malaria resistance due to ankyrin-1 deficiency is caused by the intraerythrocytic death of P. chabaudi parasites.

Ectopic mineralization of cartilage and collagen-rich tendons and ligaments in Enpp1asj-2J mice.

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Zhang, Jieyu; Dyment, Nathaniel A; Rowe, David W; Siu, Sarah Y; Sundberg, John P; Uitto, Jouni; Li, Qiaoli

2016-03-15

Generalized arterial calcification of infancy (GACI), an autosomal recessive disorder caused by mutations in the ENPP1 gene, manifests with extensive mineralization of the cardiovascular system. A spontaneous asj-2J mutant mouse has been characterized as a model for GACI. Previous studies focused on phenotypic characterization of skin and vascular tissues. This study further examined the ectopic mineralization phenotype of cartilage, collagen-rich tendons and ligaments in this mouse model. The mice were placed on either control diet or the "acceleration diet" for up to 12 weeks of age. Soft connective tissues, such as ear (elastic cartilage) and trachea (hyaline cartilage), were processed for standard histology. Assessment of ectopic mineralization in articular cartilage and fibrocartilage as well as tendons and ligaments which are attached to long bones were performed using a novel cryo-histological method without decalcification. These analyses demonstrated ectopic mineralization in cartilages as well as tendons and ligaments in the homozygous asj-2J mice at 12 weeks of age, with the presence of immature osteophytes displaying alkaline phosphatase and tartrate-resistant acid phosphatase activities as early as at 6 weeks of age. Alkaline phosphatase activity was significantly increased in asj-2J mouse serum as compared to wild type mice, indicating increased bone formation rate in these mice. Together, these data highlight the key role of ENPP1 in regulating calcification of both soft and skeletal tissues.

Nanoformulated cell-penetrating survivin mutant and its dual actions

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Sriramoju B

2014-07-01

Full Text Available Bhasker Sriramoju, Rupinder K Kanwar, Jagat R Kanwar Nanomedicine Laboratory of Immunology and Molecular Biomedical Research (NLIMBR, School of Medicine, Faculty of Health, Deakin University, Geelong, Australia Abstract: In this study, we investigated the differential actions of a dominant-negative survivin mutant (SurR9-C84A against cancerous SK-N-SH neuroblastoma cell lines and differentiated SK-N-SH neurons. In both the cases, the mutant protein displayed dual actions, where its effects were cytotoxic toward cancerous cells and proliferative toward the differentiated neurons. This can be explained by the fact that tumorous (undifferentiated SK-N-SH cells have a high endogenous survivin pool and upon treatment with mutant SuR9-C84A causes forceful survivin expression. These events significantly lowered the microtubule dynamics and stability, eventually leading to apoptosis. In the case of differentiated SK-N-SH neurons that express negligible levels of wild-type survivin, the mutant indistinguishably behaved in a wild-type fashion. It also favored cell-cycle progression, forming the chromosome-passenger complex, and stabilized the microtubule-organizing center. Therefore, mutant SurR9-C84A represents a novel therapeutic with its dual actions (cytotoxic toward tumor cells and protective and proliferative toward neuronal cells, and hence finds potential applications against a variety of neurological disorders. In this study, we also developed a novel poly(lactic-co-glycolic acid nanoparticulate formulation to surmount the hurdles associated with the delivery of SurR9-C84A, thus enhancing its effective therapeutic outcome. Keywords: survivin mutant, neurological disorders, protein therapeutics, inhibitor of apoptosis protein family, poly(lactic-co-glycolic acid

Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in mice.

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Monteiro, Gaby E R; Jansen van Vuren, Petrus; Wichgers Schreur, Paul J; Odendaal, Lieza; Clift, Sarah J; Kortekaas, Jeroen; Paweska, Janusz T

2018-04-02

The NSs protein encoded by the S segment of Rift Valley fever virus (RVFV) is the major virulence factor, counteracting the host innate antiviral defence. It contains five highly conserved cysteine residues at positions 39, 40, 149, 178 and 194, which are thought to stabilize the tertiary and quaternary structure of the protein. Here, we report significant differences between clinical, virological, histopathological and host gene responses in BALB/c mice infected with wild-type RVFV (wtRVFV) or a genetic mutant having a double cysteine-to-serine substitution at residues 39 and 40 of the NSs protein (RVFV-C39S/C40S). Mice infected with the wtRVFV developed a fatal acute disease; characterized by high levels of viral replication, severe hepatocellular necrosis, and massive up-regulation of transcription of genes encoding type I and -II interferons (IFN) as well as pro-apoptotic and pro-inflammatory cytokines. The RVFV-C39S/C40S mutant did not cause clinical disease and its attenuated virulence was consistent with virological, histopathological and host gene expression findings in BALB/c mice. Clinical signs in mice infected with viruses containing cysteine-to-serine substitutions at positions 178 or 194 were similar to those occurring in mice infected with the wtRVFV, while a mutant containing a substitution at position 149 caused mild, non-fatal disease in mice. As mutant RVFV-C39S/C40S showed an attenuated phenotype in mice, the molecular mechanisms behind this attenuation were further investigated. The results show that two mechanisms are responsible for the attenuation; (1) loss of the IFN antagonistic propriety characteristic of the wtRVFV NSs and (2) the inability of the attenuated mutant to degrade Proteine Kinase R (PKR). Copyright © 2018. Published by Elsevier B.V.

Lipid metabolism and body composition in Gclm(-/-) mice

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Kendig, Eric L. [Department of Environmental Health, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati, OH 45267 (United States); Center for Environmental Genetics, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati, OH 45267 (United States); Chen, Ying [Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, Aurora, CO 80045 (United States); Krishan, Mansi; Johansson, Elisabet; Schneider, Scott N. [Department of Environmental Health, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati, OH 45267 (United States); Genter, Mary Beth; Nebert, Daniel W. [Department of Environmental Health, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati, OH 45267 (United States); Center for Environmental Genetics, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati, OH 45267 (United States); Shertzer, Howard G., E-mail: shertzhg@ucmail.uc.edu [Department of Environmental Health, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati, OH 45267 (United States); Center for Environmental Genetics, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati, OH 45267 (United States)

2011-12-15

In humans and experimental animals, high fat diets (HFD) are associated with risk factors for metabolic diseases, such as excessive weight gain and adiposity, insulin resistance and fatty liver. Mice lacking the glutamate-cysteine ligase modifier subunit gene (Gclm(-/-)) and deficient in glutathione (GSH), are resistant to HFD-mediated weight gain. Herein, we evaluated Gclm-associated regulation of energy metabolism, oxidative stress, and glucose and lipid homeostasis. C57BL/6J Gclm(-/-) mice and littermate wild-type (WT) controls received a normal diet or an HFD for 11 weeks. HFD-fed Gclm(-/-) mice did not display a decreased respiratory quotient, suggesting that they are unable to process lipid for metabolism. Although dietary energy consumption and intestinal lipid absorption were unchanged in Gclm(-/-) mice, feeding these mice an HFD did not produce excess body weight nor fat storage. Gclm(-/-) mice displayed higher basal metabolic rates resulting from higher activities of liver mitochondrial NADH-CoQ oxidoreductase, thus elevating respiration. Although Gclm(-/-) mice exhibited strong systemic and hepatic oxidative stress responses, HFD did not promote glucose intolerance or insulin resistance. Furthermore, HFD-fed Gclm(-/-) mice did not develop fatty liver, likely resulting from very low expression levels of genes encoding lipid metabolizing enzymes. We conclude that Gclm is involved in the regulation of basal metabolic rate and the metabolism of dietary lipid. Although Gclm(-/-) mice display a strong oxidative stress response, they are protected from HFD-induced excessive weight gain and adipose deposition, insulin resistance and steatosis. -- Highlights: Black-Right-Pointing-Pointer A high fat diet does not produce body weight and fat gain in Gclm(-/-) mice. Black-Right-Pointing-Pointer A high fat diet does not induce steatosis or insulin resistance in Gclm(-/-) mice. Black-Right-Pointing-Pointer Gclm(-/-) mice have high basal metabolism and mitochondrial

Genome-wide analysis of the Pho regulon in a pstCA mutant of Citrobacter rodentium.

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Catherine Cheng

Full Text Available The phosphate-specific transport operon, pstSCAB-phoU, of Gram-negative bacteria is an essential part of the Pho regulon. Its key roles are to encode a high-affinity inorganic phosphate transport system and to prevent activation of PhoB in phosphate-rich environments. In general, mutations in pstSCAB-phoU lead to the constitutive expression of the Pho regulon. Previously, we constructed a pstCA deletion mutant of Citrobacter rodentium and found it to be attenuated for virulence in mice, its natural host. This attenuation was dependent on PhoB or PhoB-regulated gene(s because a phoB mutation restored virulence for mice to the pstCA mutant. To investigate how downstream genes may contribute to the virulence of C. rodentium, we used microarray analysis to investigate global gene expression of C. rodentium strain ICC169 and its isogenic pstCA mutant when grown in phosphate-rich medium. Overall 323 genes of the pstCA mutant were differentially expressed by at least 1.5-fold compared to the wild-type C. rodentium. Of these 145 were up-regulated and 178 were down-regulated. Differentially expressed genes included some involved in phosphate homoeostasis, cellular metabolism and protein metabolism. A large number of genes involved in stress responses and of unknown function were also differentially expressed, as were some virulence-associated genes. Up-regulated virulence-associated genes in the pstCA mutant included that for DegP, a serine protease, which appeared to be directly regulated by PhoB. Down-regulated genes included those for the production of the urease, flagella, NleG8 (a type III-secreted protein and the tad focus (which encodes type IVb pili in Yersinia enterocolitica. Infection studies using C57/BL6 mice showed that DegP and NleG8 play a role in bacterial virulence. Overall, our study provides evidence that Pho is a global regulator of gene expression in C. rodentium and indicates the presence of at least two previously unrecognized

Hemoglobin of mice with radiation-induced mutations at the hemoglobin loci

Energy Technology Data Exchange (ETDEWEB)

Popp, R A; Stratton, L P; Hawley, D K; Effron, K [Oak Ridge National Lab., TN (USA)

1979-01-15

Chemical analyses were done on the abnormal hemoglobins of the five (101 x SEC)F/sub 1/ offspring of X- irradiated adult SEC mice to determine which hemoglobin genes were expressed in each hemoglobin variant. Three offspring of irradiated SEC males did not express either of the two kinds of ..cap alpha..-chains normally found in all SEC mice. The deficient ..cap alpha..-chain synthesis caused these mice to exhibit an ..cap alpha..-thalassemia similar to human ..cap alpha..-thalassemia. Scanning electron microscopy was used to show that many erythrocytes of mice with ..cap alpha..-thalassemia have bizarre shapes; e.g. many erythrocytes appeared flattened or had thorny projections (acanthocytes). One mutant with a tandem duplication of a segment of chromosome 7 (site of locus determining ..beta..-chain structure) produced twice as much SEC as 101 ..beta..-chain polypeptides. One mutant that probably arose by non-disjunction of chromosome 7's in its unirradiated 101 mother and loss of chromosome 7 from the gamete of its irradiated SEC father did not express the SEC ..beta..-chain gene.

Hemoglobin of mice with radiation-induced mutations at the hemoglobin loci

International Nuclear Information System (INIS)

Popp, R.A.; Stratton, L.P.; Hawley, D.K.; Effron, K.

1979-01-01

Chemical analyses were done on the abnormal hemoglobins of the five (101 x SEC)F 1 offspring of X- irradiated adult SEC mice to determine which hemoglobin genes were expressed in each hemoglobin variant. Three offspring of irradiated SEC males did not express either of the two kinds of α-chains normally found in all SEC mice. The deficient α-chain synthesis caused these mice to exhibit an α-thalassemia similar to human α-thalassemia. Scanning electron microscopy was used to show that many erythrocytes of mice with α-thalassemia have bizarre shapes; e.g. many erythrocytes appeared flattened or had thorny projections (acanthocytes). One mutant with a tandem duplication of a segment of chromosome 7 (site of locus determining β-chain structure) produced twice as much SEC as 101 β-chain polypeptides. One mutant that probably arose by non-disjunction of chromosome 7's in its unirradiated 101 mother and loss of chromosome 7 from the gamete of its irradiated SEC father did not express the SEC β-chain gene. (author)

Phosphorylation of the Mdm2 oncoprotein by the c-Abl tyrosine kinase regulates p53 tumor suppression and the radiosensitivity of mice.

Science.gov (United States)

Carr, Michael I; Roderick, Justine E; Zhang, Hong; Woda, Bruce A; Kelliher, Michelle A; Jones, Stephen N

2016-12-27

The p53 tumor suppressor acts as a guardian of the genome by preventing the propagation of DNA damage-induced breaks and mutations to subsequent generations of cells. We have previously shown that phosphorylation of the Mdm2 oncoprotein at Ser394 by the ATM kinase is required for robust p53 stabilization and activation in cells treated with ionizing radiation, and that loss of Mdm2 Ser394 phosphorylation leads to spontaneous tumorigenesis and radioresistance in Mdm2 S394A mice. Previous in vitro data indicate that the c-Abl kinase phosphorylates Mdm2 at the neighboring residue (Tyr393) in response to DNA damage to regulate p53-dependent apoptosis. In this present study, we have generated an Mdm2 mutant mouse (Mdm2 Y393F ) to determine whether c-Abl phosphorylation of Mdm2 regulates the p53-mediated DNA damage response or p53 tumor suppression in vivo. The Mdm2 Y393F mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Although apoptosis is unaltered in these mice, they recover more rapidly from radiation-induced bone marrow ablation and are more resistant to whole-body radiation-induced lethality. These data reveal an in vivo role for c-Abl phosphorylation of Mdm2 in regulation of p53 tumor suppression and bone marrow failure. However, c-Abl phosphorylation of Mdm2 Tyr393 appears to play a lesser role in governing Mdm2-p53 signaling than ATM phosphorylation of Mdm2 Ser394. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2 Y393F/S394A mice and Mdm2 S394A mice display similar phenotypes.

Pharmacokinetic properties of radiolabeled mutant Interleukin-2v: a PET imaging study.

Science.gov (United States)

Hartimath, Siddesh V; Manuelli, Valeria; Zijlma, Rolf; Signore, Alberto; Nayak, Tapan K; Freimoser-Grundschober, Anne; Klein, Christian; Dierckx, Rudi A J O; de Vries, Erik F J

2018-01-23

Interleukin-2 (IL2) is a cytokine that can stimulate cytotoxic immune cells to attack infected and malignant cells. Unfortunately, IL2 can also cause serious immune-related toxicity. Recently, a mutant of IL2 (IL2v) with abolished CD25 binding, increased plasma half-life and less toxicity was engineered. Unlike wild-type IL2 (wt-IL2), mutant IL2v does not bind to the α-subunit (CD25) of the high affinity IL2αβγ receptor, but only to its β and γ subunit. Here, we investigated the biological properties of IL2v and compared with the wt-IL2 using fluorine-18 and PET. [ 18 F]FB-IL2v binds specifically to IL2 receptors (IL2R) on activated human peripheral blood monocytes (hPBMCs) and is cleared mainly by the kidneys (Balb/c mice). [ 18 F]FB-IL2v PET studies in SCID mice injected with hPBMCs revealed high uptake in the implant (0.85 ± 0.15 SUV), which was significantly reduced after pretreatment with wt-IL2 or mutant IL2v (SUV 0.26 ± 0.1 and 0.46 ± 0.1, p FB-IL2v to IL2R was reversible. The volume of distribution (V T ) and the non-displaceable binding potential (BP nd ) of mutant [ 18 F]FB-IL2v in the implant were approximately 3 times lower than those of wild-type [ 18 F]FB-IL2 ( p FB-IL2v in the implant ( p FB-IL2 binds stronger to IL2R and has faster kinetics than [18F]FB-IL2v, which makes it less suitable as a therapeutic drug. [ 18 F]FB-IL2v, on the other hand, seems to have better properties for use as a therapeutic drug.

GH dysfunction in Engrailed-2 knockout mice, a model for autism spectrum disorders

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Giovanni eProvenzano

2014-09-01

Full Text Available Insulin-like growth factor 1 (IGF-1 signaling promotes brain development and plasticity. Altered IGF-1 expression has been associated to autism spectrum disorders (ASD. IGF-1 levels were found increased in the blood and decreased in the cerebrospinal fluid of ASD children. Accordingly, IGF-1 treatment can rescue behavioral deficits in mouse models of ASD, and IGF-1 trials have been proposed for ASD children. IGF-1 is mainly synthesized in the liver, and its synthesis is dependent on growth hormone (GH produced in the pituitary gland. GH also modulates cognitive functions, and altered levels of GH have been detected in ASD patients.Here we analyzed the expression of GH, IGF-1, their receptors and regulatory hormones in the neuroendocrine system of adult male mice lacking the homeobox transcription factor Engrailed-2 (En2-/- mice. En2-/- mice display ASD-like behaviors (social interactions, defective spatial learning, increased seizure susceptibility accompanied by relevant neuropathological changes (loss of cerebellar and forebrain inhibitory neurons. Recent studies showed that En2 modulates IGF-1 activity during postnatal cerebellar development.We found that GH mRNA expression was markedly deregulated throughout the neuroendocrine axis in En2-/- mice, as compared to wild-type (WT controls. In mutant mice, GH mRNA levels were significantly increased in the pituitary gland, blood and liver, whereas decreased levels were detected in the hippocampus. These changes were paralleled by decreased levels of GH protein in the hippocampus but not other tissues of En2-/- mice. IGF-1 mRNA was significantly up-regulated in the liver and down-regulated in the En2-/- hippocampus, but no differences were detected in the levels of IGF-1 protein between the two genotypes. Our data strengthen the notion that altered GH levels in the hippocampus may be involved in learning disabilities associated to ASD.

Is auxin involved in the induction of genetic instability in barley homeotic double mutants?

Science.gov (United States)

Šiukšta, Raimondas; Vaitkūnienė, Virginija; Rančelis, Vytautas

2018-02-01

The triggers of genetic instability in barley homeotic double mutants are tweaky spike -type mutations associated with an auxin imbalance in separate spike phytomeres. Barley homeotic tweaky spike;Hooded (tw;Hd) double mutants are characterized by an inherited instability of spike and flower development, which is absent in the single parental constituents. The aim of the present study was to show that the trigger of genetic instability in the double mutants is the tw mutations, which are associated with an auxin imbalance in the developing spikes. Their pleiotropic effects on genes related to spike/flower development may cause the genetic instability of double mutants. The study of four double-mutant groups composed of different mutant alleles showed that the instability arose only if the mutant allele tw was a constituent of the double mutants. Application of auxin inhibitors and 2,4-dichlorophenoxyacetic acid (2,4-D) demonstrated the relationship of the instability of the double mutants and the phenotype of the tw mutants to auxin imbalance. 2,4-D induced phenocopies of the tw mutation in wild-type plants and rescued the phenotypes of three allelic tw mutants. The differential display (dd-PCR) method allowed the identification of several putative candidate genes in tw that may be responsible for the initiation of instability in the double mutants by pleiotropic variations of their expression in the tw mutant associated with auxin imbalance in the developing spikes. The results of the present study linked the genetic instability of homeotic double mutants with an auxin imbalance caused by one of the constituents (tw). The genetic instability of the double mutants in relation to auxin imbalance was studied for the first time. A matrocliny on instability expression was also observed.

Accumulation of pathogenic ΔmtDNA induced deafness but not diabetic phenotypes in mito-mice

International Nuclear Information System (INIS)

Nakada, Kazuto; Sato, Akitsugu; Sone, Hideyuki; Kasahara, Atsuko; Ikeda, Katsuhisa; Kagawa, Yasuo; Yonekawa, Hiromichi; Hayashi, Jun-Ichi

2004-01-01

Mito-mice carrying various proportions of deletion mutant mtDNA (ΔmtDNA) were generated by introduction of the ΔmtDNA from cultured cells into fertilized eggs of C57BL/6J (B6) strain mice. Great advantages of mito-mice are that they share exactly the same nuclear-genome background, and that their genetic variations are restricted to proportions of pathogenic ΔmtDNA. Since accumulation of ΔmtDNA to more than 75% induced respiration defects, the disease phenotypes observed exclusively in mito-mice carrying more than 75% ΔmtDNA should be due to accumulated ΔmtDNA. In this study, we focused on the expressions of hearing loss and diabetic phenotypes, since these common age-associated abnormalities have sometimes been reported to be inherited maternally and to be associated with pathogenic mutant mtDNAs. The results showed that accumulation of exogenously introduced ΔmtDNA was responsible for hearing loss, but not for expression of diabetic phenotypes in mito-mice

Defects in ultrasonic vocalization of cadherin-6 knockout mice.

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Ryoko Nakagawa

Full Text Available BACKGROUND: Although some molecules have been identified as responsible for human language disorders, there is still little information about what molecular mechanisms establish the faculty of human language. Since mice, like songbirds, produce complex ultrasonic vocalizations for intraspecific communication in several social contexts, they can be good mammalian models for studying the molecular basis of human language. Having found that cadherins are involved in the vocal development of the Bengalese finch, a songbird, we expected cadherins to also be involved in mouse vocalizations. METHODOLOGY/PRINCIPAL FINDINGS: To examine whether similar molecular mechanisms underlie the vocalizations of songbirds and mammals, we categorized behavioral deficits including vocalization in cadherin-6 knockout mice. Comparing the ultrasonic vocalizations of cadherin-6 knockout mice with those of wild-type controls, we found that the peak frequency and variations of syllables were differed between the mutant and wild-type mice in both pup-isolation and adult-courtship contexts. Vocalizations during male-male aggression behavior, in contrast, did not differ between mutant and wild-type mice. Open-field tests revealed differences in locomotors activity in both heterozygote and homozygote animals and no difference in anxiety behavior. CONCLUSIONS/SIGNIFICANCE: Our results suggest that cadherin-6 plays essential roles in locomotor activity and ultrasonic vocalization. These findings also support the idea that different species share some of the molecular mechanisms underlying vocal behavior.

The ferrous iron transporter FtrABCD is required for the virulence of Brucella abortus 2308 in mice.

Science.gov (United States)

Elhassanny, Ahmed E M; Anderson, Eric S; Menscher, Evan A; Roop, R Martin

2013-06-01

Iron transport has been linked to the virulence of Brucella strains in both natural and experimental hosts. The genes designated BAB2_0837-0840 in the Brucella abortus 2308 genome sequence are predicted to encode a CupII-type ferrous iron transporter homologous to the FtrABCD transporter recently described in Bordetella. To study the role of the Brucella FtrABCD in iron transport, an isogenic ftrA mutant was constructed from B. abortus 2308. Compared with the parental strain, the B. abortus ftrA mutant displays a decreased capacity to use non-haem iron sources in vitro, a growth defect in a low iron medium that is enhanced at pH 6, and studies employing radiolabelled FeCl3 confirmed that FtrABCD transports ferrous iron. Transcription of the ftrA gene is induced in B. abortus 2308 in response to iron deprivation and exposure to acid pH, and similar to other Brucella iron acquisition genes that have been examined the iron-responsiveness of ftrA is dependent upon the iron response regulator Irr. The B. abortus ftrA mutant exhibits significant attenuation in both cultured murine macrophages and experimentally infected mice, supporting the proposition that ferrous iron is a critical iron source for these bacteria in the mammalian host. © 2013 John Wiley & Sons Ltd.

Pilot study of large-scale production of mutant pigs by ENU mutagenesis.

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Hai, Tang; Cao, Chunwei; Shang, Haitao; Guo, Weiwei; Mu, Yanshuang; Yang, Shulin; Zhang, Ying; Zheng, Qiantao; Zhang, Tao; Wang, Xianlong; Liu, Yu; Kong, Qingran; Li, Kui; Wang, Dayu; Qi, Meng; Hong, Qianlong; Zhang, Rui; Wang, Xiupeng; Jia, Qitao; Wang, Xiao; Qin, Guosong; Li, Yongshun; Luo, Ailing; Jin, Weiwu; Yao, Jing; Huang, Jiaojiao; Zhang, Hongyong; Li, Menghua; Xie, Xiangmo; Zheng, Xuejuan; Guo, Kenan; Wang, Qinghua; Zhang, Shibin; Li, Liang; Xie, Fei; Zhang, Yu; Weng, Xiaogang; Yin, Zhi; Hu, Kui; Cong, Yimei; Zheng, Peng; Zou, Hailong; Xin, Leilei; Xia, Jihan; Ruan, Jinxue; Li, Hegang; Zhao, Weiming; Yuan, Jing; Liu, Zizhan; Gu, Weiwang; Li, Ming; Wang, Yong; Wang, Hongmei; Yang, Shiming; Liu, Zhonghua; Wei, Hong; Zhao, Jianguo; Zhou, Qi; Meng, Anming

2017-06-22

N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research.

Fps/Fes and Fer protein-tyrosinekinases play redundant roles in regulating hematopoiesis.

Science.gov (United States)

Senis, Yotis A; Craig, Andrew W B; Greer, Peter A

2003-08-01

The highly related protein-tyrosine kinases Fps (also called Fes) and Fer are sole members of a subfamily of kinases. In this study, knock-in mice harboring kinase-inactivating mutations in both fps and fer alleles were used to assess functional redundancy between Fps and Fer kinases in regulating hematopoiesis. Mice harboring kinase-inactivating mutations in fps and fer alleles were generated previously. Compound homozygous mice were bred that lack both Fps and Fer kinase activities and progeny were analyzed for potential defects in viability and fertility. Potential differences in hematopoiesis were analyzed by lineage analysis of bone marrow cells, peripheral blood counts, and hematopoietic progenitor cell colony-forming assays. Mice devoid of both Fps and Fer kinase activities were viable and displayed reduced fertility. Circulating levels of neutrophils, erythrocytes, and platelets were elevated in compound mutant mice compared to wild-type controls, suggesting that hematopoiesis is deregulated in the absence of Fps and Fer kinases. Compound mutant mice also showed reduced overall bone marrow cellularity, and lineage analysis revealed elevated CD11b(hi)Ly-6G(lo) myeloid cells, which may reflect increased granulocyte progenitors. Although no differences in the overall number of granulocyte/monocyte colony-forming progenitors were observed, qualitative differences in myeloid colonies from compound mutant mice suggested a role for Fps and Fer kinases in regulating cell-cell adhesion or a skewing in cellularity of colonies. Mice lacking both Fps and Fer kinase activities develop normally, show reduced fertility, and display defects in hematopoiesis, thus providing evidence for functional redundancy between Fps and Fer kinases in regulating hematopoiesis.

Toxicologic study of electromagnetic radiation emitted by television and video display screens and cellular telephones on chickens and mice

International Nuclear Information System (INIS)

Bastide, M.; Youbicier-Simo, B.J.; Lebecq, J.C.; Giaimis, J.; Youbicier-Simo, B.J.

2001-01-01

The effects of continuous exposure of chick embryos and young chickens to the electromagnetic fields (EMFs) emitted by video display units (VDUs) and GSM cell phone radiation, either the whole spectrum emitted or attenuated by a copper gauze, were investigated. Permanent exposure to the EMFs radiated by a VDU was associated with significantly increased fetal loss (47-68%) and markedly depressed levels of circulating specific antibodies (lgG), corticosterone and melatonin. We have also shown that under chronic exposure conditions, GSM cell phone radiation was harmful to chick embryos, stressful for healthy mice and, in this species, synergistic with cancer insofar as it depleted stress hormones. The same pathological results were observed after substantial reduction of the microwaves radiated from the cell phone by attenuating them with a copper gauze. (author)

Analysis of fast neutron-generated mutants at the Arabidopsis thaliana HY4 locus

International Nuclear Information System (INIS)

Bruggemann, E.; Handwerger, K.; Essex, C.; Storz, G.

1996-01-01

Ionizing radiation is expected to produce mutants with deletions or other chromosomal rearrangements. These mutants are useful for a variety of purposes, such as creating null alleles and cloning genes whose existence is known only from their mutant phenotype; however, only a few mutations generated by ionizing radiation have been characterized at the molecular level in Arabidopsis thaliana. Twenty fast neutron-generated alleles of the Arabidopsis HY4 locus, which encodes a blue light receptor, CRY1, were isolated and characterized. Nine of the mutant alleles displayed normal genetic behavior. The other 11 mutant alleles were poorly transmitted through the male gametophyte and were lethal in homozygous plants. Southern blot analysis demonstrated that alleles of the first group generally contain small or moderate-sized deletions at HY4, while alleles of the second group contain large deletions at this locus. These results demonstrate that fast neutrons can produce a range of deletions at a single locus in Arabidopsis. Many of these deletions would be suitable for cloning by genomic subtraction or representational difference analysis. The results also suggest the presence of an essential locus adjacent to HY4. (author)

Functional consequences of integrin gene mutations in mice

DEFF Research Database (Denmark)

Bouvard, D; Brakebusch, C; Gustafsson, E

2001-01-01

Integrins are cell-surface receptors responsible for cell attachment to extracellular matrices and to other cells. The application of mouse genetics has significantly increased our understanding of integrin function in vivo. In this review, we summarize the phenotypes of mice carrying mutant inte...

Oxygen Association-Dissociation and Stability Analysis on Mouse Hemoglobins with Mutant α- and β-Globins

Science.gov (United States)

D'Surney, S. J.; Popp, R. A.

1992-01-01

Oxygen association-dissociation and hemoglobin stability analysis were performed on mouse hemoglobins with amino acid substitutions in an α-globin (α89, His to Leu) and a β-globin (β59, Lys to Ile). The variant α-globin, designated chain 5(m) in the Hba(g2) haplotype, had a high oxygen affinity and was stable. The variant β-globin, (β(s2)) of the Hbb(s2) haplotype, also had an elevated oxygen affinity and in addition was moderately unstable in 19% isopropanol. Hemoglobins from the expected nine (Hba(g2)/Hba(g2);Hbb(s)/Hbb(s) X Hba(a)/Hba(a);Hbb(s2)/Hbb(s2)) F(2) genotypes can be grouped into five classes of P(50) values characterized by strict additivity and dependency on mutant globin gene dosage; physiologically, both globin variants gave indistinguishable effects on oxygen affinity. The hemoglobin of normal mice (Hba(a)/Hba(a);Hbb(s)/Hbb(s)) had a P(50) = 40 mm Hg and the hemoglobin of Hba(g2)/Hba(g2);Hbb(s2)/Hbb(s2) F(2) mice had a P(50) = 25 mm Hg (human P(50) = 26 mm Hg). Peripheral blood from Hba(g2)/Hba(g2);Hbb(s)/Hbb(s), Hba(a)/Hba(a);Hbb(s2)/Hbb(s2) and Hba(g2)/Hba(g2);Hbb(s2)/Hbb(s2) mice exhibited normal hematological values except for a slightly higher hematocrit for Hba(g2)/Hba(g2);Hbb(s)/Hbb(s) and Hba(g2)/Hba(g2);Hbb(s2)/Hbb(s2) mice, slightly elevated red cell counts for mice of the three mutant genotypes, and significantly lower values for the mean corpuscular volume and mean corpuscular hemoglobin for Hba(g2)/Hba(g2);Hbb(s2)/Hbb(s2) mice. PMID:1427042

Neurexin Dysfunction in Adult Neurons Results in Autistic-like Behavior in Mice

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Luis G. Rabaneda

2014-07-01

Full Text Available Autism spectrum disorders (ASDs comprise a group of clinical phenotypes characterized by repetitive behavior and social and communication deficits. Autism is generally viewed as a neurodevelopmental disorder where insults during embryonic or early postnatal periods result in aberrant wiring and function of neuronal circuits. Neurexins are synaptic proteins associated with autism. Here, we generated transgenic βNrx1ΔC mice in which neurexin function is selectively impaired during late postnatal stages. Whole-cell recordings in cortical neurons show an impairment of glutamatergic synaptic transmission in the βNrx1ΔC mice. Importantly, mutant mice exhibit autism-related symptoms, such as increased self-grooming, deficits in social interactions, and altered interaction for nonsocial olfactory cues. The autistic-like phenotype of βNrx1ΔC mice can be reversed after removing the mutant protein in aged animals. The defects resulting from disruption of neurexin function after the completion of embryonic and early postnatal development suggest that functional impairment of mature circuits can trigger autism-related phenotypes.

Oral treatment with Cu(II)(atsm) increases mutant SOD1 in vivo but protects motor neurons and improves the phenotype of a transgenic mouse model of amyotrophic lateral sclerosis.

Science.gov (United States)

Roberts, Blaine R; Lim, Nastasia K H; McAllum, Erin J; Donnelly, Paul S; Hare, Dominic J; Doble, Philip A; Turner, Bradley J; Price, Katherine A; Lim, Sin Chun; Paterson, Brett M; Hickey, James L; Rhoads, Timothy W; Williams, Jared R; Kanninen, Katja M; Hung, Lin W; Liddell, Jeffrey R; Grubman, Alexandra; Monty, Jean-Francois; Llanos, Roxana M; Kramer, David R; Mercer, Julian F B; Bush, Ashley I; Masters, Colin L; Duce, James A; Li, Qiao-Xin; Beckman, Joseph S; Barnham, Kevin J; White, Anthony R; Crouch, Peter J

2014-06-04

Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry. These analyses revealed most SOD1 in the spinal cord tissue of the SOD1G37R mice was Cu deficient. Treating with Cu(II)(atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched (65)Cu(II)(atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from Cu(II)(atsm) to SOD1, suggesting the improved locomotor function and survival of the Cu(II)(atsm)-treated SOD1G37R mice involved, at least in part, the ability of the compound to improve the Cu content of the mutant SOD1. This was supported by improved survival of SOD1G37R mice that expressed the human gene for the Cu uptake protein CTR1. Improving the metal content of mutant SOD1 in vivo with Cu(II)(atsm) did not decrease levels of misfolded SOD1. These outcomes indicate the metal content of SOD1 may be a greater determinant of the toxicity of the protein in mutant SOD1-associated forms of ALS than the mutations themselves. Improving the metal content of SOD1 therefore represents a valid therapeutic strategy for treating ALS caused by SOD1. Copyright © 2014 the authors 0270-6474/14/348021-11$15.00/0.

Deoxynybomycins inhibit mutant DNA gyrase and rescue mice infected with fluoroquinolone-resistant bacteria.

Science.gov (United States)

Parkinson, Elizabeth I; Bair, Joseph S; Nakamura, Bradley A; Lee, Hyang Y; Kuttab, Hani I; Southgate, Emma H; Lezmi, Stéphane; Lau, Gee W; Hergenrother, Paul J

2015-04-24

Fluoroquinolones are one of the most commonly prescribed classes of antibiotics, but fluoroquinolone resistance (FQR) is widespread and increasing. Deoxynybomycin (DNM) is a natural-product antibiotic with an unusual mechanism of action, inhibiting the mutant DNA gyrase that confers FQR. Unfortunately, isolation of DNM is difficult and DNM is insoluble in aqueous solutions, making it a poor candidate for development. Here we describe a facile chemical route to produce DNM and its derivatives. These compounds possess excellent activity against FQR methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci clinical isolates and inhibit mutant DNA gyrase in-vitro. Bacteria that develop resistance to DNM are re-sensitized to fluoroquinolones, suggesting that resistance that emerges to DNM would be treatable. Using a DNM derivative, the first in-vivo efficacy of the nybomycin class is demonstrated in a mouse infection model. Overall, the data presented suggest the promise of DNM derivatives for the treatment of FQR infections.

Pervasive Genotypic Mosaicism in Founder Mice Derived from Genome Editing through Pronuclear Injection.

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Daniel Oliver

Full Text Available Genome editing technologies, especially the Cas9/CRISPR system, have revolutionized biomedical research over the past several years. Generation of novel alleles has been simplified to unprecedented levels, allowing for rapid expansion of available genetic tool kits for researchers. However, the issue of genotypic mosaicism has become evident, making stringent analyses of the penetrance of genome-edited alleles essential. Here, we report that founder mice, derived from pronuclear injection of ZFNs or a mix of guidance RNAs and Cas9 mRNAs, display consistent genotypic mosaicism for both deletion and insertion alleles. To identify founders with greater possibility of transmitting the mutant allele through the germline, we developed an effective germline genotyping method. The awareness of the inherent genotypic mosaicism issue with genome editing will allow for a more efficient implementation of the technologies, and the germline genotyping method will save valuable time and resources.

In Vivo Differences in the Virulence, Pathogenicity, and Induced Protective Immunity of wboA Mutants from Genetically Different Parent Brucella spp.

Science.gov (United States)

Wang, Zhen; Niu, Jianrui; Wang, Shuangshan

2013-01-01

To explore the effects of the genetic background on the characteristics of wboA gene deletion rough mutants generated from different parent Brucella sp. strains, we constructed the rough-mutant strains Brucella melitensis 16 M-MB6, B. abortus 2308-SB6, B. abortus S19-RB6, and B. melitensis NI-NB6 and evaluated their survival, pathogenicity, and induced protective immunity in mice and sheep. In mice, the survival times of the four mutants were very different in the virulence assay, from less than 6 weeks for B. abortus S19-RB6 to 11 weeks for B. abortus 2308-SB6 and B. melitensis NI-NB6. However, B. abortus S19-RB6 and B. melitensis 16 M-MB6, with a shorter survival time in mice, offered better protection against challenges with B. abortus 2308 in protection tests than B. abortus 2308-SB6 and B. melitensis NI-NB6. It seems that the induced protective immunity of each mutant might not be associated with its survival time in vivo. In the cross-protection assay, both B. melitensis 16 M-MB6 and B. abortus S19-RB6 induced greater protection against homologous challenges than heterologous challenges. When pregnant sheep were inoculated with B. abortus S19-RB6 and B. melitensis 16 M-MB6, B. abortus S19-RB6 did not induce abortion, whereas B. melitensis 16 M-MB6 did. These results demonstrated the differences in virulence, pathogenicity, and protective immunity in vivo in the wboA deletion mutants from genetically different parent Brucella spp. and also indicated that future rough vaccine strain development could be promising if suitable parent Brucella strains and/or genes were selected. PMID:23239800

A Genome-wide Gene-Expression Analysis and Database in Transgenic Mice during Development of Amyloid or Tau Pathology

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Mar Matarin

2015-02-01

Full Text Available We provide microarray data comparing genome-wide differential expression and pathology throughout life in four lines of “amyloid” transgenic mice (mutant human APP, PSEN1, or APP/PSEN1 and “TAU” transgenic mice (mutant human MAPT gene. Microarray data were validated by qPCR and by comparison to human studies, including genome-wide association study (GWAS hits. Immune gene expression correlated tightly with plaques whereas synaptic genes correlated negatively with neurofibrillary tangles. Network analysis of immune gene modules revealed six hub genes in hippocampus of amyloid mice, four in common with cortex. The hippocampal network in TAU mice was similar except that Trem2 had hub status only in amyloid mice. The cortical network of TAU mice was entirely different with more hub genes and few in common with the other networks, suggesting reasons for specificity of cortical dysfunction in FTDP17. This Resource opens up many areas for investigation. All data are available and searchable at http://www.mouseac.org.

Preference for and discrimination of paintings by mice.

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Shigeru Watanabe

Full Text Available I measured preference for paintings (Renoir vs. Picasso or Kandinsky vs. Mondrian in mice. In general mice did not display a painting preference except for two mice: one preferred Renoir to Picasso, and the other preferred Kandinsky to Mondrian. Thereafter, I examined discrimination of paintings with new mice. When exposure to paintings of one artist was associated with an injection of morphine (3.0 mg/kg, mice displayed conditioned preference for those paintings, showing discrimination of paintings by Renoir from those by Picasso, and paintings by Kandinsky from those by Mondrian after the conditioning. They also exhibited generalization of the preference to novel paintings of the artists. After conditioning with morphine for a set of paintings consisting of two artists, mice showed discrimination between two sets of paintings also from the two artists but not in association with morphine. These results suggest that mice can discriminate not only between an artist's style but also among paintings of the same artist. When mice were trained to discriminate a pair of paintings by Kandinsky and Renoir in an operant chamber equipped with a touch screen, they showed transfer of the discrimination to new pairs of the artists, but did not show transfer of discrimination of paintings by other artists, suggesting generalization.

Binding of phage displayed Bacillus subtilis lipase A to a phosphonate suicide inhibitor

NARCIS (Netherlands)

Dröge, M.J; Ruggeberg, C.J.; van der Sloot, Almer Martinus; Schimmel, J.; Dijkstra, Durk; Verhaert, R.M D; Reetz, M.T.; Quax, Wim; Droge, MJ; Dijkstra, DS

2003-01-01

Phage display can be used as a protein engineering tool to select proteins with desirable binding properties from a library of randomly constructed mutants. Here, we describe the development of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has marked properties

Aortic wall damage in mice unable to synthesize ascorbic acid.

Science.gov (United States)

Maeda, N; Hagihara, H; Nakata, Y; Hiller, S; Wilder, J; Reddick, R

2000-01-18

By inactivating the gene for L-gulono-gamma-lactone oxidase, a key enzyme in ascorbic acid synthesis, we have generated mice that, like humans, depend on dietary vitamin C. Regular chow, containing about 110 mg/kg of vitamin C, is unable to support the growth of the mutant mice, which require L-ascorbic acid supplemented in their drinking water (330 mg/liter). Upon withdrawal of supplementation, plasma and tissue ascorbic acid levels decreased to 10-15% of normal within 2 weeks, and after 5 weeks the mutants became anemic, began to lose weight, and die. Plasma total antioxidative capacities were approximately 37% normal in homozygotes after feeding the unsupplemented diet for 3-5 weeks. As plasma ascorbic acid decreased, small, but significant, increases in total cholesterol and decreases in high density lipoprotein cholesterol were observed. The most striking effects of the marginal dietary vitamin C were alterations in the wall of aorta, evidenced by the disruption of elastic laminae, smooth muscle cell proliferation, and focal endothelial desquamation of the luminal surface. Thus, marginal vitamin C deficiency affects the vascular integrity of mice unable to synthesize ascorbic acid, with potentially profound effects on the pathogenesis of vascular diseases. Breeding the vitamin C-dependent mice with mice carrying defined genetic mutations will provide numerous opportunities for systematic studies of the role of antioxidants in health and disease.

Saccharomyces cerevisiae mutants with enhanced induced mutation and altered mitotic gene conversion.

Science.gov (United States)

Ivanov, E L; Kovaltzova, S V; Korolev, V G

1989-08-01

We have developed a method to isolate yeast (Saccharomyces cerevisiae) mutants with enhanced induced mutagenesis based on nitrous acid-induced reversion of the ade2-42 allele. Six mutants have been isolated and designated him (high induced mutagenesis), and 4 of them were studied in more detail. The him mutants displayed enhanced reversion of the ade2-42 allele, either spontaneous or induced by nitrous acid, UV light, and the base analog 6-N-hydroxylaminopurine, but not by gamma-irradiation. It is worth noting that the him mutants turned out not to be sensitive to the lethal effects of the mutagens used. The enhancement in mutation induced by nitrous acid, UV light, and 6-N-hydroxylaminopurine has been confirmed in a forward-mutation assay (induction of mutations in the ADE1, ADE2 genes). The latter agent revealed the most apparent differences between the him mutants and the wild-type strain and was, therefore, chosen for the genetic analysis of mutants, him mutations analyzed behaved as a single Mendelian trait; complementation tests indicated 3 complementation groups (HIM1, HIM2, and HIM3), each containing 1 mutant allele. Uracil-DNA glycosylase activity was determined in crude cell extracts, and no significant differences between the wild-type and him strains were detected. Spontaneous mitotic gene conversion at the ADE2 locus is altered in him1 strains, either increased or decreased, depending on the particular heteroallelic combination. Genetic evidence strongly suggests him mutations to be involved in a process of mismatch correction of molecular heteroduplexes.

Effects of UV-B radiation on a hereditary suture cataract in mice

International Nuclear Information System (INIS)

Forker, Carina; Wegener, Alfred

1997-01-01

UV-B (290-320 nm, λ max = 305 nm) radiation and the Cat2 ns (suture cataract) mutation in mice affect both the anterior lens epithelium and the formation of the suture. A low dose of UV-B radiation (2.2 Jcm -2 ) induces similar anterior subcapsular and cortical lens opacities in wild type as in heterozygous mutant mice. The UV-B treatment of the mutant lenses, however, leads to an increase in the number of epithelial cell layers in the anterior central part as compared to the wild type indicating a more severe form of the cataract formation in mutants. In addition, mutants demonstrate a predisposition for a rupture of the posterior lens capsule, because from 2.9 Jcm -2 and higher, this phenomenon could always be observed in the UV-B treated mutants, but never in the treated wild type mice. The protein biochemical analyses were performed by gel electrophoresis and isoelectric focusing of extracts of total lenses or from defined areas of the lens (lens slice technique). These covered the patterns of those proteins already synthesized before irradiation, which in irradiated lenses in no case evidenced a difference to the untreated control, neither in the wild type nor in the mutants. In contrast, by analysing specifically those proteins, which are synthesized after irradiation, in both treated groups a protein with a molecular mass of about 31 kDa becomes discernable in both treated groups. In addition, the cataractous lenses demonstrate a significantly enhanced overall synthesis of water-soluble proteins after irradiation, which might promote the rupture of the posterior capsule at the posterior pole. The present study offers for the first time the possibility to discriminate between endogeneous (genetic) effects and exogeneous (environmental) effects in cataractogenesis and to study their interactive effects. The first set of experiments demonstrated a clear intensification of the hereditary cataract by the UV-B treatment. The study supports the hypothesis that

Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

International Nuclear Information System (INIS)

Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M.; Wehnert, Manfred; Huebner, Stefan

2009-01-01

Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.

Akt2/LDLr double knockout mice display impaired glucose tolerance and develop more complex atherosclerotic plaques than LDLr knockout mice

NARCIS (Netherlands)

Rensing, Katrijn L.; de Jager, Saskia C. A.; Stroes, Erik S.; Vos, Mariska; Twickler, Marcel Th B.; Dallinga-Thie, Geesje M.; de Vries, Carlie J. M.; Kuiper, Johan; Bot, Ilze; von der Thüsen, Jan H.

2014-01-01

To characterize the phenotype of Akt2/low-density-lipoprotein receptor double knockout (dKO) (Akt2/LDLr dKO) mice with respect to insulin resistance and features of atherosclerotic plaque progression. Metabolic profile and atherosclerotic plaque progression were compared between LDLr KO mice and

Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors.

Science.gov (United States)

Regales, Lucia; Balak, Marissa N; Gong, Yixuan; Politi, Katerina; Sawai, Ayana; Le, Carl; Koutcher, Jason A; Solit, David B; Rosen, Neal; Zakowski, Maureen F; Pao, William

2007-08-29

The EGFR T790M mutation confers acquired resistance to kinase inhibitors in human EGFR mutant lung adenocarcinoma, is occasionally detected before treatment, and may confer genetic susceptibility to lung cancer. To study further its role in lung tumorigenesis, we developed mice with inducible expression in type II pneumocytes of EGFR(T790M) alone or together with a drug-sensitive L858R mutation. Both transgenic lines develop lung adenocarcinomas that require mutant EGFR for tumor maintenance but are resistant to an EGFR kinase inhibitor. EGFR(L858R+T790M)-driven tumors are transiently targeted by hsp90 inhibition. Notably, EGFR(T790M)-expressing animals develop tumors with longer latency than EGFR(L858R+T790M)-bearing mice and in the absence of additional kinase domain mutations. These new mouse models of mutant EGFR-dependent lung adenocarcinomas provide insight into clinical observations. The models should also be useful for developing improved therapies for patients with lung cancers harboring EGFR(T790M) alone or in conjunction with drug-sensitive EGFR kinase domain mutations.

Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors.

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Lucia Regales

2007-08-01

Full Text Available The EGFR T790M mutation confers acquired resistance to kinase inhibitors in human EGFR mutant lung adenocarcinoma, is occasionally detected before treatment, and may confer genetic susceptibility to lung cancer.To study further its role in lung tumorigenesis, we developed mice with inducible expression in type II pneumocytes of EGFR(T790M alone or together with a drug-sensitive L858R mutation. Both transgenic lines develop lung adenocarcinomas that require mutant EGFR for tumor maintenance but are resistant to an EGFR kinase inhibitor. EGFR(L858R+T790M-driven tumors are transiently targeted by hsp90 inhibition. Notably, EGFR(T790M-expressing animals develop tumors with longer latency than EGFR(L858R+T790M-bearing mice and in the absence of additional kinase domain mutations.These new mouse models of mutant EGFR-dependent lung adenocarcinomas provide insight into clinical observations. The models should also be useful for developing improved therapies for patients with lung cancers harboring EGFR(T790M alone or in conjunction with drug-sensitive EGFR kinase domain mutations.

Sequence analysis of laci mutations obtained from lung cells of radon-exposed big blue trademark transgenic mice

International Nuclear Information System (INIS)

Layton, A.D.; Cross, F.T.; Steigler, G.L.; Stillwell, L.S.; Jostes, R.F.; Lutze, L.H.

1994-01-01

We have exposed Big Blue trademark transgenic mice by inhalation to 320, 640 and 960 Working Level Months (WLM) of radon progeny. Mice were sacrificed after 3, 6 and 9 days; the time periods required to obtain the exposures. Control mice were also sacrificed at each time interval. In each case all tissues were excised, flash frozen in liquid nitrogen, and stored at -80 degrees C for further analysis. Twelve lacI mutations have been isolated from the lung tissue of a mouse from the 960-WLM exposure group; the lacI genes from these mutants have been sequenced. Sequence data indicate that three of the mutants have a C;G deletion at BP 978 and are possibly clonal in origin. Two mutants have multiple events within the gene: one has a an A:T to C:G transversion and a C:G insertion separated by 291 BPs; the second has a G:C to A:T transition as well as an A:T deletion followed by 6 base pairs downstream by a T:A insertion. Other mutations include a single G:C to A:T transition, a two base pair deletion, and a C:G to T:A transition. Mutant plaques are being evaluated from individual mice at other dose levels. Time course experiments are also planned. These studies will help define the molecular fine structure of mutations induced by high-LET radiation exposure

Perception of sweet taste is important for voluntary alcohol consumption in mice.

Science.gov (United States)

Blednov, Y A; Walker, D; Martinez, M; Levine, M; Damak, S; Margolskee, R F

2008-02-01

To directly evaluate the association between taste perception and alcohol intake, we used three different mutant mice, each lacking a gene expressed in taste buds and critical to taste transduction: alpha-gustducin (Gnat3), Tas1r3 or Trpm5. Null mutant mice lacking any of these three genes showed lower preference score for alcohol and consumed less alcohol in a two-bottle choice test, as compared with wild-type littermates. These null mice also showed lower preference score for saccharin solutions than did wild-type littermates. In contrast, avoidance of quinine solutions was less in Gnat3 or Trpm5 knockout mice than in wild-type mice, whereas Tas1r3 null mice were not different from wild type in their response to quinine solutions. There were no differences in null vs. wild-type mice in their consumption of sodium chloride solutions. To determine the cause for reduction of ethanol intake, we studied other ethanol-induced behaviors known to be related to alcohol consumption. There were no differences between null and wild-type mice in ethanol-induced loss of righting reflex, severity of acute ethanol withdrawal or conditioned place preference for ethanol. Weaker conditioned taste aversion (CTA) to alcohol in null mice may have been caused by weaker rewarding value of the conditioned stimulus (saccharin). When saccharin was replaced by sodium chloride, no differences in CTA to alcohol between knockout and wild-type mice were seen. Thus, deletion of any one of three different genes involved in detection of sweet taste leads to a substantial reduction of alcohol intake without any changes in pharmacological actions of ethanol.

Induced protein polymorphisms and nutritional quality of gamma irradiation mutants of sorghum

Energy Technology Data Exchange (ETDEWEB)

Mehlo, Luke, E-mail: LMehlo@csir.co.za [CSIR Biosciences, Meiring Naude Road, P.O. Box 395, Pretoria 0001 (South Africa); Mbambo, Zodwa [CSIR Biosciences, Meiring Naude Road, P.O. Box 395, Pretoria 0001 (South Africa); Microbiology Discipline, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Private Bag X54001, Durban 4000 (South Africa); Bado, Souleymane [Plant Breeding and Genetics Laboratory – Joint FAO/IAEA Agriculture and Biotechnology Laboratory, International Atomic Energy Agency Laboratories, A-2444 Seibersdorf (Austria); Lin, Johnson [Microbiology Discipline, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Private Bag X54001, Durban 4000 (South Africa); Moagi, Sydwell M.; Buthelezi, Sindisiwe; Stoychev, Stoyan; Chikwamba, Rachel [CSIR Biosciences, Meiring Naude Road, P.O. Box 395, Pretoria 0001 (South Africa)

2013-09-15

Highlights: • We analyse kafirin protein polymorphisms induced by gamma irradiation in sorghum. • One mutant with suppressed kafirins in the endosperm accumulated them in the germ. • Kafirin polymorphisms were associated with high levels of free amino acids. • Nutritional value of sorghum can be improved significantly by induced mutations. - Abstract: Physical and biochemical analysis of protein polymorphisms in seed storage proteins of a mutant population of sorghum revealed a mutant with redirected accumulation of kafirin proteins in the germ. The change in storage proteins was accompanied by an unusually high level accumulation of free lysine and other essential amino acids in the endosperm. This mutant further displayed a significant suppression in the synthesis and accumulation of the 27 kDa γ-, 24 kDa α-A1 and the 22 kDa α-A2 kafirins in the endosperm. The suppression of kafirins was counteracted by an upsurge in the synthesis and accumulation of albumins, globulins and other proteins. The data collectively suggest that sorghum has huge genetic potential for nutritional biofortification and that induced mutations can be used as an effective tool in achieving premium nutrition in staple cereals.

Induced protein polymorphisms and nutritional quality of gamma irradiation mutants of sorghum

International Nuclear Information System (INIS)

Mehlo, Luke; Mbambo, Zodwa; Bado, Souleymane; Lin, Johnson; Moagi, Sydwell M.; Buthelezi, Sindisiwe; Stoychev, Stoyan; Chikwamba, Rachel

2013-01-01

Highlights: • We analyse kafirin protein polymorphisms induced by gamma irradiation in sorghum. • One mutant with suppressed kafirins in the endosperm accumulated them in the germ. • Kafirin polymorphisms were associated with high levels of free amino acids. • Nutritional value of sorghum can be improved significantly by induced mutations. - Abstract: Physical and biochemical analysis of protein polymorphisms in seed storage proteins of a mutant population of sorghum revealed a mutant with redirected accumulation of kafirin proteins in the germ. The change in storage proteins was accompanied by an unusually high level accumulation of free lysine and other essential amino acids in the endosperm. This mutant further displayed a significant suppression in the synthesis and accumulation of the 27 kDa γ-, 24 kDa α-A1 and the 22 kDa α-A2 kafirins in the endosperm. The suppression of kafirins was counteracted by an upsurge in the synthesis and accumulation of albumins, globulins and other proteins. The data collectively suggest that sorghum has huge genetic potential for nutritional biofortification and that induced mutations can be used as an effective tool in achieving premium nutrition in staple cereals

Hematopoietic stem cell function in motheaten mice

International Nuclear Information System (INIS)

Shultz, L.D.; Bailey, C.L.; Coman, D.R.

1983-01-01

Mice homozygous for the autosomal recessive mutation ''motheaten'' have normal numbers of multipotential hematopoietic stem cells in the bone marrow and spleen as determined by spleen colony assay. Histologic examination shows no qualitative abnormality in morphology of stem cell colonies in recipients of bone marrow or spleen cells from motheaten mice. Despite the apparently normal ontogeny, distribution, and differentiative capacity of CFU stem cells, bone marrow and spleen cells from motheaten mice fail to save congenic +/+ lethally gamma-irradiated hosts. This impaired lifesparing capacity is not due to defective self-renewal but appears to be due in part to pulmonary hemorrhage from alveolar capillaries in the gamma-irradiated hosts. Treatment of motheaten mice with 500 R gamma-irradiation followed by reconstitution with normal bone marrow cells increases the lifespan of this mutant to 10 months of age. The early onset of pneumonitis and subsequent short lifespan of motheaten mice is determined at the level of progenitor cells in the bone marrow

Identification of early flowering mutants from the cut chrysanthemum variety 'Jinba' and research on the physiological characteristics

International Nuclear Information System (INIS)

Cai Haiyan; Wen Lizhu; Zheng Chengshu; Sun Xia; Li Yingying

2013-01-01

In this study, the morphological characteristics the early flower mutants induced by EMS from the cut chrysanthemum variety 'Jinba' were characterized, and ISSR-PCR was used for molecular identification of mutants. The photosynthetic characteristics, stomatal and vascular bundle cross-section for the mutants were also measured. The florescence of mutants appeared about 59 days earlier than that of controls. The plant height, leaves and flower diameter were all smaller than those of controls, but they grew well. From the PCR amplification, the differences between the mutants and the control were in the present or absent of the amplified bands. The photosynthetic characteristics, such as the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and intercellular CO_2 concentration were all significantly lower than the controls. The number of mutant's stomata significantly reduced, and some stomatas appeared variation. The vascular bundles including primary phloem and primary xylem displayed difference in the size and shape. All these work lays a foundation for breeding the early flowering of excellent chrysanthemum and further characterization of the early flowering genes. (authors)

A tetravalent virus-like particle vaccine designed to display domain III of dengue envelope proteins induces multi-serotype neutralizing antibodies in mice and macaques which confer protection against antibody dependent enhancement in AG129 mice.

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Viswanathan Ramasamy

2018-01-01

Full Text Available Dengue is one of the fastest spreading vector-borne diseases, caused by four antigenically distinct dengue viruses (DENVs. Antibodies against DENVs are responsible for both protection as well as pathogenesis. A vaccine that is safe for and efficacious in all people irrespective of their age and domicile is still an unmet need. It is becoming increasingly apparent that vaccine design must eliminate epitopes implicated in the induction of infection-enhancing antibodies.We report a Pichia pastoris-expressed dengue immunogen, DSV4, based on DENV envelope protein domain III (EDIII, which contains well-characterized serotype-specific and cross-reactive epitopes. In natural infection, <10% of the total neutralizing antibody response is EDIII-directed. Yet, this is a functionally relevant domain which interacts with the host cell surface receptor. DSV4 was designed by in-frame fusion of EDIII of all four DENV serotypes and hepatitis B surface (S antigen and co-expressed with unfused S antigen to form mosaic virus-like particles (VLPs. These VLPs displayed EDIIIs of all four DENV serotypes based on probing with a battery of serotype-specific anti-EDIII monoclonal antibodies. The DSV4 VLPs were highly immunogenic, inducing potent and durable neutralizing antibodies against all four DENV serotypes encompassing multiple genotypes, in mice and macaques. DSV4-induced murine antibodies suppressed viremia in AG129 mice and conferred protection against lethal DENV-4 virus challenge. Further, neither murine nor macaque anti-DSV4 antibodies promoted mortality or inflammatory cytokine production when passively transferred and tested in an in vivo dengue disease enhancement model of AG129 mice.Directing the immune response to a non-immunodominant but functionally relevant serotype-specific dengue epitope of the four DENV serotypes, displayed on a VLP platform, can help minimize the risk of inducing disease-enhancing antibodies while eliciting effective tetravalent

Novel chimeric virus-like particles vaccine displaying MERS-CoV receptor-binding domain induce specific humoral and cellular immune response in mice.

Science.gov (United States)

Wang, Chong; Zheng, Xuexing; Gai, Weiwei; Wong, Gary; Wang, Hualei; Jin, Hongli; Feng, Na; Zhao, Yongkun; Zhang, Weijiao; Li, Nan; Zhao, Guoxing; Li, Junfu; Yan, Jinghua; Gao, Yuwei; Hu, Guixue; Yang, Songtao; Xia, Xianzhu

2017-04-01

Middle East respiratory syndrome coronavirus (MERS-CoV) has continued spreading since its emergence in 2012 with a mortality rate of 35.6%, and is a potential pandemic threat. Prophylactics and therapies are urgently needed to address this public health problem. We report here the efficacy of a vaccine consisting of chimeric virus-like particles (VLP) expressing the receptor binding domain (RBD) of MERS-CoV. In this study, a fusion of the canine parvovirus (CPV) VP2 structural protein gene with the RBD of MERS-CoV can self-assemble into chimeric, spherical VLP (sVLP). sVLP retained certain parvovirus characteristics, such as the ability to agglutinate pig erythrocytes, and structural morphology similar to CPV virions. Immunization with sVLP induced RBD-specific humoral and cellular immune responses in mice. sVLP-specific antisera from these animals were able to prevent pseudotyped MERS-CoV entry into susceptible cells, with neutralizing antibody titers reaching 1: 320. IFN-γ, IL-4 and IL-2 secreting cells induced by the RBD were detected in the splenocytes of vaccinated mice by ELISpot. Furthermore, mice inoculated with sVLP or an adjuvanted sVLP vaccine elicited T-helper 1 (Th1) and T-helper 2 (Th2) cell-mediated immunity. Our study demonstrates that sVLP displaying the RBD of MERS-CoV are promising prophylactic candidates against MERS-CoV in a potential outbreak situation. Copyright © 2017 Elsevier B.V. All rights reserved.

Endochondral Ossification Is Accelerated in Cholinesterase-Deficient Mice and in Avian Mesenchymal Micromass Cultures.

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Janine Spieker

Full Text Available Most components of the cholinergic system are detected in skeletogenic cell types in vitro, yet the function of this system in skeletogenesis remains unclear. Here, we analyzed endochondral ossification in mutant murine fetuses, in which genes of the rate-limiting cholinergic enzymes acetyl- (AChE, or butyrylcholinesterase (BChE, or both were deleted (called here A-B+, A+B-, A-B-, respectively. In all mutant embryos bone growth and cartilage remodeling into mineralizing bone were accelerated, as revealed by Alcian blue (A-blu and Alizarin red (A-red staining. In A+B- and A-B- onset of mineralization was observed before E13.5, about 2 days earlier than in wild type and A-B+ mice. In all mutants between E18.5 to birth A-blu staining disappeared from epiphyses prematurely. Instead, A-blu+ cells were dislocated into diaphyses, most pronounced so in A-B- mutants, indicating additive effects of both missing ChEs in A-B- mutant mice. The remodeling effects were supported by in situ hybridization (ISH experiments performed on cryosections from A-B- mice, in which Ihh, Runx2, MMP-13, ALP, Col-II and Col-X were considerably decreased, or had disappeared between E18.5 and P0. With a second approach, we applied an improved in vitro micromass model from chicken limb buds that allowed histological distinction between areas of cartilage, apoptosis and mineralization. When treated with the AChE inhibitor BW284c51, or with nicotine, there was decrease in cartilage and accelerated mineralization, suggesting that these effects were mediated through nicotinic receptors (α7-nAChR. We conclude that due to absence of either one or both cholinesterases in KO mice, or inhibition of AChE in chicken micromass cultures, there is increase in cholinergic signalling, which leads to increased chondroblast production and premature mineralization, at the expense of incomplete chondrogenic differentiation. This emphasizes the importance of cholinergic signalling in cartilage and

Isolated cytochrome c oxidase deficiency in G93A SOD1 mice overexpressing CCS protein.

Science.gov (United States)

Son, Marjatta; Leary, Scot C; Romain, Nadine; Pierrel, Fabien; Winge, Dennis R; Haller, Ronald G; Elliott, Jeffrey L

2008-05-02

G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.

A Yersinia pestis tat mutant is attenuated in bubonic and small-aerosol pneumonic challenge models of infection but not as attenuated by intranasal challenge.

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Joel Bozue

Full Text Available Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

Lipid metabolism and body composition in Gclm(−/−) mice

International Nuclear Information System (INIS)

Kendig, Eric L.; Chen, Ying; Krishan, Mansi; Johansson, Elisabet; Schneider, Scott N.; Genter, Mary Beth; Nebert, Daniel W.; Shertzer, Howard G.

2011-01-01

In humans and experimental animals, high fat diets (HFD) are associated with risk factors for metabolic diseases, such as excessive weight gain and adiposity, insulin resistance and fatty liver. Mice lacking the glutamate–cysteine ligase modifier subunit gene (Gclm(−/−)) and deficient in glutathione (GSH), are resistant to HFD-mediated weight gain. Herein, we evaluated Gclm-associated regulation of energy metabolism, oxidative stress, and glucose and lipid homeostasis. C57BL/6J Gclm(−/−) mice and littermate wild-type (WT) controls received a normal diet or an HFD for 11 weeks. HFD-fed Gclm(−/−) mice did not display a decreased respiratory quotient, suggesting that they are unable to process lipid for metabolism. Although dietary energy consumption and intestinal lipid absorption were unchanged in Gclm(−/−) mice, feeding these mice an HFD did not produce excess body weight nor fat storage. Gclm(−/−) mice displayed higher basal metabolic rates resulting from higher activities of liver mitochondrial NADH-CoQ oxidoreductase, thus elevating respiration. Although Gclm(−/−) mice exhibited strong systemic and hepatic oxidative stress responses, HFD did not promote glucose intolerance or insulin resistance. Furthermore, HFD-fed Gclm(−/−) mice did not develop fatty liver, likely resulting from very low expression levels of genes encoding lipid metabolizing enzymes. We conclude that Gclm is involved in the regulation of basal metabolic rate and the metabolism of dietary lipid. Although Gclm(−/−) mice display a strong oxidative stress response, they are protected from HFD-induced excessive weight gain and adipose deposition, insulin resistance and steatosis. -- Highlights: ► A high fat diet does not produce body weight and fat gain in Gclm(−/−) mice. ► A high fat diet does not induce steatosis or insulin resistance in Gclm(−/−) mice. ► Gclm(−/−) mice have high basal metabolism and mitochondrial oxygen consumption. �

Dietary restriction but not angiotensin II type 1 receptor blockade improves DNA damage-related vasodilator dysfunction in rapidly aging Ercc1Δ/- mice.

Science.gov (United States)

Wu, Haiyan; van Thiel, Bibi S; Bautista-Niño, Paula K; Reiling, Erwin; Durik, Matej; Leijten, Frank P J; Ridwan, Yanto; Brandt, Renata M C; van Steeg, Harry; Dollé, Martijn E T; Vermeij, Wilbert P; Hoeijmakers, Jan H J; Essers, Jeroen; van der Pluijm, Ingrid; Danser, A H Jan; Roks, Anton J M

2017-08-01

DNA damage is an important contributor to endothelial dysfunction and age-related vascular disease. Recently, we demonstrated in a DNA repair-deficient, prematurely aging mouse model ( Ercc1 Δ/- mice) that dietary restriction (DR) strongly increases life- and health span, including ameliorating endothelial dysfunction, by preserving genomic integrity. In this mouse mutant displaying prominent accelerated, age-dependent endothelial dysfunction we investigated the signaling pathways involved in improved endothelium-mediated vasodilation by DR, and explore the potential role of the renin-angiotensin system (RAS). Ercc1 Δ/- mice showed increased blood pressure and decreased aortic relaxations to acetylcholine (ACh) in organ bath experiments. Nitric oxide (NO) signaling and phospho-Ser 1177 -eNOS were compromised in Ercc1 Δ / - DR improved relaxations by increasing prostaglandin-mediated responses. Increase of cyclo-oxygenase 2 and decrease of phosphodiesterase 4B were identified as potential mechanisms. DR also prevented loss of NO signaling in vascular smooth muscle cells and normalized angiotensin II (Ang II) vasoconstrictions, which were increased in Ercc1 Δ/- mice. Ercc1 Δ/ - mutants showed a loss of Ang II type 2 receptor-mediated counter-regulation of Ang II type 1 receptor-induced vasoconstrictions. Chronic losartan treatment effectively decreased blood pressure, but did not improve endothelium-dependent relaxations. This result might relate to the aging-associated loss of treatment efficacy of RAS blockade with respect to endothelial function improvement. In summary, DR effectively prevents endothelium-dependent vasodilator dysfunction by augmenting prostaglandin-mediated responses, whereas chronic Ang II type 1 receptor blockade is ineffective. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Mice with missense and nonsense NF1 mutations display divergent phenotypes compared with human neurofibromatosis type I

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Kairong Li

2016-07-01

Full Text Available Neurofibromatosis type 1 (NF1 is a common genetic disorder characterized by the occurrence of nerve sheath tumors and considerable clinical heterogeneity. Some translational studies have been limited by the lack of animal models available for assessing patient-specific mutations. In order to test therapeutic approaches that might restore function to the mutated gene or gene product, we developed mice harboring NF1 patient-specific mutations including a nonsense mutation (c.2041C>T; p.Arg681* and a missense mutation (c.2542G>C; p.Gly848Arg. The latter is associated with the development of multiple plexiform neurofibromas along spinal nerve roots. We demonstrate that the human nonsense NF1Arg681* and missense NF1Gly848Arg mutations have different effects on neurofibromin expression in the mouse and each recapitulates unique aspects of the NF1 phenotype, depending upon the genetic context when assessed in the homozygous state or when paired with a conditional knockout allele. Whereas the missense Nf1Gly848Arg mutation fails to produce an overt phenotype in the mouse, animals homozygous for the nonsense Nf1Arg681* mutation are not viable. Mice with one Nf1Arg681* allele in combination with a conditional floxed Nf1 allele and the DhhCre transgene (Nf14F/Arg681*; DhhCre display disorganized nonmyelinating axons and neurofibromas along the spinal column, which leads to compression of the spinal cord and paralysis. This model will be valuable for preclinical testing of novel nonsense suppression therapies using drugs to target in-frame point mutations that create premature termination codons in individuals with NF1.

Age-dependent pattern of cerebellar susceptibility to bilirubin neurotoxicity in vivo in mice

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Bortolussi, Giulia; Baj, Gabriele; Vodret, Simone; Viviani, Giulia; Bittolo, Tamara; Muro, Andrés F.

2014-01-01

Neonatal jaundice is caused by high levels of unconjugated bilirubin. It is usually a temporary condition caused by delayed induction of UGT1A1, which conjugates bilirubin in the liver. To reduce bilirubin levels, affected babies are exposed to phototherapy (PT), which converts toxic bilirubin into water-soluble photoisomers that are readily excreted out. However, in some cases uncontrolled hyperbilirubinemia leads to neurotoxicity. To study the mechanisms of bilirubin-induced neurological damage (BIND) in vivo, we generated a mouse model lacking the Ugt1a1 protein and, consequently, mutant mice developed jaundice as early as 36 hours after birth. The mutation was transferred into two genetic backgrounds (C57BL/6 and FVB/NJ). We exposed mutant mice to PT for different periods and analyzed the resulting phenotypes from the molecular, histological and behavioral points of view. Severity of BIND was associated with genetic background, with 50% survival of C57BL/6‑Ugt1−/− mutant mice at postnatal day 5 (P5), and of FVB/NJ-Ugt1−/− mice at P11. Life-long exposure to PT prevented cerebellar architecture alterations and rescued neuronal damage in FVB/NJ-Ugt1−/− but not in C57BL/6-Ugt1−/− mice. Survival of FVB/NJ-Ugt1−/− mice was directly related to the extent of PT treatment. PT treatment of FVB/NJ-Ugt1−/− mice from P0 to P8 did not prevent bilirubin-induced reduction in dendritic arborization and spine density of Purkinje cells. Moreover, PT treatment from P8 to P20 did not rescue BIND accumulated up to P8. However, PT treatment administered in the time-window P0–P15 was sufficient to obtain full rescue of cerebellar damage and motor impairment in FVB/NJ-Ugt1−/− mice. The possibility to modulate the severity of the phenotype by PT makes FVB/NJ-Ugt1−/− mice an excellent and versatile model to study bilirubin neurotoxicity, the role of modifier genes, alternative therapies and cerebellar development during high bilirubin conditions. PMID

Behavioural effects of high fat diet in a mutant mouse model for the schizophrenia risk gene neuregulin 1

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Holm-hansen, S.; Low, J. K.; Zieba, J.

2016-01-01

on the behavioural phenotype of test mice and attenuated particular cognitive deficits of Nrg1 mutant females. This topic requires further investigations thereby also considering other dietary factors of relevance for schizophrenia as well as interactive effects of diet with medication and sex....

The fat mass and obesity associated gene FTO functions in the brain to regulate postnatal growth in mice.

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Xue Gao

2010-11-01

Full Text Available FTO (fat mass and obesity associated was identified as an obesity-susceptibility gene by several independent large-scale genome association studies. A cluster of SNPs (single nucleotide polymorphism located in the first intron of FTO was found to be significantly associated with obesity-related traits, such as body mass index, hip circumference, and body weight. FTO encodes a protein with a novel C-terminal α-helical domain and an N-terminal double-strand β-helix domain which is conserved in Fe(II and 2-oxoglutarate-dependent oxygenase family. In vitro, FTO protein can demethylate single-stranded DNA or RNA with a preference for 3-methylthymine or 3-methyluracil. Its physiological substrates and function, however, remain to be defined. Here we report the generation and analysis of mice carrying a conditional deletion allele of Fto. Our results demonstrate that Fto plays an essential role in postnatal growth. The mice lacking Fto completely display immediate postnatal growth retardation with shorter body length, lower body weight, and lower bone mineral density than control mice, but their body compositions are relatively normal. Consistent with the growth retardation, the Fto mutant mice have reduced serum levels of IGF-1. Moreover, despite the ubiquitous expression of Fto, its specific deletion in the nervous system results in similar phenotypes as the whole body deletion, indicating that Fto functions in the central nerve system to regulate postnatal growth.

CYP 2E1 mutant mice are resistant to DDC-induced enhancement of MPTP toxicity.

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Viaggi, C; Vaglini, F; Pardini, C; Sgadò, P; Caramelli, A; Corsini, G U

2007-01-01

In order to reach a deeper insight into the mechanism of diethyldithiocarbamate (DDC)-induced enhancement of MPTP toxicity in mice, we showed that CYP450 (2E1) inhibitors, such as diallyl sulfide (DAS) or phenylethylisothiocyanate (PIC), also potentiate the selective DA neuron degeneration in C57/bl mice. Furthermore we showed that CYP 2E1 is present in the brain and in the basal ganglia of mice (Vaglini et al., 2004). However, because DAS and PIC are not selective CYP 2E1 inhibitors and in order to provide direct evidence for CYP 2E1 involvement in the enhancement of MPTP toxicity, CYP 2E1 knockout mice (GONZ) and wild type animals (SVI) of the same genetic background were treated with MPTP or the combined DDC + MPTP treatment. In CYP 2E1 knockout mice, DDC pretreatment completely fails to enhance MPTP toxicity, although enhancement of MPTP toxicity was regularly present in the SVI control animals. The immunohistochemical study confirms our results and suggests that CYP 2E1 may have a detoxifying role.

Mutant p53 protein localized in the cytoplasm inhibits autophagy.

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Morselli, Eugenia; Tasdemir, Ezgi; Maiuri, Maria Chiara; Galluzzi, Lorenzo; Kepp, Oliver; Criollo, Alfredo; Vicencio, José Miguel; Soussi, Thierry; Kroemer, Guido

2008-10-01

The knockout, knockdown or chemical inhibition of p53 stimulates autophagy. Moreover, autophagy-inducing stimuli such as nutrient depletion, rapamycin or lithium cause the depletion of cytoplasmic p53, which in turn is required for the induction of autophagy. Here, we show that retransfection of p53(-/-) HCT 116 colon carcinoma cells with wild type p53 decreases autophagy down to baseline levels. Surprisingly, one third among a panel of 22 cancer-associated p53 single amino acid mutants also inhibited autophagy when transfected into p53(-/-) cells. Those variants of p53 that preferentially localize to the cytoplasm effectively repressed autophagy, whereas p53 mutants that display a prominently nuclear distribution failed to inhibit autophagy. The investigation of a series of deletion mutants revealed that removal of the DNA-binding domain from p53 fails to interfere with its role in the regulation of autophagy. Altogether, these results identify the cytoplasmic localization of p53 as the most important feature for p53-mediated autophagy inhibition. Moreover, the structural requirements for the two biological activities of extranuclear p53, namely induction of apoptosis and inhibition of autophagy, are manifestly different.

The nude mutant gene Foxn1 is a HOXC13 regulatory target during hair follicle and nail differentiation.

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Potter, Christopher S; Pruett, Nathanael D; Kern, Michael J; Baybo, Mary Ann; Godwin, Alan R; Potter, Kathleen A; Peterson, Ron L; Sundberg, John P; Awgulewitsch, Alexander

2011-04-01

Among the Hox genes, homeobox C13 (Hoxc13) has been shown to be essential for proper hair shaft differentiation, as Hoxc13 gene-targeted (Hoxc13(tm1Mrc)) mice completely lack external hair. Because of the remarkable overt phenotypic parallels to the Foxn1(nu) (nude) mutant mice, we sought to determine whether Hoxc13 and forkhead box N1 (Foxn1) might act in a common pathway of hair follicle (HF) differentiation. We show that the alopecia exhibited by both the Hoxc13(tm1Mrc) and Foxn1(nu) mice is because of strikingly similar defects in hair shaft differentiation and that both mutants suffer from a severe nail dystrophy. These phenotypic similarities are consistent with the extensive overlap between Hoxc13 and Foxn1 expression patterns in the HF and the nail matrix. Furthermore, DNA microarray analysis of skin from Hoxc13(tm1Mrc) mice identified Foxn1 as significantly downregulated along with numerous hair keratin genes. This Foxn1 downregulation apparently reflects the loss of direct transcriptional control by HOXC13 as indicated by our results obtained through co-transfection and chromatin immunoprecipitation (ChIP) assays. As presented in the discussion, these data support a regulatory model of keratinocyte differentiation in which HOXC13-dependent activation of Foxn1 is part of a regulatory cascade controlling the expression of terminal differentiation markers.

Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate.

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Sorrenson, Brie; Suetani, Rachel J; Williams, Michael J A; Bickley, Vivienne M; George, Peter M; Jones, Gregory T; McCormick, Sally P A

2013-01-01

Mutations in the ATP-binding cassette transporter A1 (ABCA1) are a major cause of decreased HDL cholesterol (HDL-C), which infers an increased risk of cardiovascular disease (CVD). Many ABCA1 mutants show impaired localization to the plasma membrane. The aim of this study was to investigate whether the chemical chaperone, sodium 4-phenylbutyrate (4-PBA) could improve cellular localization and function of ABCA1 mutants. Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Treatment restored localization to the plasma membrane and increased cholesterol efflux function for the majority of mutants. Treatment with 4-PBA also increased ABCA1 protein expression in all transfected cell lines. In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Our study is the first to investigate the effect of the chemical chaperone, 4-PBA on ABCA1 and shows that it is capable of restoring plasma membrane localization and enhancing the cholesterol efflux function of mutant ABCA1s both in vitro and ex vivo. These results suggest 4-PBA may warrant further investigation as a potential therapy for increasing cholesterol efflux and HDL-C levels.

ENU mutagenesis reveals a novel phenotype of reduced limb strength in mice lacking fibrillin 2.

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Gaynor Miller

2010-02-01

Full Text Available Fibrillins 1 (FBN1 and 2 (FBN2 are components of microfibrils, microfilaments that are present in many connective tissues, either alone or in association with elastin. Marfan's syndrome and congenital contractural arachnodactyly (CCA result from dominant mutations in the genes FBN1 and FBN2 respectively. Patients with both conditions often present with specific muscle atrophy or weakness, yet this has not been reported in the mouse models. In the case of Fbn1, this is due to perinatal lethality of the homozygous null mice making measurements of strength difficult. In the case of Fbn2, four different mutant alleles have been described in the mouse and in all cases syndactyly was reported as the defining phenotypic feature of homozygotes.As part of a large-scale N-ethyl-N-nitrosourea (ENU mutagenesis screen, we identified a mouse mutant, Mariusz, which exhibited muscle weakness along with hindlimb syndactyly. We identified an amber nonsense mutation in Fbn2 in this mouse mutant. Examination of a previously characterised Fbn2-null mutant, Fbn2(fp, identified a similar muscle weakness phenotype. The two Fbn2 mutant alleles complement each other confirming that the weakness is the result of a lack of Fbn2 activity. Skeletal muscle from mutants proved to be abnormal with higher than average numbers of fibres with centrally placed nuclei, an indicator that there are some regenerating muscle fibres. Physiological tests indicated that the mutant muscle produces significantly less maximal force, possibly as a result of the muscles being relatively smaller in Mariusz mice.These findings indicate that Fbn2 is involved in integrity of structures required for strength in limb movement. As human patients with mutations in the fibrillin genes FBN1 and FBN2 often present with muscle weakness and atrophy as a symptom, Fbn2-null mice will be a useful model for examining this aspect of the disease process further.

Skin wound healing in MMP2-deficient and MMP2 / plasminogen double-deficient mice

DEFF Research Database (Denmark)

Frøssing, Signe; Rønø, Birgitte; Hald, Andreas

2010-01-01

-sensitive MMPs during wound healing. To address whether MMP2 is accountable for the galardin-induced healing deficiency in wildtype and Plg-deficient mice, incisional skin wounds were generated in MMP2 single-deficient mice and in MMP2/Plg double-deficient mice and followed until healed. Alternatively, tissue...... was isolated 7 days post wounding for histological and biochemical analyses. No difference was found in the time from wounding to overt gross restoration of the epidermal surface between MMP2-deficient and wildtype control littermate mice. MMP2/Plg double-deficient mice were viable and fertile, and displayed...... an unchallenged general phenotype resembling that of Plg-deficient mice, including development of rectal prolapses. MMP2/Plg double-deficient mice displayed a slight increase in the wound length throughout the healing period compared with Plg-deficient mice. However, the overall time to complete healing...

Ultraviolet-endonuclease activity in cell extracts of Saccharomyces cerevisiae mutants defective in excision of pyrimidine dimers

International Nuclear Information System (INIS)

Bekker, M.L.; Kaboev, O.K.; Akhmedov, A.T.; Luchkina, L.A.

1980-01-01

Cell-free extracts of ultraviolet-sensitive mutants of Saccharomyces cerevisiae defective in excision of pyrimidine dimers, rad1, rad2, rad3, rad4, rad10, and rad16, as well as the extracts of the wild-type strain RAD+, display ultraviolet-endonuclease activity

Impaired associative fear learning in mice with complete loss or haploinsufficiency of AMPA GluR1 receptors

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Michael Feyder

2007-12-01

Full Text Available There is compelling evidence that L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA glutamate receptors containing the GluR1 subunit contribute to the molecular mechanisms associated with learning. AMPA GluR1 glutamate receptor knockout mice (KO exhibit abnormal hippocampal and amygdala plasticity, and deficits on various assays for cognition including Pavlovian fear conditioning. Here we examined associative fear learning in mice with complete absence (KO or partial loss (heterozygous mutant, HET of GluR1 on multiple fear conditioning paradigms. After multi-trial delay or trace conditioning, KO displayed impaired tone and context fear recall relative to WT, whereas HET were normal. After one-trial delay conditioning, both KO and HET showed impaired tone and context recall. HET and KO showed normal nociceptive sensitivity in the hot plate and tail flick tests. These data demonstrate that the complete absence of GluR1 subunit-containing receptors prevents the formation of associative fear memories, while GluR1 haploinsufficiency is sufficient to impair one-trial fear learning. These findings support growing evidence of a major role for GluR1-containing AMPA receptors in amygdalamediated forms of learning and memory.

T-cell-mediated immunity to lymphocytic choriomeningitis virus in beta2-integrin (CD18)- and ICAM-1 (CD54)-deficient mice

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Christensen, Jan Pravsgaard; Marker, O; Thomsen, Allan Randrup

1996-01-01

The T-cell response to lymphocytic choriomeningitis virus was studied in mice with deficient expression of beta2-integrins or ICAM-1. In such mice, the generation of virus-specific cytotoxic T lymphocytes was only slightly impaired and bystander activation was as extensive as that observed in wild-type...... mice. T-cell-mediated inflammation, assessed as primary footpad swelling and susceptibility to intracerebral infection, was slightly compromised only in beta2-integrin-deficient mice. However, adoptive immunization of mutant mice soon after local infection did reveal a reduced capacity to support...... the inflammatory reaction, indicating that under conditions of more limited immune activation both molecules do play a role in formation of the inflammatory exudate. Finally, virus control was found to be somewhat impaired in both mutant strains. In conclusion, our results indicate that although LFA-1-ICAM-1...

Combinatorial RNA Interference Therapy Prevents Selection of Pre-existing HBV Variants in Human Liver Chimeric Mice

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Shih, Yao-Ming; Sun, Cheng-Pu; Chou, Hui-Hsien; Wu, Tzu-Hui; Chen, Chun-Chi; Wu, Ping-Yi; Enya Chen, Yu-Chen; Bissig, Karl-Dimiter; Tao, Mi-Hua

2015-01-01

Selection of escape mutants with mutations within the target sequence could abolish the antiviral RNA interference activity. Here, we investigated the impact of a pre-existing shRNA-resistant HBV variant on the efficacy of shRNA therapy. We previously identified a highly potent shRNA, S1, which, when delivered by an adeno-associated viral vector, effectively inhibits HBV replication in HBV transgenic mice. We applied the “PICKY” software to systemically screen the HBV genome, then used hydrodynamic transfection and HBV transgenic mice to identify additional six highly potent shRNAs. Human liver chimeric mice were infected with a mixture of wild-type and T472C HBV, a S1-resistant HBV variant, and then treated with a single or combined shRNAs. The presence of T472C mutant compromised the therapeutic efficacy of S1 and resulted in replacement of serum wild-type HBV by T472C HBV. In contrast, combinatorial therapy using S1 and P28, one of six potent shRNAs, markedly reduced titers for both wild-type and T472C HBV. Interestingly, treatment with P28 alone led to the emergence of escape mutants with mutations in the P28 target region. Our results demonstrate that combinatorial RNAi therapy can minimize the escape of resistant viral mutants in chronic HBV patients. PMID:26482836

Establishment of screening technique for mutant cell and analysis of base sequence in the mutation

International Nuclear Information System (INIS)

Sofuni, Toshio; Nomi, Takehiko; Yamada, Masami; Masumura, Kenichi

2000-01-01

This research project aimed to establish an easy and quick detection method for radiation-induced mutation using molecular-biological techniques and an effective analyzing method for the molecular changes in base sequence. In this year, Spi mutants derived from γ-radiation exposed mouse were analyzed by PCR method and DNA sequence method. Male transgenic mice were exposed to γ-ray at 5,10, 50 Gy and the transgene was taken out from the genome DNA from the spleen in vivo packaging method. Spi mutant plaques were obtained by infecting the recovered phage to E. coli. Sequence analysis for the mutants was made using ALFred DNA sequencer and SequiTherm TM Long-Red Cycle sequencing kit. Sequence analysis was carried out for 41 of 50 independent Spi mutants obtained. The deletions were classified into 4 groups; Group 1 included 15 mutants that were characterized with a large deletion (43 bp-10 kb) with a short homologous sequence. Group 2 included 11 mutants of a large deletion having no homologous sequence at the connecting region. Group 3 included 11 mutants having a short deletion of less than 20 bp, which occurred in the non-repetitive sequence of gam gene and possibly caused by oxidative breakage of DNA or recombination of DNA fragment produced by the breakage. Group 4 included 4 mutants having deletions as short as 20 bp or less in the repetitive sequence of gam gene, resulting in an alteration of the reading frame. Thus, the synthesis of Gam protein was terminated by the appearance of TGA between code 13 and 14 of redB gene, leading to inactivation of gam gene and redBA gene. These results indicated that most of Spi mutants had a deletion in red/gam region and the deletions in more than half mutants occurred in homologous sequences as short as 8 bp. (M.N.)

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

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Kermany, Mohammad [St. Jude Children' s Research Hospital; Parker, Lisan [St. Jude Children' s Research Hospital; Guo, Yun-Kai [St. Jude Children' s Research Hospital; Miller, Darla R [ORNL; Swanson, Douglas J [ORNL; Yoo, Tai-June [Neuroscience Institute, Memphis, TN; Goldowitz, Daniel [University of Tennessee Health Science Center, Memphis; Zuo, Jian [St. Jude Children' s Research Hospital

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.

The Effect of Vitamin E on the Survival Rate of unc-13 Caenorhabditis elegans mutants under Oxidative Stress

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Jessica Porcelan

2012-01-01

Full Text Available Caenorhabditis elegans unc-13 mutants express decreased neuronal activity and thus are a good model strain for examining defective nervous systems. These unc-13 mutants as well as wild type N2 strains, show rapid mortality when under oxidative stress. However, the antioxidant vitamin E may prolong survival in unc-13 mutant and N2 strains under oxidative stress. The addition of vitamin E to organisms under oxidative stress has a protective effect in both N2 and unc-13 C. elegans strains. Interestingly, vitamin E resulted in a greater increase in survival rate in N2 worms than with unc-13 mutant worms. While both strains displayed lower mortality rates with the addition of vitamin E, this finding suggests that vitamin E more efficiently increases survival rates of C. elegans with typical nervous system function. The efficacy of vitamin E implies that use of antioxidants may lessen the damage caused by oxidative stress in both N2 and mutant worms.

Phenotypic effects of repeated psychosocial stress during adolescence in mice mutant for the schizophrenia risk gene neuregulin-1: a putative model of gene × environment interaction.

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Desbonnet, Lieve; O'Tuathaigh, Colm; Clarke, Gerard; O'Leary, Claire; Petit, Emilie; Clarke, Niamh; Tighe, Orna; Lai, Donna; Harvey, Richard; Cryan, John F; Dinan, Timothy G; Waddington, John L

2012-05-01

There is a paucity of animal models by which the contributions of environmental and genetic factors to the pathobiology of psychosis can be investigated. This study examined the individual and combined effects of chronic social stress during adolescence and deletion of the schizophrenia risk gene neuregulin-1 (NRG1) on adult mouse phenotype. Mice were exposed to repeated social defeat stress during adolescence and assessed for exploratory behaviour, working memory, sucrose preference, social behaviour and prepulse inhibition in adulthood. Thereafter, in vitro cytokine responses to mitogen stimulation and corticosterone inhibition were assayed in spleen cells, with measurement of cytokine and brain-derived neurotrophic factor (BDNF) mRNA in frontal cortex, hippocampus and striatum. NRG1 mutants exhibited hyperactivity, decreased anxiety, impaired sensorimotor gating and reduced preference for social novelty. The effects of stress on exploratory/anxiety-related parameters, spatial working memory, sucrose preference and basal cytokine levels were modified by NRG1 deletion. Stress also exerted varied effect on spleen cytokine response to concanavalin A and brain cytokine and BDNF mRNA expression in NRG1 mutants. The experience of psychosocial stress during adolescence may trigger further pathobiological features that contribute to the development of schizophrenia, particularly in those with underlying NRG1 gene abnormalities. This model elaborates the importance of gene × environment interactions in the etiology of schizophrenia. Copyright © 2012 Elsevier Inc. All rights reserved.

Aerogenic vaccination with a Burkholderia mallei auxotroph protects against aerosol-initiated glanders in mice.

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Ulrich, Ricky L; Amemiya, Kei; Waag, David M; Roy, Chad J; DeShazer, David

2005-03-14

Burkholderia mallei is an obligate mammalian pathogen that causes the zoonotic disease glanders. Two live attenuated B. mallei strains, a capsule mutant and a branched-chain amino acid auxotroph, were evaluated for use as vaccines against aerosol-initiated glanders in mice. Animals were aerogenically vaccinated and serum samples were obtained before aerosol challenge with a high-dose (>300 times the LD50) of B. mallei ATCC 23344. Mice vaccinated with the capsule mutant developed a Th2-like Ig subclass antibody response and none survived beyond 5 days. In comparison, the auxotrophic mutant elicited a Th1-like Ig subclass antibody response and 25% of the animals survived for 1 month postchallenge. After a low-dose (5 times the LD50) aerosol challenge, the survival rates of auxotroph-vaccinated and unvaccinated animals were 50 and 0%, respectively. Thus, live attenuated strains that promote a Th1-like Ig response may serve as promising vaccine candidates against aerosol infection with B. mallei.

Oral Immunization Against Candidiasis Using Lactobacillus casei Displaying Enolase 1 from Candida albicans.

Science.gov (United States)

Shibasaki, Seiji; Karasaki, Miki; Tafuku, Senji; Aoki, Wataru; Sewaki, Tomomitsu; Ueda, Mitsuyoshi

2014-01-01

Candidiasis is a common fungal infection that is prevalent in immunocompromised individuals. In this study, an oral vaccine against Candida albicans was developed by using the molecular display approach. Enolase 1 protein (Eno1p) of C. albicans was expressed on the Lactobacillus casei cell surface by using poly-gamma-glutamic acid synthetase complex A from Bacillus subtilis as an anchoring protein. The Eno1p-displaying L. casei cells were used to immunize mice, which were later challenged with a lethal dose of C. albicans. The data indicated that the vaccine elicited a strong IgG response and increased the survival rate of the vaccinated mice. Furthermore, L. casei acted as a potent adjuvant and induced high antibody titers that were comparable to those induced by strong adjuvants such as the cholera toxin. Overall, the molecular display method can be used to rapidly develop vaccines that can be conveniently administered and require minimal processing.

Intraflagellar transporter protein (IFT27), an IFT25 binding partner, is essential for male fertility and spermiogenesis in mice.

Science.gov (United States)

Zhang, Yong; Liu, Hong; Li, Wei; Zhang, Zhengang; Shang, Xuejun; Zhang, David; Li, Yuhong; Zhang, Shiyang; Liu, Junpin; Hess, Rex A; Pazour, Gregory J; Zhang, Zhibing

2017-12-01

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27: Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survived to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymides contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the "9+2″ axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the

Intraflagellar Transporter Protein (IFT27), an IFT25 binding partner, Is Essential For Male Fertility and Spermiogenesis In Mice

Science.gov (United States)

Zhang, Yong; Liu, Hong; Li, Wei; Zhang, Zhengang; Shang, Xuejun; Zhang, David; Li, Yuhong; Zhang, Shiyang; Liu, Junpin; Hess, Rex A; Pazour, Gregory J; Zhang, Zhibing

2017-01-01

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27:Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survive to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymis contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the “9+2” axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the conditional

Cell type-specific deficiency of c-kit gene expression in mutant mice of mi/mi genotype.

Science.gov (United States)

Isozaki, K.; Tsujimura, T.; Nomura, S.; Morii, E.; Koshimizu, U.; Nishimune, Y.; Kitamura, Y.

1994-01-01

The mi locus of mice encodes a novel member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors (hereafter called mi factor). In addition to microphthalmus, osteopetrosis, and lack of melanocytes, mice of mi/mi genotype are deficient in mast cells. Since the c-kit receptor tyrosine kinase plays an important role in the development of mast cells, and since the c-kit expression by cultured mast cells from mi/mi mice is deficient in both mRNA and protein levels, the mast cell deficiency of mi/mi mice has been attributed at least in part to the deficient expression of c-kit. However, it remained to be examined whether the c-kit expression was also deficient in tissues of mi/mi mice. In the present study, we examined the c-kit expression by mi/mi skin mast cells using in situ hybridization and immunohistochemistry. Moreover, we examined the c-kit expression by various cells other than mast cells in tissues of mi/mi mice. We found that the c-kit expression was deficient in mast cells but not in erythroid precursors, testicular germ cells, and neurons of mi/mi mice. This suggested that the regulation of the c-kit transcription by the mi factor was dependent on cell types. Mice of mi/mi genotype appeared to be a useful model to analyze the function of transcription factors in the whole-animal level. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7524330

Leukemogenic Ptpn11 allele causes defective erythropoiesis in mice.

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Tatiana Usenko

Full Text Available Src homology 2 (SH2 domain-containing phosphatase 2 (SHP2, encoded by PTPN11, regulates signaling networks and cell fate in many tissues. Expression of oncogenic PTPN11 in the hematopoietic compartment causes myeloproliferative neoplasm (MPN in humans and mice. However, the stage-specific effect(s of mutant Ptpn11 on erythroid development have remained unknown. We found that expression of an activated, leukemogenic Ptpn11 allele, Ptpn11D61Y, specifically in the erythroid lineage causes dyserythropoiesis in mice. Ptpn11D61Y progenitors produce excess cKIT+ CD71+ Ter119- cells and aberrant numbers of cKITl° CD71+ erythroblasts. Mutant erythroblasts show elevated activation of ERK, AKT and STAT3 in response to EPO stimulation, and MEK inhibitor treatment blocks Ptpn11D61Y-evoked erythroid hyperproliferation in vitro. Thus, the expression of oncogenic Ptpn11 causes dyserythropoiesis in a cell-autonomous manner in vivo.

Cell-extrinsic defective lymphocyte development in Lmna(-/- mice.

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J Scott Hale

2010-04-01

Full Text Available Mutations in the LMNA gene, which encodes all A-type lamins, result in a variety of human diseases termed laminopathies. Lmna(-/- mice appear normal at birth but become runted as early as 2 weeks of age and develop multiple tissue defects that mimic some aspects of human laminopathies. Lmna(-/- mice also display smaller spleens and thymuses. In this study, we investigated whether altered lymphoid organ sizes are correlated with specific defects in lymphocyte development.Lmna(-/- mice displayed severe age-dependent defects in T and B cell development which coincided with runting. Lmna(-/- bone marrow reconstituted normal T and B cell development in irradiated wild-type recipients, driving generation of functional and self-MHC restricted CD4(+ and CD8(+ T cells. Transplantation of Lmna(-/- neonatal thymus lobes into syngeneic wild-type recipients resulted in good engraftment of thymic tissue and normal thymocyte development.Collectively, these data demonstrate that the severe defects in lymphocyte development that characterize Lmna(-/- mice do not result directly from the loss of A-type lamin function in lymphocytes or thymic stroma. Instead, the immune defects in Lmna(-/- mice likely reflect indirect damage, perhaps resulting from prolonged stress due to the striated muscle dystrophies that occur in these mice.

Calorie restriction and dwarf mice in gerontological research.

Science.gov (United States)

McKee Alderman, J; DePetrillo, Michael A; Gluesenkamp, Angela M; Hartley, Antonia C; Verhoff, S Veronica; Zavodni, Katherine L; Combs, Terry P

2010-01-01

What aging process is delayed by calorie restriction (CR) and mutations that produce long-lived dwarf mice? From 1935 until 1996, CR was the only option for increasing the maximum lifespan of laboratory rodents. In 1996, the mutation producing the Ames dwarf mouse (Prop-1(-/-)) was reported to increase lifespan. Since 1996, other gene mutations that cause dwarfism or lower body weight have been reported to increase the lifespan of mice. The recent discovery of long-lived mutant dwarf mice provides an opportunity to investigate common features between CR and dwarf models. Both CR and dwarf mutations increase insulin sensitivity. Elevated insulin sensitivity reduces oxidative stress, a potential cause of aging. The elevation of liver insulin sensitivity by the hormone adiponectin in CR and long-lived dwarf mice can lower endogenous glucose production and raise fatty acid oxidation. Adiponectin reduction of plasma glucose in CR and long-lived dwarf mice can thereby lower age-related increases in oxidative damage and cancer. Copyright 2009 S. Karger AG, Basel.

Brief report on development of indigofera pseudotinctoria mats high flavonoid mutant and anti-oxidation of its exacts

International Nuclear Information System (INIS)

Shen Xiaoxia; Mei Shufang; Shu Xiaoli; Wu Dianxing

2010-01-01

Mutant high in flavonoid was successfully developed after the dry seeds of Indigofera pseudotinctoria Mats were irradiated by 300 Gy 60 Co gamma rays. The contents of flavonoid in different tissues of mutant line MJ-HF1 were all higher than that of the wild type, especially in the seeds and leaves, which was 5.89 and 1.46 times of the wild type. Anti-oxidation testing showed that the flavonoid exacts from MJ-HF1 could decrease the contents of malondialdehyde(MDA) and increase the activities of superoxide dismutase (SOD) in the aged white mice in a 30-day feeding test. (authors)

Multiple non-cell-autonomous defects underlie neocortical callosal dysgenesis in Nfib-deficient mice

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Sunn Nana

2009-12-01

Full Text Available Abstract Background Agenesis of the corpus callosum is associated with many human developmental syndromes. Key mechanisms regulating callosal formation include the guidance of axons arising from pioneering neurons in the cingulate cortex and the development of cortical midline glial populations, but their molecular regulation remains poorly characterised. Recent data have shown that mice lacking the transcription factor Nfib exhibit callosal agenesis, yet neocortical callosal neurons express only low levels of Nfib. Therefore, we investigate here how Nfib functions to regulate non-cell-autonomous mechanisms of callosal formation. Results Our investigations confirmed a reduction in glial cells at the midline in Nfib-/- mice. To determine how this occurs, we examined radial progenitors at the cortical midline and found that they were specified correctly in Nfib mutant mice, but did not differentiate into mature glia. Cellular proliferation and apoptosis occurred normally at the midline of Nfib mutant mice, indicating that the decrease in midline glia observed was due to deficits in differentiation rather than proliferation or apoptosis. Next we investigated the development of callosal pioneering axons in Nfib-/- mice. Using retrograde tracer labelling, we found that Nfib is expressed in cingulate neurons and hence may regulate their development. In Nfib-/- mice, neuropilin 1-positive axons fail to cross the midline and expression of neuropilin 1 is diminished. Tract tracing and immunohistochemistry further revealed that, in late gestation, a minor population of neocortical axons does cross the midline in Nfib mutants on a C57Bl/6J background, forming a rudimentary corpus callosum. Finally, the development of other forebrain commissures in Nfib-deficient mice is also aberrant. Conclusion The formation of the corpus callosum is severely delayed in the absence of Nfib, despite Nfib not being highly expressed in neocortical callosal neurons. Our

Imaging Primary Lung Cancers in Mice to Study Radiation Biology

International Nuclear Information System (INIS)

Kirsch, David G.; Grimm, Jan; Guimaraes, Alexander R.; Wojtkiewicz, Gregory R.; Perez, Bradford A.; Santiago, Philip M.; Anthony, Nikolas K.; Forbes, Thomas; Doppke, Karen

2010-01-01

Purpose: To image a genetically engineered mouse model of non-small-cell lung cancer with micro-computed tomography (micro-CT) to measure tumor response to radiation therapy. Methods and Materials: The Cre-loxP system was used to generate primary lung cancers in mice with mutation in K-ras alone or in combination with p53 mutation. Mice were serially imaged by micro-CT, and tumor volumes were determined. A comparison of tumor volume by micro-CT and tumor histology was performed. Tumor response to radiation therapy (15.5 Gy) was assessed with micro-CT. Results: The tumor volume measured with free-breathing micro-CT scans was greater than the volume calculated by histology. Nevertheless, this imaging approach demonstrated that lung cancers with mutant p53 grew more rapidly than lung tumors with wild-type p53 and also showed that radiation therapy increased the doubling time of p53 mutant lung cancers fivefold. Conclusions: Micro-CT is an effective tool to noninvasively measure the growth of primary lung cancers in genetically engineered mice and assess tumor response to radiation therapy. This imaging approach will be useful to study the radiation biology of lung cancer.

In Vivo Measurements in Mice in the Bion-M 1 Mission