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Sample records for adult wild-type wt

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2015-08-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2014-11-01

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

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

    PubMed

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

    2014-11-01

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

  7. Adolescent social defeat disturbs adult aggression-related impulsivity in wild-type rats.

    PubMed

    Coppens, Caroline M; Coolen, Alex; de Boer, Sietse F; Koolhaas, Jaap M

    2014-10-01

    Adolescence is generally considered as a developmental period during which adverse social experiences may have lasting consequences in terms of an increased vulnerability to affective disorders. This study aimed at determining the individual susceptibility to adolescent social stress using a rat model. We used rats of the Wild-type Groningen strain, which are characterized by a broad variation in adult levels of aggression and impulsivity. We hypothesized that experience of social defeat in adolescence results in heightened aggression and impulsivity levels in adulthood. In contrast to our expectation, adolescent social defeat did not lead to a difference in the average adult level of aggression and impulsivity, but the significant correlation between offensive aggression and impulsivity found in control animals was not present in animals defeated during adolescence.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2016-01-01

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

  10. Genomic markers of panitumumab resistance including ERBB2/HER2 in a phase II study of KRAS wild-type (wt) metastatic colorectal cancer (mCRC)

    PubMed Central

    Barry, Garrett S.; Cheang, Maggie C.; Chang, Hector Li; Kennecke, Hagen F.

    2016-01-01

    A prospective study was conducted to identify biomarkers associated with resistance to panitumumab monotherapy in patients with metastatic colorectal cancer (mCRC). Patients with previously treated, codon 12/13 KRAS wt, mCRC were prospectively administered panitumumab 6 mg/kg IV q2weeks. Of 34 panitumumab-treated patients, 11 (32%) had progressive disease at 8 weeks and were classified as non-responders. A Nanostring nCounter-based assay identified a 5-gene expression signature (ERBB2, MLPH, IRX3, MYRF, and KLK6) associated with panitumumab resistance (P = 0.001). Immunohistochemistry and in situ hybridization determined that the HER2 (ERBB2) protein was overexpressed in 4/11 non-responding and 0/21 responding cases (P = 0.035). Two non-responding tumors had ERBB2 gene amplification only, and one demonstrated both ERBB2 amplification and mutation. A non-codon 12/13 KRAS mutation occurred in one panitumumab-resistant patient and was mutually exclusive with ERBB2/HER2 abnormalities. This study identifies a 5-gene signature associated with non-response to single agent panitumumab, including a subgroup of non-responders with evidence of aberrant ERBB2/HER2 signaling. KRAS wt tumors resistant to EGFRi may be identified by gene signature analysis, and the HER2 pathway plays an important role in resistance to therapy. PMID:26980732

  11. Wt1 dictates the fate of fetal and adult Leydig cells during development in the mouse testis.

    PubMed

    Wen, Qing; Zheng, Qiao-Song; Li, Xi-Xia; Hu, Zhao-Yuan; Gao, Fei; Cheng, C Yan; Liu, Yi-Xun

    2014-12-15

    Wilms' tumor 1 (Wt1) is a tumor suppressor gene encoding ∼24 zinc finger transcription factors. In the mammalian testis, Wt1 is expressed mostly by Sertoli cells (SCs) involved in testis development, spermatogenesis, and adult Leydig cell (ALC) steroidogenesis. Global knockout (KO) of Wt1 is lethal in mice due to defects in embryogenesis. Herein, we showed that Wt1 is involved in regulating fetal Leydig cell (FLC) degeneration and ALC differentiation during testicular development. Using Wt1(-/flox);Amh-Cre mice that specifically deleted Wt1 in the SC vs. age-matched wild-type (WT) controls, FLC-like-clusters were found in Wt1-deficient testes that remained mitotically active from postnatal day 1 (P1) to P56, and no ALC was detected at these ages. Leydig cells in mutant adult testes displayed morphological features of FLC. Also, FLC-like cells in adult mutant testes had reduced expression in ALC-associated genes Ptgds, Sult1e1, Vcam1, Hsd11b1, Hsd3b6, and Hsd17b3 but high expression of FLC-associated genes Thbs2 and Hsd3b1. Whereas serum LH and testosterone level in mutant mice were not different from controls, intratesticular testosterone level was significantly reduced. Deletion of Wt1 gene also perturbed the expression of steroidogenic enzymes Star, P450c17, Hsd3b6, Hsd3b1, Hsd17b1, and Hsd17b3. FLCs in adult mutant testes failed to convert androstenedione to testosterone due to a lack of Hsd17b3, and this defect was rescued by coculturing with fetal SCs. In summary, FLC-like cells in mutant testes are putative FLCs that remain mitotically active in adult mice, illustrating that Wt1 dictates the fate of FLC and ALC during postnatal testis development.

  12. Sod1 gene ablation in adult mice leads to physiological changes at the neuromuscular junction similar to changes that occur in old wild-type mice.

    PubMed

    Ivannikov, Maxim V; Van Remmen, Holly

    2015-07-01

    Reactive oxygen species (ROS) are believed to be important mediators of muscle atrophy and weakness in aging and many degenerative conditions. However, the mechanisms and physiological processes specifically affected by elevated ROS in neuromuscular units that contribute to muscle weakness during aging are not well defined. Here we investigate the effects of chronic oxidative stress on neurotransmission and excitation-contraction (EC) coupling mechanisms in the levator auris longus (LAL) muscle from young (4-8 months) and old (22-28 months) wild-type mice and young adult Cu-Zn superoxide dismutase 1 knockout (Sod1(-/-)) mice. The frequency of spontaneous neurotransmitter release and the amplitude of evoked neurotransmitter release in young Sod1(-/-) and old wild-type LAL neuromuscular junctions were significantly reduced from the young wild-type values, and those declines were mirrored by decreases in synaptic vesicle pool size. Presynaptic cytosolic calcium concentration and mitochondrial calcium uptake amplitudes showed substantial increases in stimulated young Sod1(-/-) and old axon terminals. Surprisingly, LAL muscle fibers from old mice showed a greater excitability than fibers from either young wild-type or young Sod1(-/-) LAL. Both evoked excitatory junction potential (EJP) and spontaneous mini EJP amplitudes were considerably higher in LAL muscles from old mice than in fibers from young Sod1(-/-) LAL muscle. Despite a greater excitability, sarcoplasmic calcium influx in both old wild-type and young Sod1(-/-) LAL muscle fibers was significantly less. Sarcoplasmic reticulum calcium levels were also reduced in both old wild-type and young Sod1(-/-) mice, but the difference was not statistically significant in muscle fibers from old wild-type mice. The protein ratio of triad calcium channels RyR1/DHPR was not different in all groups. However, fibers from both young Sod1(-/-) and old mice had substantially elevated levels of protein carbonylation and S

  13. Forebrain microglia from wild-type but not adult 5xFAD mice prevent amyloid-β plaque formation in organotypic hippocampal slice cultures.

    PubMed

    Hellwig, Sabine; Masuch, Annette; Nestel, Sigrun; Katzmarski, Natalie; Meyer-Luehmann, Melanie; Biber, Knut

    2015-01-01

    The role of microglia in amyloid-β (Aβ) deposition is controversial. In the present study, an organotypic hippocampal slice culture (OHSC) system with an in vivo-like microglial-neuronal environment was used to investigate the potential contribution of microglia to Aβ plaque formation. We found that microglia ingested Aβ, thereby preventing plaque formation in OHSCs. Conversely, Aβ deposits formed rapidly in microglia-free wild-type slices. The capacity to prevent Aβ plaque formation was absent in forebrain microglia from young adult but not juvenile 5xFamilial Alzheimer's disease (FAD) mice. Since no loss of Aβ clearance capacity was observed in both wild-type and cerebellar microglia from 5xFAD animals, the high Aβ1-42 burden in the forebrain of 5xFAD animals likely underlies the exhaustion of microglial Aβ clearance capacity. These data may therefore explain why Aβ plaque formation has never been described in wild-type mice, and point to a beneficial role of microglia in AD pathology. We also describe a new method to study Aβ plaque formation in a cell culture setting.

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

    PubMed

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

    2014-08-11

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

  15. Restoration of cocaine stimulation and reward by reintroducing wild type dopamine transporter in adult knock-in mice with a cocaine-insensitive dopamine transporter.

    PubMed

    Wu, Haiyin; O'Neill, Brian; Han, Dawn D; Thirtamara-Rajamani, Keerthi; Wang, Yanlin; Gu, Howard H

    2014-11-01

    In previous studies, we generated knock-in mice with a cocaine-insensitive dopamine transporter (DAT-CI mice) and found cocaine does not stimulate locomotion or produce reward in these mice, indicating DAT inhibition is necessary for cocaine stimulation and reward. However, DAT uptake is reduced in DAT-CI mice and thus the lack of cocaine responses could be due to adaptive changes. To test this, we used adeno-associated virus (AAV) to reintroduce the cocaine-sensitive wild type DAT (AAV-DATwt) back into adult DAT-CI mice, which restores cocaine inhibition of DAT in affected brain regions but does not reverse the adaptive changes. In an earlier study we showed that AAV-DATwt injections in regions covering the lateral nucleus accumbens (NAc) and lateral caudate-putamen (CPu) restored cocaine stimulation but not cocaine reward. In the current study, we expanded the AAV-DATwt infected areas to cover the olfactory tubercle (Tu) and the ventral midbrain (vMB) containing the ventral tegmental area (VTA) and substantia nigra (SN) in addition to CPu and NAc with multiple injections. These mice displayed the restoration of both locomotor stimulation and cocaine reward. We further found that AAV-DATwt injection in the vMB alone was sufficient to restore both cocaine stimulation and reward in DAT-CI mice. AAV injected in the VTA and SN resulted in DATwt expression and distribution to the DA terminal regions. In summary, cocaine induced locomotion and reward can be restored in fully developed DAT-CI mice, and cocaine inhibition of DAT expressed in dopaminergic neurons originated from the ventral midbrain mediates cocaine reward and stimulation.

  16. Restoration of Cocaine Stimulation and Reward by Reintroducing Wild Type Dopamine Transporter in Adult Knock-in Mice with a Cocaine-Insensitive Dopamine Transporter

    PubMed Central

    Wu, Haiyin; O’Neill, Brian; Han, Dawn D.; Thirtamara-Rajamani, Keerthi; Wang, Yanlin; Gu, Howard H.

    2014-01-01

    In previous studies, we generated knock-in mice with a cocaine-insensitive dopamine transporter (DAT-CI mice) and found cocaine does not stimulate locomotion or produce reward in these mice, indicating DAT inhibition is necessary for cocaine stimulation and reward. However, DAT uptake is reduced in DAT-CI mice and thus the lack of cocaine responses could be due to adaptive changes. To test this, we used adeno-associated virus (AAV) to reintroduce the cocaine-sensitive wild type DAT (AAV-DATwt) back into adult DAT-CI mice, which restores cocaine inhibition of DAT in affected brain regions but does not reverse the adaptive changes. In an earlier study we showed that AAV-DATwt injections in regions covering the lateral nucleus accumbens (NAc) and lateral caudate-putamen (CPu) restored cocaine stimulation but not cocaine reward. In the current study, we expanded the AAV-DATwt infected areas to cover the olfactory tubercle (Tu) and the ventral midbrain (vMB) containing the ventral tegmental area (VTA) and substantia nigra (SN) in addition to CPu and NAc with multiple injections. These mice displayed the restoration of both locomotor stimulation and cocaine reward. We further found that AAV-DATwt injection in the vMB alone was sufficient to restore both cocaine stimulation and reward in DAT-CI mice. AAV injected in the VTA and SN resulted in DATwt expression and distribution to the DA terminal regions. In summary, cocaine induced locomotion and reward can be restored in fully developed DAT-CI mice, and cocaine inhibition of DAT expressed in dopaminergic neurons originated from the ventral midbrain mediates cocaine reward and stimulation. PMID:24835281

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

    PubMed Central

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

    2009-01-01

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

  18. Acute Multiple Organ Failure in Adult Mice Deleted for the Developmental Regulator Wt1

    PubMed Central

    Chau, You-Ying; Brownstein, David; Mjoseng, Heidi; Lee, Wen-Chin; Buza-Vidas, Natalija; Nerlov, Claus; Jacobsen, Sten Eirik; Perry, Paul; Berry, Rachel; Thornburn, Anna; Sexton, David; Morton, Nik; Hohenstein, Peter; Freyer, Elisabeth; Samuel, Kay; van't Hof, Rob; Hastie, Nicholas

    2011-01-01

    There is much interest in the mechanisms that regulate adult tissue homeostasis and their relationship to processes governing foetal development. Mice deleted for the Wilms' tumour gene, Wt1, lack kidneys, gonads, and spleen and die at mid-gestation due to defective coronary vasculature. Wt1 is vital for maintaining the mesenchymal–epithelial balance in these tissues and is required for the epithelial-to-mesenchyme transition (EMT) that generates coronary vascular progenitors. Although Wt1 is only expressed in rare cell populations in adults including glomerular podocytes, 1% of bone marrow cells, and mesothelium, we hypothesised that this might be important for homeostasis of adult tissues; hence, we deleted the gene ubiquitously in young and adult mice. Within just a few days, the mice suffered glomerulosclerosis, atrophy of the exocrine pancreas and spleen, severe reduction in bone and fat, and failure of erythropoiesis. FACS and culture experiments showed that Wt1 has an intrinsic role in both haematopoietic and mesenchymal stem cell lineages and suggest that defects within these contribute to the phenotypes we observe. We propose that glomerulosclerosis arises in part through down regulation of nephrin, a known Wt1 target gene. Protein profiling in mutant serum showed that there was no systemic inflammatory or nutritional response in the mutant mice. However, there was a dramatic reduction in circulating IGF-1 levels, which is likely to contribute to the bone and fat phenotypes. The reduction of IGF-1 did not result from a decrease in circulating GH, and there is no apparent pathology of the pituitary and adrenal glands. These findings 1) suggest that Wt1 is a major regulator of the homeostasis of some adult tissues, through both local and systemic actions; 2) highlight the differences between foetal and adult tissue regulation; 3) point to the importance of adult mesenchyme in tissue turnover. PMID:22216009

  19. Safety and immunogenicity of a recombinant protein influenza A vaccine in adult human volunteers and protective efficacy against wild-type H1N1 virus challenge.

    PubMed

    Fries, L F; Dillon, S B; Hildreth, J E; Karron, R A; Funkhouser, A W; Friedman, C J; Jones, C S; Culleton, V G; Clements, M L

    1993-03-01

    A recombinant influenza A vaccine (D protein), comprising a carboxy-terminal sequence from the hemagglutinin HA2 subunit of A/Puerto Rico/8/34 virus (H1N1, A/PR/34) fused to 81 amino-terminal residues of the NS1 nonstructural protein, has previously protected mice against influenza A challenge by inducing H1N1/H2N2 cross-reactive cytotoxic T cells (CTL) without hemagglutination-inhibiting (HI) or neutralizing antibody. In our dose-escalating study, the vaccine was safe in humans and induced both IgG and T cell proliferative responses to D protein but little antibody to A/PR/34 or A/Kawasaki/8/86 (H1N1, A/KW/86) viruses. Among an additional group of A/KW/86-seronegative volunteers immunized with 500 micrograms of D protein, none had a rise in serum HI or neutralizing antibody to A/KW/86, 20% had minimal IgG responses to A/KW/86 by EIA, and a minority had any increase in A/KW/86-specific CTL activity. However, viral shedding and clinical illness score were reduced in vaccines relative to A/KW/86-seronegative unimmunized controls after intranasal challenge with wild-type A/KW/86. D protein immunization conferred significant protective immunity not currently explained by any of the immune parameters measured.

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

    PubMed

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

    2015-03-01

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

  1. Production of Cystatin C Wild Type and Stabilized Mutants

    PubMed Central

    Szymanska, Aneta; Lindstrom, Veronica; Grubb, Anders

    2010-01-01

    Cystatin C is produced in all nucleated cells. It has various functions and biological activities. Researchers are focused on its role in kidney diseases as a marker of glomerular filtration but also as a very important link in development of amyloid diseases. This work describes expression and purification of both wild type (wt) and stabilized form (stab 1 and 2) of wt cystatin C and amyloid-forming L68Q mutant of cystatin C. The recombinant cystatin C can be used in projects requiring pure cystatin C to examine models of dimerization and fibrils formation as well as a standard in clinical tests.

  2. Gender differences between hypocretin/orexin knockout and wild type mice: age, body weight, body composition, metabolic markers, leptin and insulin resistance.

    PubMed

    Ramanathan, Lalini; Siegel, Jerome M

    2014-12-01

    Female hypocretin knockout (Hcrt KO) mice have increased body weight despite decreased food intake compared to wild type (WT) mice. In order to understand the nature of the increased body weight, we carried out a detailed study of Hcrt KO and WT, male, and female mice. Female KO mice showed consistently higher body weight than WT mice, from 4 to 20 months (20-60%). Fat, muscle, and free fluid levels were all significantly higher in adult (7-9 months) as well as old (18-20 months) female KO mice compared to age-matched WT mice. Old male KO mice showed significantly higher fat content (150%) compared to age-matched WT mice, but no significant change in body weight. Respiratory quotient (-19%) and metabolic rates (-14%) were significantly lower in KO mice compared to WT mice, regardless of gender or age. Female KO mice had significantly higher serum leptin levels (191%) than WT mice at 18-20 months, but no difference between male mice were observed. Conversely, insulin resistance was significantly higher in both male (73%) and female (93%) KO mice compared to age- and sex-matched WT mice. We conclude that absence of the Hcrt peptide has gender-specific effects. In contrast, Hcrt-ataxin mice and human narcoleptics, with loss of the whole Hcrt cell, show weight gain in both sexes.

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

    PubMed Central

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

    2008-01-01

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

  4. Overexpression of the wild-type SPT1 subunit lowers desoxysphingolipid levels and rescues the phenotype of HSAN1.

    PubMed

    Eichler, Florian S; Hornemann, Thorsten; McCampbell, Alex; Kuljis, Dika; Penno, Anke; Vardeh, Daniel; Tamrazian, Eric; Garofalo, Kevin; Lee, Ho-Joon; Kini, Lohit; Selig, Martin; Frosch, Matthew; Gable, Ken; von Eckardstein, Arnold; Woolf, Clifford J; Guan, Guiman; Harmon, Jeffrey M; Dunn, Teresa M; Brown, Robert H

    2009-11-18

    Mutations in the SPTLC1 subunit of serine palmitoyltransferase (SPT) cause an adult-onset, hereditary sensory, and autonomic neuropathy type I (HSAN1). We previously reported that mice bearing a transgene-expressing mutant SPTLC1 (tgSPTLC1(C133W)) show a reduction in SPT activity and hyperpathia at 10 months of age. Now analyzed at a later age, we find these mice develop sensory loss with a distal small fiber neuropathy and peripheral myelinopathy. This phenotype is largely reversed when these mice are crossed with transgenic mice overexpressing wild-type SPTLC1 showing that the mutant SPTLC1 protein is not inherently toxic. Simple loss of SPT activity also cannot account for the HSAN1 phenotype, since heterozygous SPTLC1 knock-out mice have reduced SPT activity but are otherwise normal. Rather, the presence of two newly identified, potentially deleterious deoxysphingoid bases in the tgSPTLC1(C133W), but not in the wild-type, double-transgenic tgSPTLC1(WT + C133W) or SPTLC1(+/-) mice, suggests that the HSAN1 mutations alter amino acid selectivity of the SPT enzyme such that palmitate is condensed with alanine and glycine, in addition to serine. This observation is consistent with the hypothesis that HSAN1 is the result of a gain-of-function mutation in SPTLC1 that leads to accumulation of a toxic metabolite. PMID:19923297

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

    PubMed

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

    2015-10-15

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

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

    PubMed

    Stanić, Snezana; Pavković-Lucic, Sofija

    2005-01-01

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

  7. SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling in commercial and wild-type turkey leukocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases (PTK) and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on days...

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

    SciTech Connect

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

    1989-04-01

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

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

  11. Maternal and pup genotype contribution to growth in wild-type and tau mutant Syrian hamsters.

    PubMed

    Oklejewicz, M; Pen, I; Durieux, G C; Daan, S

    2001-07-01

    The single gene mutation tau in the Syrian hamster-apart from its effect on the circadian organization of locomotor activity--has a pronounced influence on body weight. In this study we investigate the impact of maternal and pup genotypes at the tau-locus on the growth rate of pups. Homozygous tau mutant hamsters (circadian period of 20 hours) had lower growth rates and adult body weights than wild-type hamsters, whereas heterozygous tau mutants (circadian period of 22 hours) were intermediate. In addition, heterozygous pups from heterozygous dams grew heavier than those from wild-type and homozygous dams. The effect of maternal genotype was further evaluated in a cross-foster design, where wild-type and homozygous mutant pups were fostered at birth to either wild-type or homozygous mutant dams. At all ages, the maternal tau genotype had a negative effect on body weight, whereas the pup tau genotype had a positive effect during the preweaning period and a negative effect afterward.

  12. The fusion protein of wild-type canine distemper virus is a major determinant of persistent infection

    SciTech Connect

    Plattet, Philippe; Rivals, Jean-Paul; Zuber, BenoIt; Brunner, Jean-Marc; Zurbriggen, Andreas; Wittek, Riccardo . E-mail: Riccardo.Wittek@unil.ch

    2005-07-05

    The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system but infects cell lines very inefficiently. In contrast, the genetically more distant Onderstepoort CDV vaccine strain (OP-CDV) induces extensive syncytia formation. Here, we investigated the roles of wild-type fusion (F{sub WT}) and attachment (H{sub WT}) proteins in Vero cells expressing, or not, the canine SLAM receptor by transfection experiments and by studying recombinants viruses expressing different combinations of wild-type and OP-CDV glycoproteins. We show that low fusogenicity is not due to a defect of the envelope proteins to reach the cell surface and that H{sub WT} determines persistent infection in a receptor-dependent manner, emphasizing the role of SLAM as a potent enhancer of fusogenicity. However, importantly, F{sub WT} reduced cell-to-cell fusion independently of the cell surface receptor, thus demonstrating that the fusion protein of the neurovirulent A75/17-CDV strain plays a key role in determining persistent infection.

  13. Brucella abortus ΔrpoE1 confers protective immunity against wild type challenge in a mouse model of brucellosis.

    PubMed

    Willett, Jonathan W; Herrou, Julien; Czyż, Daniel M; Cheng, Jason X; Crosson, Sean

    2016-09-30

    The Brucella abortus general stress response (GSR) system regulates activity of the alternative sigma factor, σ(E1), which controls transcription of approximately 100 genes and is required for persistence in a BALB/c mouse chronic infection model. We evaluated the host response to infection by a B. abortus strain lacking σ(E1) (ΔrpoE1), and identified pathological and immunological features that distinguish ΔrpoE1-infected mice from wild-type (WT), and that correspond with clearance of ΔrpoE1 from the host. ΔrpoE1 infection was indistinguishable from WT in terms of splenic bacterial burden, inflammation and histopathology up to 6weeks post-infection. However, Brucella-specific serum IgG levels in ΔrpoE1-infected mice were 5 times higher than WT by 4weeks post-infection, and remained significantly higher throughout the course of a 12-week infection. Total IgG and Brucella-specific IgG levels peaked strongly in ΔrpoE1-infected mice at 6weeks, which correlated with reduced splenomegaly and bacterial burden relative to WT-infected mice. Given the difference in immune response to infection with wild-type and ΔrpoE1, we tested whether ΔrpoE1 confers protective immunity to wild-type challenge. Mice immunized with ΔrpoE1 completely resisted WT infection and had significantly higher serum titers of Brucella-specific IgG, IgG2a and IFN-γ after WT challenge relative to age-matched naïve mice. We conclude that immunization of BALB/c mice with the B. abortus GSR pathway mutant, ΔrpoE1, elicits an adaptive immune response that confers significant protective immunity against WT infection.

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

    PubMed Central

    Battistini, Elena; Noli, Enrico

    2009-01-01

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

  15. Differences in Establishment of Persistence of Vaccine and Wild Type Rubella Viruses in Fetal Endothelial Cells

    PubMed Central

    Perelygina, Ludmila; Adebayo, Adebola; Metcalfe, Maureen; Icenogle, Joseph

    2015-01-01

    Both wild type (WT) and vaccine rubella virus (RV) can pass through the placenta to infect a human fetus, but only wtRV routinely causes pathology. To investigate possible reasons for this, we compared establishment of persistence of wtRV and RA27/3 vaccine strains in fetal endothelial cells. We showed that yields of RA27/3 and wtRV were similar after the first round of replication, but then only vaccine-infected cultures went through a crisis characterized by partial cell loss and gradual decline of virus titer followed by recovery and establishment of persistent cultures with low levels of RA27/3 secretion. We compared various steps of virus replication, but we were unable to identify changes, which might explain the 2-log difference in RA27/3 and wtRV yields in persistently infected cultures. Whole genome sequencing did not reveal selection of virus variants in either the wtRV or RA27/3 cultures. Quantitative single-cell analysis of RV replication by in situ hybridization detected, on average, 1–4 copies of negative-strand RNA and ~50 copies of positive-strand genomic RNA in cells infected with both vaccine and WT viruses. The distinct characteristics of RA27/3 replication were the presence of large amounts of negative-strand RV RNA and RV dsRNA at the beginning of the crisis and the accumulation of high amounts of genomic RNA in a subpopulation of infected cells during crisis and persistence. These results suggest that RA27/3 can persist in fetal endothelial cells, but the characteristics of persistence and mechanisms for the establishment and maintenance of persistence are different from wtRV. PMID:26177032

  16. Mutant and wild-type alpha-synuclein interact with mitochondrial cytochrome C oxidase.

    PubMed

    Elkon, Hanock; Don, Jermy; Melamed, Eldad; Ziv, Ilan; Shirvan, Anat; Offen, Daniel

    2002-06-01

    Alpha-synuclein, a presynaptic protein, was found to be the major component in the Lewy bodies (LB) in both inherited and sporadic Parkinson's disease (PD). Furthermore, rare mutations of alpha-synuclein cause autosomal-dominant PD. However, it is unknown how alpha-synuclein is involved in the pathogenesis of nigral degeneration in PD. In this study, we examine the protein-protein interactions of wild-type and mutant (A53T) a-synuclein with adult human brain cDNA expression library using the yeast two-hybrid technique. We found that both normal and mutant alpha-synuclein specifically interact with the mitochondrial complex IV enzyme, cytochrome C oxidase (COX). Wild-type and mutant alpha-synuclein genes were further fused with c-Myc tag and translated in rabbit reticulocyte lysate. Using anti-c-Myc antibody, we demonstrated that both wild-type and mutant alpha-synuclein, coimmunoprecipitated with COX. We also showed that potassium cyanide, a selective COX inhibitor, synergistically enhanced the sensitivity of SH-SY5Y neuroblastoma cells to dopamine-induced cell death. In conclusion, we found specific protein-protein interactions of alpha-synuclein, a major LB protein, to COX, a key enzyme of the mithochondrial respiratory system. This interaction suggests that alpha-synuclein aggregation may contribute to enhance the mitochondrial dysfunction, which might be a key factor in the pathogenesis of PD.

  17. Drought stress-induced compositional changes in tolerant transgenic rice and its wild type.

    PubMed

    Nam, Kyong-Hee; Kim, Do-Young; Shin, Hee Jae; Nam, Ki Jung; An, Joo Hee; Pack, In-Soon; Park, Jung-Ho; Jeong, Soon-Chun; Kim, Ho Bang; Kim, Chang-Gi

    2014-06-15

    Comparing well-watered versus deficit conditions, we evaluated the chemical composition of grains harvested from wild-type (WT) and drought-tolerant, transgenic rice (Oryza sativa L.). The latter had been developed by inserting AtCYP78A7, which encodes a cytochrome P450 protein. Two transgenic Lines, '10B-5' and '18A-4', and the 'Hwayoung' WT were grown under a rainout shelter. After the harvested grains were polished, their levels of key components, including proximates, amino acids, fatty acids, minerals and vitamins were analysed to determine the effect of watering system and genotype. Drought treatment significantly influenced the levels of some nutritional components in both transgenic and WT grains. In particular, the amounts of lignoceric acid and copper in the WT decreased by 12.6% and 39.5%, respectively, by drought stress, whereas those of copper and potassium in the transgenics rose by 88.1-113.3% and 10.4-11.9%, respectively, under water-deficit conditions.

  18. Targeting Mdmx to treat breast cancers with wild-type p53

    PubMed Central

    Haupt, S; Buckley, D; Pang, J-MB; Panimaya, J; Paul, P J; Gamell, C; Takano, E A; Ying Lee, Y; Hiddingh, S; Rogers, T-M; Teunisse, A F A S; Herold, M J; Marine, J-C; Fox, S B; Jochemsen, A; Haupt, Y

    2015-01-01

    The function of the tumor suppressor p53 is universally compromised in cancers. It is the most frequently mutated gene in human cancers (reviewed). In cases where p53 is not mutated, alternative regulatory pathways inactivate its tumor suppressive functions. This is primarily achieved through elevation in the expression of the key inhibitors of p53: Mdm2 or Mdmx (also called Mdm4) (reviewed). In breast cancer (BrCa), the frequency of p53 mutations varies markedly between the different subtypes, with basal-like BrCas bearing a high frequency of p53 mutations, whereas luminal BrCas generally express wild-type (wt) p53. Here we show that Mdmx is unexpectedly highly expressed in normal breast epithelial cells and its expression is further elevated in most luminal BrCas, whereas p53 expression is generally low, consistent with wt p53 status. Inducible knockdown (KD) of Mdmx in luminal BrCa MCF-7 cells impedes the growth of these cells in culture, in a p53-dependent manner. Importantly, KD of Mdmx in orthotopic xenograft transplants resulted in growth inhibition associated with prolonged survival, both in a preventative model and also in a treatment model. Growth impediment in response to Mdmx KD was associated with cellular senescence. The growth inhibitory capacity of Mdmx KD was recapitulated in an additional luminal BrCa cell line MPE600, which expresses wt p53. Further, the growth inhibitory capacity of Mdmx KD was also demonstrated in the wt p53 basal-like cell line SKBR7 line. These results identify Mdmx growth dependency in wt p53 expressing BrCas, across a range of subtypes. Based on our findings, we propose that Mdmx targeting is an attractive strategy for treating BrCas harboring wt p53. PMID:26181202

  19. H1-antihistamines exacerbate high-fat diet-induced hepatic steatosis in wild-type but not in apolipoprotein E knockout mice.

    PubMed

    Raveendran, Vineesh V; Kassel, Karen M; Smith, Donald D; Luyendyk, James P; Williams, Kurt J; Cherian, Rachel; Reed, Gregory A; Flynn, Colleen A; Csanaky, Iván L; Lickteig, Andrew L; Pratt-Hyatt, Matthew J; Klaassen, Curtis D; Dileepan, Kottarappat N

    2014-07-15

    We examined the effects of two over-the-counter H1-antihistamines on the progression of fatty liver disease in male C57Bl/6 wild-type and apolipoprotein E (ApoE)-/- mice. Mice were fed a high-fat diet (HFD) for 3 mo, together with administration of either cetirizine (4 mg/kg body wt) or fexofenadine (40 mg/kg body wt) in drinking water. Antihistamine treatments increased body weight gain, gonadal fat deposition, liver weight, and hepatic steatosis in wild-type mice but not in ApoE-/- mice. Lobular inflammation, acute inflammation, and necrosis were not affected by H1-antihistamines in either genotype. Serum biomarkers of liver injury tended to increase in antihistamine-treated wild-type mice. Serum level of glucose was increased by fexofenadine, whereas lipase was increased by cetirizine. H1-antihistamines reduced the mRNA expression of ApoE and carbohydrate response element-binding protein in wild-type mice, without altering the mRNA expression of sterol regulatory element-binding protein 1c, fatty acid synthase, or ApoB100, in either genotype. Fexofenadine increased both triglycerides and cholesterol ester, whereas cetirizine increased only cholesterol ester in liver, with a concomitant decrease in serum triglycerides by both antihistamines in wild-type mice. Antihistamines increased hepatic levels of conjugated bile acids in wild-type mice, with the effect being significant in fexofenadine-treated animals. The increase was associated with changes in the expression of organic anion transport polypeptide 1b2 and bile salt export pump. These results suggest that H1-antihistamines increase the progression of fatty liver disease in wild-type mice, and there seems to be an association between the severity of disease, presence of ApoE, and increase in hepatic bile acid levels.

  20. Analysis of purified Wild type and mutant adenovirus particles by SILAC based quantitative proteomics

    PubMed Central

    Alqahtani, Ali; Heesom, Kate; Bramson, Jonathan L.; Curiel, David; Ugai, Hideyo

    2014-01-01

    We used SILAC (stable isotope labelling of amino acids in cell culture) and high-throughput quantitative MS mass spectrometry to analyse the protein composition of highly purified WT wild type adenoviruses, mutant adenoviruses lacking an internal protein component (protein V) and recombinant adenoviruses of the type commonly used in gene therapy, including one virus that had been used in a clinical trial. We found that the viral protein abundance and composition were consistent across all types of virus examined except for the virus lacking protein V, which also had reduced amounts of another viral core protein, protein VII. In all the samples analysed we found no evidence of consistent packaging or contamination with cellular proteins. We believe this technique is a powerful method to analyse the protein composition of this important gene therapy vector and genetically engineered or synthetic virus-like particles. The raw data have been deposited at proteomexchange, identifer PXD001120. PMID:25096814

  1. Comparative transcriptomic analysis of silkwormBmovo-1 and wild type silkworm ovary

    PubMed Central

    Xue, Renyu; Hu, Xiaolong; Zhu, Liyuan; Cao, Guangli; Huang, Moli; Xue, Gaoxu; Song, Zuowei; Lu, Jiayu; Chen, Xueying; Gong, Chengliang

    2015-01-01

    The detailed molecular mechanism of Bmovo-1 regulation of ovary size is unclear. To uncover the mechanism of Bmovo-1 regulation of ovarian development and oogenesis using RNA-Seq, we compared the transcriptomes of wild type (WT) and Bmovo-1-overexpressing silkworm (silkworm+Bmovo-1) ovaries. Using a pair-end Illumina Solexa sequencing strategy, 5,296,942 total reads were obtained from silkworm+Bmovo-1 ovaries and 6,306,078 from WT ovaries. The average read length was about 100 bp. Clean read ratios were 98.79% for silkworm+Bmovo-1 and 98.87% for WT silkworm ovaries. Comparative transcriptome analysis showed 123 upregulated and 111 downregulated genes in silkworm+Bmovo-1 ovaries. These differentially expressed genes were enriched in the extracellular and extracellular spaces and involved in metabolism, genetic information processing, environmental information processing, cellular processes and organismal systems. Bmovo-1 overexpression in silkworm ovaries might promote anabolism for ovarian development and oogenesis and oocyte proliferation and transport of nutrients to ovaries by altering nutrient partitioning, which would support ovary development. Excessive consumption of nutrients for ovary development alters nutrient partitioning and deters silk protein synthesis. PMID:26643037

  2. Selective modulation of wild type receptor functions by mutants of G-protein-coupled receptors.

    PubMed

    Le Gouill, C; Parent, J L; Caron, C A; Gaudreau, R; Volkov, L; Rola-Pleszczynski, M; Stanková, J

    1999-04-30

    Members of the G-protein-coupled receptor (GPCR) family are involved in most aspects of higher eukaryote biology, and mutations in their coding sequence have been linked to several diseases. In the present study, we report that mutant GPCR can affect the functional properties of the co-expressed wild type (WT) receptor. Mutants of the human platelet-activating factor receptor that fail to show any detectable ligand binding (N285I and K298stop) or coupling to a G-protein (D63N, D289A, and Y293A) were co-expressed with the WT receptor in Chinese hamster ovary and COS-7 cells. In this context, N285I and K298stop mutant receptors inhibited 3H-WEB2086 binding and surface expression. Co-transfection with D63N resulted in a constitutively active receptor phenotype. Platelet-activating factor-induced inositol phosphate production in cells transfected with a 1:1 ratio of WT:D63N was higher than with the WT cDNA alone but was abolished with a 1:3 ratio. We confirmed that these findings could be extended to other GPCRs by showing that co-expression of the WT C-C chemokine receptor 2b with a carboxyl-terminal deletion mutant (K311stop), resulted in a decreased affinity and responsiveness to MCP-1. A better understanding of this phenomenon could lead to important tools for the prevention or treatment of certain diseases. PMID:10212233

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

    PubMed

    Wang, Yunbiao; Ezemaduka, Anastasia N

    2014-04-01

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

  4. Porphyrin Interactions with Wild Type and Mutant Mouse Ferrochelatase

    SciTech Connect

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

    1999-05-19

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

  5. Salmonella induces SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling pathways in commercial and wild-type turkey leukocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on d...

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

    PubMed

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

    2016-08-01

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

  7. PHEX Mimetic (SPR4-Peptide) Corrects and Improves HYP and Wild Type Mice Energy-Metabolism

    PubMed Central

    Zelenchuk, Lesya V.; Hedge, Anne-Marie; Rowe, Peter S. N.

    2014-01-01

    Context PHEX or DMP1 mutations cause hypophosphatemic-rickets and altered energy metabolism. PHEX binds to DMP1-ASARM-motif to form a complex with α5β3 integrin that suppresses FGF23 expression. ASARM-peptides increase FGF23 by disrupting the PHEX-DMP1-Integrin complex. We used a 4.2 kDa peptide (SPR4) that binds to ASARM-peptide/motif to study the DMP1-PHEX interaction and to assess SPR4 for the treatment of energy metabolism defects in HYP and potentially other bone-mineral disorders. Design Subcutaneously transplanted osmotic pumps were used to infuse SPR4-peptide or vehicle (VE) into wild-type mice (WT) and HYP-mice (PHEX mutation) for 4 weeks. Results SPR4 partially corrected HYP mice hypophosphatemia and increased serum 1.25(OH)2D3. Serum FGF23 remained high and PTH was unaffected. WT-SPR4 mice developed hypophosphatemia and hypercalcemia with increased PTH, FGF23 and 1.25(OH)2D3. SPR4 increased GAPDH HYP-bone expression 60× and corrected HYP-mice hyperglycemia and hypoinsulinemia. HYP-VE serum uric-acid (UA) levels were reduced and SPR4 infusion suppressed UA levels in WT-mice but not HYP-mice. SPR4 altered leptin, adiponectin, and sympathetic-tone and increased the fat mass/weight ratio for HYP and WT mice. Expression of perlipin-2 a gene involved in obesity was reduced in HYP-VE and WT-SPR4 mice but increased in HYP-SPR4 mice. Also, increased expression of two genes that inhibit insulin-signaling, ENPP1 and ESP, occurred with HYP-VE mice. In contrast, SPR4 reduced expression of both ENPP1 and ESP in WT mice and suppressed ENPP1 in HYP mice. Increased expression of FAM20C and sclerostin occurred with HYP-VE mice. SPR4 suppressed expression of FAM20C and sclerostin in HYP and WT mice. Conclusions ASARM peptides and motifs are physiological substrates for PHEX and modulate osteocyte PHEX-DMP1-α5β3-integrin interactions and thereby FGF23 expression. These interactions also provide a nexus that regulates bone and energy metabolism. SPR4 suppression of

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

    PubMed Central

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

    2006-01-01

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

  9. Gravitropism and development of wild-type and starch-deficient mutants of Arabidopsis during spaceflight

    NASA Technical Reports Server (NTRS)

    Kiss, J. Z.; Katembe, W. J.; Edelmann, R. E.

    1998-01-01

    The "starch-statolith" hypothesis has been used by plant physiologists to explain the gravity perception mechanism in higher plants. In order to help resolve some of the controversy associated with ground-based research that has supported this theory, we performed a spaceflight experiment during the January 1997 mission of the Space Shuttle STS-81. Seedlings of wild-type (WT) Arabidopsis, two reduced-starch strains, and a starchless mutant were grown in microgravity and then given a gravity stimulus on a centrifuge. In terms of development in space, germination was greater than 90% for seeds in microgravity, and flight seedlings were smaller (60% in total length) compared to control plants grown on the ground and to control plants on a rotating clinostat. Seedlings grown in space had two structural features that distinguished them from the controls: a greater density of root hairs and an anomalous hypocotyl hook structure. However, the slower growth and morphological changes observed in the flight seedlings may be due to the effects of ethylene present in the spacecraft. Nevertheless, during the flight hypocotyls of WT seedlings responded to a unilateral 60 min stimulus provided by a 1-g centrifuge while those of the starch-deficient strains did not. Thus the strain with the greatest amount of starch responded to the stimulus given in flight and therefore, these data support the starch-statolith model for gravity sensing.

  10. Rootcap structure in wild type and in a starchless mutant of Arabidopsis

    NASA Technical Reports Server (NTRS)

    Sack, F. D.; Kiss, J. Z.

    1989-01-01

    Rootcaps of the wild type (WT) and of a starchless, gravitropic mutant (TC7) of Arabidopsis thaliana L. were examined by electron microscopy to identify cellular polarities with respect to gravity. In columella cells, nuclei are located proximally, and the nuclear envelope is continuous with endoplasmic reticulum (ER) that is in turn connected to nearby plasmodesmata. Impregnation of ER with osmium ferricyanide revealed numerous contacts between columella plastids and ER in both genotypes. ER is present mostly in the outer regions of the columella protoplast except in older columella cells that are developing into peripheral cells. In vertical roots, only columella cells that are intermediate in development (story 2 cells) have a higher surface density (S) of ER in the distal compared to proximal regions of the cell. The distal but not the proximal S of the ER is constant throughout columella development. Plastids are less sedimented in TC7 columella cells compared to those of the WT. It is hypothesized that plastid contact with the ER plays a role in gravity perception in both genotypes.

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

    SciTech Connect

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

    1990-08-01

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

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

    PubMed

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

    2016-04-01

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

  13. Identification of enzymes and quantification of metabolic fluxes in the wild type and in a recombinant aspergillus oryzae strain

    PubMed

    Pedersen; Carlsen; Nielsen

    1999-01-01

    Two alpha-amylase-producing strains of Aspergillus oryzae, a wild-type strain and a recombinant containing additional copies of the alpha-amylase gene, were characterized with respect to enzyme activities, localization of enzymes to the mitochondria or cytosol, macromolecular composition, and metabolic fluxes through the central metabolism during glucose-limited chemostat cultivations. Citrate synthase and isocitrate dehydrogenase (NAD) activities were found only in the mitochondria, glucose-6-phosphate dehydrogenase and glutamate dehydrogenase (NADP) activities were found only in the cytosol, and isocitrate dehydrogenase (NADP), glutamate oxaloacetate transaminase, malate dehydrogenase, and glutamate dehydrogenase (NAD) activities were found in both the mitochondria and the cytosol. The measured biomass components and ash could account for 95% (wt/wt) of the biomass. The protein and RNA contents increased linearly with increasing specific growth rate, but the carbohydrate and chitin contents decreased. A metabolic model consisting of 69 fluxes and 59 intracellular metabolites was used to calculate the metabolic fluxes through the central metabolism at several specific growth rates, with ammonia or nitrate as the nitrogen source. The flux through the pentose phosphate pathway increased with increasing specific growth rate. The fluxes through the pentose phosphate pathway were 15 to 26% higher for the recombinant strain than for the wild-type strain.

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

    PubMed Central

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

    1999-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2007-06-01

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

  18. Aggression and aspects of impulsivity in wild-type rats.

    PubMed

    Coppens, Caroline M; de Boer, Sietse F; Buwalda, Bauke; Koolhaas, Jaap M

    2014-01-01

    Aggression is closely related to impulsive behavior both in humans and in animals. To avoid potential negative consequences, aggressive behavior is kept in control by strong inhibitory mechanisms. Failure of these inhibitory mechanisms results in violent behavior. In the present experiments, we investigated whether aggressive behavior is related to impulsive behavior. Furthermore, we investigated if violent behavior can be distinguished from "normal" aggressive behavior in terms of impulsivity levels. We used rats of the wild-type Groningen strain, rats of this strain differ widely in their level of offensive aggression expressed toward an unfamiliar intruder male, ranging from no aggression at all to very high levels of intense and sometimes violent behavior. Violent behavior was displayed by some of the animals that were given repeated winning experience. We used behavioral performance in an unpredictable operant conditioning paradigm for food reinforcement (variable interval 15) and performance in a differential-reinforcement of low rate (DRL-60s) responding as determinants for impulsivity. We predicted that offensive aggression is correlated with behavioral flexibility measured by the VI-15 procedure and that aggressive behavior is characterized by low behavioral inhibition on the DRL task. In addition we expected that violent animals would be characterized by extremely low levels of behavioral inhibition on the DRL task. We showed that the level of offensive aggression indeed positively correlated with VI-15 performance. In addition, we showed that behavioral performance on the DRL procedure is similar in low and high aggressive rats. However, violent animals can be dissociated by a lower efficiency of lever pressing on a DRL-60s schedule of reinforcement.

  19. Dietary supplementation with ipriflavone decreases hepatic iron stores in wild type mice.

    PubMed

    Patchen, Bonnie; Koppe, Tiago; Cheng, Aaron; Seo, Young Ah; Wessling-Resnick, Marianne; Fraenkel, Paula G

    2016-09-01

    Hepcidin, a peptide produced in the liver, decreases intestinal iron absorption and macrophage iron release by causing degradation of the iron exporter, ferroportin. Because its levels are inappropriately low in patients with iron overload syndromes, hepcidin is a potential drug target. We previously conducted a chemical screen that revealed ipriflavone, an orally available small molecule, as a potent inducer of hepcidin expression. To evaluate ipriflavone's effect on iron homeostasis, we placed groups of 5-week old wild type or thalassemia intermedia (Hbb(Th3+/-)) mice on a soy-free, iron-sufficient diet, AIN-93G containing 220mg iron and 0-750mgipriflavone/kg of food for 50days. Ipriflavone 500mg/kg significantly reduced liver iron stores and intestinal ferroportin expression in WT mice, while increasing the ratio of hepcidin transcript levels to liver iron stores. Ipriflavone supplementation in Hbb(Th3+/-) mice failed to alleviate iron overload and was associated with a milder reduction in intestinal ferroportin and a failure to alter the ratio of hepcidin transcript levels to liver iron stores or splenic expression of the hepcidin-regulatory hormone, erythroferrone. These data suggest that dietary supplementation with ipriflavone alone would not be sufficient to treat iron overload in thalassemia intermedia. PMID:27519943

  20. DR4 specific TRAIL variants are more efficacious than wild-type TRAIL in pancreatic cancer.

    PubMed

    Yu, Rui; Albarenque, Stella Maris; Cool, Robbert H; Quax, Wim J; Mohr, Andrea; Zwacka, Ralf M

    2014-01-01

    Current treatment modalities for pancreatic carcinoma afford only modest survival benefits. TRAIL, as a potent and specific inducer of apoptosis in cancer cells, would be a promising new treatment option. However, since not all pancreatic cancer cells respond to TRAIL, further improvements and optimizations are still needed. One strategy to improve the effectiveness of TRAIL-based therapies is to specifically target one of the 2 cell death inducing TRAIL-receptors, TRAIL-R1 or TRAIL-R2 to overcome resistance. To this end, we designed constructs expressing soluble TRAIL (sTRAIL) variants that were rendered specific for either TRAIL-R1 or TRAIL-R2 by amino acid changes in the TRAIL ectodomain. When we expressed these constructs, including wild-type sTRAIL (sTRAIL(wt)), TRAIL-R1 (sTRAIL(DR4)) and TRAIL-R2 (sTRAIL(DR5)) specific variants, in 293 producer cells we found all to be readily expressed and secreted into the supernatant. These supernatants were subsequently transferred onto target cancer cells and apoptosis measured. We found that the TRAIL-R1 specific variant had higher apoptosis-inducing activity in human pancreatic carcinoma Colo357 cells as well as PancTu1 cells that were additionally sensitized by targeting of XIAP. Finally, we tested TRAIL-R1 specific recombinant TRAIL protein (rTRAIL(DR4)) on Colo357 xenografts in nude mice and found them to be more efficacious than rTRAIL(wt). Our results demonstrate the benefits of synthetic biological approaches and show that TRAIL-R1 specific variants can potentially enhance the therapeutic efficacy of TRAIL-based therapies in pancreatic cancer, suggesting that they can possibly become part of individualized and tumor specific combination treatments in the future. PMID:25482930

  1. Prolonged ethanol administration depletes mitochondrial DNA in MnSOD-overexpressing transgenic mice, but not in their wild type littermates

    SciTech Connect

    Larosche, Isabelle; Choumar, Amal; Fromenty, Bernard; Letteron, Philippe; Abbey-Toby, Adje; Van Remmen, Holly; Epstein, Charles J.; Richardson, Arlan; Feldmann, Gerard; Pessayre, Dominique; Mansouri, Abdellah

    2009-02-01

    Alcohol consumption increases reactive oxygen species formation and lipid peroxidation, whose products can damage mitochondrial DNA (mtDNA) and alter mitochondrial function. A possible role of manganese superoxide dismutase (MnSOD) on these effects has not been investigated. To test whether MnSOD overexpression modulates alcohol-induced mitochondrial alterations, we added ethanol to the drinking water of transgenic MnSOD-overexpressing (TgMnSOD) mice and their wild type (WT) littermates for 7 weeks. In TgMnSOD mice, alcohol administration further increased the activity of MnSOD, but decreased cytosolic glutathione as well as cytosolic glutathione peroxidase activity and peroxisomal catalase activity. Whereas ethanol increased cytochrome P-450 2E1 and mitochondrial ROS generation in both WT and TgMnSOD mice, hepatic iron, lipid peroxidation products and respiratory complex I protein carbonyls were only increased in ethanol-treated TgMnSOD mice but not in WT mice. In ethanol-fed TgMnSOD mice, but not ethanol-fed WT mice, mtDNA was depleted, and mtDNA lesions blocked the progress of polymerases. The iron chelator, DFO prevented hepatic iron accumulation, lipid peroxidation, protein carbonyl formation and mtDNA depletion in alcohol-treated TgMnSOD mice. Alcohol markedly decreased the activities of complexes I, IV and V of the respiratory chain in TgMnSOD, with absent or lesser effects in WT mice. There was no inflammation, apoptosis or necrosis, and steatosis was similar in ethanol-treated WT and TgMnSOD mice. In conclusion, prolonged alcohol administration selectively triggers iron accumulation, lipid peroxidation, respiratory complex I protein carbonylation, mtDNA lesions blocking the progress of polymerases, mtDNA depletion and respiratory complex dysfunction in TgMnSOD mice but not in WT mice.

  2. Green tea extract as a treatment for patients with wild-type transthyretin amyloidosis: an observational study

    PubMed Central

    Siepen, Fabian aus dem; Bauer, Ralf; Aurich, Matthias; Buss, Sebastian J; Steen, Henning; Altland, Klaus; Katus, Hugo A; Kristen, Arnt V

    2015-01-01

    Background Causative treatment of patients with wild-type transthyretin amyloid cardiomyopathy (wtATTR-CM) is lacking. Recent reports indicate the potential use of epigallocatechin-3-gallate (EGCG), the most abundant catechin in green tea, to inhibit amyloid fibril formation. We sought to investigate changes of cardiac function and morphology in patients with wtATTR-CM after consumption of green tea extract (GTE). Methods Twenty-five male patients (71 [64; 80] years) with wtATTR-CM were submitted to clinical examination, echocardiography, cardiac magnetic resonance imaging (cMRI) (n=14), and laboratory testing before and after daily consumption of GTE capsules containing 600 mg epigallocatechin-3-gallate for at least 12 months. Results A significant decrease of left ventricular (LV) myocardial mass by 6% (196 [100; 247] vs 180 [85; 237] g; P=0.03) by cMRI and total cholesterol by 8.4% (191 [118; 267] vs 173 [106; 287] mg/dL; P=0.006) was observed after a 1-year period of GTE consumption. LV ejection fraction by cMRI (53% [33%; 69%] vs 54% [28%; 71%]; P=0.75), LV wall thickness (17 [13; 21] vs 18 [14; 25] mm; P=0.1), and mitral annular plane systolic excursion (10 [5; 23] vs 8 [4; 13] mm; P=0.3) by echocardiography remained unchanged. Conclusion This study supports LV mass stabilization in patients with wtATTR-CM consuming GTE potentially indicating amyloid fibril reduction. PMID:26673202

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

    PubMed

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

    2012-02-28

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

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

    PubMed

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

    2012-02-28

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

  5. Wild-Type Measles Virus with the Hemagglutinin Protein of the Edmonston Vaccine Strain Retains Wild-Type Tropism in Macaques

    PubMed Central

    Nagata, Noriyo; Kato, Sei-ich; Ami, Yasushi; Suzaki, Yuriko; Suzuki, Tadaki; Sato, Yuko; Tsunetsugu-Yokota, Yasuko; Mori, Kazuyasu; Van Nguyen, Nguyen; Kimura, Hideki; Nagata, Kyosuke

    2012-01-01

    A major difference between vaccine and wild-type strains of measles virus (MV) in vitro is the wider cell specificity of vaccine strains, resulting from the receptor usage of the hemagglutinin (H) protein. Wild-type H proteins recognize the signaling lymphocyte activation molecule (SLAM) (CD150), which is expressed on certain cells of the immune system, whereas vaccine H proteins recognize CD46, which is ubiquitously expressed on all nucleated human and monkey cells, in addition to SLAM. To examine the effect of the H protein on the tropism and attenuation of MV, we generated enhanced green fluorescent protein (EGFP)-expressing recombinant wild-type MV strains bearing the Edmonston vaccine H protein (MV-EdH) and compared them to EGFP-expressing wild-type MV strains. In vitro, MV-EdH replicated in SLAM+ as well as CD46+ cells, including primary cell cultures from cynomolgus monkey tissues, whereas the wild-type MV replicated only in SLAM+ cells. However, in macaques, both wild-type MV and MV-EdH strains infected lymphoid and respiratory organs, and widespread infection of MV-EdH was not observed. Flow cytometric analysis indicated that SLAM+ lymphocyte cells were infected preferentially with both strains. Interestingly, EGFP expression of MV-EdH in tissues and lymphocytes was significantly weaker than that of the wild-type MV. Taken together, these results indicate that the CD46-binding activity of the vaccine H protein is important for determining the cell specificity of MV in vitro but not the tropism in vivo. They also suggest that the vaccine H protein attenuates MV growth in vivo. PMID:22238320

  6. Polymerase C1 levels and poly(R-3-hydroxyalkanoate) synthesis in wild-type and recombinant Pseudomonas strains.

    PubMed Central

    Kraak, M N; Smits, T H; Kessler, B; Witholt, B

    1997-01-01

    A functional antibody highly specific for polymerase C1 of Pseudomonas oleovorans GPo1 was raised and used to determine polymerase C1 levels in in vivo experiments. The polymerase C1 antibodies did not show a cross-reaction with polymerase C2 of P. oleovorans. In wild-type P. oleovorans GPo1 and Pseudomonas putida KT2442, amounts of 0.075 and 0.06% polymerase relative to total protein, respectively, were found. P. oleovorans GPo1(pGEc405), which contained additional copies of the polymerase C1-encoding gene under the control of its native promoter, contained 0.5% polymerase C1 relative to total protein. Polymerase C1 reached 10% of total cell protein when the polymerase C1-encoding gene was overexpressed through the P(alk) promoter in P. oleovorans GPo1(pET702, pGEc74). Amounts of poly(R-3-hydroxyalkanoate) (PHA) increased significantly under non-nitrogen-limiting conditions when additional polymerase C1 was expressed in P. oleovorans. Whereas P. oleovorans produced 34% (wt/wt) PHA under these conditions, a PHA level of 64% (wt/wt) could be reached for P. oleovorans GPo1(pGEc405) and a PHA level of 52% (wt/wt) could be reached for P. oleovorans GPo1(pET702, pGEc74) after induction, compared to a PHA level of 13% for the uninduced control. All recombinant Pseudomonas strains containing additional polymerase C1 showed small changes in their PHA composition. Larger amounts of 3-hydroxyhexanoate monomer and smaller amounts of 3-hydroxyoctanoate and -decanoate were found compared to those of the wild type. Two different methods were developed to quantify rates of incorporation of new monomers into preexisting PHA granules. P. oleovorans GPo1 cells grown under nitrogen-limiting conditions showed growth stage-dependent incorporation rates. The highest PHA synthesis rates of 9.5 nmol of C8/C6 monomers/mg of cell dry weight (CDW)/min were found during the mid-stationary phase, which equals a rate of production of 80 g of PHA/kg of CDW/h. PMID:9260937

  7. Rapid Phosphoproteomic Effects of Abscisic Acid (ABA) on Wild-Type and ABA Receptor-Deficient A. thaliana Mutants*

    PubMed Central

    Minkoff, Benjamin B.; Stecker, Kelly E.; Sussman, Michael R.

    2015-01-01

    Abscisic acid (ABA)1 is a plant hormone that controls many aspects of plant growth, including seed germination, stomatal aperture size, and cellular drought response. ABA interacts with a unique family of 14 receptor proteins. This interaction leads to the activation of a family of protein kinases, SnRK2s, which in turn phosphorylate substrates involved in many cellular processes. The family of receptors appears functionally redundant. To observe a measurable phenotype, four of the fourteen receptors have to be mutated to create a multilocus loss-of-function quadruple receptor (QR) mutant, which is much less sensitive to ABA than wild-type (WT) plants. Given these phenotypes, we asked whether or not a difference in ABA response between the WT and QR backgrounds would manifest on a phosphorylation level as well. We tested WT and QR mutant ABA response using isotope-assisted quantitative phosphoproteomics to determine what ABA-induced phosphorylation changes occur in WT plants within 5 min of ABA treatment and how that phosphorylation pattern is altered in the QR mutant. We found multiple ABA-induced phosphorylation changes that occur within 5 min of treatment, including three SnRK2 autophosphorylation events and phosphorylation on SnRK2 substrates. The majority of robust ABA-dependent phosphorylation changes observed were partially diminished in the QR mutant, whereas many smaller ABA-dependent phosphorylation changes observed in the WT were not responsive to ABA in the mutant. A single phosphorylation event was increased in response to ABA treatment in both the WT and QR mutant. A portion of the discovery data was validated using selected reaction monitoring-based targeted measurements on a triple quadrupole mass spectrometer. These data suggest that different subsets of phosphorylation events depend upon different subsets of the ABA receptor family to occur. Altogether, these data expand our understanding of the model by which the family of ABA receptors directs

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

    PubMed Central

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

    2016-01-01

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

  9. Computational classification of different wild-type zebrafish strains based on their variation in light-induced locomotor response.

    PubMed

    Gao, Yuan; Zhang, Gaonan; Jelfs, Beth; Carmer, Robert; Venkatraman, Prahatha; Ghadami, Mohammad; Brown, Skye A; Pang, Chi Pui; Leung, Yuk Fai; Chan, Rosa H M; Zhang, Mingzhi

    2016-02-01

    Zebrafish larvae display a rapid and characteristic swimming behaviour after abrupt light onset or offset. This light-induced locomotor response (LLR) has been widely used for behavioural research and drug screening. However, the locomotor responses have long been shown to be different between different wild-type (WT) strains. Thus, it is critical to define the differences in the WT LLR to facilitate accurate interpretation of behavioural data. In this investigation, we used support vector machine (SVM) models to classify LLR data collected from three WT strains: AB, TL and TLAB (a hybrid of AB and TL), during early embryogenesis, from 3 to 9 days post-fertilisation (dpf). We analysed both the complete dataset and a subset of the data during the first 30after light change. This initial period of activity is substantially driven by vision, and is also known as the visual motor response (VMR). The analyses have resulted in three major conclusions: First, the LLR is different between the three WT strains, and at different developmental stages. Second, the distinguishable information in the VMR is comparable to, if not better than, the full dataset for classification purposes. Third, the distinguishable information of WT strains in the light-onset response differs from that in the light-offset response. While the classification accuracies were higher for the light-offset than light-onset response when using the complete LLR dataset, a reverse trend was observed when using a shorter VMR dataset. Together, our results indicate that one should use caution when extrapolating interpretations of LLR/VMR obtained from one WT strain to another.

  10. Molecular alterations and expression of succinate dehydrogenase complex in wild-type KIT/PDGFRA/BRAF gastrointestinal stromal tumors

    PubMed Central

    Celestino, Ricardo; Lima, Jorge; Faustino, Alexandra; Vinagre, João; Máximo, Valdemar; Gouveia, António; Soares, Paula; Manuel Lopes, José

    2013-01-01

    Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract, disclosing somatic KIT, PDGFRA and BRAF mutations. Loss of function of succinate dehydrogenase (SDH) complex is an alternative molecular mechanism in GISTs, namely in carriers of germline mutations of the SDH complex that develop Carney–Stratakis dyad characterized by multifocal GISTs and multicentric paragangliomas (PGLs). We studied a series of 25 apparently sporadic primary wild-type (WT) KIT/PDGFRA/BRAF GISTs occurring in patients without personal or familial history of PGLs, re-evaluated clinicopathological features and analyzed molecular alterations and immunohistochemistry expression of SDH complex. As control, we used a series of well characterized 49 KIT/PDGFRA/BRAF-mutated GISTs. SDHB expression was absent in 20% and SDHB germline mutations were detected in 12% of WT GISTs. Germline SDHB mutations were significantly associated to younger age at diagnosis. A significant reduction in SDHB expression in WT GISTs was found when compared with KIT/PDGFRA/BRAF-mutated GISTs. No significant differences were found when comparing DOG-1 and c-KIT expression in WT, SDHB-mutated and KIT/PDGFRA/BRAF-mutated GISTs. Our results confirm the occurrence of germline SDH genes mutations in isolated, apparently sporadic WT GISTs. WT KIT/PDGFRA/BRAF GISTs without SDHB or SDHA/SDHB expression may correspond to Carney–Stratakis dyad or Carney triad. Most importantly, the possibility of PGLs (Carney–Stratakis dyad) and/or pulmonary chondroma (Carney triad) should be addressed in these patients and their kindred. PMID:22948025

  11. Molecular alterations and expression of succinate dehydrogenase complex in wild-type KIT/PDGFRA/BRAF gastrointestinal stromal tumors.

    PubMed

    Celestino, Ricardo; Lima, Jorge; Faustino, Alexandra; Vinagre, João; Máximo, Valdemar; Gouveia, António; Soares, Paula; Lopes, José Manuel

    2013-05-01

    Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract, disclosing somatic KIT, PDGFRA and BRAF mutations. Loss of function of succinate dehydrogenase (SDH) complex is an alternative molecular mechanism in GISTs, namely in carriers of germline mutations of the SDH complex that develop Carney-Stratakis dyad characterized by multifocal GISTs and multicentric paragangliomas (PGLs). We studied a series of 25 apparently sporadic primary wild-type (WT) KIT/PDGFRA/BRAF GISTs occurring in patients without personal or familial history of PGLs, re-evaluated clinicopathological features and analyzed molecular alterations and immunohistochemistry expression of SDH complex. As control, we used a series of well characterized 49 KIT/PDGFRA/BRAF-mutated GISTs. SDHB expression was absent in 20% and SDHB germline mutations were detected in 12% of WT GISTs. Germline SDHB mutations were significantly associated to younger age at diagnosis. A significant reduction in SDHB expression in WT GISTs was found when compared with KIT/PDGFRA/BRAF-mutated GISTs. No significant differences were found when comparing DOG-1 and c-KIT expression in WT, SDHB-mutated and KIT/PDGFRA/BRAF-mutated GISTs. Our results confirm the occurrence of germline SDH genes mutations in isolated, apparently sporadic WT GISTs. WT KIT/PDGFRA/BRAF GISTs without SDHB or SDHA/SDHB expression may correspond to Carney-Stratakis dyad or Carney triad. Most importantly, the possibility of PGLs (Carney-Stratakis dyad) and/or pulmonary chondroma (Carney triad) should be addressed in these patients and their kindred. PMID:22948025

  12. Evaluation of Electrical Impedance as a Biomarker of Myostatin Inhibition in Wild Type and Muscular Dystrophy Mice

    PubMed Central

    Sanchez, Benjamin; Li, Jia; Yim, Sung; Pacheck, Adam; Widrick, Jeffrey J.; Rutkove, Seward B.

    2015-01-01

    Objectives Non-invasive and effort independent biomarkers are needed to better assess the effects of drug therapy on healthy muscle and that affected by muscular dystrophy (mdx). Here we evaluated the use of multi-frequency electrical impedance for this purpose with comparison to force and histological parameters. Methods Eight wild-type (wt) and 10 mdx mice were treated weekly with RAP-031 activin type IIB receptor at a dose of 10 mg kg−1 twice weekly for 16 weeks; the investigators were blinded to treatment and disease status. At the completion of treatment, impedance measurements, in situ force measurements, and histology analyses were performed. Results As compared to untreated animals, RAP-031 wt and mdx treated mice had greater body mass (18% and 17%, p < 0.001 respectively) and muscle mass (25% p < 0.05 and 22% p < 0.001, respectively). The Cole impedance parameters in treated wt mice, showed a 24% lower central frequency (p < 0.05) and 19% higher resistance ratio (p < 0.05); no significant differences were observed in the mdx mice. These differences were consistent with those seen in maximum isometric force, which was greater in the wt animals (p < 0.05 at > 70 Hz), but not in the mdx animals. In contrast, maximum force normalized by muscle mass was unchanged in the wt animals and lower in the mdx animals by 21% (p < 0.01). Similarly, myofiber size was only non-significantly higher in treated versus untreated animals (8% p = 0.44 and 12% p = 0.31 for wt and mdx animals, respectively). Conclusions Our findings demonstrate electrical impedance of muscle reproduce the functional and histological changes associated with myostatin pathway inhibition and do not reflect differences in muscle size or volume. This technique deserves further study in both animal and human therapeutic trials. PMID:26485280

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

    DOE PAGES

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

    2016-03-23

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

  14. Rapid Phosphoproteomic Effects of Abscisic Acid (ABA) on Wild-Type and ABA Receptor-Deficient A. thaliana Mutants.

    PubMed

    Minkoff, Benjamin B; Stecker, Kelly E; Sussman, Michael R

    2015-05-01

    Abscisic acid (ABA)¹ is a plant hormone that controls many aspects of plant growth, including seed germination, stomatal aperture size, and cellular drought response. ABA interacts with a unique family of 14 receptor proteins. This interaction leads to the activation of a family of protein kinases, SnRK2s, which in turn phosphorylate substrates involved in many cellular processes. The family of receptors appears functionally redundant. To observe a measurable phenotype, four of the fourteen receptors have to be mutated to create a multilocus loss-of-function quadruple receptor (QR) mutant, which is much less sensitive to ABA than wild-type (WT) plants. Given these phenotypes, we asked whether or not a difference in ABA response between the WT and QR backgrounds would manifest on a phosphorylation level as well. We tested WT and QR mutant ABA response using isotope-assisted quantitative phosphoproteomics to determine what ABA-induced phosphorylation changes occur in WT plants within 5 min of ABA treatment and how that phosphorylation pattern is altered in the QR mutant. We found multiple ABA-induced phosphorylation changes that occur within 5 min of treatment, including three SnRK2 autophosphorylation events and phosphorylation on SnRK2 substrates. The majority of robust ABA-dependent phosphorylation changes observed were partially diminished in the QR mutant, whereas many smaller ABA-dependent phosphorylation changes observed in the WT were not responsive to ABA in the mutant. A single phosphorylation event was increased in response to ABA treatment in both the WT and QR mutant. A portion of the discovery data was validated using selected reaction monitoring-based targeted measurements on a triple quadrupole mass spectrometer. These data suggest that different subsets of phosphorylation events depend upon different subsets of the ABA receptor family to occur. Altogether, these data expand our understanding of the model by which the family of ABA receptors directs

  15. [Drug susceptibility of wild-type and mutant H7N9 neuraminidase to zanamivir and oseltamivir].

    PubMed

    Wei, Yan-Nan; Zhang, Chao; Chen, Qing; Guo, Ying

    2014-07-01

    This study aimed to investigate the drug susceptibility of wild-type and mutant avian influenza A (H7N9) virus neuraminidase (NA) to oseltamivir and zanamivir. Codon optimized DNA of H7N9 (A/ Hangzhou/1/2013) NA was synthesized and constructed into the pcDNA3.1/His vector (NA(H7N9-WT)). Mutant NA(H7N9-H274Y) and NA(H7N9-R292K) plasmids were constructed by directed mutagenesis PCR using NA(H7N9-WT) plasmid as the template followed by sequencing. NA plasmids were transfected into 293T cells and cell lysates containing NAs were collected 48 h post-transfection. Wild-type and mutant NAs were analyzed by Western blotting and their activities were tested by the 4-MUNANA-based assay. All three NAs were expressed and enzymatic activities were confirmed. The effects of oseltamivir and zanamivir on all three NAs were then tested. It showed that the half maximal inhibitory concentrations (IC50s) of oseltamivir carboxylate on NA(H7N9-WT), NA(H7N9-H274Y) and NA(H7N9-R292K) were 1.6 nM, 15.1 nM, and > 1 000 nM with fold changes of 9 and > 625, respectively. The IC50 values of zanamivir on NA(H7N9-WT), NA(H7N9-H274Y), and NA(H7N9-R292K) were 1.1 nM, 1.4 nM, and 38.0 nM with fold changes of 1.3 and 34, respectively. These results indicated that oseltamivir and zanamivir could significantly inhibit NA(H7N9-WT). NA(H7N9-R292K) showed high-level resistance to both drugs (34-fold and 625-fold) and NA(H7N9-H274Y) was sensitive to both (1.3-fold and 9-fold). These results indicated that both oseltamivir and zanamivir could be used for patients infected with the H7N9 virus. However, when patients carried the H7N9 virus with a NA R292K mutation, other medications would be preferred over oseltamivir or zanamivir. PMID:25272593

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-09-01

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

  18. Estimation of the wild-type minimum inhibitory concentration value distribution.

    PubMed

    Jaspers, Stijn; Aerts, Marc; Verbeke, Geert; Beloeil, Pierre-Alexandre

    2014-01-30

    Antimicrobial resistance has become one of the main public health burdens of the last decades, and monitoring the development and spread of non-wild-type isolates has therefore gained increased interest. Monitoring is performed based on the minimum inhibitory concentration (MIC) values, which are collected through the application of dilution experiments. In order to account for the unobserved population heterogeneity of wild-type and non-wild-type isolates, mixture models are extremely useful. Instead of estimating the entire mixture globally, it was our major aim to provide an estimate for the wild-type first component only. The characteristics of this first component are not expected to change over time, once the wild-type population has been confidently identified for a given antimicrobial. With this purpose, we developed a new method based on the multinomial distribution, and we carry out a simulation study to study the properties of the new estimator. Because the new approach fits within the likelihood framework, we can compare distinct distributional assumptions in order to determine the most suitable distribution for the wild-type population. We determine the optimal parameters based on the AIC criterion, and attention is also paid to the model-averaged approach using the Akaike weights. The latter is thought to be very suitable to derive specific characteristics of the wild-type distribution and to determine limits for the wild-type MIC range. In this way, the new method provides an elegant means to compare distinct distributional assumptions and to quantify the wild-type MIC distribution of specific antibiotic-bacterium combinations.

  19. A nonimmunogenic sarcoma transduced with the cDNA for interferon gamma elicits CD8+ T cells against the wild-type tumor: correlation with antigen presentation capability

    PubMed Central

    1992-01-01

    To be recognized by CD8+ T lymphocytes, target cells must process and present peptide antigens in the context of major histocompatibility complex (MHC) class I molecules. The nonimmunogenic, low class I- expressing, methylcholanthrene (MCA)-induced murine sarcoma cell line, MCA 101, is a poor presenter of endogenously generated viral antigens to specific CD8+ T lymphocytes and cannot be used to generate tumor infiltrating lymphocytes (TIL). Since interferon gamma (IFN-gamma) has been shown to upregulate three sets of molecules important for antigen processing and presentation, we retrovirally transduced wild-type MCA 101 (101.WT) tumor with the mIFN-gamma cDNA to create the 101.NAT cell line. Unlike 101.WT, some clones of retrovirally transduced 101.NAT tumor expressed high levels of class I, and could be used to generate CD8+ TIL. More importantly, these TIL were therapeutic in vivo against established pulmonary metastases from the wild-type tumor. Although not uniformly cytotoxic amongst several separate cultures, these TIL did specifically release cytokines (IFN-gamma and tumor necrosis factor- alpha) in response to 101.WT targets. 101.WT's antigen presentation deficit was also reversed by gene modification with mIFN-gamma cDNA. 101.NAT had a greatly improved capacity to present viral antigens to CD8+ cytotoxic T lymphocytes. These findings show that a nonimmunogenic tumor, incapable of generating a CD8+ T cell immune response, could be gene-modified to generate a therapeutically useful immune response against the wild-type tumor. This strategy may be useful in developing treatments for tumor histologies not thought to be susceptible to T cell-based immunotherapy. PMID:1588273

  20. Differential composition of culture supernatants from wild-type Brucella abortus and its isogenic virB mutants.

    PubMed

    Delpino, M Victoria; Comerci, Diego J; Wagner, Mary Ann; Eschenbrenner, Michel; Mujer, Cesar V; Ugalde, Rodolfo A; Fossati, Carlos A; Baldi, Pablo C; Delvecchio, Vito G

    2009-07-01

    The virB genes coding type IV secretion system are necessary for the intracellular survival and replication of Brucella spp. In this study, extracellular proteins from B. abortus 2308 (wild type, WT) and its isogenic virB10 polar mutant were compared. Culture supernatants harvested in the early stationary phase were concentrated and subjected to 2D electrophoresis. Spots present in the WT strain but absent in the virB10 mutant (differential spots) were considered extracellular proteins released in a virB-related manner, and were identified by MALDI-TOF analysis and matching with Brucella genomes. Among the 11 differential proteins identified, DnaK chaperone (Hsp70), choloylglycine hydrolase (CGH) and a peptidyl-prolyl cis-trans isomerase (PPIase) were chosen for further investigation because of their homology with extracellular and/or virulence factors from other bacteria. The three proteins were obtained in recombinant form and specific monoclonal antibodies (mAbs) were prepared. By Western blot with these mAbs, the three proteins were detected in supernatants from the WT but not in those from the virB10 polar mutant or from strains carrying non-polar mutations in virB10 or virB11 genes. These results suggest that the expression of virB genes affects the extracellular release of DnaK, PPIase and CGH, and possibly other proteins from B. abortus.

  1. Safety and Immunogenicity of DNA Vaccines Encoding Ebolavirus and Marburgvirus Wild-Type Glycoproteins in a Phase I Clinical Trial

    PubMed Central

    Sarwar, Uzma N.; Costner, Pamela; Enama, Mary E.; Berkowitz, Nina; Hu, Zonghui; Hendel, Cynthia S.; Sitar, Sandra; Plummer, Sarah; Mulangu, Sabue; Bailer, Robert T.; Koup, Richard A.; Mascola, John R.; Nabel, Gary J.; Sullivan, Nancy J.; Graham, Barney S.; Ledgerwood, Julie E.

    2015-01-01

    Background Ebolavirus and Marburgvirus cause severe hemorrhagic fever with high mortality and are potential bioterrorism agents. There are no available vaccines or therapeutic agents. Previous clinical trials evaluated transmembrane-deleted and point-mutation Ebolavirus glycoproteins (GPs) in candidate vaccines. Constructs evaluated in this trial encode wild-type (WT) GP from Ebolavirus Zaire and Sudan species and the Marburgvirus Angola strain expressed in a DNA vaccine. Methods The VRC 206 study evaluated the safety and immunogenicity of these DNA vaccines (4 mg administered intramuscularly by Biojector) at weeks 0, 4, and 8, with a homologous boost at or after week 32. Safety evaluations included solicited reactogenicity and coagulation parameters. Primary immune assessment was done by means of GP-specific enzyme-linked immunosorbent assay. Results The vaccines were well tolerated, with no serious adverse events; 80% of subjects had positive enzyme-linked immunosorbent assay results (≥30) at week 12. The fourth DNA vaccination boosted the immune responses. Conclusions The investigational Ebolavirus and Marburgvirus WT GP DNA vaccines were safe, well tolerated, and immunogenic in this phase I study. These results will further inform filovirus vaccine research toward a goal of inducing protective immunity by using WT GP antigens in candidate vaccine regimens. Clinical Trials Registration NCT00605514. PMID:25225676

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

    PubMed Central

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

    2016-01-01

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

  3. Leaf hydraulic conductance varies with vein anatomy across Arabidopsis thaliana wild-type and leaf vein mutants.

    PubMed

    Caringella, Marissa A; Bongers, Franca J; Sack, Lawren

    2015-12-01

    Leaf venation is diverse across plant species and has practical applications from paleobotany to modern agriculture. However, the impact of vein traits on plant performance has not yet been tested in a model system such as Arabidopsis thaliana. Previous studies analysed cotyledons of A. thaliana vein mutants and identified visible differences in their vein systems from the wild type (WT). We measured leaf hydraulic conductance (Kleaf ), vein traits, and xylem and mesophyll anatomy for A. thaliana WT (Col-0) and four vein mutants (dot3-111 and dot3-134, and cvp1-3 and cvp2-1). Mutant true leaves did not possess the qualitative venation anomalies previously shown in the cotyledons, but varied quantitatively in vein traits and leaf anatomy across genotypes. The WT had significantly higher mean Kleaf . Across all genotypes, there was a strong correlation of Kleaf with traits related to hydraulic conductance across the bundle sheath, as influenced by the number and radial diameter of bundle sheath cells and vein length per area. These findings support the hypothesis that vein traits influence Kleaf , indicating the usefulness of this mutant system for testing theory that was primarily established comparatively across species, and supports a strong role for the bundle sheath in influencing Kleaf .

  4. Gene Mutation Analysis in EGFR Wild Type NSCLC Responsive to Erlotinib: Are There Features to Guide Patient Selection?

    PubMed Central

    Ulivi, Paola; Delmonte, Angelo; Chiadini, Elisa; Calistri, Daniele; Papi, Maximilian; Mariotti, Marita; Verlicchi, Alberto; Ragazzini, Angela; Capelli, Laura; Gamboni, Alessandro; Puccetti, Maurizio; Dubini, Alessandra; Burgio, Marco Angelo; Casanova, Claudia; Crinò, Lucio; Amadori, Dino; Dazzi, Claudio

    2014-01-01

    Tyrosine kinase inhibitors (TKIs) are very efficacious in non-small-cell lung cancer (NSCLC) patients harboring activating Epidermal Growth Factor Receptor (EGFR) mutations. However, about 10% of EGFR wild type (wt) patients respond to TKI, with unknown molecular mechanisms of sensitivity. We considered a case series of 34 EGFR wt NSCLC patients responsive to erlotinib after at least one line of therapy. Responsive patients were matched with an equal number of non-responsive EGFR wt patients. A panel of 26 genes, for a total of 214 somatic mutations, was analyzed by MassARRAY® System (Sequenom, San Diego, CA, USA). A 15% KRAS mutation was observed in both groups, with a prevalence of G12C in non-responders (80% vs. 40% in responders). NOTCH1, p53 and EGFR-resistance-related mutations were found more frequently in non-responders, whereas EGFR-sensitizing mutations and alterations in genes involved in proliferation pathways were more frequent in responders. In conclusion, our findings indicate that p53, NOTCH1 and exon 20 EGFR mutations seem to be related to TKI resistance. KRAS mutations do not appear to influence the TKI response, although G12C mutation is more frequent in non-responders. Finally, the use of highly sensitive methodologies could lead to the identification of under-represented EGFR mutations potentially associated with TKI sensitivity. PMID:25561229

  5. Protein flexibility and conformational state: a comparison of collective vibrational modes of wild-type and D96N bacteriorhodopsin.

    PubMed

    Whitmire, S E; Wolpert, D; Markelz, A G; Hillebrecht, J R; Galan, J; Birge, R R

    2003-08-01

    Far infrared (FIR) spectral measurements of wild-type (WT) and D96N mutant bacteriorhodopsin thin films have been carried out using terahertz time domain spectroscopy as a function of hydration, temperature, and conformational state. The results are compared to calculated spectra generated via normal mode analyses using CHARMM. We find that the FIR absorbance is slowly increasing with frequency and without strong narrow features over the range of 2-60 cm(-1) and up to a resolution of 0.17 cm(-1). The broad absorption shifts in frequency with decreasing temperature as expected with a strongly anharmonic potential and in agreement with neutron inelastic scattering results. Decreasing hydration shifts the absorption to higher frequencies, possibly resulting from decreased coupling mediated by the interior water molecules. Ground-state FIR absorbances have nearly identical frequency dependence, with the mutant having less optical density than the WT. In the M state, the FIR absorbance of the WT increases whereas there is no change for D96N. These results represent the first measurement of FIR absorbance change as a function of conformational state.

  6. WT1 regulates the development of the posterior taste field.

    PubMed

    Gao, Yankun; Toska, Eneda; Denmon, Dane; Roberts, Stefan G E; Medler, Kathryn F

    2014-06-01

    Despite the importance of taste in determining nutrient intake, our understanding of the processes that control the development of the peripheral taste system is lacking. Several early regulators of taste development have been identified, including sonic hedgehog, bone morphogenetic protein 4 and multiple members of the Wnt/β-catenin signaling pathway. However, the regulation of these factors, including their induction, remains poorly understood. Here, we identify a crucial role for the Wilms' tumor 1 protein (WT1) in circumvallate (CV) papillae development. WT1 is a transcription factor that is important in the normal development of multiple tissues, including both the olfactory and visual systems. In mice, WT1 expression is detectable by E12.5, when the CV taste placode begins to form. In mice lacking WT1, the CV fails to develop normally and markers of early taste development are dysregulated compared with wild type. We demonstrate that expression of the WT1 target genes Lef1, Ptch1 and Bmp4 is significantly reduced in developing tongue tissue derived from Wt1 knockout mice and that, in normal tongue, WT1 is bound to the promoter regions of these genes. Moreover, siRNA knockdown of WT1 in cultured taste cells leads to a reduction in the expression of Lef1 and Ptch1. Our data identify WT1 as a crucial transcription factor in the development of the CV through the regulation of multiple signaling pathways that have established roles in the formation and patterning of taste placodes.

  7. Two weeks of metformin treatment enhances mitochondrial respiration in skeletal muscle of AMPK kinase dead but not wild type mice.

    PubMed

    Kristensen, Jonas M; Larsen, Steen; Helge, Jørn W; Dela, Flemming; Wojtaszewski, Jørgen F P

    2013-01-01

    Metformin is used as an anti-diabetic drug. Metformin ameliorates insulin resistance by improving insulin sensitivity in liver and skeletal muscle. Reduced mitochondrial content has been reported in type 2 diabetic muscles and it may contribute to decreased insulin sensitivity characteristic for diabetic muscles. The molecular mechanism behind the effect of metformin is not fully clarified but inhibition of complex I in the mitochondria and also activation of the 5'AMP activated protein kinase (AMPK) has been reported in muscle. Furthermore, both AMPK activation and metformin treatment have been associated with stimulation of mitochondrial function and biogenesis. However, a causal relationship in skeletal muscle has not been investigated. We hypothesized that potential effects of in vivo metformin treatment on mitochondrial function and protein expressions in skeletal muscle are dependent upon AMPK signaling. We investigated this by two weeks of oral metformin treatment of muscle specific kinase dead α(2) (KD) AMPK mice and wild type (WT) littermates. We measured mitochondrial respiration and protein activity and expressions of key enzymes involved in mitochondrial carbohydrate and fat metabolism and oxidative phosphorylation. Mitochondrial respiration, HAD and CS activity, PDH and complex I-V and cytochrome c protein expression were all reduced in AMPK KD compared to WT tibialis anterior muscles. Surprisingly, metformin treatment only enhanced respiration in AMPK KD mice and thereby rescued the respiration defect compared to the WT mice. Metformin did not influence protein activities or expressions in either WT or AMPK KD mice.We conclude that two weeks of in vivo metformin treatment enhances mitochondrial respiration in the mitochondrial deficient AMPK KD but not WT mice. The improvement seems to be unrelated to AMPK, and does not involve changes in key mitochondrial proteins.

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

    PubMed

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

    2012-01-01

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

  9. Resolution of Specific Nucleotide Mismatches by Wild-Type and AZT-Resistant Reverse Transcriptases during HIV-1 Replication.

    PubMed

    Kharytonchyk, Siarhei; King, Steven R; Ndongmo, Clement B; Stilger, Krista L; An, Wenfeng; Telesnitsky, Alice

    2016-06-01

    A key contributor to HIV-1 genetic variation is reverse transcriptase errors. Some mutations result because reverse transcriptase (RT) lacks 3' to 5' proofreading exonuclease and can extend mismatches. However, RT also excises terminal nucleotides to a limited extent, and this activity contributes to AZT resistance. Because HIV-1 mismatch resolution has been studied in vitro but only indirectly during replication, we developed a novel system to study mismatched base pair resolution during HIV-1 replication in cultured cells using vectors that force template switching at defined locations. These vectors generated mismatched reverse transcription intermediates, with proviral products diagnostic of mismatch resolution mechanisms. Outcomes for wild-type (WT) RT and an AZT-resistant (AZT(R)) RT containing a thymidine analog mutation set-D67N, K70R, D215F, and K219Q-were compared. AZT(R) RT did not excise terminal nucleotides more frequently than WT, and for the majority of tested mismatches, both WT and AZT(R) RTs extended mismatches in more than 90% of proviruses. However, striking enzyme-specific differences were observed for one mispair, with WT RT preferentially resolving dC-rC pairs either by excising the mismatched base or switching templates prematurely, while AZT(R) RT primarily misaligned the primer strand, causing deletions via dislocation mutagenesis. Overall, the results confirmed HIV-1 RT's high capacity for mismatch extension during virus replication and revealed dramatic differences in aberrant intermediate resolution repertoires between WT and AZT(R) RTs on one mismatched replication intermediate. Correlating mismatch extension frequencies observed here with reported viral mutation rates suggests a complex interplay of nucleotide discrimination and mismatch extension drives HIV-1 mutagenesis. PMID:27075671

  10. First Demonstration of Positive Allosteric-like Modulation at the Human Wild Type Translocator Protein (TSPO).

    PubMed

    Narlawar, Rajeshwar; Werry, Eryn L; Scarf, Alana M; Hanani, Raphy; Chua, Sook Wern; King, Victoria A; Barron, Melissa L; Martins, Ralph N; Ittner, Lars M; Rendina, Louis M; Kassiou, Michael

    2015-11-12

    We show that changing the number and position of nitrogen atoms in the heteroatomic core of a pyrazolopyrimidine acetamide is sufficient to induce complex binding to wild type human TSPO. Only compounds with this complex binding profile lacked intrinsic effect on glioblastoma proliferation but positively modulated the antiproliferative effects of a synthetic TSPO ligand. To the best of our knowledge this is the first demonstration of allosteric-like interaction at the wild type human TSPO.

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

    PubMed Central

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

    2016-01-01

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

  12. Single cell monitoring of growth arrest and morphological changes induced by transfer of wild-type p53 alleles to glioblastoma cells.

    PubMed Central

    Van Meir, E G; Roemer, K; Diserens, A C; Kikuchi, T; Rempel, S A; Haas, M; Huang, H J; Friedmann, T; de Tribolet, N; Cavenee, W K

    1995-01-01

    Mutation of the p53 tumor suppressor gene is one of the earliest identified genetic lesions during malignant progression of human astrocytomas. To assess the functional significance of these mutations, wild-type (WT) p53 genes were introduced into glioblastoma cell lines having mutant, WT, or null endogenous p53 alleles. Populations of cells with mutant or null endogenous p53 alleles and exogenous WT p53 were spontaneously selected in culture for cells expressing only mutant p53 or no p53, which then displayed a growth and tumorigenic phenotype identical to the parental cells. To determine the phenotypic consequences of WT p53 expression before the occurrence of mutations, we developed a single cell assay to monitor WT p53-dependent transcription activity. Transfer and expression of exogenous WT p53 genes to cells with endogenous mutant or deleted, but not WT, p53 alleles caused growth arrest and morphological changes, including increased cell size and acquisition of multiple nuclei. This supports the hypothesis that genetic lesions of the p53 gene play an important role in the genesis of astrocytomas. Furthermore, the high sensitivity of the episomal single cell reporter strategy developed here has potential clinical applications in the rapid screening of patients for germ-line mutations of the p53 gene or any other gene with known targets for transcriptional transactivation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7862624

  13. Reactivation of wild-type and mutant p53 by tryptophanolderived oxazoloisoindolinone SLMP53-1, a novel anticancer small-molecule

    PubMed Central

    Soares, Joana; Raimundo, Liliana; Pereira, Nuno A.L.; Monteiro, Ângelo; Gomes, Sara; Bessa, Cláudia; Pereira, Clara; Queiroz, Glória; Bisio, Alessandra; Fernandes, João; Gomes, Célia; Reis, Flávio; Gonçalves, Jorge; Inga, Alberto; Santos, Maria M.M.; Saraiva, Lucília

    2016-01-01

    Restoration of the p53 pathway, namely by reactivation of mutant (mut) p53, represents a valuable anticancer strategy. Herein, we report the identification of the enantiopure tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a novel reactivator of wild-type (wt) and mut p53, using a yeast-based screening strategy. SLMP53-1 has a p53-dependent anti-proliferative activity in human wt and mut p53R280K-expressing tumor cells. Additionally, SLMP53-1 enhances p53 transcriptional activity and restores wt-like DNA binding ability to mut p53R280K. In wt/mut p53-expressing tumor cells, SLMP53-1 triggers p53 transcription-dependent and mitochondrial apoptotic pathways involving BAX, and wt/mut p53 mitochondrial translocation. SLMP53-1 inhibits the migration of wt/mut p53-expressing tumor cells, and it shows promising p53-dependent synergistic effects with conventional chemotherapeutics. In xenograft mice models, SLMP53-1 inhibits the growth of wt/mut p53-expressing tumors, but not of p53-null tumors, without apparent toxicity. Collectively, besides the potential use of SLMP53-1 as anticancer drug, the tryptophanol-derived oxazoloisoindolinone scaffold represents a promissing starting point for the development of effective p53-reactivating drugs. PMID:26735173

  14. Amplification-free In Situ KRAS Point Mutation Detection at 60 copies/mL in Urine in a Background of 1000-fold Wild Type

    PubMed Central

    KirimLi, Ceyhun E.; Shih, Wei-Heng; Shih, Wan Y.

    2016-01-01

    We have examined in situ detection of single-nucleotide KRAS mutation in urine using a (Pb(Mg1/3Nb2/3)O3)0.65(PbTiO3)0.35 (PMN-PT) piezoelectric plate sensor (PEPS) coated with a 17-nucleotide (nt) locked nucleic acid (LNA) probe DNA complementary to the KRAS mutation. To enhance in situ mutant (MT) DNA detection specificity against the wild type (WT), the detection was carried out in a flow with a flow rate of 4 mL/min and at 63°C with the PEPS vertically situated at the center of the flow in which both the temperature and the flow impingement force discriminated the wild type. Under such conditions, PEPS was shown to specifically detect KRAS MT in situ with 60 copies/mL analytical sensitivity in a background of clinically-relevant 1000-fold more WT in 30 min without DNA isolation, amplification, or labeling. For validation, the detection was followed with detection in a mixture of blue MT fluorescent reporter microspheres (FRMs) (MT FRMs) that bound to only the captured MT and orange WT FRMs that bound to only the captured WT. Microscopic examinations showed that the captured blue MT FRMs still outnumbered the orange WT FRMs by a factor of 4 to 1 even though WT was 1000-fold of MT in urine. Finally, multiplexed specific mutation detection was demonstrated using a 6-PEPS array each with a probe DNA targeting one of the 6 codon-12 KRAS mutations. PMID:26783561

  15. Amplification-free in situ KRAS point mutation detection at 60 copies per mL in urine in a background of 1000-fold wild type.

    PubMed

    Kirimli, Ceyhun E; Shih, Wei-Heng; Shih, Wan Y

    2016-02-21

    We have examined the in situ detection of a single-nucleotide KRAS mutation in urine using a (Pb(Mg1/3Nb2/3)O3)0.65(PbTiO3)0.35 (PMN-PT) piezoelectric plate sensor (PEPS) coated with a 17-nucleotide (nt) locked nucleic acid (LNA) probe DNA complementary to the KRAS mutation. To enhance the in situ mutant (MT) DNA detection specificity against the wild type (WT), detection was carried out in a flow with a flow rate of 4 mL min(-1) and at 63 °C with the PEPS vertically situated at the center of the flow in which both the temperature and the flow impingement force discriminated the wild type. Under such conditions, PEPS was shown to specifically detect KRAS MT in situ with 60 copies per mL analytical sensitivity in a background of clinically-relevant 1000-fold more WT in 30 min without DNA isolation, amplification, or labeling. For validation, this detection was followed with detection in a mixture of blue MT fluorescent reporter microspheres (FRMs) (MT FRMs) that bound to only the captured MT and orange WT FRMs that bound to only the captured WT. Microscopic examinations showed that the captured blue MT FRMs still outnumbered the orange WT FRMs by a factor of 4 to 1 even though WT was 1000-fold of MT in urine. Finally, multiplexed specific mutation detection was demonstrated using a 6-PEPS array each with a probe DNA targeting one of the 6 codon-12 KRAS mutations. PMID:26783561

  16. Comparative Transcriptome of Wild Type and Selected Strains of the Microalgae Tisochrysis lutea Provides Insights into the Genetic Basis, Lipid Metabolism and the Life Cycle

    PubMed Central

    Carrier, Gregory; Garnier, Matthieu; Le Cunff, Loïc; Bougaran, Gaël; Probert, Ian; De Vargas, Colomban; Corre, Erwan; Cadoret, Jean-Paul; Saint-Jean, Bruno

    2014-01-01

    The applied exploitation of microalgae cultures has to date almost exclusively involved the use of wild type strains, deposited over decades in dedicated culture collections. Concomitantly, the concept of improving algae with selection programs for particular specific purposes is slowly emerging. Studying since a decade an economically and ecologically important haptophyte Tisochrysis lutea (Tiso), we took advantage of the availability of wild type (Tiso-Wt) and selected (Tiso-S2M2) strains to conduct a molecular variations study. This endeavour presented substantial challenges: the genome assembly was not yet available, the life cycle unknown and genetic diversity of Tiso-Wt poorly documented. This study brings the first molecular data in order to set up a selection strategy for that microalgae. Following high-throughput Illumina sequencing, transcriptomes of Tiso-Wt and Tiso-S2M2 were de novo assembled and annotated. Genetic diversity between both strains was analyzed and revealed a clear conservation, while a comparison of transcriptomes allowed identification of polymorphisms resulting from the selection program. Of 34,374 transcripts, 291 were differentially expressed and 165 contained positional polymorphisms (SNP, Indel). We focused on lipid over-accumulation of the Tiso-S2M2 strain and 8 candidate genes were identified by combining analysis of positional polymorphism, differential expression levels, selection signature and by study of putative gene function. Moreover, genetic analysis also suggests the existence of a sexual cycle and genetic recombination in Tisochrysis lutea. PMID:24489800

  17. Differential Transcriptome Networks between IDO1-Knockout and Wild-Type Mice in Brain Microglia and Macrophages

    PubMed Central

    Gonzalez-Pena, Dianelys; Nixon, Scott E.; Southey, Bruce R.; Lawson, Marcus A.; McCusker, Robert H.; Hernandez, Alvaro G.; Dantzer, Robert; Kelley, Keith W.; Rodriguez-Zas, Sandra L.

    2016-01-01

    Microglia in the brain and macrophages in peripheral organs are cell types responsible for immune response to challenges. Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunomodulatory enzyme of the tryptophan pathway that is expressed in the brain. The higher activity of IDO1 in response to immune challenge has been implicated in behavioral disorders. The impact of IDO1 depletion on the microglia transcriptome has not been studied. An investigation of the transcript networks in the brain microglia from IDO1-knockout (IDO1-KO) mice was undertaken, relative to peripheral macrophages and to wild-type (WT) mice under unchallenged conditions. Over 105 transcript isoforms were differentially expressed between WT and IDO1-KO within cell type. Within microglia, Saa3 and Irg1 were over-expressed in IDO1-KO relative to WT. Within macrophages, Csf3 and Sele were over-expressed in IDO1-KO relative to WT. Among the genes differentially expressed between strains, enriched biological processes included ion homeostasis and ensheathment of neurons within microglia, and cytokine and chemokine expression within macrophages. Over 11,110 transcript isoforms were differentially expressed between microglia and macrophages and of these, over 10,800 transcripts overlapped between strains. Enriched biological processes among the genes over- and under-expressed in microglia relative to macrophages included cell adhesion and apoptosis, respectively. Detected only in microglia or macrophages were 421 and 43 transcript isoforms, respectively. Alternative splicing between cell types based on differential transcript isoform abundance was detected in 210 genes including Phf11d, H2afy, and Abr. Across strains, networks depicted a predominance of genes under-expressed in microglia relative to macrophages that may be a precursor for the different response of both cell types to challenges. The detected transcriptome differences enhance the understanding of the role of IDO1 in the microglia transcriptome

  18. Potentiation of a tumor cell susceptibility to autologous CTL killing by restoration of wild-type p53 function.

    PubMed

    Thiery, Jérôme; Dorothée, Guillaume; Haddada, Hedi; Echchakir, Hamid; Richon, Catherine; Stancou, Rodica; Vergnon, Isabelle; Benard, Jean; Mami-Chouaib, Fathia; Chouaib, Salem

    2003-06-15

    Inactivation of p53 has been implicated in many types of tumors particularly in non-small cell lung carcinoma, one of the most common cancers in which p53 mutation has been frequently identified. The aim of this study was to investigate the influence of p53 status on the regulation of tumor susceptibility to specific CTL-mediated cell death. For this purpose, we used a cytotoxic T lymphocyte clone, Heu127, able to lyse the human autologous lung carcinoma cell line, IGR-Heu, in a HLA-A2-restricted manner. Direct genomic DNA sequencing revealed that IGR-Heu expresses a mutated p53 at codon 132 of the exon 5 which results in the loss of p53 capacity to induce the expression of the p53-regulated gene product p21(waf/CIP1). Initial experiments demonstrated that IGR-Heu was resistant to Fas, TNF, and TRAIL apoptotic pathways. This correlated with the lack of p55 TNFRI, Fas, DR4, and DR5 expression. The effect of wild-type (wt) p53 restoration on the sensitization of IGR-Heu to autologous CTL clone lysis was investigated following infection of the tumor cell line with a recombinant adenovirus encoding the wt p53 (Adwtp53). We demonstrate that the restoration of wt p53 expression and function resulted in a significant potentiation of target cell susceptibility to CTL-mediated lysis. The wt p53-induced optimization of tumor cell killing by specific CTL involves at least in part Fas-mediated pathway via induction of CD95 expression by tumor cells but does not appear to interfere with granzyme B cytotoxic pathway. PMID:12794118

  19. Wild-type Human γD-crystallin Promotes Aggregation of Its Oxidation-mimicking, Misfolding-prone W42Q Mutant*

    PubMed Central

    Serebryany, Eugene; King, Jonathan A.

    2015-01-01

    Non-native protein conformers generated by mutation or chemical damage template aggregation of wild-type, undamaged polypeptides in diseases ranging from amyotrophic lateral sclerosis to cancer. We tested for such interactions in the natively monomeric human eye lens protein γd-crystallin, whose aggregation leads to cataract disease. The oxidation-mimicking W42Q mutant of γd-crystallin formed non-native polymers starting from a native-like state under physiological conditions. Aggregation occurred in the temperature range 35–45 °C, in which the mutant protein began to lose the native conformation of its N-terminal domain. Surprisingly, wild-type γd-crystallin promoted W42Q polymerization in a catalytic manner, even at mutant concentrations too low for homogeneous nucleation to occur. The presence of wild-type protein also downshifted the temperature range of W42Q aggregation. W42Q aggregation required formation of a non-native intramolecular disulfide bond but not intermolecular cross-linking. Transient WT/W42Q binding may catalyze this oxidative misfolding event in the mutant. That a more stable variant in a mixture can specifically promote aggregation of a less stable one rationalizes how extensive aggregation of rare damaged polypeptides can occur during the course of aging. PMID:25787081

  20. Resistance and gain-of-resistance phenotypes in cancers harboring wild-type p53

    PubMed Central

    Martinez-Rivera, Michelle; Siddik, Zahid H.

    2012-01-01

    Chemotherapy is the bedrock for the clinical management of cancer, and the tumor suppressor p53 has a central role in this therapeutic modality. This protein facilitates favorable antitumor drug response through a variety of key cellular functions, including cell cycle arrest, senescence, and apoptosis. These functions essentially cease once p53 becomes mutated, as occurs in ~50% of cancers, and some p53 mutants even exhibit gain-of-function effects, which lead to greater drug resistance. However, it is becoming increasingly evident that resistance is also seen in cancers harboring wild-type p53. In this review, we discuss how wild-type p53 is inactivated to render cells resistant to antitumor drugs. This may occur through various mechanisms, including an increase in proteasomal degradation, defects in post-translational modification, and downstream defects in p53 target genes. We also consider evidence that the resistance seen in wild-type p53 cancers can be substantially greater than that seen in mutant p53 cancers, and this poses a far greater challenge for efforts to design strategies that increase drug response in resistant cancers already primed with wild-type p53. Because the mechanisms contributing to this wild-type p53 “gain-of-resistance” phenotype are largely unknown, a concerted research effort is needed to identify the underlying basis for the occurrence of this phenotype and, in parallel, to explore the possibility that the phenotype may be a product of wild-type p53 gain-of-function effects. Such studies are essential to lay the foundation for a rational therapeutic approach in the treatment of resistant wild-type p53 cancers. PMID:22227014

  1. Registered report: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.

    PubMed

    Bhargava, Ajay; Pelech, Steven; Woodard, Ben; Kerwin, John; Maherali, Nimet

    2016-01-01

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from 'RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth' by Hatzivassiliou and colleagues, published in Nature in 2010 (Hatzivassiliou et al., 2010). Hatzivassiliou and colleagues examined the paradoxical response of RAF-WT tumors to treatment with RAF inhibitors. The key experiments being replicated include Figure 1A, in which the original authors demonstrated that treatment of a subset of BRAF(WT) tumor cell lines with RAF small molecule inhibitors resulted in an increase in cell viability, Figure 2B, which reported that RAF inhibitor activation of the MAPK pathway was dependent on CRAF but not BRAF, and Figure 4A, where the dimerization of BRAF and CRAF was modulated by the RAF inhibitor PLX4720, but not GDC-0879. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.

  2. Differential morphology and transcriptome profile between the incompletely fused carpels ovary and its wild-type in maize.

    PubMed

    Li, Hongping; Wu, Yufeng; Zhao, Yali; Hu, Xiuli; Chang, Jianfeng; Wang, Qun; Dong, Pengfei; Zhang, Moubiao; Li, Chaohai

    2016-01-01

    We have isolated a new mutation in maize, incompletely fused carpels (ifc), which results in an open stylar canal on the ovary and an incomplete pericarp at the top of the kernel. The maize ovary derives from the fusion of three carpels; however, the molecular networks regulating maize carpel fusion remain largely unclear. In this study, RNA sequencing (RNA-seq) was performed on wild-type (WT) and ifc ovaries that were collected after carpel fusion defects could be morphologically distinguished. In total, 877 differentially expressed genes were identified. Functional analysis revealed overexpression of genes related to "DNA binding", "transcription regulation", "hormones", and "stress responses". Among the 88 differentially expressed transcription factor (TF) genes, five showed a high degree of conservation (77.7-88.0% amino acid identity) of their conserved domains with genes associated with carpel fusion deficiency in Arabidopsis thaliana, suggesting that these five genes might control carpel fusion in maize. In addition, 30 genes encoding components of hormone synthesis and signaling pathways were differentially expressed between ifc and WT ovaries, indicating complex hormonal regulation during carpel fusion. These results help elucidate the underlying mechanisms that regulate carpel fusion, supporting the functional analysis of genes involved in producing this phenotype. PMID:27587343

  3. Differential morphology and transcriptome profile between the incompletely fused carpels ovary and its wild-type in maize

    PubMed Central

    Li, Hongping; Wu, Yufeng; Zhao, Yali; Hu, Xiuli; Chang, Jianfeng; Wang, Qun; Dong, Pengfei; Zhang, Moubiao; Li, Chaohai

    2016-01-01

    We have isolated a new mutation in maize, incompletely fused carpels (ifc), which results in an open stylar canal on the ovary and an incomplete pericarp at the top of the kernel. The maize ovary derives from the fusion of three carpels; however, the molecular networks regulating maize carpel fusion remain largely unclear. In this study, RNA sequencing (RNA-seq) was performed on wild-type (WT) and ifc ovaries that were collected after carpel fusion defects could be morphologically distinguished. In total, 877 differentially expressed genes were identified. Functional analysis revealed overexpression of genes related to “DNA binding”, “transcription regulation”, “hormones”, and “stress responses”. Among the 88 differentially expressed transcription factor (TF) genes, five showed a high degree of conservation (77.7–88.0% amino acid identity) of their conserved domains with genes associated with carpel fusion deficiency in Arabidopsis thaliana, suggesting that these five genes might control carpel fusion in maize. In addition, 30 genes encoding components of hormone synthesis and signaling pathways were differentially expressed between ifc and WT ovaries, indicating complex hormonal regulation during carpel fusion. These results help elucidate the underlying mechanisms that regulate carpel fusion, supporting the functional analysis of genes involved in producing this phenotype. PMID:27587343

  4. Antigen spreading-induced CD8+T cells confer protection against the lethal challenge of wild-type malignant mesothelioma by eliminating myeloid-derived suppressor cells

    PubMed Central

    Lee, Boon Kiat; Tang, Jiansong; Wu, Xilin; Cheung, Ka-Wai; Lok Lo, Nathan Tin; Man, Kwan; Liu, Li; Chen, Zhiwei

    2015-01-01

    A key focus in cancer immunotherapy is to investigate the mechanism of efficacious vaccine responses. Using HIV-1 GAG-p24 in a model PD1-based DNA vaccine, we recently reported that vaccine-elicited CD8+ T cells conferred complete prevention and therapeutic cure of AB1-GAG malignant mesothelioma in immunocompetent BALB/c mice. Here, we further investigated the efficacy and correlation of protection on the model vaccine-mediated antigen spreading against wild-type AB1 (WT-AB1) mesothelioma. We found that this vaccine was able to protect mice completely from three consecutive lethal challenges of AB1-GAG mesothelioma. Through antigen spreading these animals also developed tumor-specific cytotoxic CD8+ T cells, but neither CD4+ T cells nor antibodies, rejecting WT-AB1 mesothelioma. A majority of these protected mice (90%) were also completely protected against the lethal WT-AB1 challenge. Adoptive cell transfer experiments further demonstrated that antigen spreading-induced CD8+ T cells conferred efficacious therapeutic effects against established WT-AB1 mesothelioma and prevented the increase of exhausted PD-1+ and Tim-3+ CD8+ T cells. A significant inverse correlation was found between the frequency of functional PD1−Tim3− CD8+ T cells and that of MDSCs or tumor mass in vivo. Mechanistically, we found that WT-AB1 mesothelioma induced predominantly polymorphonuclear (PMN) MDSCs in vivo. In co-cultures with efficacious CD8+ T cells, a significant number of PMN-MDSCs underwent apoptosis in a dose-dependent way. Our findings indicate that efficacious CD8+ T cells capable of eliminating both tumor cells and MDSCs are likely necessary for fighting wild-type malignant mesothelioma. PMID:26431275

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

    PubMed

    Verbeek, Peter; Iwamoto, Toshitaka; Murakami, Noboru

    2008-01-28

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

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

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

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

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

    PubMed

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

    1997-06-01

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

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

    PubMed

    Verbeek, Peter; Iwamoto, Toshitaka; Murakami, Noboru

    2008-01-28

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

  9. [Physiological differences between HPS/PHI over-expressing transgenic and wild-type geraniums under formaldehyde stress revealed by FTIR analysis].

    PubMed

    Tang, Li-juan; Zhang, Ya-nan; Song, Zhong-bang; Zhang, Wei; Huang, Shu-shi; Li, Kun-zhi; Chen, Li-mei

    2012-05-01

    In the present study, FTIR was used to analyze changes in chemical component contents and spectra characters of 3-hexulose-6-phosphate synthase/6-phosphate-3-hexuloisomerase (HPS/PHI) over-expressing transgenic and wild-type (WT) geraniums under formaldehyde (HCHO) stress to examine if FTIR could be a new method for identification of phenotypic differences between the transgenic plants with a photosynthetic HCHO-assimilation pathway and the WT plants. The WT and transgenic geranium plants were treated with 4 mmol x L(-1) HCHO for 0, 1, 2, 3 and 4 days, respectively. The comparison of FTIR spectral characteristics at different time points between the transgenic and WT plants indicated that the contents of carbohydrate, proteins and aliphatic compounds were significantly higher than those in the WT plants after 4 days of HCHO-treatment. This may be due to installation of the photosynthetic HCHO-assimilation pathway in the transgenic geranium, which enhanced its ability to metabolize and assimilate HCHO, thus allowed more HCHO to be fixed to 6-phosphate fructose, and then entered assimilation pathways for synthesis of a variety of intracellular components. The results suggest that FTIR can be a new method to identify the phenotypic differences between transgenic plants with a photosynthetic HCHO-assimilation pathway and WT plants.

  10. Comparative studies of senescence-related enzymes in the cotyledon of chlorophyll b-deficient mutant and its wild type oilseed rape during senescence.

    PubMed

    Qin, Tingting; Fu, Jingfeng; Zhang, Nianhui; Du, Linfang

    2006-09-01

    The change patterns of senescence-related enzymes during cotyledon senescence were studied in a chlorophyll (Chl) b-deficient mutant type (MT, Cr3529) and its wild type (WT) of Brassica napus L. seedlings. The fresh weight on the basis of cotyledon number initially increased till 20 days after planting (DAP) and then kept relative constant. The protein content decreased sharply since 20 DAP and Chl content reduced since 10 DAP in both types; however the rate of degradation in protein and Chl in the MT was slower than that in the WT since 20 DAP. Superoxide dismutase (SOD; E.C.1.15.1.1) activity declined in the WT but increased in the MT since 20 DAP. Activity of peroxidase (POD; E.C.1.11.1.7) increased markedly after 20 DAP in both types. Esterase (EST; E.C.3.1.1.1) activity increased in both types since 10 DAP, whereas at 40 DAP it was much lower in the MT than that in the WT. In addition, bands patterns of SOD, POD and EST isozymes were changed during cotyledon development in both types, but some differences were observed. Cu/ZnSODs activities were higher in the MT at 40 DAP as compared with the WT. These results showed that day 20 was the turning point during the cotyledon development and the senescence in the MT cotyledon was slower than that in the WT. PMID:22980198

  11. Genome-Wide Identification of the Transcription Factors Involved in Citrus Fruit Ripening from the Transcriptomes of a Late-Ripening Sweet Orange Mutant and Its Wild Type

    PubMed Central

    Wu, Juxun; Fu, Lili; Yi, Hualin

    2016-01-01

    Fruit ripening is a genetically programmed process. Transcription factors (TFs) play key roles in plant development and ripening by temporarily and spatially regulating the transcription of their target genes. In this study, a total of 159 TFs were identified from a spontaneous late-ripening mutant 'Fengwan' (C. sinensis L. Osbeck) sweet orange (MT) and its wild-type counterpart ('Fengjie 72–1', WT) along the ripening period via the Transcription Factor Prediction of PlantTFDB 3.0. Fifty-two differentially expressed TFs were identified between MT and WT; 92 and 120 differentially expressed TFs were identified in WT and MT, respectively. The Venn diagram analysis showed that 16 differentially expressed TFs were identified between MT and WT and during the ripening of WT and MT. These TFs were primarily assigned to the families of C2H2, Dof, bHLH, ERF, MYB, NAC and LBD. Particularly, the number of TFs of the ERF family was the greatest between MT and WT. According to the results of the WGCNA analysis, a weighted correlation network analysis tool, several important TFs correlated to abscisic acid (ABA), citric acid, fructose, glucose and sucrose were identified, such as RD26, NTT, GATA7 and MYB21/62/77. Hierarchical cluster analysis and the expression analysis conducted at five fruit ripening stages further validated the pivotal TFs that potentially function during orange fruit development and ripening. PMID:27104786

  12. Genome-Wide Identification of the Transcription Factors Involved in Citrus Fruit Ripening from the Transcriptomes of a Late-Ripening Sweet Orange Mutant and Its Wild Type.

    PubMed

    Wu, Juxun; Fu, Lili; Yi, Hualin

    2016-01-01

    Fruit ripening is a genetically programmed process. Transcription factors (TFs) play key roles in plant development and ripening by temporarily and spatially regulating the transcription of their target genes. In this study, a total of 159 TFs were identified from a spontaneous late-ripening mutant 'Fengwan' (C. sinensis L. Osbeck) sweet orange (MT) and its wild-type counterpart ('Fengjie 72-1', WT) along the ripening period via the Transcription Factor Prediction of PlantTFDB 3.0. Fifty-two differentially expressed TFs were identified between MT and WT; 92 and 120 differentially expressed TFs were identified in WT and MT, respectively. The Venn diagram analysis showed that 16 differentially expressed TFs were identified between MT and WT and during the ripening of WT and MT. These TFs were primarily assigned to the families of C2H2, Dof, bHLH, ERF, MYB, NAC and LBD. Particularly, the number of TFs of the ERF family was the greatest between MT and WT. According to the results of the WGCNA analysis, a weighted correlation network analysis tool, several important TFs correlated to abscisic acid (ABA), citric acid, fructose, glucose and sucrose were identified, such as RD26, NTT, GATA7 and MYB21/62/77. Hierarchical cluster analysis and the expression analysis conducted at five fruit ripening stages further validated the pivotal TFs that potentially function during orange fruit development and ripening. PMID:27104786

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

    PubMed

    Cordner, Z A; Tamashiro, K L K

    2016-01-01

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

  14. Electrical Impedance Myography to Detect the Effects of Electrical Muscle Stimulation in Wild Type and Mdx Mice

    PubMed Central

    Li, Jia; Yim, Sung; Pacheck, Adam; Sanchez, Benjamin; Rutkove, Seward B.

    2016-01-01

    Objective Tools to better evaluate the impact of therapy on nerve and muscle disease are needed. Electrical impedance myography (EIM) is sensitive to neuromuscular disease progression as well as to therapeutic interventions including myostatin inhibition and antisense oligonucleotide-based treatments. Whether the technique identifies the impact of electrical muscle stimulation (EMS) is unknown. Methods Ten wild-type (wt) C57B6 mice and 10 dystrophin-deficient (mdx) mice underwent 2 weeks of 20 min/day EMS on left gastrocnemius and sham stimulation on the right gastrocnemius. Multifrequency EIM data and limb girth were obtained before and at the conclusion of the protocol. Muscle weight, in situ force measurements, and muscle fiber histology were also assessed at the conclusion of the study. Results At the time of sacrifice, muscle weight was greater on the EMS-treated side than on the sham-stimulated side (p = 0.018 for wt and p = 0.007 for mdx). Similarly, in wt animals, EIM parameters changed significantly compared to baseline (resistance (p = 0.009), reactance (p = 0.0003) and phase (p = 0.002); these changes were due in part to reductions in the EIM values on the EMS-treated side and elevations on the sham-simulated side. Mdx animals showed analogous but non-significant changes (p = 0.083, p = 0.064, and p = 0.57 for resistance, reactance and phase, respectively). Maximal isometric force trended higher on the stimulated side in wt animals only (p = 0.06). Myofiber sizes in wt animals were also larger on the stimulated side than on the sham-stimulated side (p = 0.034); no significant difference was found in the mdx mice (p = 0.79). Conclusion EIM is sensitive to stimulation-induced muscle alterations in wt animals; similar trends are also present in mdx mice. The mechanisms by which these EIM changes develop, however, remains uncertain. Possible explanations include longer-term trophic effects and shorter-term osmotic effects. PMID:26986564

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  16. Effects of Mechanical Overloading on the Properties of Soleus Muscle Fibers, with or without Damage in MDX and Wild Type Mice

    NASA Astrophysics Data System (ADS)

    Terada, Masahiro; Kawano, Fuminori; Ohira, Takashi; Oke, Yoshihiko; Nakai, Naoya; Ohira, Yoshinobu

    2008-06-01

    Effects of mechanical overloading on the characteristics of regenerating or not-regenerating soleus muscle fibers were studied. The muscle fibers of mdx mice were characterized by the localization of myonuclei. Muscle damage was also induced in wild type (WT) mice by injection of cardiotoxin (CTX) into soleus muscle. Overloading was applied for 14 days to the left soleus muscle in mdx and intact and CTX-injected WT mice by removing the distal tendons of plantaris and gastrocnemius muscles. The contralateral muscle served as the normal control. These animals were then allowed ambulation recovery in the cage. Central myonuclei were noted in many fibers of mdx and CTX-injected mice with or without overloading. In general, the fibers with central nuclei were considered as regenerating fibers. The fibers with more central nuclei were increased in mdx mice, but the fibers with more peripheral nuclei were increased in CTX-injected WT mice by overloading. The muscle satellite cells, neuromuscular junctions (NMJ), and myonuclei were stained. Most of the properties, such as number of myonuclei and satellite cells, size of NMJ, and fiber length, were not influenced by mechanical overloading in all mice. Approximately 0.6% branched fibers were seen in the intact soleus of mdx mice, although these fibers were not detected in WT mice. However, the percentage of these fibers was increased by overloading especially in mdx mice (~50% vs. ~2.5% in WT). In CTX-injected WT mice, these fibers were ~15% with or without overloading. The fiber cross sectional area in normal WT, but not in mdx and CTX-injected WT mice, was increased by overloading (p<0.05). These results suggested that the functional overload induced muscle damage in mdx mice, but promoted the regeneration in CTX-injected WT mice.

  17. Transcriptional repression in normal human keratinocytes by wild-type and mutant p53.

    PubMed

    Alvarez-Salas, L M; Velazquez, A; Lopez-Bayghen, E; Woodworth, C D; Garrido, E; Gariglio, P; DiPaolo, J A

    1995-05-01

    Wild-type p53 is a nuclear phosphoprotein that inhibits cell proliferation and represses transcriptionally most TATA box-containing promoters in transformed or tumor-derived cell lines. This study demonstrates that p53 alters transcription of the long control region (LCR) of human papillomavirus type 18 (HPV-18). Wild-type and mutant p53 143Val to Ala repressed the HPV-18 LCR promoter in normal human keratinocytes, the natural host cell for HPV infections. Repression by wild-type p53 was also observed in C-33A cells and in an HPV-16-immortalized cell line with an inducible wild-type p53. However, when C-33A cells were cotransfected with the HPV-18 LCR and mutant 143Val to Ala, repression did not occur. Mutant p53 135Cys to Ser did not induce repression in either normal human keratinocytes or in the C-33A line; although like 143Val to Ala, it is thought to affect the DNA binding activity of the wild-type protein. The ability of mutant p53 143Val to Ala to inactivate the HPV early promoter in normal cells (by approximately 60% reduction) suggests that this mutant may be able to associate with wild-type p53 and interact with TATA box-binding proteins. Therefore, these results demonstrate that the transcriptional activities of p53 mutants may be dependent upon the cell type assayed and the form of its endogenous p53. Furthermore, normal human keratinocytes represent an alternative model for determining the activities of p53 mutants.

  18. Intravenous ascorbate improves spatial memory in middle-aged APP/PSEN1 and wild type mice.

    PubMed

    Kennard, John A; Harrison, Fiona E

    2014-05-01

    The present study investigated the effects of a single intravenous (i.v.) dose of Vitamin C (ascorbate, ASC) on spatial memory in APP/PSEN1 mice, an Alzheimer's disease model. First, we confirmed the uptake time course in ASC-depleted gulo (-/-) mice, which cannot synthesize ASC. Differential tissue uptake was seen based on ASC transporter distribution. Liver (SVCT1 and SVCT2) ASC was elevated at 30, 60 and 120 min post-treatment (125 mg/kg, i.v.), whereas spleen (SVCT2) ASC increased at 60 and 120 min. There was no detectable change in cortical (SVCT2 at choroid plexus, and neurons) ASC within the 2-h interval, although the cortex preferentially retained ASC. APP/PSEN1 and wild type (WT) mice at three ages (3, 9, or 20 months) were treated with ASC (125 mg/kg, i.v.) or saline 45 min before testing on the Modified Y-maze, a two-trial task of spatial memory. Memory declined with age and ASC treatment improved performance in 9-month-old APP/PSEN1 and WT mice. APP/PSEN1 mice displayed no behavioral impairment relative to WT controls. Although dopamine and metabolite DOPAC decreased in the nucleus accumbens with age, and improved spatial memory was correlated with increased dopamine in saline treated mice, acute ASC treatment did not alter monoamine levels in the nucleus accumbens. These data show that the Modified Y-maze is sensitive to age-related deficits, but not additional memory deficits due to amyloid pathology in APP/PSEN1 mice. They also suggest improvements in short-term spatial memory were not due to changes in the neuropathological features of AD or monoamine signaling.

  19. Wild-Type p53 Enhances Efficiency of Simian Virus 40 Large-T-Antigen-Induced Cellular Transformation▿

    PubMed Central

    Hermannstädter, Andrea; Ziegler, Christine; Kühl, Marion; Deppert, Wolfgang; Tolstonog, Genrich V.

    2009-01-01

    Abortive infection of BALB/c mouse embryo fibroblasts differing in p53 gene status (p53+/+ versus p53−/−) with simian virus 40 (SV40) revealed a quantitatively and qualitatively decreased transformation efficiency in p53−/− cells compared to p53+/+ cells, suggesting a supportive effect of wild-type (wt) p53 in the SV40 transformation process. SV40 transformation efficiency also was low in immortalized p53−/− BALB/c 10-1 cells but could be restored to approximately the level in immortalized p53+/+ BALB/c 3T3 cells by reconstituting wt p53, but not mutant p53 (mutp53), expression. Stable expression of large T antigen (LT) in p53+/+ 3T3 cells resulted in full transformation, while LT expression in p53−/− 10-1 cells could not promote growth in suspension or in soft agar to a significant extent. The helper effect of wt p53 is mediated by its cooperation with LT and resides in the p53 N terminus, as an N-terminally truncated p53 (ΔNp53) could not rescue the p53-null phenotype. The p53 N terminus serves as a scaffold for recruiting transcriptional regulators like p300/CBP and Mdm2 into the LT-p53 complex. Consequently, LT affected global and specific gene expression in p53+/+ cells significantly more than in p53−/− cells. Our data suggest that recruitment of transcriptional regulators into the LT-p53 complex may help to modify cellular gene expression in response to the needs of cellular transformation. PMID:19625393

  20. Intravenous ascorbate improves spatial memory in middle-aged APP/PSEN1 and wild type mice

    PubMed Central

    Kennard, John A.; Harrison, Fiona E.

    2014-01-01

    The present study investigated the effects of a single intravenous (i.v.) dose of Vitamin C (ascorbate, ASC) on spatial memory in APP/PSEN1 mice, an Alzheimer's disease model. First, we confirmed the uptake time course in ASC-depleted gulo (−/−) mice, which cannot synthesize ASC. Differential tissue uptake was seen based on ASC transporter distribution. Liver (SVCT1 & SVCT2) ASC was elevated at 30, 60 and 120 min post-treatment (125 mg/kg, i.v.), whereas spleen (SVCT2) ASC increased at 60 and 120 min. There was no detectable change in cortical (SVCT2 at choroid plexus, and neurons) ASC within the 2-hour interval, although the cortex preferentially retained ASC. APP/PSEN1 and wild type (WT) mice at three ages (3, 9, or 20 months) were treated with ASC (125 mg/kg, i.v.) or saline 45 min before testing on the Modified Y-maze, a two-trial task of spatial memory. Memory declined with age and ASC treatment improved performance in 9 month-old APP/PSEN1 and WT mice. APP/PSEN1 mice displayed no behavioral impairment relative to WT controls. Although dopamine and metabolite DOPAC decreased in the nucleus accumbens with age, and improved spatial memory was correlated with increased dopamine in saline treated mice, acute ASC treatment did not alter monoamine levels in the nucleus accumbens. These data show that the Modified Y-maze is sensitive to age-related deficits, but not additional memory deficits due to amyloid pathology in APP/PSEN1 mice. They also suggest improvements in short-term spatial memory were not due to changes in the neuropathological features of AD or monoamine signaling. PMID:24508240

  1. Chronic allergic inflammation causes vascular remodeling and pulmonary hypertension in BMPR2 hypomorph and wild-type mice.

    PubMed

    Mushaben, Elizabeth M; Hershey, Gurjit Khurana; Pauciulo, Michael W; Nichols, William C; Le Cras, Timothy D

    2012-01-01

    Loss-of-function mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene have been identified in patients with heritable pulmonary arterial hypertension (PAH); however, disease penetrance is low, suggesting additional factors play a role. Inflammation is associated with PAH and vascular remodeling, but whether allergic inflammation triggers vascular remodeling in individuals with BMPR2 mutations is unknown. Our goal was to determine if chronic allergic inflammation would induce more severe vascular remodeling and PAH in mice with reduced BMPR-II signaling. Groups of Bmpr2 hypomorph and wild-type (WT) Balb/c/Byj mice were exposed to house dust mite (HDM) allergen, intranasally for 7 or 20 weeks to generate a model of chronic inflammation. HDM exposure induced similar inflammatory cell counts in all groups compared to controls. Muscularization of pulmonary arterioles and arterial wall thickness were increased after 7 weeks HDM, more severe at 20 weeks, but similar in both groups. Right ventricular systolic pressure (RVSP) was measured by direct cardiac catheterization to assess PAH. RVSP was similarly increased in both HDM exposed groups after 20 weeks compared to controls, but not after 7 weeks. Airway hyperreactivity (AHR) to methacholine was also assessed and interestingly, at 20 weeks, was more severe in HDM exposed Bmpr2 hypomorph mice versus WT. We conclude that chronic allergic inflammation caused PAH and while the severity was mild and similar between WT and Bmpr2 hypomorph mice, AHR was enhanced with reduced BMPR-II signaling. These data suggest that vascular remodeling and PAH resulting from chronic allergic inflammation occurs independently of BMPR-II pathway alterations.

  2. Wild-type human p53 transactivates the human proliferating cell nuclear antigen promoter

    SciTech Connect

    Shivakumar, C.V.; Brown, D.R.; Deb, S.; Deb, S.P.

    1995-12-01

    The p53 tumor suppressor protein negatively regulates cell growth and somatic mutations in the p53 gene lead to uncontrolled cell growth and oncogenesis. This report describes research which demonstrates, using a number of different cell lines, that at low levels, wild-type p53 transactivates the human proliferating cell nuclear antigen (PCNA) promoter. When expressed at similar levels, tumor-derived p53 mutants did not transactivate the PCNA promoter. It also reports the identification of a wild-type human p53-binding site on the human PCNA promote. 84 refs., 5 figs, 3 tabs.

  3. Wild-Type p53 Binds to the TATA-Binding Protein and Represses Transcription

    NASA Astrophysics Data System (ADS)

    Seto, Edward; Usheva, Anny; Zambetti, Gerard P.; Momand, Jamil; Horikoshi, Nobuo; Weinmann, Roberto; Levine, Arnold J.; Shenk, Thomas

    1992-12-01

    p53 activates transcription of genes with a p53 response element, and it can repress genes lacking the element. Here we demonstrate that wild-type but not mutant p53 inhibits transcription in a HeLa nuclear extract from minimal promoters. Wild-type but not mutant p53 binds to human TATA-binding protein (TBP). p53 does not bind to yeast TBP, and it cannot inhibit transcription in a HeLa extract where yeast TBP substitutes for human TBP. These results suggest a model in which p53 binds to TBP and interferes with transcriptional initiation.

  4. WT1 mutations in T-ALL.

    PubMed

    Tosello, Valeria; Mansour, Marc R; Barnes, Kelly; Paganin, Maddalena; Sulis, Maria Luisa; Jenkinson, Sarah; Allen, Christopher G; Gale, Rosemary E; Linch, David C; Palomero, Teresa; Real, Pedro; Murty, Vundavalli; Yao, Xiaopan; Richards, Susan M; Goldstone, Anthony; Rowe, Jacob; Basso, Giuseppe; Wiernik, Peter H; Paietta, Elisabeth; Pieters, Rob; Horstmann, Martin; Meijerink, Jules P P; Ferrando, Adolfo A

    2009-07-30

    The molecular mechanisms involved in disease progression and relapse in T-cell acute lymphoblastic leukemia (T-ALL) are poorly understood. We used single nucleotide polymorphism array analysis to analyze paired diagnostic and relapsed T-ALL samples to identify recurrent genetic alterations in T-ALL. This analysis showed that diagnosis and relapsed cases have common genetic alterations, but also that relapsed samples frequently lose chromosomal markers present at diagnosis, suggesting that relapsed T-ALL emerges from an ancestral clone different from the major leukemic population at diagnosis. In addition, we identified deletions and associated mutations in the WT1 tumor suppressor gene in 2 of 9 samples. Subsequent analysis showed WT1 mutations in 28 of 211 (13.2%) of pediatric and 10 of 85 (11.7%) of adult T-ALL cases. WT1 mutations present in T-ALL are predominantly heterozygous frameshift mutations resulting in truncation of the C-terminal zinc finger domains of this transcription factor. WT1 mutations are most prominently found in T-ALL cases with aberrant rearrangements of the oncogenic TLX1, TLX3, and HOXA transcription factor oncogenes. Survival analysis demonstrated that WT1 mutations do not confer adverse prognosis in pediatric and adult T-ALL. Overall, these results identify the presence of WT1 mutations as a recurrent genetic alteration in T-ALL. PMID:19494353

  5. Adsorption of rare earth ions onto the cell walls of wild-type and lipoteichoic acid-defective strains of Bacillus subtilis.

    PubMed

    Moriwaki, Hiroshi; Koide, Remi; Yoshikawa, Ritsuko; Warabino, Yuya; Yamamoto, Hiroki

    2013-04-01

    The aim of this study is to investigate the potential of cell walls of wild-type and lipoteichoic acid-defective strains of Bacillus subtilis 168 to adsorb rare earth ions. Freeze-dried cell powders prepared from both strains were used for the evaluation of adsorption ability for the rare earth ions, namely, La(III), Eu(III), and Tm(III). The rare earth ions were efficiently adsorbed onto powders of both wild-type strain (WT powder) and lipoteichoic acid-defective strain (∆LTA powder) at pH 3. The maximum adsorption capacities for Tm(III) by WT and ∆LTA powders were 43 and 37 mg g(-1), respectively. Removal (in percent) of Tm(III), La(III), and Eu(III) from aqueous solution by WT powder was greater than by ∆LTA powder. These results indicate that rare earth ions are adsorbed to functional groups, such as phosphate and carboxyl groups, of lipoteichoic acid. We observed coagulated ∆LTA powder in the removal of rare earth ions (1-20 mg L(-1)) from aqueous solution. In contrast, sedimentation of WT powder did not occur under the same conditions. This unique feature of ∆LTA powder may be caused by the difference of the distribution between lipoteichoic acid and wall teichoic acid. It appears that ∆LTA powder is useful for removal of rare earth ions by adsorption, because aggregation allows for rapid separation of the adsorbent by filtration.

  6. Innate interferon response in macrophage and epithelial cells infected with wild-type compared to DNA adenine methylase and flagellin mutant Salmonella enterica serovar Typhimurium.

    PubMed

    Simon, Raphael; Samuel, Charles E

    2007-04-01

    Salmonella enterica serovar Typhimurium is highly virulent and mediates robust interferon (IFN)-stimulated gene (ISG) induction, whereas bacterial mutants that lack the DNA adenine methylase (Dam) are attenuated, elicit a reduced ISG activation profile, and establish immunity to murine typhoid fever. We show here that in contrast to observations in mice, infection of macrophage cell cultures with either wild-type (WT) or dam(-) mutant Salmonella resulted in surprisingly similar kinetics and amplitudes of induction of IFN-beta, the type I IFN-alpha,beta beacon gene Mx, and the type II IFN-gamma beacon inducible nitric oxide synthase (iNOS). Likewise, activation of NF-kappaB-dependent gene expression in epithelial cells was comparable with WT and dam(-) mutant Salmonella. In contrast, the flagellin-deficient flhC(-) mutant did not activate NF-kappaB in epithelial cells but activated ISG expression comparable to that of WT Salmonella in macrophage cells. WT and dam(-) strains displayed a similar Toll-like receptor 5 (TLR5)-dependent NF-kappaB activation, whereas the flhC(-) mutant lacked this activity. UV-inactivated Salmonella elicited similar ISG induction to that of viable Salmonella in macrophages and mediated the establishment of a functional antiviral state but displayed decreased cytocidal activity. These results establish that the inherent IFN system-inducing capacities of dam(-) and WT Salmonella strains in cultured macrophage and epithelial cells, unlike the mouse, are indistinguishable.

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

    SciTech Connect

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

    2006-07-01

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

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

    SciTech Connect

    Cuthill, S.; Poellinger, L.

    1988-04-19

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

  9. Tendon fascicle gliding in wild type, heterozygous, and lubricin knockout mice.

    PubMed

    Kohrs, Ross T; Zhao, Chunfeng; Sun, Yu-Long; Jay, Gregory D; Zhang, Ling; Warman, Matthew L; An, Kai-Nan; Amadio, Peter C

    2011-03-01

    The objective of this study was to investigate the role of lubricin in the lubrication of tendon fascicles. Lubricin, a glycoprotein, lubricates cartilage and tendon surfaces, but the function of lubricin within the tendon fascicle is unclear. We developed a novel method to assess the gliding resistance of a single fascicle in a mouse tail model and used it to test the hypothesis that gliding resistance would be increased in lubricin knockout mice. Thirty-six mouse tails were used from 12 wild type, 12 heterozygous, and 12 lubricin knockout mice. A 15 mm long fascicle segment was pulled proximally after being divided distally. The peak resistance during fascicle pullout and the fascicle perimeter were measured. Lubricin expression was evaluated by immunohistochemistry. The peak gliding resistance in the lubricin knockout mice was significantly higher than in the wild type (p < 0.05). Fascicles from heterozygous mice were intermediate in value, but not significantly different from either wild type or lubricin knockout fascicles in peak gliding resistance. No significant difference was found in fascicle perimeter among the three groups. No correlation was observed between fascicle perimeter and gliding resistance. While lubricin was detected by immunostaining on the fascicle surface in wild type and heterozygous mice, lubricin was not detectable in the tendons of knockout mice. We conclude that the absence of lubricin is associated with increased interfascicular friction and that lubricin may play an important role in interfascicular lubrication.

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

    EPA Science Inventory

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

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

    PubMed

    Berry, D; Volz, P A

    1979-10-01

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

  12. Comparative Effects of Wild-type, bmr-6, bmr-12 and Stacked Sorghum: Sorghum Stover Digestibility

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparative Effects of Wild-type, bmr-6, bmr-12 and Stacked Sorghum: Sorghum Stover Digestibility H. M. Dann,1 A. M. DiCerbo,1 J. F. Pedersen,2 and R. J. Grant1 1 William H. Miner Agricultural Research Institute, Chazy, NY 2 USDA, ARS, NPA Wheat, Sorghum and Forage Research, University of Nebraska, ...

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

    EPA Science Inventory

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

  14. Chloroplast parameters differ in wild type and transgenic poplars overexpressing gsh1 in the cytosol.

    PubMed

    Ivanova, L A; Ronzhina, D A; Ivanov, L A; Stroukova, L V; Peuke, A D; Rennenberg, H

    2009-07-01

    Poplar mutants overexpressing the bacterial genes gsh1 or gsh2 encoding the enzymes of glutathione biosynthesis are among the best-characterised transgenic plants. However, this characterisation originates exclusively from laboratory studies, and the performance of these mutants under field conditions is largely unknown. Here, we report a field experiment in which the wild-type poplar hybrid Populus tremula x P. alba and a transgenic line overexpressing the bacterial gene gsh1 encoding gamma-glutamylcysteine synthetase in the cytosol were grown for 3 years at a relatively clean (control) field site and a field site contaminated with heavy metals. Aboveground biomass accumulation was slightly smaller in transgenic compared to wild-type plants; soil contamination significantly decreased biomass accumulation in both wild-type and transgenic plants by more than 40%. Chloroplasts parameters, i.e., maximal diameter, projection area and perimeter, surface area and volume, surface/volume ratio and a two-dimensional form coefficient, were found to depend on plant type, leaf tissue and soil contamination. The greatest differences between wild and transgenic poplars were observed at the control site. Under these conditions, chloroplast sizes in palisade tissue of transgenic poplar significantly exceeded those of the wild type. In contrast to the wild type, palisade chloroplast volume exceeded that of spongy chloroplasts in transgenic poplars at both field sites. Chlorophyll content per chloroplast was the same in wild and transgenic poplars. Apparently, the increase in chloroplast volume was not connected to changes in the photosynthetic centres. Chloroplasts of transgenic poplar at the control site were more elongated in palisade cells and close to spherical in spongy mesophyll chloroplasts. At the contaminated site, palisade and spongy cell chloroplasts of leaves from transgenic trees and the wild type were the same shape. Transgenic poplars also had a smaller chloroplast

  15. Registered report: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth

    PubMed Central

    Bhargava, Ajay; Pelech, Steven; Woodard, Ben; Kerwin, John; Maherali, Nimet

    2016-01-01

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from 'RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth' by Hatzivassiliou and colleagues, published in Nature in 2010 (Hatzivassiliou et al., 2010). Hatzivassiliou and colleagues examined the paradoxical response of RAF-WT tumors to treatment with RAF inhibitors. The key experiments being replicated include Figure 1A, in which the original authors demonstrated that treatment of a subset of BRAFWT tumor cell lines with RAF small molecule inhibitors resulted in an increase in cell viability, Figure 2B, which reported that RAF inhibitor activation of the MAPK pathway was dependent on CRAF but not BRAF, and Figure 4A, where the dimerization of BRAF and CRAF was modulated by the RAF inhibitor PLX4720, but not GDC-0879. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife. DOI: http://dx.doi.org/10.7554/eLife.09976.001 PMID:26882073

  16. Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

    PubMed Central

    Aprile-Garcia, Fernando; Metzger, Michael W.; Paez-Pereda, Marcelo; Stadler, Herbert; Acuña, Matías; Liberman, Ana C.; Senin, Sergio A.; Gerez, Juan; Hoijman, Esteban; Refojo, Damian; Mitkovski, Mišo; Panhuysen, Markus; Stühmer, Walter; Holsboer, Florian; Deussing, Jan M.; Arzt, Eduardo

    2016-01-01

    The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations. PMID:26986975

  17. Brown adipose tissue dynamics in wild-type and UCP1-knockout mice: in vivo insights with magnetic resonance.

    PubMed

    Grimpo, Kirsten; Völker, Maximilian N; Heppe, Eva N; Braun, Steve; Heverhagen, Johannes T; Heldmaier, Gerhard

    2014-03-01

    We used noninvasive magnetic resonance imaging (MRI) and magnetic resonance spectroscopy to compare interscapular brown adipose tissue (iBAT) of wild-type (WT) and uncoupling protein 1 (UCP1)-knockout mice lacking UCP1-mediated nonshivering thermogenesis (NST). Mice were sequentially acclimated to an ambient temperature of 30°C, 18°C, and 5°C. We detected a remodeling of iBAT and a decrease in its lipid content in all mice during cold exposure. Ratios of energy-rich phosphates (ATP/ADP, phosphocreatine/ATP) in iBAT were maintained stable during noradrenergic stimulation of thermogenesis in cold- and warm-adapted mice and no difference between the genotypes was observed. As free fatty acids (FFAs) serve as fuel for thermogenesis and activate UCP1 for uncoupling of oxidative phosphorylation, brown adipose tissue is considered to be a main acceptor and consumer of FFAs. We measured a major loss of FFAs from iBAT during noradrenergic stimulation of thermogenesis. This mobilization of FFAs was observed in iBAT of WT mice as well as in mice lacking UCP1. The high turnover and the release of FFAs from iBAT suggests an enhancement of lipid metabolism, which in itself contributes to the sympathetically activated NST and which is independent from uncoupled respiration mediated by UCP1. Our study demonstrates that MRI, besides its potential for visualizing and quantification of fat tissue, is a valuable tool for monitoring functional in vivo processes like lipid and phosphate metabolism during NST. PMID:24343897

  18. Experimental Support for the Ecoimmunity Theory: Distinct Phenotypes of Nonlymphocytic Cells in SCID and Wild-Type Mice.

    PubMed

    Ochayon, David E; Baranovski, Boris M; Malkin, Peter; Schuster, Ronen; Kalay, Noa; Ben-Hamo, Rotem; Sloma, Ido; Levinson, Justin; Brazg, Jared; Efroni, Sol; Lewis, Eli C; Nevo, Uri

    2016-01-01

    Immune tolerance toward "self" is critical in multiple immune disorders. While there are several mechanisms to describe the involvement of immune cells in the process, the role of peripheral tissue cells in that context is not yet clear. The theory of ecoimmunity postulates that interactions between immune and tissue cells represent a predator-prey relationship. A lifelong interaction, shaped mainly during early ontogeny, leads to selection of nonimmune cell phenotypes. Normally, therefore, nonimmune cells that evolve alongside an intact immune system would be phenotypically capable of evading immune responses, and cells whose phenotype falls short of satisfying this steady state would expire under hostile immune responses. This view was supported until recently by experimental evidence showing an inferior endurance of severe combined immunodeficiency (SCID)-derived pancreatic islets when engrafted into syngeneic immune-intact wild-type (WT) mice, relative to islets from WT. Here we extend the experimental exploration of ecoimmunity by searching for the presence of the phenotypic changes suggested by the theory. Immune-related phenotypes of islets, spleen, and bone marrow immune cells were determined, as well as SCID and WT nonlymphocytic cells. Islet submass grafting was performed to depict syngeneic graft functionality. Islet cultures were examined under both resting and inflamed conditions for expression of CD40 and major histocompatibility complex (MHC) class I/II and release of interleukin-1α (IL-1α), IL-1β, IL-6, tumor necrosis factor-α (TNF-α), IL-10, and insulin. Results depict multiple pathways that appear to be related to the sculpting of nonimmune cells by immune cells; 59 SCID islet genes displayed relative expression changes compared with WT islets. SCID cells expressed lower tolerability to inflammation and higher levels of immune-related molecules, including MHC class I. Accordingly, islets exhibited a marked increase in insulin release upon

  19. Relationship between heat-labile enterotoxin secretion capacity and virulence in wild type porcine-origin enterotoxigenic Escherichia coli strains.

    PubMed

    Wijemanne, Prageeth; Xing, Jun; Berberov, Emil M; Marx, David B; Francis, David H; Moxley, Rodney A

    2015-01-01

    Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system (T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had the highest LT secretion level all porcine-origin WT strains tested (P<0.05). The level of LT secretion (concentration in supernatants at 6-h culture) explained 92% of the variation in time-to-a-moribund-condition (R2 = 0.92, P<0.0001) in gnotobiotic piglets inoculated with either strain 3030-2, or an ETEC strain of lesser virulence (2534-86), or a non-enterotoxigenic WT strain (G58-1). All 16 porcine ETEC strains were positive by PCR analysis for the T2SS genes, gspD and gspK, and bioinformatic analysis of 4 porcine-origin strains for which complete genomic sequences were available revealed a T2SS with a high degree of homology to that of H10407. Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18. Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml. This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and

  20. Differences among lesions with exon 19, exon 21 EGFR mutations and wild types in surgically resected non-small cell lung cancer

    PubMed Central

    Jin, Ying; Chen, Ming; Yu, Xinmin

    2016-01-01

    The clinical behavior of patients with advanced non-small cell lung cancer (NSCLC) differ between epidermal growth factor receptor (EGFR) exon 19 deletion (Ex19) and EGFR exon 21 L858R mutation (Ex21). This study aimed to evaluate whether these differences exist in surgically resected NSCLC. A total of 198 patients with surgically resected NSCLC harbouring Ex19 (n = 53), Ex21 (n = 51), and EGFR wild-type (Wt) (n = 94) were analyzed. The clinicopathological features, laboratory parameters, recurrent sites and disease-free survival (DFS) were compared according to mutational EGFR status. Ex21 occurred more frequently in female (p < 0.001), never-smokers (p < 0.001), adenocarcinoma (p < 0.001), low grade (p = 0.013) than Wt lesions. Ex19 occurred more frequently in female (p = 0.016), never-smokers (p = 0.008), adenocarcinoma (p < 0.001), low grade (p = 0.025) than Wt lesions. Ex 21 lesions (p = 0.026) had larger lepidic components than Wt lesions. Wt lesions had larger mucinous variant components than Ex21 lesions (p = 0.045) and Ex19 lesions (p = 0.015). Ex21 lesions were associated with lower pretreatment neutrophil: lymphocyte ratio (NLR) than Wt lesions (p = 0.017). The recurrent sites and DFS were similar among patients with Wt, Ex19 and Ex21. PMID:27527915

  1. G551D-CFTR needs more bound actin than wild-type CFTR to maintain its presence in plasma membranes.

    PubMed

    Trouvé, Pascal; Kerbiriou, Mathieu; Teng, Ling; Benz, Nathalie; Taiya, Mehdi; Le Hir, Sophie; Férec, Claude

    2015-08-01

    Cystic Fibrosis is due to mutations in the CFTR gene. The missense mutation G551D (approx. 5% of cases) encodes a CFTR chloride channel with normal cell surface expression but with an altered chloride channel activity, leading to a severe phenotype. Our aim was to identify specific interacting proteins of G551D-CFTR which could explain the channel defect. Wild-type CFTR (Wt-CFTR) was co-immunoprecipitated from stably transfected HeLa cells and resolved by 2D gel electrophoresis. Among the detected spots, one was expressed at a high level. Mass Spectrometry revealed that it corresponded to actin which is known to be involved in the CFTR's channel function. To assess whether actin could be involved in the altered G551D-CFTR function, its basal expression was studied. Because actin expression was the same in wt- and in G551D-CFTR expressing cells, its interaction with both wt- and G551D-CFTR was studied by co-immunoprecipitation, and we found that a higher amount of actin was bound onto G551D-CFTR than onto Wt-CFTR. The role of actin upon wt- and G551D-CFTR function was further studied by patch-clamp experiments after cytochalasin D treatment of the cells. We found a decrease of the very weak currents in G551D-CFTR expressing cells. Because a higher amount of actin is bound onto G551D-CFTR than onto Wt-CFTR, it is likely to be not involved in the mutated CFTR's defect. Nevertheless, because actin is necessary to maintain the very weak global currents observed in G551D-CFTR expressing HeLa cells, we conclude that more actin is necessary to maintain G551D-CFTR in the plasma membrane than for Wt-CFTR.

  2. Wild Type p53 gene sensitizes rat C6 glioma cells to HSV-TK/ACV treatment in vitro and in vivo.

    PubMed

    Huang, Qiang; Xia, Zhibo; You, Yongping; Pu, Peiyu

    2010-12-01

    Suicide gene therapy using herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV), has been extensively tested for the treatment of glioma. Our previous study showed that exogenous wild type p53 (wt-p53) enhanced the anti-tumor effect of HSV-TK/GCV therapy. However, the use of GCV is hindered by its low penetration to the brain and its toxicity when used at higher dose. In the present study, we used another pro-drug, acyclovir (ACV), and examined the therapeutic efficacy of HSV-TK/ACV combining with wt-p53 in C6 glioma cells. We observed that wt-p53 combined with HSV-TK/ACV resulted in the super-additive anti-tumor effect in vitro. Exogenous wt-p53 significantly enhanced the sensitivity of TK positive C6 cells to ACV in vitro. Our in vivo experiment demonstrated that the effect of wt-p53 and HSV-TK/ACV combination therapy was better than that of HSV-TK/ACV alone. The survival time of tumor-bearing rats treated with wt-p53 in combination with HSV-TK/ACV was also significantly prolonged than those treated with HSV-TK/ACV alone. These results suggest that wt-p53 can enhance the therapeutic efficacy of HSV-TK/ACV both in vitro and in vivo. These findings are considerably valuable with the respect of using less toxic ACV as prodrug. This novel strategy could provide benefit to HSV-TK/prodrug gene therapy.

  3. Endoglin Deficiency in Bone Marrow Is Sufficient to Cause Cerebrovascular Dysplasia in the Adult Mouse after VEGF Stimulation

    PubMed Central

    Choi, Eun-Jung; Walker, Espen J.; Degos, Vincent; Jun, Kristine; Kuo, Robert; Pile-Spellman, John; Su, Hua; Young, William L.

    2013-01-01

    Background and Purpose Bone marrow-derived cells (BMDCs) home to vascular endothelial growth factor (VEGF)-induced brain angiogenic foci, and VEGF induces cerebrovascular dysplasia in adult endoglin heterozygous (Eng+/−) mice. We hypothesized that Eng+/− BMDCs cause cerebrovascular dysplasia in the adult mouse after VEGF stimulation. Methods BM transplantation was performed using adult wild-type (WT) and Eng+/− mice as donors/recipients. An adeno-associated viral vector expressing VEGF (AAV-VEGF) was injected into the basal ganglia 4 weeks after transplantation. Vascular density, dysplasia index (vessels >15 μm/100 vessels), and BMDCs in the angiogenic foci were analyzed. Results The dysplasia index of WT/Eng+/− BM mice was higher than WT/WT BM mice (p<0.001) and was similar to Eng+/−/Eng+/− BM mice (p=0.2). Dysplasia in Eng+/− mice was partially rescued by WT BM (p<0.001). WT/WT BM and WT/Eng+/− BM mice had similar numbers of BMDCs in the angiogenic foci (p=0.4), most of which were CD68+. Eng+/− monocytes/macrophages expressed less matrix metalloproteinase-9 and Notch1. Conclusions ENG-deficient BMDCs are sufficient for VEGF to induce vascular dysplasia in the adult mouse brain. Our data support a previously unrecognized role of BM in the development of cerebrovascular malformations. PMID:23306322

  4. Mobility and subcellular localization of endogenous, gene-edited Tau differs from that of over-expressed human wild-type and P301L mutant Tau

    PubMed Central

    Di Xia; Gutmann, Julia M.; Götz, Jürgen

    2016-01-01

    Alzheimer’s disease (AD) and a subset of frontotemporal dementia termed FTLD-Tau are characterized by a massive, yet incompletely characterized and understood redistribution of Tau. To establish a framework for understanding this pathology, we used the genome-editing tool TALEN and generated Tau-mEOS2 knock-in mice to determine the mobility and subcellular localization of endogenous Tau in hippocampal cultures. We analysed Tau in axons, dendrites and spines at three stages of maturation using live-cell imaging, photo-conversion and FRAP assays. Tau-mEOS2 cultures were compared with those over-expressing EGFP-tagged forms of human wild-type (hWT-Tau) and P301L mutant Tau (hP301L-Tau), modelling Tau accumulation in AD and FTLD-Tau, respectively. In developing neurons, Tau-mEOS2 followed a proximo-distal gradient in axons and a subcellular distribution similar to that of endogenous Tau in neurons obtained from wild-type mice, which were abolished, when either hWT-Tau or hP301L-Tau was over-expressed. For the three conditions, FRAP analysis revealed a similar mobility in dendrites compared with axons; however, Tau-mEOS2 was less mobile than hWT-Tau and hP301L-Tau and the mobile fraction was smaller, possibly reflecting less efficient microtubule binding of Tau when over-expressed. Together, our study presents Tau-mEOS2 mice as a novel tool for the study of Tau in a physiological and a pathological context. PMID:27378256

  5. DNA-binding dependent and independent functions of WT1 protein during human hematopoiesis

    SciTech Connect

    Svensson, Emelie; Eriksson, Helena; Gekas, Christos; Olofsson, Tor; Richter, Johan; Gullberg, Urban . E-mail: urban.gullberg@hematologi.lu.se

    2005-08-01

    The Wilms tumor gene 1 (WT1) encodes a zinc-finger-containing transcription factor highly expressed in immature hematopoietic progenitor cells. Overexpression and presence of somatic mutations in acute leukemia indicate a role for WT1 in the pathogenesis of leukemia. CD34{sup +} progenitor cells were transduced with one splice variant of human WT1 without the KTS insert in the zinc-finger domain, WT1(+/-), and with a deleted mutant of WT1 lacking the entire zinc-finger region, WT1(delZ), thus incapable of binding DNA. We show that inhibition of erythroid colony formation and differentiation is absolutely dependent on the DNA-binding zinc-finger domain of WT1. Unexpectedly, however, WT1(delZ) was equally effective as wild type protein in the reduction of myeloid clonogenic growth as well as in stimulation of myeloid differentiation, as judged by the expression of cell surface CD11b. Expression of neither WT1(+/-) nor WT1(delZ) upregulated mRNA for the cdk inhibitor p21{sup Waf1/Cip1} or p27{sup Kip1}. Our results demonstrate that WT1 affects proliferation and differentiation in erythroid and myeloid cells by different molecular mechanisms, and suggest that mutations affecting the zinc-finger domain of WT1 could interfere with normal differentiation in the pathogenesis of leukemia.

  6. Transcriptomic Insights into the Response of Placenta and Decidua Basalis to the CpG Oligodeoxynucleotide Stimulation in Non-Obese Diabetic Mice and Wild-Type Controls

    PubMed Central

    Liu, Xiao-Rui; Guo, Yu-Na; Qin, Chuan-Mei; Qin, Xiao-Li; Tao, Fei; Su, Fei; Tian, Fu-Ju; Zhang, Yan; Lin, Yi

    2016-01-01

    Intrauterine infection is one of the most frequent causes of miscarriage. CpG oligodeoxynucleotide (CpG ODN) can mimic intrauterine infection. CpG ODN-induced embryo-resorption was observed consistently in the NK-cell deficient non-obese diabetic (NOD) mice but not in the wild-type (WT) mice. To elucidate the molecular mechanisms of differential pregnancy outcomes, differentially expressed genes (DEGs) in the placenta and decidua basalis was revealed by RNA-Seq with CpG ODN or control ODN treatment. Common DEGs in the WT and NOD mice were enriched in antimicrobial/antibacterial humoral responses that may be activated as a primary response to bacterial infection. The susceptibility to CpG ODN-induced embryo-resorption in the NOD mice might mainly be attributed to M1 macrophage polarization and the immunodeficient status, such as the down-regulation in antigen processing and presentation, allograft rejection, and natural killer cell mediated cytotoxicity. In contrast, the WT mice with normal immune systems could activate multiple immune responses and be resistant to CpG ODN-induced embryo-resorption, such as M2 macrophage differentiation and activation regulated by complement component C1q and peroxisome proliferation-activated receptor (PPAR) signaling pathways. Collectively, this study suggests that the immunodeficient status of NOD mice and the macrophage polarization regulated by C1q and PPAR signaling might be the basis for differential pregnancy outcomes between the NOD and WT mice. PMID:27527166

  7. CAR-Engineered NK Cells Targeting Wild-Type EGFR and EGFRvIII Enhance Killing of Glioblastoma and Patient-Derived Glioblastoma Stem Cells

    PubMed Central

    Han, Jianfeng; Chu, Jianhong; Keung Chan, Wing; Zhang, Jianying; Wang, Youwei; Cohen, Justus B.; Victor, Aaron; Meisen, Walter H.; Kim, Sung-hak; Grandi, Paola; Wang, Qi-En; He, Xiaoming; Nakano, Ichiro; Chiocca, E. Antonio; Glorioso III, Joseph C.; Kaur, Balveen; Caligiuri, Michael A.; Yu, Jianhua

    2015-01-01

    Glioblastoma (GB) remains the most aggressive primary brain malignancy. Adoptive transfer of chimeric antigen receptor (CAR)-modified immune cells has emerged as a promising anti-cancer approach, yet the potential utility of CAR-engineered natural killer (NK) cells to treat GB has not been explored. Tumors from approximately 50% of GB patients express wild-type EGFR (wtEGFR) and in fewer cases express both wtEGFR and the mutant form EGFRvIII; however, previously reported CAR T cell studies only focus on targeting EGFRvIII. Here we explore whether both wtEGFR and EGFRvIII can be effectively targeted by CAR-redirected NK cells to treat GB. We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells. EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-γ production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner. In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival. These findings support intracranial administration of NK-92-EGFR-CAR cells represents a promising clinical strategy to treat GB. PMID:26155832

  8. Limited In Vivo Production of Type I or Type III Interferon After Infection of Macaques with Vaccine or Wild-Type Strains of Measles Virus

    PubMed Central

    Shivakoti, Rupak; Hauer, Debra; Adams, Robert J.; Lin, Wen-Hsuan W.; Duprex, William Paul; de Swart, Rik L.

    2015-01-01

    The innate immune response to viral infections often includes induction of types I and III interferons (IFNs) and production of antiviral proteins. Measles is a severe virus-induced rash disease, but in vitro studies suggest that in the absence of defective interfering RNAs, neither wild-type (WT) nor vaccine strains of measles virus (MeV) induce IFN. To determine whether IFN is produced in vivo, we studied tissues from macaques infected with vaccine or WT strains of MeV using quantitative reverse transcriptase–polymerase chain reaction to assess levels of IFN and IFN-stimulated gene (ISG) mRNAs and a flow cytometry-based bioassay to assess levels of biologically active IFN. There was little to no induction of type I IFN, type III IFN, Mx, or ISG56 mRNAs in monkeys infected with vaccine or WT MeV and no IFN detection by bioassay. Therefore, the innate responses to infection with vaccine or WT strains of MeV are not dependent on IFN production. PMID:25517681

  9. Differential regulation of insulin-like growth factor-I receptor gene expression by wild type and mutant androgen receptor in prostate cancer cells.

    PubMed

    Schayek, Hagit; Seti, Hila; Greenberg, Norman M; Sun, Shihua; Werner, Haim; Plymate, Stephen R

    2010-07-29

    The progression of prostate cancer from an organ-confined, androgen-sensitive disease to a metastatic one is associated with dysregulation of androgen receptor (AR)-regulated target genes and with a decrease in insulin-like growth factor-I receptor (IGF-IR) expression. To investigate the differential effects of wild type (wt) and mutant AR on IGF-IR levels we employed a series of isogenic prostate-derived cell lines and human xenografts. We show that basal and phosphorylated IGF-IR levels progressively decreased as prostate cancer cells became more tumorigenic and metastatic. In addition, we show that wt, but not mutant, AR along with dihydrotestosterone treatment increased IGF-IR promoter activity and endogenous IGF-IR levels. ChIP analysis show enhanced AR binding to the IGF-IR promoter in AR-overexpressing cells. Finally, wt AR-overexpressing cells display an enhanced proliferation rate. In summary, we provide evidence that activated wt AR enhances IGF-IR transcription in prostate cancer cells via a mechanism that involves AR binding to the IGF-IR promoter. AR mutations alter the ability of the mutated protein to regulate IGF-IR expression. Our results suggest that prostate cancer progression is associated with a decrease in IGF-IR expression that could be the result of impaired ability of AR to stimulate IGF-IR gene expression. PMID:20417685

  10. Antimicrobial susceptibility in Escherichia coli of human and avian origin--a comparison of wild-type distributions.

    PubMed

    Sjölund, M; Bengtsson, S; Bonnedahl, J; Hernandez, J; Olsen, B; Kahlmeter, G

    2009-05-01

    In the present study, the antimicrobial susceptibilities of 97 Escherichia coli isolates from birds, and 100 clinical isolates from blood cultures, were determined by disk diffusion. The wild-type distributions were defined by the normalized resistance interpretation method. It is shown that the avian and clinical inhibition zone diameter distributions of wild-type E. coli are indistinguishable.

  11. A mutant chaperone converts a wild-type protein into a tumor-specific antigen.

    PubMed

    Schietinger, Andrea; Philip, Mary; Yoshida, Barbara A; Azadi, Parastoo; Liu, Hui; Meredith, Stephen C; Schreiber, Hans

    2006-10-13

    Monoclonal antibodies have become important therapeutic agents against certain cancers. Many tumor-specific antigens are mutant proteins that are predominantly intracellular and thus not readily accessible to monoclonal antibodies. We found that a wild-type transmembrane protein could be transformed into a tumor-specific antigen. A somatic mutation in the chaperone gene Cosmc abolished function of a glycosyltransferase, disrupting O-glycan Core 1 synthesis and creating a tumor-specific glycopeptidic neo-epitope consisting of a monosaccharide and a specific wild-type protein sequence. This epitope induced a high-affinity, highly specific, syngeneic monoclonal antibody with antitumor activity. Such tumor-specific glycopeptidic neo-epitopes represent potential targets for monoclonal antibody therapy.

  12. Wild-type minimal inhibitory concentration distributions in bacteria of animal origin in Argentina.

    PubMed

    Pantozzi, Florencia L; Ibar, Mariela P; Nievas, Victorio F; Vigo, Germán B; Moredo, Fabiana A; Giacoboni, Gabriela I

    2014-01-01

    The aim of this study was to determine the antimicrobial resistance profiles of indicator bacteria isolated from domestic animal feces. Minimal inhibitory concentration (MIC) was determined by agar dilution. Interpretative criteria on the basis of wild-type MIC distributions and epidemiological cutoff values (ECOFF or ECV) were used according to the 'European Committee on Antimicrobial Susceptibility Testing' (EUCAST) data. Results from 237 isolates of Escherichia coli showed reduced susceptibility for ampicillin, streptomycin and tetracycline, the antimicrobials commonly used in intensive breeding of pigs and hens. Regarding all the species of the genus Enterococcus spp., there are only ECOFF or ECV for vancomycin. Of the 173 Enterococcus spp. isolated, only one showed reduced susceptibility to vancomycin and was classified as 'non-wild-type' (NWT) population. This is the first report in Argentina showing data of epidemiological cutoff values in animal bacteria.

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

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

  14. Fatal wild-type varicella-zoster virus encephalitis without a rash in a vaccinated child.

    PubMed

    Ibraheem, Mam; Marin, Mona; Leung, Jessica; Bryce, Clare H; Schmid, D Scott; Zaki, Sherif R; Drew, Clifton; Liu, Lindy; Smelser, Chad

    2013-02-01

    Encephalitis associated with varicella-zoster virus, rare among children in the varicella vaccine era, has generally been associated with a rash. We report fatal wild-type varicella-zoster virus encephalitis without a rash in a child who had received 1 dose of varicella vaccine. Varicella-zoster virus encephalitis should be considered in the differential diagnosis for children presenting with acute neurologic symptoms, even vaccine recipients.

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

    SciTech Connect

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

    2014-11-07

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

  16. Transcriptomic analysis of Clostridium thermocellum Populus hydrolysate-tolerant mutant strain shows increased cellular efficiency in response to Populus hydrolysate compared to the wild type strain

    PubMed Central

    2014-01-01

    Background The thermophilic, anaerobic bacterium, Clostridium thermocellum is a model organism for consolidated processing due to its efficient fermentation of cellulose. Constituents of dilute acid pretreatment hydrolysate are known to inhibit C. thermocellum and other microorganisms. To evaluate the biological impact of this type of hydrolysate, a transcriptomic analysis of growth in hydrolysate-containing medium was conducted on 17.5% v/v Populus hydrolysate-tolerant mutant (PM) and wild type (WT) strains of C. thermocellum. Results In two levels of Populus hydrolysate medium (0% and 10% v/v), the PM showed both gene specific increases and decreases of gene expression compared to the wild-type strain. The PM had increased expression of genes in energy production and conversion, and amino acid transport and metabolism in both standard and 10% v/v Populus hydrolysate media. In particular, expression of the histidine metabolism increased up to 100 fold. In contrast, the PM decreased gene expression in cell division and sporulation (standard medium only), cell defense mechanisms, cell envelope, cell motility, and cellulosome in both media. The PM downregulated inorganic ion transport and metabolism in standard medium but upregulated it in the hydrolysate media when compared to the WT. The WT differentially expressed 1072 genes in response to the hydrolysate medium which included increased transcription of cell defense mechanisms, cell motility, and cellulosome, and decreased expression in cell envelope, amino acid transport and metabolism, inorganic ion transport and metabolism, and lipid metabolism, while the PM only differentially expressed 92 genes. The PM tolerates up to 17.5% v/v Populus hydrolysate and growth in it elicited 489 genes with differential expression, which included increased expression in energy production and conversion, cellulosome production, and inorganic ion transport and metabolism and decreased expression in transcription and cell

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

    PubMed

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

    2013-12-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed

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

    2000-02-01

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

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

    PubMed Central

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

    2009-01-01

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

  2. Developmentally controlled telomere addition in wild-type and mutant paramecia.

    PubMed Central

    Forney, J D; Blackburn, E H

    1988-01-01

    We analyzed sites of macronuclear telomere addition at a single genetic locus in Paramecium tetraurelia. We showed that in homozygous wild-type cells, differential genomic processing during macronuclear development resulted in the A surface antigen gene being located 8, 13, or 26 kilobases upstream from a macronuclear telomere. We describe variable rearrangements that occurred at the telomere 8 kilobases from the A gene. A mutant (d48) that forms a telomere near the 5' end of the A gene was also analyzed. This mutant was shown to create simple terminal deletions; telomeric repeats were added directly to the truncated wild-type A gene sequence. In both the mutant and wild-type cells, the telomeric sequences (a mixture of C4A2 and C3A3 repeats) were added to various sequences within a specific 200- to 500-base-pair region rather than to a single site. No similarities were found in the primary sequences surrounding the telomere addition sites. The mutation in d48 changed the region of telomere addition at the A gene locus; this is the first example in ciliates of a mutation that affects the site of telomere addition. Images PMID:3336360

  3. Effect of Fluorosis on Liver Cells of VC Deficient and Wild Type Mice

    PubMed Central

    Wei, Wei; Jiao, Yan; Ma, Yonghui; Stuart, John M.; Li, Xiudian; Zhao, Fusheng; Wang, Lishi; Sun, DianJun

    2014-01-01

    For decades, mouse and other rodents have been used for the study of oxidative or related studies such as the effect of fluoride. It is known that rodents normally synthesize their own vitamin C (VC) due to the presence of a key enzyme in ascorbic acid synthesis, l-gulono-lactone-γ-oxidase (Gulo), while humans do not have the capacity of VC synthesis due to the deletion of most parts of the GULO gene. The spontaneous fracture (sfx) mouse recently emerged as a model for study of VC deficiency. We investigated the effect of fluoride on liver cells from wild type Balb/c and sfx mice. We found that activities of SOD, GPx, and CAT were reduced in both wild type and sfx mice; however, the amount of reduction in the sfx cells is more than that in Balb/c cells. In addition, while both cells increased MDA, the increase in the sfx cells is greater than that in Balb/c cells. Gene networks of Sod, Gpx, and Cat in the liver of humans and mice are also different. Our study suggests that reaction to fluoride in vitamin C deficient mice might be different from that of wild type mice. PMID:24693236

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

    NASA Technical Reports Server (NTRS)

    Moore, R.

    1989-01-01

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

  5. Gravity-dependent differentiation and root coils in Arabidopsis thaliana wild type and phospholipase-A-I knockdown mutant grown on the International Space Station.

    PubMed

    Scherer, G F E; Pietrzyk, P

    2014-01-01

    Arabidopsis roots on 45° tilted agar in 1-g grow in wave-like figures. In addition to waves, formation of root coils is observed in several mutants compromised in gravitropism and/or auxin transport. The knockdown mutant ppla-I-1 of patatin-related phospholipase-A-I is delayed in root gravitropism and forms increased numbers of root coils. Three known factors contribute to waving: circumnutation, gravisensing and negative thigmotropism. In microgravity, deprivation of wild type (WT) and mutant roots of gravisensing and thigmotropism and circumnutation (known to slow down in microgravity, and could potentially lead to fewer waves or increased coiling in both WT and mutant). To resolve this, mutant ppla-I-1 and WT were grown in the BIOLAB facility in the International Space Station. In 1-g, roots of both types only showed waving. In the first experiment in microgravity, the mutant after 9 days formed far more coils than in 1-g but the WT also formed several coils. After 24 days in microgravity, in both types the coils were numerous with slightly more in the mutant. In the second experiment, after 9 days in microgravity only the mutant formed coils and the WT grew arcuated roots. Cell file rotation (CFR) on the mutant root surface in microgravity decreased in comparison to WT, and thus was not important for coiling. Several additional developmental responses (hypocotyl elongation, lateral root formation, cotyledon expansion) were found to be gravity-influenced. We tentatively discuss these in the context of disturbances in auxin transport, which are known to decrease through lack of gravity.

  6. Gravity-dependent differentiation and root coils in Arabidopsis thaliana wild type and phospholipase-A-I knockdown mutant grown on the International Space Station.

    PubMed

    Scherer, G F E; Pietrzyk, P

    2014-01-01

    Arabidopsis roots on 45° tilted agar in 1-g grow in wave-like figures. In addition to waves, formation of root coils is observed in several mutants compromised in gravitropism and/or auxin transport. The knockdown mutant ppla-I-1 of patatin-related phospholipase-A-I is delayed in root gravitropism and forms increased numbers of root coils. Three known factors contribute to waving: circumnutation, gravisensing and negative thigmotropism. In microgravity, deprivation of wild type (WT) and mutant roots of gravisensing and thigmotropism and circumnutation (known to slow down in microgravity, and could potentially lead to fewer waves or increased coiling in both WT and mutant). To resolve this, mutant ppla-I-1 and WT were grown in the BIOLAB facility in the International Space Station. In 1-g, roots of both types only showed waving. In the first experiment in microgravity, the mutant after 9 days formed far more coils than in 1-g but the WT also formed several coils. After 24 days in microgravity, in both types the coils were numerous with slightly more in the mutant. In the second experiment, after 9 days in microgravity only the mutant formed coils and the WT grew arcuated roots. Cell file rotation (CFR) on the mutant root surface in microgravity decreased in comparison to WT, and thus was not important for coiling. Several additional developmental responses (hypocotyl elongation, lateral root formation, cotyledon expansion) were found to be gravity-influenced. We tentatively discuss these in the context of disturbances in auxin transport, which are known to decrease through lack of gravity. PMID:24373011

  7. Next-generation sequencing facilitates quantitative analysis of wild-type and Nrl−/− retinal transcriptomes

    PubMed Central

    Brooks, Matthew J.; Rajasimha, Harsha K.; Roger, Jerome E.

    2011-01-01

    Purpose Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis. Methods Retinal mRNA profiles of 21-day-old wild-type (WT) and neural retina leucine zipper knockout (Nrl−/−) mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl−/− mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl−/− retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions Our study represents the first detailed analysis of retinal

  8. Genetic relationships and epidemiological links between wild type 1 poliovirus isolates in Pakistan and Afghanistan

    PubMed Central

    2012-01-01

    Background/Aim Efforts have been made to eliminate wild poliovirus transmission since 1988 when the World Health Organization began its global eradication campaign. Since then, the incidence of polio has decreased significantly. However, serotype 1 and serotype 3 still circulate endemically in Pakistan and Afghanistan. Both countries constitute a single epidemiologic block representing one of the three remaining major global reservoirs of poliovirus transmission. In this study we used genetic sequence data to investigate transmission links among viruses from diverse locations during 2005-2007. Methods In order to find the origins and routes of wild type 1 poliovirus circulation, polioviruses were isolated from faecal samples of Acute Flaccid Paralysis (AFP) patients. We used viral cultures, two intratypic differentiation methods PCR, ELISA to characterize as vaccine or wild type 1 and nucleic acid sequencing of entire VP1 region of poliovirus genome to determine the genetic relatedness. Results One hundred eleven wild type 1 poliovirus isolates were subjected to nucleotide sequencing for genetic variation study. Considering the 15% divergence of the sequences from Sabin 1, Phylogenetic analysis by MEGA software revealed that active inter and intra country transmission of many genetically distinct strains of wild poliovirus type 1 belonged to genotype SOAS which is indigenous in this region. By grouping wild type 1 polioviruses according to nucleotide sequence homology, three distinct clusters A, B and C were obtained with multiple chains of transmission together with some silent circulations represented by orphan lineages. Conclusion Our results emphasize that there was a persistent transmission of wild type1 polioviruses in Pakistan and Afghanistan during 2005-2007. The epidemiologic information provided by the sequence data can contribute to the formulation of better strategies for poliomyelitis control to those critical areas, associated with high risk

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

    SciTech Connect

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

    2008-07-03

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

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

    SciTech Connect

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

    2011-11-15

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

  11. Suppressed Accumulation of Cerebral Amyloid β Peptides in Aged Transgenic Alzheimer’s Disease Mice by Transplantation with Wild-Type or Prostaglandin E2 Receptor Subtype 2-Null Bone Marrow

    PubMed Central

    Keene, C. Dirk; Chang, Rubens C.; Lopez-Yglesias, Americo H.; Shalloway, Bryan R.; Sokal, Izabella; Li, Xianwu; Reed, Patrick J.; Keene, Lisa M.; Montine, Kathleen S.; Breyer, Richard M.; Rockhill, Jason K.; Montine, Thomas J.

    2010-01-01

    A complex therapeutic challenge for Alzheimer’s disease (AD) is minimizing deleterious aspects of microglial activation while maximizing beneficial actions, including phagocytosis/clearance of amyloid β (Aβ) peptides. One potential target is selective suppression of microglial prostaglandin E2 receptor subtype 2 (EP2) function, which influences microglial phagocytosis and elaboration of neurotoxic cytokines. To test this hypothesis, we transplanted bone marrow cells derived from wild-type mice or mice homozygous deficient for EP2 (EP2−/−) into lethally irradiated 5-month-old wild-type or APPswe-PS1ΔE9 double transgenic AD mouse model recipients. We found that cerebral engraftment by bone marrow transplant (BMT)-derived wild-type or EP2−/− microglia was more efficient in APPswe-PS1ΔE9 than in wild-type mice, and APPswe-PS1ΔE9 mice that received EP2−/− BMT had increased cortical microglia compared with APPswe-PS1ΔE9 mice that received wild-type BMT. We found that myeloablative irradiation followed by bone marrow transplant-derived microglia engraftment, rather than cranial irradiation or BMT alone, was responsible for the approximate one-third reduction in both Aβ plaques and potentially more neurotoxic soluble Aβ species. An additional 25% reduction in cerebral cortical Aβ burden was achieved in mice that received EP2−/− BMT compared with mice that received wild-type BMT. Our results provide a foundation for an adult stem cell-based therapy to suppress soluble Aβ peptide and plaque accumulation in the cerebrum of patients with AD. PMID:20522650

  12. [Construction of the new Escherichia coli K-12 wild-type strain with improved growth characteristics for application in metabolic engineering].

    PubMed

    Biriukova, I V; Krylov, A A; Kiseleva, E M; Minaeva, N I; Mashko, S V

    2010-03-01

    MG1655 of Escherichia coli K-12 is frequently used in metabolic engineering as the wild-type strain. However, its two mutations, ilvG and rph-1 provide a negative effect on culture growth. The "polar effect" of rph-1 decreases the level of pyrE expression, causing partial auxotrophy for pyrimidines. Mutation ilvG leading to the appearance of Val(S) phenotype causes retardation of cell growth rate on media containing amino acids. In this work, the substitution of two loci in the genome of MG1655 with the recovery of the wild-type phenotype was accomplished. Gene rph(wt) from the chromosome of E. coli TG1 was marked via Red-dependent integration of DNA fragment carrying lambda attL-Cm(R)-lambda attR and transduced with phage P1 into MG1655; later, the Cm(R) marker was removed with the use of lambda Xis/Int recombinase. Parallel to this procedure, a spontaneous Val(R) mutant of E. coli MG1655 yielding colonies of maximal size on M9 medium with glucose in the presence of Val (50 microg/ml) was isolated. It was shown that a nucleotide deletion in the isolated Val(R) strain had been generated in the region of the identified E. coli K-12 ilvG mutation, which led to the recovery of the reading frame and active protein synthesis. This mutation named ilvG-15, which is the only reason for the Val(R) phenotype in the obtained strain, was transferred to MG1655-rph(wt) using cotransduction, by analogy to the transfer of rph(wt). Evaluation of rates of aerobically growing cells (microm, hour(-1)) on M9 medium with glucose produced the following values: 0.56, 0.69, and 0.73 for strains MG1655, MG1655-rph(wt), and MG1655-(rph(wt), ilvG-15), respectively.

  13. Target binding to S100B reduces dynamic properties and increases Ca2+-binding affinity for wild type and EF-hand mutant proteins

    PubMed Central

    Liriano, Melissa A.; Varney, Kristen M.; Wright, Nathan T.; Hoffman, Cassandra L.; Toth, Eric A.; Ishima, Rieko; Weber, David J.

    2012-01-01

    Mutations in the second EF-hand (D61N, D63N, D65N, E72A) of S100B were used to study its Ca2+-binding and dynamic properties in the absence and presence of abound target, TRTK-12. With D63NS100B as an exception (D63NKD = 50 ± 9 µM), Ca2+-binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 (D61NKD = 412 ± 67 µM; D65NKD = 968 ± 171 µM; E72AKD = 471 ± 133 µM), when compared to wild-type protein (WTKD = 56 ± 9 µM). For the TRTK-12 complexes, the Ca2+-binding affinity to wild type (WT+TRTKKD = 12 ± 10 µM) and the EF2 mutants were increased by 5- to 19-fold versus in the absence of target (D61N+TRTKKD = 29 ± 1.2 µM; D63N+TRTKKD = 10 ± 2.2 µM; D65N+TRTKKD = 73 ± 4.4 µM; E72A+TRTKKD = 18 ± 3.7 µM). In addition, Rex, as measured using relaxation dispersion for side chain 15N resonances of Asn63 (D63NS100B) was reduced upon TRTK-12 binding when measured by nuclear magnetic resonance (NMR). Likewise, backbone motions on multiple time scales (ps-ms) throughout wild type, D61NS100B D63NS100B, and D65NS100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca2+-bound (2.0 Å) and TRTK-bound (1.2 Å) D63NS100B showed no change in Ca2+ coordination, so these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca2+-binding affinity in solution. Thus, a model for how S100B-TRTK12 complex formation increases Ca2+ binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12. PMID:22824086

  14. Activation of nicotinic α(7) acetylcholine receptor enhances long term potentation in wild type mice but not in APP(swe)/PS1ΔE9 mice.

    PubMed

    Söderman, Andreas; Mikkelsen, Jens D; West, Mark J; Christensen, Ditte Z; Jensen, Morten S

    2011-01-10

    Amyloid β (Aβ) plays a central role in Alzheimer's disease (AD) and binds to the nicotinic α(7) receptor (α(7) nAChR). Little is known about the degree to which the binding of Aβ to the α(7) nAChR influences the role of this receptor in long-term potentiation (LTP), however. We have studied the effect of the partial α(7) nAChR agonist SSR180711 on hippocampal slice preparations from normal wild type (Wt) and APP(swe)/PS1ΔE9 transgenic (Tg) mice. In the hippocampal slices from the 6 months old Wt mice, the application of both nicotine (5μM) and SSR180711 (300nM) resulted in a significant enhancement of LTP expressed in area CA1. However, in the Tg mice the application of SSR180711 did not result in an increase in LTP beyond control levels. The amount of binding of the α(7) nAChR ligand 125-I-α-bungarotoxin was not different between in Tg and Wt mice. These findings indicate that the α(7) nAChR is functionally blocked in the hippocampal neurons, downstream of the α(7) nAChR, and that this is likely due to an interaction between the receptor and Aβ, which leads to changes in LTP.

  15. Wild-type and mutant AvrA- Salmonella induce broadly similar immune pathways in the chicken ceca with key differences in signaling intermediates and inflammation.

    PubMed

    Arsenault, Ryan J; Genovese, Kenneth J; He, Haiqi; Wu, Huixia; Neish, Andrew S; Kogut, Michael H

    2016-02-01

    Salmonella enterica serovar Typhimurium (ST) is a serious infectious disease throughout the world, and a major reservoir for Salmonella is chicken. Chicken infected with Salmonella do not develop clinical disease, this may be the result of important host interactions with key virulence proteins. To study this, we inoculated chicken with mutant Salmonella Typhimurium that lacked the virulence protein AvrA (AvrA(-)). AvrA is referred to as an avirulence factor, as it moderates the host immune response. The lack of the AvrA virulence gene in ST resulted in reduced weight gain, enhanced persistence and greater extraintestinal organ invasion in chickens, as compared to wild-type (WT) ST. Kinome analysis was performed on inoculated cecal tissue. The majority of the signal transduction pathways induced by AvrA(-) and WT ST were similar; however, we observed alterations in innate immune system signaling. In addition, a leukocyte migration pathway was altered by AvrA(-) ST that may allow greater gut barrier permeability and invasion by the mutant. Cytokine expression did not appear significantly altered at 7 d post-inoculation; at 14 d post-inoculation, there was an observed increase in the expression of anti-inflammatory IL-10 in the WT inoculated ceca. This study is the first to describe mutant AvrA(-) ST infection of chicken and provides further insight into the Salmonella responses observed in chicken relative to other species such as humans and cattle. PMID:26574031

  16. Wild-type and mutant AvrA- Salmonella induce broadly similar immune pathways in the chicken ceca with key differences in signaling intermediates and inflammation.

    PubMed

    Arsenault, Ryan J; Genovese, Kenneth J; He, Haiqi; Wu, Huixia; Neish, Andrew S; Kogut, Michael H

    2016-02-01

    Salmonella enterica serovar Typhimurium (ST) is a serious infectious disease throughout the world, and a major reservoir for Salmonella is chicken. Chicken infected with Salmonella do not develop clinical disease, this may be the result of important host interactions with key virulence proteins. To study this, we inoculated chicken with mutant Salmonella Typhimurium that lacked the virulence protein AvrA (AvrA(-)). AvrA is referred to as an avirulence factor, as it moderates the host immune response. The lack of the AvrA virulence gene in ST resulted in reduced weight gain, enhanced persistence and greater extraintestinal organ invasion in chickens, as compared to wild-type (WT) ST. Kinome analysis was performed on inoculated cecal tissue. The majority of the signal transduction pathways induced by AvrA(-) and WT ST were similar; however, we observed alterations in innate immune system signaling. In addition, a leukocyte migration pathway was altered by AvrA(-) ST that may allow greater gut barrier permeability and invasion by the mutant. Cytokine expression did not appear significantly altered at 7 d post-inoculation; at 14 d post-inoculation, there was an observed increase in the expression of anti-inflammatory IL-10 in the WT inoculated ceca. This study is the first to describe mutant AvrA(-) ST infection of chicken and provides further insight into the Salmonella responses observed in chicken relative to other species such as humans and cattle.

  17. Metallothionein-I/II null mice are more sensitive than wild-type mice to the hepatotoxic and nephrotoxic effects of chronic oral or injected inorganic arsenicals.

    PubMed

    Liu, J; Liu, Y; Goyer, R A; Achanzar, W; Waalkes, M P

    2000-06-01

    Metallothionein (MT) is a low-molecular-weight, sulfhydryl-rich, metal-binding protein that can protect against the toxicity of cadmium, mercury, and copper. However, the role of MT in arsenic (As)-induced toxicity is less certain. To better define the ability of MT to modify As toxicity, MT-I/II knockout (MT-null) mice and the corresponding wild-type mice (WT) were exposed to arsenite [As(III)] or arsenate [As(V)] either through the drinking water for 48 weeks, or through repeated sc injections (5 days/week) for 15 weeks. Chronic As exposure increased tissue MT concentrations (2-5-fold) in the WT but not in MT-null mice. Arsenic by both routes produced damage to the liver (fatty infiltration, inflammation, and focal necrosis) and kidney (tubular cell vacuolization, inflammatory cell infiltration, and interstitial fibrosis) in both MT-null and WT mice. However, in MT-null mice, the pathological lesions were more frequent and severe when compared to WT mice. This was confirmed biochemically, in that, at the higher oral doses of As, blood urea nitrogen (BUN) levels were increased more in MT-null mice (60%) than in WT mice (30%). Chronic As exposures produced 2-10 fold elevation of serum interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha levels, with greater increases seen by repeated injections than by oral exposure, and again, MT-null mice had higher serum cytokines than WT mice after As exposure. Repeated As injections also decreased hepatic glutathione (GSH) by 35%, but GSH-peroxidase and GSH-reductase were minimally affected. MT-null mice were more sensitive than WT mice to the effect of GSH depletion by As(V). Hepatic caspase-3 activity was increased (2-3-fold) in both WT and MT-null mice, indicative of apoptotic cell death. In summary, chronic inorganic As exposure produced injuries to multiple organs, and MT-null mice are generally more susceptible than WT mice to As-induced toxicity regardless of route of exposure, suggesting that MT could be a

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-08-01

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

  20. Inhibition of Nitric Oxide Synthase 1 Induces Salt-Sensitive Hypertension in Nitric Oxide Synthase 1α Knockout and Wild-Type Mice.

    PubMed

    Wang, Ximing; Chandrashekar, Kiran; Wang, Lei; Lai, En Yin; Wei, Jin; Zhang, Gensheng; Wang, Shaohui; Zhang, Jie; Juncos, Luis A; Liu, Ruisheng

    2016-04-01

    We recently showed that α, β, and γ splice variants of neuronal nitric oxide synthase (NOS1) expressed in the macula densa and NOS1β accounts for most of the NO generation. We have also demonstrated that the mice with deletion of NOS1 specifically from the macula densa developed salt-sensitive hypertension. However, the global NOS1 knockout (NOS1KO) strain is neither hypertensive nor salt sensitive. This global NOS1KO strain is actually an NOS1αKO model. Consequently, we hypothesized that inhibition of NOS1β in NOS1αKO mice induces salt-sensitive hypertension. NOS1αKO and C57BL/6 wild-type (WT) mice were implanted with telemetry transmitters and divided into 7-nitroindazole (10 mg/kg/d)-treated and nontreated groups. All of the mice were fed a normal salt (0.4% NaCl) diet for 5 days, followed by a high-salt diet (4% NaCl). NO generation by the macula densa was inhibited by >90% in WT and NOS1αKO mice treated with 7-nitroindazole. Glomerular filtration rate in conscious mice was increased by ≈ 40% after a high-salt diet in both NOS1αKO and WT mice. In response to acute volume expansion, glomerular filtration rate, diuretic and natriuretic response were significantly blunted in the WT and knockout mice treated with 7-nitroindazole. Mean arterial pressure had no significant changes in mice fed a high-salt diet, but increased ≈ 15 mm Hg similarly in NOS1αKO and WT mice treated with 7-nitroindazole. We conclude that NOS1β, but not NOS1α, plays an important role in control of sodium excretion and hemodynamics in response to either an acute or a chronic salt loading.

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

    PubMed

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

    2015-08-01

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

  2. Bone Turnover in Wild Type and Pleiotrophin-Transgenic Mice Housed for Three Months in the International Space Station (ISS)

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2016-06-01

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

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

    PubMed Central

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

    2010-01-01

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

  5. Zika virus infection during the period of maximal brain growth causes microcephaly and corticospinal neuron apoptosis in wild type mice

    PubMed Central

    Huang, Wen-Chin; Abraham, Rachy; Shim, Byoung-Shik; Choe, Hyeryun; Page, Damon T.

    2016-01-01

    Zika virus (ZIKV) infection in pregnant women has been established as a cause of microcephaly in newborns. Here we test the hypothesis that neurodevelopmental stages when the brain is undergoing rapid growth are particularly vulnerable to the effects of ZIKV infection. We injected ZIKV intracranially into wild type C57BL/6 mice at two different time points: early postnatal development, when the brain is growing at its maximal rate, and at weaning, when the brain has largely reached adult size. Both time points showed widespread immunoreactivity for ZIKV and cleaved caspase 3 (CC3, a marker of apoptosis) throughout the brain. However, in early postnatal ZIKV injected mice, some brain areas and cell types display particularly large increases in apoptosis that we did not observe in older animals. Corticospinal pyramidal neurons, a cell type implicated in human microcephaly associated with ZIKV infection, are an example of one such cell type. Proliferating cells in the ventricular zone stem cell compartment are also depleted. These findings are consistent with the hypothesis that periods of rapid brain growth are especially susceptible to neurodevelopmental effects of ZIKV infection, and establish a valuable model to investigate mechanisms underlying neurodevelopmental effects of ZIKV infection and explore candidate therapeutics. PMID:27713505

  6. Tropomodulin 1 directly controls thin filament length in both wild-type and tropomodulin 4-deficient skeletal muscle

    PubMed Central

    Gokhin, David S.; Ochala, Julien; Domenighetti, Andrea A.; Fowler, Velia M.

    2015-01-01

    The sarcomeric tropomodulin (Tmod) isoforms Tmod1 and Tmod4 cap thin filament pointed ends and functionally interact with the leiomodin (Lmod) isoforms Lmod2 and Lmod3 to control myofibril organization, thin filament lengths, and actomyosin crossbridge formation in skeletal muscle fibers. Here, we show that Tmod4 is more abundant than Tmod1 at both the transcript and protein level in a variety of muscle types, but the relative abundances of sarcomeric Tmods are muscle specific. We then generate Tmod4−/− mice, which exhibit normal thin filament lengths, myofibril organization, and skeletal muscle contractile function owing to compensatory upregulation of Tmod1, together with an Lmod isoform switch wherein Lmod3 is downregulated and Lmod2 is upregulated. However, RNAi depletion of Tmod1 from either wild-type or Tmod4−/− muscle fibers leads to thin filament elongation by ∼15%. Thus, Tmod1 per se, rather than total sarcomeric Tmod levels, controls thin filament lengths in mouse skeletal muscle, whereas Tmod4 appears to be dispensable for thin filament length regulation. These findings identify Tmod1 as the key direct regulator of thin filament length in skeletal muscle, in both adult muscle homeostasis and in developmentally compensated contexts. PMID:26586224

  7. Does the early social environment affect structure and consistency of personality in wild-type male's rat?

    PubMed

    Gracceva, Giulia; Koolhaas, Jaap M; Groothuis, Ton G G

    2011-09-01

    Animal personality has been extensively studied from a functional and evolutionary point of view. Less attention has been paid to the development of personality, its phenotypic plasticity, and the influence of manipulation of early environmental factors. Here we describe the effects of manipulating the sex ratio of the litter, at postnatal day (pnd) 3, in wild-type rats, on personality traits in adulthood. We measured the treatment effects on aggression, defensive burying, and open field behavior at pnd 90 and 120, as well as on their contextual generality, and stability over time (differential and structural consistency). Main effects of litter composition were found on open field behavior at pnd 120 but not on the other behaviors. Since correlations between behaviors changed over time irrespective of the specific treatment, whereas in previous studies on unmanipulated litters this was not the case we suggest that early handling may disrupt adult personality traits. Overall the data indicate that personality is less stable over time that often assumed, having both proximate and ultimate implications.

  8. Rearing in Seawater Mesocosms Improves the Spawning Performance of Growth Hormone Transgenic and Wild-Type Coho Salmon

    PubMed Central

    Leggatt, Rosalind A.; Hollo, Tanya; Vandersteen, Wendy E.; McFarlane, Kassandra; Goh, Benjamin; Prevost, Joelle; Devlin, Robert H.

    2014-01-01

    Growth hormone (GH) transgenes can significantly accelerate growth rates in fish and cause associated alterations to their physiology and behaviour. Concern exists regarding potential environmental risks of GH transgenic fish, should they enter natural ecosystems. In particular, whether they can reproduce and generate viable offspring under natural conditions is poorly understood. In previous studies, GH transgenic salmon grown under contained culture conditions had lower spawning behaviour and reproductive success relative to wild-type fish reared in nature. However, wild-type salmon cultured in equal conditions also had limited reproductive success. As such, whether decreased reproductive success of GH transgenic salmon is due to the action of the transgene or to secondary effects of culture (or a combination) has not been fully ascertained. Hence, salmon were reared in large (350,000 L), semi-natural, seawater tanks (termed mesocosms) designed to minimize effects of standard laboratory culture conditions, and the reproductive success of wild-type and GH transgenic coho salmon from mesocosms were compared with that of wild-type fish from nature. Mesocosm rearing partially restored spawning behaviour and success of wild-type fish relative to culture rearing, but remained lower overall than those reared in nature. GH transgenic salmon reared in the mesocosm had similar spawning behaviour and success as wild-type fish reared in the mesocosm when in full competition and without competition, but had lower success in male-only competition experiments. There was evidence of genotype×environmental interactions on spawning success, so that spawning success of transgenic fish, should they escape to natural systems in early life, cannot be predicted with low uncertainty. Under the present conditions, we found no evidence to support enhanced mating capabilities of GH transgenic coho salmon compared to wild-type salmon. However, it is clear that GH transgenic salmon are

  9. Use of SLAM and PVRL4 and identification of pro-HB-EGF as cell entry receptors for wild type phocine distemper virus.

    PubMed

    Melia, Mary M; Earle, John Philip; Abdullah, Haniah; Reaney, Katherine; Tangy, Frederic; Cosby, Sara Louise

    2014-01-01

    Signalling lymphocyte activation molecule (SLAM) has been identified as an immune cell receptor for the morbilliviruses, measles (MV), canine distemper (CDV), rinderpest and peste des petits ruminants (PPRV) viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4), also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV) remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt) MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG) and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF),for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO) cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation. PMID:25171206

  10. Use of SLAM and PVRL4 and identification of pro-HB-EGF as cell entry receptors for wild type phocine distemper virus.

    PubMed

    Melia, Mary M; Earle, John Philip; Abdullah, Haniah; Reaney, Katherine; Tangy, Frederic; Cosby, Sara Louise

    2014-01-01

    Signalling lymphocyte activation molecule (SLAM) has been identified as an immune cell receptor for the morbilliviruses, measles (MV), canine distemper (CDV), rinderpest and peste des petits ruminants (PPRV) viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4), also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV) remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt) MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG) and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF),for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO) cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation.

  11. Positron emission tomographic imaging of the cannabinoid type 1 receptor system with [¹¹C]OMAR ([¹¹C]JHU75528): improvements in image quantification using wild-type and knockout mice.

    PubMed

    Herance, Raúl; Rojas, Santiago; Abad, Sergio; Jiménez, Xavier; Gispert, Juan Domingo; Millán, Olga; Martín-García, Elena; Burokas, Aurelijus; Serra, Miquel Àngel; Maldonado, Rafael; Pareto, Deborah

    2011-12-01

    In this study, we assessed the feasibility of using positron emission tomography (PET) and the tracer [¹¹C]OMAR ([¹¹C]JHU75528), an analogue of rimonabant, to study the brain cannabinoid type 1 (CB1) receptor system. Wild-type (WT) and CB1 knockout (KO) animals were imaged at baseline and after pretreatment with blocking doses of rimonabant. Brain uptake in WT animals was higher (50%) than in KO animals in baseline conditions. After pretreatment with rimonabant, WT uptake lowered to the level of KO animals. The results of this study support the feasibility of using PET with the radiotracer [¹¹C]JHU75528 to image the brain CB1 receptor system in mice. In addition, this methodology can be used to assess the effect of new drugs in preclinical studies using genetically manipulated animals.

  12. Effects of female pheromones on gonadotropin-releasing hormone gene expression and luteinizing hormone release in male wild-type and oestrogen receptor-alpha knockout mice.

    PubMed

    Gore, A C; Wersinger, S R; Rissman, E F

    2000-12-01

    Pheromones are an important class of environmental cues that affect the hypothalamic-pituitary-gonadal axis in a variety of vertebrate species, including humans. When male mice contact female-soiled bedding, or urine, they display a reflexive luteinizing hormone (LH) surge within 30 min. Aside from the requirement that males have gonads to show this response, the physiological mechanisms that underlie this pituitary response are unknown. In this experiment, we asked if female pheromones acted at the level of gonadotropin-releasing hormone (GnRH) gene expression to affect this hormone response. In addition, we also examined the contribution of one of the oestrogen receptors (ERalpha) by studying this neuroendocrine reflex in wild-type and oestrogen receptor-alpha knockout (ERalphaKO) males. Both ERalphaKO and wild-type males showed the expected LH surge, 45 and 90 min after contact with female pheromones. Males housed in clean bedding or bedding soiled by another adult male did not display the LH elevation. Interestingly, this dramatic change in LH concentrations was not accompanied by any alterations in GnRH mRNA expression or levels of primary transcript in the preoptic area-anterior hypothalamus. The one exception to this was a significant increase in GnRH mRNA expression in tissue collected from wild-type males exposed to bedding from another male. This is particularly intriguing since LH was not elevated in these males. These data replicate and extend our previous finding that ERalphaKO males do exhibit an LH surge in response to female pheromones. Thus, this neuroendocrine response is regulated by a steroid receptor other than ERalpha and does not require alterations in GnRH mRNA expression.

  13. Insights into wild-type and mutant p53 functions provided by genetically engineered mice.

    PubMed

    Donehower, Lawrence A

    2014-06-01

    Recent whole-exome sequencing studies of numerous human cancers have now conclusively shown that the TP53 tumor-suppressor gene is the most frequently mutated gene in human cancers. Despite extensive studies of the TP53 gene and its encoded protein (p53), our understanding of how TP53 mutations contribute to cancer initiation and progression remain incomplete. Genetically engineered mice with germline or inducible Trp53 somatic mutations have provided important insights into the mechanisms by which different types of p53 mutation influence cancer development. Trp53 germline mutations that alter specific p53 structural domains or posttranslation modification sites have benefitted our understanding of wild-type p53 functions in a whole organism context. Moreover, genetic approaches to reestablish functional wild-type p53 to p53-deficient tissues and tumors have increased our understanding of the therapeutic potential of restoring functional p53 signaling to cancers. This review outlines many of the key insights provided by the various categories of Trp53 mutant mice that have been generated by multiple genetic engineering approaches.

  14. Accelerated Telomere Shortening and Replicative Senescence in Human Fibroblasts Overexpressing Mutant and Wild Type Lamin A

    PubMed Central

    Huang, Shurong; Risques, Rosa Ana; Martin, George M.; Rabinovitch, Peter S.; Oshima, Junko

    2008-01-01

    LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. To our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectible WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes. PMID:17870066

  15. Proteomic response to physiological fermentation stresses in a wild-type wine strain of Saccharomyces cerevisiae.

    PubMed Central

    Trabalzini, Lorenza; Paffetti, Alessandro; Scaloni, Andrea; Talamo, Fabio; Ferro, Elisa; Coratza, Grazietta; Bovalini, Lucia; Lusini, Paola; Martelli, Paola; Santucci, Annalisa

    2003-01-01

    We report a study on the adaptive response of a wild-type wine Saccharomyces cerevisiae strain, isolated from natural spontaneous grape must, to mild and progressive physiological stresses due to fermentation. We observed by two-dimensional electrophoresis how the yeast proteome changes during glucose exhaustion, before the cell enters its complete stationary phase. On the basis of their identification, the proteins representing the S. cerevisiae proteomic response to fermentation stresses were divided into three classes: repressed proteins, induced proteins and autoproteolysed proteins. In an overall view, the proteome adaptation of S. cerevisiae at the time of glucose exhaustion seems to be directed mainly against the effects of ethanol, causing both hyperosmolarity and oxidative responses. Stress-induced autoproteolysis is directed mainly towards specific isoforms of glycolytic enzymes. Through the use of a wild-type S. cerevisiae strain and PMSF, a specific inhibitor of vacuolar proteinase B, we could also distinguish the specific contributions of the vacuole and the proteasome to the autoproteolytic process. PMID:12401115

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

    PubMed

    Chen, Kuan-Hui Ethan; Walker, Ameae M

    2016-06-01

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

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

    PubMed

    Zhang, Jiapu; Liu, David D W

    2011-06-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  20. The mechanical properties of tail tendon fascicles from lubricin knockout, wild type and heterozygous mice.

    PubMed

    Reuvers, John; Thoreson, Andrew R; Zhao, Chunfeng; Zhang, Ling; Jay, Gregory D; An, Kai-Nan; Warman, Matthew L; Amadio, Peter C

    2011-10-01

    The purpose of this study was to analyze the effects of lubricin on tendon stiffness and viscoelasticity. A total of 36 mice were tested with 12 mice in each of the following groups: lubricin knock-out ⁻/⁻, heterozygous ⁺/⁻ and wild-type ⁺/⁺. A ramp test was used to determine the elastic modulus by pulling the fascicles to 2.5% strain amplitude at a rate of 0.05 mm/s. Then, followed by a relaxation test that pulled the fascicles to 5% strain amplitude at a rate of 2 mm/s. The fascicles were allowed to relax for 2 min at the maximum strain and a single-cycle relaxation ratio was used to characterize viscoelastic properties. There was no significant difference in the Young's modulus between the three groups (p > 0.05), but the knockout mice had a significantly (p < 0.05) lower relaxation ratio than the wild type mice. Based on these data, we concluded that lubricin expression has an effect on the viscoelastic properties of tendon fascicles. The clinical significance of this finding, if any, remains to be demonstrated.

  1. The mechanical properties of tail tendon fascicles from lubricin knockout, wild type and heterozygous mice

    PubMed Central

    Reuvers, John; Thoreson, Andrew R.; Zhao, Chunfeng; Zhang, Ling; Jay, Gregory D.; An, Kai-Nan; Warman, Matthew L.; Amadio, Peter C.

    2014-01-01

    The purpose of this study was to analyze the effects of lubricin on tendon stiffness and viscoelasticity. A total of 36 mice were tested with 12 mice in each of the following groups: lubricin knock-out (−/−), heterozygous (+/−) and wild-type (+/+). A ramp test was used to determine the elastic modulus by pulling the fascicles to 2.5% strain amplitude at a rate of 0.05 mm/s. Then, followed by a relaxation test that pulled the fascicles to 5% strain amplitude at a rate of 2 mm/s. The fascicles were allowed to relax for 2 min at the maximum strain and a single-cycle relaxation ratio was used to characterize viscoelastic properties. There was no significant difference in the Young’s modulus between the three groups (p > 0.05), but the knockout mice had a significantly (p < 0.05) lower relaxation ratio than the wild type mice. Based on these data, we concluded that lubricin expression has an effect on the viscoelastic properties of tendon fascicles. The clinical significance of this finding, if any, remains to be demonstrated. PMID:21821131

  2. Mycoplasma gallisepticum infection in wild-type turkeys living in close contact with domestic fowl.

    PubMed

    Jessup, D A; DaMassa, A J; Lewis, R; Jones, K R

    1983-12-01

    Mycoplasma gallisepticum was isolated from 2 wild-type turkeys (Meleagris gallopavo) and 1 domestic turkey living in close contact on a farm in Tehama County, California. Sinusitis was detected in 2 of 14 wild-type turkeys and in 1 of 12 feral broad-breasted bronze turkeys, but in none of several chickens on the premises. The entire mixed flock was captured, sinus aspirates were collected from affected birds, and blood samples were obtained from all birds for serologic testing. Blood samples also were obtained from 10 domestic turkeys on adjacent premises from which breeding stock had been borrowed. The M gallisepticum isolated from sinus aspirates was typed and inoculated into susceptible chickens, resulting in airsacculitis. California wild turkeys with and without histories of exposure to domestic fowl and wild turkeys shipped into California from Texas for release were tested for antibodies to M gallisepticum, using the plate agglutination test. Evidence of M gallisepticum infection was not found in wild turkeys at any location other than the original premises.

  3. Craniofacial statistical deformation models of wild-type mice and Crouzon mice

    NASA Astrophysics Data System (ADS)

    Ólafsdóttir, Hildur; Darvann, Tron A.; Ersbøll, Bjarne K.; Hermann, Nuno V.; Oubel, Estanislao; Larsen, Rasmus; Frangi, Alejandro F.; Larsen, Per; Perlyn, Chad A.; Morriss-Kay, Gillian M.; Kreiborg, Sven

    2007-03-01

    Crouzon syndrome is characterised by premature fusion of cranial sutures and synchondroses leading to craniofacial growth disturbances. The gene causing the syndrome was discovered approximately a decade ago and recently the first mouse model of the syndrome was generated. In this study, a set of Micro CT scans of the heads of wild-type (normal) mice and Crouzon mice were investigated. Statistical deformation models were built to assess the anatomical differences between the groups, as well as the within-group anatomical variation. Following the approach by Rueckert et al. we built an atlas using B-spline-based nonrigid registration and subsequently, the atlas was nonrigidly registered to the cases being modelled. The parameters of these registrations were then used as input to a PCA. Using different sets of registration parameters, different models were constructed to describe (i) the difference between the two groups in anatomical variation and (ii) the within-group variation. These models confirmed many known traits in the wild-type and Crouzon mouse craniofacial anatomy. However, they also showed some new traits.

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

    PubMed

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

    2006-03-01

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

  5. Genetic recombination of tick-borne flaviviruses among wild-type strains.

    PubMed

    Norberg, Peter; Roth, Anette; Bergström, Tomas

    2013-06-01

    Genetic recombination has been suggested to occur in mosquito-borne flaviviruses. In contrast, tick-borne flaviviruses have been thought to evolve in a clonal manner, although recent studies suggest that recombination occurs also for these viruses. We re-analyzed the data and found that previous conclusions on wild type recombination were probably falsely drawn due to misalignments of nucleotide sequences, ambiguities in GenBank sequences, or different laboratory culture histories suggestive of recombination events in laboratory. To evaluate if reliable predictions of wild type recombination of tick-borne flaviviruses can be made, we analyzed viral strains sequenced exclusively for this study, and other flavivirus sequences retrieved from GenBank. We detected genetic signals supporting recombination between viruses within the three clades of TBEV-Eu, TBEV-Sib and TBEV-Fe, respectively. Our results suggest that the tick-borne encephalitis viruses may undergo recombination under natural conditions, but that geographic barriers restrict most recombination events to involve only closely genetically related viruses.

  6. Glucocorticoid-regulated glycoprotein maturation in wild-type and mutant rat cell lines

    PubMed Central

    1986-01-01

    Glucocorticoid hormones can regulate the posttranslational maturation of mouse mammary tumor virus (MTV) precursor polyproteins in M1.54, a stably infected rat hepatoma cell line. We have used complement- mediated cytolysis to recover variants of M1.54 that fail to express MTV cell surface glycoproteins in a hormone-regulated manner (Firestone, G.L., and K.R. Yamamoto, 1983, Mol. Cell. Biol., 3:149- 160). One such clonal isolate, CR4, is similar to wild-type with respect to synthesis of MTV mRNAs, production of the MTV glycoprotein precursor (gPr74env) and a glycosylated maturation product (gp51), and hormone-induced processing of two MTV phosphoproteins. In contrast, three viral cell surface glycoproteins (gp78, gp70, and gp32) and one extracellular species (gp70s), which derive from gPr74env in glucocorticoid-treated wild-type cells, fail to appear in CR4. CR4 showed no apparent alterations in proliferation rate, cell shape, or expression of total functional mRNA and bulk glycoproteins. We conclude that the genetic lesion in CR4 defines a highly selective hormone- regulated glycoprotein maturation pathway that alters the fate of a restricted subset of precursor species. PMID:3023398

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

    PubMed

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

    2015-11-01

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

  8. Structural insights into conformational stability of both wild-type and mutant EZH2 receptor

    PubMed Central

    Aier, Imlimaong; Varadwaj, Pritish Kumar; Raj, Utkarsh

    2016-01-01

    Polycomb group (PcG) proteins have been observed to maintain the pattern of histone by methylation of the histone tail responsible for the gene expression in various cellular processes, of which enhancer of zeste homolog 2 (EZH2) acts as tumor suppressor. Overexpression of EZH2 results in hyper activation found in a variety of cancer. Point mutation on two important residues were induced and the results were compared between the wild type and mutant EZH2. The mutation of Y641 and A677 present in the active region of the protein alters the interaction of the top ranked compound with the newly modeled binding groove of the SET domain, giving a GLIDE score of −12.26 kcal/mol, better than that of the wild type at −11.664 kcal/mol. In depth analysis were carried out for understanding the underlying molecular mechanism using techniques viz. molecular dynamics, principal component analysis, residue interaction network and free energy landscape analysis, which showed that the mutated residues changed the overall conformation of the system along with the residue-residue interaction network. The insight from this study could be of great relevance while designing new compounds for EZH2 enzyme inhibition and the effect of mutation on the overall binding mechanism of the system. PMID:27713574

  9. Co-fibrillogenesis of Wild-type and D76N β2-Microglobulin: THE CRUCIAL ROLE OF FIBRILLAR SEEDS.

    PubMed

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

    2016-04-29

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-06-01

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

  11. The Wilms' tumor gene Wt1 is required for normal development of the retina.

    PubMed

    Wagner, Kay-Dietrich; Wagner, Nicole; Vidal, Valerie P I; Schley, Gunnar; Wilhelm, Dagmar; Schedl, Andreas; Englert, Christoph; Scholz, Holger

    2002-03-15

    The Wilms' tumor gene Wt1 is known for its important functions during genitourinary and mesothelial formation. Here we show that Wt1 is necessary for neuronal development in the vertebrate retina. Mouse embryos with targeted disruption of Wt1 exhibit remarkably thinner retinas than age-matched wild-type animals. A large fraction of retinal ganglion cells is lost by apoptosis, and the growth of optic nerve fibers is severely disturbed. Strikingly, expression of the class IV POU-domain transcription factor Pou4f2 (formerly Brn-3b), which is critical for the survival of most retinal ganglion cells, is lost in Wt1(-/-) retinas. Forced expression of Wt1 in cultured cells causes an up-regulation of Pou4f2 mRNA. Moreover, the Wt1(-KTS) splice variant can activate a reporter construct carrying 5'-regulatory sequences of the human POU4F2. The lack of Pou4f2 and the ocular defects in Wt1(-/-) embryos are rescued by transgenic expression of a 280 kb yeast artificial chromosome carrying the human WT1 gene. Taken together, our findings demonstrate a continuous requirement for Wt1 in normal retina formation with a critical role in Pou4f2-dependent ganglion cell differentiation.

  12. Subtype-selective induction of wild-type p53 and apoptosis, but not cell cycle arrest, by human somatostatin receptor 3.

    PubMed

    Sharma, K; Patel, Y C; Srikant, C B

    1996-12-01

    Somatostatin (SST) exerts direct antiproliferative effects in tumor cells, triggering either growth arrest or apoptosis. The cellular actions of SST are transduced through a family of five distinct somatostatin receptor subtypes (SSTR1-5). Whereas growth inhibition has been reported to follow stimulation of protein tyrosine phosphatase via SSTR2 or inhibition of Ca2+ channels via SSTR5 in heterologous expression systems, the subtype selectivity for signaling apoptosis has not been investigated. The tumor suppressor protein p53 and the protooncogene product c-Myc regulate cell cycle progression (growth factors present) or apoptosis (growth factors absent). The p53-induced G1 arrest requires induction of p21, an inhibitor of cyclin-dependent kinases, whereas apoptosis requires induction of Bax. c-Myc is capable of abrogating p53-induced G1 arrest by interfering with the inhibitory action of p21 on cyclin-dependent kinases. We have, therefore, investigated the regulation of p53, p21, c-Myc, and Bax and cellular apoptosis in relation to cell cycle progression in CHO-K1 cells stably expressing individual human SSTR1-5. We demonstrate that apoptosis is signaled uniquely through human SSTR3 and is associated with dephosphorylation-dependent conformational change in wild-type (wt) p53 as well as induction of Bax. The induction of wt p53 occurs rapidly and precedes the onset of apoptosis. We show that the increase in wt p53 is not associated with the induction of p21 or c-Myc when octreotide-induced apoptosis becomes evident, suggesting that such apoptosis does not require G1 arrest and is not c-Myc dependent. These findings provide the first evidence for hormonal induction of wt p53-associated apoptosis via G protein-coupled receptor in a subtype-selective manner.

  13. Expression of the Prion Protein Family Member Shadoo Causes Drug Hypersensitivity That Is Diminished by the Coexpression of the Wild Type Prion Protein.

    PubMed

    Nyeste, Antal; Bencsura, Petra; Vida, István; Hegyi, Zoltán; Homolya, László; Fodor, Elfrieda; Welker, Ervin

    2016-02-26

    The prion protein (PrP) seems to exert both neuroprotective and neurotoxic activities. The toxic activities are associated with the C-terminal globular parts in the absence of the flexible N terminus, specifically the hydrophobic domain (HD) or the central region (CR). The wild type prion protein (PrP-WT), having an intact flexible part, exhibits neuroprotective qualities by virtue of diminishing many of the cytotoxic effects of these mutant prion proteins (PrPΔHD and PrPΔCR) when coexpressed. The prion protein family member Doppel, which possesses a three-dimensional fold similar to the C-terminal part of PrP, is also harmful to neuronal and other cells in various models, a phenotype that can also be eliminated by the coexpression of PrP-WT. In contrast, another prion protein family member, Shadoo (Sho), a natively disordered protein possessing structural features similar to the flexible N-terminal tail of PrP, exhibits PrP-WT-like protective properties. Here, we report that, contrary to expectations, Sho expression in SH-SY5Y or HEK293 cells induces the same toxic phenotype of drug hypersensitivity as PrPΔCR. This effect is exhibited in a dose-dependent manner and is also counteracted by the coexpression of PrP-WT. The opposing effects of Shadoo in different model systems revealed here may be explored to help discern the relationship of the various toxic activities of mutant PrPs with each other and the neurotoxic effects seen in neurodegenerative diseases, such as transmissible spongiform encephalopathy and Alzheimer disease.

  14. Expression of the Prion Protein Family Member Shadoo Causes Drug Hypersensitivity That Is Diminished by the Coexpression of the Wild Type Prion Protein.

    PubMed

    Nyeste, Antal; Bencsura, Petra; Vida, István; Hegyi, Zoltán; Homolya, László; Fodor, Elfrieda; Welker, Ervin

    2016-02-26

    The prion protein (PrP) seems to exert both neuroprotective and neurotoxic activities. The toxic activities are associated with the C-terminal globular parts in the absence of the flexible N terminus, specifically the hydrophobic domain (HD) or the central region (CR). The wild type prion protein (PrP-WT), having an intact flexible part, exhibits neuroprotective qualities by virtue of diminishing many of the cytotoxic effects of these mutant prion proteins (PrPΔHD and PrPΔCR) when coexpressed. The prion protein family member Doppel, which possesses a three-dimensional fold similar to the C-terminal part of PrP, is also harmful to neuronal and other cells in various models, a phenotype that can also be eliminated by the coexpression of PrP-WT. In contrast, another prion protein family member, Shadoo (Sho), a natively disordered protein possessing structural features similar to the flexible N-terminal tail of PrP, exhibits PrP-WT-like protective properties. Here, we report that, contrary to expectations, Sho expression in SH-SY5Y or HEK293 cells induces the same toxic phenotype of drug hypersensitivity as PrPΔCR. This effect is exhibited in a dose-dependent manner and is also counteracted by the coexpression of PrP-WT. The opposing effects of Shadoo in different model systems revealed here may be explored to help discern the relationship of the various toxic activities of mutant PrPs with each other and the neurotoxic effects seen in neurodegenerative diseases, such as transmissible spongiform encephalopathy and Alzheimer disease. PMID:26721882

  15. Wild-type and specific mutant androgen receptor mediates transcription via 17β-estradiol in sex hormone-sensitive cancer cells.

    PubMed

    Susa, Takao; Ikaga, Reina; Kajitani, Takashi; Iizuka, Masayoshi; Okinaga, Hiroko; Tamamori-Adachi, Mimi; Okazaki, Tomoki

    2015-07-01

    We previously encountered regulatory processes wherein dihydrotestosterone (DHT) exerted its inhibitory effect on parathyroid hormone-related protein (PTHrP) gene repression through the estrogen receptor (ER)α, but not the androgen receptor (AR), in breast cancer MCF-7 cells. Here, we investigated whether such aberrant ligand-nuclear receptor (NR) interaction is present in prostate cancer LNCaP cells. First, we confirmed that LNCaP cells expressed large amounts of AR at negligible levels of ERα/β or progesterone receptor. Both suppression of PTHrP and activation of prostate-specific antigen genes were observed after independent administration of 17β-estradiol (E2), DHT, or R5020. Consistent with the notion that the LNCaP AR lost its ligand specificity due to a mutation (Thr-Ala877), experiments with siRNA targeting the respective NR revealed that the AR monopolized the role of the mediator of shared hormone-dependent regulation, which was invariably associated with nuclear translocation of this mutant AR. Microarray analysis of gene regulation by DHT, E2, or R5020 disclosed that more than half of the genes downstream of the AR (Thr-Ala877) overlapped in the LNCaP cells. Of particular interest, we realized that the AR (wild-type [wt]) and AR (Thr-Ala877) were equally responsible for the E2-AR interactions. Fluorescence microscopy experiments demonstrated that both EGFP-AR (wt) and EGFP-AR (Thr-Ala877) were exclusively localized within the nucleus after E2 or DHT treatment. Furthermore, reporter assays revealed that some other cancer cells exhibited aberrant E2-AR (wt) signaling similar to that in the LNCaP cells. We herein postulate the presence of entangled interactions between wt AR and E2 in certain hormone-sensitive cancer cells.

  16. Impressive long-term disease stabilization by nilotinib in two pretreated patients with KIT/PDGFRA wild-type metastatic gastrointestinal stromal tumours.

    PubMed

    Pantaleo, Maria Abbondanza; Nannini, Margherita; Saponara, Maristella; Gnocchi, Chiara; Di Scioscio, Valerio; Lolli, Cristian; Catena, Fausto; Astolfi, Annalisa; Di Battista, Monica; Biasco, Guido

    2012-06-01

    KIT/PDGFRA wild-type (WT) gastrointestinal stromal tumours (GISTs) showed a response rate to imatinib ranging from 0 to 25%. Nilotinib is a new-generation tyrosine kinase inhibitor that has demonstrated clinical activity in pretreated GIST patients. At present, no correlation between nilotinib activity and clinical/pathological/molecular features is available. We report on two WT GIST patients resistant to imatinib and sunitinib, and enrolled in the CAMN107A2201 study who achieved an impressive disease control by nilotinib. Both patients have germ-line mutations in the SDHA gene. In April 2004, a 39-year-old woman presented gastric GIST with multiple liver metastases and was treated with imatinib 400 mg/day, followed by imatinib 800 mg/day and then sunitinib. In August 2007, because of disease progression, she was enrolled in the CAMN107A2201 study and assigned to the nilotinib 800 mg/day arm. In March 2005, a 27-year-old woman started imatinib 600 mg/day and then sunitinib for gastric GIST with multiple liver and lung metastases. In October 2007, because of disease progression, she was enrolled in the CAMN107A2201 study and assigned to the nilotinib 800 mg/day arm. One patient still showed stable disease after 46 months of treatment according to the Response Evaluation Criteria In Solid Tumors, and a partial response after 9 months according to Choi's criteria. The other patient still showed stable disease after 42 months according to Response Evaluation Criteria In Solid Tumors. At present, they continue to receive nilotinib. We report very long-term disease stabilization under nilotinib treatment in two pretreated WT GIST patients. In-vitro studies and clinical analyses are warranted to evaluate a potential correlation between nilotinib activity and WT genotype or other clinical/pathological features.

  17. Experimental investigation of magneto-aerotaxis on wild-type magnetotactic bacteria in sediment

    NASA Astrophysics Data System (ADS)

    Mao, X.; Egli, R.

    2012-12-01

    Magnetotactic bacteria (MB) synthesize chains of magnetic particles, called magnetosomes, which provide a magnetic dipole that passively aligns the cells along the geomagnetic field. Flagellar propulsion allows MB to swim straight along field lines in what is known as magnetotaxis. The flagellum rotation sense is controlled by the chemical environment, so that MB can efficiently move across chemically stratified environments to reach the so-called oxic-anoxic interface (OAI). This combination of oriented swimming controlled by chemical (oxygen) sensing is called magneto-aerotaxis (Frankel 1997). Experiments with MB cultures show that magnetic spirilla can change instantaneously the swimming direction, while the behaviour of cocci depends on a sort of 'internal state' dictated by their original location with respect to the OAI. Here, we present first results the magneto-aerotactic behaviour of wild-type MB living in microcosms created with sediment retrieved from lake Chiemsee (Bavaria, Germany). In these microcosms, a stable population of MB (mainly unidentified strains of cocci, and Magnetobacterium Bavaricum) occur in the upmost few cm below the sediment surface, with maximum concentrations just below the OAI. We tested the reaction of this MB population to changes in chemical conditions by putting the microcosm inside a glove box with controlled oxygen-free atmospheres (N2 and CO2). A new equilibrium was reached within few weeks, with the OAI first moving upward and then disappearing. The depth distribution and swimming direction of MB was tested during and after the formation of a new equilibrium. We were never able to observe swimming directions consistent with bacteria moving upward in the sediment, as it was the case with cultured cocci in Frankel [1997], even long time after the entire sediment column became completely anoxic. Nevertheless, the disappearance of the OAI was accompanied by a slight but significant decrease of the total MB population

  18. Quantitative analysis of wild-type and V600E mutant BRAF proteins in colorectal carcinoma using immunoenrichment and targeted mass spectrometry.

    PubMed

    Chen, Hang; Hsiao, Yung-Chin; Chiang, Sum-Fu; Wu, Chia-Chun; Lin, Yu-Tsun; Liu, Hsuan; Zhao, Hong; Chen, Jinn-Shiun; Chang, Yu-Sun; Yu, Jau-Song

    2016-08-24

    The BRAF V600E mutation is one of the most common mutations implicated in the development of several types of cancer including colorectal cancer (CRC), where it is associated with aggressive disease phenotypes and poor outcomes. The status of the BRAF V600E mutation is frequently determined by direct DNA sequencing. However, no previous study has sought to quantify the BRAF V600E protein in cancer specimens. Here, we evaluated immunoenrichment coupled with two MS-based quantitative techniques, namely multiple reaction monitoring (MRM) and single ion monitoring conjugated accurate inclusion mass screening (SIM-AIMS), to detect and precisely quantify wild-type (WT) and V600E mutant BRAF proteins in DNA sequence-confirmed CRC tissue specimens. WT and V600E BRAF proteins were immunoprecipitated from a CRC cell line (HT-29), and their representative peptides ((592)IGDFGLATVK(601) and (592)IGDFGLATEK(601), respectively) were confirmed by LC-MS/MS analysis and then quantified by MRM or SIM-AIMS with spiked stable isotope-labeled peptide standards. Both assays worked well for measuring WT BRAF from different amounts of HT-29 cell lysates, but the MRM assay was more sensitive than SIM-AIMS assay for quantifying lower levels of V600E BRAF. In protein extracts (2 mg) from 11 CRC tissue specimens, the MRM assay could measure WT BRAF in all 11 cases (0.32-1.66 ng) and the V600E BRAF in two cases (0.1-0.13 ng; mutant-to-WT ratio, 0.16-0.17). The SIM-AIMS assay could also detect WT and V600E BRAF in CRC specimens, but the measured levels of both targets were lower than those determined by MRM assay. Collectively, this study provides an effective method to precisely quantify WT and V600E BRAF proteins in complex biological samples using immunoenrichment-coupled targeted MS. Since the V600E BRAF protein has emerged as an important therapeutic target for cancer, the developed assay should facilitate future BRAF-related basic and clinical studies. PMID:27497007

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

    PubMed Central

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

    2013-01-01

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

  20. Changes of alternative oxidase activity, capacity and protein content in leaves of Cucumis sativus wild-type and MSC16 mutant grown under different light intensities.

    PubMed

    Florez-Sarasa, Igor; Ostaszewska, Monika; Galle, Alexander; Flexas, Jaume; Rychter, Anna M; Ribas-Carbo, Miquel

    2009-12-01

    In vitro studies demonstrated that alternative oxidase (AOX) is biochemically regulated by a sulfhydryl-disulfide system, interaction with alpha-ketoacids, ubiquinone pool redox state and protein content among others. However, there is still scarce information about the in vivo regulation of the AOX. Cucumis sativus wild-type (WT) and MSC16 mutant plants were grown under two different light intensities and were used to analyze the relationship between the amount of leaf AOX protein and its in vivo capacity and activity at night and day periods. WT and MSC16 plants presented lower total respiration (V(t)), cytochrome oxidase pathway (COP) activity (v(cyt)) and alternative oxidase pathway (AOP) activity (v(alt)) when grown at low light (LL), although growth light intensity did not change the amount of cytochrome oxidase (COX) nor AOX protein. Changes of v(cyt) related to growing light conditions suggested a substrate availability and energy demand control. On the other hand, the total amount of AOX protein present in the tissue does not play a role in the regulation neither of the capacity nor of the activity of the AOP in vivo. Soluble carbohydrates were directly related to the activity of the AOP. However, although differences in soluble sugar contents mostly regulate the capacity of the AOP at different growth light intensities, additional regulatory mechanisms are necessary to fully explain the observed results.

  1. Urokinase receptor-deficient mice mount an innate immune response to and clarify respiratory viruses as efficiently as wild-type mice.

    PubMed

    Ramos, Manuel; Lao, Yolanda; Eguiluz, César; Del Val, Margarita; Martínez, Isidoro

    2015-01-01

    The plasminogen activator receptor (uPAR) is required for lung infiltration by innate immune cells in respiratory bacterial infections. In order to verify if this held true for respiratory viruses, wild type (WT) and uPAR knockout (uPAR(-/-)) mice were inoculated intranasally with the human respiratory syncytial virus (HRSV) and the influenza A virus. At several days post-infection (dpi), viral titers in the lungs were determined while cell infiltrates in the bronchoalveolar lavage (BAL) were analyzed by flow cytometry. In the case of influenza A, body weight loss and mortality were also monitored. Only minor differences were observed between infected WT and uPAR(-/-) mice, primarily in influenza virus replication and pathology. These results indicate that uPAR does not play a major role in limiting virus replication or in orchestrating the innate immune response against HRSV or influenza infections in mice. This suggests that there are fundamental differences in the immune control of the viral infections studied here and those caused by bacteria. PMID:26115163

  2. Urokinase receptor-deficient mice mount an innate immune response to and clarify respiratory viruses as efficiently as wild-type mice

    PubMed Central

    Ramos, Manuel; Lao, Yolanda; Eguiluz, César; Del Val, Margarita; Martínez, Isidoro

    2015-01-01

    The plasminogen activator receptor (uPAR) is required for lung infiltration by innate immune cells in respiratory bacterial infections. In order to verify if this held true for respiratory viruses, wild type (WT) and uPAR knockout (uPAR−/−) mice were inoculated intranasally with the human respiratory syncytial virus (HRSV) and the influenza A virus. At several days post-infection (dpi), viral titers in the lungs were determined while cell infiltrates in the bronchoalveolar lavage (BAL) were analyzed by flow cytometry. In the case of influenza A, body weight loss and mortality were also monitored. Only minor differences were observed between infected WT and uPAR−/− mice, primarily in influenza virus replication and pathology. These results indicate that uPAR does not play a major role in limiting virus replication or in orchestrating the innate immune response against HRSV or influenza infections in mice. This suggests that there are fundamental differences in the immune control of the viral infections studied here and those caused by bacteria. PMID:26115163

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

    PubMed

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

    2013-10-01

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

  4. Changes in polyphenol and sugar concentrations in wild type and genetically modified Nicotiana langsdorffii Weinmann in response to water and heat stress.

    PubMed

    Ancillotti, Claudia; Bogani, Patrizia; Biricolti, Stefano; Calistri, Elisa; Checchini, Leonardo; Ciofi, Lorenzo; Gonnelli, Cristina; Del Bubba, Massimo

    2015-12-01

    In this study wild type Nicotiana langsdorffii plants were genetically transformed by the insertion of the rat gene (gr) encoding the glucocorticoid receptor or the rolC gene and exposed to water and heat stress. Water stress was induced for 15 days by adding 20% PEG 6000 in the growth medium, whereas the heat treatment was performed at 50 °C for 2 h, after that a re-growing capability study was carried out. The plant response to stress was investigated by determining electrolyte leakage, dry weight biomass production and water content. These data were evaluated in relation to antiradical activity and concentrations of total polyphenols, selected phenolic compounds and some soluble sugars, as biochemical indicators of metabolic changes due to gene insertion and/or stress treatments. As regards the water stress, the measured physiological parameters evidenced an increasing stress level in the order rolC < gr < WT plants (e.g. about 100% and 50% electrolyte leakage increase in WT and gr samples, respectively) and complied with the biochemical pattern, which consisted in a general decrease of antiradical activity and phenolics, together with an increase in sugars. As regard heat stress, electrolyte leakage data were only in partial agreement with the re-growing capability study. In fact, according to this latter evaluation, gr was the genotype less affected by the heat shock. In this regard, sugars and especially phenolic compounds are informative of the long-term effects due to heat shock treatment.

  5. Analysis of cuticular wax constituents and genes that contribute to the formation of 'glossy Newhall', a spontaneous bud mutant from the wild-type 'Newhall' navel orange.

    PubMed

    Liu, Dechun; Yang, Li; Zheng, Qiong; Wang, Yuechen; Wang, Minli; Zhuang, Xia; Wu, Qi; Liu, Chuanfu; Liu, Shanbei; Liu, Yong

    2015-08-01

    Navel orange (Citrus sinensis [L.] Osbeck) fruit surfaces contain substantial quantities of cuticular waxes, which have important eco-physiological roles, such as water retention and pathogen defense. The wax constituents of ripe navel orange have been studied in various reports, while the wax changes occurring during fruit development and the molecular mechanism underlying their biosynthesis/export have not been investigated. Recently, we reported a spontaneous bud mutant from the wild-type (WT) 'Newhall' Navel orange. This mutant displayed unusual glossy fruit peels and was named 'glossy Newhall' (MT). In this study, we compared the developmental profiles of the epicuticular and intracuticular waxes on the WT and MT fruit surfaces. The formation of epicuticular wax crystals on the navel orange surface was shown to be dependent on the accumulation of high amounts of aliphatic wax components with trace amounts of terpenoids. In sharp contrast, the underlying intracuticular wax layers have relatively low concentrations of aliphatic wax components but high concentrations of cyclic wax compounds, especially terpenoids at the late fruit developmental stages. Our work also showed that many genes that are involved in wax biosynthesis and export pathways were down-regulated in MT fruit peels, leading to a decrease in aliphatic wax component amounts and the loss of epicuticular wax crystals, ultimately causing the glossy phenotype of MT fruits.

  6. Genome Sequence of SG33 Strain and Recombination between Wild-Type and Vaccine Myxoma Viruses

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2014-08-15

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

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

    PubMed

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

    2016-01-01

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

  9. Adeno-Associated Virus Enhances Wild-Type and Oncolytic Adenovirus Spread

    PubMed Central

    Laborda, Eduardo; Puig-Saus, Cristina; Cascalló, Manel; Chillón, Miguel

    2013-01-01

    Abstract The contamination of adenovirus (Ad) stocks with adeno-associated viruses (AAV) is usually unnoticed, and it has been associated with lower Ad yields upon large-scale production. During Ad propagation, AAV contamination needs to be detected routinely by polymerase chain reaction without symptomatic suspicion. In this study, we describe that the coinfection of either Ad wild type 5 or oncolytic Ad with AAV results in a large-plaque phenotype associated with an accelerated release of Ad from coinfected cells. This accelerated release was accompanied with the expected decrease in Ad yields in two out of three cell lines tested. Despite this lower Ad yield, coinfection with AAV accelerated cell death and enhanced the cytotoxicity mediated by Ad propagation. Intratumoral coinjection of Ad and AAV in two xenograft tumor models improved antitumor activity and mouse survival. Therefore, we conclude that accidental or intentional AAV coinfection has important implications for Ad-mediated virotherapy. PMID:24020980

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

    PubMed

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

    2010-02-01

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

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

    PubMed

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

    2010-02-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  14. Organophosphonate Utilization by the Wild-Type Strain of Penicillium notatum

    PubMed Central

    Bujacz, B.; Wieczorek, P.; Krzysko-Lupicka, T.; Golab, Z.; Lejczak, B.; Kavfarski, P.

    1995-01-01

    We studied the biodegradation of compounds containing phosphorus-to-carbon bonds by using a wild-type strain of Penicillium notatum. The substrate specificity of this strain was studied, and we found that it is able to utilize structurally diverse organophosphonates as sole sources of phosphorus. This ability seems to be inducible, as indicated by the presence of a lag phase during growth. A popular herbicide, glyphosate, inhibited fungal growth, but it was also degraded by the fungus if it was applied in sublethal doses. This indicates that P. notatum may play an important role in biodegradation of organophosphonates. The strain which we used did not metabolize any of the phosphonates which we tested when they were used as sole carbon or nitrogen sources. PMID:16535094

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

    PubMed

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

    2016-02-19

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

  16. Comparison of growth and pubertal progression in wild type female rats with different bedding types

    PubMed Central

    Kang, Byung Ho; Kim, Shin-Hee; Jung, Kyung A; Kim, So Youn; Chung, Sung-Hoon; Park, Young Shil; Yoon, Kyung Lim

    2015-01-01

    Purpose Endocrine-disrupting chemicals interfere with the endocrine system and therefore affect growth and pubertal progression. The study aim was to compare the growth and pubertal progression in wild-type female rats with different bedding types. Methods Twenty 5-week-old female wild-type Sprague Dawley rats were randomly assigned to two groups with different bedding types: one group received wood shaving bedding, while a second group received corncob bedding. We determined crown-rump length and body weight as anthropometric measurements and assessed the serum growth hormone (GH) and estradiol levels. The gh1 mRNA expression levels were compared using quantitative real time transcription polymerase chain reaction. The estrous cycle was evaluated by vaginal smear. Results The anthropometric measurements were not significantly different between the two groups. The mean relative expression of the gh1 gene was lower in the corncob bedding group than that in the wood shaving group (P=0.768). Meanwhile serum GH and estradiol were increased in the wood shaving bedding group; however this difference was not statistically significant. The time to first estrus and the length of the estrous cycle were increased in the corncob bedding group; the proportion of normal estrous cycles was also decreased. These findings indicate irregularities in the estrous cycle. Conclusion Endocrine-disrupting chemicals in corncob bedding might be associated with time to first estrus and length of the estrous cycle. Therefore, the type of bedding should be considered as a factor affecting pubertal progression in rodents. PMID:25883928

  17. An Ultra-Violet Tolerant Wild-Type Strain of Melanin-Producing Bacillus thuringiensis

    PubMed Central

    Sansinenea, Estibaliz; Salazar, Francisco; Ramirez, Melanie; Ortiz, Aurelio

    2015-01-01

    Background: Bacillus thuringiensis is the most successful biological control agent used in agriculture, forestry and mosquito control. However, the insecticidal activity of the B. thuringiensis formulation is not very stable and rapidly loses its biological activity under field conditions, due to the ultraviolet radiation in sunlight. Melanin is known to absorb radiation therefore photo protection of B. thuringiensis based on melanin has been extensively studied. Objectives: The aim of this study was to find a wild type strain of naturally melanin-producing B. thuringiensis to avoid any mutation or manipulation that can affect the Cry protein content. Materials and Methods: Bacillus thuringiensis strains were isolated from soils of different States of Mexico and pigment extraction was followed by lowering the pH to 2 using 1N HCl. Pigment was characterized by some chemical tests based on its solubility, bleaching by H2O2 and flocculation with FeCl3, and using an Infrared (IR) spectrum. Ultraviolet (UV) irradiation experiment was performed to probe the melanin efficacy. Results: ELI52 strain of B. thuringiensis was confirmed to naturally produce melanin. The Cry protein analysis suggested that ELI52 is probably a B. thuringiensis subsp. israelensis strain with toxic activity against the Diptera order of insects. Ultra Violet protection efficacy of melanin was probed counting total viable colonies after UV radiation and comparing the results with the non-producing melanin strain L-DOPA (L-3, 4-dihydroxyphenylalanine) was also detected in the culture. ELI52 strain showed an antagonistic effect over some common bacteria from the environment. Conclusions: ELI52 wild-type strain of B. thuringiensis is a good bio-insecticide that produces melanin with UV-resistance that is probably toxic against the Diptera order of insects and can inhibit the growth of other environmental bacteria. PMID:26421136

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

    PubMed

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

    2016-07-15

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

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2009-11-01

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

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

    PubMed Central

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

    1999-01-01

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

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

    PubMed

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

    2015-07-13

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

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

    PubMed

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

    2015-01-01

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

  4. Identification of a new androgen receptor (AR) co-regulator BUD31 and related peptides to suppress wild-type and mutated AR-mediated prostate cancer growth via peptide screening and X-ray structure analysis

    PubMed Central

    Hsu, Cheng-Lung; Liu, Jai-Shin; Wu, Po-Long; Guan, Hong-Hsiang; Chen, Yuh-Ling; Lin, An-Chi; Ting, Huei-Ju; Pang, See-Tong; Yeh, Shauh-Der; Ma, Wen-Lung; Chen, Chung-Jung; Wu, Wen-Guey; Chang, Chawnshang

    2014-01-01

    Treatment with individual anti-androgens is associated with the development of hot-spot mutations in the androgen receptor (AR), including T877A (hydroxyflutamide [HF]) and W741(C/L) (bicalutamide [CDX]). Here, we found that anti-androgens bound mt-ARs (HF-T877A-AR-LBD and CDX-W741L-AR-LBD) have similar binary structure to the 5α-dihydrotestosterone (DHT) bound wild type (wt) AR (DHT-wt-AR-LBD). Phage display revealed that these ARs bound to similar peptides, including BUD31, containing an Fxx(F/H/L/W/Y)Y motif cluster with Tyr in the +5 position. Structural analyses of the AR-LBD-BUD31 complex at 2.1 Å resolution revealed formation of an extra hydrogen bond between the Tyr+5 residue of the peptide and Gln733 of the AR AF2 domain, suggesting that peptides with Fxx(F/H/L/W/Y)Y motifs can interact with wt or mutated ARs. Functional studies showed that BUD31-related peptides suppressed transactivation of both DHT-wt-AR and HF-T877A-AR by interrupting AR N- and C-terminal interactions, thereby inhibiting wt and mutant AR-mediated prostate cancer cell growth. Collectively, these results suggest the combination of peptide screening and X-ray structure analysis as a new strategy for developing anti-androgens that simultaneously suppress both wt and mutated AR function. PMID:25091737

  5. Specific in situ hepatitis B viral double mutation (HBVDM) detection in urine with 60 copies ml(-1) analytical sensitivity in a background of 250-fold wild type without DNA isolation and amplification.

    PubMed

    Kirimli, Ceyhun E; Shih, Wei-Heng; Shih, Wan Y

    2015-03-01

    We have examined in situ detection of hepatitis B virus 1762T/1764A double mutation (HBVDM) in urine using a (Pb(Mg(1/3)Nb(2/3))O3)(0.65)(PbTiO3)(0.35) (PMN-PT) piezoelectric plate sensor (PEPS) coated with a 16-nucleotide (nt) probe DNA (pDNA) complementary to the HBVDM. The in situ mutation (MT) detection was carried out in a flow with the PEPS vertically situated at the center of the flow in a background of wild type (WT). For validation, this detection was followed by detection in the mixture of MT fluorescent reporter microspheres (FRMs) (MT FRMs) and WT FRMs that emitted different fluorescence colours and were designed to specifically bind to MT and WT, respectively. At 30 °C and 4 ml min(-1), a PEPS was shown to specifically detect HBVDM in situ with 60 copies ml(-1) analytical sensitivity in a background of clinically-relevant 250-fold more WT in 30 min without DNA isolation, amplification, or labelling as validated by the visualization of the captured MT FRMs and WT FRMs following FRM detection where the captured MT FRMs outnumbered the WT FRMs by a factor of 5 to 1. PMID:25599103

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

    PubMed

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

    2015-11-24

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

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

    PubMed Central

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

    2015-01-01

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

  8. 13C NMR and fluorescence analysis of tryptophan dynamics in wild-type and two single-Trp variants of Escherichia coli thioredoxin.

    PubMed Central

    Kemple, M D; Yuan, P; Nollet, K E; Fuchs, J A; Silva, N; Prendergast, F G

    1994-01-01

    The rotational motion of tryptophan side chains in oxidized and reduced wild-type (WT) Escherichia coli thioredoxin and in two single-tryptophan variants of E. coli thioredoxin was studied in solution in the temperature range 20-50 degrees C from 13C-NMR relaxation rate measurements at 75.4 and 125.7 MHz and at 20 degrees C from steady-state and time-resolved trp fluorescence anisotropy measurements. Tryptophan enriched with 13C at the delta 1 and epsilon 3 sites of the indole ring was incorporated into WT thioredoxin and into two single-trp mutants, W31F and W28F, in which trp-28 or trp-31 of WT thioredoxin was replaced, respectively, with phenylalanine. The NMR relaxation data were interpreted using the Lipari and Szabo "model-free" approach (G. Lipari and A. Szabo. 1982. J. Amer. Chem. Soc. 104:4546-4559) with trp steady-state anisotropy data included for the variants at 20 degrees C. Values for the correlation time for the overall rotational motion (tau m) from NMR of oxidized and reduced WT thioredoxin at 35 degrees C agree well with those given by Stone et al. (Stone, M. J., K. Chandrasekhar, A. Holmgren, P. E. Wright, and H. J. Dyson. 1993. Biochemistry. 32:426-435) from 15N NMR relaxation rates, and the dependence of tau m on viscosity and temperature was in accord with the Stokes-Einstein relationship. Order parameters (S2) near 1 were obtained for the trp side chains in the WT proteins even at 50 degrees C. A slight increase in the amplitude of motion (decrease in S2) of trp-31, which is near the protein surface, but not of trp-28, which is partially buried in the protein matrix, was observed in reduced relative to oxidized WT thioredoxin. For trp-28 in W31F, order parameters near 1 (S2 > or = 0.8) at 20 degrees C were found, whereas trp-31 in W28F yielded the smallest order parameters (S2 approximately 0.6) of any of the cases. Analysis of time-resolved anisotropy decays in W28F and W31F yielded S2 values in good agreement with NMR, but gave tau m values

  9. Effects of temperature and growth hormone on individual growth trajectories of wild-type and transgenic coho salmon Oncorhynchus kisutch.

    PubMed

    Lõhmus, M; Björklund, M; Sundström, L F; Devlin, R H

    2010-02-01

    In this study, individual growth patterns of wild-type and growth-enhanced coho salmon Oncorhynchus kisutch at 8, 12 and 16 degrees C water temperature were followed. Despite large differences among individuals in growth rates, there was generally little variation in the shape of the growth curves among O. kisutch individuals of both genotypes and at all temperatures. Typically, individuals that were relatively large initially were also relatively large at the end of the growth period. The limitation in variation was more pronounced in the growth-enhanced O. kisutch than in the wild type, where the relative size of some individuals reared at 12 and 8 degrees C changed by the end of the trial. As a warmer temperature seems to decrease the plasticity of growth trajectories in wild-type fish, it is possible that global warming will influence the ability of wild fish to adapt their growth to changing conditions.

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

    PubMed

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

    2015-09-01

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

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

    PubMed

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

    2016-07-25

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

  12. Enigmatic in vivo iduronate-2-sulfatase (IDS) mutant transcript correction to wild-type in Hunter syndrome.

    PubMed

    Lualdi, Susanna; Tappino, Barbara; Di Duca, Marco; Dardis, Andrea; Anderson, Christopher J; Biassoni, Roberto; Thompson, Peter W; Corsolini, Fabio; Di Rocco, Maja; Bembi, Bruno; Regis, Stefano; Cooper, David N; Filocamo, Mirella

    2010-04-01

    Sequence analysis of the X-linked iduronate-2-sulfatase (IDS) gene in two Hunter syndrome patients revealed a lack of concordance between IDS genomic DNA and cDNA. These individuals were found to be hemizygous respectively for a nonsense mutation [c.22C>T;p.R8X] and a frameshift micro-insertion [c.10insT;p.P4Sfs] in their genomic DNA. However, both wild-type and mutant IDS sequences were evident upon cDNA analysis. Similar discrepant results were also obtained in a third unrelated patient carrying the same p.R8X mutation. Since both p.R8X mutations were inherited from carrier mothers, somatic mosaicism could be excluded. Although the presence of wild-type IDSmRNA-transcripts was confirmed in all three patients by restriction enzyme digestion, clone sequencing, pyrosequencing and single nucleotide primer extension (SNuPE), no wild-type IDS genomic sequence was detectable. The relative abundance of wild-type and mutation-bearing IDS-transcripts in different tissues was quantified by SNuPE. Although IDS transcript levels, as measured by real-time PCR, were reduced (51-71% normal) in these patients, some wild-type IDS protein was detectable by western blotting. Various possible explanations for these unprecedented findings (e.g. accidental contamination, artefactual in vitro nucleotide misincorporation, malsegregation of an extra maternal X-chromosome) were explored and experimentally excluded. PCR-based discriminant assay and segregation analysis of a linked IDS polymorphism (rs1141608) also served to exclude the presence of IDS cDNA derived from the maternal wild-type chromosome. Although it remains to be formally demonstrated by direct experimentation, the intriguing possibility arises that we have observed the in vivo correction of heritable gene lesions at the RNA level operating via a correction mechanism akin to RNA-editing. (c) 2010 Wiley-Liss, Inc.

  13. Gene expression patterns in the hippocampus during the development and aging of Glud1 (Glutamate Dehydrogenase 1) transgenic and wild type mice

    PubMed Central

    2014-01-01

    Background Extraneuronal levels of the neurotransmitter glutamate in brain rise during aging. This is thought to lead to synaptic dysfunction and neuronal injury or death. To study the effects of glutamate hyperactivity in brain, we created transgenic (Tg) mice in which the gene for glutamate dehydrogenase (Glud1) is over-expressed in neurons and in which such overexpression leads to excess synaptic release of glutamate. In this study, we analyzed whole genome expression in the hippocampus, a region important for learning and memory, of 10 day to 20 month old Glud1 and wild type (wt) mice. Results During development, maturation and aging, both Tg and wt exhibited decreases in the expression of genes related to neurogenesis, neuronal migration, growth, and process elongation, and increases in genes related to neuro-inflammation, voltage-gated channel activity, and regulation of synaptic transmission. Categories of genes that were differentially expressed in Tg vs. wt during development were: synaptic function, cytoskeleton, protein ubiquitination, and mitochondria; and, those differentially expressed during aging were: synaptic function, vesicle transport, calcium signaling, protein kinase activity, cytoskeleton, neuron projection, mitochondria, and protein ubiquitination. Overall, the effects of Glud1 overexpression on the hippocampus transcriptome were greater in the mature and aged than the young. Conclusions Glutamate hyperactivity caused gene expression changes in the hippocampus at all ages. Some of these changes may result in premature brain aging. The identification of these genomic expression differences is important in understanding the effects of glutamate dysregulation on neuronal function during aging or in neurodegenerative diseases. PMID:24593767

  14. Comparing Gene Expression during Cadmium Uptake and Distribution: Untreated versus Oral Cd-Treated Wild-Type and ZIP14 Knockout Mice

    PubMed Central

    Jorge-Nebert, Lucia F.; Gálvez-Peralta, Marina; Landero Figueroa, Julio; Somarathna, Maheshika; Hojyo, Shintaro; Fukada, Toshiyuki; Nebert, Daniel W.

    2015-01-01

    The nonessential metal cadmium (Cd) is toxic only after entering the cell. Proteins possibly relevant to intracellular Cd accumulation include the divalent metal transporter-1 (DMT1) and all 14 zinc-like iron-like protein (ZIP) importers, 10 zinc transporter (ZnT) exporters, and metallothionein chaperones MT1 and MT2. Comparing oral Cd-treated ZIP14 knockout (KO) with wild-type (WT) mice, we predicted Cd uptake and distribution would be diminished in the KO—because ZIP14 is very highly expressed in GI tract and liver; this was indeed observed for Cd content in liver. However, the reverse was found in kidney and lung from 6 or 12 h through 10 days of Cd exposure; at these times, Cd accumulation was unexpectedly greater in KO than WT mice; mRNA levels of the 27 above-mentioned genes were thus examined in proximal small intestine (PSI) versus kidney to see if these paradoxical effects could be explained by substantial alterations in any of the other 26 genes. PSI genes highly expressed in untreated WT animals included seven ZIP and five ZnT transporters, DMT1, MT1, and MT2; kidney genes included 11 ZIP and 7 ZnT transporters, DMT1, MT1, and MT2. Over 10 days of oral Cd, a bimodal response was seen for Cd content in PSI and for various mRNAs; initially, acute effects caused by the toxic metal; subsequently, the up- or down-regulation of important genes presumably to combat the sustained adversity. These data underscore the complex interplay between the gastrointestinal tract and renal proteins that might be relevant to Cd uptake and distribution in animals exposed to oral Cd. PMID:25294218

  15. Reactive oxygen species- and nitric oxide-mediated lung inflammation and mitochondrial dysfunction in wild-type and iNOS-deficient mice exposed to diesel exhaust particles.

    PubMed

    Zhao, Hongwen; Ma, Joseph K; Barger, Mark W; Mercer, Robert R; Millecchia, Lyndell; Schwegler-Berry, Diane; Castranova, Vince; Ma, Jane Y

    2009-01-01

    Pulmonary responses to diesel exhaust particles (DEP) exposure are mediated through enhanced production of reactive oxygen species (ROS) and nitric oxide (NO) by alveolar macrophages (AM). The current study examined the differential roles of ROS and NO in DEP-induced lung injury using C57B/6J wild-type (WT) and inducible NO synthase knockout (iNOS KO) mice. Mice exposed by pharyngeal aspiration to DEP or carbon black particles (CB) (35 mg/kg) showed an inflammatory profile that included neutrophil infiltration, increased lactate dehydrogenase (LDH) activity, and elevated albumin content in bronchoalveolar lavage fluid (BALF) at 1, 3, and 7 d postexposure. The organic extract of DEP (DEPE) did not induce an inflammatory response. Comparing WT to iNOS KO mice, the results show that NO enhanced DEP-induced neutrophils infiltration and plasma albumin content in BALF and upregulated the production of the pro-inflammatory cytokine interleukin 12 (IL-12) by AM. DEP-exposed AM from iNOS KO mice displayed diminished production of IL-12 and, in response to ex vivo lipopolysaccharide (LPS) challenge, decreased production of IL-12 but increased production of IL-10 when compared to cells from WT mice. DEP, CB, but not DEPE, induced DNA damage and mitochondria dysfunction in AM, however, that is independent of cellular production of NO. These results demonstrate that DEP-induced immune/inflammatory responses in mice are regulated by both ROS- and NO-mediated pathways. NO did not affect ROS-mediated mitochondrial dysfunction and DNA damage but upregulated IL-12 and provided a counterbalance to the ROS-mediated adaptive stress response that downregulates IL-12 and upregulates IL-10. PMID:19267316

  16. Age-Related Changes in Pre- and Postsynaptic Partners of the Cholinergic C-Boutons in Wild-Type and SOD1G93A Lumbar Motoneurons

    PubMed Central

    Milan, Léa; Courtand, Gilles; Cardoit, Laura; Masmejean, Frédérique; Barrière, Grégory; Cazalets, Jean-René; Garret, Maurice; Bertrand, Sandrine S.

    2015-01-01

    Large cholinergic synaptic terminals known as C-boutons densely innervate the soma and proximal dendrites of motoneurons that are prone to neurodegeneration in amyotrophic lateral sclerosis (ALS). Studies using the Cu/Zn-superoxide dismutase (SOD1) mouse model of ALS have generated conflicting data regarding C-bouton alterations exhibited during ALS pathogenesis. In the present work, a longitudinal study combining immunohistochemistry, biochemical approaches and extra- and intra-cellular electrophysiological recordings revealed that the whole spinal cholinergic system is modified in the SOD1 mouse model of ALS compared to wild type (WT) mice as early as the second postnatal week. In WT motoneurons, both C-bouton terminals and associated M2 postsynaptic receptors presented a complex age-related dynamic that appeared completely disrupted in SOD1 motoneurons. Indeed, parallel to C-bouton morphological alterations, analysis of confocal images revealed a clustering process of M2 receptors during WT motoneuron development and maturation that was absent in SOD1 motoneurons. Our data demonstrated for the first time that the lamina X cholinergic interneurons, the neuronal source of C-boutons, are over-abundant in high lumbar segments in SOD1 mice and are subject to neurodegeneration in the SOD1 animal model. Finally, we showed that early C-bouton system alterations have no physiological impact on the cholinergic neuromodulation of newborn motoneurons. Altogether, these data suggest a complete reconfiguration of the spinal cholinergic system in SOD1 spinal networks that could be part of the compensatory mechanisms established during spinal development. PMID:26305672

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

    PubMed

    Markvartová, V; Vozeh, F

    2008-01-01

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

  18. Establishment of three cell lines from Chinese giant salamander and their sensitivities to the wild-type and recombinant ranavirus.

    PubMed

    Yuan, Jiang-Di; Chen, Zhong-Yuan; Huang, Xing; Gao, Xiao-Chan; Zhang, Qi-Ya

    2015-06-12

    Known as lethal pathogens, Ranaviruses have been identified in diseased fish, amphibians (including Chinese giant salamander Andrias davidianus, the world's largest amphibian) and reptiles, causing organ necrosis and systemic hemorrhage. Here, three Chinese giant salamander cell lines, thymus cell line (GSTC), spleen cell line (GSSC) and kidney cell line (GSKC) were initially established. Their sensitivities to ranaviruses, wild-type Andrias davidianus ranavirus (ADRV) and recombinant Rana grylio virus carrying EGFP gene (rRGV-EGFP) were tested. Temporal transcription pattern of ranavirus major capsid protein (MCP), fluorescence and electron microscopy observations showed that both the wild-type and recombinant ranavirus could replicate in the cell lines.

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

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

  20. Trifluoperazine rescues human dopaminergic cells from wild-type α-synuclein-induced toxicity.

    PubMed

    Höllerhage, Matthias; Goebel, Joachim N; de Andrade, Anderson; Hildebrandt, Tobias; Dolga, Amalia; Culmsee, Carsten; Oertel, Wolfgang H; Hengerer, Bastian; Höglinger, Günter U

    2014-07-01

    Parkinson's disease (PD) is the most frequent neurodegenerative movement disorder. Presently, there is no causal therapy available to slow down or halt disease progression. The presynaptic protein alpha-synuclein aggregates to form intraneuronal Lewy bodies in PD. It is generally believed that intermediates on the way from monomers to the large aggregates would mediate neurotoxicity, but the precise species and mechanism responsible for neuronal death are controversially debated. To study alpha-synuclein-mediated toxicity, we developed a new model in which moderate overexpression of wild-type alpha-synuclein led to gradual death of human postmitotic dopaminergic neurons. In accordance with findings in postmortem PD brains, small oligomeric species occurred and the autophagic flux was impaired in our model. The phenothiazine neuroleptic trifluoperazine, an activator of macroautophagy, selectively reduced one particular alpha-synuclein species and rescued cells. Inversely, blocking of autophagy led to an accumulation of this oligomeric species and increased cell death. These data show that activation of autophagy is a promising approach to protect against alpha-synuclein pathology and likely acts by targeting one specific alpha-synuclein species.

  1. Cellular Distribution and Functions of Wild-Type and Constitutively Activated Dictyostelium PakBV⃞

    PubMed Central

    de la Roche, Marc; Mahasneh, Amjad; Lee, Sheu-Fen; Rivero, Francisco; Côté, Graham P.

    2005-01-01

    Dictyostelium PakB, previously termed myosin I heavy chain kinase, is a member of the p21-activated kinase (PAK) family. Two-hybrid assays showed that PakB interacts with Dictyostelium Rac1a/b/c, RacA (a RhoBTB protein), RacB, RacC, and RacF1. Wild-type PakB displayed a cytosolic distribution with a modest enrichment at the leading edge of migrating cells and at macropinocytic and phagocytic cups, sites consistent with a role in activating myosin I. PakB fused at the N terminus to green fluorescent protein was proteolyzed in cells, resulting in removal of the catalytic domain. C-terminal truncated PakB and activated PakB lacking the p21-binding domain strongly localized to the cell cortex, to macropinocytic cups, to the posterior of migrating cells, and to the cleavage furrow of dividing cells. These data indicate that in its open, active state, the N terminus of PakB forms a tight association with cortical actin filaments. PakB-null cells displayed no significant behavioral defects, but cells expressing activated PakB were unable to complete cytokinesis when grown in suspension and exhibited increased rates of phagocytosis and pinocytosis. PMID:15509655

  2. Probing flagellar promoter occupancy in wild-type and mutant Caulobacter crescentus by chromatin immunoprecipitation.

    PubMed

    Davis, Nicole J; Viollier, Patrick H

    2011-06-01

    In the asymmetric predivisional cell of Caulobacter crescentus, TipF and TipN mark the cellular pole for future flagellar development. TipF is essential for motility and contains a cyclic-di-GMP phosphodiesterase-like (EAL) domain that is necessary for proper function. TipN is localized to the flagellar pole before TipF and is essential for the proper placement of the flagellum in C. crescentus. Using β-galactosidase promoter-probe assays and quantitative chromatin immunoprecipitation, we investigated the influence of the C. crescentus flagellar assembly regulator TipF on flagellar gene transcription. We compared the transcriptional activity of class II-fliF-lacZ, class III-flgE-lacZ, and class IV-fljL-lacZ fusions in a ΔtipF mutant with that of other flagellar mutants and the wild-type strain. We subsequently verified the in vivo occupancy of the fliF, flgE, and fljL flagellar promoters by the flagellar regulators CtrA, FlbD, and FliX in addition to RNA polymerase. We deduce that TipF contributes to proper expression of flagellar genes in C. crescentus by acting both within and outside of the canonical flagellar gene expression hierarchy.

  3. Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin.

    PubMed

    Fiaschi, Luigi; Di Palo, Benedetta; Scarselli, Maria; Pozzi, Clarissa; Tomaszewski, Kelly; Galletti, Bruno; Nardi-Dei, Vincenzo; Arcidiacono, Letizia; Mishra, Ravi P N; Mori, Elena; Pallaoro, Michele; Falugi, Fabiana; Torre, Antonina; Fontana, Maria Rita; Soriani, Marco; Bubeck Wardenburg, Juliane; Grandi, Guido; Rappuoli, Rino; Ferlenghi, Ilaria; Bagnoli, Fabio

    2016-06-01

    Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus.

  4. Wild-Type Transthyretin Cardiac Amyloidosis: Novel Insights From Advanced Imaging.

    PubMed

    Narotsky, David L; Castano, Adam; Weinsaft, Jonathan W; Bokhari, Sabahat; Maurer, Mathew S

    2016-09-01

    Amyloidosis is caused by extracellular deposition of abnormal protein fibrils, resulting in destruction of tissue architecture and impairment of organ function. The most common forms of systemic amyloidosis are light-chain and transthyretin-related (ATTR). ATTR can result from an autosomal dominant hereditary transmission of mutated genes in the transthyretin or from a wild-type form of disease (ATTRwt), previously known as senile cardiac amyloidosis. With the aging of the worldwide population, ATTRwt will emerge as the most common type of cardiac amyloidosis that clinicians encounter. Diagnosis of systemic amyloidosis is often delayed, either because of the false assumption that it is a rare disease, or because of misdiagnosis as a result of mistaking it with other conditions. Clinicians must integrate clinical clues from history, physical examination, and common diagnostic tests to raise suspicion for ATTRwt. The historical gold standard for diagnosis of cardiac amyloid is endomyocardial biopsy analysis with pathological distinction of precursor protein type, but this method often results in delayed diagnosis because of the limited availability of expertise to perform and interpret the endomyocardial biopsy specimen. Emerging noninvasive imaging modalities provide easier, accurate screening for ATTRwt. These modalities include advanced echocardiography, using strain imaging and the myocardial contraction fraction; nuclear scintigraphy, which can differentiate between ATTR and light-chain cardiac amyloid; and cardiac magnetic resonance imaging, using extracellular volume measurement, late gadolinium enhancement, and distinct T1 mapping. These novel approaches reveal insights into the prevalence, clinical course, morphological effects, and prognosis of ATTRwt. PMID:27568874

  5. Molecular analysis of mutant and wild type alcohol dehydrogenase alleles from Drosophila

    SciTech Connect

    Batzer, M.A.

    1988-01-01

    Wild type alcohol dehydrogenase polypeptides (ADH) from Drosophila melanogaster transformants were examined using western blots and polyclonal antiserum specific for Drosophila melanogaster ADH. Mutants induced in Drosophila spermatozoa at the alcohol dehydrogenase (Adh) locus using X-rays, 1-ethyl-1-nitrosourea (ENU) or ethyl methanesulfonate (EMS) were characterized using genetic complementation tests, western blots, Southern blots, northern blots and enzymatic amplification of the Adh locus. Genetic complementation tests showed that 22/30 X-ray-induced mutants, and 3/13 ENU and EMS induced mutants were multi-locus deficiencies. Western blot analysis of the intragenic mutations showed that 4/7 X-ray-induced mutants produced detectable polypeptides, one of which was normal in molecular weight and charge. In contrast 8/10 intragenic ENU and EMS induced mutants produced normal polypeptides. Southern blot analysis showed that 5/7 intragenic X-ray induced mutants and all 10 of the intragenic ENU and EMS induced mutants were normal with respect to the alleles they were derived from.

  6. Quantification of gait parameters in freely walking wild type and sensory deprived Drosophila melanogaster

    PubMed Central

    Mendes, César S; Bartos, Imre; Akay, Turgay; Márka, Szabolcs; Mann, Richard S

    2013-01-01

    Coordinated walking in vertebrates and multi-legged invertebrates such as Drosophila melanogaster requires a complex neural network coupled to sensory feedback. An understanding of this network will benefit from systems such as Drosophila that have the ability to genetically manipulate neural activities. However, the fly's small size makes it challenging to analyze walking in this system. In order to overcome this limitation, we developed an optical method coupled with high-speed imaging that allows the tracking and quantification of gait parameters in freely walking flies with high temporal and spatial resolution. Using this method, we present a comprehensive description of many locomotion parameters, such as gait, tarsal positioning, and intersegmental and left-right coordination for wild type fruit flies. Surprisingly, we find that inactivation of sensory neurons in the fly's legs, to block proprioceptive feedback, led to deficient step precision, but interleg coordination and the ability to execute a tripod gait were unaffected. DOI: http://dx.doi.org/10.7554/eLife.00231.001 PMID:23326642

  7. Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin

    PubMed Central

    Fiaschi, Luigi; Di Palo, Benedetta; Scarselli, Maria; Pozzi, Clarissa; Tomaszewski, Kelly; Galletti, Bruno; Nardi-Dei, Vincenzo; Arcidiacono, Letizia; Mishra, Ravi P. N.; Mori, Elena; Pallaoro, Michele; Falugi, Fabiana; Torre, Antonina; Fontana, Maria Rita; Soriani, Marco; Bubeck Wardenburg, Juliane; Grandi, Guido; Rappuoli, Rino

    2016-01-01

    Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus. PMID:27030589

  8. Wild-type p53 controls cell motility and invasion by dual regulation of MET expression

    PubMed Central

    Hwang, Chang-Il; Matoso, Andres; Corney, David C.; Flesken-Nikitin, Andrea; Körner, Stefanie; Wang, Wei; Boccaccio, Carla; Thorgeirsson, Snorri S.; Comoglio, Paolo M.; Hermeking, Heiko; Nikitin, Alexander Yu.

    2011-01-01

    Recent observations suggest that p53 mutations are responsible not only for growth of primary tumors but also for their dissemination. However, mechanisms involved in p53-mediated control of cell motility and invasion remain poorly understood. By using the primary ovarian surface epithelium cell culture, we show that conditional inactivation of p53 or expression of its mutant forms results in overexpression of MET receptor tyrosine kinase, a crucial regulator of invasive growth. At the same time, cells acquire increased MET-dependent motility and invasion. Wild-type p53 negatively regulates MET expression by two mechanisms: (i) transactivation of MET-targeting miR-34, and (ii) inhibition of SP1 binding to MET promoter. Both mechanisms are not functional in p53 absence, but mutant p53 proteins retain partial MET promoter suppression. Accordingly, MET overexpression, cell motility, and invasion are particularly high in p53-null cells. These results identify MET as a critical effector of p53 and suggest that inhibition of MET may be an effective antimetastatic approach to treat cancers with p53 mutations. These results also show that the extent of advanced cancer traits, such as invasion, may be determined by alterations in individual components of p53/MET regulatory network. PMID:21831840

  9. Evaluation of short-interfering RNAs treatment in experimental rabies due to wild-type virus.

    PubMed

    Appolinario, Camila Michele; Allendorf, Susan Dora; Peres, Marina Gea; Fonseca, Clovis Reynaldo; Vicente, Acacia Ferreira; Antunes, João Marcelo Azevedo de Paula; Pantoja, José Carlos Figueiredo; Megid, Jane

    2015-01-01

    We have evaluated the efficacy of short-interfering RNAs targeting the nucleoprotein gene and also the brain immune response in treated and non-treated infected mice. Mice were inoculated with wild-type virus, classified as dog (hv2) or vampire bat (hv3) variants and both groups were treated or left as controls. No difference was observed in the lethality rate between treated and non-treated groups, although clinical evaluation of hv2 infected mice showed differences in the severity of clinical disease (p=0.0006). Evaluation of brain immune response 5 days post-inoculation in treated hv2 group showed no difference among the analyzed genes, whereas after 10 days post-inoculation there was increased expression of 2',5'-oligoadenylate synthetase 1, tumor necrosis factor alpha, interleukin 12, interferon gamma, and C-X-C motif chemokine 10 associated with higher expression of N gene in the same period (p<0.0001). In hv2 non-treated group only higher interferon beta expression was found at day 5. The observed differences in results of the immune response genes between treated and non-treated groups is not promising as they had neither impact on mortality nor even a reduction in the expression of N gene in siRNA treated animals. This finding suggests that the use of pre-designed siRNA alone may not be useful in rabies treatment. PMID:26254692

  10. Profile of Cytokines and Chemokines Triggered by Wild-Type Strains of Rabies Virus in Mice.

    PubMed

    Appolinário, Camila Michele; Allendorf, Susan Dora; Peres, Marina Gea; Ribeiro, Bruna Devidé; Fonseca, Clóvis R; Vicente, Acácia Ferreira; Antunes, João Marcelo A de Paula; Megid, Jane

    2016-02-01

    Rabies is a lethal infectious disease that causes 55,000 human deaths per year and is transmitted by various mammalian species, such as dogs and bats. The host immune response is essential for avoiding viral progression and promoting viral clearance. Cytokines and chemokines are crucial in the development of an immediate antiviral response; the rabies virus (RABV) attempts to evade this immune response. The virus's capacity for evasion is correlated with its pathogenicity and the host's inflammatory response, with highly pathogenic strains being the most efficient at hijacking the host's defense mechanisms and thereby decreasing inflammation. The purpose of this study was to evaluate the expression of a set of cytokine and chemokine genes that are related to the immune response in the brains of mice inoculated intramuscularly or intracerebrally with two wild-type strains of RABV, one from dog and the other from vampire bat. The results demonstrated that the gene expression profile is intrinsic to the specific rabies variant. The prompt production of cytokines and chemokines seems to be more important than their levels of expression for surviving a rabies infection. PMID:26711511

  11. Subcellular potassium and sodium distribution in Saccharomyces cerevisiae wild-type and vacuolar mutants.

    PubMed

    Herrera, Rito; Álvarez, María C; Gelis, Samuel; Ramos, José

    2013-09-15

    Living cells accumulate potassium (K⁺) to fulfil multiple functions. It is well documented that the model yeast Saccharomyces cerevisiae grows at very different concentrations of external alkali cations and keeps high and low intracellular concentrations of K⁺ and sodium (Na⁺) respectively. However less attention has been paid to the study of the intracellular distribution of these cations. The most widely used experimental approach, plasma membrane permeabilization, produces incomplete results, since it usually considers only cytoplasm and vacuoles as compartments where the cations are present in significant amounts. By isolating and analysing the main yeast organelles, we have determined the subcellular location of K⁺ and Na⁺ in S. cerevisiae. We show that while vacuoles accumulate most of the intracellular K⁺ and Na⁺, the cytosol contains relatively low amounts, which is especially relevant in the case of Na⁺. However K⁺ concentrations in the cytosol are kept rather constant during the K⁺-starvation process and we conclude that, for that purpose, vacuolar K⁺ has to be rapidly mobilized. We also show that this intracellular distribution is altered in four different mutants with impaired vacuolar physiology. Finally, we show that both in wild-type and vacuolar mutants, nuclei contain and keep a relatively constant and important percentage of total intracellular K⁺ and Na⁺, which most probably is involved in the neutralization of negative charges.

  12. Evaluation of short-interfering RNAs treatment in experimental rabies due to wild-type virus.

    PubMed

    Appolinario, Camila Michele; Allendorf, Susan Dora; Peres, Marina Gea; Fonseca, Clovis Reynaldo; Vicente, Acacia Ferreira; Antunes, João Marcelo Azevedo de Paula; Pantoja, José Carlos Figueiredo; Megid, Jane

    2015-01-01

    We have evaluated the efficacy of short-interfering RNAs targeting the nucleoprotein gene and also the brain immune response in treated and non-treated infected mice. Mice were inoculated with wild-type virus, classified as dog (hv2) or vampire bat (hv3) variants and both groups were treated or left as controls. No difference was observed in the lethality rate between treated and non-treated groups, although clinical evaluation of hv2 infected mice showed differences in the severity of clinical disease (p=0.0006). Evaluation of brain immune response 5 days post-inoculation in treated hv2 group showed no difference among the analyzed genes, whereas after 10 days post-inoculation there was increased expression of 2',5'-oligoadenylate synthetase 1, tumor necrosis factor alpha, interleukin 12, interferon gamma, and C-X-C motif chemokine 10 associated with higher expression of N gene in the same period (p<0.0001). In hv2 non-treated group only higher interferon beta expression was found at day 5. The observed differences in results of the immune response genes between treated and non-treated groups is not promising as they had neither impact on mortality nor even a reduction in the expression of N gene in siRNA treated animals. This finding suggests that the use of pre-designed siRNA alone may not be useful in rabies treatment.

  13. Fluorescent Trimethoprim Conjugate Probes To Assess Drug Accumulation in Wild Type and Mutant Escherichia coli

    PubMed Central

    2016-01-01

    Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (ΔtolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment. PMID:27737551

  14. Intraperitoneal Infection of Wild-Type Mice with Synthetically Generated Mammalian Prion.

    PubMed

    Wang, Xinhe; McGovern, Gillian; Zhang, Yi; Wang, Fei; Zha, Liang; Jeffrey, Martin; Ma, Jiyan

    2015-07-01

    The prion hypothesis postulates that the infectious agent in transmissible spongiform encephalopathies (TSEs) is an unorthodox protein conformation based agent. Recent successes in generating mammalian prions in vitro with bacterially expressed recombinant prion protein provide strong support for the hypothesis. However, whether the pathogenic properties of synthetically generated prion (rec-Prion) recapitulate those of naturally occurring prions remains unresolved. Using end-point titration assay, we showed that the in vitro prepared rec-Prions have infectious titers of around 104 LD50/μg. In addition, intraperitoneal (i.p.) inoculation of wild-type mice with rec-Prion caused prion disease with an average survival time of 210-220 days post inoculation. Detailed pathological analyses revealed that the nature of rec-Prion induced lesions, including spongiform change, disease specific prion protein accumulation (PrP-d) and the PrP-d dissemination amongst lymphoid and peripheral nervous system tissues, the route and mechanisms of neuroinvasion were all typical of classical rodent prions. Our results revealed that, similar to naturally occurring prions, the rec-Prion has a titratable infectivity and is capable of causing prion disease via routes other than direct intra-cerebral challenge. More importantly, our results established that the rec-Prion caused disease is pathogenically and pathologically identical to naturally occurring contagious TSEs, supporting the concept that a conformationally altered protein agent is responsible for the infectivity in TSEs.

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

    PubMed Central

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

    2013-01-01

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

  16. Wild-type p53 binds to MYC promoter G-quadruplex

    PubMed Central

    Petr, Marek; Helma, Robert; Polášková, Alena; Krejčí, Aneta; Dvořáková, Zuzana; Kejnovská, Iva; Navrátilová, Lucie; Adámik, Matej; Vorlíčková, Michaela; Brázdová, Marie

    2016-01-01

    G-quadruplexes are four-stranded nucleic acid structures that are implicated in the regulation of transcription, translation and replication. Genome regions enriched in putative G-quadruplex motifs include telomeres and gene promoters. Tumour suppressor p53 plays a critical role in regulatory pathways leading to cell cycle arrest, DNA repair and apoptosis. In addition to transcriptional regulation mediated via sequence-specific DNA binding, p53 can selectively bind various non-B DNA structures. In the present study, wild-type p53 (wtp53) binding to G-quadruplex formed by MYC promoter nuclease hypersensitive element (NHE) III1 region was investigated. Wtp53 binding to MYC G-quadruplex is comparable to interaction with specific p53 consensus sequence (p53CON). Apart from the full-length wtp53, its isolated C-terminal region (aa 320–393) as well, is capable of high-affinity MYC G-quadruplex binding, suggesting its critical role in this type of interaction. Moreover, wtp53 binds to MYC promoter region containing putative G-quadruplex motif in two wtp53-expressing cell lines. The results suggest that wtp53 binding to G-quadruplexes can take part in transcriptional regulation of its target genes. PMID:27634752

  17. Ultrastructural analysis of wild type and mutant Drosophila melanogaster using helium ion microscopy.

    PubMed

    Boseman, Adam; Nowlin, Kyle; Ashraf, Sarmadia; Yang, Jijin; Lajeunesse, Dennis

    2013-08-01

    Insects have evolved numerous adaptations to survive a variety of environmental conditions. Given that the primary interface between insects and the environment is mediated through their skin or cuticle, many of these adaptations are found in extraordinary cuticle diversity both in morphology and structure. Not all of these adaptions manifest themselves in changes in the chemical composition of the cuticle but rather as elaborations of the surface structures of the cuticle. Typically the examination of these micro- and nanoscale structures has been performed using scanning electron microscopy (SEM). Typically, in order to decrease surface charging and increase resolution, an obscuring conductive layer is applied to the sample surface, but this layer limits the ability to identify nanoscale surface structures. In this paper we use a new technology, helium ion microscopy (HIM) to examine surface structures on the cuticle of wild type and mutant Drosophila. Helium ion microscopy permits high resolution imaging of biological samples without the need for coating. We compare HIM to traditional SEM and demonstrate certain advantages of this type of microscopy, with our focus being high resolution characterization of nanostructures on the cuticle of Drosophila melanogaster and potentially other biological specimens.

  18. Comparative analysis of leg and antenna development in wild-type and homeotic Drosophila melanogaster.

    PubMed

    Cummins, Mark; Pueyo, Jose I; Greig, Steve A; Couso, Juan Pablo

    2003-07-01

    The insect leg and antenna are thought to be homologous structures, evolved from a common ancestral appendage. The homeotic transformations of antenna to leg in Drosophila produced by mutation of the Hox gene Antennapedia are position-specific, such that every particular antenna structure is transformed into a specific leg counterpart. This has been taken to suggest that the developmental programmes of these two appendages are still similar. In particular, the mechanisms for the specification of a cell's position within the appendage would be identical, only their interpretation would be different and subject to homeotic gene control. Here we explore the degree of conservation between the developmental programmes of leg and antenna in Drosophila and other dipterans, in wild-type and homeotic conditions. Most of the appendage pattern-forming genes are active in both appendages, and their expression domains are partially conserved. However, the regulatory relationships and interactions between these genes are different, and in fact cells change their expression while undergoing homeotic transformation. Thus, the positional information, and the mechanisms which generate it, are not strictly conserved between leg and antenna; and homeotic genes alter the establishment of positional clues, not only their interpretation. The partial conservation of pattern-forming genes in both appendages ensures a predictable re-specification of positional clues, producing the observed positional specificity of homeotic transformations.

  19. WILD-TYPE GROSS LEUKEMIA VIRUS AND THE PATHOGENESIS OF THE GLOMERULONEPHRITIS OF NEW ZEALAND MICE

    PubMed Central

    Mellors, Robert C.; Shirai, Toshikazu; Aoki, Tadao; Huebner, Robert J.; Krawczynski, Krzysztof

    1971-01-01

    The pathogenesis of the spontaneous glomerulonephritis of NZB and (NZB x NZW) F1 hybrid mice is related at least in part to the formation of natural antibody against antigens of the G (Gross) system, and apparently to the deposition in the glomeruli of immune complexes of G natural antibody with G soluble antigen (GSA), type-specific antigen specified by wild-type Gross leukemia virus. G natural antibody and GSA are detectable in the acid-buffer eluate of the kidneys of NZB mice during the course of the glomerulonephritis. (NZB x NZW) F1 hybrid mice develop glomerulonephritis and produce GSA and free G natural antibody earlier in life than do NZB mice. The proteinuria manifestation of the gomerulonephritis of (NZB x NZW) F1 hybrid mice becomes increasingly prevalent as GSA undergoes immune elimination from the circulation. Gross leukemia virus-specified antigens together with bound immunoglobulins are located in the glomerular lesions of (NZB x NZW) F1 hybrid mice, both in the mesangium as observed in NZB mice and also in the wall of the peripheral capillary loops of the glomeruli. PMID:4924198

  20. Profile of Cytokines and Chemokines Triggered by Wild-Type Strains of Rabies Virus in Mice.

    PubMed

    Appolinário, Camila Michele; Allendorf, Susan Dora; Peres, Marina Gea; Ribeiro, Bruna Devidé; Fonseca, Clóvis R; Vicente, Acácia Ferreira; Antunes, João Marcelo A de Paula; Megid, Jane

    2016-02-01

    Rabies is a lethal infectious disease that causes 55,000 human deaths per year and is transmitted by various mammalian species, such as dogs and bats. The host immune response is essential for avoiding viral progression and promoting viral clearance. Cytokines and chemokines are crucial in the development of an immediate antiviral response; the rabies virus (RABV) attempts to evade this immune response. The virus's capacity for evasion is correlated with its pathogenicity and the host's inflammatory response, with highly pathogenic strains being the most efficient at hijacking the host's defense mechanisms and thereby decreasing inflammation. The purpose of this study was to evaluate the expression of a set of cytokine and chemokine genes that are related to the immune response in the brains of mice inoculated intramuscularly or intracerebrally with two wild-type strains of RABV, one from dog and the other from vampire bat. The results demonstrated that the gene expression profile is intrinsic to the specific rabies variant. The prompt production of cytokines and chemokines seems to be more important than their levels of expression for surviving a rabies infection.

  1. A comparative study of cytokinins in caryopsis development in the maize miniature 1 seed mutant and its wild type

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report here a comparative developmental profile of cytokinins, both total quantity and diversity of various forms, in relation to cell size, cell number and endoreduplication in developing caryopses of a cell wall invertase-deficient miniature1 (mn1) seed mutant and its wild type, Mn1, genotype. ...

  2. Responses of Wild-Type and Resistant Strains of the Hyperthermophilic Bacterium Thermotoga maritima to Chloramphenicol Challenge▿ †

    PubMed Central

    Montero, Clemente I.; Johnson, Matthew R.; Chou, Chung-Jung; Conners, Shannon B.; Geouge, Sarah G.; Tachdjian, Sabrina; Nichols, Jason D.; Kelly, Robert M.

    2007-01-01

    Transcriptomes and growth physiologies of the hyperthermophile Thermotoga maritima and an antibiotic-resistant spontaneous mutant were compared prior to and following exposure to chloramphenicol. While the wild-type response was similar to that of mesophilic bacteria, reduced susceptibility of the mutant was attributed to five mutations in 23S rRNA and phenotypic preconditioning to chloramphenicol. PMID:17557852

  3. Long-Term Treatment with Erlotinib for EGFR Wild-Type Non-Small Cell Lung Cancer: A Case Report.

    PubMed

    Polychronidou, Genovefa; Papakotoulas, Pavlos

    2013-01-01

    The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib are known to have greater efficacy in EGFR mutation-positive non-small cell lung cancer (NSCLC), although erlotinib also has activity in wild-type disease. We report the successful long-term maintenance treatment of a patient with EGFR wild-type NSCLC with gefitinib and later erlotinib. The patient (male; 44 years old; smoker) was diagnosed with EGFR wild-type NSCLC after computer tomography had revealed a mediastinal mass, and histology and mutation testing had identified the tumor as an EGFR wild-type grade 3 adenocarcinoma. The patient received multiple rounds of chemotherapy, followed by gefitinib maintenance (3 years). Later on, he received erlotinib maintenance and developed a persistent rash (grade 1/2) that lasted throughout the treatment. The patient's condition has remained stable on erlotinib for more than 5 years, with no evidence of progression. We describe the patient's disease course and treatment in the context of EGFR TKI therapy and the prognostic factors for long-term clinical outcomes of NSCLC, including the development of erlotinib-induced rash.

  4. Cytoplasmic localization of wild-type p53 in glioblastomas correlates with expression of vimentin and glial fibrillary acidic protein.

    PubMed Central

    Sembritzki, Olivier; Hagel, Christian; Lamszus, Katrin; Deppert, Wolfgang; Bohn, Wolfgang

    2002-01-01

    Cytoplasmic accumulation of wild-type p53 in tumor cells indicates that the tumor suppressor is inactive with regard to growth suppressive functions. Whether this occurs randomly during tumor development or characterizes a certain tumor cell subset is not known. Here we assayed primary glioblastomas for expression and subcellular localization of p53 and determined a correlation with expression of intermediate filament proteins characterizing glial cell development. Sixty-nine percent of the tumors were p53 positive in immunohistochemistry. A significant number of tumors (23%) accumulated wild-type p53 in the cytoplasm, which correlated with the presence of vimentin and glial fibrillary acidic protein, except for 1 case. Tumors with exclusive nuclear p53 contained none or only one of these intermediate filament proteins. In an alternative approach, tumors positive for glial fibrillary acidic protein were screened for expression of p53 and vimentin. Thirty-eight percent of these tumors showed cytoplasmic p53, and all of those also expressed vimentin. Tumors with only nuclear p53 were vimentin negative, except for 1 case. No mutation was detected in p53 exons 5 to 8 in tumors with cytoplasmic p53, suggesting that they express wild-type p53. The data indicate that a cytoplasmic accumulation of wild-type p53 in human primary glioblastomas correlates with a certain intermediate filament protein expression, suggesting that it identifies a certain subset of tumors. PMID:12084347

  5. Receptor usage and differential downregulation of CD46 by measles virus wild-type and vaccine strains.

    PubMed

    Schneider-Schaulies, J; Schnorr, J J; Brinckmann, U; Dunster, L M; Baczko, K; Liebert, U G; Schneider-Schaulies, S; ter Meulen, V

    1995-04-25

    Recently, two cell surface molecules, CD46 and moesin, have been found to be functionally associated with measles virus (MV) infectivity of cells. We investigated the receptor usage of MV wild-type, subacute sclerosing panencephalitis, and vaccine strains and their effect on the down-regulation of CD46 after infection. We found that the infection of human cell lines with all 19 MV strains tested was inhibitable with antibodies against CD46. In contrast, not all strains of MV led to the downregulation of CD46 following infection. The group of CD46 non-downregulating strains comprised four lymphotropic wild-type isolates designated AB, DF, DL, and WTF. Since the downregulation of CD46 is caused by interaction with newly synthesized MV hemagglutinin (MV-H), we tested the capability of recombinant MV-H proteins to downregulate CD46. Recombinant MV-H proteins of MV strains Edmonston, Halle, and CM led to the down-regulation of CD46, whereas those of DL and WTF did not. This observed differential downregulation by different MV strains has profound consequences, since lack of CD46 on the cell surface leads to susceptibility of cells to complement lysis. These results suggest that lymphotropic wild-type strains of MV which do not downregulate CD46 may have an advantage for replication in vivo. The relatively weak immune response against attenuated vaccine strains of MV compared with wild-type strains might be related to this phenomenon. PMID:7732009

  6. How much more would KNM-WT 15000 have grown?

    PubMed

    Ruff, Christopher B; Burgess, M Loring

    2015-03-01

    Because of its completeness, the juvenile Homo ergaster/erectus KNM-WT 15000 has played an important role in studies of the evolution of body form in Homo. Early attempts to estimate his adult body size used modern human growth models. However, more recent evidence, particularly from the dentition, suggests that he may have had a more chimpanzee-like growth trajectory. Here we re-estimate his adult stature and body mass using ontogenetic data derived from four African ape taxa: Pan troglodytes troglodytes, Pan troglodytes schweinfurthii, Pan paniscus, and Gorilla gorilla gorilla. The average percentage change in femoral and tibial lengths and femoral head breadth between individuals at the same stage of dental development as KNM-WT 15000 - eruption of M2s but not M3s - and adult individuals with fully fused long bone epiphyses, was determined. Results were then applied to KNM-WT 15000, and his adult size estimated from skeletal dimensions using modern human prediction formulae. Using this approach, adult stature best estimates of 176-180 cm and body mass best estimates of 80-83 kg were obtained. These estimates are close to those estimated directly from longitudinal changes in body length and body mass between 8 and 12 years of age in chimpanzees, the suggested chronological equivalent to KNM-WT 15000's remaining growth period. Thus, even using an African ape growth model, it is likely that KNM-WT 15000 would have attained close to 180 cm in stature (without a slight reduction for his lower cranial height) and 80 kg in body mass as an adult. Other evidence from the East African Early Pleistocene indicates that KNM-WT 15000 was not unusually large-bodied for his time period. PMID:25449954

  7. Candida albicans Als3p is required for wild-type biofilm formation on silicone elastomer surfaces

    PubMed Central

    Zhao, Xiaomin; Daniels, Karla J.; Oh, Soon-Hwan; Green, Clayton B.; Yeater, Kathleen M.; Soll, David R.; Hoyer, Lois L.

    2007-01-01

    Candida albicans ALS3 encodes a large cell-surface glycoprotein that has adhesive properties. Immunostaining of cultured C. albicans germ tubes showed that Als3p is distributed diffusely across the germ tube surface. Two-photon laser scanning microscopy of model catheter biofilms grown using a PALS3-green fluorescent protein (GFP) reporter strain showed GFP production in hyphae throughout the biofilm structure while biofilms grown using a PTPI1-GFP reporter strain showed GFP in both hyphae and yeast-form cells. Model catheter biofilms formed by an als3Δ/als3Δ strain were weakened structurally and had approximately half the biomass of a wild-type biofilm. Reintegration of a wild-type ALS3 allele restored biofilm mass and wild-type biofilm structure. Production of an Als3p-Agα1p fusion protein under control of the ALS3 promoter in the als3Δ/als3Δ strain restored some of the wild-type biofilm structural features, but not the wild-type biofilm mass. Despite its inability to restore wild-type biofilm mass, the Als3p-Agα1p fusion protein mediated adhesion of the als3Δ/als3Δ C. albicans strain to human buccal epithelial cells (BECs). The adhesive role of the Als3p N-terminal domain was further demonstrated by blocking adhesion of C. albicans to BECs with immunoglobulin reactive against the Als3p N-terminal sequences. Together, these data suggest that portions of Als3p that are important for biofilm formation may be different from those that are important in BEC adhesion, and that Als3p may have multiple functions in biofilm formation. Overexpression of ALS3 in an efg1Δ/efg1Δ strain that was deficient for filamentous growth and biofilm formation resulted in growth of elongated C. albicans cells, even under culture conditions that do not favour filamentation. In the catheter biofilm model, the ALS3 overexpression strain formed biofilm with a mass similar to that of a wild-type control. However, C. albicans cells in the biofilm had yeast-like morphology. This

  8. Fluoroquinolone interactions with Mycobacterium tuberculosis gyrase: Enhancing drug activity against wild-type and resistant gyrase

    PubMed Central

    Aldred, Katie J.; Kerns, Robert J.; Berger, James M.; Osheroff, Neil

    2016-01-01

    Mycobacterium tuberculosis is a significant source of global morbidity and mortality. Moxifloxacin and other fluoroquinolones are important therapeutic agents for the treatment of tuberculosis, particularly multidrug-resistant infections. To guide the development of new quinolone-based agents, it is critical to understand the basis of drug action against M. tuberculosis gyrase and how mutations in the enzyme cause resistance. Therefore, we characterized interactions of fluoroquinolones and related drugs with WT gyrase and enzymes carrying mutations at GyrAA90 and GyrAD94. M. tuberculosis gyrase lacks a conserved serine that anchors a water–metal ion bridge that is critical for quinolone interactions with other bacterial type II topoisomerases. Despite the fact that the serine is replaced by an alanine (i.e., GyrAA90) in M. tuberculosis gyrase, the bridge still forms and plays a functional role in mediating quinolone–gyrase interactions. Clinically relevant mutations at GyrAA90 and GyrAD94 cause quinolone resistance by disrupting the bridge–enzyme interaction, thereby decreasing drug affinity. Fluoroquinolone activity against WT and resistant enzymes is enhanced by the introduction of specific groups at the C7 and C8 positions. By dissecting fluoroquinolone–enzyme interactions, we determined that an 8-methyl-moxifloxacin derivative induces high levels of stable cleavage complexes with WT gyrase and two common resistant enzymes, GyrAA90V and GyrAD94G. 8-Methyl-moxifloxacin was more potent than moxifloxacin against WT M. tuberculosis gyrase and displayed higher activity against the mutant enzymes than moxifloxacin did against WT gyrase. This chemical biology approach to defining drug–enzyme interactions has the potential to identify novel drugs with improved activity against tuberculosis. PMID:26792518

  9. Selection for Evi1 activation in myelomonocytic leukemia induced by hyperactive signaling through wild-type NRas.

    PubMed

    Wolf, S; Rudolph, C; Morgan, M; Büsche, G; Salguero, G; Stripecke, R; Schlegelberger, B; Baum, C; Modlich, U

    2013-06-20

    Activation of NRas signaling is frequently found in human myeloid leukemia and can be induced by activating mutations as well as by mutations in receptors or signaling molecules upstream of NRas. To study NRas-induced leukemogenesis, we retrovirally overexpressed wild-type NRas in a murine bone marrow transplantation (BMT) model in C57BL/6J mice. Overexpression of wild-type NRas caused myelomonocytic leukemias ∼3 months after BMT in the majority of mice. A subset of mice (30%) developed malignant histiocytosis similar to mice that received mutationally activated NRas(G12D)-expressing bone marrow. Aberrant Ras signaling was demonstrated in cells expressing mutationally active or wild-type NRas, as increased activation of Erk and Akt was observed in both models. However, more NRas(G12D) were found to be in the activated, GTP-bound state in comparison with wild-type NRas. Consistent with observations reported for primary human myelomonocytic leukemia cells, Stat5 activation was also detected in murine leukemic cells. Furthermore, clonal evolution was detected in NRas wild-type-induced leukemias, including expansion of clones containing activating vector insertions in known oncogenes, such as Evi1 and Prdm16. In vitro cooperation of NRas and Evi1 improved long-term expansion of primary murine bone marrow cells. Evi1-positive cells upregulated Bcl-2 and may, therefore, provide anti-apoptotic signals that collaborate with the NRas-induced proliferative effects. As activation of Evi1 has been shown to coincide with NRAS mutations in human acute myeloid leukemia, our murine model recapitulates crucial events in human leukemogenesis. PMID:22847614

  10. Only minor differences in renal osteodystrophy features between wild-type and sclerostin knockout mice with chronic kidney disease.

    PubMed

    Cejka, Daniel; Parada-Rodriguez, Diego; Pichler, Stefanie; Marculescu, Rodrig; Kramer, Ina; Kneissel, Michaela; Gross, Thomas; Reisinger, Andreas; Pahr, Dieter; Monier-Faugere, Marie-Claude; Haas, Martin; Malluche, Hartmut H

    2016-10-01

    Renal osteodystrophy affects the majority of patients with advanced chronic kidney disease (CKD) and is characterized by progressive bone loss. This study evaluated the effects of sclerostin knockout on bone in a murine model of severe, surgically induced CKD in both sclerostin knockout and wild-type mice. Mice of both genotypes with normal kidney function served as controls. Tibiae were analyzed using micro-computed tomography, and lumbar vertebrae were analyzed by histomorphometry. Results were tested for statistical significance by 2-way ANOVA to investigate whether bone of the knockout mice reacted differently to CKD compared with bone of wild-type mice. In the tibiae, there was no difference after creation of CKD between wild-type and knockout animals for cortical thickness or cross-sectional moment of inertia. Increases in cortical porosity induced by CKD differed significantly between genotypes in the tibial metaphysis but not in the diaphysis. In the trabecular compartment, no difference in reaction to CKD between genotypes was found for bone volume, trabecular number, trabecular thickness, and trabecular separation. In the lumbar vertebrae, significant differences in response to CKD between wild-type and knockout mice were seen for both bone volume and trabecular thickness. Osteoblast parameters did not differ significantly, whereas osteoclast numbers significantly increased in the wild-type but significantly decreased in knockout mice with CKD. No differences in response to CKD between genotypes were found for bone formation rate or mineral apposition rate. Thus, complete absence of sclerostin has only minor effects on CKD-induced bone loss in mice. PMID:27528549

  11. Research on the ultrafast fluorescence property of thylakoid membranes of the wild-type and mutant rice

    NASA Astrophysics Data System (ADS)

    Ren, Zhao-Yu; Xu, Xiao-Ming; Wang, Shui-Cai; Xin, Yue-Yong; He, Jun-Fang; Hou, Xun

    2003-10-01

    A high yielding rice variety mutant (Oryza sativa L., Zhenhui 249) with low chlorophyll b (Chl b) has been discovered in natural fields. It has a quality character controlled by a pair of recessive genes (nuclear gene). The partial loss of Chl b in content affects the efficiency of light harvest in a light harvest complex (LHC), thus producing the difference of the exciting energy transfer and the efficiency of photochemistry conversion between the mutant and wild-type rice in photosynthetic unit. The efficiency of utilizing light energy is higher in the mutant than that in the wild-type rice relatively. For further discussion of the above-mentioned difference and learning about the mechanism of the increase in the photochemical efficiency of the mutant, the pico-second resolution fluorescence spectrum measurement with delay-frame-scanning single photon counting technique is adopted. Thylakoid membranes of the mutant and the wild-type rice are excited by an Ar+ laser with a pulse width of 120 ps, repetition rate of 4 MHz and wavelength of 514 nm. Compared with the time and spectrum property of exciting fluorescence, conclusions of those ultrafast dynamic experiments are: 1) The speeds of the exciting energy transferred in photo-system I are faster than that in photo-system II in both samples. 2) The speeds of the exciting energy transfer of mutant sample are faster than those of the wild-type. This might be one of the major reasons why the efficiency of photosynthesis is higher in mutant than that in the wild-type rice.

  12. MicroRNA-based Therapeutic Strategies for Targeting Mutant and Wild Type RAS in Cancer

    PubMed Central

    Sharma, Sriganesh B.; Ruppert, J. Michael

    2015-01-01

    MicroRNAs (miRs) have been causally implicated in the progression and development of a wide variety of cancers. miRs modulate the activity of key cell signaling networks by regulating the translation of pathway component proteins. Thus, the pharmacological targeting of miRs that regulate cancer cell signaling networks, either by promoting (using miR-supplementation) or by suppressing (using anti-sense oligonucleotide based strategies) miR activity is an area of intense research. The RAS-Extracellular signal regulated kinase (ERK) pathway represents a major miR-regulated signaling network that endows cells with some of the classical hallmarks of cancer, and is often inappropriately activated in malignancies by somatic genetic alteration through point mutation or alteration of gene copy number. In addition, recent progress indicates that many tumors may be deficient in GTPase activating proteins (GAPs) due to the collaborative action of oncogenic microRNAs. Recent studies also suggest that in tumors harboring a mutant RAS allele there is a critical role for wild type RAS proteins in determining overall RAS-ERK pathway activity. Together, these two advances comprise a new opportunity for therapeutic intervention. In this review, we evaluate miR-based therapeutic strategies for modulating RAS-ERK signaling in cancers, in particular for more direct modulation of RAS-GTP levels, with the potential to complement current strategies in order to yield more durable treatment responses. To this end, we discuss the potential for miR-based therapies focused on three prominent miRs including the pan-RAS regulator let-7 and the GAP regulator comprised of miR-206 and miR-21 (miR-206/21). PMID:26284568

  13. Crystal structure of wild-type human thrombin in the Na+-free state

    PubMed Central

    Johnson, Daniel J. D.; Adams, Ty E.; Li, Wei; Huntington, James A.

    2005-01-01

    Regulation of thrombin activity is critical for haemostasis and the prevention of thrombosis. Thrombin has several procoagulant substrates, including fibrinogen and platelet receptors, and essential cofactors for stimulating its own formation. However, thrombin is also capable of serving an anticoagulant function by activating protein C. The specificity of thrombin is primarily regulated by binding to the cofactor TM (thrombomodulin), but co-ordination of Na+ can also affect thrombin activity. The Na+-free form is often referred to as ‘slow’ because of reduced rates of cleavage of procoagulant substrates, but the slow form is still capable of rapid activation of protein C in the presence of TM. The molecular basis of the slow proteolytic activity of thrombin has remained elusive, in spite of two decades of solution studies and many published crystallographic structures. In the present paper, we report the first structure of wild-type unliganded human thrombin grown in the absence of co-ordinating Na+. The Na+-binding site is observed in a highly ordered position 6 Å (1 Å=0.1 nm) removed from that seen in the Na+-bound state. The movement of the Na+ loop results in non-catalytic hydrogen-bonding in the active site and blocking of the S1 and S2 substrate-binding pockets. Similar, if more dramatic, changes were observed in a previous structure of the constitutively slow thrombin variant E217K. The slow behaviour of thrombin in solutions devoid of Na+ can now be understood in terms of an equilibrium between an inert species, represented by the crystal structure described in the present paper, and an active form, where the addition of Na+ populates the active state. PMID:16201969

  14. Assessing benzene-induced toxicity on wild type Euglena gracilis Z and its mutant strain SMZ.

    PubMed

    Peng, Cheng; Arthur, Dionne M; Sichani, Homa Teimouri; Xia, Qing; Ng, Jack C

    2013-11-01

    Benzene is a representative member of volatile organic compounds and has been widely used as an industrial solvent. Groundwater contamination of benzene may pose risks to human health and ecosystems. Detection of benzene in the groundwater using chemical analysis is expensive and time consuming. In addition, biological responses to environmental exposures are uninformative using such analysis. Therefore, the aim of this study was to employ a microorganism, Euglena gracilis (E. gracilis) as a putative model to monitor the contamination of benzene in groundwater. To this end, we examined the wild type of E. gracilis Z and its mutant form, SMZ in their growth rate, morphology, chlorophyll content, formation of reactive oxygen species (ROS) and DNA damage in response to benzene exposure. The results showed that benzene inhibited cell growth in a dose response manner up to 48 h of exposure. SMZ showed a greater sensitivity compared to Z in response to benzene exposure. The difference was more evident at lower concentrations of benzene (0.005-5 μM) where growth inhibition occurred in SMZ but not in Z cells. We found that benzene induced morphological changes, formation of lipofuscin, and decreased chlorophyll content in Z strain in a dose response manner. No significant differences were found between the two strains in ROS formation and DNA damage by benzene at concentrations affecting cell growth. Based on these results, we conclude that E. gracilis cells were sensitive to benzene-induced toxicities for certain endpoints such as cell growth rate, morphological change, depletion of chlorophyll. Therefore, it is a potentially suitable model for monitoring the contamination of benzene and its effects in the groundwater.

  15. Vitamin D2-enriched button mushroom (Agaricus bisporus) improves memory in both wild type and APPswe/PS1dE9 transgenic mice.

    PubMed

    Bennett, Louise; Kersaitis, Cindy; Macaulay, Stuart Lance; Münch, Gerald; Niedermayer, Garry; Nigro, Julie; Payne, Matthew; Sheean, Paul; Vallotton, Pascal; Zabaras, Dimitrios; Bird, Michael

    2013-01-01

    Vitamin D deficiency is widespread, affecting over 30% of adult Australians, and increasing up to 80% for at-risk groups including the elderly (age>65). The role for Vitamin D in development of the central nervous system is supported by the association between Vitamin D deficiency and incidence of neurological and psychiatric disorders including Alzheimer's disease (AD). A reported positive relationship between Vitamin D status and cognitive performance suggests that restoring Vitamin D status might provide a cognitive benefit to those with Vitamin D deficiency. Mushrooms are a rich source of ergosterol, which can be converted to Vitamin D2 by treatment with UV light, presenting a new and convenient dietary source of Vitamin D2. We hypothesised that Vitamin D2-enriched mushrooms (VDM) could prevent the cognitive and pathological abnormalities associated with dementia. Two month old wild type (B6C3) and AD transgenic (APPSwe/PS1dE9) mice were fed a diet either deficient in Vitamin D2 or a diet which was supplemented with VDM, containing 1±0.2 µg/kg (∼54 IU/kg) vitamin D2, for 7 months. Effects of the dietary intervention on memory were assessed pre- and post-feeding. Brain sections were evaluated for amyloid β (Aβ) plaque loads and inflammation biomarkers using immuno-histochemical methods. Plasma vitamin D metabolites, Aβ40, Aβ42, calcium, protein and cholesterol were measured using biochemical assays. Compared with mice on the control diet, VDM-fed wild type and AD transgenic mice displayed improved learning and memory, had significantly reduced amyloid plaque load and glial fibrillary acidic protein, and elevated interleukin-10 in the brain. The results suggest that VDM might provide a dietary source of Vitamin D2 and other bioactives for preventing memory-impairment in dementia. This study supports the need for a randomised clinical trial to determine whether or not VDM consumption can benefit cognitive performance in the wider population. PMID:24204618

  16. Complete sequence of three different biotypes of tomato spotted wilt virus (wild type, tomato Sw-5 resistance-breaking and pepper Tsw resistance-breaking) from Spain.

    PubMed

    Debreczeni, Diana E; López, Carmelo; Aramburu, José; Darós, José Antonio; Soler, Salvador; Galipienso, Luis; Falk, Bryce W; Rubio, Luis

    2015-08-01

    Tomato spotted wilt virus (TSWV) occurs worldwide and causes production losses in many important horticultural crops such as tomato and pepper. Breeding resistant cultivars has been the most successful method so far for TSWV disease control, but only two genes have been found to confer resistance against a wide spectrum of TSWV isolates: Sw-5 in tomato and Tsw in pepper. However, TSWV resistance-breaking isolates have emerged in different countries a few years after using resistant cultivars. In this paper, we report the first complete nucleotide sequences of three Spanish TSWV isolates with different biotypes according to their abilities to overcome resistance: LL-N.05 (wild type, WT), Pujol1TL3 (Sw-5 resistance breaking, SBR) and PVR (Tsw resistance-breaking, TBR). The genome of these TSWV isolates consisted of three segments: L (8913-8914 nt), M (4752-4825 nt) and (S 2924-2961 nt). Variations in nucleotide sequences and genomic RNA lengths among the different virus biotypes are reported here. Phylogenetic analysis of the five TSWV open reading frames showed evidence of reassortment between genomic segments of LL-N.05 and Pujol1TL3, which was supported by analysis with different recombination-detecting algorithms.

  17. Wild-type and mutant p53 differentially regulate NADPH oxidase 4 in TGF-β-mediated migration of human lung and breast epithelial cells

    PubMed Central

    Boudreau, H E; Casterline, B W; Burke, D J; Leto, T L

    2014-01-01

    Background: Transforming growth factor-beta (TGF-β) induces the epithelial-to-mesenchymal transition (EMT) leading to increased cell plasticity at the onset of cancer cell invasion and metastasis. Mechanisms involved in TGF-β-mediated EMT and cell motility are unclear. Recent studies showed that p53 affects TGF-β/SMAD3-mediated signalling, cell migration, and tumorigenesis. We previously demonstrated that Nox4, a Nox family NADPH oxidase, is a TGF-β/SMAD3-inducible source of reactive oxygen species (ROS) affecting cell migration and fibronectin expression, an EMT marker, in normal and metastatic breast epithelial cells. Our present study investigates the involvement of p53 in TGF-β-regulated Nox4 expression and cell migration. Methods: We investigated the effect of wild-type p53 (WT-p53) and mutant p53 proteins on TGF-β-regulated Nox4 expression and cell migration. Nox4 mRNA and protein, ROS production, cell migration, and focal adhesion kinase (FAK) activation were examined in three different cell models based on their p53 mutational status. H1299, a p53-null lung epithelial cell line, was used for heterologous expression of WT-p53 or mutant p53. In contrast, functional studies using siRNA-mediated knockdown of endogenous p53 were conducted in MDA-MB-231 metastatic breast epithelial cells that express p53-R280K and MCF-10A normal breast cells that have WT-p53. Results: We found that WT-p53 is a potent suppressor of TGF-β-induced Nox4, ROS production, and cell migration in p53-null lung epithelial (H1299) cells. In contrast, tumour-associated mutant p53 proteins (R175H or R280K) caused enhanced Nox4 expression and cell migration in both TGF-β-dependent and TGF-β-independent pathways. Moreover, knockdown of endogenous mutant p53 (R280K) in TGF-β-treated MDA-MB-231 metastatic breast epithelial cells resulted in decreased Nox4 protein and reduced phosphorylation of FAK, a key regulator of cell motility. Expression of WT-p53 or dominant-negative Nox4

  18. Changes in polyphenol and sugar concentrations in wild type and genetically modified Nicotiana langsdorffii Weinmann in response to water and heat stress.

    PubMed

    Ancillotti, Claudia; Bogani, Patrizia; Biricolti, Stefano; Calistri, Elisa; Checchini, Leonardo; Ciofi, Lorenzo; Gonnelli, Cristina; Del Bubba, Massimo

    2015-12-01

    In this study wild type Nicotiana langsdorffii plants were genetically transformed by the insertion of the rat gene (gr) encoding the glucocorticoid receptor or the rolC gene and exposed to water and heat stress. Water stress was induced for 15 days by adding 20% PEG 6000 in the growth medium, whereas the heat treatment was performed at 50 °C for 2 h, after that a re-growing capability study was carried out. The plant response to stress was investigated by determining electrolyte leakage, dry weight biomass production and water content. These data were evaluated in relation to antiradical activity and concentrations of total polyphenols, selected phenolic compounds and some soluble sugars, as biochemical indicators of metabolic changes due to gene insertion and/or stress treatments. As regards the water stress, the measured physiological parameters evidenced an increasing stress level in the order rolC < gr < WT plants (e.g. about 100% and 50% electrolyte leakage increase in WT and gr samples, respectively) and complied with the biochemical pattern, which consisted in a general decrease of antiradical activity and phenolics, together with an increase in sugars. As regard heat stress, electrolyte leakage data were only in partial agreement with the re-growing capability study. In fact, according to this latter evaluation, gr was the genotype less affected by the heat shock. In this regard, sugars and especially phenolic compounds are informative of the long-term effects due to heat shock treatment. PMID:26410575

  19. Cellular immunity survey against urinary tract infection using pVAX/fimH cassette with mammalian and wild type codon usage as a DNA vaccine

    PubMed Central

    Bagherpour, Ghasem; Khoramabadi, Nima; Fallah Mehrabadi, Jalil; Mahdavi, Mehdi; Halabian, Raheleh; Amin, Mohsen; Izadi Mobarakeh, Jalal; Einollahi, Behzad

    2014-01-01

    Purpose FimH (the adhesion fragment of type 1 fimbriae) is implicated in uropathogenic Escherichia coli (UPEC) attachment to epithelial cells through interaction with mannose. Recently, some studies have found that UPEC can thrive intracellularly causing recurrent urinary tract infection (UTI). Almost all vaccines have been designed to induce antibodies against UPEC. Yet, the humoral immune response is not potent enough to overcome neither the primary UTI nor recurrent infections. However, DNA vaccines offer the possibility of inducing cell mediated immune responses and may be a promising preventive tool. Materials and Methods In this study, we employed two different open reading frames within mammalian (mam) and wild type (wt) codons of fimH gene. Optimized fragments were cloned in pVAX-1. Expression of the protein in COS-7 was confirmed by western blot analysis after assessing pVAX/fimH(mam) and pVAX/fimH(wt). The constructs were injected to BALB/c mice at plantar surface of feet followed by electroporation. Results The mice immunized with both constructs following booster injection with recombinant FimH showed increased interferon-γ and interleukin-12 responses significantly higher than non-immunized ones (p<0.05). The immunized mice were challenged with UPEC and then the number of bacteria recovered from the immunized mice was compared with the non-immunized ones. Decreased colony count in immunized mice along with cytokine responses confirmed the promising immune response by the DNA vaccines developed in this study. Conclusion In conclusion, DNA vaccines of UPEC proteins may confer some levels of protection which can be improved by multiple constructs or boosters. PMID:25003092

  20. Expression pattern of immediate early genes in the cerebellum of D1R KO, D2R KO, and wild type mice under vestibular-controlled activity.

    PubMed

    Nakamura, Toru; Sato, Asako; Kitsukawa, Takashi; Sasaoka, Toshikuni; Yamamori, Tetsuo

    2015-01-01

    We previously reported the different motor abilities of D1R knockout (KO), D2R KO and wild-type (WT) mice. To understand the interaction between the cerebellum and the striatal direct and indirect pathways, we examined the expression patterns of immediate early genes (IEG) in the cerebellum of these three genotypes of mice. In the WT naive mice, there was little IEG expression. However, we observed a robust expression of c-fos mRNA in the vermis and hemisphere after running rota-rod tasks. In the vermis, c-fos was expressed throughout the lobules except lobule 7, and also in crus 1 of the ansiform lobule (Crus1), copula of the pyramis (Cop) and most significantly in the flocculus in the hemisphere. jun-B was much less expressed but more preferentially expressed in Purkinje cells. In addition, we observed significant levels of c-fos and jun-B expressions after handling mice, and after the stationary rota-rod task in naive mice. Surprisingly, we observed significant expression of c-fos and jun-B even 30 min after single weighing. Nonetheless, certain additional c-fos and jun-B expressions were observed in three genotypes of the mice that experienced several sessions of motor tasks 24 h after stationary rota-rod task and on days 1 and 5 after rota-rod tasks, but no significant differences in expressions after the running rota-rod tasks were observed among the three genotypes. In addition, there may be some differences 24 h after the stationary rota-rod task between the naive mice and the mice that experienced several sessions of motor tasks.

  1. Expression pattern of immediate early genes in the cerebellum of D1R KO, D2R KO, and wild type mice under vestibular-controlled activity

    PubMed Central

    Nakamura, Toru; Sato, Asako; Kitsukawa, Takashi; Sasaoka, Toshikuni; Yamamori, Tetsuo

    2015-01-01

    We previously reported the different motor abilities of D1R knockout (KO), D2R KO and wild-type (WT) mice. To understand the interaction between the cerebellum and the striatal direct and indirect pathways, we examined the expression patterns of immediate early genes (IEG) in the cerebellum of these three genotypes of mice. In the WT naive mice, there was little IEG expression. However, we observed a robust expression of c-fos mRNA in the vermis and hemisphere after running rota-rod tasks. In the vermis, c-fos was expressed throughout the lobules except lobule 7, and also in crus 1 of the ansiform lobule (Crus1), copula of the pyramis (Cop) and most significantly in the flocculus in the hemisphere. jun-B was much less expressed but more preferentially expressed in Purkinje cells. In addition, we observed significant levels of c-fos and jun-B expressions after handling mice, and after the stationary rota-rod task in naive mice. Surprisingly, we observed significant expression of c-fos and jun-B even 30 min after single weighing. Nonetheless, certain additional c-fos and jun-B expressions were observed in three genotypes of the mice that experienced several sessions of motor tasks 24 h after stationary rota-rod task and on days 1 and 5 after rota-rod tasks, but no significant differences in expressions after the running rota-rod tasks were observed among the three genotypes. In addition, there may be some differences 24 h after the stationary rota-rod task between the naive mice and the mice that experienced several sessions of motor tasks. PMID:26137459

  2. Induced Apoptosis Investigation in Wild-type and FLT3-ITD Acute Myeloid Leukemia Cells by Nanochannel Electroporation and Single-cell qRT-PCR.

    PubMed

    Gao, Keliang; Huang, Xiaomeng; Chiang, Chi-Ling; Wang, Xinmei; Chang, Lingqian; Boukany, Pouyan; Marcucci, Guido; Lee, Robert; Lee, Ly James

    2016-05-01

    Nanochannel electroporation (NEP) was applied to deliver precise dosages of myeloid cell leukemia-1 (Mcl-1)-specific siRNA and molecular beacons to two types of acute myeloid leukemia (AML) cells, FMS-like tyrosine kinase-3 wild-type (WT) and internal tandem duplications (ITD) type at the single-cell level. NEP, together with single-cell quantitative reverse transcription PCR, led to an observation showing nearly 20-folds more Mcl-1 siRNA than MCL1 mRNA were required to induce cell death for both cell lines and patient blasts, i.e., ~8,800 siRNAs for ~500 ± 50 mRNAs in ITD cells and ~6,000 siRNAs for ~300 ± 50 mRNAs in WT cells. A time-lapse study revealed that >75% MCL1 mRNA was downregulated within 1 hour after delivery of a small amount of siRNA. However, additional siRNA was required to inhibit the newly transcribed mRNA for >12 hours until the cell lost its ability of self-protection recovery. A multidelivery strategy of low doses and short delivery interval, which require 77% less siRNA and has the potential of lower side effects and clinical cost, was as effective as a single high-dose siRNA delivery. Our method provides a viable analytical tool to investigate gene silencing at the single-cell level for oligonucleotide-based therapy.

  3. Computational Studies of a Mechanism for Binding and Drug Resistance in the Wild Type and Four Mutations of HIV-1 Protease with a GRL-0519 Inhibitor.

    PubMed

    Hu, Guodong; Ma, Aijing; Dou, Xianghua; Zhao, Liling; Wang, Jihua

    2016-01-01

    Drug resistance of mutations in HIV-1 protease (PR) is the most severe challenge to the long-term efficacy of HIV-1 PR inhibitor in highly active antiretroviral therapy. To elucidate the molecular mechanism of drug resistance associated with mutations (D30N, I50V, I54M, and V82A) and inhibitor (GRL-0519) complexes, we have performed five molecular dynamics (MD) simulations and calculated the binding free energies using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method. The ranking of calculated binding free energies is in accordance with the experimental data. The free energy spectra of each residue and inhibitor interaction for all complexes show a similar binding model. Analysis based on the MD trajectories and contribution of each residues show that groups R2 and R3 mainly contribute van der Waals energies, while groups R1 and R4 contribute electrostatic interaction by hydrogen bonds. The drug resistance of D30N can be attributed to the decline in binding affinity of residues 28 and 29. The size of Val50 is smaller than Ile50 causes the residue to move, especially in chain A. The stable hydrophobic core, including the side chain of Ile54 in the wild type (WT) complex, became unstable in I54M because the side chain of Met54 is flexible with two alternative conformations. The binding affinity of Ala82 in V82A decreases relative to Val82 in WT. The present study could provide important guidance for the design of a potent new drug resisting the mutation inhibitors. PMID:27240358

  4. Effects of long-term treatment with pioglitazone on cognition and glucose metabolism of PS1-KI, 3xTg-AD, and wild-type mice.

    PubMed

    Masciopinto, F; Di Pietro, N; Corona, C; Bomba, M; Pipino, C; Curcio, M; Di Castelnuovo, A; Ciavardelli, D; Silvestri, E; Canzoniero, L M T; Sekler, I; Pandolfi, A; Sensi, S L

    2012-12-20

    In this study, we investigated the effects of long-term (9-month) treatment with pioglitazone (PIO; 20 mg/kg/d) in two animal models of Alzheimer's disease (AD)-related neural dysfunction and pathology: the PS1-KI(M146V) (human presenilin-1 (M146V) knock-in mouse) and 3xTg-AD (triple transgenic mouse carrying AD-linked mutations) mice. We also investigated the effects on wild-type (WT) mice. Mice were monitored for body mass changes, fasting glycemia, glucose tolerance, and studied for changes in brain mitochondrial enzyme activity (complexes I and IV) as well as energy metabolism (lactate dehydrogenase (LDH)). Cognitive effects were investigated with the Morris water maze (MWM) test and the object recognition task (ORT). Behavioral analysis revealed that PIO treatment promoted positive cognitive effects in PS1-KI female mice. These effects were associated with normalization of peripheral gluco-regulatory abnormalities that were found in untreated PS1-KI females. PIO-treated PS1-KI females also showed no statistically significant alterations in brain mitochondrial enzyme activity but significantly increased reverse LDH activity.PIO treatment produced no effects on cognition, glucose metabolism, or mitochondrial functioning in 3xTg-AD mice. Finally, PIO treatment promoted enhanced short-term memory performance in WT male mice, a group that did not show deregulation of glucose metabolism but that showed decreased activity of complex I in hippocampal and cortical mitochondria. Overall, these results indicate metabolically driven cognitive-enhancing effects of PIO that are differentially gender-related among specific genotypes.

  5. Functional Recovery of AQP2 Recessive Mutations Through Hetero-Oligomerization with Wild-Type Counterpart.

    PubMed

    El Tarazi, Abdulah; Lussier, Yoann; Da Cal, Sandra; Bissonnette, Pierre; Bichet, Daniel G

    2016-01-01

    Aquaporin-2 (AQP2) is a homotetrameric water channel responsible for the final water reuptake in the kidney. Mutations in the protein induce nephrogenic diabetes insipidus (NDI), which challenges the water balance by producing large urinary volumes. Although recessive AQP2 mutations are believed to generate non-functional and monomeric proteins, the literature identifies several mild mutations which suggest the existence of mixed wt/mut tetramers likely to carry function in heterozygotes. Using Xenopus oocytes, we tested this hypothesis and found that mild mutants (V24A, D150E) can associate with wt-AQP2 in mixed heteromers, providing clear functional gain in the process (62 ± 17% and 63 ± 17% increases, respectively), conversely to the strong monomeric R187C mutant which fails to associate with wt-AQP2. In kidney cells, both V24A and D150E display restored targeting while R187C remains in intracellular stores. Using a collection of mutations to expand recovery analyses, we demonstrate that inter-unit contacts are central to this recovery process. These results not only present the ground data for the functional recovery of recessive AQP2 mutants through heteromerization, which prompt to revisit the accepted NDI model, but more importantly describe a general recovery process that could impact on all multimeric systems where recessive mutations are found. PMID:27641679

  6. Functional Recovery of AQP2 Recessive Mutations Through Hetero-Oligomerization with Wild-Type Counterpart

    PubMed Central

    El Tarazi, Abdulah; Lussier, Yoann; Da Cal, Sandra; Bissonnette, Pierre; Bichet, Daniel G.

    2016-01-01

    Aquaporin-2 (AQP2) is a homotetrameric water channel responsible for the final water reuptake in the kidney. Mutations in the protein induce nephrogenic diabetes insipidus (NDI), which challenges the water balance by producing large urinary volumes. Although recessive AQP2 mutations are believed to generate non-functional and monomeric proteins, the literature identifies several mild mutations which suggest the existence of mixed wt/mut tetramers likely to carry function in heterozygotes. Using Xenopus oocytes, we tested this hypothesis and found that mild mutants (V24A, D150E) can associate with wt-AQP2 in mixed heteromers, providing clear functional gain in the process (62 ± 17% and 63 ± 17% increases, respectively), conversely to the strong monomeric R187C mutant which fails to associate with wt-AQP2. In kidney cells, both V24A and D150E display restored targeting while R187C remains in intracellular stores. Using a collection of mutations to expand recovery analyses, we demonstrate that inter-unit contacts are central to this recovery process. These results not only present the ground data for the functional recovery of recessive AQP2 mutants through heteromerization, which prompt to revisit the accepted NDI model, but more importantly describe a general recovery process that could impact on all multimeric systems where recessive mutations are found. PMID:27641679

  7. Wild-Type Phosphoribosylpyrophosphate Synthase (PRS) from Mycobacterium tuberculosis: A Bacterial Class II PRS?

    PubMed Central

    Breda, Ardala; Martinelli, Leonardo K. B.; Bizarro, Cristiano V.; Rosado, Leonardo A.; Borges, Caroline B.; Santos, Diógenes S.; Basso, Luiz A.

    2012-01-01

    The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of Pi. ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of Pi would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of Mt

  8. Wild-type phosphoribosylpyrophosphate synthase (PRS) from Mycobacterium tuberculosis: a bacterial class II PRS?

    PubMed

    Breda, Ardala; Martinelli, Leonardo K B; Bizarro, Cristiano V; Rosado, Leonardo A; Borges, Caroline B; Santos, Diógenes S; Basso, Luiz A

    2012-01-01

    The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of P(i). ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of P(i) would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of

  9. Amino acid uptake profiling of wild type and recombinant Streptomyces lividans TK24 batch fermentations.

    PubMed

    D'Huys, Pieter-Jan; Lule, Ivan; Van Hove, Sven; Vercammen, Dominique; Wouters, Christine; Bernaerts, Kristel; Anné, Jozef; Van Impe, Jan F M

    2011-04-10

    Streptomyces lividans is considered an interesting host for the secretory production of heterologous proteins. To obtain a good secretion yield of heterologous proteins, the availability of suitable nitrogen sources in the medium is required. Often, undefined mixtures of amino acids are used to improve protein yields. However, the understanding of amino acid utilization as well as their contribution to the heterologous protein synthesis is poor. In this paper, amino acid utilization by wild type and recombinant S. lividans TK24 growing on a minimal medium supplemented with casamino acids is profiled by intensive analysis of the exometabolome (metabolic footprint) as a function of time. Dynamics of biomass, substrates, by-products and heterologous protein are characterized, analyzed and compared. As an exemplary protein mouse Tumor Necrosis Factor Alpha (mTNF-α) is considered. Results unveil preferential glutamate and aspartate assimilation, together with glucose and ammonium, but the associated high biomass growth rate is unfavorable for protein production. Excretion of organic acids as well as alanine is observed. Pyruvate and alanine overflow point at an imbalance between carbon and nitrogen catabolism and biosynthetic fluxes. Lactate secretion is probably related to clump formation. Heterologous protein production induces a slowdown in growth, denser clump formation and a shift in metabolism, as reflected in the altered substrate requirements and overflow pattern. Besides glutamate and aspartate, most amino acids are catabolized, however, their exact contribution in heterologous protein production could not be seized from macroscopic quantities. The metabolic footprints presented in this paper provide a first insight into the impact and relevance of amino acids on biomass growth and protein production. Type and availability of substrates together with biomass growth rate and morphology affect the protein secretion efficiency and should be optimally controlled

  10. Developmental expression of wild-type and mutant presenilin-1 in hippocampal neurons from transgenic mice: evidence for novel species-specific properties of human presenilin-1.

    PubMed Central

    Lévesque, L.; Annaert, W.; Craessaerts, K.; Mathews, P. M.; Seeger, M.; Nixon, R. A.; Van Leuven, F.; Gandy, S.; Westaway, D.; St George-Hyslop, P.; De Strooper, B.; Fraser, P. E.

    1999-01-01

    Presenilins 1 (PS1) and 2 (PS2) are multispanning transmembrane proteins associated with familial Alzheimer disease (FAD). They are developmentally regulated, being expressed at highest levels during neuronal differentiation and are sustained at a lower level throughout life. We investigated the distribution and metabolism of endogenous murine PS1 as well as human wild-type (wtPS1) and the familial AD Met146Leu (M146L) mutant presenilins in dissociated cultures of hippocampal neurons derived from control and transgenic mice. We found that the PS1 endoproteolytic fragments and, to a lesser extent, the full-length protein, were expressed as early as day 3 post-plating. Both species increased until the cells were fully differentiated at day 12. Confocal microscopy revealed that presenilin is present in the Golgi and endoplasmic reticulum and, as in punctate, vesicle-like structures within developing neurites and growth cones. Using a human-specific PS1 antibody, we were able to independently examine the distribution of the transgenic protein which, although similar to the endogenous, showed some unique qualities. These included (i) some heterogeneity in the proteolytic fragments of human PS1; (ii) significantly reduced levels of full-length human PS1, possibly as a result of preferential processing; and (iii) a more discrete intracellular distribution of human PS1. Colocalization with organelle-specific proteins revealed that PS1 was located in a diffuse staining pattern in the MAP2-positive dendrites and in a punctate manner in GAP43-positive axons. PS1 showed considerable overlap with GAP43, particularly at the growth cones. Similar patterns of PS1 distribution were detected in cultures derived from transgenic animals expressing human wild-type or mutant presenilins. The studies demonstrate that mutant presenilins are not grossly different in their processing or distribution within cultured neurons, which may represent more physiological models as compared to

  11. Cadmium tolerance, cysteine and thiol peptide levels in wild type and chromium-tolerant strains of Scenedesmus acutus (Chlorophyceae).

    PubMed

    Torricelli, Elena; Gorbi, Gessica; Pawlik-Skowronska, Barbara; Di Toppi, Luigi Sanità; Corradi, Maria Grazia

    2004-07-14

    Two strains of the unicellular green alga Scenedesmus acutus with different sensitivity to hexavalent chromium were compared for their tolerance of cadmium, by means of growth and recovery tests, and determination of cysteine, reduced glutathione and phytochelatin content, after short-term exposure to various cadmium concentrations (from 1.125 to 27 microM). Growth experiments showed that, after 7-day treatments with cadmium, the chromium-tolerant strain reached a significantly higher cell density and, after 24-h exposure to Cd, was able to resume growth significantly better than the wild type. Constitutive level of cysteine was higher in the chromium-tolerant strain, while glutathione levels were similar in the two strains. The higher content of cysteine and the maintenance of both reduced glutathione and phytochelatin high levels in the presence of cadmium, support the higher cadmium co-tolerance of the chromium-tolerant strain in comparison with the wild type one. PMID:15177949

  12. EXAFS of Klebsiella pneumoniae nitrogenase MoFe protein from wild-type and nif V mutant strains

    SciTech Connect

    Eidsness, M.K.; Flank, A.M.; Smith, B.E.; Flood, A.C.; Garner, C.D.; Cramer. S.P.

    1986-05-14

    The enzyme nitrogenase catalyzes the biological reduction of N/sub 2/ to NH/sub 3/. In Klebsiella pneumoniae a cluster of 17 genes in seven transcriptional units has been associated with nitrogen fixation. The nitrogenase enzyme from the nif V mutants is relatively ineffective at dinitrogen reduction, is more efficient than the wild-type enzyme at HCN reduction, and has its hydrogen evolution activity inhibited up to 80% by CO. This altered substrate specificity has been shown to be associated with the iron-molybdenum cofactor, FeMo-co, of the enzyme. X-ray absorption spectroscopy has been a valuable tool for probing the molybdenum environment of wild-type nitrogenase, and the authors report here similar studies on the Nif V/sup -/ enzyme.

  13. Phospholamban mutants compete with wild type for SERCA binding in living cells

    SciTech Connect

    Gruber, Simon J.; Haydon, Suzanne; Thomas, David D.

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer PLB phosphorylation in HEK cells increased FRET between YFP-PLB and CFP-SERCA. Black-Right-Pointing-Pointer Competition: Expressing loss-of-function PLB mutants in the system decreased FRET. Black-Right-Pointing-Pointer The FRET assay could screen potential therapeutic PLB mutants to activate SERCA. -- Abstract: We have used fluorescent fusion proteins stably expressed in HEK cells to detect directly the interaction between the sarcoplasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLB) in living cells, in order to design PLB mutants for gene therapy. Ca{sup 2+} cycling in muscle cells depends strongly on SERCA. Heart failure (HF), which contributes to 12% of US deaths, typically exhibits decreased SERCA activity, and several potential therapies for HF aim to increase SERCA activity. We are investigating the use of LOF-PLB mutants (PLB{sub M}) as gene therapy vectors to increase SERCA activity. Active SERCA1a and WT-PLB, tagged at their N termini with fluorescent proteins (CFP and YFP), were coexpressed in stable HEK cell lines, and fluorescence resonance energy transfer (FRET) was used to detect their interaction directly. Phosphorylation of PLB, induced by forskolin, caused an increase in FRET from CFP-SERCA to YFP-PLB, indicating that SERCA inhibition can be relieved without dissociation of the complex. This suggests that a LOF mutant might bind to SERCA with sufficient affinity to complete effectively with WT-PLB, thus relieving SERCA inhibition. Therefore, we transiently expressed a series of PLB{sub M} in the CFP-SERCA/YFP-PLB cell line, and found decreased FRET, implying competition between PLB{sub M} and WT-PLB for binding to SERCA. These results establish this FRET assay as a rapid and quantitative means of screening PLB{sub M} for optimization of gene therapy to activate SERCA, as needed for gene therapy in HF.

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

    PubMed

    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.

  15. Full Genome Sequence-Based Comparative Study of Wild-Type and Vaccine Strains of Infectious Laryngotracheitis Virus from Italy.

    PubMed

    Piccirillo, Alessandra; Lavezzo, Enrico; Niero, Giulia; Moreno, Ana; Massi, Paola; Franchin, Elisa; Toppo, Stefano; Salata, Cristiano; Palù, Giorgio

    2016-01-01

    Infectious laryngotracheitis (ILT) is an acute and highly contagious respiratory disease of chickens caused by an alphaherpesvirus, infectious laryngotracheitis virus (ILTV). Recently, full genome sequences of wild-type and vaccine strains have been determined worldwide, but none was from Europe. The aim of this study was to determine and analyse the complete genome sequences of five ILTV strains. Sequences were also compared to reveal the similarity of strains across time and to discriminate between wild-type and vaccine strains. Genomes of three ILTV field isolates from outbreaks occurred in Italy in 1980, 2007 and 2011, and two commercial chicken embryo origin (CEO) vaccines were sequenced using the 454 Life Sciences technology. The comparison with the Serva genome showed that 35 open reading frames (ORFs) differed across the five genomes. Overall, 54 single nucleotide polymorphisms (SNPs) and 27 amino acid differences in 19 ORFs and two insertions in the UL52 and ORFC genes were identified. Similarity among the field strains and between the field and the vaccine strains ranged from 99.96% to 99.99%. Phylogenetic analysis revealed a close relationship among them, as well. This study generated data on genomic variation among Italian ILTV strains revealing that, even though the genetic variability of the genome is well conserved across time and between wild-type and vaccine strains, some mutations may help in differentiating among them and may be involved in ILTV virulence/attenuation. The results of this study can contribute to the understanding of the molecular bases of ILTV pathogenicity and provide genetic markers to differentiate between wild-type and vaccine strains. PMID:26890525

  16. Full Genome Sequence-Based Comparative Study of Wild-Type and Vaccine Strains of Infectious Laryngotracheitis Virus from Italy

    PubMed Central

    Niero, Giulia; Moreno, Ana; Massi, Paola; Franchin, Elisa; Toppo, Stefano; Salata, Cristiano; Palù, Giorgio

    2016-01-01

    Infectious laryngotracheitis (ILT) is an acute and highly contagious respiratory disease of chickens caused by an alphaherpesvirus, infectious laryngotracheitis virus (ILTV). Recently, full genome sequences of wild-type and vaccine strains have been determined worldwide, but none was from Europe. The aim of this study was to determine and analyse the complete genome sequences of five ILTV strains. Sequences were also compared to reveal the similarity of strains across time and to discriminate between wild-type and vaccine strains. Genomes of three ILTV field isolates from outbreaks occurred in Italy in 1980, 2007 and 2011, and two commercial chicken embryo origin (CEO) vaccines were sequenced using the 454 Life Sciences technology. The comparison with the Serva genome showed that 35 open reading frames (ORFs) differed across the five genomes. Overall, 54 single nucleotide polymorphisms (SNPs) and 27 amino acid differences in 19 ORFs and two insertions in the UL52 and ORFC genes were identified. Similarity among the field strains and between the field and the vaccine strains ranged from 99.96% to 99.99%. Phylogenetic analysis revealed a close relationship among them, as well. This study generated data on genomic variation among Italian ILTV strains revealing that, even though the genetic variability of the genome is well conserved across time and between wild-type and vaccine strains, some mutations may help in differentiating among them and may be involved in ILTV virulence/attenuation. The results of this study can contribute to the understanding of the molecular bases of ILTV pathogenicity and provide genetic markers to differentiate between wild-type and vaccine strains. PMID:26890525

  17. Siderophore receptor PupA as a marker to monitor wild-type Pseudomonas putida WCS358 in natural environments.

    PubMed Central

    Raaijmakers, J M; Bitter, W; Punte, H L; Bakker, P A; Weisbeek, P J; Schippers, B

    1994-01-01

    For application of genetically engineered fluorescent Pseudomonas spp., specific markers are required for monitoring of wild-type Pseudomonas strains and their genetically modified derivatives in natural environments. In this study, the specific siderophore receptor PupA of plant growth-promoting Pseudomonas putida WCS358 was used as a marker to monitor wild-type strain WCS358. After introduction into natural soil and rhizosphere environments, strain WCS358 could be recovered efficiently on a medium amended with 300 microM pseudobactin 358. Although low population densisties of indigenous pseudomonads (less than or equal to 10(3)/g of soil or root) were recovered on the pseudobactin 358-amended medium, subsequent agglutination assays with a WCS358-specific polyclonal antiserum enabled accurate monitoring of populations of wild-type strain WCS358 over a range of approximately 10(3) to 10(7) CFU/g of soil or root. Genetic analysis of the background population by PCR and Southern hybridization revealed that natural occurrence of the pupA gene was limited to a very small number of indigenous Pseudomonas spp. which are very closely related to P. putida WCS358. The PupA marker system enabled the study of differences in rhizosphere colonization among wild-type strain WCS358, rifampin-resistant derivative WCS358rr, and Tn5 mutant WCS358::xylE. Chromosomally mediated rifampin resistance did not affect the colonizing ability of P. putida WCS358. However, Tn5 mutant WCS358::xylE colonized the radish rhizosphere significantly less than did its parental strain. Images PMID:8017914

  18. Efficient quiescent infection of normal human diploid fibroblasts with wild-type herpes simplex virus type 1.

    PubMed

    McMahon, Robert; Walsh, Derek

    2008-10-01

    Quiescent infection of cultured cells with herpes simplex virus type 1 (HSV-1) provides an important, amenable means of studying the molecular mechanics of a nonproductive state that mimics key aspects of in vivo latency. To date, establishing high-multiplicity nonproductive infection of human cells with wild-type HSV-1 has proven challenging. Here, we describe simple culture conditions that established a cell state in normal human diploid fibroblasts that supported efficient quiescent infection using wild-type virus and exhibited many important properties of the in vivo latent state. Despite the efficient production of immediate early (IE) proteins ICP4 and ICP22, the latter remained unprocessed, and viral late gene products were only transiently and inefficiently produced. This low level of virus activity in cultures was rapidly suppressed as the nonproductive state was established. Entry into quiescence was associated with inefficient production of the viral trans-activating protein ICP0, and the accumulation of enlarged nuclear PML structures normally dispersed during productive infection. Lytic replication was rapidly and efficiently restored by exogenous expression of HSV-1 ICP0. These findings are in agreement with previous models in which quiescence was established with HSV mutants disrupted in their expression of IE gene products that included ICP0 and, importantly, provide a means to study cellular mechanisms that repress wild-type viral functions to prevent productive replication. We discuss this model in relation to existing systems and its potential as a simple tool to study the molecular mechanisms of quiescent infection in human cells using wild-type HSV-1.

  19. Functional independence of monomeric CHIP28 water channels revealed by expression of wild-type mutant heterodimers.

    PubMed

    Shi, L B; Skach, W R; Verkman, A S

    1994-04-01

    CHIP28 is a major water transporting protein in erythrocytes and kidney which forms tetramers in membranes (Verbavatz, J. M., Brown, D., Sabolic, I., Valenti, G., Ausiello, D. A., Van Hoek, A. N., Ma, T., and Verkman, A. S. (1993) J. Cell Biol. 123, 605-618). To determine whether CHIP28 monomers function independently, chimeric cDNA dimers were constructed which contained wild-type CHIP28 in series with either wild-type CHIP28, a non-water transporting CHIP28 mutant (C189W), or a functional but mercurial-insensitive CHIP28 mutant (C189S). Transcribed cRNAs were injected in Xenopus oocytes and plasma membrane expression was assayed by quantitative immunofluorescence. Water channel function was measured by osmotically induced swelling. CHIP28 homo- and heterodimers were targeted to the oocyte plasma membrane and functioned as water channels. Relative osmotic water permeability (Pf) values (normalized for plasma membrane expression of monomeric subunits) were: 1.0 (CHIP28 monomer), 0.0 (C189W), 1.07 (C189S), 1.10 (CHIP28-CHIP28 dimer) and 0.52 (CHIP28-C189W). The increase in oocyte Pf was linearly related to plasma membrane expression of wild-type CHIP28 and C189S subunits. HgCl2 (0.3 mM) inhibited channel-mediated Pf in oocytes expressing wild-type CHIP28 monomers and dimers by 85-90%, but did not inhibit Pf in oocytes expressing C189S. HgCl2 inhibited Pf in oocytes expressing CHIP28-C189S dimers by 44 +/- 7%, consistent with one mercurial-sensitive and one insensitive subunit in the heterodimer. These results indicate that despite their assembly in tetramers, monomeric CHIP28 subunits function independently as water channels. PMID:7511600

  20. Assessment of allele-specific gene silencing by RNA interference with mutant and wild-type reporter alleles.

    PubMed

    Ohnishi, Yusuke; Tokunaga, Katsushi; Kaneko, Kiyotoshi; Hohjoh, Hirohiko

    2006-02-28

    Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically suppressing the expression of alleles associated with disease. To realize such allele-specific RNAi (ASPRNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital, but is also difficult. Here, we show ASP-RNAi against the Swedish- and London-type amyloid precursor protein (APP) variants related to familial Alzheimer's disease using two reporter alleles encoding the Photinus and Renilla luciferase genes and carrying mutant and wild-type allelic sequences in their 3'-untranslated regions. We examined the effects of siRNA duplexes against the mutant alleles in allele-specific gene silencing and off-target silencing against the wild-type allele under heterozygous conditions, which were generated by cotransfecting the reporter alleles and siRNA duplexes into cultured human cells. Consistently, the siRNA duplexes determined to confer ASP-RNAi also inhibited the expression of the bona fide mutant APP and the production of either amyloid beta 40- or 42-peptide in Cos-7 cells expressing both the full-length Swedish- and wild-type APP alleles. The present data suggest that the system with reporter alleles may permit the preclinical assessment of siRNA duplexes conferring ASP-RNAi, and thus contribute to the design and selection of the most suitable of such siRNA duplexes.

  1. Response to metal stress of Nicotiana langsdorffii plants wild-type and transgenic for the rat glucocorticoid receptor gene.

    PubMed

    Fuoco, Roger; Bogani, Patrizia; Capodaglio, Gabriele; Del Bubba, Massimo; Abollino, Ornella; Giannarelli, Stefania; Spiriti, Maria Michela; Muscatello, Beatrice; Doumett, Saer; Turetta, Clara; Zangrando, Roberta; Zelano, Vincenzo; Buiatti, Marcello

    2013-05-01

    Recently our findings have shown that the integration of the gene coding for the rat gluco-corticoid receptor (GR receptor) in Nicotiana langsdorffii plants induced morphophysiological effects in transgenic plants through the modification of their hormonal pattern. Phytohormones play a key role in plant responses to many different biotic and abiotic stresses since a modified hormonal profile up-regulates the activation of secondary metabolites involved in the response to stress. In this work transgenic GR plants and isogenic wild type genotypes were exposed to metal stress by treating them with 30ppm cadmium(II) or 50ppm chromium(VI). Hormonal patterns along with changes in key response related metabolites were then monitored and compared. Heavy metal up-take was found to be lower in the GR plants. The transgenic plants exhibited higher values of S-abscisic acid (S-ABA) and 3-indole acetic acid (IAA), salicylic acid and total polyphenols, chlorogenic acid and antiradical activity, compared to the untransformed wild type plants. Both Cd and Cr treatments led to an increase in hormone concentrations and secondary metabolites only in wild type plants. Analysis of the results suggests that the stress responses due to changes in the plant's hormonal system may derive from the interaction between the GR receptor and phytosteroids, which are known to play a key role in plant physiology and development.

  2. Cellular oxidative stress and the control of apoptosis by wild-type p53, cytotoxic compounds, and cytokines.

    PubMed Central

    Lotem, J; Peled-Kamar, M; Groner, Y; Sachs, L

    1996-01-01

    Apoptosis induced by wild-type p53 or cytotoxic compounds in myeloid leukemic cells can be inhibited by the cytokines interleukin 6, interleukin 3, granulocyte-macrophage colony-stimulating factor, and interferon gamma and by antioxidants. The antioxidants and cytokines showed a cooperative protective effect against induction of apoptosis. Cells with a higher intrinsic level of peroxide production showed a higher sensitivity to induction of apoptosis and required a higher cytokine concentration to inhibit apoptosis. Decreasing the intrinsic oxidative stress in cells by antioxidants thus inhibited apoptosis, whereas increasing this intrinsic stress by adding H2O2 enhanced apoptosis. Induction of apoptosis by wild-type p53 was not preceded by increased peroxide production or lipid peroxidation and the protective effect of cytokines was not associated with a decrease in these properties. The results indicate that the intrinsic degree of oxidative stress can regulate cell susceptibility to wild-type p53-dependent and p53-independent induction of apoptosis and the ability of cytokines to protect cells against apoptosis. Images Fig. 1 Fig. 3 Fig. 4 PMID:8799172

  3. Cellular Oxidative Stress and the Control of Apoptosis by Wild-Type p53, Cytotoxic Compounds, and Cytokines

    NASA Astrophysics Data System (ADS)

    Lotem, Joseph; Peled-Kamar, Mira; Groner, Yoram; Sachs, Leo

    1996-08-01

    Apoptosis induced by wild-type p53 or cytotoxic compounds in myeloid leukemic cells can be inhibited by the cytokines interleukin 6, interleukin 3, granulocyte-macrophage colony-stimulating factor, and interferon γ and by antioxidants. The antioxidants and cytokines showed a cooperative protective effect against induction of apoptosis. Cells with a higher sensitivity to induction of apoptosis and required a higher cytokine concentration to inhibit apoptosis. Decreasing the intrinsic oxidative stress in cells by antioxidants thus inhibited apoptosis, whereas increasing this intrinsic stress by adding H2O2 enhanced apoptosis. Induction of apoptosis by wild-type p53 was not preceded by increased peroxide production or lipid peroxidation and the protective effect of cytokines was not associated with a decrease in these properties. The results indicate that the intrinsic degree of oxidative stress can regulate cell susceptibility to wild-type p53-dependent and p53-independent induction of apoptosis and the ability of cytokines to protect cells against apoptosis.

  4. AAV delivery of wild-type rhodopsin preserves retinal function in a mouse model of autosomal dominant retinitis pigmentosa.

    PubMed

    Mao, Haoyu; James, Thomas; Schwein, Alison; Shabashvili, Arseniy E; Hauswirth, William W; Gorbatyuk, Marina S; Lewin, Alfred S

    2011-05-01

    Autosomal dominant retinitis pigmentosa (ADRP) is frequently caused by mutations in RHO, the gene for rod photoreceptor opsin. Earlier, a study on mice carrying mutated rhodopsin transgenes on either RHO + / +  or RHO + /- backgrounds suggested that the amount of wild-type rhodopsin affected survival of photoreceptors. Therefore, we treated P23H RHO transgenic mice with adeno-associated virus serotype 5 (AAV5) expressing a cDNA clone of the rhodopsin gene (RHO301) that expressed normal opsin from the mouse opsin promoter. Analysis of the electroretinogram (ERG) demonstrated that increased expression of RHO301 slowed the rate of retinal degeneration in P23H mice: at 6 months, a-wave amplitudes were increased by 100% and b-wave amplitudes by 79%. In contrast, nontransgenic mice injected with AAV5 RHO301 demonstrated a decrease in the ERG, confirming the damaging effect of rhodopsin overproduction in normal photoreceptors. In P23H mice, the increase in the ERG amplitudes was correlated with improvement of retinal structure: the thickness of the outer nuclear layer in RHO301-treated eyes was increased by 80% compared with control eyes. These findings suggest that the wild-type RHO gene can be delivered to rescue retinal degeneration in mice carrying a RHO mutation and that increased production of normal rhodopsin can suppress the effect of the mutated protein. These findings make it possible to treat ADRP caused by different mutations of RHO with the expression of wild-type RHO.

  5. Metabolic Engineering of Light and Dark Biochemical Pathways in Wild-Type and Mutant Strains of Synechocystis PCC 6803 for Maximal, 24-Hour Production of Hydrogen Gas

    SciTech Connect

    Ely, Roger L.; Chaplen, Frank W.R.

    2014-03-11

    This project used the cyanobacterial species Synechocystis PCC 6803 to pursue two lines of inquiry, with each line addressing one of the two main factors affecting hydrogen (H2) production in Synechocystis PCC 6803: NADPH availability and O2 sensitivity. H2 production in Synechocystis PCC 6803 requires a very high NADPH:NADP+ ratio, that is, the NADP pool must be highly reduced, which can be problematic because several metabolic pathways potentially can act to raise or lower NADPH levels. Also, though the [NiFe]-hydrogenase in PCC 6803 is constitutively expressed, it is reversibly inactivated at very low O2 concentrations. Largely because of this O2 sensitivity and the requirement for high NADPH levels, a major portion of overall H2 production occurs under anoxic conditions in the dark, supported by breakdown of glycogen or other organic substrates accumulated during photosynthesis. Also, other factors, such as N or S limitation, pH changes, presence of other substances, or deletion of particular respiratory components, can affect light or dark H2 production. Therefore, in the first line of inquiry, under a number of culture conditions with wild type (WT) Synechocystis PCC 6803 cells and a mutant with impaired type I NADPH-dehydrogenase (NDH-1) function, we used H2 production profiling and metabolic flux analysis, with and without specific inhibitors, to examine systematically the pathways involved in light and dark H2 production. Results from this work provided rational bases for metabolic engineering to maximize photobiological H2 production on a 24-hour basis. In the second line of inquiry, we used site-directed mutagenesis to create mutants with hydrogenase enzymes exhibiting greater O2 tolerance. The research addressed the following four tasks: 1. Evaluate the effects of various culture conditions (N, S, or P limitation; light/dark; pH; exogenous organic carbon) on H2 production profiles of WT cells and an NDH-1 mutant; 2. Conduct metabolic flux analyses for

  6. Homo- and hetero-dimerization of human UDP-glucuronosyltransferase 2B7 (UGT2B7) wild type and its allelic variants affect zidovudine glucuronidation activity.

    PubMed

    Yuan, Lingmin; Qian, Sainan; Xiao, Yongsheng; Sun, Hongying; Zeng, Su

    2015-05-01

    Most human UDP-glucuronosyltransferase (UGT; EC 2.4.1.17) genes contain non-synonymous single nucleotide polymorphisms (nsSNPs) which cause amino acid substitutions. Allelic variants caused by nsSNPs may exhibit absent or reduced enzyme activity. UGT2B7 is one of the most important UGTs that glucuronidates abundant endobiotics and xenobiotics, such as estriol, morphine, and anticancer drugs. Three nsSNPs, UGT2B7*71S (211G>T), UGT2B7*2 (802C>T) and UGT2B7*5 (1192G>A) are observed in the UGT2B7 gene, and they code for allozymes UGT2B7*71S (A71S), UGT2B7*2 (H268Y), and UGT2B7*5 (D398N). UGT2B7 has been observed to form oligomers that affect its enzymatic activity and in this study, we investigated protein-protein interactions among UGT2B7 allozymes wild type (WT), A71S, H268Y and D398N, by performing a systematic quantitative fluorescence resonance energy transfer (FRET) analysis in combination with co-immunoprecipitation assay. Quantitative FRET analysis revealed that UGT2B7 allozymes formed homo- and hetero-dimers and showed distinct features in donor-acceptor distances. Both codon 71 and codon 268 in the N-terminal domain were involved in the dimeric interaction. Co-immunoprecipitation experiments also proved that UGT2B7 allozymes formed stable dimers. The glucuronidation activities of homo- and hetero-dimers were further tested with zidovudine as the substrate. An increase in activity was observed when WT hetero-dimerized with A71S compared with homo-dimers, while both H268Y and D398N impaired the activity of WT and A71S by forming hetero-dimers. In addition, zidovudine glucuronidation activity is associated with FRET distance. These findings provide insights into the consequences of amino acid substitution in UGT2B7 on zidovudine glucuronidation and the association between protein-protein interaction and glucuronidation activity. PMID:25770680

  7. Efficacy of Carboplatin Alone and in Combination with ABT888 in Intracranial Murine Models of BRCA-Mutated and BRCA-Wild-Type Triple-Negative Breast Cancer.

    PubMed

    Karginova, Olga; Siegel, Marni B; Van Swearingen, Amanda E D; Deal, Allison M; Adamo, Barbara; Sambade, Maria J; Bazyar, Soha; Nikolaishvili-Feinberg, Nana; Bash, Ryan; O'Neal, Sara; Sandison, Katie; Parker, Joel S; Santos, Charlene; Darr, David; Zamboni, William; Lee, Yueh Z; Miller, C Ryan; Anders, Carey K

    2015-04-01

    Patients with breast cancer brain metastases have extremely limited survival and no approved systemic therapeutics. Triple-negative breast cancer (TNBC) commonly metastasizes to the brain and predicts poor prognosis. TNBC frequently harbors BRCA mutations translating to platinum sensitivity potentially augmented by additional suppression of DNA repair mechanisms through PARP inhibition. We evaluated brain penetrance and efficacy of carboplatin ± the PARP inhibitor ABT888, and investigated gene-expression changes in murine intracranial TNBC models stratified by BRCA and molecular subtype status. Athymic mice were inoculated intracerebrally with BRCA-mutant: SUM149 (basal), MDA-MB-436 (claudin-low); or BRCA-wild-type (wt): MDA-MB-468 (basal), MDA-MB-231BR (claudin-low). TNBC cells were treated with PBS control [intraperitoneal (IP), weekly], carboplatin (50 mg/kg/wk, IP), ABT888 (25 mg/kg/d, oral gavage), or their combination. DNA damage (γ-H2AX), apoptosis (cleaved caspase-3, cC3), and gene expression were measured in intracranial tumors. Carboplatin ± ABT888 significantly improved survival in BRCA-mutant intracranial models compared with control, but did not improve survival in BRCA-wt intracranial models. Carboplatin + ABT888 revealed a modest survival advantage versus carboplatin in BRCA-mutant models. ABT888 yielded a marginal survival benefit in the MDA-MB-436, but not in the SUM149 model. BRCA-mutant SUM149 expression of γ-H2AX and cC3 proteins was elevated in all treatment groups compared with control, whereas BRCA-wt MDA-MB-468 cC3 expression did not increase with treatment. Carboplatin treatment induced common gene-expression changes in BRCA-mutant models. Carboplatin ± ABT888 penetrates the brain and improves survival in BRCA-mutant intracranial TNBC models with corresponding DNA damage and gene-expression changes. Combination therapy represents a potential promising treatment strategy for patients with TNBC brain metastases warranting further

  8. Development of a High-Yield Live Attenuated H7N9 Influenza Virus Vaccine That Provides Protection against Homologous and Heterologous H7 Wild-Type Viruses in Ferrets

    PubMed Central

    Baz, Mariana; Lu, Janine; Paskel, Myeisha; Santos, Celia; Subbarao, Kanta; Jin, Hong; Matsuoka, Yumiko

    2014-01-01

    ABSTRACT Live attenuated H7N9 influenza vaccine viruses that possess the hemagglutinin (HA) and neuraminidase (NA) gene segments from the newly emerged wild-type (wt) A/Anhui/1/2013 (H7N9) and six internal protein gene segments from the cold-adapted influenza virus A/Ann Arbor/6/60 (AA ca) were generated by reverse genetics. The reassortant virus containing the original wt A/Anhui/1/2013 HA and NA sequences replicated poorly in eggs. Multiple variants with amino acid substitutions in the HA head domain that improved viral growth were identified by viral passage in eggs and MDCK cells. The selected vaccine virus containing two amino acid changes (N133D/G198E) in the HA improved viral titer by more than 10-fold (reached a titer of 108.6 fluorescent focus units/ml) without affecting viral antigenicity. Introduction of these amino acid changes into an H7N9 PR8 reassortant virus also significantly improved viral titers and HA protein yield in eggs. The H7N9 ca vaccine virus was immunogenic in ferrets. A single dose of vaccine conferred complete protection of ferrets from homologous wt A/Anhui/1/2013 (H7N9) and nearly complete protection from heterologous wt A/Netherlands/219/2003 (H7N7) challenge infection. Therefore, this H7N9 live attenuated influenza vaccine (LAIV) candidate has been selected for vaccine manufacture and clinical evaluation to protect humans from wt H7N9 virus infection. IMPORTANCE In response to the recent avian H7N9 influenza virus infection in humans, we developed a live attenuated H7N9 influenza vaccine (LAIV) with two amino acid substitutions in the viral HA protein that improved vaccine yield by 10-fold in chicken embryonated eggs, the substrate for vaccine manufacture. The two amino acids also improved the antigen yield for inactivated H7N9 vaccines, demonstrating that this finding could great facilitate the efficiency of H7N9 vaccine manufacture. The candidate H7N9 LAIV was immunogenic and protected ferrets against homologous and heterologous

  9. Analysis of dibenzo[def,p]chrysene-deoxyadenosine adducts in wild-type and cytochrome P450 1b1 knockout mice using stable-isotope dilution UHPLC-MS/MS.

    PubMed

    Harper, Tod A; Morré, Jeff; Lauer, Fredine T; McQuistan, Tammie J; Hummel, Jessica M; Burchiel, Scott W; Williams, David E

    2015-04-01

    The polycyclic aromatic hydrocarbon (PAH), dibenzo[def,p]chrysene (DBC; also known as dibenzo[a,l]pyrene), is a potent carcinogen in animal models and a class 2A human carcinogen. Recent investigations into DBC-mediated toxicity identified DBC as a potent immunosuppressive agent similar to the well-studied immunotoxicant 7,12-dimethylbenz[a]anthracene (DMBA). DBC, like DMBA, is bioactivated by cytochrome P450 (CYP) 1B1 and forms the reactive metabolite DBC-11,12-diol-13,14-epoxide (DBCDE). DBCDE is largely responsible for the genotoxicity associated with DBC exposure. The immunosuppressive properties of several PAHs are also linked to genotoxic mechanisms. Therefore, this study was designed to identify DBCDE-DNA adduct formation in the spleen and thymus of wild-type and cytochrome P450 1b1 (Cyp1b1) knockout (KO) mice using a highly sensitive stable-isotope dilution UHPLC-MS/MS method. Stable-isotope dilution UHPLC-MS/MS identified the major DBC adducts (±)-anti-cis-DBCDE-dA and (±)-anti-trans-DBCDE-dA in the lung, liver, and spleen of both WT and Cyp1b1 KO mice. However, adduct formation in the thymus was below the level of quantitation for our method. Additionally, adduct formation in Cyp1b1 KO mice was significantly reduced compared to wild-type (WT) mice receiving DBC via oral gavage. In conclusion, the current study identifies for the first time DBCDE-dA adducts in the spleen of mice supporting the link between genotoxicity and immunosuppression, in addition to supporting previous studies identifying Cyp1b1 as the primary CYP involved in DBC bioactivation to DBCDE. The high levels of DBC-DNA adducts identified in the spleen, along with the known high levels of Cyp1b1 expression in this organ, supports further investigation into DBC-mediated immunotoxicity.

  10. Analysis of dibenzo[def,p]chrysene-deoxyadenosine adducts in wild-type and cytochrome P450 1b1 knockout mice using stable-isotope dilution UHPLC-MS/MS.

    PubMed

    Harper, Tod A; Morré, Jeff; Lauer, Fredine T; McQuistan, Tammie J; Hummel, Jessica M; Burchiel, Scott W; Williams, David E

    2015-04-01

    The polycyclic aromatic hydrocarbon (PAH), dibenzo[def,p]chrysene (DBC; also known as dibenzo[a,l]pyrene), is a potent carcinogen in animal models and a class 2A human carcinogen. Recent investigations into DBC-mediated toxicity identified DBC as a potent immunosuppressive agent similar to the well-studied immunotoxicant 7,12-dimethylbenz[a]anthracene (DMBA). DBC, like DMBA, is bioactivated by cytochrome P450 (CYP) 1B1 and forms the reactive metabolite DBC-11,12-diol-13,14-epoxide (DBCDE). DBCDE is largely responsible for the genotoxicity associated with DBC exposure. The immunosuppressive properties of several PAHs are also linked to genotoxic mechanisms. Therefore, this study was designed to identify DBCDE-DNA adduct formation in the spleen and thymus of wild-type and cytochrome P450 1b1 (Cyp1b1) knockout (KO) mice using a highly sensitive stable-isotope dilution UHPLC-MS/MS method. Stable-isotope dilution UHPLC-MS/MS identified the major DBC adducts (±)-anti-cis-DBCDE-dA and (±)-anti-trans-DBCDE-dA in the lung, liver, and spleen of both WT and Cyp1b1 KO mice. However, adduct formation in the thymus was below the level of quantitation for our method. Additionally, adduct formation in Cyp1b1 KO mice was significantly reduced compared to wild-type (WT) mice receiving DBC via oral gavage. In conclusion, the current study identifies for the first time DBCDE-dA adducts in the spleen of mice supporting the link between genotoxicity and immunosuppression, in addition to supporting previous studies identifying Cyp1b1 as the primary CYP involved in DBC bioactivation to DBCDE. The high levels of DBC-DNA adducts identified in the spleen, along with the known high levels of Cyp1b1 expression in this organ, supports further investigation into DBC-mediated immunotoxicity. PMID:25868132

  11. Analysis of Dibenzo[def,p]chrysene-Deoxyadenosine Adducts in Wild-Type and Cytochrome P450 1b1 Knockout Mice using Stable-Isotope Dilution UHPLC-MS/MS

    PubMed Central

    Harper, Tod A.; Morré, Jeff; Lauer, Fredine T.; McQuistan, Tammie J.; Hummel, Jessica M.; Burchiel, Scott W.; Williams, David E.

    2015-01-01

    The polycyclic aromatic hydrocarbon (PAH), dibenzo[def,p]chrysene (DBC; also known as dibenzo[a,l]pyrene), is a potent carcinogen in animal models and a class 2A human carcinogen. Recent investigations into DBC-mediated toxicity identified DBC as a potent immunosuppressive agent similar to the well-studied immunotoxicant 7,12-dimethylbenz[a]anthracene (DMBA). DBC, like DMBA, is bioactivated by cytochrome P450 (CYP) 1B1 and forms the reactive metabolite DBC-11,12-diol-13,14-epoxide (DBCDE). DBCDE is largely responsible for the genotoxicity associated with DBC exposure. The immunosuppressive properties of several PAHs are also linked to genotoxic mechanisms. Therefore, this study was designed to identify DBCDE-DNA adduct formation in the spleen and thymus of wild-type and cytochrome P450 1b1 (Cyp1b1) knockout (KO) mice using a highly sensitive stable-isotope dilution UHPLC-MS/MS method. Stable-isotope dilution UHPLC-MS/MS identified the major DBC adducts (±)-anti-cis-DBCDE-dA and (±)-anti-trans-DBCDE-dA in the lung, liver, and spleen of both WT and Cyp1b1 KO mice. However, adduct formation in the thymus was below the level of quantitation for our method. Additionally, adduct formation in Cyp1b1 KO mice was significantly reduced compared to wild-type (WT) mice receiving DBC via oral gavage. In conclusion, the current study identifies for the first time DBCDE-dA adducts in the spleen of mice supporting the link between genotoxicity and immunosuppression, in addition to supporting previous studies identifying Cyp1b1 as the primary CYP involved in DBC bioactivation to DBCDE. The high levels of DBC-DNA adducts identified in the spleen, along with the known high levels of Cyp1b1 expression in this organ, supports further investigation into DBC-mediated immunotoxicity. PMID:25868132

  12. Phytoextraction potential of wild type and 35S-gshI transgenic poplar trees (Populus x Canescens) for environmental pollutants herbicide paraquat, salt sodium, zinc sulfate and nitric oxide in vitro.

    PubMed

    Gyulai, G; Bittsánszky, A; Szabó, Z; Waters, L; Gullner, G; Kampfl, G; Heltai, G; Komíves, T

    2014-01-01

    Phytoextraction potentials of two transgenic (TR) poplar (Populus x canescens) clones TRggs11 and TRlgl6 were compared with that of wild-type (WT) following exposure to paraquat, zinc sulfate, common salt and nitric oxide (NO), using a leaf-disc system incubated for 21 days on EDTA-containing nutritive WPM media in vitro. Glutathione (GSH) contents of leaf discs of TRlgl6 and TRggs11 showed increments to 296% and 190%, respectively, compared with WT. NO exposure led to a twofold GSH content in TRlgl6, which was coupled with a significantly increased sulfate uptake when exposed to 10(-3) M ZnSO4. The highest mineral contents of Na, Zn, Mn, Cu, and Mo was observed in the TRggs11 clone. Salt-induced activity of catalase enzyme increased in both TR clones significantly compared with WT under NaCl (0.75% and 1.5%) exposure. The in silico sequence analyses of gsh1 genes revealed that P. x canadensis and Salix sachalinensis show the closest sequence similarity to that of P. x canescens, which predicted an active GSH production with high phytoextraction potentials of these species with indication for their use where P. x canescens can not be grown.

  13. Thermal conductivity of U-20 wt.%Pu-2 wt.%Am-10 wt.%Zr alloy

    NASA Astrophysics Data System (ADS)

    Nishi, Tsuyoshi; Nakajima, Kunihisa; Takano, Masahide; Kurata, Masaki; Arita, Yuji

    2015-09-01

    The authors fabricated U-20 wt.%Pu-2 wt.%Am-10 wt.%Zr alloys and evaluated the heat capacity and thermal conductivity. The heat capacity was measured between 335 and 827 K by a drop calorimetry. The thermal diffusivity was measured between 300 and 1073 K by a laser flash method. The thermal conductivity was evaluated from the measured thermal diffusivity and heat capacity. The thermal conductivity of U-20 wt.%Pu-2 wt.%Am-10 wt.%Zr alloy was slightly higher than literature values of U-Pu-Zr and U-Pu-MA-Zr alloys. The thermal conductivity was formulated for use in practical design for Generation IV fast reactors.

  14. The Inability of Wild-Type Rabies Virus To Activate Dendritic Cells Is Dependent on the Glycoprotein and Correlates with Its Low Level of the De Novo-Synthesized Leader RNA

    PubMed Central

    Yang, Yang; Huang, Ying; Gnanadurai, Clement W.; Cao, Shengbo; Liu, Xueqin

    2014-01-01

    ABSTRACT Dendritic cells (DCs) are the most efficient antigen-presenting cells, playing a key role in the adaptive immune responses to viral infections. Our studies demonstrate that wild-type (wt) rabies virus (RABV) does not activate DCs. Adoptive transfer of DCs primed with wt RABV did not activate DCs, stimulate virus neutralizing antibodies (VNA), or protect recipients against challenge. However, adoptive transfer of DCs primed with laboratory-attenuated RABV resulted in DC activation, production of VNA, and protection against challenge. In vitro studies with recombinant RABV (laboratory-attenuated RABV expressing the glycoprotein or the phosphoprotein from wt RABV) demonstrate that DC activation is dependent on the glycoprotein and involves the IPS-1 pathway. Furthermore, binding to and entry into DCs by wt RABV is severely blocked, and the copy number of de novo-synthesized leader RNA was two logs lower in DCs infected with the wt than in DCs treated with laboratory-attenuated RABV. However, transient transfection of DCs with synthesized leader RNA from either wt or attenuated RABV is capable of activating DCs in a dose-dependent manner. Thus, the inability of wt RABV to activate DCs correlates with its low level of the de novo-synthesized leader RNA. IMPORTANCE Rabies remains a public health threat, with more than 55,000 fatalities each year around the world. Since DCs play a key role in the adaptive immune responses to viral infections, we investigated the ability of rabies virus (RABV) to activate DCs. It was found that the adoptive transfer of DCs primed with wt RABV did not activate DCs, stimulate VNA, or protect mice against lethal challenge. However, laboratory-attenuated RABV mediates the activation of DCs via the IPS-1 pathway and is glycoprotein dependent. We further show that wt RABV evades DC-mediated immune activation by inefficient binding/entry into DCs and as a result of a reduced level of de novo-synthesized leader RNA. These findings may

  15. Kinetic, mechanistic, and structural modeling studies of truncated wild-type leucine-rich repeat kinase 2 and the G2019S mutant.

    PubMed

    Liu, Min; Kang, Stephanie; Ray, Soumya; Jackson, Justin; Zaitsev, Alexandra D; Gerber, Scott A; Cuny, Gregory D; Glicksman, Marcie A

    2011-11-01

    Leucine-rich repeat kinase 2 (LRRK2), a large and complex protein that possesses two enzymatic properties, kinase and GTPase, is one of the major genetic factors in Parkinson's disease (PD). Here, we characterize the kinetic and catalytic mechanisms of truncated wild-type (t-wt) LRRK2 and its most common mutant, G2019S (t-G2019S), with a structural interpretation of the kinase domain. First, the substitution of threonine with serine in the LRRKtide peptide results in a much less efficient substrate as demonstrated by a 26-fold decrease in k(cat) and a 6-fold decrease in binding affinity. The significant decrease in k(cat) is attributed to a slow chemical transfer step as evidenced by the inverse solvent kinetic isotope effect in the proton inventory and pL (pH or pD)-dependent studies. The shape of the proton inventory and pL profile clearly signals the involvement of a general base (pK(a) = 7.5) in the catalysis with a low fractionation factor in the ground state. We report for the first time that the increased kinase activity of the G2019S mutant is substrate-dependent. Homology modeling of the kinase domain (open and closed forms) and structural analysis of the docked peptide substrates suggest that electrostatic interactions play an important role in substrate recognition, which is affected by G2019S and may directly influence the kinetic properties of the enzyme. Finally, the GTPase activity of the t-G2019S mutant was characterized, and the mutation modestly decreases GTPase activity without significantly affecting GTP binding affinity. PMID:21961647

  16. Methylation of arsenic by recombinant human wild-type arsenic (+ 3 oxidation state) methyltransferase and its methionine 287 threonine (M287T) polymorph: Role of glutathione

    SciTech Connect

    Ding, Lan; Saunders, R. Jesse; Drobná, Zuzana; Walton, Felecia S.; Xun, Pencheng; Thomas, David J.; Stýblo, Miroslav

    2012-10-01

    Arsenic (+ 3 oxidation state) methyltransferase (AS3MT) is the key enzyme in the pathway for methylation of arsenicals. A common polymorphism in the AS3MT gene that replaces a threonyl residue in position 287 with a methionyl residue (AS3MT/M287T) occurs at a frequency of about 10% among populations worldwide. Here, we compared catalytic properties of recombinant human wild-type (wt) AS3MT and AS3MT/M287T in reaction mixtures containing S-adenosylmethionine, arsenite (iAs{sup III}) or methylarsonous acid (MAs{sup III}) as substrates and endogenous or synthetic reductants, including glutathione (GSH), a thioredoxin reductase (TR)/thioredoxin (Trx)/NADPH reducing system, or tris (2-carboxyethyl) phosphine hydrochloride (TCEP). With either TR/Trx/NADPH or TCEP, wtAS3MT or AS3MT/M287T catalyzed conversion of iAs{sup III} to MAs{sup III}, methylarsonic acid (MAs{sup V}), dimethylarsinous acid (DMAs{sup III}), and dimethylarsinic acid (DMAs{sup V}); MAs{sup III} was converted to DMAs{sup III} and DMAs{sup V}. Although neither enzyme required GSH to support methylation of iAs{sup III} or MAs{sup III}, addition of 1 mM GSH decreased K{sub m} and increased V{sub max} estimates for either substrate in reaction mixtures containing TR/Trx/NADPH. Without GSH, V{sub max} and K{sub m} values were significantly lower for AS3MT/M287T than for wtAS3MT. In the presence of 1 mM GSH, significantly more DMAs{sup III} was produced from iAs{sup III} in reactions catalyzed by the M287T variant than in wtAS3MT-catalyzed reactions. Thus, 1 mM GSH modulates AS3MT activity, increasing both methylation rates and yield of DMAs{sup III}. AS3MT genotype exemplified by differences in regulation of wtAS3MT and AS3MT/M287T-catalyzed reactions by GSH may contribute to differences in the phenotype for arsenic methylation and, ultimately, to differences in the disease susceptibility in individuals chronically exposed to inorganic arsenic. -- Highlights: ► Human AS3MT and AS3MT(M287T) require a dithiol

  17. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    SciTech Connect

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; Rathore, Rajendra; Ramchandran, Ramani

    2014-12-18

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.

  18. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    DOE PAGES

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; et al

    2014-12-18

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function.more » We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.« less

  19. Flipping in the Pore: Discovery of Dual Inhibitors That Bind in Different Orientations to the Wild-Type versus the Amantadine-Resistant S31N Mutant of the Influenza A Virus M2 Proton Channel

    PubMed Central

    2015-01-01

    Influenza virus infections lead to numerous deaths and millions of hospitalizations each year. One challenge facing anti-influenza drug development is the heterogeneity of the circulating influenza viruses, which comprise several strains with variable susceptibility to antiviral drugs. For example, the wild-type (WT) influenza A viruses, such as the seasonal H1N1, tend to be sensitive to antiviral drugs, amantadine and rimantadine, while the S31N mutant viruses, such as the pandemic 2009 H1N1 (H1N1pdm09) and seasonal H3N2, are resistant to this class of drugs. Thus, drugs targeting both WT and the S31N mutant are highly desired. We report our design of a novel class of dual inhibitors along with their ion channel blockage and antiviral activities. The potency of the most active compound 11 in inhibiting WT and the S31N mutant influenza viruses is comparable with that of amantadine in inhibiting WT influenza virus. Solution NMR studies and molecular dynamics (MD) simulations of drug-M2 interactions supported our design hypothesis: namely, the dual inhibitor binds in the WT M2 channel with an aromatic group facing down toward the C-terminus, while the same drug binds in the S31N M2 channel with its aromatic group facing up toward the N-terminus. The flip-flop mode of drug binding correlates with the structure–activity relationship (SAR) and has paved the way for the next round of rational design of broad-spectrum antiviral drugs. PMID:25470189

  20. De novo Comparative Transcriptome Analysis of Acremonium chrysogenum: High-Yield and Wild-Type Strains of Cephalosporin C Producer

    PubMed Central

    Liu, Yan; Xie, Liping; Gong, Guihua; Zhang, Wei; Zhu, Baoquan; Hu, Youjia

    2014-01-01

    β-lactam antibiotics are widely used in clinic. Filamentous fungus Acremonium chrysogenum is an important industrial fungus for the production of CPC, one of the major precursors of β-lactam antibiotics. Although its fermentation yield has been bred significantly over the past decades, little is known regarding molecular changes between the industrial strain and the wild type strain. This limits the possibility to improve CPC production further by molecular breeding. Comparative transcriptome is a powerful tool to understand the molecular mechanisms of CPC industrial high yield producer compared to wild type. A total of 57 million clean sequencing reads with an average length of 100 bp were generated from Illumina sequencing platform. 22,878 sequences were assembled. Among the assembled unigenes, 9502 were annotated and 1989 annotated sequences were assigned to 121 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) database. Furthermore, we compared the transcriptome differences between a high-yield and a wild-type strain during fermentation. A total of 4329 unigenes with significantly different transcription level were identified, among which 1737 were up-regulated and 2592 were down-regulated. 24 pathways were subsequently determined which involve glycerolipid metabolism, galactose metabolism, and pyrimidine metabolism. We also examined the transcription levels of 18 identified genes, including 11 up-regulated genes and 7 down-regulated genes using reverse transcription quantitative -PCR (RT-qPCR). The results of RT-qPCR were consistent with the Illumina sequencing. In this study, the Illumina sequencing provides the most comprehensive sequences for gene expression profile of Acremonium chrysogenum and allows de novo transcriptome assembly while lacking genome information. Comparative analysis of RNA-seq data reveals the complexity of the transcriptome in the fermentation of different yield strains. This is an important

  1. Extracellular enzyme activities during lignocellulose degradation by Streptomyces spp. : a comparative study of wild-type and genetically manipulated strains

    SciTech Connect

    Ramachandra, M.; Crawford, D.L.; Pometto, A.L. III

    1987-12-01

    The wild-type ligninolytic actinomycete Streptomyces viridosporus T7A and two genetically manipulated strains with enhanced abilities to produce a water-soluble lignin degradation intermediate, an acid-precipitable polymeric lignin (APPL), were grown on lignocellulose in solid-state fermentation cultures. Culture filtrates were periodically collected, analyzed for APPL, and assayed for extracellular lignocellulose-catabolizing enzyme activities. Two APPL-overproducing strains, UV irradiation mutant T7A-81 and protoplast fusion recombinant SR-10, had higher and longer persisting peroxidase, esterase, and endoglucanase activities than did the wild-type strain T7A. Results implicated one or more of these enzymes in lignin solubilization. Only mutant T7A-81 had higher xylanase activity than the wild type. The peroxidase was induced by both lignocellulose and APPL. This extracellular enzyme has some similarities to previously described ligninases in fungi. This is the first report of such an enzyme in Streptomyces spp. Four peroxidase isozymes were present, and all catalyzed the oxidation of 3,4-dihydroxyphenylalanine, while one also catalyzed hydrogen peroxide-dependent oxidation of homoprotocatechuic acid and caffeic acid. Three constitutive esterase isozymes were produced which differed in substrate specificity toward ..cap alpha..-naphthyl acetate and ..cap alpha..-naphthyl butyrate. Three endoglucanase bands, which also exhibited a low level of xylanase activity, were identified on polyacrylamide gels as was one xylanase-specific band. There were no major differences in the isoenzymes produced by the different strains. The probable role of each enzyme in lignocellulose degradation is discussed.

  2. Photosystem II Activity of Wild Type Synechocystis PCC 6803 and Its Mutants with Different Plastoquinone Pool Redox States.

    PubMed

    Voloshina, O V; Bolychevtseva, Y V; Kuzminov, F I; Gorbunov, M Y; Elanskaya, I V; Fadeev, V V

    2016-08-01

    To assess the role of redox state of photosystem II (PSII) acceptor side electron carriers in PSII photochemical activity, we studied sub-millisecond fluorescence kinetics of the wild type Synechocystis PCC 6803 and its mutants with natural variability in the redox state of the plastoquinone (PQ) pool. In cyanobacteria, dark adaptation tends to reduce PQ pool and induce a shift of the cyanobacterial photosynthetic apparatus to State 2, whereas illumination oxidizes PQ pool, leading to State 1 (Mullineaux, C. W., and Holzwarth, A. R. (1990) FEBS Lett., 260, 245-248). We show here that dark-adapted Ox(-) mutant with naturally reduced PQ is characterized by slower QA(-) reoxidation and O2 evolution rates, as well as lower quantum yield of PSII primary photochemical reactions (Fv/Fm) as compared to the wild type and SDH(-) mutant, in which the PQ pool remains oxidized in the dark. These results indicate a large portion of photochemically inactive PSII reaction centers in the Ox(-) mutant after dark adaptation. While light adaptation increases Fv/Fm in all tested strains, indicating PSII activation, by far the greatest increase in Fv/Fm and O2 evolution rates is observed in the Ox(-) mutant. Continuous illumination of Ox(-) mutant cells with low-intensity blue light, that accelerates QA(-) reoxidation, also increases Fv/Fm and PSII functional absorption cross-section (590 nm); this effect is almost absent in the wild type and SDH(-) mutant. We believe that these changes are caused by the reorganization of the photosynthetic apparatus during transition from State 2 to State 1. We propose that two processes affect the PSII activity during changes of light conditions: 1) reversible inactivation of PSII, which is associated with the reduction of electron carriers on the PSII acceptor side in the dark, and 2) PSII activation under low light related to the increase in functional absorption cross-section at 590 nm.

  3. Photosystem II Activity of Wild Type Synechocystis PCC 6803 and Its Mutants with Different Plastoquinone Pool Redox States.

    PubMed

    Voloshina, O V; Bolychevtseva, Y V; Kuzminov, F I; Gorbunov, M Y; Elanskaya, I V; Fadeev, V V

    2016-08-01

    To assess the role of redox state of photosystem II (PSII) acceptor side electron carriers in PSII photochemical activity, we studied sub-millisecond fluorescence kinetics of the wild type Synechocystis PCC 6803 and its mutants with natural variability in the redox state of the plastoquinone (PQ) pool. In cyanobacteria, dark adaptation tends to reduce PQ pool and induce a shift of the cyanobacterial photosynthetic apparatus to State 2, whereas illumination oxidizes PQ pool, leading to State 1 (Mullineaux, C. W., and Holzwarth, A. R. (1990) FEBS Lett., 260, 245-248). We show here that dark-adapted Ox(-) mutant with naturally reduced PQ is characterized by slower QA(-) reoxidation and O2 evolution rates, as well as lower quantum yield of PSII primary photochemical reactions (Fv/Fm) as compared to the wild type and SDH(-) mutant, in which the PQ pool remains oxidized in the dark. These results indicate a large portion of photochemically inactive PSII reaction centers in the Ox(-) mutant after dark adaptation. While light adaptation increases Fv/Fm in all tested strains, indicating PSII activation, by far the greatest increase in Fv/Fm and O2 evolution rates is observed in the Ox(-) mutant. Continuous illumination of Ox(-) mutant cells with low-intensity blue light, that accelerates QA(-) reoxidation, also increases Fv/Fm and PSII functional absorption cross-section (590 nm); this effect is almost absent in the wild type and SDH(-) mutant. We believe that these changes are caused by the reorganization of the photosynthetic apparatus during transition from State 2 to State 1. We propose that two processes affect the PSII activity during changes of light conditions: 1) reversible inactivation of PSII, which is associated with the reduction of electron carriers on the PSII acceptor side in the dark, and 2) PSII activation under low light related to the increase in functional absorption cross-section at 590 nm. PMID:27677553

  4. Novel software for analysis of root gravitropism: comparative response patterns of Arabidopsis wild-type and axr1 seedlings

    NASA Technical Reports Server (NTRS)

    Ishikawa, H.; Evans, M. L.

    1997-01-01

    In an earlier study (Evans, Ishikawa & Estelle 1994, Planta 194, 215-222) we used a video digitizer system to compare the kinetics of auxin action on root elongation in wild-type seedlings and seedlings of auxin response mutants of Arabidopsis thaliana (L.) Heynh. We have since modified the system software to allow determination of elongation on opposite sides of vertical or gravistimulated roots and to allow continuous measurement of the angle of orientation of sequential subsections of the root during the response. We used this technology to compare the patterns of differential growth that generate curvature in roots of the Columbia ecotype and in the mutants axr1-3, axr1-12 and axr2, which show reduced gravitropic responsiveness and reduced sensitivity to inhibition by auxin. The pattern of differential growth during gravitropism differed in roots of wild-type and axr1 seedlings. In wild-type roots, initial curvature resulted from differential inhibition of elongation in the distal elongation zone (DEZ). This was followed by an acceleration of elongation along the top side of the DEZ. In roots of axr1-3, curvature resulted from differential stimulation of elongation whereas in roots of axr1-12 the response was variable. Roots of axr2 did not exhibit gravitropic curvature. The observation that the pattern of differential growth causing curvature is dramatically altered by a change in sensitivity to auxin is consistent with the classical Cholodny-Went theory of gravitropism which maintains that differential growth patterns induced by gravistimulation are mediated primarily by gravi-induced shifts in auxin distribution. The new technology introduced with this report allows automated determination of stimulus response patterns in the small but experimentally popular roots of Arabidopsis.

  5. Accumulation of a bioactive benzoisochromanequinone compound kalafungin by a wild type antitumor-medermycin-producing streptomycete strain.

    PubMed

    Lü, Jin; He, Qiang; Huang, Luyao; Cai, Xiaofeng; Guo, Wenwen; He, Jing; Zhang, Lili; Li, Aiying

    2015-01-01

    Medermycin and kalafungin, two antibacterial and antitumor antibiotics isolated from different streptomycetes, share an identical polyketide skeleton core. The present study reported the discovery of kalafungin in a medermycin-producing streptomycete strain for the first time. A mutant strain obtained through UV mutagenesis showed a 3-fold increase in the production of this antibiotic, compared to the wild type strain. Heterologous expression experiments suggested that its production was severely controlled by the gene cluster for medermycin biosynthesis. In all, these findings suggested that kalafungin and medermycin could be accumulated by the same streptomycete and share their biosynthetic pathway to some extent in this strain. PMID:25695632

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

    PubMed Central

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

    1998-01-01

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

  7. Accumulation of a Bioactive Benzoisochromanequinone Compound Kalafungin by a Wild Type Antitumor-Medermycin-Producing Streptomycete Strain

    PubMed Central

    Lü, Jin; He, Qiang; Huang, Luyao; Cai, Xiaofeng; Guo, Wenwen; He, Jing; Zhang, Lili; Li, Aiying

    2015-01-01

    Medermycin and kalafungin, two antibacterial and antitumor antibiotics isolated from different streptomycetes, share an identical polyketide skeleton core. The present study reported the discovery of kalafungin in a medermycin-producing streptomycete strain for the first time. A mutant strain obtained through UV mutagenesis showed a 3-fold increase in the production of this antibiotic, compared to the wild type strain. Heterologous expression experiments suggested that its production was severely controlled by the gene cluster for medermycin biosynthesis. In all, these findings suggested that kalafungin and medermycin could be accumulated by the same streptomycete and share their biosynthetic pathway to some extent in this strain. PMID:25695632

  8. Accumulation of a bioactive benzoisochromanequinone compound kalafungin by a wild type antitumor-medermycin-producing streptomycete strain.

    PubMed

    Lü, Jin; He, Qiang; Huang, Luyao; Cai, Xiaofeng; Guo, Wenwen; He, Jing; Zhang, Lili; Li, Aiying

    2015-01-01

    Medermycin and kalafungin, two antibacterial and antitumor antibiotics isolated from different streptomycetes, share an identical polyketide skeleton core. The present study reported the discovery of kalafungin in a medermycin-producing streptomycete strain for the first time. A mutant strain obtained through UV mutagenesis showed a 3-fold increase in the production of this antibiotic, compared to the wild type strain. Heterologous expression experiments suggested that its production was severely controlled by the gene cluster for medermycin biosynthesis. In all, these findings suggested that kalafungin and medermycin could be accumulated by the same streptomycete and share their biosynthetic pathway to some extent in this strain.

  9. Inhibitory activity of oxyresveratrol on wild-type and drug-resistant varicella-zoster virus replication in vitro.

    PubMed

    Sasivimolphan, Pattaraporn; Lipipun, Vimolmas; Likhitwitayawuid, Kittisak; Takemoto, Masaya; Pramyothin, Pornpen; Hattori, Masao; Shiraki, Kimiyasu

    2009-10-01

    The anti-herpes simplex virus (HSV) compound, oxyresveratrol, purified from a Thai traditional medicinal plant of Artocarpus lakoocha, was evaluated for its anti-varicella-zoster virus (VZV) activity. This compound exhibited IC(50) values (50%-inhibitory concentrations for virus plaque formation) of 12.82, 12.80, 12.99 and 12.82 microg/ml against wild type, thymidine kinase-deficient and two types of DNA polymerase mutants with acyclovir-resistance, respectively. Thus oxyresveratrol showed a broad spectrum of anti-VZV activity with a mechanism of action different from that of acyclovir.

  10. Induction of lytic pathways in T cell clones derived from wild-type or protein tyrosine kinase Fyn mutant mice.

    PubMed

    Lancki, D W; Fields, P; Qian, D; Fitch, F W

    1995-08-01

    The OVA-reactive CD4+ Th1 clones and alloreactive CD8+ clones derived from wild-type or fyn-/- mice serve as model systems which have allowed us to investigate several aspects of the molecular events associated with T cell-mediated cytotoxicity, including 1) the differential utilization of two distinct cytolytic pathways by CD4+ Th1 clones and CD8+ CTL, 2) a comparison of the pathways of lysis induced by stimulation of the TCR or by alternative stimuli, 3) the requirement of Fyn for derivation of antigen-specific T-cell clones having properties of CD4+ Th1 and CD8+ CTL cells 4) the differential requirement of Fyn in the induction of responses by TCR and the alternative stimuli. Stimulation through the TCR, either by APC bearing relevant antigen or by immobilized anti-CD3 mAb, resulted in comparable levels of target cell lysis by clones from both wild-type and fyn-/- mice. These clones also utilize the Fas pathway to lyse target cells. Thus, Fyn does not appear to be required for expression of the Fas pathway when triggered through the TCR. In contrast, lysis of target cells by T-cell clones lacking Fyn was deficient when stimulated through Thy-1 or Ly-6C (using mAb) or with Con A or phorbol ester as compared to clones derived from wild-type mice. The basis for the defect in response to stimulation through the GPI-linked molecules appears to be a signaling defect which affects all of the functional responses we measured, while the defect in response to Con A stimulation appears to affect lysis but not lymphokine production. Thus, Fyn expression is selectively required for efficient activation of the Fas pathway of lysis through Thy-1, Ly-6C, and by Con A or phorbol ester in these T-cell clones. CD8+ clones derived from fyn-/- mutant mice, like clones derived from wild-type mice, display antigen-specific lysis, and appear to express perforin message and perforin protein. A Ca(++)-dependent (presumably perforin/exocytosis) component and Fas component of lysis was

  11. Events Surrounding the Early Development of Euglena Chloroplasts: 14. Biosynthesis of Cytochrome c-552 in Wild Type and Mutant Cells.

    PubMed

    Freyssinet, G; Harris, G C; Nasatir, M; Schiff, J A

    1979-05-01

    Lack of a suitable assay has thwarted attempts to measure cytochrome c-552 in dark-grown wild type cells of Euglena gracilis var. bacillaris in mutants and in other situations where the concentrations are low. Purification methods are described based on electrofocusing which provide a cytochrome c-552 preparation homogeneous enough to elicit a single reactive antibody in rabbits; this antibody is then used as a specific and sensitive assay for cytochrome c-552. Dark-grown cells of wild type and of mutants O(1)BS, O(2)BX, G(1)BU and P(1)BXL (which make normal sized chloroplasts with abnormal internal structure in the light) have 0.02 to 0.1 x 10(-11) micromoles of cytochrome c-552 per cell, 10 to 150 times less than light-grown cells. Light-grown cells of these mutants and of wild type show a ratio of chlorophyll to cytochrome of about 300 (mole to mole). Cytochrome c-552 is undetectable in dark-grown Y(1)BXD, Y(3)BUD, and W(34)ZUD which cannot carry plastid development beyond the proplastid in light; the light-grown cells of these mutants have levels of cytochrome similar to or lower than dark-grown wild type cells. Cytochrome c-552 is undetectable in light- and dark-grown mutants in which plastid DNA is undetectable (such as Y(2)BUL, W(3)BUL, W(8)BHL, and W(10)BSmL) consistent with the view, but not proving, that this molecule may be coded, at least in part, in plastid DNA. During light-induced chloroplast development in resting cells, cytochrome c-552 formation behaves in all respects like chlorophyll except that the dark-grown cells contain low amounts of the cytochrome c-552 but lack chlorophyll. Thus, both cytochrome c-552 and chlorophyll show the same lag period even when the length is changed by nutritional manipulation; preillumination largely eliminates the lag in the formation of both molecules, cycloheximide and streptomycin both inhibit the biosynthesis of chlorophyll and cytochrome c-552 in the same manner, and the formation of both during chloroplast

  12. Different dynamic movements of wild-type and pathogenic VCPs and their cofactors to damaged mitochondria in a Parkin-mediated mitochondrial quality control system.

    PubMed

    Kimura, Yoko; Fukushi, Junpei; Hori, Seiji; Matsuda, Noriyuki; Okatsu, Kei; Kakiyama, Yukie; Kawawaki, Junko; Kakizuka, Akira; Tanaka, Keiji

    2013-12-01

    VCP/p97 is a hexameric ring-shaped AAA(+) ATPase that participates in various ubiquitin-associated cellular functions. Mis-sense mutations in VCP gene are associated with the pathogenesis of two inherited diseases: inclusion body myopathy associated with Paget's disease of the bone and front-temporal dementia (IBMPFD) and familial amyotrophic lateral sclerosis (ALS). These pathogenic VCPs have higher affinities for several cofactors, including Npl4, Ufd1 and p47. In Parkin-dependent mitochondrial quality control systems, VCP migrates to damaged mitochondria (e.g., those treated with uncouplers) to aid in the degradation of mitochondrial outer membrane proteins and to eliminate mitochondria. We showed that endogenous Npl4 and p47 also migrate to mitochondria after uncoupler treatment, and Npl4, Ufd1 or p47 silencing causes defective mitochondria clearance after uncoupler treatment. Moreover, pathogenic VCPs show impaired migration to mitochondria, and the exogenous pathogenic VCP expression partially inhibits Npl4 and p47 localization to mitochondria. These results suggest that the increased affinities of pathogenic VCPs for these cofactors cause the impaired movement of pathogenic VCPs. In adult flies, exogenous expression of wild-type VCP, but not pathogenic VCPs, reduces the number of abnormal mitochondria in muscles. Failure of pathogenic VCPs to function on damaged mitochondria may be related to the pathogenesis of IBMPFD and ALS.

  13. Vitamin D2-Enriched Button Mushroom (Agaricus bisporus) Improves Memory in Both Wild Type and APPswe/PS1dE9 Transgenic Mice

    PubMed Central

    Bennett, Louise; Kersaitis, Cindy; Macaulay, Stuart Lance; Münch, Gerald; Niedermayer, Garry; Nigro, Julie; Payne, Matthew; Sheean, Paul; Vallotton, Pascal; Zabaras, Dimitrios; Bird, Michael

    2013-01-01

    Vitamin D deficiency is widespread, affecting over 30% of adult Australians, and increasing up to 80% for at-risk groups including the elderly (age>65). The role for Vitamin D in development of the central nervous system is supported by the association between Vitamin D deficiency and incidence of neurological and psychiatric disorders including Alzheimer’s disease (AD). A reported positive relationship between Vitamin D status and cognitive performance suggests that restoring Vitamin D status might provide a cognitive benefit to those with Vitamin D deficiency. Mushrooms are a rich source of ergosterol, which can be converted to Vitamin D2 by treatment with UV light, presenting a new and convenient dietary source of Vitamin D2. We hypothesised that Vitamin D2-enriched mushrooms (VDM) could prevent the cognitive and pathological abnormalities associated with dementia. Two month old wild type (B6C3) and AD transgenic (APPSwe/PS1dE9) mice were fed a diet either deficient in Vitamin D2 or a diet which was supplemented with VDM, containing 1±0.2 µg/kg (∼54 IU/kg) vitamin D2, for 7 months. Effects of the dietary intervention on memory were assessed pre- and post-feeding. Brain sections were evaluated for amyloid β (Aβ) plaque loads and inflammation biomarkers using immuno-histochemical methods. Plasma vitamin D metabolites, Aβ40, Aβ42, calcium, protein and cholesterol were measured using biochemical assays. Compared with mice on the control diet, VDM-fed wild type and AD transgenic mice displayed improved learning and memory, had significantly reduced amyloid plaque load and glial fibrillary acidic protein, and elevated interleukin-10 in the brain. The results suggest that VDM might provide a dietary source of Vitamin D2 and other bioactives for preventing memory-impairment in dementia. This study supports the need for a randomised clinical trial to determine whether or not VDM consumption can benefit cognitive performance in the wider population. PMID

  14. Evaluation of antibody-dependent cell-mediated cytotoxicity activity and cetuximab response in KRAS wild-type metastatic colorectal cancer patients

    PubMed Central

    Lo Nigro, Cristiana; Ricci, Vincenzo; Vivenza, Daniela; Monteverde, Martino; Strola, Giuliana; Lucio, Francesco; Tonissi, Federica; Miraglio, Emanuela; Granetto, Cristina; Fortunato, Mirella; Merlano, Marco Carlo

    2016-01-01

    AIM: To investigate the prognostic role of invariant natural killer T (iNKT) cells and antibody-dependent cell-mediated cytotoxicity (ADCC) in wild type KRAS metastatic colorectal cancer (mCRC) patients treated with cetuximab. METHODS: Forty-one KRAS wt mCRC patients, treated with cetuximab and irinotecan-based chemotherapy in II and III lines were analyzed. Genotyping of single nucleotide polymorphism (SNP)s in the FCGR2A, FCGR3A and in the 3’ untranslated regions of KRAS and mutational analysis for KRAS, BRAF and NRAS genes was determined either by sequencing or allelic discrimination assays. Enriched NK cells were obtained from lymphoprep-peripheral blood mononuclear cell and iNKT cells were defined by co-expression of CD3, TCRVα24, TCRVβ11. ADCC was evaluated as ex vivo NK-dependent activity, measuring lactate dehydrogenase release. RESULTS: At basal, mCRC patients performing ADCC activity above the median level (71%) showed an improved overall survival (OS) compared to patients with ADCC below (median 16 vs 8 mo; P = 0.026). We did not find any significant correlation of iNKT cells with OS (P = 0.19), albeit we observed a trend to a longer survival after 10 mo in patients with iNKT above median basal level (0.382 cells/microliter). Correlation of OS and progression-free survival (PFS) with interesting SNPs involved in ADCC ability revealed not to be significant. Patients carrying alleles both with A in FCGR2A and TT in FCGR3A presented a trend of longer PFS (median 9 vs 5 mo; P = 0.064). Chemotherapy impacted both iNKT cells and ADCC activity. Their prognostic values get lost when we analysed them after 2 and 4 mo of treatment. CONCLUSION: Our results suggest a link between iNKT cells, basal ADCC activity, genotypes in FCGR2A and FCGR3A, and efficacy of cetuximab in KRAS wt mCRC patients. PMID:26909137

  15. Formation of DNA adducts in wild-type and transgenic mice expressing human sulfotransferases 1A1 and 1A2 after oral exposure to furfuryl alcohol

    PubMed Central

    Høie, Anja Hortemo; Monien, Bernhard Hans; Sakhi, Amrit Kaur; Glatt, Hansruedi; Hjertholm, Hege; Husøy, Trine

    2015-01-01

    Furfuryl alcohol (FFA) is present in many heat-treated foods as a result of its formation via dehydration of pentoses. It is also used legally as a flavouring agent. In an inhalation study conducted in the National Toxicology Program, FFA showed some evidence of carcinogenic activity in rats and mice. FFA was generally negative in conventional genotoxicity assays, which suggests that it may be a non-genotoxic carcinogen. However, it was recently found that FFA is mutagenic in Salmonella strains expressing appropriate sulfotransferases (SULTs), such as human or mouse SULT1A1. The same DNA adducts that were formed by FFA in these strains, mainly N 2-((furan-2-yl)methyl)-2′-deoxyguanosine (N 2-MF-dG), were also detected in tissues of FFA-exposed mice and even in human lung specimens. In the present study, a single oral dose of FFA (250mg/kg body weight) or saline was administered to FVB/N mice and transgenic mice expressing human SULT1A1/1A2 on the FVB/N background. The transgenic mice were used, since human and mouse SULT1A1 substantially differ in substrate specificity and tissue distribution. DNA adducts were studied in liver, kidney, proximal and distal small intestine as well as colon, using isotope-dilution ultra performance liquid chromatography (UPLC–MS/MS). Surprisingly, low levels of adducts that may represent N 2-MF-dG were detected even in tissues of untreated mice. FFA exposure enhanced the adduct levels in colon and liver, but not in the remaining investigated tissues of wild-type (wt) mice. The situation was similar in transgenic mice, except that N 2-MF-dG levels were also strongly enhanced in the proximal small intestine. These different results between wt and transgenic mice may be attributed to the fact that human SULT1A1, but not the orthologous mouse enzyme, is strongly expressed in the small intestine. PMID:25904584

  16. Phosphate Homeostasis in Conditions of Phosphate Proficiency and Limitation in the Wild Type and the phoP Mutant of Streptomyces lividans.

    PubMed

    Smirnov, Aleksey; Esnault, Catherine; Prigent, Magali; Holland, Ian Barry; Virolle, Marie-Joelle

    2015-01-01

    Phosphate, as a constituent of the high energy molecules, ATP/GTP and polyphosphate, plays a crucial role in most of the metabolic processes of living organisms. Therefore, the adaptation to low Pi availability is a major challenge for bacteria. In Streptomyces, this adaptation is tightly controlled by the two component PhoR/PhoP system. In this study, the free intracellular Pi, ATP, ADP and polyP content of the wild type and the phoP mutant strain of S. lividans TK24 were analyzed at discrete time points throughout growth in Pi replete and limited media. PolyP length and content was shown to be directly related to the Pi content of the growth medium. In Pi repletion, ATP and high molecular weight (HMW) polyP contents were higher in the phoP mutant than in the WT strain. This supports the recently proposed repressive effect of PhoP on oxidative phosphorylation. High oxidative phosphorylation activity might also have a direct or indirect positive impact on HMW polyP synthesis. In Pi sufficiency as in Pi limitation, the degradation of these polymers was shown to be clearly delayed in the phoP mutant, indicating PhoP dependent expression of the enzymes involved in this degradation. The efficient storage of Pi as polyphosphate and/or its inefficient degradation in Pi in the phoP mutant resulted in low levels of free Pi and ATP that are likely to be, at least in part, responsible for the very poor growth of this mutant in Pi limitation. Furthermore, short polyP was shown to be present outside the cell, tightly bound to the mycelium via electrostatic interactions involving divalent cations. Less short polyP was found to be associated with the mycelium of the phoP mutant than with that of the WT strain, indicating that generation and externalization of these short polyP molecules was directly or indirectly dependent on PhoP.

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

    PubMed

    Goldmann, W H; Ezzell, R M

    1996-07-10

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

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

    PubMed

    Goldmann, W H; Ezzell, R M

    1996-07-10

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

  19. Combination of cetuximab and PP242 synergistically suppress the progression of wild-type KRAS colorectal carcinoma.

    PubMed

    Cheng, Lei; Xia, Zuguang; Bian, Xinyu; Li, Guangchao; Hu, Jing; Cao, Ya; Wang, Qing; Qian, Xiaoping

    2015-01-01

    Mammalian target of rapamycin (mTOR) has been shown to be overactive in human colorectal cancer, but the first-generation mTOR inhibitor, rapamycin, has failed to show clinical efficacy against colorectal cancer. On the other hand, although the second-generation mTOR inhibitor, PP242, has exerted substantial efficacy, it was revealed that independent inhibition by PP242 was transient, which could lead to positive-feedback loop to EGFR. Using wild-type KRAS colorectal cancer cells as models, we investigate the treatment efficacy of a widely used anti-EGFR monoclonal antibody, cetuximab, and PP242, alone or in combination in vitro and in vivo. Results of cell viability assays confirmed the synergistic inhibitory effect of PP242 and cetuximab on the survival of Caco-2 and HT-29 cells. Moreover, the ability of cancer-cell invasion and proliferation was also significantly inhibited by the combination therapy when compared with cetuximab or PP242 alone. Interestingly, the percentage of CD44-positive cancer cells was substantially decreased by the combination therapy in comparison with PP242 alone through fluorescence-activated cell sorting. The growth of cancer stem-like cell spheres in vitro was also maximally inhibited by combination therapy, in terms of either diameter or number. More importantly, the efficacy of combination therapy was more prominent than either drug alone in established tumor xenografts. These findings supported the potential use of combination therapy of PP242 and cetuximab against wild-type KRAS colorectal carcinomas.

  20. Discovery of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin in wild-type cells

    PubMed Central

    Morkunas, Bernardas; Gal, Balint; Galloway, Warren R J D; Hodgkinson, James T; Ibbeson, Brett M; Sing Tan, Yaw; Welch, Martin

    2016-01-01

    Summary Pyocyanin is a small molecule produced by Pseudomonas aeruginosa that plays a crucial role in the pathogenesis of infections by this notorious opportunistic pathogen. The inhibition of pyocyanin production has been identified as an attractive antivirulence strategy for the treatment of P. aeruginosa infections. Herein, we report the discovery of an inhibitor of pyocyanin production in cultures of wild-type P. aeruginosa which is based around a 4-alkylquinolin-2(1H)-one scaffold. To the best of our knowledge, this is the first reported example of pyocyanin inhibition by a compound based around this molecular framework. The compound may therefore be representative of a new structural sub-class of pyocyanin inhibitors, which could potentially be exploited in in a therapeutic context for the development of critically needed new antipseudomonal agents. In this context, the use of wild-type cells in this study is notable, since the data obtained are of direct relevance to native situations. The compound could also be of value in better elucidating the role of pyocyanin in P. aeruginosa infections. Evidence suggests that the active compound reduces the level of pyocyanin production by inhibiting the cell–cell signalling mechanism known as quorum sensing. This could have interesting implications; quorum sensing regulates a range of additional elements associated with the pathogenicity of P. aeruginosa and there is a wide range of other potential applications where the inhibition of quorum sensing is desirable. PMID:27559393

  1. Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium graminearum.

    PubMed

    Jansen, Carin; von Wettstein, Diter; Schäfer, Wilhelm; Kogel, Karl-Heinz; Felk, Angelika; Maier, Frank J

    2005-11-15

    Fusarium head blight epidemics of wheat and barley cause heavy economic losses to farmers due to yield decreases and production of mycotoxin that renders the grain useless for flour and malt products. No highly resistant cultivars are available at present. Hyphae of germinating fungal spores use different paths of infection: After germination at the extruded tip of an ovary, the hyphae travel along the epicarp in the space between the lemma and palea. Infection of the developing kernel proceeds through the epicarp, successively destroying the layers of the fruit coat and finally the starch and protein accumulating endosperm. Hyphae reaching the rachis proceed to apically located developing kernels. Using a constitutively green fluorescence protein-expressing Fusarium wild-type strain, and its knockout mutant, preventing trichothecene synthesis, we demonstrate that trichothecenes are not a virulence factor during infection through the fruit coat. In the absence of trichothecenes, the fungus is blocked by the development of heavy cell wall thickenings in the rachis node of Nandu wheat, a defense inhibited by the mycotoxin. In barley hyphae of both wild-type and the trichothecene knockout mutant, are inhibited at the rachis node and rachilla, limiting infection of adjacent florets through the phloem and along the surface of the rachis. Effective resistance to Fusarium head blight requires expression of genes that combat these different pathways of infection.

  2. Lymphocyte invasion in IC10/Basal-like breast tumors is associated with wild-type TP53

    PubMed Central

    Quigley, David; Silwal-Pandit, Laxmi; Dannenfelser, Ruth; Langerød, Anita; Vollan, Hans Kristian Moen; Vaske, Charles; Siegel, Josie Ursini; Troyanskaya, Olga; Chin, Suet-Feung; Caldas, Carlos; Balmain, Allan

    2014-01-01

    Lymphocytic infiltration is associated with better prognosis in several epithelial malignancies including breast cancer. The tumor suppressor TP53 is mutated in approximately 30% of breast adenocarcinomas, with varying frequency across molecular subtypes. In this study of 1,420 breast tumors, we tested for interaction between TP53 mutation status and tumor subtype determined by PAM50 and Integrative Cluster analysis. In Integrative Cluster 10 (IC10)/Basal-like breast cancer we identify an association between lymphocytic infiltration, determined by an expression score, and retention of wild-type TP53. The expression-derived score agreed with the degree of lymphocytic infiltration assessed by pathological review, and application of the Nanodissect algorithm was suggestive of this infiltration being primarily of cytotoxic T lymphocytes (CTL). Elevated expression of this CTL signature was associated with longer survival in IC10/Basal-like tumors. These findings identify a new link between the TP53 pathway and the adaptive immune response in ER-negative breast tumors, suggesting a connection between TP53 inactivation and failure of tumor immunosurveillance. IMPLICATIONS The association of lymphocytic invasion of estrogen receptor-negative breast tumors with the retention of wild-type TP53 implies a novel protective connection between TP53 function and tumor immunosurveillance. PMID:25351767

  3. Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium graminearum

    PubMed Central

    Jansen, Carin; von Wettstein, Diter; Schäfer, Wilhelm; Kogel, Karl-Heinz; Felk, Angelika; Maier, Frank J.

    2005-01-01

    Fusarium head blight epidemics of wheat and barley cause heavy economic losses to farmers due to yield decreases and production of mycotoxin that renders the grain useless for flour and malt products. No highly resistant cultivars are available at present. Hyphae of germinating fungal spores use different paths of infection: After germination at the extruded tip of an ovary, the hyphae travel along the epicarp in the space between the lemma and palea. Infection of the developing kernel proceeds through the epicarp, successively destroying the layers of the fruit coat and finally the starch and protein accumulating endosperm. Hyphae reaching the rachis proceed to apically located developing kernels. Using a constitutively green fluorescence protein-expressing Fusarium wild-type strain, and its knockout mutant, preventing trichothecene synthesis, we demonstrate that trichothecenes are not a virulence factor during infection through the fruit coat. In the absence of trichothecenes, the fungus is blocked by the development of heavy cell wall thickenings in the rachis node of Nandu wheat, a defense inhibited by the mycotoxin. In barley hyphae of both wild-type and the trichothecene knockout mutant, are inhibited at the rachis node and rachilla, limiting infection of adjacent florets through the phloem and along the surface of the rachis. Effective resistance to Fusarium head blight requires expression of genes that combat these different pathways of infection. PMID:16263921

  4. Intra-host competition between nef-defective escape mutants and wild-type human immunodeficiency virus type 1.

    PubMed Central

    Altes, H K; Jansen, V A

    2000-01-01

    Various forms of nef genes with deletions at conserved positions along the sequence have been reported to persist in human immunodeficiency virus type 1 infected patients. We investigate the forces maintaining such variants in the proviral population. The main selection pressures are preservation of function and host immune response. The crippled Nef protein might have fewer epitopes, and as such be less visible to the specific immune response, but it will lose some function. Does a trade-off between avoidance of the immune response and loss of function explain the dynamics of the crippled virus found in the patients? To answer this question, we formulated a deterministic model of the virus-host interactions. We found that when the crippled protein presents few epitopes and suffers little loss of function, the two viral types can coexist. Otherwise, the wild-type comes to prevail. The mutant form might initially dominate, but as the selective pressure by the CD84+ T cells decreases over the course of infection, the advantage for the crippled form of losing epitopes disappears. Hence, we go from a situation of coexistence of wild-type and mutant, to a situation of only full-length nef. The results are discussed in the context of the suggested use of live attenuated vaccines having deletions in nef. PMID:10687825

  5. Phylogenetic analysis of the haemagglutinin gene of current wild-type canine distemper viruses from South Africa: lineage Africa.

    PubMed

    Woma, Timothy Y; van Vuuren, Moritz; Bosman, Ana-Mari; Quan, Melvyn; Oosthuizen, Marinda

    2010-07-14

    There are no reports of CDV isolations in southern Africa, and although CDV is said to have geographically distinct lineages, molecular information of African strains has not yet been documented. Viruses isolated in cell cultures were subjected to reverse transcription-polymerase chain reaction (RT-PCR), and the complete H gene was sequenced and phylogenetically analysed with other strains from GenBank. Phylogenetic comparisons of the complete H gene of CDV isolates from different parts of the world (available in GenBank) with wild-type South African isolates revealed nine clades. All South African isolates form a separate African clade of their own and thus are clearly separated from the American, European, Asian, Arctic and vaccine virus clades. It is likely that only the 'African lineage' of CDV may be circulating in South Africa currently, and the viruses isolated from dogs vaccinated against CDV are not the result of reversion to virulence of vaccine strains, but infection with wild-type strains.

  6. Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium graminearum.

    PubMed

    Jansen, Carin; von Wettstein, Diter; Schäfer, Wilhelm; Kogel, Karl-Heinz; Felk, Angelika; Maier, Frank J

    2005-11-15

    Fusarium head blight epidemics of wheat and barley cause heavy economic losses to farmers due to yield decreases and production of mycotoxin that renders the grain useless for flour and malt products. No highly resistant cultivars are available at present. Hyphae of germinating fungal spores use different paths of infection: After germination at the extruded tip of an ovary, the hyphae travel along the epicarp in the space between the lemma and palea. Infection of the developing kernel proceeds through the epicarp, successively destroying the layers of the fruit coat and finally the starch and protein accumulating endosperm. Hyphae reaching the rachis proceed to apically located developing kernels. Using a constitutively green fluorescence protein-expressing Fusarium wild-type strain, and its knockout mutant, preventing trichothecene synthesis, we demonstrate that trichothecenes are not a virulence factor during infection through the fruit coat. In the absence of trichothecenes, the fungus is blocked by the development of heavy cell wall thickenings in the rachis node of Nandu wheat, a defense inhibited by the mycotoxin. In barley hyphae of both wild-type and the trichothecene knockout mutant, are inhibited at the rachis node and rachilla, limiting infection of adjacent florets through the phloem and along the surface of the rachis. Effective resistance to Fusarium head blight requires expression of genes that combat these different pathways of infection. PMID:16263921

  7. Utilization of Whole-Cell MALDI-TOF Mass Spectrometry to Differentiate Burkholderia pseudomallei Wild-Type and Constructed Mutants.

    PubMed

    Niyompanich, Suthamat; Srisanga, Kitima; Jaresitthikunchai, Janthima; Roytrakul, Sittiruk; Tungpradabkul, Sumalee

    2015-01-01

    Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS) has been widely adopted as a useful technology in the identification and typing of microorganisms. This study employed the whole-cell MALDI-TOF MS to identify and differentiate wild-type and mutants containing constructed single gene mutations of Burkholderia pseudomallei, a pathogenic bacterium causing melioidosis disease in both humans and animals. Candidate biomarkers for the B. pseudomallei mutants, including rpoS, ppk, and bpsI isolates, were determined. Taxon-specific and clinical isolate-specific biomarkers of B. pseudomallei were consistently found and conserved across all average mass spectra. Cluster analysis of MALDI spectra of all isolates exhibited separate distribution. A total of twelve potential mass peaks discriminating between wild-type and mutant isolates were identified using ClinProTools analysis. Two peaks (m/z 2721 and 2748 Da) were specific for the rpoS isolate, three (m/z 3150, 3378, and 7994 Da) for ppk, and seven (m/z 3420, 3520, 3587, 3688, 4623, 4708, and 5450 Da) for bpsI. Our findings demonstrated that the rapid, accurate, and reproducible mass profiling technology could have new implications in laboratory-based rapid differentiation of extensive libraries of genetically altered bacteria. PMID:26656930

  8. An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein.

    PubMed

    Irving, James A; Miranda, Elena; Haq, Imran; Perez, Juan; Kotov, Vadim R; Faull, Sarah V; Motamedi-Shad, Neda; Lomas, David A

    2015-05-15

    A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro molecular scale to the induction of mutant-like oligomerization in a wild-type protein. Using the common pathogenic E342K (Z) variant of α1-antitrypsin as antigen-whose native state is susceptible to the formation of a proto-oligomeric intermediate-we have produced a mAb (5E3) that increases the rate of oligomerization of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognizes a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerization intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerization by either the pathogenic E342K mutation or the binding of mAb5E3 occurs without affecting the energetic barrier to polymerization. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the E342K mutant, by facilitating the conformational interchange between these two states.

  9. Isolation and characterization of DNA sequences that are specifically bound by wild-type p53 protein.

    PubMed Central

    Foord, O; Navot, N; Rotter, V

    1993-01-01

    Wild-type p53 was shown to function as a transcription factor. The N-terminal region of the protein contains the transcription activation domain, while the C terminus is responsible for DNA binding. Localization of the DNA-binding domain of the p53 protein to the highly conserved carboxy-terminal region suggests that the interaction of p53 with DNA is important for its function. We have developed a strategy for studying the DNA sequence specificity of p53-DNA binding that is based on random sequence selection. We report here on the isolation of murine genomic DNA clones that are specifically bound by the wild-type p53 protein but are not bound by mutant p53 protein forms. The isolated p53 target gene contains the unique DNA-binding sequence GACACTGGTCACACTTGGCTGCTTAGGAAT. This fragment exhibits promoter activity as measured by its capacity to activate transcription of the chloramphenicol acetyltransferase reporter gene. Our results suggest that p53 directly binds DNA and functions as a typical transcription factor. Images PMID:8441383

  10. Physcomitrella patens auxin conjugate synthetase (GH3) double knockout mutants are more resistant to Pythium infection than wild type.

    PubMed

    Mittag, Jennifer; Šola, Ivana; Rusak, Gordana; Ludwig-Müller, Jutta

    2015-07-01

    Auxin homeostasis is involved in many different plant developmental and stress responses. The auxin amino acid conjugate synthetases belonging to the GH3 family play major roles in the regulation of free indole-3-acetic acid (IAA) levels and the moss Physcomitrella patens has two GH3 genes in its genome. A role for IAA in several angiosperm--pathogen interactions was reported, however, in a moss--oomycete pathosystem it had not been published so far. Using GH3 double knockout lines we have investigated the role of auxin homeostasis during the infection of P. patens with the two oomycete species, Pythium debaryanum and Pythium irregulare. We show that infection with P. debaryanum caused stronger disease symptoms than with P. irregulare. Also, P. patens lines harboring fusion constructs of an auxin-inducible promoter from soybean (GmGH3) with a reporter (ß-glucuronidase) showed higher promoter induction after P. debaryanum infection than after P. irregulare, indicating a differential induction of the auxin response. Free IAA was induced upon P. debaryanum infection in wild type by 1.6-fold and in two GH3 double knockout (GH3-doKO) mutants by 4- to 5-fold. All GH3-doKO lines showed a reduced disease symptom progression compared to wild type. Since P. debaryanum can be inhibited in growth on medium containing IAA, these data might indicate that endogenous high auxin levels in P. patens GH3-doKO mutants lead to higher resistance against the oomycete.

  11. Accelerated telomere shortening and replicative senescence in human fibroblasts overexpressing mutant and wild-type lamin A

    SciTech Connect

    Huang Shurong; Risques, Rosa Ana; Martin, George M.; Rabinovitch, Peter S.; Oshima, Junko

    2008-01-01

    LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. To our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectable WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes.

  12. A recombinant measles vaccine virus expressing wild-type glycoproteins: consequences for viral spread and cell tropism.

    PubMed

    Johnston, I C; ter Meulen, V; Schneider-Schaulies, J; Schneider-Schaulies, S

    1999-08-01

    Wild-type, lymphotropic strains of measles virus (MV) and tissue culture-adapted MV vaccine strains possess different cell tropisms. This observation has led to attempts to identify the viral receptors and to characterize the functions of the MV glycoproteins. We have functionally analyzed the interactions of MV hemagglutinin (H) and fusion (F) proteins of vaccine (Edmonston) and wild-type (WTF) strains in different combinations in transfected cells. Cell-cell fusion occurs when both Edmonston F and H proteins are expressed in HeLa or Vero cells. The expression of WTF glycoproteins in HeLa cells did not result in syncytia, yet they fused efficiently with cells of lymphocytic origin. To further investigate the role of the MV glycoproteins in virus cell entry and also the role of other viral proteins in cell tropism, we generated recombinant vaccine MVs containing one or both glycoproteins from WTF. These viruses were viable and grew similarly in lymphocytic cells. Recombinant viruses expressing the WTFH protein showed a restricted spread in HeLa cells but spread efficiently in Vero cells. Parental WTF remained restricted in both cell types. Therefore, not only differential receptor usage but also other cell-specific factors are important in determining MV cell tropism. PMID:10400788

  13. The Role of WT1 in Embryonic Development and Normal Organ Homeostasis.

    PubMed

    Wilm, Bettina; Muñoz-Chapuli, Ramon

    2016-01-01

    The Wilms' tumor suppressor gene 1 (Wt1) is critically involved in a number of developmental processes in vertebrates, including cell differentiation, control of the epithelial/mesenchymal phenotype, proliferation, and apoptosis. Wt1 proteins act as transcriptional and post-transcriptional regulators, in mRNA splicing and in protein-protein interactions. Furthermore, Wt1 is involved in adult tissue homeostasis, kidney function, and cancer. For these reasons, Wt1 function has been extensively studied in a number of animal models to establish its spatiotemporal expression pattern and the developmental fate of the cells expressing this gene. In this chapter, we review the developmental anatomy of Wt1, collecting information about its dynamic expression in mesothelium, kidney, gonads, cardiovascular system, spleen, nervous system, lung, and liver. We also describe the adult expression of Wt1 in kidney podocytes, gonads, mesothelia, visceral adipose tissue, and a small fraction of bone marrow cells. We have reviewed the available animal models for Wt1-expressing cell lineage analysis, including direct Wt1 expression reporters and systems for permanent Wt1 lineage tracing, based on constitutive or inducible Cre recombinase expression under control of a Wt1 promoter. Finally we provide a number of laboratory protocols to be used with these animal models in order to assess reporter expression. PMID:27417957

  14. Liposome-mediated transfection of wild-type P53 DNA into human prostate cancer cells is improved by low-frequency ultrasound combined with microbubbles

    PubMed Central

    BAI, WEN-KUN; ZHANG, WEI; HU, BING; YING, TAO

    2016-01-01

    Prostate cancer is a common type of cancer in elderly men. The aim of the present study was to evaluate the effects of ultrasound exposure in combination with SonoVue microbubbles on liposome-mediated transfection of wild-type P53 genes into human prostate cancer cells. PC-3 human prostate cancer cells were exposed to ultrasound; duty cycle was controlled at 20% (2 sec on, 8 sec off) for 5 min with and without SonoVue microbubble echo-contrast agent using a digital sonifier (frequency, 21 kHz; intensity, 46 mW/cm2). The cells were divided into eight groups, as follows: Group A (SonoVue + wild-type P53), group B (ultrasound + wild-type P53), group C (SonoVue + ultrasound + wild-type P53), group D (liposome + wild-type P53), group E (liposome + SonoVue + wild-type P53), group F (liposome + wild-type P53 + ultrasound), group G (liposome + wild-type P53 + ultrasound + SonoVue) and the control group (wild-type P53). Following treatment, a hemocytometer was used to measure cell lysis, reverse transcription-quantitative polymerase chain reaction and western blotting were performed to detect P53 gene transfection efficiency, Cell Counting Kit-8 was employed to reveal cell proliferation and Annexin V/propidium iodide staining was used to determine cell apoptosis. Cell lysis was minimal in each group. Wild-type P53 gene and protein expression were significantly increased in the PC-3 cells in group G compared with the control and all other groups (P<0.01). Cell proliferation was significantly suppressed in group G compared with the control group and all other groups (P<0.01). Cell apoptosis levels in group G were significantly improved compared with the control group and all other groups (P<0.01). Thus, the results of the present study indicate that the use of low-frequency and low-energy ultrasound in combination with SonoVue microbubbles may be a potent physical method for increasing liposome gene delivery efficiency. PMID:27313702

  15. Drug-induced apoptosis in hepatoma cells is mediated by the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wild-type p53.

    PubMed Central

    Müller, M; Strand, S; Hug, H; Heinemann, E M; Walczak, H; Hofmann, W J; Stremmel, W; Krammer, P H; Galle, P R

    1997-01-01

    Chemotherapeutic drugs are cytotoxic by induction of apoptosis in drug-sensitive cells. We investigated the mechanism of bleomycin-induced cytotoxicity in hepatoma cells. At concentrations present in the sera of patients during therapy, bleomycin induced transient accumulation of nuclear wild-type (wt) p53 and upregulated expression of cell surface CD95 (APO-1/Fas) receptor in hepatoma cells carrying wt p53 (HepG2). Bleomycin did not increase CD95 in hepatoma cells with mutated p53 (Huh7) or in hepatoma cells which were p53-/- (Hep3B). In addition, sensitivity towards CD95-mediated apoptosis was also increased in wt p53 positive HepG2 cells. Microinjection of wt p53 cDNA into HepG2 cells had the same effect. In contrast, bleomycin did not enhance susceptibility towards CD95-mediated apoptosis in Huh7 and in Hep3B cells. Furthermore, bleomycin treatment of HepG2 cells increased CD95 ligand (CD95L) mRNA expression. Most notably, bleomycin-induced apoptosis in HepG2 cells was almost completely inhibited by antibodies which interfere with CD95 receptor/ligand interaction. These data suggest that apoptosis induced by bleomycin is mediated, at least in part, by p53-dependent stimulation of the CD95 receptor/ligand system. The same applies to other anti-cancer drugs such as cisplatin and methotrexate. These data may have major consequences for drug treatment of cancer and the explanation of drug sensitivity and resistance. PMID:9022073

  16. Curcumin-Induced Heme Oxygenase-1 Expression Prevents H2O2-Induced Cell Death in Wild Type and Heme Oxygenase-2 Knockout Adipose-Derived Mesenchymal Stem Cells

    PubMed Central

    Cremers, Niels A. J.; Lundvig, Ditte M. S.; van Dalen, Stephanie C. M.; Schelbergen, Rik F.; van Lent, Peter L. E. M.; Szarek, Walter A.; Regan, Raymond F.; Carels, Carine E.; Wagener, Frank A. D. T. G.

    2014-01-01

    Mesenchymal stem cell (MSC) administration is a promising adjuvant therapy to treat tissue injury. However, MSC survival after administration is often hampered by oxidative stress at the site of injury. Heme oxygenase (HO) generates the cytoprotective effector molecules biliverdin/bilirubin, carbon monoxide (CO) and iron/ferritin by breaking down heme. Since HO-activity mediates anti-apoptotic, anti-inflammatory, and anti-oxidative effects, we hypothesized that modulation of the HO-system affects MSC survival. Adipose-derived MSCs (ASCs) from wild type (WT) and HO-2 knockout (KO) mice were isolated and characterized with respect to ASC marker expression. In order to analyze potential modulatory effects of the HO-system on ASC survival, WT and HO-2 KO ASCs were pre-treated with HO-activity modulators, or downstream effector molecules biliverdin, bilirubin, and CO before co-exposure of ASCs to a toxic dose of H2O2. Surprisingly, sensitivity to H2O2-mediated cell death was similar in WT and HO-2 KO ASCs. However, pre-induction of HO-1 expression using curcumin increased ASC survival after H2O2 exposure in both WT and HO-2 KO ASCs. Simultaneous inhibition of HO-activity resulted in loss of curcumin-mediated protection. Co-treatment with glutathione precursor N-Acetylcysteine promoted ASC survival. However, co-incubation with HO-effector molecules bilirubin and biliverdin did not rescue from H2O2-mediated cell death, whereas co-exposure to CO-releasing molecules-2 (CORM-2) significantly increased cell survival, independently from HO-2 expression. Summarizing, our results show that curcumin protects via an HO-1 dependent mechanism against H2O2-mediated apoptosis, and likely through the generation of CO. HO-1 pre-induction or administration of CORMs may thus form an attractive strategy to improve MSC therapy. PMID:25299695

  17. Systems genomics of metabolic phenotypes in wild-type Drosophila melanogaster.

    PubMed

    Reed, Laura K; Lee, Kevin; Zhang, Zhi; Rashid, Lubna; Poe, Amy; Hsieh, Benjamin; Deighton, Nigel; Glassbrook, Norm; Bodmer, Rolf; Gibson, Greg

    2014-06-01

    Systems biology is an approach to dissection of complex traits that explicitly recognizes the impact of genetic, physiological, and environmental interactions in the generation of phenotypic variation. We describe comprehensive transcriptional and metabolic profiling in Drosophila melanogaster across four diets, finding little overlap in modular architecture. Genotype and genotype-by-diet interactions are a major component of transcriptional variation (24 and 5.3% of the total variation, respectively) while there were no main effects of diet (<1%). Genotype was also a major contributor to metabolomic variation (16%), but in contrast to the transcriptome, diet had a large effect (9%) and the interaction effect was minor (2%) for the metabolome. Yet specific principal components of these molecular phenotypes measured in larvae are strongly correlated with particular metabolic syndrome-like phenotypes such as pupal weight, larval sugar content and triglyceride content, development time, and cardiac arrhythmia in adults. The second principal component of the metabolomic profile is especially informative across these traits with glycine identified as a key loading variable. To further relate this physiological variability to genotypic polymorphism, we performed evolve-and-resequence experiments, finding rapid and replicated changes in gene frequency across hundreds of loci that are specific to each diet. Adaptation to diet is thus highly polygenic. However, loci differentially transcribed across diet or previously identified by RNAi knockdown or expression QTL analysis were not the loci responding to dietary selection. Therefore, loci that respond to the selective pressures of diet cannot be readily predicted a priori from functional analyses.

  18. Structure and expression of wild-type and suppressible alleles of the Drosophila purple gene

    SciTech Connect

    Kim, Nacksung |; Park, Dongkook; Yim, John

    1996-04-01

    Viable mutant alleles of purple (pr), such as pr{sup bw}, exhibit mutant eye colors. This reflects low 6-pyruvoyl tetrahydropterin (PTP) synthase activity required for pigment synthesis. PTP synthase is also required for synthesis of the enzyme cofactor biopterin; presumably this is why some pr alleles are lethal. The pr{sup bw} eye color phenotype is suppressed by suppressor of sable [su(s)] mutations. The pr gene was cloned to explore the mechanism of this suppression. pr produces two PTP synthase mRNAs: one constitutively from a distal promoter and one in late pupae and young adult heads from a proximal promoter. The latter presumably supports eye pigment synthesis. The pr{sup bw} allele has a 412 retrotransposon in an intron spliced from both mRNAs. However, the head-specific mRNA is reduced > 10-fold in pr{sup bw} and is restored by a su(s) mutation, while the constitutive transcript is barely affected. The Su(s) protein probably alters processing of RNA containing 412. Because the intron containing 412 is the first in the head-specific mRNA and the second in the constitutive mRNA, binding of splicing machinery to nascent transcripts before the 412 insertion is transcribed may preclude the effects of Su(s) protein. 43 refs., 9 figs.

  19. First-line cetuximab-based chemotherapies for patients with advanced or metastatic KRAS wild-type colorectal cancer

    PubMed Central

    Uemura, Mamoru; Kim, Ho Min; Hata, Tsuyoshi; Sakata, Kazuya; Okuyama, Masaki; Takemoto, Hiroyoshi; Fujii, Hitoshi; Fukuzaki, Takayuki; Morita, Tetsushi; Hata, Taishi; Takemasa, Ichiro; Satoh, Taroh; Mizushima, Tsunekazu; Doki, Yuichiro; Mori, Maski

    2016-01-01

    Colorectal cancer (CRC) is one of the most commonly occurring cancers worldwide. A burgeoning number of studies have demonstrated that the addition of cetuximab to another standard first-line regimen markedly improves the outcome of CRC treatment. However, at present, the efficacy and safety of cetuximab-based combination chemotherapy has not been well described in Japan. The aim of the present study was to evaluate the efficacy and safety of first-line chemotherapies that included cetuximab for patients with advanced or metastatic Kirsten rat sarcoma viral oncogene homolog (KRAS) wild-type CRC in Japan. This prospective multicenter observational study was conducted at 13 affiliated medical institutions. A total of 64 patients were enrolled between 2010 and 2013. The patients met the following criteria for eligibility: i) histologically confirmed, advanced or metastatic KRAS wild-type CRC; and ii) cetuximab-based chemotherapies administered as a first-line treatment. First-line cetuximab-based treatments were administered as follows: 29 patients (45.3%) received a combination of infusional fluorouracil, leucovorin and oxaliplatin; 14 patients (21.9%) received a combination of capecitabine and oxaliplatin; and 10 patients (15.6%) received a combination of infusional fluorouracil, leucovorin and irinotecan. The overall response rate (including complete plus partial responses) was 50% (32/64 patients). Initially, 48 lesions were diagnosed as unresectable. Among those, 13 lesions (27.1%) were converted to a resectable status following cetuximab-based combination chemotherapy treatments. The median overall survival time and the progression-free survival time were 1,189 and 359 days, respectively. The most frequent grade 3/4 adverse event was neutropenia, which occurred in 20.3% of the patients. The incidence of grade 3/4 skin toxicity was 17.2% (11/64 patients). Cetuximab-based therapies may represent a promising first-line regimen for patients with advanced or

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

    PubMed Central

    2011-01-01

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

  1. Genetic Characterization of the Hemagglutinin Genes of Wild-Type Measles Virus Circulating in China, 1993–2009

    PubMed Central

    Zhu, Zhen; Liu, Chunyu; Mao, Naiying; Ji, Yixin; Wang, Huiling; Jiang, Xiaohong; Li, Chongshan; Tang, Wei; Feng, Daxing; Wang, Changyin; Zheng, Lei; Lei, Yue; Ling, Hua; Zhao, Chunfang; Ma, Yan; He, Jilan; Wang, Yan; Li, Ping; Guan, Ronghui; Zhou, Shujie; Zhou, Jianhui; Wang, Shuang; Zhang, Hong; Zheng, Huanying; Liu, Leng; Ma, Hemuti; Guan, Jing; Lu, Peishan; Feng, Yan; Zhang, Yanjun; Zhou, Shunde; Xiong, Ying; Ba, Zhuoma; Chen, Hui; Yang, Xiuhui; Bo, Fang; Ma, Yujie; Liang, Yong; Lei, Yake; Gu, Suyi; Liu, Wei; Chen, Meng; Featherstone, David; Jee, Youngmee; Bellini, William J.; Rota, Paul A.; Xu, Wenbo

    2013-01-01

    Background China experienced several large measles outbreaks in the past two decades, and a series of enhanced control measures were implemented to achieve the goal of measles elimination. Molecular epidemiologic surveillance of wild-type measles viruses (MeV) provides valuable information about the viral transmission patterns. Since 1993, virologic surveillnace has confirmed that a single endemic genotype H1 viruses have been predominantly circulating in China. A component of molecular surveillance is to monitor the genetic characteristics of the hemagglutinin (H) gene of MeV, the major target for virus neutralizing antibodies. Principal Findings Analysis of the sequences of the complete H gene from 56 representative wild-type MeV strains circulating in China during 1993–2009 showed that the H gene sequences were clustered into 2 groups, cluster 1 and cluster 2. Cluster1 strains were the most frequently detected cluster and had a widespread distribution in China after 2000. The predicted amino acid sequences of the H protein were relatively conserved at most of the functionally significant amino acid positions. However, most of the genotype H1 cluster1 viruses had an amino acid substitution (Ser240Asn), which removed a predicted N-linked glycosylation site. In addition, the substitution of Pro397Leu in the hemagglutinin noose epitope (HNE) was identified in 23 of 56 strains. The evolutionary rate of the H gene of the genotype H1 viruses was estimated to be approximately 0.76×10−3 substitutions per site per year, and the ratio of dN to dS (dN/dS) was <1 indicating the absence of selective pressure. Conclusions Although H genes of the genotype H1 strains were conserved and not subjected to selective pressure, several amino acid substitutions were observed in functionally important positions. Therefore the antigenic and genetic properties of H genes of wild-type MeVs should be monitored as part of routine molecular surveillance for measles in China. PMID

  2. RNA Sequencing Identifies Multiple Fusion Transcripts, Differentially Expressed Genes, and Reduced Expression of Immune Function Genes in BRAF (V600E) Mutant vs BRAF Wild-Type Papillary Thyroid Carcinoma

    PubMed Central

    Chindris, Ana-Maria; Asmann, Yan W.; Casler, John D.; Serie, Daniel J.; Reddi, Honey V.; Cradic, Kendall W.; Rivera, Michael; Grebe, Stefan K.; Necela, Brian M.; Eberhardt, Norman L.; Carr, Jennifer M.; McIver, Bryan; Copland, John A.; Aubrey Thompson, E.

    2014-01-01

    Context: The BRAF V600E mutation (BRAF-MUT) confers an aggressive phenotype in papillary thyroid carcinoma, but unidentified additional genomic abnormalities may be required for full phenotypic expression. Objective: RNA sequencing (RNA-Seq) was performed to identify genes differentially expressed between BRAF-MUT and BRAF wild-type (BRAF-WT) tumors and to correlate changes to patient clinical status. Design: BRAF-MUT and BRAF-WT tumors were identified in patients with T1N0 and T2–3N1 tumors evaluated in a referral medical center. Gene expression levels were determined (RNA-Seq) and fusion transcripts were detected. Multiplexed capture/detection and digital counting of mRNA transcripts (nCounter, NanoString Technologies) validated RNA-Seq data for immune system-related genes. Patients: BRAF-MUT patients included nine women, three men; nine were TNM stage I and three were stage III. Three (25%) had tumor infiltrating lymphocytes. BRAF-WT included five women, three men; all were stage I, and five (62.5%) had tumor infiltrating lymphocytes. Results: RNA-Seq identified 560 of 13 085 genes differentially expressed between BRAF-MUT and BRAF-WT tumors. Approximately 10% of these genes were related to MetaCore immune function pathways; 51 were underexpressed in BRAF-MUT tumors, whereas 4 (HLAG, CXCL14, TIMP1, IL1RAP) were overexpressed. The four most differentially overexpressed immune genes in BRAF-WT tumors (IL1B; CCL19; CCL21; CXCR4) correlated with lymphocyte infiltration. nCounter confirmed the RNA-Seq expression level data. Eleven different high-confidence fusion transcripts were detected (four interchromosomal; seven intrachromosomal) in 13 of 20 tumors. All in-frame fusions were validated by RT-PCR. Conclusion: BRAF-MUT papillary thyroid cancers have reduced expression of immune/inflammatory response genes compared with BRAF-WT tumors and correlate with lymphocyte infiltration. In contrast, HLA-G and CXCL14 are overexpressed in BRAF-MUT tumors. Sixty-five percent

  3. Wild-Type U2AF1 Antagonizes the Splicing Program Characteristic of U2AF1-Mutant Tumors and Is Required for Cell Survival

    PubMed Central

    Fei, Dennis Liang; Motowski, Hayley; Chatrikhi, Rakesh; Gao, Shaojian; Kielkopf, Clara L.; Varmus, Harold

    2016-01-01

    We have asked how the common S34F mutation in the splicing factor U2AF1 regulates alternative splicing in lung cancer, and why wild-type U2AF1 is retained in cancers with this mutation. A human lung epithelial cell line was genetically modified so that U2AF1S34F is expressed from one of the two endogenous U2AF1 loci. By altering levels of mutant or wild-type U2AF1 in this cell line and by analyzing published data on human lung adenocarcinomas, we show that S34F-associated changes in alternative splicing are proportional to the ratio of S34F:wild-type gene products and not to absolute levels of either the mutant or wild-type factor. Preferential recognition of specific 3′ splice sites in S34F-expressing cells is largely explained by differential in vitro RNA-binding affinities of mutant versus wild-type U2AF1 for those same 3′ splice sites. Finally, we show that lung adenocarcinoma cell lines bearing U2AF1 mutations do not require the mutant protein for growth in vitro or in vivo. In contrast, wild-type U2AF1 is required for survival, regardless of whether cells carry the U2AF1S34F allele. Our results provide mechanistic explanations of the magnitude of splicing changes observed in U2AF1-mutant cells and why tumors harboring U2AF1 mutations always retain an expressed copy of the wild-type allele. PMID:27776121

  4. Detailed O-glycomics of the Muc2 mucin from colon of wild-type, core 1- and core 3-transferase-deficient mice highlights differences compared with human MUC2

    PubMed Central

    Thomsson, Kristina A; Holmén-Larsson, Jessica M; Ångström, Jonas; Johansson, Malin EV; Xia, Lijun; Hansson, Gunnar C

    2012-01-01

    The heavily O-glycosylated mucin MUC2 constitutes the major protein in the mucosal layer that acts as a physical barrier protecting the epithelial layer in the colon. In this study, Muc2 was purified from mucosal scrapings from the colon of wild-type (WT) mice, core 3 transferase knockout (C3Gnt−/−) mice and intestinal epithelial cell-specific core 1 knockout (IEC C1Galt1−/−) mice. The Muc2 O-glycans were released by reductive β-elimination and analyzed with liquid chromatography-mass spectrometry in the negative-ion mode. Muc2 from the distal colon of WT and C3Gnt−/− knockout mice carried a mixture of core 1- or core 2-type glycans, whereas Muc2 from IEC C1Galt1−/− mice carried highly sialylated core 3- and core 4-type glycans. A large portion of NeuAc in all mouse models was positioned on disialylated N-acetyllactosamine units, an epitope not reported on human colonic MUC2. Mass spectra and proton NMR spectroscopy revealed an abundant NeuAc linked to internally positioned N-acetylglucosamine on colonic murine Muc2, which also differs markedly from human MUC2. Our results highlight that murine colonic Muc2 O-glycosylation is substantially different from human MUC2, which could be one explanation for the different commensal microbiota of these two species. PMID:22581805

  5. Comparative Analysis of miRNAs and Their Target Transcripts between a Spontaneous Late-Ripening Sweet Orange Mutant and Its Wild-Type Using Small RNA and Degradome Sequencing

    PubMed Central

    Wu, Juxun; Zheng, Saisai; Feng, Guizhi; Yi, Hualin

    2016-01-01

    Fruit ripening in citrus is not well-understood at the molecular level. Knowledge of the regulatory mechanism of citrus fruit ripening at the post-transcriptional level in particular is lacking. Here, we comparatively analyzed the miRNAs and their target genes in a spontaneous late-ripening mutant, “Fengwan” sweet orange (MT) (Citrus sinensis L. Osbeck), and its wild-type counterpart (“Fengjie 72-1,” WT). Using high-throughput sequencing of small RNAs and RNA degradome tags, we identified 107 known and 21 novel miRNAs, as well as 225 target genes. A total of 24 miRNAs (16 known miRNAs and 8 novel miRNAs) were shown to be differentially expressed between MT and WT. The expression pattern of several key miRNAs and their target genes during citrus fruit development and ripening stages was examined. Csi-miR156k, csi-miR159, and csi-miR166d suppressed specific transcription factors (GAMYBs, SPLs, and ATHBs) that are supposed to be important regulators involved in citrus fruit development and ripening. In the present study, miRNA-mediated silencing of target genes was found under complicated and sensitive regulation in citrus fruit. The identification of miRNAs and their target genes provide new clues for future investigation of mechanisms that regulate citrus fruit ripening. PMID:27708662

  6. Structure of wild-type yeast RNA polymerase II and location of Rpb4 and Rpb7.

    PubMed

    Jensen, G J; Meredith, G; Bushnell, D A; Kornberg, R D

    1998-04-15

    The three-dimensional structure of wild-type yeast RNA polymerase II has been determined at a nominal resolution of 24 A. A difference map between this structure and that of the polymerase lacking subunits Rpb4 and Rpb7 showed these two subunits forming part of the floor of the DNA-binding (active center) cleft, and revealed a slight inward movement of the protein domain surrounding the cleft. Surface plasmon resonance measurements showed that Rpb4 and Rpb7 stabilize a minimal pre-initiation complex containing promoter DNA, TATA box-binding protein (TBP), transcription factor TFIIB and the polymerase. These findings suggest that Rpb4 and Rpb7 play a role in coupling the entry of DNA into the active center cleft to closure of the cleft. Such a role can explain why these subunits are necessary for promoter-specific transcription in vitro and for a normal stress response in vivo.

  7. Comparison of fluorescence properties of wild type and the W15F mutant of horse liver alcohol dehydrogenase

    NASA Astrophysics Data System (ADS)

    Eftink, Maurice R.; Wong, Cing-Yuen; Park, Doo-Hong; Shearer, Gretchen L.; Plapp, Bryce V.

    1994-08-01

    Horse liver alcohol dehydrogenase is a homodimeric protein; each subunit has two tryptophan residues that are in distinctly different microenvironments. Trp-15 is located on the surface and Trp-314 is buried at the intersubunit interface. Steady-state and time-resolved fluorescence and phosphorescence studies have enabled the assignment of parameters, e.g., quantum yield, emission maximum, decay times, to the individual tryptophan residues of the protein. We have prepared, by site-directed mutagenesis, the mutated W15F protein and have characterized its fluorescence properties. We show that the Trp-314 of the mutant experiences an apolar microenvironment, but that the fluorescence decay and exposure to solute quenchers of the mutant are somewhat different than was expected from the assignments for the wild type.

  8. Comparison of the Proteomes of Three Yeast Wild Type Strains: CEN.PK2, FY1679 and W303

    PubMed Central

    Larsen, Peter Mose; Blomberg, Anders; Görg, Angelika; Roepstorff, Peter; Norbeck, Joakim; Fey, Stephen John

    2001-01-01

    Yeast deletion strains created during gene function analysis projects very often show drastic phenotypic differences depending on the genetic background used. These results indicate the existence of important molecular differences between the CEN.PK2, FY1679 and W303 wild type strains. To characterise these differences we have compared the protein expression levels between CEN.PK2, FY1679 and W303 strains using twodimensional gel electrophoresis and identified selected proteins by mass spectrometric analysis. We have found that FY1679 and W303 strains are more similar to each other than to the CEN.PK2 strain. This study identifies 62 proteins that are differentially expressed between the strains and provides a valuable source of data for the interpretation of yeast mutant phenotypes observed in CEN.PK2, FY1679 and W303 strains. PMID:18628919

  9. Expression profiling of wild type and β-catenin gene disrupted human BxPC-3 pancreatic adenocarcinoma cells.

    PubMed

    Olsen, Petter Angell; Lund, Kaja; Krauss, Stefan

    2015-06-01

    To study the role of WNT/β-catenin signaling in pancreatic adenocarcinoma, human BxPC-3 cell lines deficient of the central canonical WNT signaling protein β-catenin were established by using zinc-finger nuclease mediated targeted genomic disruption of the β-catenin gene (CTNNB1). Comparison of the global transcription levels in wild type cells with two β-catenin gene disrupted clones identified 85 transcripts that were the most differentially regulated. Gene ontology (GO) term enrichment analysis of these transcripts identified "cell adhesion" as the most significantly enriched GO term. Here we describe the data from the transcription profiling analysis published in the article "Implications of Targeted Genomic Disruption of β-Catenin in BxPC-3 Pancreatic Adenocarcinoma Cells" [1]. Data have been deposited to the Gene Expression Omnibus (GEO) database repository with the dataset identifier GSE63072. PMID:26484203

  10. A comparison of nucleotide sequences of measles virus L genes derived from wild-type viruses and SSPE brain tissues.

    PubMed

    Komase, K; Rima, B K; Pardowitz, I; Kunz, C; Billeter, M A; ter Meulen, V; Baczko, K

    1995-04-20

    The nucleotide sequences of the large protein (L) gene derived from two wild-type measles viruses (MV) and two SSPE brain-derived viruses have been determined. All sequences have single large open reading frames encoding 2183 amino acid residues. The deduced L proteins are well conserved and the proposed functional domains which have been identified for rhabdo- and paramyxoviruses are completely conserved in all strains. The degree of variability of L proteins is the lowest of all structural proteins of MV, reflecting its role in virus reproduction and persistence. Biased hypermutation was not observed in the L genes derived from SSPE brain tissue. None of the nucleotide changes can be associated with the attenuated phenotype of the Edmonston vaccine viruses. PMID:7747453

  11. High-throughput functional screening of steroid substrates with wild-type and chimeric P450 enzymes.

    PubMed

    Urban, Philippe; Truan, Gilles; Pompon, Denis

    2014-01-01

    The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation. PMID:25243177

  12. High-Throughput Functional Screening of Steroid Substrates with Wild-Type and Chimeric P450 Enzymes

    PubMed Central

    Truan, Gilles; Pompon, Denis

    2014-01-01

    The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation. PMID:25243177

  13. Atm heterozygous mice are more sensitive to radiation-induced cataracts than are their wild-type counterparts

    NASA Technical Reports Server (NTRS)

    Worgul, Basil V.; Smilenov, Lubomir; Brenner, David J.; Junk, Anna; Zhou, Wei; Hall, Eric J.

    2002-01-01

    It is important to know whether the human population includes genetically predisposed radiosensitive subsets. In vitro studies have shown that cells from individuals homozygous for ataxia telangiectasia (A-T) are much more radiosensitive than cells from unaffected individuals. Although cells heterozygous for the ATM gene (ATM(+/-)) may be slightly more radiosensitive in vitro, it remained to be determined whether the greater susceptibility of ATM(+/-) cells translates into an increased sensitivity for late effects in vivo, though there is a suggestion that radiotherapy patients that are heterozygous for the ATM gene may be more at risk of developing late normal tissue damage. We chose cataractogenesis in the lens as a means to assay for the effects of ATM deficiency in a late-responding tissue. One eye of wild-type, Atm heterozygous and homozygous knockout mice was exposed to 0.5-, 1.0-, 2.0-, or 4.0-Gy x rays. The animals were followed weekly for cataract development by conventional slit-lamp biomicroscopy. Cataract development in the animals of all three groups was strongly dependent on dose. The lenses of homozygous mice were the first to opacify at any given dose. Most important in the present context is that cataracts appeared earlier in the heterozygous versus wild-type animals. The data suggest that ATM heterozygotes in the human population may also be radiosensitive. This may influence the choice of individuals destined to be exposed to higher than normal doses of radiation, such as astronauts, and may also suggest that radiotherapy patients who are ATM heterozygotes could be predisposed to increased late normal tissue damage.

  14. Spectroscopic studies of wild-type and mutant "zinc finger" peptides: determinants of domain folding and structure.

    PubMed

    Párraga, G; Horvath, S; Hood, L; Young, E T; Klevit, R E

    1990-01-01

    The "zinc finger" model [Miller, J., McLachlan, A. D. & Klug, A. (1985) EMBO J. 4, 1609-1614; Brown, R. S., Sander, C. & Argos, P. (1985) FEBS Lett. 186, 271-274] makes both specific structural and specific functional predictions about zinc finger consensus sequences that can be tested with a combination of genetic, molecular biological, and biophysical techniques. The yeast transcription factor ADR1 contains two adjacent zinc finger domains; genetic and deletion analyses showed that amino acid substitutions and deletions in the zinc finger domains resulted in the loss of protein activity. To test the structural and folding predictions of the zinc finger model, peptides encompassing each of the ADR1 fingers were synthesized (ADR1a and ADR1b) as well as a mutant finger peptide (del138) deleted for a single amino acid residue. The folding and metal-binding characteristics of these were assessed by 1H nuclear magnetic resonance (NMR) and visible spectroscopy. While a single unique conformational species was detected for the two wild-type peptides upon tetrahedral binding of zinc, the deletion peptide did not bind zinc with tetrahedral geometry, nor did it fold into a zinc finger domain. The metal-binding and folding results found with the mutant peptide were similar to those obtained when thiol alkylation or imidazole protonation of the wild-type peptides was performed. These data indicate that ligand spacing and both thiol and imidazole participation in zinc binding are specific and necessary requirements for zinc finger folding, which provides direct support for the initial predictions of the model.

  15. Spectroscopic studies of wild-type and mutant "zinc finger" peptides: determinants of domain folding and structure.

    PubMed Central

    Párraga, G; Horvath, S; Hood, L; Young, E T; Klevit, R E

    1990-01-01

    The "zinc finger" model [Miller, J., McLachlan, A. D. & Klug, A. (1985) EMBO J. 4, 1609-1614; Brown, R. S., Sander, C. & Argos, P. (1985) FEBS Lett. 186, 271-274] makes both specific structural and specific functional predictions about zinc finger consensus sequences that can be tested with a combination of genetic, molecular biological, and biophysical techniques. The yeast transcription factor ADR1 contains two adjacent zinc finger domains; genetic and deletion analyses showed that amino acid substitutions and deletions in the zinc finger domains resulted in the loss of protein activity. To test the structural and folding predictions of the zinc finger model, peptides encompassing each of the ADR1 fingers were synthesized (ADR1a and ADR1b) as well as a mutant finger peptide (del138) deleted for a single amino acid residue. The folding and metal-binding characteristics of these were assessed by 1H nuclear magnetic resonance (NMR) and visible spectroscopy. While a single unique conformational species was detected for the two wild-type peptides upon tetrahedral binding of zinc, the deletion peptide did not bind zinc with tetrahedral geometry, nor did it fold into a zinc finger domain. The metal-binding and folding results found with the mutant peptide were similar to those obtained when thiol alkylation or imidazole protonation of the wild-type peptides was performed. These data indicate that ligand spacing and both thiol and imidazole participation in zinc binding are specific and necessary requirements for zinc finger folding, which provides direct support for the initial predictions of the model. PMID:2104978

  16. Computational characterization for catalytic activities of human CD38's wild type, E226 and E146 mutants.

    PubMed

    Nguyen, My H; Dang, Van U; Luu, Boi V

    2010-06-01

    A series of the complexes of human CD38's wild type, E226 and E146 mutants as well have been simulated. The biosoftwares well simulate the penetration of nicotinamide-adenine-dinucleotide (NAD) into the active site. The nicotinamide end of NAD penetrates deep into the active site consistent with cleavage of the nicotinamide-glycosidic bond which is the first step of catalysis creating a Michaelis complex regarded as the intermediate product of NAD cyclase and hydrolysis reaction. The breaking down hydrogen bond between 2'-3' OH ribosyl and the residues replaced Glu(226) makes NAD to be less constrained in active site and nicotinamide (NA) becomes more difficult to be cleaved and eliminates the mutant catalytic activities. The large majority of the substrate NAD is hydrolyzed to ADPR while the conversion of NAD to cADPR is not the dominant reaction catalyzed by wild-type human CD38. The more strongly kept ribosyl group by hydrogen bonds the more NADase and the less cyclase activity. Breaking hydrogen bonds of ribosyl 2'- and 3'-OH by mutation will loosen it to promote the cyclase. The cyclic adenosine diphosphate-ribose (cADPR) could also penetrate deeply into active site to make some hydrogen bonds with Glu(146) and Glu(226); however, its docking poses are affected by a residue located at the entrance of the catalytic pocket (Lys(129)). These results are in good agreement with the previous crystallographic analysis and the experiments quantified the catalytic activities of human CD38 and its mutants.

  17. A Killed, Genetically Engineered Derivative of a Wild-Type Extraintestinal Pathogenic E. coli strain is a Vaccine Candidate

    PubMed Central

    Russo, Thomas A.; Beanan, Janet M.; Olson, Ruth; Genagon, Stacy A.; MacDonald, Ulrike; Cope, John J.; Davidson, Bruce A.; Johnston, Brian; Johnson, James R.

    2007-01-01

    Infections due to extraintestinal pathogenic E. coli (ExPEC) result in significant morbidity, mortality and increased healthcare costs. An efficacious vaccine against ExPEC would be desirable. In this report we explore the use of killed-whole E. coli as a vaccine immunogen. Given the diversity of capsule and O-antigens in ExPEC we have hypothesized that alternative targets are viable vaccine candidates. We have also hypothesized that immunization with a genetically engineered strain that is deficient in the capsule and O-antigen will generate a greater immune response against antigens other than the capsular and O-antigen epitopes than a wild-type strain. Lastly, we hypothesize that mucosal immunization with killed E. coli has the potential to generate a significant immune response. In this study we demonstrated that nasal immunization with a formalin-killed ExPEC derivative deficient in capsule and O-antigen results in a significantly greater overall humoral response compared to its wild-type derivative (which demonstrates that capsule and/or the O-antigen impede the development of an optimal humoral immune response) and a significantly greater immune response against non-capsular and O-antigen epitopes. These antibodies also bound to a subset of heterologous ExPEC strains and enhanced neutrophil-mediated bactericidal activity against the homologous and a heterologous strain. Taken together these studies support the concept that formalin-killed genetically engineered ExPEC derivatives are whole cell vaccine candidates to prevent infections due to ExPEC. PMID:17306426

  18. Combination of cetuximab and PP242 synergistically suppress the progression of wild-type KRAS colorectal carcinoma

    PubMed Central

    Cheng, Lei; Xia, Zuguang; Bian, Xinyu; Li, Guangchao; Hu, Jing; Cao, Ya; Wang, Qing; Qian, Xiaoping

    2015-01-01

    Mammalian target of rapamycin (mTOR) has been shown to be overactive in human colorectal cancer, but the first-generation mTOR inhibitor, rapamycin, has failed to show clinical efficacy against colorectal cancer. On the other hand, although the second-generation mTOR inhibitor, PP242, has exerted substantial efficacy, it was revealed that independent inhibition by PP242 was transient, which could lead to positive-feedback loop to EGFR. Using wild-type KRAS colorectal cancer cells as models, we investigate the treatment efficacy of a widely used anti-EGFR monoclonal antibody, cetuximab, and PP242, alone or in combination in vitro and in vivo. Results of cell viability assays confirmed the synergistic inhibitory effect of PP242 and cetuximab on the survival of Caco-2 and HT-29 cells. Moreover, the ability of cancer-cell invasion and proliferation was also significantly inhibited by the combination therapy when compared with cetuximab or PP242 alone. Interestingly, the percentage of CD44-positive cancer cells was substantially decreased by the combination therapy in comparison with PP242 alone through fluorescence-activated cell sorting. The growth of cancer stem-like cell spheres in vitro was also maximally inhibited by combination therapy, in terms of either diameter or number. More importantly, the efficacy of combination therapy was more prominent than either drug alone in established tumor xenografts. These findings supported the potential use of combination therapy of PP242 and cetuximab against wild-type KRAS colorectal carcinomas. PMID:26586952

  19. Manipulation of cellular GSH biosynthetic capacity via TAT-mediated protein transduction of wild-type or a dominant-negative mutant of glutamate cysteine ligase alters cell sensitivity to oxidant-induced cytotoxicity

    SciTech Connect

    Backos, Donald S.; Brocker, Chad N.; Franklin, Christopher C.

    2010-02-15

    The glutathione (GSH) antioxidant defense system plays a central role in protecting mammalian cells against oxidative injury. Glutamate cysteine ligase (GCL) is the rate-limiting enzyme in GSH biosynthesis and is a heterodimeric holoenzyme composed of catalytic (GCLC) and modifier (GCLM) subunits. As a means of assessing the cytoprotective effects of enhanced GSH biosynthetic capacity, we have developed a protein transduction approach whereby recombinant GCL protein can be rapidly and directly transferred into cells when coupled to the HIV TAT protein transduction domain. Bacterial expression vectors encoding TAT fusion proteins of both GCL subunits were generated and recombinant fusion proteins were synthesized and purified to near homogeneity. The TAT-GCL fusion proteins were capable of heterodimerization and formation of functional GCL holoenzyme in vitro. Exposure of Hepa-1c1c7 cells to the TAT-GCL fusion proteins resulted in the time- and dose-dependent transduction of both GCL subunits and increased cellular GCL activity and GSH levels. A heterodimerization-competent, enzymatically deficient GCLC-TAT mutant was also generated in an attempt to create a dominant-negative suppressor of GCL. Transduction of cells with a catalytically inactive GCLC(E103A)-TAT mutant decreased cellular GCL activity in a dose-dependent manner. TAT-mediated manipulation of cellular GCL activity was also functionally relevant as transduction with wild-type GCLC(WT)-TAT or mutant GCLC(E103A)-TAT conferred protection or enhanced sensitivity to H{sub 2}O{sub 2}-induced cell death, respectively. These findings demonstrate that TAT-mediated transduction of wild-type or dominant-inhibitory mutants of the GCL subunits is a viable means of manipulating cellular GCL activity to assess the effects of altered GSH biosynthetic capacity.

  20. PCR bias toward the wild-type k-ras and p53 sequences: implications for PCR detection of mutations and cancer diagnosis.

    PubMed

    Barnard, R; Futo, V; Pecheniuk, N; Slattery, M; Walsh, T

    1998-10-01

    PCR-based cancer diagnosis requires detection of rare mutations in k-ras, p53 or other genes. The assumption has been that mutant and wild-type sequences amplify with near equal efficiency, so that they are eventually present in proportions representative of the starting material. Work on factor IX suggests that this assumption is invalid for one case of near-sequence identity. To test the generality of this phenomenon and its relevance to cancer diagnosis, primers distant from point mutations in p53 and k-ras were used to amplify wild-type and mutant sequences from these genes. A substantial bias against PCR amplification of mutants was observed for two regions of the p53 gene and one region of k-ras. For k-ras and p53, bias was observed when the wild-type and mutant sequences were amplified separately or when mixed in equal proportions before PCR. Bias was present with proofreading and non-proofreading polymerases. Mutant and wild-type segments of the factor V, cystic fibrosis transmembrane conductance regulator and prothrombin genes were amplified and did not exhibit PCR bias. Therefore, the assumption of equal PCR efficiency for point mutant and wild-type sequences is invalid in several systems. Quantitative or diagnostic PCR will require validation for each locus, and enrichment strategies may be needed to optimize detection of mutants. PMID:9793653

  1. Accelerating Influenza Research: Vaccines, Antivirals, Immunomodulators and Monoclonal Antibodies. The Manufacture of a New Wild-Type H3N2 Virus for the Human Viral Challenge Model

    PubMed Central

    Fullen, Daniel J.; Noulin, Nicolas; Catchpole, Andrew; Fathi, Hosnieh; Murray, Edward J.; Mann, Alex; Eze, Kingsley; Balaratnam, Ganesh; Borley, Daryl W.; Gilbert, Anthony; Lambkin-Williams, Rob

    2016-01-01

    Background Influenza and its associated diseases are a major cause of morbidity and mortality. The United States Advisory Committee on Immunization Practices recommends influenza vaccination for everyone over 6 months of age. The failure of the flu vaccine in 2014–2015 demonstrates the need for a model that allows the rapid development of novel antivirals, universal/intra-seasonal vaccines, immunomodulators, monoclonal antibodies and other novel treatments. To this end we manufactured a new H3N2 influenza virus in compliance with Good Manufacturing Practice for use in the Human Viral Challenge Model. Methods and Strain Selection We chose an H3N2 influenza subtype, rather than H1N1, given that this strain has the most substantial impact in terms of morbidity or mortality annually as described by the Centre for Disease Control. We first subjected the virus batch to rigorous adventitious agent testing, confirmed the virus to be wild-type by Sanger sequencing and determined the virus titres appropriate for human use via the established ferret model. We built on our previous experience with other H3N2 and H1N1 viruses to develop this unique model. Human Challenge and Conclusions We conducted an initial safety and characterisation study in healthy adult volunteers, utilising our unique clinical quarantine facility in London, UK. In this study we demonstrated this new influenza (H3N2) challenge virus to be both safe and pathogenic with an appropriate level of disease in volunteers. Furthermore, by inoculating volunteers with a range of different inoculum titres, we established the minimum infectious titre required to achieve reproducible disease whilst ensuring a sensitive model that can be translated to design of subsequent field based studies. Trial Registration ClinicalTrials.gov NCT02525055 PMID:26761707

  2. The effect of dietary prebiotics and probiotics on body weight, large intestine indices, and fecal bile acid profile in wild type and IL10-/- mice.

    PubMed

    Kuo, Shiu-Ming; Merhige, Patricia M; Hagey, Lee R

    2013-01-01

    Previous studies have suggested roles of probiotics and prebiotics on body weight management and intestinal function. Here, the effects of a dietary prebiotic, inulin (50 mg/g diet), and probiotic, Bfidobacterium animalis subsp. lactis (Bb12) (final dose verified at 10(5) colony forming unit (cfu)/g diet, comparable to human consumption), were determined separately and in combination in mice using cellulose-based AIN-93G diets under conditions allowed for the growth of commensal bacteria. Continuous consumption of Bb12 and/or inulin did not affect food intake or body, liver, and spleen weights of young and adult mice. Fecal bile acid profiles were determined by nanoESI-MS/MS tandem mass spectrometry. In the presence of inulin, more bacterial deconjugation of taurine from primary bile acids was observed along with an increased cecal weight. Consumption of inulin in the absence or presence of Bb12 also increased the villus cell height in the proximal colon along with a trend of higher bile acid sulfation by intestinal cells. Feeding Bb12 alone at the physiological dose did not affect bile acid deconjugation and had little effect on other intestinal indices. Although interleukin (IL)10-null mice are susceptible to enterocolitis, they maintained the same body weight as the wild type mice under our specific pathogen-free housing condition and showed no signs of inflammation. Nevertheless, they had smaller cecum suggesting a mildly compromised intestinal development even before the disease manifestation. Our results are consistent with the notion that dietary factors such as prebiotics play important roles in the growth of intestinal microbiota and may impact on the intestinal health. In addition, fecal bile acid profiling could potentially be a non-invasive tool in monitoring the intestinal environment. PMID:23555939

  3. Genotype differences in anxiety and fear learning and memory of WT and ApoE4 mice associated with enhanced generation of hippocampal reactive oxygen species.

    PubMed

    Villasana, Laura E; Weber, Sydney; Akinyeke, Tunde; Raber, Jacob

    2016-09-01

    Apolipoprotein E (apoE), involved in cholesterol and lipid metabolism, also influences cognitive function and injury repair. In humans, apoE is expressed in three isoforms. E4 is a risk factor for age-related cognitive decline and Alzheimer's disease, particularly in women. E4 might also be a risk factor for developing behavioral and cognitive changes following (56) Fe irradiation, a component of the space environment astronauts are exposed to during missions. These changes might be related to enhanced generation of reactive oxygen species (ROS). In this study, we compared the behavioral and cognitive performance of sham-irradiated and irradiated wild-type (WT) mice and mice expressing the human E3 or E4 isoforms, and assessed the generation of ROS in hippocampal slices from these mice. E4 mice had greater anxiety-like and conditioned fear behaviors than WT mice, and these genotype differences were associated with greater levels of ROS in E4 than WT mice. The greater generation of ROS in the hippocampus of E4 than WT mice might contribute to their higher anxiety levels and enhanced fear conditioning. In E4, but not WT, mice, phorbol-12-myristate-13-acetate-treated hippocampal slices showed more dihydroxy ethidium oxidation in sham-irradiated than irradiated mice and hippocampal heme oxygenase-1 levels were higher in irradiated than sham-irradiated E4 mice. Mice with apolipoprotein E4 (E4), a risk factor for Alzheimer's disease, have greater anxiety-like and conditioned fear behaviors than wild-type (WT) mice. Generation of reactive oxygen species (ROS, in red) 3 months following (56) Fe irradiation, a component of the space environment astronauts are exposed to, is more pronounced in the hippocampus of E4 than WT mice. In E4, but not WT, mice, hippocampal levels of the oxidative stress-relevant marker heme oxygenase-1 are higher in irradiated than sham-irradiated E4 mice. PMID:27412623

  4. Genotype differences in anxiety and fear learning and memory of WT and ApoE4 mice associated with enhanced generation of hippocampal reactive oxygen species.

    PubMed

    Villasana, Laura E; Weber, Sydney; Akinyeke, Tunde; Raber, Jacob

    2016-09-01

    Apolipoprotein E (apoE), involved in cholesterol and lipid metabolism, also influences cognitive function and injury repair. In humans, apoE is expressed in three isoforms. E4 is a risk factor for age-related cognitive decline and Alzheimer's disease, particularly in women. E4 might also be a risk factor for developing behavioral and cognitive changes following (56) Fe irradiation, a component of the space environment astronauts are exposed to during missions. These changes might be related to enhanced generation of reactive oxygen species (ROS). In this study, we compared the behavioral and cognitive performance of sham-irradiated and irradiated wild-type (WT) mice and mice expressing the human E3 or E4 isoforms, and assessed the generation of ROS in hippocampal slices from these mice. E4 mice had greater anxiety-like and conditioned fear behaviors than WT mice, and these genotype differences were associated with greater levels of ROS in E4 than WT mice. The greater generation of ROS in the hippocampus of E4 than WT mice might contribute to their higher anxiety levels and enhanced fear conditioning. In E4, but not WT, mice, phorbol-12-myristate-13-acetate-treated hippocampal slices showed more dihydroxy ethidium oxidation in sham-irradiated than irradiated mice and hippocampal heme oxygenase-1 levels were higher in irradiated than sham-irradiated E4 mice. Mice with apolipoprotein E4 (E4), a risk factor for Alzheimer's disease, have greater anxiety-like and conditioned fear behaviors than wild-type (WT) mice. Generation of reactive oxygen species (ROS, in red) 3 months following (56) Fe irradiation, a component of the space environment astronauts are exposed to, is more pronounced in the hippocampus of E4 than WT mice. In E4, but not WT, mice, hippocampal levels of the oxidative stress-relevant marker heme oxygenase-1 are higher in irradiated than sham-irradiated E4 mice.

  5. Benzo[a]pyrene (BP) DNA adduct formation in DNA repair–deficient p53 haploinsufficient [Xpa(−/−)p53(+/−)] and wild-type mice fed BP and BP plus chlorophyllin for 28 days

    PubMed Central

    Poirier, Miriam C.

    2012-01-01

    We have evaluated DNA damage (DNA adduct formation) after feeding benzo[a]pyrene (BP) to wild-type (WT) and cancer-susceptible Xpa(−/−)p53(+/−) mice deficient in nucleotide excision repair and haploinsufficient for the tumor suppressor p53. DNA damage was evaluated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ES-MS/MS), which measures r7,t8,t9-trihydroxy-c-10-(N 2-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG), and a chemiluminescence immunoassay (CIA), using anti-r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)–DNA antiserum, which measures both BPdG and the other stable BP-DNA adducts. When mice were fed 100 ppm BP for 28 days, BP-induced DNA damage measured in esophagus, liver and lung was typically higher in Xpa(−/−)p53(+/−) mice, compared with WT mice. This result is consistent with the previously observed tumor susceptibility of Xpa(−/−)p53(+/−) mice. BPdG, the major DNA adduct associated with tumorigenicity, was the primary DNA adduct formed in esophagus (a target tissue in the mouse), whereas total BP-DNA adducts predominated in higher levels in the liver (a non-target tissue in the mouse). In an attempt to lower BP-induced DNA damage, we fed the WT and Xpa(−/−)p53(+/−) mice 0.3% chlorophyllin (CHL) in the BP-containing diet for 28 days. The addition of CHL resulted in an increase of BP–DNA adducts in esophagus, liver and lung of WT mice, a lowering of BPdG in esophagi of WT mice and livers of Xpa(−/−)p53(+/−) mice and an increase of BPdG in livers of WT mice. Therefore, the addition of CHL to a BP-containing diet showed a lack of consistent chemoprotective effect, indicating that oral CHL administration may not reduce PAH–DNA adduct levels consistently in human organs. PMID:22828138

  6. Effects of ascorbic acid on carcinogenicity and acute toxicity of nickel subsulfide, and on tumor transplants growth in gulonolactone oxidase knock-out mice and wild-type C57BL mice

    SciTech Connect

    Kasprzak, Kazimierz S.; Diwan, Bhalchandra A.; Kaczmarek, Monika Z.; Logsdon, Daniel L.; Fivash, Mathew J.; Salnikow, Konstantin

    2011-11-15

    The aim of this study was to test a hypothesis that ascorbate depletion could enhance carcinogenicity and acute toxicity of nickel. Homozygous L-gulono- < gamma > -lactone oxidase gene knock-out mice (Gulo-/- mice) unable to produce ascorbate and wild-type C57BL mice (WT mice) were injected intramuscularly with carcinogenic nickel subsulfide (Ni{sub 3}S{sub 2}), and observed for the development of injection site tumors for 57 weeks. Small pieces of one of the induced tumors were transplanted subcutaneously into separate groups of Gulo-/- and WT mice and the growth of these tumors was measured for up to 3 months. The two strains of mice differed significantly with regard to (1) Ni{sub 3}S{sub 2} carcinogenesis: Gulo-/- mice were 40% more susceptible than WT mice; and (2) transplanted tumors development: Gulo-/- mice were more receptive to tumor growth than WT mice, but only in terms of a much shorter tumor latency; later in the exponential phase of growth, the growth rates were the same. And, with adequate ascorbate supplementation, the two strains were equally susceptible to acute toxicity of Ni{sub 3}S{sub 2}. Statistically significant effects of dietary ascorbate dosing levels were the following: (1) reduction in ascorbate supplementation increased acute toxicity of Ni{sub 3}S{sub 2} in Gulo-/- mice; (2) ascorbate supplementation extended the latency of transplanted tumors in WT mice. In conclusion, the lack of endogenous ascorbate synthesis makes Gulo-/- mice more susceptible to Ni{sub 3}S{sub 2} carcinogenesis. Dietary ascorbate tends to attenuate acute toxicity of Ni{sub 3}S{sub 2} and to extend the latency of transplanted tumors. The latter effects may be of practical importance to humans and thus deserve further studies. -- Highlights: Black-Right-Pointing-Pointer Ascorbate depletion enhances carcinogenicity and acute toxicity of nickel. Black-Right-Pointing-Pointer Gulo-/- mice unable to synthesize ascorbate were used in this study. Black

  7. Reductive Elimination of H2 Activates Nitrogenase to Reduce the N≡N Triple Bond: Characterization of the E4(4H) Janus Intermediate in Wild-Type Enzyme.

    PubMed

    Lukoyanov, Dmitriy; Khadka, Nimesh; Yang, Zhi-Yong; Dean, Dennis R; Seefeldt, Lance C; Hoffman, Brian M

    2016-08-24

    We proposed a reductive elimination/oxidative addition (re/oa) mechanism for reduction of N2 to 2NH3 by nitrogenase, based on identification of a freeze-trapped intermediate of the α-70(Val→Ile) MoFe protein as the Janus intermediate that stores four reducing equivalents on FeMo-co as two [Fe-H-Fe] bridging hydrides (denoted E4(4H)). The mechanism postulates that obligatory re of the hydrides as H2 drives reduction of N2 to a state (denoted E4(2N2H)) with a moiety at the diazene (HN═NH) reduction level bound to the catalytic FeMo-co. EPR/ENDOR/photophysical measurements on wild type (WT) MoFe protein now establish this mechanism. They show that a state freeze-trapped during N2 reduction by WT MoFe is the same Janus intermediate, thereby establishing the α-70(Val→Ile) intermediate as a reliable guide to mechanism. Monitoring the Janus state in WT MoFe during N2 reduction under mixed-isotope condition, H2O buffer/D2, and the converse, establishes that the bridging hydrides/deuterides do not exchange with solvent during enzymatic turnover, thereby solving longstanding puzzles. Relaxation of E4(2N2H) to the WT resting-state is shown to occur via oa of H2 and release of N2 to form Janus, followed by sequential release of two H2, demonstrating the kinetic reversibility of the re/oa equilibrium. Relative populations of E4(2N2H)/E4(4H) freeze-trapped during WT turnover furthermore show that the reversible re/oa equilibrium between [E4(4H) + N2] and [E4(2N2H) + H2] is ∼ thermoneutral (ΔreG(0) ∼ -2 kcal/mol), whereas, by itself, hydrogenation of N2(g) is highly endergonic. These findings demonstrate that (i) re/oa accounts for the historical Key Constraints on mechanism, (ii) that Janus is central to N2 reduction by WT enzyme, which (iii) indeed occurs via the re/oa mechanism. Thus, emerges a picture of the central mechanistic steps by which nitrogenase carries out one of the most challenging chemical transformations in biology. PMID:27529724

  8. Perturbation of Auxin Homeostasis by Overexpression of Wild-Type IAA15 Results in Impaired Stem Cell Differentiation and Gravitropism in Roots

    PubMed Central

    Yan, Da-Wei; Wang, Jing; Yuan, Ting-Ting; Hong, Li-Wei; Gao, Xiang; Lu, Ying-Tang

    2013-01-01

    Aux/IAAs interact with auxin response factors (ARFs) to repress their transcriptional activity in the auxin signaling pathway. Previous studies have focused on gain-of-function mutations of domain II and little is known about whether the expression level of wild-type Aux/IAAs can modulate auxin homeostasis. Here we examined the perturbation of auxin homeostasis by ectopic expression of wild-type IAA15. Root gravitropism and stem cell differentiation were also analyzed. The transgenic lines were less sensitive to exogenous auxin and exhibited low-auxin phenotypes including failures in gravity response and defects in stem cell differentiation. Overexpression lines also showed an increase in auxin concentration and reduced polar auxin transport. These results demonstrate that an alteration in the expression of wild-type IAA15 can disrupt auxin homeostasis. PMID:23472140

  9. Effects of Bio-Au Nanoparticles on Electrochemical Activity of Shewanella oneidensis Wild Type and ΔomcA/mtrC Mutant

    NASA Astrophysics Data System (ADS)

    Wu, Ranran; Cui, Li; Chen, Lixiang; Wang, Chao; Cao, Changli; Sheng, Guoping; Yu, Hanqing; Zhao, Feng

    2013-11-01

    Both Shewanella oneidensis MR-1 wild type and its mutant ΔomcA/mtrC are capable of transforming AuIII into Au nanoparticles (AuNPs). Cyclic voltammetry reveals a decrease in redox current after the wild type is exposed to AuIII but an increase in oxidation current for the mutant. The peak current of the wild type is much higher than that of the mutant before the exposure of AuIII, but lower than that of the mutant after the formation of AuNPs. This suggests that damage to the electron transfer chain in the mutant could be repaired by AuNPs to a certain extent. Spectroscopy and SDS-PAGE analysis indicate a decrease in cell protein content after the formation of AuNPs, which provides a convenient way to detect intracellular information on cells.

  10. Effects of bio-Au nanoparticles on electrochemical activity of Shewanella oneidensis wild type and ΔomcA/mtrC mutant.

    PubMed

    Wu, Ranran; Cui, Li; Chen, Lixiang; Wang, Chao; Cao, Changli; Sheng, Guoping; Yu, Hanqing; Zhao, Feng

    2013-11-22

    Both Shewanella oneidensis MR-1 wild type and its mutant ΔomcA/mtrC are capable of transforming Au(III) into Au nanoparticles (AuNPs). Cyclic voltammetry reveals a decrease in redox current after the wild type is exposed to Au(III) but an increase in oxidation current for the mutant. The peak current of the wild type is much higher than that of the mutant before the exposure of Au(III), but lower than that of the mutant after the formation of AuNPs. This suggests that damage to the electron transfer chain in the mutant could be repaired by AuNPs to a certain extent. Spectroscopy and SDS-PAGE analysis indicate a decrease in cell protein content after the formation of AuNPs, which provides a convenient way to detect intracellular information on cells.

  11. Detection and differentiation of wild-type and vaccine strains of canine distemper virus by a duplex reverse transcription polymerase chain reaction.

    PubMed

    Dong, X Y; Li, W H; Zhu, J L; Liu, W J; Zhao, M Q; Luo, Y W; Chen, J D

    2015-01-01

    Canine distemper virus (CDV) is the cause of canine distemper (CD) which is a severe and highly contagious disease in dogs. In the present study, a duplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of CDV. Four primers were designed to detect and discriminate the two viruses by generating 638- and 781-bp cDNA products, respectively. Furthermore, the duplex RT-PCR method was used to detect 67 field samples suspected of CD from Guangdong province in China. Results showed that, 33 samples were to be wild-type-like. The duplex RT-PCR method exhibited high specificity and sensitivity which could be used to effectively detect and differentiate wild-type and vaccine CDV, indicating its use for clinical detection and epidemiological surveillance. PMID:27175171

  12. Detection and differentiation of wild-type and vaccine strains of canine distemper virus by a duplex reverse transcription polymerase chain reaction

    PubMed Central

    Dong, X. Y.; Li, W. H.; Zhu, J. L.; Liu, W. J.; Zhao, M. Q.; Luo, Y. W.; Chen, J. D.

    2015-01-01

    Canine distemper virus (CDV) is the cause of canine distemper (CD) which is a severe and highly contagious disease in dogs. In the present study, a duplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of CDV. Four primers were designed to detect and discriminate the two viruses by generating 638- and 781-bp cDNA products, respectively. Furthermore, the duplex RT-PCR method was used to detect 67 field samples suspected of CD from Guangdong province in China. Results showed that, 33 samples were to be wild-type-like. The duplex RT-PCR method exhibited high specificity and sensitivity which could be used to effectively detect and differentiate wild-type and vaccine CDV, indicating its use for clinical detection and epidemiological surveillance. PMID:27175171

  13. Nogo Receptor Signaling Restricts Adult Neural Plasticity by Limiting Synaptic AMPA Receptor Delivery

    PubMed Central

    Jitsuki, Susumu; Nakajima, Waki; Takemoto, Kiwamu; Sano, Akane; Tada, Hirobumi; Takahashi-Jitsuki, Aoi; Takahashi, Takuya

    2016-01-01

    Experience-dependent plasticity is limited in the adult brain, and its molecular and cellular mechanisms are poorly understood. Removal of the myelin-inhibiting signaling protein, Nogo receptor (NgR1), restores adult neural plasticity. Here we found that, in NgR1-deficient mice, whisker experience-driven synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) insertion in the barrel cortex, which is normally complete by 2 weeks after birth, lasts into adulthood. In vivo live imaging by two-photon microscopy revealed more AMPAR on the surface of spines in the adult barrel cortex of NgR1-deficient than on those of wild-type (WT) mice. Furthermore, we observed that whisker stimulation produced new spines in the adult barrel cortex of mutant but not WT mice, and that the newly synthesized spines contained surface AMPAR. These results suggest that Nogo signaling limits plasticity by restricting synaptic AMPAR delivery in coordination with anatomical plasticity. PMID:26472557

  14. Steady state fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1) and its active site mutants.

    PubMed

    Sonawane, Prashant; Vishwakarma, Rishi Kishore; Singh, Somesh; Gaikwad, Sushama; Khan, Bashir M

    2014-05-01

    Fluorescence quenching and time resolved fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1), a multitryptophan protein from Leucaena leucocephala and 10 different active site mutants were carried out to investigate tryptophan environment. The enzyme showed highest affinity for feruloyl CoA (K(a)  = 3.72 × 10(5) M(-1)) over other CoA esters and cinnamaldehydes, as determined by fluorescence spectroscopy. Quenching of the fluorescence by acrylamide for wild type and active site mutants was collisional with almost 100% of the tryptophan fluorescence accessible under native condition and remained same after denaturation of protein with 6 M GdnHCl. In wild type Ll-CCRH1, the extent of quenching achieved with iodide (f(a) = 1.0) was significantly higher than cesium ions (f(a) = 0.33) suggesting more density of positive charge around surface of trp conformers under native conditions. Denaturation of wild type protein with 6 M GdnHCl led to significant increase in the quenching with cesium (f(a) = 0.54), whereas quenching with iodide ion was decreased (f(a) = 0.78), indicating reorientation of charge density around trp from positive to negative and heterogeneity in trp environment. The Stern-Volmer plots for wild type and mutants Ll-CCRH1 under native and denatured conditions, with cesium ion yielded biphasic quenching profiles. The extent of quenching for cesium and iodide ions under native and denatured conditions observed in active site mutants was significantly different from wild type Ll-CCRH1 under the same conditions. Thus, single substitution type mutations of active site residues showed heterogeneity in tryptophan microenvironment and differential degree of conformation of protein under native or denatured conditions. PMID:24322526

  15. After a cold conditioning swim, UCP2-deficient mice are more able to defend against the cold than wild type mice.

    PubMed

    Abdelhamid, Ramy E; Kovács, Katalin J; Nunez, Myra G; Larson, Alice A

    2014-08-01

    Uncoupling protein 2 (UCP2) is widely distributed throughout the body including the brain, adipose tissue and skeletal muscles. In contrast to UCP1, UCP2 does not influence resting body temperature and UCP2-deficient (-/-) mice have normal thermoregulatory responses to a single exposure to cold ambient temperatures. Instead, UCP2-deficient mice are more anxious, exhibit anhedonia and have higher circulating corticosterone than wild type mice. To test the possible role of UCP2 in depressive behavior we exposed UCP2-deficient and wild type mice to a cold (26°C) forced swim and simultaneously measured rectal temperatures during and after the swim. The time that UCP2-deficient mice spent immobile did not differ from wild type mice and all mice floated more on day 2. However, UCP2-deficient mice were more able to defend against the decrease in body temperature during a second daily swim at 26°C than wild type mice (area under the curve for wild type mice: 247.0±6.4; for UCP2-deficient mice: 284.4±3.8, P<0.0001, Student's t test). The improved thermoregulation of wild type mice during a second swim at 26°C correlated with their greater immobility whereas defense against the warmth during a swim at 41°C correlated better with greater immobility of UCP2-deficient mice. Together these data indicate that while the lack of UCP2 has no acute effect on body temperature, UCP2 may inhibit rapid improvements in defense against cold, in contrast to UCP1, whose main function is to promote thermogenesis. PMID:24952267

  16. After a cold conditioning swim, UCP2-deficient mice are more able to defend against the cold than wild type mice.

    PubMed

    Abdelhamid, Ramy E; Kovács, Katalin J; Nunez, Myra G; Larson, Alice A

    2014-08-01

    Uncoupling protein 2 (UCP2) is widely distributed throughout the body including the brain, adipose tissue and skeletal muscles. In contrast to UCP1, UCP2 does not influence resting body temperature and UCP2-deficient (-/-) mice have normal thermoregulatory responses to a single exposure to cold ambient temperatures. Instead, UCP2-deficient mice are more anxious, exhibit anhedonia and have higher circulating corticosterone than wild type mice. To test the possible role of UCP2 in depressive behavior we exposed UCP2-deficient and wild type mice to a cold (26°C) forced swim and simultaneously measured rectal temperatures during and after the swim. The time that UCP2-deficient mice spent immobile did not differ from wild type mice and all mice floated more on day 2. However, UCP2-deficient mice were more able to defend against the decrease in body temperature during a second daily swim at 26°C than wild type mice (area under the curve for wild type mice: 247.0±6.4; for UCP2-deficient mice: 284.4±3.8, P<0.0001, Student's t test). The improved thermoregulation of wild type mice during a second swim at 26°C correlated with their greater immobility whereas defense against the warmth during a swim at 41°C correlated better with greater immobility of UCP2-deficient mice. Together these data indicate that while the lack of UCP2 has no acute effect on body temperature, UCP2 may inhibit rapid improvements in defense against cold, in contrast to UCP1, whose main function is to promote thermogenesis.

  17. A surface plasmon resonance assay for measurement of neuraminidase inhibition, sensitivity of wild-type influenza neuraminidase and its H274Y mutant to the antiviral drugs zanamivir and oseltamivir.

    PubMed

    Somasundaram, Balaji; Fee, Conan J; Fredericks, Rayleen; Watson, Andrew J A; Fairbanks, Antony J; Hall, Richard J

    2015-09-01

    Antiviral resistance is currently monitored by a labelled enzymatic assay, which can give inconsistent results because of the short half-life of the labelled product, and variations in assay conditions. In this paper, we describe a competitive surface plasmon resonance (SPR) inhibition assay for measuring the sensitivities of wild-type neuraminidase (WT NA) and the H274Y (histidine 274 tyrosine) NA mutant to antiviral drugs. The two NA isoforms were expressed in High-five™ (Trichoplusia ni) insect cells. A spacer molecule (1,6-hexanediamine (HDA)) was conjugated to the 7-hydroxyl group of zanamivir, and the construct (HDA-zanamivir) was immobilized onto a SPR sensor chip to obtain a final immobilization response of 431 response units. The immobilized HDA-zanamivir comprised a bio-specific ligand for the WT and mutant proteins. The effects of the natural substrate (sialic acid) and two inhibitors (zanamivir and oseltamivir) on NA binding to the immobilized ligand were studied. The processed SPR data was analysed to determine 50% inhibitory concentrations (IC50-spr ), using a log dose-response curve fit. Although both NA isoforms had almost identical IC50-spr values for sialic acid (WT = 5.5 nM; H274Y mutant = 3.25 nM) and zanamivir (WT = 2.16 nM; H274Y mutant = 2.42 nM), there were significant differences between the IC50-spr values obtained for the WT (7.7 nM) and H274Y mutant (256 nM) NA in the presence of oseltamivir, indicating that oseltamivir has a reduced affinity for the H274Y mutant. The SPR inhibition assay strategy presented in this work could be applied for the rapid screening of newly emerging variants of NA for their sensitivity to antiviral drugs.

  18. A randomized, placebo-controlled, phase 1/2 study of tivantinib (ARQ 197) in combination with irinotecan and cetuximab in patients with metastatic colorectal cancer with wild-type KRAS who have received first-line systemic therapy.

    PubMed

    Eng, Cathy; Bessudo, Alberto; Hart, Lowell L; Severtsev, Aleksey; Gladkov, Oleg; Müller, Lothar; Kopp, Mikhail V; Vladimirov, Vladimir; Langdon, Robert; Kotiv, Bogdan; Barni, Sandro; Hsu, Ching; Bolotin, Ellen; von Roemeling, Reinhard; Schwartz, Brian; Bendell, Johanna C

    2016-07-01

    Cetuximab in combination with an irinotecan-containing regimen is a standard treatment in patients with KRAS wild-type (KRAS WT), metastatic colorectal cancer (mCRC). We investigated the addition of the oral MET inhibitor tivantinib to cetuximab + irinotecan (CETIRI) based on preclinical evidence that activation of the MET pathway may confer resistance to anti-EGFR therapy. Previously treated patients with KRAS WT advanced or mCRC were enrolled. The phase 1, open-label 3 + 3, dose-escalation study evaluated the safety and maximally tolerated dose of tivantinib plus CETIRI. The phase 2, randomized, double-blinded, placebo-controlled study of biweekly CETIRI plus tivantinib or placebo was restricted to patients who had received only one prior line of chemotherapy. The phase 2 primary endpoint was progression-free survival (PFS). The recommended phase 2 dose was tivantinib (360 mg/m(2) twice daily) with biweekly cetuximab (500 mg/m(2)) and irinotecan (180 mg/m(2)). Among 117 patients evaluable for phase 2 analysis, no statistically significant PFS difference was observed: 8.3 months on tivantinib vs. 7.3 months on placebo (HR, 0.85; 95% confidence interval, 0.55-1.33; P = 0.38). Subgroup analyses trended in favor of tivantinib in patients with MET-High tumors by immunohistochemistry, PTEN-Low tumors, or those pretreated with oxaliplatin, but subgroups were too small to draw conclusions. Neutropenia, diarrhea, nausea and rash were the most frequent severe adverse events in tivantinib-treated patients. The combination of tivantinib and CETIRI was well tolerated but did not significantly improve PFS in previously treated KRAS WT mCRC. Tivantinib may be more active in specific subgroups. PMID:26891420

  19. Phosphorylation at Ser-15 and Ser-392 in mutant p53 molecules from tumors is altered compared to wild-type p53

    SciTech Connect

    Ullrich, S.J.; Sakaguchi, K.; Fiscella, M.; Appella, E. ); Lees-Miller, S.P.; Anderson, C.W. ); Mercer, W.E. )

    1993-07-01

    The product of the p53 gene suppresses cell growth and plays a critical role in suppressing development of human tumors. p53 protein binds DNA, activates transcription, and can be phosphorylated at N- and C-terminal sites. Previously, wild-type p53 was shown to be hyperphosphorylated compared to mutant p53 during p53-mediated growth arrest in vivo. Here we show that Ser-15 and Ser-9 in the N-terminal transactivation domain of wild-type human p53 are phosphorylated in vivo in cells derived from the human glioblastoma line T98G. In [IIe[sup 237

  20. Farnesol restores wild-type colony morphology to 96% of Candida albicans colony morphology variants recovered following treatment with mutagens.

    PubMed

    Jensen, Ellen C; Hornby, Jacob M; Pagliaccetti, Nicole E; Wolter, Chuleeon M; Nickerson, Kenneth W; Atkin, Audrey L

    2006-04-01

    Candida albicans is a diploid fungus that undergoes a morphological transition between budding yeast, hyphal, and pseudohyphal forms. The morphological transition is strongly correlated with virulence and is regulated in part by quorum sensing. Candida albicans produces and secretes farnesol that regulates the yeast to mycelia morphological transition. Mutants that fail to synthesize or respond to farnesol could be locked in the filamentous mode. To test this hypothesis, a collection of C. albicans mutants were isolated that have altered colony morphologies indicative of the presence of hyphal cells under environmental conditions where C. albicans normally grows only as yeasts. All mutants were characterized for their ability to respond to farnesol. Of these, 95.9% fully or partially reverted to wild-type morphology on yeast malt (YM) agar plates supplemented with farnesol. All mutants that respond to farnesol regained their hyphal morphology when restreaked on YM plates without farnesol. The observation that farnesol remedial mutants are so common (95.9%) relative to mutants that fail to respond to farnesol (4.1%) suggests that farnesol activates and (or) induces a pathway that can override many of the morphogenesis defects in these mutants. Additionally, 9 mutants chosen at random were screened for farnesol production. Two mutants failed to produce detectable levels of farnesol. PMID:16699554

  1. Isolation and characterization of liver epithelial cell lines from wild-type and mutant TgN737Rpw mice.

    PubMed Central

    Richards, W. G.; Yoder, B. K.; Isfort, R. J.; Detilleux, P. G.; Foster, C.; Neilsen, N.; Woychik, R. P.; Wilkinson, J. E.

    1997-01-01

    The Tg737 gene encodes a tetratricopeptide repeat containing protein that, when disrupted in TgN737Rpw mutant mice, results in pleiotropic phenotypes that include the proliferation of epithelial cells. In the kidney and liver, this causes a phenotype that resembles autosomal recessive polycystic kidney disease. In the liver, the affected epithelial cells morphologically and immunologically resemble oval cells. Here we describe the isolation, culture, and characterization of epithelial cell lines derived from the livers of wild-type, heterozygous, and homozygous TgN737Rpw mice. Essentially homogeneous cell cultures were established and the expression of liver markers was examined by reverse transcriptase polymerase chain reaction and by immunohistochemistry. All of the cell lines reacted to the A6 antibody that was raised against mouse oval cells and expressed markers seen in oval cells. Cells transplanted into the interscapular fat pads of isogenic mice formed well defined ductular structures. Furthermore, in transfection experiments, we have demonstrated the involvement of Tg737 in cellular proliferation. Images Figure 1 Figure 2 Figure 3 PMID:9094975

  2. Sex and Immunogen-Specific Benefits of Immunotherapy Targeting Islet Amyloid Polypeptide in Transgenic and Wild-Type Mice

    PubMed Central

    Krishnamurthy, Pavan K.; Rajamohamedsait, Hameetha B.; Gonzalez, Veronica; Rajamohamedsait, Wajitha J.; Ahmed, Nawal; Krishnaswamy, Senthilkumar; Sigurdsson, Einar M.

    2016-01-01

    Type 2 diabetes mellitus is characterized by the deposition of islet amyloid polypeptide (IAPP) as amyloid in islets, a process thought to be toxic to β-cells. To determine the feasibility of targeting these aggregates therapeutically, we vaccinated transgenic (Tg) mice that overexpress human IAPP and were fed a high-fat diet to promote their diabetic phenotype. Our findings indicate that prophylactic vaccination with IAPP and its derivative IAPP7-19-TT, protects wild-type female mice, but not males, from obesity-induced early mortality, and the derivative showed a strong trend for prolonging the lifespan of Tg females but not males. Furthermore, IAPP7-19-TT-immunized Tg females cleared a glucose bolus more efficiently than controls, while IAPP-immunized Tg females showed an impaired ability to clear a glucose bolus compared to their adjuvant injected Tg controls. Interestingly, IAPP or IAPP7-19-TT treatments had no effect on glucose clearance in Tg males. Overall, these beneficial effects of IAPP targeted immunization depend on Tg status, sex, and immunogen. Hence, future studies in this field should carefully consider these variables that clearly affect the therapeutic outcome. In conclusion, IAPP targeting immunotherapy may have benefits in patients with type 2 diabetes. PMID:27379014

  3. Long-term continuous allopregnanolone elevation causes memory decline and hippocampus shrinkage, in female wild-type B6 mice.

    PubMed

    Bengtsson, Sara K S; Johansson, Maja; Bäckström, Torbjörn

    2016-02-01

    Chronic stress in various forms increases the risk for cognitive dysfunction, dementia and Alzheimer's disease. While the pathogenesis behind these findings is unknown, growing evidence suggests that chronic increase in neurosteroid levels, such as allopregnanolone, is part of the mechanism. We treated wild-type C57BL/6J mice with allopregnanolone for 5months, using osmotic pumps. This treatment led to moderately increased levels of allopregnanolone, equivalent to that of mild chronic stress. After an interval of no treatment for 1month, female mice showed impaired learning and memory function in the Morris water maze (MWM) in combination with diminished hippocampus weight and increased cerebellum weight, both correlating to MWM performance. Male mice showed a minor reduction in memory function and no differences in brain structure. We conclude that chronic allopregnanolone elevation can lead to cognitive dysfunction and negative brain alterations. We suggest that allopregnanolone could play a key role in the pathogenesis of stress-induced cognitive disturbances and perhaps dementia. PMID:26497250

  4. Endogenous p53 protein generated from wild-type alternatively spliced p53 RNA in mouse epidermal cells.

    PubMed Central

    Kulesz-Martin, M F; Lisafeld, B; Huang, H; Kisiel, N D; Lee, L

    1994-01-01

    We previously demonstrated that a wild-type alternatively spliced p53 (p53as) RNA exists in mouse cultured cells and normal mouse tissues at approximately 25 to 33% of the level of the major p53 RNA form. The alternative RNA transcript is 96 nucleotides longer than the major transcript as a result of alternative splicing of intron 10 sequences. The protein expected to be generated from the p53as transcript is 9 amino acids shorter than the major p53 protein and has 17 different amino acids at the carboxyl terminus. We report here that p53as protein exists in nontransformed and malignant epidermal cells and is localized to the nucleus. In addition, p53as protein is preferentially expressed during the G2 phase of the cell cycle and in cells with greater than G2 DNA content compared with the major p53 protein, which is preferentially expressed in G1. The p53as immunoreactivity is elevated and shifted to the G1 phase of the cell cycle following actinomycin D treatment of nontransformed cells but not malignant cells. In view of the dimerization and tetramerization of p53 protein which may be necessary for its DNA binding and transcriptional activation activities, the presence of p53as protein in cells has important implications for understanding the physiological function(s) of the p53 gene. Images PMID:8114705

  5. BCAT1 promotes cell proliferation through amino acid catabolism in gliomas carrying wild-type IDH1

    PubMed Central

    Park, Yoon Jung; Wang, Wei; Schlotter, Magdalena; Lindroth, Anders M; Pleier, Sabrina V; Bai, Alfa H C; Karra, Daniela; Piro, Rosario M; Felsberg, Jörg; Addington, Adele; Lemke, Dieter; Weibrecht, Irene; Hovestadt, Volker; Rolli, Claudio G; Campos, Benito; Turcan, Sevin; Sturm, Dominik; Witt, Hendrik; Chan, Timothy A; Herold-Mende, Christel; Kemkemer, Ralf; König, Rainer; Schmidt, Kathrin; Hull, William-Edmund; Pfister, Stefan M; Jugold, Manfred; Hutson, Susan M; Plass, Christoph; Okun, Jürgen G; Reifenberger, Guido; Lichter, Peter; Radlwimmer, Bernhard

    2016-01-01

    Here we show that glioblastoma express high levels of branched-chain amino acid transaminase 1 (BCAT1), the enzyme that initiates the catabolism of branched-chain amino acids (BCAAs). Expression of BCAT1 was exclusive to tumors carrying wild-type isocitrate dehydrogenase 1 (IDH1) and IDH2 genes and was highly correlated with methylation patterns in the BCAT1 promoter region. BCAT1 expression was dependent on the concentration of α-ketoglutarate substrate in glioma cell lines and could be suppressed by ectopic overexpression of mutant IDH1 in immortalized human astrocytes, providing a link between IDH1 function and BCAT1 expression. Suppression of BCAT1 in glioma cell lines blocked the excretion of glutamate and led to reduced proliferation and invasiveness in vitro, as well as significant decreases in tumor growth in a glioblastoma xenograft model. These findings suggest a central role for BCAT1 in glioma pathogenesis, making BCAT1 and BCAA metabolism attractive targets for the development of targeted therapeutic approaches to treat patients with glioblastoma. PMID:23793099

  6. Cyclodextrin Alters GABAergic Input to CA1 Pyramidal Cells in Wild-Type But Not in NPC1-Deficient Mice.

    PubMed

    Frech, Moritz J; Rabenstein, Michael; Bovensiepen, Katja; Rost, Sebastian; Rolfs, Arndt

    2015-01-01

    Niemann-Pick type C1 disease (NPC1) is a neurodegenerative disorder caused by mutations in the NPC1 gene. Actual, no causative treatment for NPC1 is available, although some drugs have been proven to be beneficial to patients, for example, 2-hydroxypropyl-β-cyclodextrin (CDX). In this study, we used the BALB/c_Nctr-Npc1m1N/-J mouse strain to study the effect of CDX, which is described to prolong the life span and to alleviate the pathogenic phenotype. By means of patch clamp recordings, we measured inhibitory postsynaptic currents (IPSCs) of CA1 pyramidal cells of CDX-treated and -untreated animals to elucidate the influence of CDX on the synaptic transmission. Surprisingly, CDX induced a significantly higher GABAergic IPSC frequency in wild-type mice than in NPC1(-/-) mice. Although the IPSCs were mainly GABAergic, we observed a significant reduction of the IPSC frequency in the presence of the glycine receptor antagonist strychnine. The effect of strychnine did not differ in untreated and treated animals, indicating that the effect of CDX was most likely not based on an interaction with glycinergic transmission machinery. However, the unexpected effect of CDX on the GABAergic synaptic transmission is of special interest as a disturbance plays, for example, a crucial role in epilepsy and, moreover, as CDX is currently under investigation as a treatment for NPC1 in humans. PMID:26392920

  7. Comparative analysis of the integument transcriptomes of the black dilute mutant and the wild-type silkworm Bombyx mori

    PubMed Central

    Wu, Songyuan; Tong, Xiaoling; Peng, Chenxing; Xiong, Gao; Lu, Kunpeng; hu, Hai; Tan, Duan; Li, Chunlin; Han, Minjin; Lu, Cheng; Dai, Fangyin

    2016-01-01

    The insect cuticle is a critical protective shell that is composed predominantly of chitin and various cuticular proteins and pigments. Indeed, insects often change their surface pigment patterns in response to selective pressures, such as threats from predators, sexual selection and environmental changes. However, the molecular mechanisms underlying the construction of the epidermis and its pigmentation patterns are not fully understood. Among Lepidoptera, the silkworm is a favorable model for color pattern research. The black dilute (bd) mutant of silkworm is the result of a spontaneous mutation; the larval body color is notably melanized. We performed integument transcriptome sequencing of the wild-type strain Dazao and the mutant strains +/bd and bd/bd. In these experiments, during an early stage of the fourth molt, a stage at which approximately 51% of genes were expressed genome wide (RPKM ≥1) in each strain. A total of 254 novel transcripts were characterized using Cuffcompare and BLAST analyses. Comparison of the transcriptome data revealed 28 differentially expressed genes (DEGs) that may contribute to bd larval melanism, including 15 cuticular protein genes that were remarkably highly expressed in the bd/bd mutant. We suggest that these significantly up-regulated cuticular proteins may promote melanism in silkworm larvae. PMID:27193628

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

    PubMed Central

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

    2015-01-01

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

  9. Melatonin enhances cold tolerance in drought-primed wild-type and abscisic acid-deficient mutant barley.

    PubMed

    Li, Xiangnan; Tan, Dun-Xian; Jiang, Dong; Liu, Fulai

    2016-10-01

    Melatonin is involved in multiple plant developmental processes and various stress responses. To explore the roles of melatonin played as well as its association with abscisic acid (ABA) in a process of drought priming-induced cold tolerance (DPICT), a wild-type barley and its ABA-deficient mutant Az34 counterpart were selected for comparison, in which the effects of melatonin application (either foliarly or rhizospherically) and/or drought priming on the cold tolerance of both types of barleys were systematically investigated. It was demonstrated that the early drought priming induced an increase of endogenous melatonin production, which is not ABA dependent. In addition, exogenously applied melatonin resulted in higher ABA concentration in the drought-primed plants than in the nonprimed plants when exposed to cold stress, indicating that ABA responded in a drought-dependent manner. The interplay of melatonin and ABA leads to plants maintaining better water status. Drought priming-induced melatonin accumulation enhanced the antioxidant capacity in both chloroplasts and mitochondria, which sustained the photosynthetic electron transport in photosynthetic apparatus of the plants under cold stress. These results suggest that the exogenous melatonin application enhances the DPICT by modulating subcellular antioxidant systems and ABA levels in barley. PMID:27299847

  10. Development of infectious clones of a wild-type Korean rabies virus and evaluation of their pathogenic potential.

    PubMed

    Park, Jun-Sun; Kim, Chi-Kyeong; Um, Ji-Hye; Ju, Young Ran; Lee, Yeong Seon; Choi, Young-Ki; Kim, Su Yeon

    2016-09-01

    Most reverse genetic (RG) systems for rabies viruses (RVs) have been constructed on the genome background of laboratory-adapted strains. In this study, we developed an RG system using a Korean wild type (KGH) strain to investigate the pathogenic potential of different strains. We developed a RG system with the KGH strain for the first time. Following the complete genome sequencing of the KGH strain, pKGH infectious clones were constructed using the CMV/T7 promoter, and HamRz and HdvRz were introduced to allow self-cleavage of the synthesized RNA. We successfully recovered the rescued virus by constructing chimeric RVs in which we replaced a part of the construct with the partial gene from the fixed RC-HL strain. The rescued viruses formed clearer and countable plaques in an immunostaining plaque assay, with a distinct plaque morphology. Furthermore, compared with the chimeric RVs, the pKGH/RCinsΔ4 strain containing the KGH strain G protein exhibited a decreased efficiency of cell-to-cell spreading in BHK-21 cells and significantly reduced (100-1000 fold) replication kinetics. However, pKGH/RCinsΔ4 strain-infected mice revealed 100% morbidity at 11days post-infection, whereas other chimeric RV strains showed no mortality. Our RG system is a useful tool for studying differences in the cell-to-cell spreading efficiency and replication with respect to the different internalization patterns of street and fixed laboratory-adapted viruses. PMID:27397101

  11. Two progressive substrates of the M-intermediate can be identified in glucose-embedded, wild-type bacteriorhodopsin.

    PubMed Central

    Vonck, J; Han, B G; Burkard, F; Perkins, G A; Glaeser, R M

    1994-01-01

    Glucose-embedded bacteriorhodopsin shows M-intermediates with different Amide I infrared bands when samples are illuminated at 240 or 260 K, in contrast with fully hydrated samples where a single M-intermediate is formed at all temperatures. In hydrated, but not in glucose-embedded specimens, the N intermediate is formed together with M at 260 K. Both Fourier transform infrared and electron diffraction data from glucose-embedded bacteriorhodopsin suggest that at 260 K a mixture is formed of the M-state that is trapped at 240 K, and a different M-intermediate (MN) that is also formed by mutant forms of bacteriorhodopsin that lack a carboxyl group at the 96 position, necessary for the M to N transition. The fact that an MN species is trapped in glucose-embedded, wild-type bacteriorhodopsin suggests that the glucose samples lack functionally important water molecules that are needed for the proton transfer aspartate 96 to the Schiff base (and, thus, to form the N-intermediate); thus, aspartate 96 is rendered ineffective as a proton donor. PMID:7811930

  12. Synthesis and utilization of fatty acids by wild-type and fatty acid auxotrophs of Caulobacter crescentus.

    PubMed Central

    Letts, V; Shaw, P; Shapiro, L; Henry, S

    1982-01-01

    The fatty acid composition of the dimorphic bacterium Caulobacter crescentus was found to consist primarily of 16- and 18-carbon fatty acids, both saturated and monounsaturated, in agreement with the findings of Chow and Schmidt (J. Gen. Microbiol. 83:359-373, 1974). In addition, two minor but as yet unidentified fatty acids were detected. Chromatographic mobilities suggested that these fatty acids may be a cyclopropane and a branched-chain fatty acid. In addition, we demonstrated that the fatty acid composition of wild-type C. crescentus can be altered by growing the cells in medium supplemented with any one of a variety of unsaturated fatty acids. Linoleic acid, a diunsaturated fatty acid which is not synthesized by C. crescentus, was incorporated into phospholipids without apparent modification. In addition, we found that C. crescentus, like Escherichia coli, synthesizes vaccenic acid (18:1 delta 11,cis) rather than oleic acid (18:1 delta 9,cis). This result allowed us to deduce that the mechanism of fatty acid desaturation in C. crescentus is anaerobic, as it is in E. coli. Finally, we examined the fatty acid biosynthesis and composition of two unsaturated fatty acid auxotrophs of C. crescentus. Neither of these mutants resembled the E. coli unsaturated fatty acid auxotrophs, which have defined enzymatic lesions in fatty acid biosynthesis. Rather, the mutants appeared to have defects relating to the complex coordination of membrane biogenesis and cell cycle events in C. crescentus. Images PMID:7107555

  13. Ethylene-Mediated Programmed Cell Death during Maize Endosperm Development of Wild-Type and shrunken2 Genotypes.

    PubMed Central

    Young, T. E.; Gallie, D. R.; DeMason, D. A.

    1997-01-01

    We characterized the progression of programmed cell death during maize (Zea mays L.) endosperm development of starchy (Su; wild-type) and shrunken2 (sh2) genotypes and tested the involve ment of ethylene in mediating this process. Histological and viability staining demonstrated that endosperm cell death was initiated earlier and progressed more rapidly in sh2 endosperm compared with Su endosperm. Internucleosomal DNA fragmentation accompanied endosperm cell death and occurred more extensively in sh2 endosperm. 1-Aminocyclopropane-1-carboxylic acid levels peaked approximately 16 d after pollination (dap) in Su endosperm and gradually decreased during subsequent development, whereas two large 1-aminocyclopropane-1-carboxylic acid peaks were observed in sh2 endosperm, the first between 16 and 20 dap and the second at 36 dap. Ethylene levels were elevated in sh2 kernels compared with Su kernels, with an initial peak 20 dap approximately 3-fold higher than in Su kernels and a second peak 36 dap approximately 5-fold higher than that in Su kernels. Ethylene treatment of Su kernels resulted in earlier and more extensive endosperm cell death and DNA fragmentation. Aminoethoxyvinylglycine treatment of sh2 kernels reduced the extent of DNA fragmentation. We conclude that ethylene is involved in triggering programmed cell death in developing maize endosperm and is responsible for the aberrant phenotype of sh2 kernels. PMID:12223841

  14. Identification of lipoxygenase (LOX) genes from legumes and their responses in wild type and cultivated peanut upon Aspergillus flavus infection

    PubMed Central

    Song, Hui; Wang, Pengfei; Li, Changsheng; Han, Suoyi; Lopez-Baltazar, Javier; Zhang, Xinyou; Wang, Xingjun

    2016-01-01

    Lipoxygenase (LOX) genes are widely distributed in plants and play crucial roles in resistance to biotic and abiotic stress. Although they have been characterized in various plants, little is known about the evolution of legume LOX genes. In this study, we identified 122 full-length LOX genes in Arachis duranensis, Arachis ipaënsis, Cajanus cajan, Cicer arietinum, Glycine max, Lotus japonicus and Medicago truncatula. In total, 64 orthologous and 36 paralogous genes were identified. The full-length, polycystin-1, lipoxygenase, alpha-toxin (PLAT) and lipoxygenase domain sequences from orthologous and paralogous genes exhibited a signature of purifying selection. However, purifying selection influenced orthologues more than paralogues, indicating greater functional conservation of orthologues than paralogues. Neutrality and effective number of codons plot results showed that natural selection primarily shapes codon usage, except for C. arietinum, L. japonicas and M. truncatula LOX genes. GCG, ACG, UCG, CGG and CCG codons exhibited low relative synonymous codon usage (RSCU) values, while CCA, GGA, GCU, CUU and GUU had high RSCU values, indicating that the latter codons are strongly preferred. LOX expression patterns differed significantly between wild-type peanut and cultivated peanut infected with Aspergillus flavus, which could explain the divergent disease resistance of wild progenitor and cultivars. PMID:27731413

  15. EPR analysis of cyanide complexes of wild-type human neuroglobin and mutants in comparison to horse heart myoglobin.

    PubMed

    Van Doorslaer, Sabine; Trandafir, Florin; Harmer, Jeffrey R; Moens, Luc; Dewilde, Sylvia

    2014-06-01

    Electron paramagnetic resonance (EPR) data reveal large differences between the ferric ((13)C-)cyanide complexes of wild-type human neuroglobin (NGB) and its H64Q and F28L point mutants and the cyanide complexes of mammalian myo- and haemoglobin. The point mutations, which involve residues comprising the distal haem pocket in NGB, induce smaller, but still significant changes, related to changes in the stabilization of the cyanide ligand. Furthermore, for the first time, the full (13)C hyperfine tensor of the cyanide carbon of cyanide-ligated horse heart myoglobin (hhMb) was determined using Davies ENDOR (electron nuclear double resonance). Disagreement of these experimental data with earlier predictions based on (13)C NMR data and a theoretical model reveal significant flaws in the model assumptions. The same ENDOR procedure allowed also partial determination of the corresponding (13)C hyperfine tensor of cyanide-ligated NGB and H64QNGB. These (13)C parameters differ significantly from those of cyanide-ligated hhMb and challenge our current theoretical understanding of how the haem environment influences the magnetic parameters obtained by EPR and NMR in cyanide-ligated haem proteins.

  16. Parametrisation of the free energy of ATP binding to wild-type and mutant Kir6.2 potassium channels.

    PubMed

    Moran, Oscar; Grottesi, Alessandro; Chadburn, Andrew J; Tammaro, Paolo

    2013-01-01

    ATP-sensitive K(+) (K(ATP)) channels, comprised of pore-forming Kir6.x and regulatory SURx subunits, play important roles in many cellular functions; because of their sensitivity to inhibition by intracellular ATP, K(ATP) channels provide a link between cell metabolism and membrane electrical activity. We constructed structural homology models of Kir6.2 and a series of Kir6.2 channels carrying mutations within the putative ATP-binding site. Computational docking was carried out to determine the conformation of ATP in its binding site. The Linear Interaction Energy (LIE) method was used to estimate the free-energy of ATP binding to wild-type and mutant Kir6.2 channels. Comparisons of the theoretical binding free energies for ATP with those determined from mutational experiments enabled the identification of the most probable conformation of ATP bound to the Kir6.2 channel. A set of LIE parameters was defined that may enable prediction of the effects of additional Kir6.2 mutations within the ATP binding site on the affinity for ATP.

  17. Comparison of transcriptomes of wild-type and isothiazolone-resistant Pseudomonas aeruginosa by using RNA-seq.

    PubMed

    Zhou, Gang; Shi, Qing-Shan; Huang, Xiao-Mo; Xie, Xiao-Bao

    2016-06-01

    Isothiazolone biocides (such as Kathon) are widely used in a variety of industrial and domestic applications. However, the mechanisms through which bacteria develop resistance to these biocides are not completely clear. A better understanding of these mechanisms can contribute to optimal use of these biocides. In this study, transcription profiles of a Kathon-resistant strain of Pseudomonas aeruginosa (Pa-R) and the wild-type strain were determined using RNA sequencing (RNA-Seq) with the Illumina HiSeq 2000 platform. RNA-Seq generated 18,657,896 sequence reads aligned to 7093 genes. In all, 1550 differently expressed genes (DEGs, log2 ratio ≥1, false discovery rate (FDR) ≤0.001) were identified, of which 482 were up-regulated and 1068 were down-regulated. Most Kathon-induced genes were involved in metabolic and cellular processes. DEGs significantly enriched nitrogen metabolism and oxidative phosphorylation pathways. In addition, Pa-R showed cross-resistance to triclosan and ciprofloxacin and showed repressed pyocyanin production. These results may improve our understanding of the resistance mechanisms of P. aeruginosa against isothiazolones, and provide insight into the development of more efficient isothiazolones.

  18. Protective responses in mice to vaccination with multiply administered cold-adapted influenza vaccine reassortants and wild-type viruses.

    PubMed

    Romanova, J R; Tannock, G A; Alexandrova, G I

    1997-01-01

    Protective responses to influenza vaccine reassortants derived from the cold-adapted (ca) donor strains A/Leningrad/134/17/57 and B/USSR/60/69 and wild-type epidemic viruses were studied in two strains of mice. Preliminary experiments revealed that, when mixtures of three viruses were inoculated intranasally to mice with 50 microliters containing 10(6) EID50 per 200 microliters (10(5.4) EID50 per mouse), interference between strains did not occur. However, interference with the growth of the influenza reassortant B/60/32/R took place if its concentration in the mixture was reduced to 10(5) (10(4.4) per mouse) or if it was inoculated at 10(6) EID50 (10(5.4) per mouse) in the presence of the influenza reassortant R/34 and two other influenza A epidemic strains; interference was unrelated to serological responses to infection with B/60/32/R. Despite evidence of interference, mice inoculated with the same mixtures in two identical doses, three weeks apart, were able to clear a challenge from each of seven homotypic and heterotypic influenza A and B strains. Heterotypic clearance of influenza A challenge viruses was greater following mixed infection, indicating that common determinants within the surface antigen glycoproteins contributed to immune responses which were broader than could be expected to be induced by parenteral vaccination.

  19. Dataset of differentially regulated proteins in HUVECs challenged with wild type and UGM1 mutant Aspergillus fumigatus strains.

    PubMed

    Neves, Gabriela Westerlund Peixoto; Curty, Nathália; Kubitschek-Barreira, Paula Helena; Fontaine, Thierry; Souza, Gustavo Henrique Martins Ferreira; Cunha, Marcel Lyra; Goldman, Gustavo H; Beauvais, Anne; Latgé, Jean-Paul; Lopes-Bezerra, Leila M

    2016-12-01

    Invasive aspergillosis is the primary opportunistic invasive fungal infection described in neutropenic hematologic patients, caused by the angioinvasive pathogen Aspergillus fumigatus. The molecular mechanisms associated with A. fumigatus infection in the vascular endothelium are poorly understood. In this context, we used a high-throughput proteomic approach to unveil the proteins modulated in HUVECs after interaction with a wild type strain and the UGM1 mutant (Δugm1) of A. fumigatus. The proteomic analysis was also performed in HUVECs challenged with a galactosaminogalactan (GAG) purified from A. fumigatus cell wall. The dataset presented here correspond to all proteins identified that fit a 2-fold change criteria (log 2 ratio ≥ 1 or ≤ -1), disregarding the statistical validation cut off, in order to supplement the research article entitled "Modifications to the composition of the hyphal outer layer of Aspergillus fumigatus modulates the HUVEC proteins associated with inflammatory and stress responses" (G.W.P. Neves, N.A. Curty, P.H. Kubitschek-Barreira, T. Fontaine, G.H.M.F. Souza, M. Lyra Cunha, G.H. Goldman, A. Beauvais, J.P. Latgé, L.M. Lopes-Bezerra, 2016) [1]. The mass spectrometry proteomic data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002823. PMID:27622208

  20. Dataset of differentially regulated proteins in HUVECs challenged with wild type and UGM1 mutant Aspergillus fumigatus strains.

    PubMed

    Neves, Gabriela Westerlund Peixoto; Curty, Nathália; Kubitschek-Barreira, Paula Helena; Fontaine, Thierry; Souza, Gustavo Henrique Martins Ferreira; Cunha, Marcel Lyra; Goldman, Gustavo H; Beauvais, Anne; Latgé, Jean-Paul; Lopes-Bezerra, Leila M

    2016-12-01

    Invasive aspergillosis is the primary opportunistic invasive fungal infection described in neutropenic hematologic patients, caused by the angioinvasive pathogen Aspergillus fumigatus. The molecular mechanisms associated with A. fumigatus infection in the vascular endothelium are poorly understood. In this context, we used a high-throughput proteomic approach to unveil the proteins modulated in HUVECs after interaction with a wild type strain and the UGM1 mutant (Δugm1) of A. fumigatus. The proteomic analysis was also performed in HUVECs challenged with a galactosaminogalactan (GAG) purified from A. fumigatus cell wall. The dataset presented here correspond to all proteins identified that fit a 2-fold change criteria (log 2 ratio ≥ 1 or ≤ -1), disregarding the statistical validation cut off, in order to supplement the research article entitled "Modifications to the composition of the hyphal outer layer of Aspergillus fumigatus modulates the HUVEC proteins associated with inflammatory and stress responses" (G.W.P. Neves, N.A. Curty, P.H. Kubitschek-Barreira, T. Fontaine, G.H.M.F. Souza, M. Lyra Cunha, G.H. Goldman, A. Beauvais, J.P. Latgé, L.M. Lopes-Bezerra, 2016) [1]. The mass spectrometry proteomic data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002823.

  1. Characterization of phenotype variations of luminescent and non-luminescent variants of Vibrio harveyi wild type and quorum sensing mutants.

    PubMed

    Hong, N T X; Baruah, K; Vanrompay, D; Bossier, P

    2016-03-01

    Vibrio harveyi, a luminescent Gram-negative motile marine bacterium, is an important pathogen responsible for causing severe diseases in shrimp, finfish and molluscs leading to severe economic losses. Non-luminescent V. harveyi obtained by culturing luminescent strains under static and dark condition were reported to alter the levels of virulence factors and metalloprotease gene and luxR expression when compared to their luminescent variants. Presently, we conducted an in vitro study aiming at the characterization of virulence-related phenotypic traits of the wild-type V. harveyi BB120 strain and its isogenic quorum sensing mutants before and after switching to the non-luminescent status. We measured the production of caseinase, haemolysin and elastase and examined swimming motility and biofilm formation. Our results showed that switching from the bioluminescent to the non-luminescent state changed the phenotypic physiology or behaviour of V. harveyi resulting in alterations in caseinase and haemolytic activities, swimming motility and biofilm formation. The switching capacity was to a large extent independent from the quorum sensing status, in that quorum sensing mutants were equally capable of making the phenotypic switch.

  2. Characterization of Hemodynamics in Great Arteries of Wild-Type Mouse Using Computational Fluid Dynamics Based on Ultrasound Images.

    PubMed

    Chen, Zhuo; Zhou, Yue; Ma, Youcai; Wang, Jingying; He, Yihua; Li, Zhian

    2016-03-01

    Hemodynamic factors in cardiovascular system are hypothesized to play a significant role in causing structural heart development. It is thus important to improve our understanding of velocity characteristics and parameters. We present such a study on wild-type mouse to characterize the vessel geometry, flow pattern, and wall shear stress in great arteries. Microultrasound imaging for small animals was used to measure blood boundary and velocity of the great arteries. Subsequently, specimens' flow boundary conditions were used for 3-dimensional reconstructions of the great artery and aortic arch dimensions, and blood flow velocity data were input into subject-specific computational fluid dynamics for modeling hemodynamics. Measurement by microultrasound imaging showed that blood velocities in the great artery and aortic arch had strong correlations with vascular sizes, whereas blood pressure had a weak trend in relation to vascular size. Wall shear stress magnitude increased when closer to arterial branches and reduced proximally in the aortic root and distally in the descending aorta, and the parameters were related to the fluid mechanics in branches in some degree. We developed a method to investigate fluid mechanics in mouse arteries, using a combination of microultrasound and computational fluid dynamics, and demonstrated its ability to reveal detailed geometric, kinematic, and fluid mechanics parameters.

  3. Unexpected mitochondrial matrix localization of Parkinson's disease-related DJ-1 mutants but not wild-type DJ-1.

    PubMed

    Kojima, Waka; Kujuro, Yuki; Okatsu, Kei; Bruno, Queliconi; Koyano, Fumika; Kimura, Mayumi; Yamano, Koji; Tanaka, Keiji; Matsuda, Noriyuki

    2016-07-01

    DJ-1 has been identified as a gene responsible for recessive familial Parkinson's disease (familial Parkinsonism), which is caused by a mutation in the PARK7 locus. Consistent with the inferred correlation between Parkinson's disease and mitochondrial impairment, mitochondrial localization of DJ-1 and its implied role in mitochondrial quality control have been reported. However, the mechanism by which DJ-1 affects mitochondrial function remains poorly defined, and the mitochondrial localization of DJ-1 is still controversial. Here, we show the mitochondrial matrix localization of various pathogenic and artificial DJ-1 mutants by multiple independent experimental approaches including cellular fractionation, proteinase K protection assays, and specific immunocytochemistry. Localization of various DJ-1 mutants to the matrix is dependent on the membrane potential and translocase activity in both the outer and the inner membranes. Nevertheless, DJ-1 possesses neither an amino-terminal alpha-helix nor a predictable matrix-targeting signal, and a post-translocation processing-derived molecular weight change is not observed. In fact, wild-type DJ-1 does not show any evidence of mitochondrial localization at all. Such a mode of matrix localization of DJ-1 is difficult to explain by conventional mechanisms and implies a unique matrix import mechanism for DJ-1 mutants. PMID:27270837

  4. A comparison of early floral ontogeny in wild-type and floral homeotic mutant phenotypes of Primula.

    PubMed

    Webster, Margaret A; Gilmartin, Philip A M

    2003-04-01

    Primula flowers are heteromorphic with individual plants producing either pin-form or thrum-form flowers. We have used scanning electron microscopy to observe early development of wild-type flowers of primrose (Primula vulgaris), cowslip (P. veris), and the polyanthus hybrid (P. x tommasinii x P. vulgaris). Floral ontogeny in Primula is different from that observed in the well-studied models Antirrhinum majus and Arabidopsis thaliana and our studies reveal morphological landmark events that define the sequence of early floral development in Primula into specific stages. Pin-form and thrum-form flowers are indistinguishable during early development with differentiation of the two floral morphs occurring beyond the differentiation of floral organs. Early ontogeny of flowers with homeotic mutant phenotypes was also studied to determine the timing of developmental reprogramming in these mutants. Phenotypes studied included Hose in Hose and Jack in the Green that develop petaloid sepals and leafy sepals, respectively, and Jackanapes plants that carry both these dominant mutations. Recessive double and semi- double flowers that produce additional whorls of petals and/or stamens in place of carpels were also studied. We describe a previously undocumented recessive Primula mutant phenotype, sepaloid, that produces sepals in place of petals and stamens, and a new non-homeotic, dominant mutant phenotype Split Perianth, in which sepals and petals fail to fuse to form the typical calyx and corolla structures. The molecular basis of these mutant phenotypes in relation to the ABC model is discussed.

  5. Comparative analysis of the integument transcriptomes of the black dilute mutant and the wild-type silkworm Bombyx mori.

    PubMed

    Wu, Songyuan; Tong, Xiaoling; Peng, Chenxing; Xiong, Gao; Lu, Kunpeng; Hu, Hai; Tan, Duan; Li, Chunlin; Han, Minjin; Lu, Cheng; Dai, Fangyin

    2016-01-01

    The insect cuticle is a critical protective shell that is composed predominantly of chitin and various cuticular proteins and pigments. Indeed, insects often change their surface pigment patterns in response to selective pressures, such as threats from predators, sexual selection and environmental changes. However, the molecular mechanisms underlying the construction of the epidermis and its pigmentation patterns are not fully understood. Among Lepidoptera, the silkworm is a favorable model for color pattern research. The black dilute (bd) mutant of silkworm is the result of a spontaneous mutation; the larval body color is notably melanized. We performed integument transcriptome sequencing of the wild-type strain Dazao and the mutant strains +/bd and bd/bd. In these experiments, during an early stage of the fourth molt, a stage at which approximately 51% of genes were expressed genome wide (RPKM ≥1) in each strain. A total of 254 novel transcripts were characterized using Cuffcompare and BLAST analyses. Comparison of the transcriptome data revealed 28 differentially expressed genes (DEGs) that may contribute to bd larval melanism, including 15 cuticular protein genes that were remarkably highly expressed in the bd/bd mutant. We suggest that these significantly up-regulated cuticular proteins may promote melanism in silkworm larvae. PMID:27193628

  6. Data set for comparison of cellular dynamics between human AAVS1 locus-modified and wild-type cells

    PubMed Central

    Mizutani, Takeomi; Haga, Hisashi; Kawabata, Kazushige

    2016-01-01

    This data article describes cellular dynamics, such as migration speed and mobility of the cytoskeletal protein, of wild-type human fibroblast cells and cells with a modified adeno-associated virus integration site 1 (AAVS1) locus on human chromosome 19. Insertion of exogenous gene into the AAVS1 locus has been conducted in recent biological researches. Previously, our data showed that the AAVS1-modification changes cellular contractile force (Mizutani et al., 2015 [1]). To assess if this AAVS1-modification affects cell migration, we compared cellular migration speed and turnover of cytoskeletal protein in human fibroblasts and fibroblasts with a green fluorescent protein gene knocked-in at the AAVS1 locus in this data article. Cell nuclei were stained and changes in their position attributable to cell migration were analyzed. Fluorescence recovery was observed after photobleaching for the fluorescent protein-tagged myosin regulatory light chain. Data here are related to the research article “Transgene Integration into the Human AAVS1 Locus Enhances Myosin II-Dependent Contractile Force by Reducing Expression of Myosin Binding Subunit 85” [1]. PMID:26937449

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

    PubMed

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

    2010-01-01

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

  8. Effects of norspermidine and spermidine on biofilm formation by potentially pathogenic Escherichia coli and Salmonella enterica wild-type strains.

    PubMed

    Nesse, Live L; Berg, Kristin; Vestby, Lene K

    2015-03-01

    Polyamines are present in all living cells. In bacteria, polyamines are involved in a variety of functions, including biofilm formation, thus indicating that polyamines may have potential in the control of unwanted biofilm. In the present study, the effects of the polyamines norspermidine and spermidine on biofilms of 10 potentially pathogenic wild-type strains of Escherichia coli serotype O103:H2, Salmonella enterica subsp. enterica serovar Typhimurium, and S. enterica serovar Agona were investigated. We found that exogenously supplied norspermidine and spermidine did not mediate disassembly of preformed biofilm of any of the E. coli and S. enterica strains. However, the polyamines did affect biofilm production. Interestingly, the two species reacted differently to the polyamines. Both polyamines reduced the amount of biofilm formed by E. coli but tended to increase biofilm formation by S. enterica. Whether the effects observed were due to the polyamines specifically targeting biofilm formation, being toxic for the cells, or maybe a combination of the two, is not known. However, there were no indications that the effect was mediated through binding to exopolysaccharides, as earlier suggested for E. coli. Our results indicate that norspermidine and spermidine do not have potential as inhibitors of S. enterica biofilm. Furthermore, we found that the commercial polyamines used contributed to the higher pH of the test medium. Failure to acknowledge and control this important phenomenon may lead to misinterpretation of the results. PMID:25595767

  9. Abscisic acid-responsive guard cell metabolomes of Arabidopsis wild-type and gpa1 G-protein mutants.

    PubMed

    Jin, Xiaofen; Wang, Rui-Sheng; Zhu, Mengmeng; Jeon, Byeong Wook; Albert, Reka; Chen, Sixue; Assmann, Sarah M

    2013-12-01

    Individual metabolites have been implicated in abscisic acid (ABA) signaling in guard cells, but a metabolite profile of this specialized cell type is lacking. We used liquid chromatography-multiple reaction monitoring mass spectrometry for targeted analysis of 85 signaling-related metabolites in Arabidopsis thaliana guard cell protoplasts over a time course of ABA treatment. The analysis utilized ∼ 350 million guard cell protoplasts from ∼ 30,000 plants of the Arabidopsis Columbia accession (Col) wild type and the heterotrimeric G-protein α subunit mutant, gpa1, which has ABA-hyposensitive stomata. These metabolomes revealed coordinated regulation of signaling metabolites in unrelated biochemical pathways. Metabolites clustered into different temporal modules in Col versus gpa1, with fewer metabolites showing ABA-altered profiles in gpa1. Ca(2+)-mobilizing agents sphingosine-1-phosphate and cyclic adenosine diphosphate ribose exhibited weaker ABA-stimulated increases in gpa1. Hormone metabolites were respon