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  1. Regulation of DAF-16-mediated Innate Immunity in Caenorhabditis elegans.

    PubMed

    Singh, Varsha; Aballay, Alejandro

    2009-12-18

    Activation of the innate immune system results in a rapid microbicidal response against microorganisms, which needs to be fine-tuned because uncontrolled immune responses can lead to infection and cancer, as well as conditions such as Crohn disease, atherosclerosis, and Alzheimer disease. Here we report that excessive activity of the conserved FOXO transcription factor DAF-16 enhances susceptibility to bacterial infections in Caenorhabditis elegans. We found that increased temperature activates not only DAF-16 nuclear import but also a control mechanism involved in DAF-16 nuclear export. The nuclear export of DAF-16 requires heat shock transcription factor HSF-1 and Hsp70/HSP-1. Furthermore, we show that increased expression of the water channel Aquoporin-1 is responsible for the deleterious consequences of excessive DAF-16-mediated immune response. These studies reveal a stress-inducible mechanism involved in the regulation of DAF-16 and indicate that uncontrolled DAF-16 activity and water homeostasis are a cause of the deleterious effects of excessive immune responses.

  2. Molecular cloning and DNA binding characterization of DAF-16 orthologs from Ancylostoma hookworms⋆

    PubMed Central

    Gao, Xin; Frank, Daniel; Hawdon, John M.

    2009-01-01

    Infective hookworm L3s encounter a host-specific signal during infection that re-initiates a suspended developmental pathway, resulting in development to the adult stage. This resumption of development in the host is analogous to recovery of developmentally arrested Caenorhabditis elegans dauer larvae in response to favorable environmental signals. Dauer recovery in C. elegans dauers and hookworm L3s is mediated by insulin-like signaling (ILS). A key output of ILS in C. elegans is the forkhead transcription factor DAF-16, which controls the expression of genes required for maintenance of the dauer stage. The similarity between recovery pathways of L3s and dauers suggests that DAF-16 functions similarly in hookworm L3 activation. To test this, orthologs of Ce-DAF-16 were isolated from the hookworms Ancylostoma caninum and Ancylostoma ceylanicum. The protein sequences of hookworm DAF-16 DNA binding domains were identical, and shared 94% identity with the b and c isoforms of Ce-DAF-16. Ac-DAF-16 expressed in HEK293 kidney cells bound strongly to the conserved DAF family binding element (DBE), but not to a random DNA sequence. Ac-DAF-16 was able to drive transcription of a reporter gene located downstream of six copies of the DBE in NIH3T3 cells under starved conditions. Addition of serum caused a decrease in reporter gene expression, indicating that DAF-16 is negatively regulated by growth factor stimulation. These data confirm the presence of DAF-16 orthologs in hookworms, and demonstrate that Ac-DAF-16 binds to and drives transcription from a conserved DAF-16 family DNA binding element. PMID:18930062

  3. C. elegans SIRT6/7 homolog SIR-2.4 promotes DAF-16 relocalization and function during stress.

    PubMed

    Chiang, Wei-Chung; Tishkoff, Daniel X; Yang, Bo; Wilson-Grady, Joshua; Yu, Xiaokun; Mazer, Travis; Eckersdorff, Mark; Gygi, Steven P; Lombard, David B; Hsu, Ao-Lin

    2012-09-01

    FoxO transcription factors and sirtuin family deacetylases regulate diverse biological processes, including stress responses and longevity. Here we show that the Caenorhabditis elegans sirtuin SIR-2.4--homolog of mammalian SIRT6 and SIRT7 proteins--promotes DAF-16-dependent transcription and stress-induced DAF-16 nuclear localization. SIR-2.4 is required for resistance to multiple stressors: heat shock, oxidative insult, and proteotoxicity. By contrast, SIR-2.4 is largely dispensable for DAF-16 nuclear localization and function in response to reduced insulin/IGF-1-like signaling. Although acetylation is known to regulate localization and activity of mammalian FoxO proteins, this modification has not been previously described on DAF-16. We find that DAF-16 is hyperacetylated in sir-2.4 mutants. Conversely, DAF-16 is acetylated by the acetyltransferase CBP-1, and DAF-16 is hypoacetylated and constitutively nuclear in response to cbp-1 inhibition. Surprisingly, a SIR-2.4 catalytic mutant efficiently rescues the DAF-16 localization defect in sir-2.4 null animals. Acetylation of DAF-16 by CBP-1 in vitro is inhibited by either wild-type or mutant SIR-2.4, suggesting that SIR-2.4 regulates DAF-16 acetylation indirectly, by preventing CBP-1-mediated acetylation under stress conditions. Taken together, our results identify SIR-2.4 as a critical regulator of DAF-16 specifically in the context of stress responses. Furthermore, they reveal a novel role for acetylation, modulated by the antagonistic activities of CBP-1 and SIR-2.4, in modulating DAF-16 localization and function.

  4. Pristionchus pacificus daf-16 is essential for dauer formation but dispensable for mouth form dimorphism.

    PubMed

    Ogawa, Akira; Bento, Gilberto; Bartelmes, Gabi; Dieterich, Christoph; Sommer, Ralf J

    2011-04-01

    The nematode Pristionchus pacificus shows two forms of phenotypic plasticity: dauer formation and dimorphism of mouth form morphologies. It can therefore serve as a model for studying the evolutionary mechanisms that underlie phenotypic plasticity. Formation of dauer larvae is observed in many other species and constitutes one of the most crucial survival strategies in nematodes, whereas the mouth form dimorphism is an evolutionary novelty observed only in P. pacificus and related nematodes. We have previously shown that the same environmental cues and steroid signaling control both dauer formation and mouth form dimorphism. Here, we examine by mutational analysis and whole-genome sequencing the function of P. pacificus (Ppa) daf-16, which encodes a forkhead transcription factor; in C. elegans, daf-16 is the target of insulin signaling and plays important roles in dauer formation. We found that mutations in Ppa-daf-16 cause strong dauer formation-defective phenotypes, suggesting that Ppa-daf-16 represents one of the evolutionarily conserved regulators of dauer formation. Upon strong dauer induction with lophenol, Ppa-daf-16 individuals formed arrested larvae that partially resemble wild-type dauer larvae, indicating that Ppa-daf-16 is also required for dauer morphogenesis. By contrast, regulation of mouth form dimorphism was unaffected by Ppa-daf-16 mutations and mutant animals responded normally to environmental cues. Our results suggest that mechanisms for dauer formation and mouth form regulation overlap partially, but not completely, and one of two key transcriptional regulators of the dauer regulatory network was either independently co-opted for, or subsequently lost by, the mouth form regulatory network.

  5. Oleanolic acid activates daf-16 to increase lifespan in Caenorhabditis elegans

    SciTech Connect

    Zhang, Jiaolong; Lu, Lulu; Zhou, Lijun

    2015-12-25

    Oleanolic acid (OA) is an active ingredient in natural plants. It has been reported to possess a variety of pharmacological activities, but very little is known about its effects of anti-aging. We investigate here whether OA has an impact on longevity in vivo, and more specifically, we have examined effects of OA on the lifespan and stress tolerance in Caenorhabditis elegans (C. elegans). Our results showed that OA could extend the lifespan, increase its stress resistance and reduce the intracellular reactive oxygen species (ROS) in wild-type worms. Moreover, we have found that OA-induced longevity may not be associated with the calorie restriction (CR) mechanism. Our mechanistic studies using daf-16 loss-of-function mutant strains (GR1307) indicated that the extension of lifespan by OA requires daf-16. In addition, OA treatment could also modulate the nuclear localization, and the quantitative real-time PCR results revealed that up-regulation of daf-16 target genes such as sod-3, hsp-16.2 and ctl-1 could prolong lifespan and increase stress response in C. elegans. This study overall uncovers the longevity effect of OA and its underpinning mechanisms. - Graphical abstract: Oleanolic acid modulates the activity of DAF-16 to promote longevity and increase stress resistance in Caenorhabditis elegans. - Highlights: • OA extends the lifespan of wild-type Caenorhabditis elegans. • OA improves the stress resistance and reduces the intracellular ROS level in C. elegans. • OA induces lifespan extension may not proceed through the CR mechanism. • OA extends the lifespan in C. elegans is modulated by daf-16.

  6. Rifampicin reduces advanced glycation end products and activates DAF-16 to increase lifespan in Caenorhabditis elegans.

    PubMed

    Golegaonkar, Sandeep; Tabrez, Syed S; Pandit, Awadhesh; Sethurathinam, Shalini; Jagadeeshaprasad, Mashanipalya G; Bansode, Sneha; Sampathkumar, Srinivasa-Gopalan; Kulkarni, Mahesh J; Mukhopadhyay, Arnab

    2015-06-01

    Advanced glycation end products (AGEs) are formed when glucose reacts nonenzymatically with proteins; these modifications are implicated in aging and pathogenesis of many age-related diseases including type II diabetes, atherosclerosis, and neurodegenerative disorders. Thus, pharmaceutical interventions that can reduce AGEs may delay age-onset diseases and extend lifespan. Using LC-MS(E), we show that rifampicin (RIF) reduces glycation of important cellular proteins in vivo and consequently increases lifespan in Caenorhabditis elegans by up to 60%. RIF analog rifamycin SV (RSV) possesses similar properties, while rifaximin (RMN) lacks antiglycation activity and therefore fails to affect lifespan positively. The efficacy of RIF and RSV as potent antiglycating agents may be attributed to the presence of a p-dihydroxyl moiety that can potentially undergo spontaneous oxidation to yield highly reactive p-quinone structures, a feature absent in RMN. We also show that supplementing rifampicin late in adulthood is sufficient to increase lifespan. For its effect on longevity, rifampicin requires DAF-18 (nematode PTEN) as well as JNK-1 and activates DAF-16, the FOXO homolog. Interestingly, the drug treatment modulates transcription of a different subset of DAF-16 target genes, those not controlled by the conserved Insulin-IGF-1-like signaling pathway. RIF failed to increase the lifespan of daf-16 null mutant despite reducing glycation, showing thereby that DAF-16 may not directly affect AGE formation. Together, our data suggest that the dual ability to reduce glycation in vivo and activate prolongevity processes through DAF-16 makes RIF and RSV effective lifespan-extending interventions. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  7. dbl-1/TGF-β and daf-12/NHR Signaling Mediate Cell-Nonautonomous Effects of daf-16/FOXO on Starvation-Induced Developmental Arrest

    PubMed Central

    Moore, Brad T.; Jordan, James M.; Maxwell, Colin S.; Schindler, Adam J.; Baugh, L. Ryan

    2015-01-01

    Nutrient availability has profound influence on development. In the nematode C. elegans, nutrient availability governs post-embryonic development. L1-stage larvae remain in a state of developmental arrest after hatching until they feed. This “L1 arrest” (or "L1 diapause") is associated with increased stress resistance, supporting starvation survival. Loss of the transcription factor daf-16/FOXO, an effector of insulin/IGF signaling, results in arrest-defective and starvation-sensitive phenotypes. We show that daf-16/FOXO regulates L1 arrest cell-nonautonomously, suggesting that insulin/IGF signaling regulates at least one additional signaling pathway. We used mRNA-seq to identify candidate signaling molecules affected by daf-16/FOXO during L1 arrest. dbl-1/TGF-β, a ligand for the Sma/Mab pathway, daf-12/NHR and daf-36/oxygenase, an upstream component of the daf-12 steroid hormone signaling pathway, were up-regulated during L1 arrest in a daf-16/FOXO mutant. Using genetic epistasis analysis, we show that dbl-1/TGF-β and daf-12/NHR steroid hormone signaling pathways are required for the daf-16/FOXO arrest-defective phenotype, suggesting that daf-16/FOXO represses dbl-1/TGF-β, daf-12/NHR and daf-36/oxygenase. The dbl-1/TGF-β and daf-12/NHR pathways have not previously been shown to affect L1 development, but we found that disruption of these pathways delayed L1 development in fed larvae, consistent with these pathways promoting development in starved daf-16/FOXO mutants. Though the dbl-1/TGF-β and daf-12/NHR pathways are epistatic to daf-16/FOXO for the arrest-defective phenotype, disruption of these pathways does not suppress starvation sensitivity of daf-16/FOXO mutants. This observation uncouples starvation survival from developmental arrest, indicating that DAF-16/FOXO targets distinct effectors for each phenotype and revealing that inappropriate development during starvation does not cause the early demise of daf-16/FOXO mutants. Overall, this study

  8. Transgenic C. elegans dauer larvae expressing hookworm phospho null DAF-16/FoxO exit dauer.

    PubMed

    Gelmedin, Verena; Brodigan, Thomas; Gao, Xin; Krause, Michael; Wang, Zhu; Hawdon, John M

    2011-01-01

    Parasitic hookworms and the free-living model nematode Caenorhabtidis elegans share a developmental arrested stage, called the dauer stage in C. elegans and the infective third-stage larva (L3) in hookworms. One of the key transcription factors that regulate entrance to and exit from developmental arrest is the forkhead transcription factor DAF-16/FoxO. During the dauer stage, DAF-16 is activated and localized in the nucleus. DAF-16 is negatively regulated by phosphorylation by the upstream kinase AKT, which causes DAF-16 to localize out of the nucleus and the worm to exit from dauer. DAF-16 is conserved in hookworms, and hypothesized to control recovery from L3 arrest during infection. Lacking reverse genetic techniques for use in hookworms, we used C. elegans complementation assays to investigate the function of Ancylostoma caninum DAF-16 during entrance and exit from L3 developmental arrest. We performed dauer switching assays and observed the restoration of the dauer phenotype when Ac-DAF-16 was expressed in temperature-sensitive dauer defective C. elegans daf-2(e1370);daf-16(mu86) mutants. AKT phosphorylation site mutants of Ac-DAF-16 were also able to restore the dauer phenotype, but surprisingly allowed dauer exit when temperatures were lowered. We used fluorescence microscopy to localize DAF-16 during dauer and exit from dauer in C. elegans DAF-16 mutant worms expressing Ac-DAF-16, and found that Ac-DAF-16 exited the nucleus during dauer exit. Surprisingly, Ac-DAF-16 with mutated AKT phosphorylation sites also exited the nucleus during dauer exit. Our results suggest that another mechanism may be involved in the regulation DAF-16 nuclear localization during recovery from developmental arrest.

  9. Non-autonomous DAF-16/FOXO activity antagonizes age-related loss of C. elegans germline stem/progenitor cells

    PubMed Central

    Qin, Zhao; Hubbard, E. Jane Albert

    2015-01-01

    Stem cells maintain tissues and organs over the lifespan of individuals. How aging influences this process is unclear. Here we investigate the effects of aging on C. elegans germline stem/progenitor cells and show that the progenitor pool is depleted over time in a manner dependent on inhibition of DAF-16/FOXO by insulin/IGF-1 signalling (IIS). Our data indicate that DAF-16/FOXO activity in certain somatic gonad cells is required for germline progenitor maintenance, and that this role is separable from the effect of DAF-16/FOXO on organismal aging. In addition, blocking germ cell flux, similar to reducing IIS, maintains germline progenitors. This effect is partially dependent on gonadal DAF-16/FOXO activity. Our results imply that (1) longevity pathways can regulate aging stem cells through anatomically separable mechanisms, (2) stem cell maintenance is not necessarily prioritized and (3) stem cell regulation can occur at the level of an entire organ system such as the reproductive system. PMID:25960195

  10. Transcriptional regulation of Caenorhabditis elegans FOXO/DAF-16 modulates lifespan

    PubMed Central

    2014-01-01

    Background Insulin/IGF-1 signaling plays a central role in longevity across phylogeny. In C. elegans, the forkhead box O (FOXO) transcription factor, DAF-16, is the primary target of insulin/IGF-1 signaling, and multiple isoforms of DAF-16 (a, b, and d/f) modulate lifespan, metabolism, dauer formation, and stress resistance. Thus far, across phylogeny modulation of mammalian FOXOs and DAF-16 have focused on post-translational regulation with little focus on transcriptional regulation. In C. elegans, we have previously shown that DAF-16d/f cooperates with DAF-16a to promote longevity. In this study, we generated transgenic strains expressing near-endogenous levels of either daf-16a or daf-16d/f, and examined temporal expression of the isoforms to further define how these isoforms contribute to lifespan regulation. Results Here, we show that DAF-16a is sensitive both to changes in gene dosage and to alterations in the level of insulin/IGF-1 signaling. Interestingly, we find that as worms age, the intestinal expression of daf-16d/f but not daf-16a is dramatically upregulated at the level of transcription. Preventing this transcriptional upregulation shortens lifespan, indicating that transcriptional regulation of daf-16d/f promotes longevity. In an RNAi screen of transcriptional regulators, we identify elt-2 (GATA transcription factor) and swsn-1 (core subunit of SWI/SNF complex) as key modulators of daf-16d/f gene expression. ELT-2 and another GATA factor, ELT-4, promote longevity via both DAF-16a and DAF-16d/f while the components of SWI/SNF complex promote longevity specifically via DAF-16d/f. Conclusions Our findings indicate that transcriptional control of C. elegans FOXO/daf-16 is an essential regulatory event. Considering the conservation of FOXO across species, our findings identify a new layer of FOXO regulation as a potential determinant of mammalian longevity and age-related diseases such as cancer and diabetes. PMID:24834345

  11. The Deubiquitylase MATH-33 Controls DAF-16 Stability and Function in Metabolism and Longevity

    PubMed Central

    Heimbucher, Thomas; Liu, Zheng; Bossard, Carine; McCloskey, Richard; Carrano, Andrea C.; Riedel, Christian G.; Tanasa, Bogdan; Klammt, Christian; Fonslow, Bryan R.; Riera, Celine E.; Lillemeier, Bjorn F.; Kemphues, Kenneth; Yates, John R.; O'Shea, Clodagh; Hunter, Tony; Dillin, Andrew

    2015-01-01

    SUMMARY One of the major determinants of aging in organisms ranging from worms to man are FOXO family transcription factors, which are downstream effectors of Insulin/IGF-1 signaling (IIS). The molecular mechanisms that actively promote DAF16/FOXO stability and function are unknown. Here we identify the deubiquitylating enzyme MATH-33 as an essential DAF-16 regulator in IIS, which stabilizes active DAF-16 protein levels and, as a consequence, influences DAF-16 functions, such as metabolism, stress response and longevity in C. elegans. MATH-33 associates with DAF-16 in cellulo and in vitro. MATH-33 functions as a deubiquitylase by actively removing ubiquitin moieties from DAF-16, thus counteracting the action of the RLE-1 E3-ubiquitin ligase. Our findings support a model in which MATH-33 promotes DAF-16 stability in response to decreased IIS by directly modulating its ubiquitylation state, suggesting that regulated oscillations in the stability of DAF-16 protein play an integral role in controlling processes such as metabolism and longevity. PMID:26154057

  12. Calycosin promotes lifespan in Caenorhabditis elegans through insulin signaling pathway via daf-16, age-1 and daf-2.

    PubMed

    Lu, Lulu; Zhao, Xuan; Zhang, Jianyong; Li, Miao; Qi, Yonghao; Zhou, Lijun

    2017-07-01

    The naturally occurring calycosin is a known antioxidant that prevents redox imbalance in organisms. However, calycosin's effect on lifespan and its physiological molecular mechanisms are not yet well understood. In this study, we demonstrated that calycosin could prolong the lifespan of Caenorhabditis elegans, and that such extension was associated with its antioxidant capability as well as its ability to enhance stress resistance and reduce ROS (reactive oxygen species) accumulation. To explore mechanisms of this longevity effect, we assessed the impact of calycosin on lifespans of insulin-signaling impaired worms: daf-2, age-1, and daf-16 mutants. We found that calycosin did not alter the lifespan of all three mutants, thereby suggesting that calycosin requires insulin signaling to promote lifespan extension. On the other hand, we observed that calycosin could enhance the nuclear translocation of the core transcription factor DAF-16/FoXO instead of the conserved stress-responsive transcription factor SKN-1/Nrf-2. This observation is consistent with the understanding that the nuclear localized DAF-16 up-regulates its downstream targets sod-3, ctl-1, and hsp-16.2. Lastly, it is also noteworthy that the longevity effect of calycosin is likely not associated with the calorie restriction mechanism. Collectively, our results strongly suggest that calycosin could function as an antioxidant to extend the lifespan of C. elegans by enhancing nucleus translocation of DAF-16 through the insulin-signaling pathway. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  13. Royal jelly promotes DAF-16-mediated proteostasis to tolerate β-amyloid toxicity in C. elegans model of Alzheimer's disease.

    PubMed

    Wang, Xiaoxia; Cao, Min; Dong, Yuqing

    2016-08-23

    Numerous studies have demonstrated that dietary intervention may promote health and help prevent Alzheimer's disease (AD). We recently reported that bee products of royal jelly (RJ) and enzyme-treated royal jelly (eRJ) were potent to promote healthy aging in C. elegans. Here, we examined whether RJ/eRJ consumption may benefit to mitigate the AD symptom in the disease model of C. elegans. Our results showed that RJ/eRJ supplementation significantly delayed the body paralysis in AD worms, suggesting the β-amyloid (Aβ) toxicity attenuation effects of RJ/eRJ. Genetic analyses suggested that RJ/eRJ-mediated alleviation of Aβ toxicity in AD worms required DAF-16, rather than HSF-1 and SKN-1, in an insulin/IGF signaling dependent manner. Moreover, RJ/eRJ modulated the transactivity of DAF-16 and dramatically improved the protein solubility in aged worms. Given protein solubility is a hallmark of healthy proteostasis, our findings demonstrated that RJ/eRJ supplementation improved proteostasis, and this promotion depended on the transactivity of DAF-16. Collectively, the present study not only elucidated the possible anti-AD mechanism of RJ/eRJ, but also provided evidence from a practical point of view to shed light on the extensive correlation of proteostasis and the prevention of neurodegenerative disorders.

  14. RNA helicase HEL-1 promotes longevity by specifically activating DAF-16/FOXO transcription factor signaling in Caenorhabditis elegans.

    PubMed

    Seo, Mihwa; Seo, Keunhee; Hwang, Wooseon; Koo, Hee Jung; Hahm, Jeong-Hoon; Yang, Jae-Seong; Han, Seong Kyu; Hwang, Daehee; Kim, Sanguk; Jang, Sung Key; Lee, Yoontae; Nam, Hong Gil; Lee, Seung-Jae V

    2015-08-04

    The homeostatic maintenance of the genomic DNA is crucial for regulating aging processes. However, the role of RNA homeostasis in aging processes remains unknown. RNA helicases are a large family of enzymes that regulate the biogenesis and homeostasis of RNA. However, the functional significance of RNA helicases in aging has not been explored. Here, we report that a large fraction of RNA helicases regulate the lifespan of Caenorhabditis elegans. In particular, we show that a DEAD-box RNA helicase, helicase 1 (HEL-1), promotes longevity by specifically activating the DAF-16/forkhead box O (FOXO) transcription factor signaling pathway. We find that HEL-1 is required for the longevity conferred by reduced insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) and is sufficient for extending lifespan. We further show that the expression of HEL-1 in the intestine and neurons contributes to longevity. HEL-1 enhances the induction of a large fraction of DAF-16 target genes. Thus, the RNA helicase HEL-1 appears to promote longevity in response to decreased IIS as a transcription coregulator of DAF-16. Because HEL-1 and IIS are evolutionarily well conserved, a similar mechanism for longevity regulation via an RNA helicase-dependent regulation of FOXO signaling may operate in mammals, including humans.

  15. Royal jelly promotes DAF-16-mediated proteostasis to tolerate β-amyloid toxicity in C. elegans model of Alzheimer's disease

    PubMed Central

    Wang, Xiaoxia; Cao, Min; Dong, Yuqing

    2016-01-01

    Numerous studies have demonstrated that dietary intervention may promote health and help prevent Alzheimer's disease (AD). We recently reported that bee products of royal jelly (RJ) and enzyme-treated royal jelly (eRJ) were potent to promote healthy aging in C. elegans. Here, we examined whether RJ/eRJ consumption may benefit to mitigate the AD symptom in the disease model of C. elegans. Our results showed that RJ/eRJ supplementation significantly delayed the body paralysis in AD worms, suggesting the β-amyloid (Aβ) toxicity attenuation effects of RJ/eRJ. Genetic analyses suggested that RJ/eRJ-mediated alleviation of Aβ toxicity in AD worms required DAF-16, rather than HSF-1 and SKN-1, in an insulin/IGF signaling dependent manner. Moreover, RJ/eRJ modulated the transactivity of DAF-16 and dramatically improved the protein solubility in aged worms. Given protein solubility is a hallmark of healthy proteostasis, our findings demonstrated that RJ/eRJ supplementation improved proteostasis, and this promotion depended on the transactivity of DAF-16. Collectively, the present study not only elucidated the possible anti-AD mechanism of RJ/eRJ, but also provided evidence from a practical point of view to shed light on the extensive correlation of proteostasis and the prevention of neurodegenerative disorders. PMID:27472466

  16. Genome-wide endogenous DAF-16/FOXO recruitment dynamics during lowered insulin signalling in C. elegans

    PubMed Central

    Kumar, Neeraj; Jagtap, Urmila; Verma, Sonia; Mukhopadhyay, Arnab

    2015-01-01

    Lowering insulin-IGF-1-like signalling (IIS) activates FOXO transcription factors (TF) to extend life span across species. To study the dynamics of FOXO chromatin occupancy under this condition in C. elegans, we report the first recruitment profile of endogenous DAF-16 and show that the response is conserved. DAF-16 predominantly acts as a transcriptional activator and binding within the 0.5 kb promoter-proximal region results in maximum induction of downstream targets that code for proteins involved in detoxification and longevity. Interestingly, genes that are activated under low IIS already have higher DAF-16 recruited to their promoters in WT. DAF-16 binds to variants of the FOXO consensus sequence in the promoter proximal regions of genes that are exclusively targeted during low IIS. We also define a set of ‘core’ direct targets, after comparing multiple studies, which tend to co-express and contribute robustly towards IIS-associated phenotypes. Additionally, we show that nuclear hormone receptor DAF-12 as well as zinc-finger TF EOR-1 may bind DNA in close proximity to DAF-16 and distinct TF classes that are direct targets of DAF-16 may be instrumental in regulating its indirect targets. Together, our study provides fundamental insights into the transcriptional biology of FOXO/DAF-16 and gene regulation downstream of the IIS pathway. PMID:26539642

  17. The C. elegans Ortholog of USP7 controls DAF-16 stability in Insulin/IGF-1-like signaling.

    PubMed

    Heimbucher, Thomas; Hunter, Tony

    2015-01-01

    FOXO family transcription factors are downstream effectors of Insulin/IGF-1 signaling (IIS) and are regulated by posttranslational modification and coregulators, including components of the ubiquitin-proteasome system (UPS). Cofactors promoting DAF-16/FOXO protein stability and function in IIS have not been described yet. In a recent study, we have identified the deubiquitylating enzyme MATH-33, the ortholog of mammalian USP7/HAUSP, as an essential DAF-16 coregulator. We found that MATH-33 actively stabilizes DAF-16 protein levels when IIS is downregulated. Here we discuss how DAF-16/FOXO transcription factors are regulated by the UPS, in particular by the interplay of E3-ubiquitin ligases and deubiquitylating enzymes, which is critical for balancing DAF-16/FOXO activity and degradation. Recent findings raise the intriguing possibility that regulated oscillations in DAF-16/FOXO steady state levels play an integral role in mechanisms controlling healthspan and lifespan extension.

  18. The longevity effect of echinacoside in Caenorhabditis elegans mediated through daf-16.

    PubMed

    Wang, Xue; Zhang, Jiaolong; Lu, Lulu; Zhou, Lijun

    2015-01-01

    Echinacoside (ECH), a natural polyphenolic compound, has been reported to possess important pharmacological activities. However, very little is known about whether or how ECH affects longevity in vivo. We have examined the effects of ECH on the life span and stress tolerance in Caenorhabditis elegans. Our studies demonstrate that the life span of wild-type worms could be extended in the presence of ECH. Furthermore, ECH was found to increase tolerance of worms to heat shock and oxidative stress, while not exerting any influence on pharyngeal pumping rate and progeny production. Our mechanistic studies indicate that supplementation of ECH increases the transcript level of daf-16. ECH treatment also modulates the nuclear localization and transcriptional activities of daf-16, thus fine tunes the expression of daf-16 target genes to promote longevity and increases stress response in C. elegans. Overall, this work reveals the longevity effect of ECH and elucidates the underpinning mechanisms.

  19. The longevity effect of cranberry extract in Caenorhabditis elegans is modulated by daf-16 and osr-1.

    PubMed

    Guha, Sujay; Cao, Min; Kane, Ryan M; Savino, Anthony M; Zou, Sige; Dong, Yuqing

    2013-10-01

    Nutraceuticals are known to have numerous health and disease preventing properties. Recent studies suggest that extracts containing cranberry may have anti-aging benefits. However, little is known about whether and how cranberry by itself promotes longevity and healthspan in any organism. Here we examined the effect of a cranberry only extract on lifespan and healthspan in Caenorhabditis elegans. Supplementation of the diet with cranberry extract (CBE) increased the lifespan in C. elegans in a concentration-dependent manner. Cranberry also increased tolerance of C. elegans to heat shock, but not to oxidative stress or ultraviolet irradiation. In addition, we tested the effect of cranberry on brood size and motility and found that cranberry did not influence these behaviors. Our mechanistic studies indicated that lifespan extension induced by CBE requires the insulin/IGF signaling pathway and DAF-16. We also found that cranberry promotes longevity through osmotic stress resistant-1 (OSR-1) and one of its downstream effectors, UNC-43, but not through SEK-1, a component of the p38 MAP kinase pathway. However, SIR-2.1 and JNK signaling pathways are not required for cranberry to promote longevity. Our findings suggest that cranberry supplementation confers increased longevity and stress resistance in C. elegans through pathways modulated by daf-16 and osr-1. This study reveals the anti-aging property of widely consumed cranberry and elucidates the underpinning mechanisms.

  20. LIN-28 balances longevity and germline stem cell number in Caenorhabditis elegans through let-7/AKT/DAF-16 axis.

    PubMed

    Wang, Dan; Hou, Lei; Nakamura, Shuhei; Su, Ming; Li, Fang; Chen, Weiyang; Yan, Yizhen; Green, Christopher D; Chen, Di; Zhang, Hong; Antebi, Adam; Han, Jing-Dong J

    2017-02-01

    The RNA-binding protein LIN-28 was first found to control developmental timing in Caenorhabditis elegans. Later, it was found to play important roles in pluripotency, metabolism, and cancer in mammals. Here we report that a low dosage of lin-28 enhanced stress tolerance and longevity, and reduced germline stem/progenitor cell number in C. elegans. The germline LIN-28-regulated microRNA let-7 was required for these effects by targeting akt-1/2 and decreasing their protein levels. AKT-1/2 and the downstream DAF-16 transcription factor were both required for the lifespan and germline stem cell effects of lin-28. The pathway also mediated dietary restriction induced lifespan extension and reduction in germline stem cell number. Thus, the LIN-28/let-7/AKT/DAF-16 axis we delineated here is a program that plays an important role in balancing reproduction and somatic maintenance and their response to the environmental energy level-a central dogma of the 'evolutionary optimization' of resource allocation that modulates aging. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  1. Towards understanding the lifespan extension by reduced insulin signaling: bioinformatics analysis of DAF-16/FOXO direct targets in Caenorhabditis elegans

    PubMed Central

    Li, Yan-Hui; Zhang, Gai-Gai

    2016-01-01

    DAF-16, the C. elegans FOXO transcription factor, is an important determinant in aging and longevity. In this work, we manually curated FOXODB http://lyh.pkmu.cn/foxodb/, a database of FOXO direct targets. It now covers 208 genes. Bioinformatics analysis on 109 DAF-16 direct targets in C. elegans found interesting results. (i) DAF-16 and transcription factor PQM-1 co-regulate some targets. (ii) Seventeen targets directly regulate lifespan. (iii) Four targets are involved in lifespan extension induced by dietary restriction. And (iv) DAF-16 direct targets might play global roles in lifespan regulation. PMID:27027346

  2. Towards understanding the lifespan extension by reduced insulin signaling: bioinformatics analysis of DAF-16/FOXO direct targets in Caenorhabditis elegans.

    PubMed

    Li, Yan-Hui; Zhang, Gai-Gai

    2016-04-12

    DAF-16, the C. elegans FOXO transcription factor, is an important determinant in aging and longevity. In this work, we manually curated FOXODB http://lyh.pkmu.cn/foxodb/, a database of FOXO direct targets. It now covers 208 genes. Bioinformatics analysis on 109 DAF-16 direct targets in C. elegans found interesting results. (i) DAF-16 and transcription factor PQM-1 co-regulate some targets. (ii) Seventeen targets directly regulate lifespan. (iii) Four targets are involved in lifespan extension induced by dietary restriction. And (iv) DAF-16 direct targets might play global roles in lifespan regulation.

  3. Myricetin-Mediated Lifespan Extension in Caenorhabditis elegans Is Modulated by DAF-16

    PubMed Central

    Büchter, Christian; Ackermann, Daniela; Havermann, Susannah; Honnen, Sebastian; Chovolou, Yvonni; Fritz, Gerhard; Kampkötter, Andreas; Wätjen, Wim

    2013-01-01

    Myricetin is a naturally occurring flavonol found in many plant based food sources. It increases the lifespan of Caenorhabditis elegans, but the molecular mechanisms are not yet fully understood. We have investigated the impact of this flavonoid on the transcription factors DAF-16 (C. elegans FoxO homologue) and SKN-1 (Nrf2 homologue), which have crucial functions in the regulation of ageing. Myricetin is rapidly assimilated by the nematode, causes a nuclear translocation of DAF-16 but not of SKN-1, and finally prolongs the mean adult lifespan of C. elegans by 32.9%. The lifespan prolongation was associated with a decrease in the accumulation of reactive oxygen species (ROS) detected by DCF. Myricetin also decreases the formation of lipofuscin, a pigment consisting of highly oxidized and cross-linked proteins that is considered as a biomarker of ageing in diverse species. The lifespan extension was completely abolished in a daf-16 loss-of-function mutant strain (CF1038). Consistently with this result, myricetin was also not able to diminish stress-induced ROS accumulation in the mutant. These results strongly indicate that the pro-longevity effect of myricetin is dependent on DAF-16 and not on direct anti-oxidative effects of the flavonoid. PMID:23736695

  4. Longevity Genes Revealed by Integrative Analysis of Isoform-Specific daf-16/FoxO Mutants of Caenorhabditis elegans

    PubMed Central

    Chen, Albert Tzong-Yang; Guo, Chunfang; Itani, Omar A.; Budaitis, Breane G.; Williams, Travis W.; Hopkins, Christopher E.; McEachin, Richard C.; Pande, Manjusha; Grant, Ana R.; Yoshina, Sawako; Mitani, Shohei; Hu, Patrick J.

    2015-01-01

    FoxO transcription factors promote longevity across taxa. How they do so is poorly understood. In the nematode Caenorhabditis elegans, the A- and F-isoforms of the FoxO transcription factor DAF-16 extend life span in the context of reduced DAF-2 insulin-like growth factor receptor (IGFR) signaling. To elucidate the mechanistic basis for DAF-16/FoxO-dependent life span extension, we performed an integrative analysis of isoform-specific daf-16/FoxO mutants. In contrast to previous studies suggesting that DAF-16F plays a more prominent role in life span control than DAF-16A, isoform-specific daf-16/FoxO mutant phenotypes and whole transcriptome profiling revealed a predominant role for DAF-16A over DAF-16F in life span control, stress resistance, and target gene regulation. Integration of these datasets enabled the prioritization of a subset of 92 DAF-16/FoxO target genes for functional interrogation. Among 29 genes tested, two DAF-16A-specific target genes significantly influenced longevity. A loss-of-function mutation in the conserved gene gst-20, which is induced by DAF-16A, reduced life span extension in the context of daf-2/IGFR RNAi without influencing longevity in animals subjected to control RNAi. Therefore, gst-20 promotes DAF-16/FoxO-dependent longevity. Conversely, a loss-of-function mutation in srr-4, a gene encoding a seven-transmembrane-domain receptor family member that is repressed by DAF-16A, extended life span in control animals, indicating that DAF-16/FoxO may extend life span at least in part by reducing srr-4 expression. Our discovery of new longevity genes underscores the efficacy of our integrative strategy while providing a general framework for identifying specific downstream gene regulatory events that contribute substantially to transcription factor functions. As FoxO transcription factors have conserved functions in promoting longevity and may be dysregulated in aging-related diseases, these findings promise to illuminate fundamental

  5. Longevity Genes Revealed by Integrative Analysis of Isoform-Specific daf-16/FoxO Mutants of Caenorhabditis elegans.

    PubMed

    Chen, Albert Tzong-Yang; Guo, Chunfang; Itani, Omar A; Budaitis, Breane G; Williams, Travis W; Hopkins, Christopher E; McEachin, Richard C; Pande, Manjusha; Grant, Ana R; Yoshina, Sawako; Mitani, Shohei; Hu, Patrick J

    2015-10-01

    FoxO transcription factors promote longevity across taxa. How they do so is poorly understood. In the nematode Caenorhabditis elegans, the A- and F-isoforms of the FoxO transcription factor DAF-16 extend life span in the context of reduced DAF-2 insulin-like growth factor receptor (IGFR) signaling. To elucidate the mechanistic basis for DAF-16/FoxO-dependent life span extension, we performed an integrative analysis of isoform-specific daf-16/FoxO mutants. In contrast to previous studies suggesting that DAF-16F plays a more prominent role in life span control than DAF-16A, isoform-specific daf-16/FoxO mutant phenotypes and whole transcriptome profiling revealed a predominant role for DAF-16A over DAF-16F in life span control, stress resistance, and target gene regulation. Integration of these datasets enabled the prioritization of a subset of 92 DAF-16/FoxO target genes for functional interrogation. Among 29 genes tested, two DAF-16A-specific target genes significantly influenced longevity. A loss-of-function mutation in the conserved gene gst-20, which is induced by DAF-16A, reduced life span extension in the context of daf-2/IGFR RNAi without influencing longevity in animals subjected to control RNAi. Therefore, gst-20 promotes DAF-16/FoxO-dependent longevity. Conversely, a loss-of-function mutation in srr-4, a gene encoding a seven-transmembrane-domain receptor family member that is repressed by DAF-16A, extended life span in control animals, indicating that DAF-16/FoxO may extend life span at least in part by reducing srr-4 expression. Our discovery of new longevity genes underscores the efficacy of our integrative strategy while providing a general framework for identifying specific downstream gene regulatory events that contribute substantially to transcription factor functions. As FoxO transcription factors have conserved functions in promoting longevity and may be dysregulated in aging-related diseases, these findings promise to illuminate fundamental

  6. The C. elegans microRNA mir-71 acts in neurons to promote germline-mediated longevity through regulation of DAF-16/FOXO

    PubMed Central

    Boulias, Konstantinos; Horvitz, H. Robert

    2012-01-01

    Summary The lifespan of Caenorhabditis elegans is controlled by signaling between the germline and the soma. Germ cell removal extends lifespan by triggering the activation of the DAF-16/FOXO transcription factor in the intestine. Here we analyze microRNA function in C. elegans aging and show that the microRNA mir-71 functions to mediate the effects of germ cell loss on lifespan. mir-71 is required for the lifespan extension caused by germline removal, and overexpression of mir-71 further extends the lifespan of animals lacking germ cells. mir-71 functions in the nervous system to facilitate the localization and transcriptional activity of DAF-16 in the intestine. Our findings reveal a novel microRNA-dependent mechanism of lifespan regulation by the germline and indicate that signaling among the gonad, the nervous system and the intestine coordinates the lifespan of the entire organism. PMID:22482727

  7. DAF-16/Forkhead Box O Transcription Factor: Many Paths to a Single Fork(head) in the Road

    PubMed Central

    Yen, Kelvin; Narasimhan, Sri Devi

    2011-01-01

    Abstract The Caenorhabditis elegans Forkhead box O transcription factor (FOXO) homolog DAF-16 functions as a central mediator of multiple biological processes such as longevity, development, fat storage, stress resistance, and reproduction. In C. elegans, similar to other systems, DAF-16 functions as the downstream target of a conserved, well-characterized insulin/insulin-like growth factor (IGF)-1 signaling pathway. This cascade is comprised of an insulin/IGF-1 receptor, which signals through a conserved PI 3-kinase/AKT pathway that ultimately downregulates DAF-16/FOXO activity. Importantly, studies have shown that multiple pathways intersect with the insulin/IGF-1 signaling pathway and impinge on DAF-16 for their regulation. Therefore, in C. elegans, the single FOXO family member, DAF-16, integrates signals from several pathways and then regulates its many downstream target genes. Antioxid. Redox Signal. 14, 623–634. PMID:20673162

  8. DAF-16 and Δ9 desaturase genes promote cold tolerance in long-lived Caenorhabditis elegans age-1 mutants.

    PubMed

    Savory, Fiona R; Sait, Steven M; Hope, Ian A

    2011-01-01

    In Caenorhabditis elegans, mutants of the conserved insulin/IGF-1 signalling (IIS) pathway are long-lived and stress resistant due to the altered expression of DAF-16 target genes such as those involved in cellular defence and metabolism. The three Δ(9) desaturase genes, fat-5, fat-6 and fat-7, are included amongst these DAF-16 targets, and it is well established that Δ(9) desaturase enzymes play an important role in survival at low temperatures. However, no assessment of cold tolerance has previously been reported for IIS mutants. We demonstrate that long-lived age-1(hx546) mutants are remarkably resilient to low temperature stress relative to wild type worms, and that this is dependent upon daf-16. We also show that cold tolerance following direct transfer to low temperatures is increased in wild type worms during the facultative, daf-16 dependent, dauer stage. Although the cold tolerant phenotype of age-1(hx546) mutants is predominantly due to the Δ(9) desaturase genes, additional transcriptional targets of DAF-16 are also involved. Surprisingly, survival of wild type adults following a rapid temperature decline is not dependent upon functional daf-16, and cellular distributions of a DAF-16::GFP fusion protein indicate that DAF-16 is not activated during low temperature stress. This suggests that cold-induced physiological defences are not specifically regulated by the IIS pathway and DAF-16, but expression of DAF-16 target genes in IIS mutants and dauers is sufficient to promote cross tolerance to low temperatures in addition to other forms of stress.

  9. DAF-16/FoxO and EGL-27/GATA promote developmental growth in response to persistent somatic DNA damage

    PubMed Central

    Babu, Vipin; Ermolaeva, Maria A.; Müller, Roman-Ulrich; Frommolt, Peter; Williams, Ashley B.; Greiss, Sebastian; Schneider, Jennifer I.; Benzing, Thomas; Schermer, Bernhard; Schumacher, Björn

    2014-01-01

    Genome maintenance defects cause complex disease phenotypes characterized by developmental failure, cancer susceptibility, and premature aging. It remains poorly understood how DNA damage responses function during organismal development and maintain tissue functionality when DNA damage accumulates with aging. Here we show that the FoxO transcription factor DAF-16 is activated in response to DNA damage during development while the DNA damage responsiveness of DAF-16 declines with aging. We find that in contrast to its established role in mediating starvation arrest, DAF-16 alleviates DNA damage-induced developmental arrest and even in the absence of DNA repair promotes developmental growth and enhances somatic tissue functionality. We demonstrate that the GATA transcription factor EGL-27 co-regulates DAF-16 target genes in response to DNA damage and together with DAF-16 promotes developmental growth. We propose that EGL-27/GATA activity specifies DAF-16 mediated DNA damage responses to enable developmental progression and to prolong tissue functioning when DNA damage persists. PMID:25419847

  10. Arsenite exposure accelerates aging process regulated by the transcription factor DAF-16/FOXO in Caenorhabditis elegans.

    PubMed

    Yu, Chan-Wei; How, Chun Ming; Liao, Vivian Hsiu-Chuan

    2016-05-01

    Arsenic is a known human carcinogen and high levels of arsenic contamination in food, soils, water, and air are of toxicology concerns. Nowadays, arsenic is still a contaminant of emerging interest, yet the effects of arsenic on aging process have received little attention. In this study, we investigated the effects and the underlying mechanisms of chronic arsenite exposure on the aging process in Caenorhabditis elegans. The results showed that prolonged arsenite exposure caused significantly decreased lifespan compared to non-exposed ones. In addition, arsenite exposure (100 μM) caused significant changes of age-dependent biomarkers, including a decrease of defecation frequency, accumulations of intestinal lipofuscin and lipid peroxidation in an age-dependent manner in C. elegans. Further evidence revealed that intracellular reactive oxygen species (ROS) level was significantly increased in an age-dependent manner upon 100 μM arsenite exposure. Moreover, the mRNA levels of transcriptional makers of aging (hsp-16.1, hsp-16.49, and hsp-70) were increased in aged worms under arsenite exposure (100 μM). Finally, we showed that daf-16 mutant worms were more sensitive to arsenite exposure (100 μM) on lifespan and failed to induce the expression of its target gene sod-3 in aged daf-16 mutant under arsenite exposure (100 μM). Our study demonstrated that chronic arsenite exposure resulted in accelerated aging process in C. elegans. The overproduction of intracellular ROS and the transcription factor DAF-16/FOXO play roles in mediating the accelerated aging process by arsenite exposure in C. elegans. This study implicates a potential ecotoxicological and health risk of arsenic in the environment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Diosgenin a phytosterol substitute for cholesterol, prolongs the lifespan and mitigates glucose toxicity via DAF-16/FOXO and GST-4 in Caenorhabditis elegans.

    PubMed

    Shanmugam, Govindan; Mohankumar, Amirthalingam; Kalaiselvi, Duraisamy; Nivitha, Sundararaj; Murugesh, Easwaran; Shanmughavel, Piramanayagam; Sundararaj, Palanisamy

    2017-09-23

    Caenorhabditis elegans is a sterol auxotroph requires minute amount of exogenous sterol for their growth and development. To culture the C. elegans, cholesterol was given as sterol molecule to maintain the optimum survival of worms. Diosgenin (DG), a plant derived steroidal saponin, structurally similar to cholesterol has been used as a precursor for the synthesis of steroidal hormones. In this study, worms were cultured with cholesterol (Cho(+)) and cholesterol-free (Cho(-)) medium with DG (5, 10 and 50μg/mL) at 20°C. It was observed that worms cultured in (Cho(-)) exhibits late egg production, reduced lipid level and short lifespan, while addition of DG overcomes all defective facts. Combinations of both cholesterol and DG further extend the lifespan (20.8%), hinder lipid level and resistance to oxidative, thermal and high glucose stress. The intracellular ROS quantification was done by flouroscenic probe H2DCF-DA and confirmed that DG had significantly reduced ROS level (35.85%). Increased lifespan of worms were observed in the medium treated with DG which activates the nuclear translocation of DAF-16/FOXO transcription factor, followed by downstream antioxidant gene sod-3 as evidenced by GFP tagged strain. The expression of Phase II detoxification enzyme GST-4 significantly (p<0.001) increased in transgenic worms exposed to DG with 50mM glucose, and storage of lipid in intestinal cells was reduced in N2 wild type worms. Genetic requirement of DG induced longevity was studied with different mutant strains of mev-1, daf-16, skn-1, and eat-2. These studies have proved that DG is a sterol source to worms and modulate the DAF-16, SOD-3 and GST-4 expression levels to extend the lifespan of worms. The present study has also highlighted the use of phytosterols as an alternative to cholesterol. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  12. Caenorhabditis elegans EAK-3 inhibits dauer arrest via nonautonomous regulation of nuclear DAF-16/FoxO activity.

    PubMed

    Zhang, Yanmei; Xu, Jinling; Puscau, Cristina; Kim, Yongsoon; Wang, Xi; Alam, Hena; Hu, Patrick J

    2008-03-15

    Insulin regulates development, metabolism, and lifespan via a conserved PI3K/Akt pathway that promotes cytoplasmic sequestration of FoxO transcription factors. The regulation of nuclear FoxO is poorly understood. In the nematode Caenorhabditis elegans, insulin-like signaling functions in larvae to inhibit dauer arrest and acts during adulthood to regulate lifespan. In a screen for genes that modulate C. elegans insulin-like signaling, we identified eak-3, which encodes a novel protein that is specifically expressed in the two endocrine XXX cells. The dauer arrest phenotype of eak-3 mutants is fully suppressed by mutations in daf-16/FoxO, which encodes the major target of C. elegans insulin-like signaling, and daf-12, which encodes a nuclear receptor regulated by steroid hormones known as dafachronic acids. eak-3 mutation does not affect DAF-16/FoxO subcellular localization but enhances expression of the direct DAF-16/FoxO target sod-3 in a daf-16/FoxO- and daf-12-dependent manner. eak-3 mutants have normal lifespans, suggesting that EAK-3 decouples insulin-like regulation of development and longevity. We propose that EAK-3 activity in the XXX cells promotes the synthesis and/or secretion of a hormone that acts in parallel to AKT-1 to inhibit the expression of DAF-16/FoxO target genes. Similar hormonal pathways may regulate FoxO target gene expression in mammals.

  13. DAF-16/FOXO regulates homeostasis of essential sleep-like behavior during larval transitions in C. elegans.

    PubMed

    Driver, Robert J; Lamb, Annesia L; Wyner, Abraham J; Raizen, David M

    2013-03-18

    Sleep homeostasis, which refers to the maintenance of sleep amount or depth following sleep deprivation, indicates that sleep and sleep-like states serve fundamental functions that cannot be bypassed [1]. Homeostasis of sleep-like behavior is observed during C. elegans lethargus, a 2-3 hr behavioral quiescent period that occurs during larval state transitions [2]. Here, we report a role for DAF-16/FOXO, a transcription factor that is active under conditions of stress [3], in the response to deprivation of lethargus quiescence. Forced locomotion during lethargus results in nuclear translocation of DAF-16. The formation of dauer larvae, a developmental state promoted by daf-16, is increased in response to quiescence deprivation. daf-16 mutants show an impaired homeostatic response to deprivation of lethargus quiescence and are hypersensitive to the lethal effects of forced locomotion during lethargus. DAF-16 expression in muscle cells, but not in neurons, is sufficient to restore a homeostatic response to deprivation of quiescence, pointing to a role for muscle in sleep homeostasis. These findings are relevant to clinical observations of altered metabolic signaling in response to sleep deprivation and suggest that these signaling pathways may act in nonneuronal tissue to regulate sleep behaviors.

  14. C. elegans EAK-3 inhibits dauer arrest via nonautonomous regulation of nuclear DAF-16/FoxO activity

    PubMed Central

    Zhang, Yanmei; Xu, Jinling; Puscau, Cristina; Kim, Yongsoon; Wang, Xi; Alam, Hena; Hu, Patrick J.

    2008-01-01

    SUMMARY Insulin regulates development, metabolism, and lifespan via a conserved PI3K/Akt pathway that promotes cytoplasmic sequestration of FoxO transcription factors. The regulation of nuclear FoxO is poorly understood. In the nematode Caenorhabditis elegans, insulin-like signaling functions in larvae to inhibit dauer arrest and acts during adulthood to regulate lifespan. In a screen for genes that modulate C. elegans insulin-like signaling, we identified eak-3, which encodes a novel protein that is specifically expressed in the two endocrine XXX cells. The dauer arrest phenotype of eak-3 mutants is fully suppressed by mutations in daf-16/FoxO, which encodes the major target of C. elegans insulin-like signaling, and daf-12, which encodes a nuclear receptor regulated by steroid hormones known as dafachronic acids. eak-3 mutation does not affect DAF-16/FoxO subcellular localization but enhances expression of the direct DAF-16/FoxO target sod-3 in a daf-16/FoxO- and daf-12-dependent manner. eak-3 mutants have normal lifespans, suggesting that EAK-3 decouples insulin-like regulation of development and longevity. We propose that EAK-3 activity in the XXX cells promotes the synthesis and/or secretion of a hormone that acts in parallel to AKT-1 to inhibit the expression of DAF-16/FoxO target genes. Similar hormonal pathways may regulate FoxO target gene expression in mammals. PMID:18241854

  15. DAF-16/FOXO regulates homeostasis of essential sleep-like behavior during larval transitions in C. elegans

    PubMed Central

    Driver, Robert J.; Lamb, Annesia L.; Wyner, Abraham J.; Raizen, David M.

    2014-01-01

    Summary Sleep homeostasis, which refers to the maintenance of sleep amount or depth following sleep deprivation, indicates that sleep and sleep-like states serve fundamental functions that cannot be bypassed [1]. Homeostasis of sleep-like behavior is observed during C. elegans lethargus, a 2-3 hour behavioral quiescent period that occurs during larval state transitions [2]. Here, we report a role for DAF-16/FOXO, a transcription factor that is active under conditions of stress [3], in the response to deprivation of lethargus quiescence. Forced locomotion during lethargus results in nuclear translocation of DAF-16. The formation of dauer larvae, a developmental state promoted by daf-16, is increased in response to quiescence deprivation. daf-16 mutants show an impaired homeostatic response to deprivation of lethargus quiescence, and are hypersensitive to the lethal effects of forced locomotion during lethargus. DAF-16 expression in muscle cells but not in neurons is sufficient to restore a homeostatic response to deprivation of quiescence, pointing to a role for muscle in sleep homeostasis. These findings are relevant to clinical observations of altered metabolic signaling in response to sleep deprivation and suggest that these signaling pathways may act in non-neuronal tissue to regulate sleep behaviors. PMID:23477722

  16. Octopamine enhances oxidative stress resistance through the fasting-responsive transcription factor DAF-16/FOXO in C. elegans.

    PubMed

    Hoshikawa, Haruka; Uno, Masaharu; Honjoh, Sakiko; Nishida, Eisuke

    2017-02-01

    Dietary restriction regimens lead to enhanced stress resistance and extended life span in many species through the regulation of fasting and/or diet-responsive mechanisms. The fasting stimulus is perceived by sensory neurons and causes behavioral and metabolic adaptations. Octopamine (OA), one of the Caenorhabditis elegans neurotransmitters, is involved in behavioral adaptations, and its levels are increased under fasting conditions. However, it remains largely unknown how OA contributes to the fasting responses. In this study, we found that OA administration enhanced organismal resistance to oxidative stress. This enhanced resistance was suppressed by a mutation of the OA receptors, SER-3 and SER-6. Moreover, we found that OA administration promoted the nuclear translocation of DAF-16, the key transcription factor in fasting responses, and that the OA-induced enhancement of stress resistance required DAF-16. Altogether, our results suggest that OA signaling, which is triggered by the absence of food, shifts the organismal state to a more protective one to prepare for environmental stresses. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  17. Royal Jelly-Mediated Prolongevity and Stress Resistance in Caenorhabditis elegans Is Possibly Modulated by the Interplays of DAF-16, SIR-2.1, HCF-1, and 14-3-3 Proteins.

    PubMed

    Wang, Xiaoxia; Cook, Lauren F; Grasso, Lindsay M; Cao, Min; Dong, Yuqing

    2015-07-01

    Recent studies suggest that royal jelly (RJ) and its related substances may have antiaging properties. However, the molecular mechanisms underlying the beneficial effects remain elusive. We report that the effects of RJ and enzyme-treated RJ (eRJ) on life span and health span in Caenorhabditis elegans (C elegans) are modulated by the sophisticated interplays of DAF-16, SIR-2.1, HCF-1, and 14-3-3 proteins. Dietary supplementation with RJ or eRJ increased C. elegans life span in a dose-dependent manner. The RJ and eRJ consumption increased the tolerance of C elegans to oxidative stress, ultraviolet irradiation, and heat shock stress. Our genetic analyses showed that RJ/eRJ-mediated life-span extension requires insulin/IGF-1 signaling and the activities of DAF-16, SIR-2.1, HCF-1, and FTT-2, a 14-3-3 protein. Earlier studies reported that DAF-16/FOXO, SIR-2.1/SIRT1, FTT-2, and HCF-1 have extensive interplays in worms and mammals. Our present findings suggest that RJ/eRJ-mediated promotion of longevity and stress resistance in C elegans is dependent on these conserved interplays. From an evolutionary point of view, this study not only provides new insights into the molecular mechanisms of RJ's action on health span promotion in C elegans, but also has imperative implications in using RJ/eRJ as nutraceuticals to delay aging and age-related disorders.

  18. C. elegans DAF-16/FOXO interacts with TGF-ß/BMP signaling to induce germline tumor formation via mTORC1 activation

    PubMed Central

    Yan, Yijian; Pfeifer, Dietmar; Donner v. Gromoff, Erika; Wang, Yimin; Maier, Wolfgang; Baumeister, Ralf

    2017-01-01

    Activation of the FOXO transcription factor DAF-16 by reduced insulin/IGF signaling (IIS) is considered to be beneficial in C. elegans due to its ability to extend lifespan and to enhance stress resistance. In the germline, cell-autonomous DAF-16 activity prevents stem cell proliferation, thus acting tumor-suppressive. In contrast, hypodermal DAF-16 causes a tumorous germline phenotype characterized by hyperproliferation of the germline stem cells and rupture of the adjacent basement membrane. Here we show that cross-talk between DAF-16 and the transforming growth factor ß (TGFß)/bone morphogenic protein (BMP) signaling pathway causes germline hyperplasia and results in disruption of the basement membrane. In addition to activating MADM/NRBP/hpo-11 gene alone, DAF-16 also directly interacts with both R-SMAD proteins SMA-2 and SMA-3 in the nucleus to regulate the expression of mTORC1 pathway. Knocking-down of BMP genes or each of the four target genes in the hypodermis was sufficient to inhibit germline proliferation, indicating a cell-non-autonomously controlled regulation of stem cell proliferation by somatic tissues. We propose the existence of two antagonistic DAF-16/FOXO functions, a cell-proliferative somatic and an anti-proliferative germline activity. Whereas germline hyperplasia under reduced IIS is inhibited by DAF-16 cell-autonomously, activation of somatic DAF-16 in the presence of active IIS promotes germline proliferation and eventually induces tumor-like germline growth. In summary, our results suggest a novel pathway crosstalk of DAF-16 and TGF-ß/BMP that can modulate mTORC1 at the transcriptional level to cause stem-cell hyperproliferation. Such cell-type specific differences may help explaining why human FOXO activity is considered to be tumor-suppressive in most contexts, but may become oncogenic, e.g. in chronic and acute myeloid leukemia. PMID:28549065

  19. MicroRNA mir-34 provides robustness to environmental stress response via the DAF-16 network in C. elegans

    PubMed Central

    Isik, Meltem; Blackwell, T. Keith; Berezikov, Eugene

    2016-01-01

    Diverse stresses and aging alter expression levels of microRNAs, suggesting a role for these posttranscriptional regulators of gene expression in stress modulation and longevity. Earlier studies demonstrated a central role for the miR-34 family in promoting cell cycle arrest and cell death following stress in human cells. However, the biological significance of this response was unclear. Here we show that in C. elegans mir-34 upregulation is necessary for developmental arrest, correct morphogenesis, and adaptation to a lower metabolic state to protect animals against stress-related damage. Either deletion or overexpression of mir-34 lead to an impaired stress response, which can largely be explained by perturbations in DAF-16/FOXO target gene expression. We demonstrate that mir-34 expression is regulated by the insulin signaling pathway via a negative feedback loop between miR-34 and DAF-16/FOXO. We propose that mir-34 provides robustness to stress response programs by controlling noise in the DAF-16/FOXO-regulated gene network. PMID:27905558

  20. Gengnianchun, a Traditional Chinese Medicine, Enhances Oxidative Stress Resistance and Lifespan in Caenorhabditis elegans by Modulating daf-16/FOXO

    PubMed Central

    2017-01-01

    Objective. Gengnianchun (GNC), a traditional Chinese medicine (TCM), is primarily used to improve declining functions related to aging. In this study, we investigated its prolongevity and stress resistance properties and explored the associated regulatory mechanism using a Caenorhabditis elegans model. Methods. Wild-type C. elegans N2 was used for lifespan analysis and oxidative stress resistance assays. Transgenic animals were used to investigate pathways associated with antioxidative stress activity. The effects of GNC on levels of reactive oxygen species (ROS) and expression of specific genes were examined. Results. GNC-treated wild-type worms showed an increase in survival time under both normal and oxidative stress conditions. GNC decreased intracellular ROS levels by 67.95%. GNC significantly enhanced the oxidative stress resistance of several mutant strains, suggesting that the protective effect of GNC is independent of the function of these genes. However, the oxidative stress resistance effect of GNC was absent in worms with daf-16 mutation. We also found upregulation of daf-16 downstream targets including sod-3 and mtl-1. Conclusions. Our findings suggest that GNC extends the lifespan of C. elegans and enhances its resistance to oxidative stress via a daf-16/FOXO-dependent pathway. This study also provides a feasible method for screening the biological mechanisms of TCMs. PMID:28400847

  1. MicroRNA mir-34 provides robustness to environmental stress response via the DAF-16 network in C. elegans.

    PubMed

    Isik, Meltem; Blackwell, T Keith; Berezikov, Eugene

    2016-12-01

    Diverse stresses and aging alter expression levels of microRNAs, suggesting a role for these posttranscriptional regulators of gene expression in stress modulation and longevity. Earlier studies demonstrated a central role for the miR-34 family in promoting cell cycle arrest and cell death following stress in human cells. However, the biological significance of this response was unclear. Here we show that in C. elegans mir-34 upregulation is necessary for developmental arrest, correct morphogenesis, and adaptation to a lower metabolic state to protect animals against stress-related damage. Either deletion or overexpression of mir-34 lead to an impaired stress response, which can largely be explained by perturbations in DAF-16/FOXO target gene expression. We demonstrate that mir-34 expression is regulated by the insulin signaling pathway via a negative feedback loop between miR-34 and DAF-16/FOXO. We propose that mir-34 provides robustness to stress response programs by controlling noise in the DAF-16/FOXO-regulated gene network.

  2. TOR signaling and rapamycin influence longevity by regulating SKN-1/Nrf and DAF-16/FoxO.

    PubMed

    Robida-Stubbs, Stacey; Glover-Cutter, Kira; Lamming, Dudley W; Mizunuma, Masaki; Narasimhan, Sri Devi; Neumann-Haefelin, Elke; Sabatini, David M; Blackwell, T Keith

    2012-05-02

    The TOR kinase, which is present in the functionally distinct complexes TORC1 and TORC2, is essential for growth but associated with disease and aging. Elucidation of how TOR influences life span will identify mechanisms of fundamental importance in aging and TOR functions. Here we show that when TORC1 is inhibited genetically in C. elegans, SKN-1/Nrf, and DAF-16/FoxO activate protective genes, and increase stress resistance and longevity. SKN-1 also upregulates TORC1 pathway gene expression in a feedback loop. Rapamycin triggers a similar protective response in C. elegans and mice, but increases worm life span dependent upon SKN-1 and not DAF-16, apparently by interfering with TORC2 along with TORC1. TORC1, TORC2, and insulin/IGF-1-like signaling regulate SKN-1 activity through different mechanisms. We conclude that modulation of SKN-1/Nrf and DAF-16/FoxO may be generally important in the effects of TOR signaling in vivo and that these transcription factors mediate an opposing relationship between growth signals and longevity.

  3. 33 CFR 157.22 - Intact stability requirements.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 2 2011-07-01 2011-07-01 false Intact stability requirements... OIL IN BULK Design, Equipment, and Installation § 157.22 Intact stability requirements. All tank ships of 5,000 DWT and above contracted after December 3, 2001 must comply with the intact stability...

  4. 33 CFR 157.22 - Intact stability requirements.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 2 2012-07-01 2012-07-01 false Intact stability requirements... OIL IN BULK Design, Equipment, and Installation § 157.22 Intact stability requirements. All tank ships of 5,000 DWT and above contracted after December 3, 2001 must comply with the intact stability...

  5. 33 CFR 157.22 - Intact stability requirements.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 2 2013-07-01 2013-07-01 false Intact stability requirements... OIL IN BULK Design, Equipment, and Installation § 157.22 Intact stability requirements. All tank ships of 5,000 DWT and above contracted after December 3, 2001 must comply with the intact stability...

  6. 33 CFR 157.22 - Intact stability requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 2 2014-07-01 2014-07-01 false Intact stability requirements... OIL IN BULK Design, Equipment, and Installation § 157.22 Intact stability requirements. All tank ships of 5,000 DWT and above contracted after December 3, 2001 must comply with the intact stability...

  7. 33 CFR 157.22 - Intact stability requirements.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Intact stability requirements... OIL IN BULK Design, Equipment, and Installation § 157.22 Intact stability requirements. All tank ships of 5,000 DWT and above contracted after December 3, 2001 must comply with the intact stability...

  8. 46 CFR 174.045 - Intact stability requirements.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Intact stability requirements. 174.045 Section 174.045 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Intact stability requirements. (a) Each unit must be designed so that the wind heeling moments (Hm) and...

  9. 46 CFR 174.045 - Intact stability requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability requirements. 174.045 Section 174.045 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Intact stability requirements. (a) Each unit must be designed so that the wind heeling moments (Hm) and...

  10. 46 CFR 174.045 - Intact stability requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact stability requirements. 174.045 Section 174.045 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Intact stability requirements. (a) Each unit must be designed so that the wind heeling moments (Hm) and...

  11. 46 CFR 174.045 - Intact stability requirements.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability requirements. 174.045 Section 174.045 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Intact stability requirements. (a) Each unit must be designed so that the wind heeling moments (Hm) and...

  12. The DAF-16 FOXO Transcription Factor Regulates natc-1 to Modulate Stress Resistance in Caenorhabditis elegans, Linking Insulin/IGF-1 Signaling to Protein N-Terminal Acetylation

    PubMed Central

    Warnhoff, Kurt; Murphy, John T.; Kumar, Sandeep; Schneider, Daniel L.; Peterson, Michelle; Hsu, Simon; Guthrie, James; Robertson, J. David; Kornfeld, Kerry

    2014-01-01

    The insulin/IGF-1 signaling pathway plays a critical role in stress resistance and longevity, but the mechanisms are not fully characterized. To identify genes that mediate stress resistance, we screened for C. elegans mutants that can tolerate high levels of dietary zinc. We identified natc-1, which encodes an evolutionarily conserved subunit of the N-terminal acetyltransferase C (NAT) complex. N-terminal acetylation is a widespread modification of eukaryotic proteins; however, relatively little is known about the biological functions of NATs. We demonstrated that loss-of-function mutations in natc-1 cause resistance to a broad-spectrum of physiologic stressors, including multiple metals, heat, and oxidation. The C. elegans FOXO transcription factor DAF-16 is a critical target of the insulin/IGF-1 signaling pathway that mediates stress resistance, and DAF-16 is predicted to directly bind the natc-1 promoter. To characterize the regulation of natc-1 by DAF-16 and the function of natc-1 in insulin/IGF-1 signaling, we analyzed molecular and genetic interactions with key components of the insulin/IGF-1 pathway. natc-1 mRNA levels were repressed by DAF-16 activity, indicating natc-1 is a physiological target of DAF-16. Genetic studies suggested that natc-1 functions downstream of daf-16 to mediate stress resistance and dauer formation. Based on these findings, we hypothesize that natc-1 is directly regulated by the DAF-16 transcription factor, and natc-1 is a physiologically significant effector of the insulin/IGF-1 signaling pathway that mediates stress resistance and dauer formation. These studies identify a novel biological function for natc-1 as a modulator of stress resistance and dauer formation and define a functionally significant downstream effector of the insulin/IGF-1 signaling pathway. Protein N-terminal acetylation mediated by the NatC complex may play an evolutionarily conserved role in regulating stress resistance. PMID:25330323

  13. Benzimidazole derivative M084 extends the lifespan of Caenorhabditis elegans in a DAF-16/FOXO-dependent way.

    PubMed

    Ding, Ai-Jun; Wu, Gui-Sheng; Tang, Bin; Hong, Xuechuan; Zhu, Michael X; Luo, Huai-Rong

    2017-02-01

    With the growth of aging population, there is increasing demand to develop strategy to improve the aging process and aging-related diseases. Benzimidazole and its derivatives are crucial heterocyclic backbone of many drugs and compounds with diverse therapeutic applications, including alleviation of aging-related diseases. Here, we investigate if the benzimidazole derivative n-butyl-[1H]-benzimidazol-2-amine (M084), a novel inhibitor of TRPC4 and TRPC5 channels and antidepressant, could affect the lifespan of Caenorhabditis elegans (C. elegans). Our results showed that M084 could extend the lifespan of C. elegans, delay age-related decline of phenotypes, and improve stress resistance. M084 could not extend the lifespan of the loss-of-function mutants of daf-16, daf-2, pdk-1, aak-2, clk-1, isp-1, sir-2.1, and skn-1. M084 could decrease the ATP level and increase the gene expression of mitochondrial unfolded protein response factors. Thus, M084 might inhibit the mitochondrial respiration, activate mitochondrial unfolded protein response and AMPK, recruite SIR-2.1 and SKN-1, and finally through the transcription factor DAF-16, delay the aging process of C. elegans. Our findings reveal the new pharmaceutical potential of benzimidazole derivatives and provide clue for developing novel anti-aging agents.

  14. Increased age reduces DAF-16 and SKN-1 signaling and the hormetic response of Caenorhabditis elegans to the xenobiotic juglone

    PubMed Central

    Przybysz, Aaron J.; Choe, Keith P.; Roberts, L. Jackson; Strange, Kevin

    2009-01-01

    Cells adapt to stressors by activating mechanisms that repair damage and protect them from further injury. Stress-induced damage accumulates with age and contributes to age associated diseases. Increased age attenuates the ability to mount a stress response, but little is known about the mechanisms by which this occurs. To begin addressing this problem, we studied hormesis in the nematode Caenorhabditis elegans. When exposed to a low concentration of the xenobiotic juglone, young worms mount a robust hormetic stress response and survive a subsequent exposure to a higher concentration of juglone that is normally lethal to naïve animals. Old worms are unable to mount this adaptive response. Microarray and RNAi analyses demonstrate that an altered transcriptional response to juglone is responsible in part for the reduced adaptation of old worms. Many genes differentially regulated in young versus old animals are known or postulated to be regulated by the FOXO homologue DAF-16 and the Nrf2 homologue SKN-1. Activation of these pathways is greatly reduced in juglone stressed old worms. DAF-16- and SKN-1-like transcription factors play highly conserved roles in regulating stress resistance and longevity genes. Our studies provide a foundation for developing a molecular understanding of how age affects cytoprotective transcriptional pathways. PMID:19428455

  15. 46 CFR 174.145 - Intact stability requirements.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability requirements. 174.145 Section 174.145 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... stability requirements. (a) In each condition of loading and operation, each vessel must be shown by design...

  16. 46 CFR 174.045 - Intact stability requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Mobile Offshore Drilling Units § 174.045 Intact stability requirements. (a) Each unit must be designed so that the wind heeling moments (Hm) and... downflooding would occur, whichever angle is less; and (3) Area (B) is the area on GRAPH 174.045 under the...

  17. 46 CFR 173.063 - Intact stability requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... PERTAINING TO VESSEL USE School Ships § 173.063 Intact stability requirements. (a) Except as provided in this... subchapter, the value of “T” is equal to the angle of heel at which the deck edge is immersed or 1/3 of the downflooding angle, whichever is less. (c) In applying the requirements of § 171.055(d) (1) and (2) of this...

  18. DAF-16 and TCER-1 Facilitate Adaptation to Germline Loss by Restoring Lipid Homeostasis and Repressing Reproductive Physiology in C. elegans.

    PubMed

    Amrit, Francis Raj Gandhi; Steenkiste, Elizabeth Marie; Ratnappan, Ramesh; Chen, Shaw-Wen; McClendon, T Brooke; Kostka, Dennis; Yanowitz, Judith; Olsen, Carissa Perez; Ghazi, Arjumand

    2016-02-01

    Elimination of the proliferating germline extends lifespan in C. elegans. This phenomenon provides a unique platform to understand how complex metazoans retain metabolic homeostasis when challenged with major physiological perturbations. Here, we demonstrate that two conserved transcription regulators essential for the longevity of germline-less adults, DAF-16/FOXO3A and TCER-1/TCERG1, concurrently enhance the expression of multiple genes involved in lipid synthesis and breakdown, and that both gene classes promote longevity. Lipidomic analyses revealed that key lipogenic processes, including de novo fatty acid synthesis, triglyceride production, desaturation and elongation, are augmented upon germline removal. Our data suggest that lipid anabolic and catabolic pathways are coordinately augmented in response to germline loss, and this metabolic shift helps preserve lipid homeostasis. DAF-16 and TCER-1 also perform essential inhibitory functions in germline-ablated animals. TCER-1 inhibits the somatic gene-expression program that facilitates reproduction and represses anti-longevity genes, whereas DAF-16 impedes ribosome biogenesis. Additionally, we discovered that TCER-1 is critical for optimal fertility in normal adults, suggesting that the protein acts as a switch supporting reproductive fitness or longevity depending on the presence or absence of the germline. Collectively, our data offer insights into how organisms adapt to changes in reproductive status, by utilizing the activating and repressive functions of transcription factors and coordinating fat production and degradation.

  19. DAF-16 and TCER-1 Facilitate Adaptation to Germline Loss by Restoring Lipid Homeostasis and Repressing Reproductive Physiology in C. elegans

    PubMed Central

    Amrit, Francis Raj Gandhi; Steenkiste, Elizabeth Marie; Ratnappan, Ramesh; Chen, Shaw-Wen; McClendon, T. Brooke; Kostka, Dennis; Yanowitz, Judith; Olsen, Carissa Perez; Ghazi, Arjumand

    2016-01-01

    Elimination of the proliferating germline extends lifespan in C. elegans. This phenomenon provides a unique platform to understand how complex metazoans retain metabolic homeostasis when challenged with major physiological perturbations. Here, we demonstrate that two conserved transcription regulators essential for the longevity of germline-less adults, DAF-16/FOXO3A and TCER-1/TCERG1, concurrently enhance the expression of multiple genes involved in lipid synthesis and breakdown, and that both gene classes promote longevity. Lipidomic analyses revealed that key lipogenic processes, including de novo fatty acid synthesis, triglyceride production, desaturation and elongation, are augmented upon germline removal. Our data suggest that lipid anabolic and catabolic pathways are coordinately augmented in response to germline loss, and this metabolic shift helps preserve lipid homeostasis. DAF-16 and TCER-1 also perform essential inhibitory functions in germline-ablated animals. TCER-1 inhibits the somatic gene-expression program that facilitates reproduction and represses anti-longevity genes, whereas DAF-16 impedes ribosome biogenesis. Additionally, we discovered that TCER-1 is critical for optimal fertility in normal adults, suggesting that the protein acts as a switch supporting reproductive fitness or longevity depending on the presence or absence of the germline. Collectively, our data offer insights into how organisms adapt to changes in reproductive status, by utilizing the activating and repressive functions of transcription factors and coordinating fat production and degradation. PMID:26862916

  20. Nutritional and hormonal factors control the gene expression of FoxOs, the mammalian homologues of DAF-16.

    PubMed

    Imae, M; Fu, Z; Yoshida, A; Noguchi, T; Kato, H

    2003-04-01

    Transcription factors of the FoxO family in mammals are orthologues of the Caenorhabditis elegans forkhead factor DAF-16, which has been characterized as a target of insulin-like signalling. Three members of this family have been identified in rodents: FoxO1, FoxO3 and FoxO4, originally termed FKHR, FKHRL1 and AFX respectively. A number of in vitro studies have revealed that FoxOs are regulated through phosphorylation in response to insulin and related growth factors, resulting in their nuclear exclusion and inactivation. To clarify the mechanisms involved in the regulation of these factors in vivo, we investigated in the present study whether or not, and if so how, their mRNA levels in rat liver respond to the stimuli of several nutritional and hormonal factors. Imposed fasting for 48 h significantly elevated mRNA levels of FoxO1 (1.5-fold), FoxO3 (1.4-fold), and FoxO4 (1.6-fold). Refeeding for 3 h recovered the induced mRNA levels of FoxO1 and FoxO3 to the control levels, but did not affect that of FoxO4. FoxO1 and FoxO4 mRNA levels were proved to be highly reflective of their protein levels measured by Western immunoblotting. Of the three FoxO genes, FoxO4 only showed altered levels of mRNA (a 1.5-fold increase) in response to a protein-free diet. Streptozotocin-induced diabetes for 28 days decreased hepatic mRNA levels of FoxO1 and FoxO3 and increased the level of FoxO4 mRNA, but short-term (7 days) diabetes had fewer effects on the expression of these genes. Insulin replacement partially restored the FoxO1 and FoxO4 mRNA levels, but had no effect on the FoxO3 mRNA level. Daily administration for 1 week of dexamethasone, a synthetic glucocorticoid, increased the mRNA levels of FoxO1 (1.8-fold) and FoxO3 (2.4-fold). These results show that the FoxO genes respond differently to nutritional and hormonal factors, suggesting a new mechanism for the regulation of FoxO-dependent gene expression by these factors. Moreover, changes of FoxO1 and FoxO4 in the nucleus in

  1. 46 CFR 178.320 - Intact stability requirements-non-sailing vessels.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability requirements-non-sailing vessels. 178... VESSELS (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Intact Stability Standards § 178.320 Intact stability requirements—non-sailing vessels. (a) As permitted by § 178.310(c) of this part, the...

  2. DAF-16/FoxO Directly Regulates an Atypical AMP-Activated Protein Kinase Gamma Isoform to Mediate the Effects of Insulin/IGF-1 Signaling on Aging in Caenorhabditis elegans

    PubMed Central

    Tullet, Jennifer M. A.; Araiz, Caroline; Sanders, Matthew J.; Au, Catherine; Benedetto, Alexandre; Papatheodorou, Irene; Clark, Emily; Schmeisser, Kathrin; Jones, Daniel; Schuster, Eugene F.; Thornton, Janet M.; Gems, David

    2014-01-01

    The DAF-16/FoxO transcription factor controls growth, metabolism and aging in Caenorhabditis elegans. The large number of genes that it regulates has been an obstacle to understanding its function. However, recent analysis of transcript and chromatin profiling implies that DAF-16 regulates relatively few genes directly, and that many of these encode other regulatory proteins. We have investigated the regulation by DAF-16 of genes encoding the AMP-activated protein kinase (AMPK), which has α, β and γ subunits. C. elegans has 5 genes encoding putative AMP-binding regulatory γ subunits, aakg-1-5. aakg-4 and aakg-5 are closely related, atypical isoforms, with orthologs throughout the Chromadorea class of nematodes. We report that ∼75% of total γ subunit mRNA encodes these 2 divergent isoforms, which lack consensus AMP-binding residues, suggesting AMP-independent kinase activity. DAF-16 directly activates expression of aakg-4, reduction of which suppresses longevity in daf-2 insulin/IGF-1 receptor mutants. This implies that an increase in the activity of AMPK containing the AAKG-4 γ subunit caused by direct activation by DAF-16 slows aging in daf-2 mutants. Knock down of aakg-4 expression caused a transient decrease in activation of expression in multiple DAF-16 target genes. This, taken together with previous evidence that AMPK promotes DAF-16 activity, implies the action of these two metabolic regulators in a positive feedback loop that accelerates the induction of DAF-16 target gene expression. The AMPK β subunit, aakb-1, also proved to be up-regulated by DAF-16, but had no effect on lifespan. These findings reveal key features of the architecture of the gene-regulatory network centered on DAF-16, and raise the possibility that activation of AMP-independent AMPK in nutritionally replete daf-2 mutant adults slows aging in C. elegans. Evidence of activation of AMPK subunits in mammals suggests that such FoxO-AMPK interactions may be evolutionarily conserved

  3. Emodin extends lifespan of Caenorhabditis elegans through insulin/IGF-1 signaling pathway depending on DAF-16 and SIR-2.1.

    PubMed

    Zhao, Xuan; Lu, Lulu; Qi, Yonghao; Li, Miao; Zhou, Lijun

    2017-10-01

    The naturally occurring anthraquinone emodin has been serving primarily as an anti-bacterial and anti-inflammatory agent. However, little is known about its potential on anti-aging. This investigation examined the effect of emodin on lifespan and focused on its physiological molecular mechanisms in vivo. Using Caenorhabditis elegans (C. elegans) as an animal model, we found emodin could extend lifespan of worms and improve their antioxidant capacity. Our mechanistic studies revealed that emodin might function via insulin/IGF-1 signaling (IIS) pathway involving, specifically the core transcription factor DAF-16. Quantitative RT-PCR results illustrated that emodin up-regulated transcription of DAF-16 target genes which express antioxidants to promote antioxidant capacity and lifespan of worms. In addition, attenuated effect in sir-2.1 mutants suggests that emodin likely functioned in a SIR-2.1-dependent manner. Our study uncovers a novel role of emodin in prolonging lifespan and supports the understanding of emodin being a beneficial dietary supplement.

  4. True navigation in migrating gulls requires intact olfactory nerves

    PubMed Central

    Wikelski, Martin; Arriero, Elena; Gagliardo, Anna; Holland, Richard A.; Huttunen, Markku J.; Juvaste, Risto; Mueller, Inge; Tertitski, Grigori; Thorup, Kasper; Wild, Martin; Alanko, Markku; Bairlein, Franz; Cherenkov, Alexander; Cameron, Alison; Flatz, Reinhard; Hannila, Juhani; Hüppop, Ommo; Kangasniemi, Markku; Kranstauber, Bart; Penttinen, Maija-Liisa; Safi, Kamran; Semashko, Vladimir; Schmid, Heidi; Wistbacka, Ralf

    2015-01-01

    During migratory journeys, birds may become displaced from their normal migratory route. Experimental evidence has shown that adult birds can correct for such displacements and return to their goal. However, the nature of the cues used by migratory birds to perform long distance navigation is still debated. In this experiment we subjected adult lesser black-backed gulls migrating from their Finnish/Russian breeding grounds (from >60°N) to Africa (to < 5°N) to sensory manipulation, to determine the sensory systems required for navigation. We translocated birds westward (1080 km) or eastward (885 km) to simulate natural navigational challenges. When translocated westwards and outside their migratory corridor birds with olfactory nerve section kept a clear directional preference (southerly) but were unable to compensate for the displacement, while intact birds and gulls with the ophthalmic branch of the trigeminal nerve sectioned oriented towards their population-specific migratory corridor. Thus, air-borne olfactory information seems to be important for migrating gulls to navigate successfully in some circumstances. PMID:26597351

  5. Antioxidative Activities of Both Oleic Acid and Camellia tenuifolia Seed Oil Are Regulated by the Transcription Factor DAF-16/FOXO in Caenorhabditis elegans.

    PubMed

    Wei, Chia-Cheng; Yen, Pei-Ling; Chang, Shang-Tzen; Cheng, Pei-Ling; Lo, Yi-Chen; Liao, Vivian Hsiu-Chuan

    2016-01-01

    Tea seed oil is a high quality edible oil, yet lacking sufficient scientific evidences to support the nutritional and medical purposes. We identified major and minor components in Camellia tenuifolia seed oil and investigated the antioxidative activity and its underlying mechanisms in Caenorhabditis elegans. The results showed that the major constitutes in C. tenuifolia seed oil were unsaturated fatty acids (~78.4%). Moreover, two minor compounds, β-amyrin and β-sitosterol, were identified and their antioxidative activity was examined. We found that oleic acid was the major constitute in C. tenuifolia seed oil and plays a key role in the antioxidative activity of C. tenuifolia seed oil in C. elegans. This study found evidences that the transcription factor DAF-16/FOXO was involved in both oleic acid- and C. tenuifolia seed oil-mediated oxidative stress resistance in C. elegans. This study suggests the potential of C. tenuifolia seed oil as nutrient or functional foods.

  6. Dataset of the human homologues and orthologues of lipid-metabolic genes identified as DAF-16 targets their roles in lipid and energy metabolism.

    PubMed

    Fan, Lavender Yuen-Nam; Saavedra-García, Paula; Lam, Eric Wing-Fai

    2017-04-01

    The data presented in this article are related to the review article entitled 'Unravelling the role of fatty acid metabolism in cancer through the FOXO3-FOXM1 axis' (Saavedra-Garcia et al., 2017) [24]. Here, we have matched the DAF-16/FOXO3 downstream genes with their respective human orthologues and reviewed the roles of these targeted genes in FA metabolism. The list of genes listed in this article are precisely selected from literature reviews based on their functions in mammalian FA metabolism. The nematode Caenorhabditis elegans gene orthologues of the genes are obtained from WormBase, the online biological database of C. elegans. This dataset has not been uploaded to a public repository yet.

  7. Antioxidative Activities of Both Oleic Acid and Camellia tenuifolia Seed Oil Are Regulated by the Transcription Factor DAF-16/FOXO in Caenorhabditis elegans

    PubMed Central

    Wei, Chia-Cheng; Yen, Pei-Ling; Chang, Shang-Tzen; Cheng, Pei-Ling; Lo, Yi-Chen; Liao, Vivian Hsiu-Chuan

    2016-01-01

    Background Tea seed oil is a high quality edible oil, yet lacking sufficient scientific evidences to support the nutritional and medical purposes. We identified major and minor components in Camellia tenuifolia seed oil and investigated the antioxidative activity and its underlying mechanisms in Caenorhabditis elegans. Principal Findings The results showed that the major constitutes in C. tenuifolia seed oil were unsaturated fatty acids (~78.4%). Moreover, two minor compounds, β-amyrin and β-sitosterol, were identified and their antioxidative activity was examined. We found that oleic acid was the major constitute in C. tenuifolia seed oil and plays a key role in the antioxidative activity of C. tenuifolia seed oil in C. elegans. Conclusions This study found evidences that the transcription factor DAF-16/FOXO was involved in both oleic acid- and C. tenuifolia seed oil-mediated oxidative stress resistance in C. elegans. This study suggests the potential of C. tenuifolia seed oil as nutrient or functional foods. PMID:27275864

  8. 46 CFR 178.310 - Intact stability requirements-general.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... chapter while under sail, as well as the requirements of § 170.170 in subchapter S in this chapter while... storm sails set and trimmed flat (if a sailing vessel as defined in § 170.055(n) of this chapter)....

  9. 46 CFR 178.310 - Intact stability requirements-general.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... chapter while under sail, as well as the requirements of § 170.170 in subchapter S in this chapter while... storm sails set and trimmed flat (if a sailing vessel as defined in § 170.055(n) of this chapter)....

  10. 46 CFR 178.310 - Intact stability requirements-general.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... chapter while under sail, as well as the requirements of § 170.170 in subchapter S in this chapter while... storm sails set and trimmed flat (if a sailing vessel as defined in § 170.055(n) of this chapter)....

  11. 46 CFR 173.063 - Intact stability requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... graphs 173.063 (a) and (b) and multiplied by the multiplier in graph 173.063(e). (3) For a vessel in service on exposed waters, “Y” and “Z” are determined from graphs 173.063 (c) and (d) and multiplied by the multiplier from graph 173.063(e). (4) To convert required numerals to units of “metric...

  12. 46 CFR 173.063 - Intact stability requirements.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... graphs 173.063 (a) and (b) and multiplied by the multiplier in graph 173.063(e). (3) For a vessel in service on exposed waters, “Y” and “Z” are determined from graphs 173.063 (c) and (d) and multiplied by the multiplier from graph 173.063(e). (4) To convert required numerals to units of “metric...

  13. 46 CFR 173.063 - Intact stability requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... graphs 173.063 (a) and (b) and multiplied by the multiplier in graph 173.063(e). (3) For a vessel in service on exposed waters, “Y” and “Z” are determined from graphs 173.063 (c) and (d) and multiplied by the multiplier from graph 173.063(e). (4) To convert required numerals to units of “metric...

  14. 46 CFR 173.063 - Intact stability requirements.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... graphs 173.063 (a) and (b) and multiplied by the multiplier in graph 173.063(e). (3) For a vessel in service on exposed waters, “Y” and “Z” are determined from graphs 173.063 (c) and (d) and multiplied by the multiplier from graph 173.063(e). (4) To convert required numerals to units of “metric...

  15. von Willebrand factor storage requires intact prosequence cleavage site.

    PubMed

    Journet, A M; Saffaripour, S; Cramer, E M; Tenza, D; Wagner, D D

    1993-02-01

    Large multimers of the adhesive glycoprotein von Willebrand factor (vWf) are stored in endothelial cells in rod-shaped granules called Weibel-Palade bodies, while small multimers are secreted constitutively. Expression of pro-vWf in other cells with a regulated pathway of secretion, results in formation of vWf-containing storage granules that have a morphology similar to Weibel-Palade bodies. vWf expressed without its prosequence is not stored. To evaluate the importance of prosequence cleavage in vWf storage, the Arg at position -1, known to be necessary for cleavage, was mutated to Gly. Transfection of this cleavage mutant into two cell lines with a regulated pathway of secretion (RIN 5F and AtT-20 cells) led to the formation of large multimers. However, treatment of the cell lysates by the enzyme endoglycosidase H (Endo-H) did not reveal significant amounts of intracellular Endo-H-resistant vWf, which indicates the absence of a pool of stored processed vWf. In addition, no Weibel-Palade body-like structure was detected in these cells by immunofluorescence labeling with anti-vWf antiserum. Electron microscopy and immunocytochemistry of RIN 5F cells expressing the pro-vWf mutant confirmed the absence of Weibel-Palade body-like structures. In addition, anti-vWf-linked gold particles were found in the ER, occasionally in rounded granules and particularly in lysosomal structures which were abundant. We conclude that the formation of large aggregates is not sufficient to induce efficient vWf storage, and that the lack of cleavage of the prosequence may direct the mutant pro-vWf molecule to a degradative pathway. Therefore, the prosequence cleavage is a requirement for vWf storage.

  16. Gaze-cueing requires intact face processing - Insights from acquired prosopagnosia.

    PubMed

    Burra, Nicolas; Kerzel, Dirk; Ramon, Meike

    2017-04-01

    Gaze-cueing is the automatic spatial orienting of attention in the direction of perceived gaze. Participants respond faster to targets located at positions congruent with the direction of gaze, compared to incongruent ones (gaze cueing effect, GCE). However, it still remains unclear whether its occurrence depends on intact integration of information from the entire eye region or face, rather than simply the presence of the eyes per se. To address this question, we investigated the GCE in PS, an extensively studied case of pure acquired prosopagnosia. In our gaze-cueing paradigm, we manipulated the duration at which cues were presented (70ms vs. 400ms) and the availability of facial information (full-face vs. eyes-only). For 70ms cue duration, we found a context-dependent dissociation between PS and controls: PS showed a GCE for eyes-only stimuli, whereas controls showed a GCE only for full-face stimuli. For 400ms cue duration, PS showed gaze-cueing independently of stimulus context, whereas in healthy controls a GCE again emerged only for full-face stimuli. Our findings suggest that attentional deployment based on the gaze direction of briefly presented faces requires intact processing of facial information, which affords salience to the eye region. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Tomato susceptibility to root-knot nematodes requires an intact jasmonic acid signaling pathway.

    PubMed

    Bhattarai, Kishor K; Xie, Qi-Guang; Mantelin, Sophie; Bishnoi, Usha; Girke, Thomas; Navarre, Duroy A; Kaloshian, Isgouhi

    2008-09-01

    Responses of resistant (Mi-1/Mi-1) and susceptible (mi-1/ mi-1) tomato (Solanum lycopersicum) to root-knot nematodes (RKNs; Meloidogyne spp.) infection were monitored using cDNA microarrays, and the roles of salicylic acid (SA) and jasmonic acid (JA) defense signaling were evaluated in these interactions. Array analysis was used to compare transcript profiles in incompatible and compatible interactions of tomato roots 24 h after RKN infestation. The jai1 and def1 tomato mutant, altered in JA signaling, and tomato transgenic line NahG, altered in SA signaling, in the presence or absence of the RKN resistance gene Mi-1, were evaluated. The array analysis identified 1,497 and 750 genes differentially regulated in the incompatible and compatible interactions, respectively. Of the differentially regulated genes, 37% were specific to the incompatible interactions. NahG affected neither Mi-1 resistance nor basal defenses to RKNs. However, jai1 reduced tomato susceptibility to RKNs while not affecting Mi-1 resistance. In contrast, the def1 mutant did not affect RKN susceptibility. These results indicate that JA-dependent signaling does not play a role in Mi-1-mediated defense; however, an intact JA signaling pathway is required for tomato susceptibility to RKNs. In addition, low levels of SA might be sufficient for basal and Mi-1 resistance to RKNs.

  18. An intact acrosome is required for the chemotactic response to progesterone in mouse spermatozoa.

    PubMed

    Guidobaldi, Hector A; Hirohashi, Noritaka; Cubilla, Marisa; Buffone, Mariano G; Giojalas, Laura C

    2017-04-01

    Mammalian sperm become fertilization-competent in the oviduct, during a process known as capacitation that involves the acquisition of the ability to exocytose the acrosome but also the chemotactic responses-both of which contribute to successful fertilization. Chemotaxis is used by spermatozoa to orient and to locate the egg; the acrosome reaction facilitates sperm binding to and fusing with the egg membrane. Mammalian spermatozoa are able to sense picomolar concentrations of progesterone, which drives chemotactic behavior. The state of the acrosome during the chemotactic response, however, is unknown. Genetically modified mouse spermatozoa were employed in a chemotaxis assay under fluorescence microscopy to evaluate their acrosome status while swimming, allowing us to elucidate the acrosome integrity of sperm responding to progesterone-induced chemotaxis. We first showed that wild-type mouse spermatozoa chemotactically respond to a gradient of progesterone, and that the genetic modifications employed do not affect the chemotactic behavior of sperm to progesterone. Next, we found that acrosome-intact, but not acrosome-reacted, spermatozoa orient and respond to picomolar concentrations of progesterone and that chemotaxis normally occurs prior to the acrosome reaction. Our results suggest that premature commitment to acrosome exocytosis leads to navigation failure, so proper control and timing of the acrosome reaction is required for fertilization success and male fertility. © 2017 Wiley Periodicals, Inc.

  19. Nucleotides in precursor tRNAs that are required intact for catalysis by RNase P RNAs.

    PubMed

    Thurlow, D L; Shilowski, D; Marsh, T L

    1991-02-25

    Precursor tRNAAsp molecules, containing a 26-base 5' leader, were treated with diethylpyrocarbonate, 50% hydrazine or anhydrous hydrazine/3M NaCl and then subjected to processing by RNase P RNAs from Escherichia coli or Bacillus subtilis. Fully processed tRNAs and material not successfully cleaved by the catalytic RNAs were analyzed for their content of chemically altered nucleotides. Several bases were identified as being required intact for optimal activity as substrate as judged by exclusion of chemically modified residues from processed molecules, and simultaneous enhancement in material that was not recognized as substrate. Such nucleotides cluster near the site of cleavage at the mature 5' end and in the T stem and loop. Purines at residues 1 and 2 adjacent to the site of cleavage, position 57 in the T loop, and site 64 in the T stem exhibited the most pronounced effects. These results suggest a model of recognition of substrate by RNase P RNAs in which the ribozyme interacts with the corner of the precursor tRNA's three dimensional structure, where the T- and D-loops are juxtaposed, and extends along the top of the molecule back towards the site of catalysis.

  20. Function of RSKS-1-AAK-2-DAF-16 signaling cascade in enhancing toxicity of multi-walled carbon nanotubes can be suppressed by mir-259 activation in Caenorhabditis elegans

    NASA Astrophysics Data System (ADS)

    Zhuang, Ziheng; Li, Min; Liu, Hui; Luo, Libo; Gu, Weidong; Wu, Qiuli; Wang, Dayong

    2016-08-01

    Caenorhabditis elegans is an important non-mammalian alternative assay model for toxicological study. Previous study has indicated that exposure to multi-walled carbon nanotubes (MWCNTs) dysregulated the transcriptional expression of mir-259. In this study, we examined the molecular basis for mir-259 in regulating MWCNTs toxicity in nematodes. Mutation of mir-259 induced a susceptible property to MWCNTs toxicity, and MWCNTs exposure induced a significant increase in mir-259::GFP in pharyngeal/intestinal valve and reproductive tract, implying that mir-259 might mediate a protection mechanisms for nematodes against MWCNTs toxicity. RSKS-1, a putative ribosomal protein S6 kinase, acted as the target for mir-259 in regulating MWCNTs toxicity, and mutation of rsks-1 suppressed the susceptible property of mir-259 mutant to MWCNTs toxicity. Moreover, mir-259 functioned in pharynx-intestinal valve and RSKS-1 functioned in pharynx to regulate MWCNTs toxicity. Furthermore, RSKS-1 regulated MWCNTs toxicity by suppressing the function of AAK-2-DAF-16 signaling cascade. Our results will strengthen our understanding the microRNAs mediated protection mechanisms for animals against the toxicity from certain nanomaterials.

  1. Co-regulation of the DAF-16 target gene, cyp-35B1/dod-13, by HSF-1 in C. elegans dauer larvae and daf-2 insulin pathway mutants.

    PubMed

    Iser, Wendy B; Wilson, Mark A; Wood, William H; Becker, Kevin; Wolkow, Catherine A

    2011-03-09

    Insulin/IGF-I-like signaling (IIS) has both cell autonomous and non-autonomous functions. In some cases, targets through which IIS regulates cell-autonomous functions, such as cell growth and metabolism, have been identified. In contrast, targets for many non-autonomous IIS functions, such as C. elegans dauer morphogenesis, remain elusive. Here, we report the use of genomic and genetic approaches to identify potential non-autonomous targets of C. elegans IIS. First, we used transcriptional microarrays to identify target genes regulated non-autonomously by IIS in the intestine or in neurons. C. elegans IIS controls expression of a number of stress response genes, which were differentially regulated by tissue-restricted IIS. In particular, expression of sod-3, a MnSOD enzyme, was not regulated by tissue-restricted IIS on the microarrays, while expression of hsp-16 genes was rescued back to wildtype by tissue restricted IIS. One IIS target regulated non-autonomously by age-1 was cyp-35B1/dod-13, encoding a cytochrome P450. Genetic analysis of the cyp-35B1 promoter showed both DAF-16 and HSF-1 are direct regulators. Based on these findings, we propose that hsf-1 may participate in the pathways mediating non-autonomous activities of age-1 in C. elegans.

  2. Function of RSKS-1-AAK-2-DAF-16 signaling cascade in enhancing toxicity of multi-walled carbon nanotubes can be suppressed by mir-259 activation in Caenorhabditis elegans

    PubMed Central

    Zhuang, Ziheng; Li, Min; Liu, Hui; Luo, Libo; Gu, Weidong; Wu, Qiuli; Wang, Dayong

    2016-01-01

    Caenorhabditis elegans is an important non-mammalian alternative assay model for toxicological study. Previous study has indicated that exposure to multi-walled carbon nanotubes (MWCNTs) dysregulated the transcriptional expression of mir-259. In this study, we examined the molecular basis for mir-259 in regulating MWCNTs toxicity in nematodes. Mutation of mir-259 induced a susceptible property to MWCNTs toxicity, and MWCNTs exposure induced a significant increase in mir-259::GFP in pharyngeal/intestinal valve and reproductive tract, implying that mir-259 might mediate a protection mechanisms for nematodes against MWCNTs toxicity. RSKS-1, a putative ribosomal protein S6 kinase, acted as the target for mir-259 in regulating MWCNTs toxicity, and mutation of rsks-1 suppressed the susceptible property of mir-259 mutant to MWCNTs toxicity. Moreover, mir-259 functioned in pharynx-intestinal valve and RSKS-1 functioned in pharynx to regulate MWCNTs toxicity. Furthermore, RSKS-1 regulated MWCNTs toxicity by suppressing the function of AAK-2-DAF-16 signaling cascade. Our results will strengthen our understanding the microRNAs mediated protection mechanisms for animals against the toxicity from certain nanomaterials. PMID:27573184

  3. Components of the Cultivated Red Seaweed Chondrus crispus Enhance the Immune Response of Caenorhabditis elegans to Pseudomonas aeruginosa through the pmk-1, daf-2/daf-16, and skn-1 Pathways

    PubMed Central

    Liu, Jinghua; Hafting, Jeff; Critchley, Alan T.; Banskota, Arjun H.

    2013-01-01

    Marine macroalgae are rich in bioactive compounds that can, when consumed, impart beneficial effects on animal and human health. The red seaweed Chondrus crispus has been reported to have a wide range of health-promoting activities, such as antitumor and antiviral activities. Using a Caenorhabditis elegans infection model, we show that C. crispus water extract (CCWE) enhances host immunity and suppresses the expression of quorum sensing (QS) and the virulence factors of Pseudomonas aeruginosa (strain PA14). Supplementation of nematode growth medium with CCWE induced the expression of C. elegans innate immune genes, such as irg-1, irg-2, F49F1.6, hsf-1, K05D8.5, F56D6.2, C29F3.7, F28D1.3, F38A1.5 ZK6.7, lys-1, spp-1, and abf-1, by more than 2-fold, while T20G5.7 was not affected. Additionally, CCWE suppressed the expression of PA14 QS genes and virulence factors, although it did not affect the growth of the bacteria. These effects correlated with a 28% reduction in the PA14-inflicted killing of C. elegans. Kappa-carrageenan (K-CGN), a major component of CCWE, was shown to play an important role in the enhancement of host immunity. Using C. elegans mutants, we identified that pmk-1, daf-2/daf-16, and skn-1 are essential in the K-CGN-induced host immune response. In view of the conservation of innate immune pathways between C. elegans and humans, the results of this study suggest that water-soluble components of C. crispus may also play a health-promoting role in higher animals and humans. PMID:24056462

  4. A 44 bp intestine-specific hermaphrodite-specific enhancer from the C. elegans vit-2 vitellogenin gene is directly regulated by ELT-2, MAB-3, FKH-9 and DAF-16 and indirectly regulated by the germline, by daf-2/insulin signaling and by the TGF-β/Sma/Mab pathway.

    PubMed

    Goszczynski, Barbara; Captan, Vasile V; Danielson, Alicia M; Lancaster, Brett R; McGhee, James D

    2016-05-01

    The Caenorhabditis elegans vitellogenin genes are transcribed in the intestine of adult hermaphrodites but not of males. A 44-bp region from the vit-2 gene promoter is able largely to reconstitute this tissue-, stage- and sex-specific-expression. This "enhancer" contains a binding site for the DM-domain factor MAB-3, the male-specific repressor of vitellogenesis, as well as an activator site that we show is the direct target of the intestinal GATA factor ELT-2. We further show that the enhancer is directly activated by the winged-helix/forkhead-factor FKH-9, (whose gene has been shown by others to be a direct target of DAF-16), by an unknown activator binding to the MAB-3 site, and by the full C. elegans TGF-β/Sma/Mab pathway acting within the intestine. The vit-2 gene has been shown by others to be repressed by the daf-2/daf-16 insulin signaling pathway, which so strongly influences aging and longevity in C. elegans. We show that the activity of the 44 bp vit-2 enhancer is abolished by loss of daf-2 but is restored by simultaneous loss of daf-16. DAF-2 acts from outside of the intestine but DAF-16 acts both from outside of the intestine and from within the intestine where it binds directly to the same non-canonical target site that interacts with FKH-9. Activity of the 44 bp vit-2 enhancer is also inhibited by loss of the germline, in a manner that is only weakly influenced by DAF-16 but that is strongly influenced by KRI-1, a key downstream effector in the pathway by which germline loss increases C. elegans lifespan. The complex behavior of this enhancer presumably allows vitellogenin gene transcription to adjust to demands of body size, germline proliferation and nutritional state but we suggest that the apparent involvement of this enhancer in aging and longevity "pathways" could be incidental. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Planarians require an intact brain to behaviorally react to cocaine, but not to react to nicotine.

    PubMed

    Pagán, O R; Deats, S; Baker, D; Montgomery, E; Wilk, G; Tenaglia, M; Semon, J

    2013-08-29

    Planarians possess a rudimentary brain with many features in common with vertebrate brains. They also display a remarkable capacity for tissue regeneration including the complete regeneration of the nervous system. Using the induction of planarian seizure-like movements (pSLMs) as a behavioral endpoint, we demonstrate that an intact nervous system is necessary for this organism to react to cocaine exposure, but not necessary to react to nicotine administration. Decapitated planarians (Girardia tigrina) display pSLMs indistinguishable from intact worms when exposed to nicotine, but cocaine-induced pSLMs are reduced by about 95% upon decapitation. Decapitated worms recover their normal sensitivity to cocaine within 5 days after head amputation. In worms where half of the brain was removed or partially dissected, the expression of cocaine-induced pSLMs was reduced by approximately 75%. Similar amputations at the level of the tail did not show a significant decrease to cocaine exposure. To the best of our knowledge, our work is the first report that explores how regenerating planarians react to the exposure of cocaine.

  6. Planarians require an intact brain to behaviorally react to cocaine, but not to react to nicotine

    PubMed Central

    Pagán, Oné R.; Deats, Sean; Baker, Debra; Montgomery, Erica; Wilk, Galia; Tenaglia, Matthew; Semon, Joshua

    2013-01-01

    Planarians possess a rudimentary brain with many features in common with vertebrate brains. They also display a remarkable capacity for tissue regeneration including the complete regeneration of the nervous system. Using the induction of planarian seizure-like movements (pSLMs) as a behavioral endpoint, we demonstrate that an intact nervous system is necessary for this organism to react to cocaine exposure, but not necessary to react to nicotine administration. Decapitated planarians (Girardia tigrina) display pSLMs indistinguishable from intact worms when exposed to nicotine, but cocaine-induced pSLMs are reduced by about 95% upon decapitation. Decapitated worms recover their normal sensitivity to cocaine within five days after head amputation. In worms where half of the brain was removed or partially dissected, the expression of cocaine-induced pSLMs was reduced by approximately 75 %. Similar amputations at the level of the tail did not show a significant decrease to cocaine exposure. To the best of our knowledge, our work is the first report that explores how regenerating planarians react to the exposure of cocaine. PMID:23684614

  7. Differential Metabolisms of Green Leaf Volatiles in Injured and Intact Parts of a Wounded Leaf Meet Distinct Ecophysiological Requirements

    PubMed Central

    Matsui, Kenji; Sugimoto, Kohichi; Mano, Jun'ichi; Ozawa, Rika; Takabayashi, Junji

    2012-01-01

    Almost all terrestrial plants produce green leaf volatiles (GLVs), consisting of six-carbon (C6) aldehydes, alcohols and their esters, after mechanical wounding. C6 aldehydes deter enemies, but C6 alcohols and esters are rather inert. In this study, we address why the ability to produce various GLVs in wounded plant tissues has been conserved in the plant kingdom. The major product in completely disrupted Arabidopsis leaf tissues was (Z)-3-hexenal, while (Z)-3-hexenol and (Z)-3-hexenyl acetate were the main products formed in the intact parts of partially wounded leaves. 13C-labeled C6 aldehydes placed on the disrupted part of a wounded leaf diffused into neighboring intact tissues and were reduced to C6 alcohols. The reduction of the aldehydes to alcohols was catalyzed by an NADPH-dependent reductase. When NADPH was supplemented to disrupted tissues, C6 aldehydes were reduced to C6 alcohols, indicating that C6 aldehydes accumulated because of insufficient NADPH. When the leaves were exposed to higher doses of C6 aldehydes, however, a substantial fraction of C6 aldehydes persisted in the leaves and damaged them, indicating potential toxicity of C6 aldehydes to the leaf cells. Thus, the production of C6 aldehydes and their differential metabolisms in wounded leaves has dual benefits. In disrupted tissues, C6 aldehydes and their α,β-unsaturated aldehyde derivatives accumulate to deter invaders. In intact cells, the aldehydes are reduced to minimize self-toxicity and allow healthy cells to survive. The metabolism of GLVs is thus efficiently designed to meet ecophysiological requirements of the microenvironments within a wounded leaf. PMID:22558466

  8. Polar localization of Escherichia coli chemoreceptors requires an intact Tol-Pal complex.

    PubMed

    Santos, Thiago M A; Lin, Ti-Yu; Rajendran, Madhusudan; Anderson, Samantha M; Weibel, Douglas B

    2014-06-01

    Subcellular biomolecular localization is critical for the metabolic and structural properties of the cell. The functional implications of the spatiotemporal distribution of protein complexes during the bacterial cell cycle have long been acknowledged; however, the molecular mechanisms for generating and maintaining their dynamic localization in bacteria are not completely understood. Here we demonstrate that the trans-envelope Tol-Pal complex, a widely conserved component of the cell envelope of Gram-negative bacteria, is required to maintain the polar positioning of chemoreceptor clusters in Escherichia coli. Localization of the chemoreceptors was independent of phospholipid composition of the membrane and the curvature of the cell wall. Instead, our data indicate that chemoreceptors interact with components of the Tol-Pal complex and that this interaction is required to polarly localize chemoreceptor clusters. We found that disruption of the Tol-Pal complex perturbs the polar localization of chemoreceptors, alters cell motility, and affects chemotaxis. We propose that the E. coli Tol-Pal complex restricts mobility of the chemoreceptor clusters at the cell poles and may be involved in regulatory mechanisms that co-ordinate cell division and segregation of the chemosensory machinery.

  9. Polar localization of Escherichia coli chemoreceptors requires an intact Tol–Pal complex

    PubMed Central

    Santos, Thiago M. A.; Lin, Ti-Yu; Rajendran, Madhusudan; Anderson, Samantha M.; Weibel, Douglas B.

    2014-01-01

    Summary Subcellular biomolecular localization is critical for the metabolic and structural properties of the cell. The functional implications of the spatiotemporal distribution of protein complexes during the bacterial cell cycle have long been acknowledged; however, the molecular mechanisms for generating and maintaining their dynamic localization in bacteria are not completely understood. Here we demonstrate that the trans-envelope Tol–Pal complex, a widely conserved component of the cell envelope of Gram-negative bacteria, is required to maintain the polar positioning of chemoreceptor clusters in Escherichia coli. Localization of the chemoreceptors was independent of phospholipid composition of the membrane and the curvature of the cell wall. Instead, our data indicate that chemoreceptors interact with components of the Tol–Pal complex and that this interaction is required to polarly localize chemoreceptor clusters. We found that disruption of the Tol–Pal complex perturbs the polar localization of chemoreceptors, alters cell motility, and affects chemotaxis. We propose that the E. coli Tol–Pal complex restricts mobility of the chemoreceptor clusters at the cell poles and may be involved in regulatory mechanisms that co-ordinate cell division and segregation of the chemosensory machinery. PMID:24720726

  10. The DDR at telomeres lacking intact shelterin does not require substantial chromatin decompaction.

    PubMed

    Timashev, Leonid A; Babcock, Hazen; Zhuang, Xiaowei; de Lange, Titia

    2017-03-15

    Telomeres are protected by shelterin, a six-subunit protein complex that represses the DNA damage response (DDR) at chromosome ends. Extensive data suggest that TRF2 in shelterin remodels telomeres into the t-loop structure, thereby hiding telomere ends from double-stranded break repair and ATM signaling, whereas POT1 represses ATR signaling by excluding RPA. An alternative protection mechanism was suggested recently by which shelterin subunits TRF1, TRF2, and TIN2 mediate telomeric chromatin compaction, which was proposed to minimize access of DDR factors. We performed superresolution imaging of telomeres in mouse cells after conditional deletion of TRF1, TRF2, or both, the latter of which results in the complete loss of shelterin. Upon removal of TRF1 or TRF2, we observed only minor changes in the telomere volume in most of our experiments. Upon codeletion of TRF1 and TRF2, the telomere volume increased by varying amounts, but even those samples exhibiting small changes in telomere volume showed DDR at nearly all telomeres. Upon shelterin removal, telomeres underwent 53BP1-dependent clustering, potentially explaining at least in part the apparent increase in telomere volume. Furthermore, chromatin accessibility, as determined by ATAC-seq (assay for transposase-accessible chromatin [ATAC] with high-throughput sequencing), was not substantially altered by shelterin removal. These results suggest that the DDR induced by shelterin removal does not require substantial telomere decompaction.

  11. Initiation of Chondrocyte Self-Assembly Requires an Intact Cytoskeletal Network

    PubMed Central

    Lee, Jennifer K.; Hu, Jerry C.Y.

    2016-01-01

    Self-assembly and self-organization have recently emerged as robust scaffold-free tissue engineering methodologies that can be used to generate various tissues, including cartilage, vessel, and liver. Self-assembly, in particular, is a scaffold-free platform for tissue engineering that does not require the input of exogenous energy to the system. Although self-assembly can generate functional tissues, most notably neocartilage, the mechanisms of self-assembly remain unclear. To study the self-assembling process, we used articular chondrocytes as a model to identify parameters that can affect this process. Specifically, the roles of cell–cell and cell–matrix adhesion molecules, surface-bound collagen, and the actin cytoskeletal network were investigated. Using time-lapse imaging, we analyzed the early stages of chondrocyte self-assembly. Within hours, chondrocytes rapidly coalesced into cell clusters before compacting to form tight cellular structures. Chondrocyte self-assembly was found to depend primarily on integrin function and secondarily on cadherin function. In addition, actin or myosin II inhibitors prevented chondrocyte self-assembly, suggesting that cell adhesion alone is not sufficient, but rather the active contractile actin cytoskeleton is essential for proper chondrocyte self-assembly and the formation of neocartilage. Better understanding of the self-assembly mechanisms allows for the rational modulation of this process toward generating neocartilages with improved properties. These findings are germane to understanding self-assembly, an emerging platform for tissue engineering of a plethora of tissues, especially as these neotissues are poised for translation. PMID:26729374

  12. Energy requirement and food intake behaviour in young adult intact male cats with and without predisposition to overweight.

    PubMed

    Wichert, Brigitta; Trossen, Julia; Uebelhart, Daniel; Wanner, Marcel; Hartnack, Sonja

    2012-01-01

    Obesity is a common problem in cats. In the experimental cat family of the institute of animal nutrition besides a "normal" lean phenotype, cats with predisposition to an overweight phenotype are present. To investigate energy requirements and food intake behaviour of intact male cats of different phenotypes, six "normal" lean cats (GL) and six cats disposed to overweight (GO) were used. At the beginning of the experiment, all cats had an ideal body condition score of 5. To reach this the GO cats had to pass a weight-loss program. Energy requirements of the cats were determined using respiration chambers, whereas the amount and frequency of food intake was measured with a feeding station recording the data automatically. Energy requirement at weight constancy of the GO cats was even on fat-free mass (FFM) significantly (P = 0.02) lower (162.6 kJ/kg FFM/d) than that of the "normal" lean cats (246 kJ/kg FFM/d). The GO cats also showed a higher food intake 34.5 ± 1.5 g dry matter/kg body weight(0.67) compared to the GL cats (24.0 ± 2.1 g dry matter/kg body weight(0.67))(P = 0.001). In conclusion quantifiable differences in food intake and behaviour in cats predisposed to overweight compared to "normal" lean cats were found.

  13. Energy Requirement and Food Intake Behaviour in Young Adult Intact Male Cats with and without Predisposition to Overweight

    PubMed Central

    Wichert, Brigitta; Trossen, Julia; Uebelhart, Daniel; Wanner, Marcel; Hartnack, Sonja

    2012-01-01

    Obesity is a common problem in cats. In the experimental cat family of the institute of animal nutrition besides a “normal” lean phenotype, cats with predisposition to an overweight phenotype are present. To investigate energy requirements and food intake behaviour of intact male cats of different phenotypes, six “normal” lean cats (GL) and six cats disposed to overweight (GO) were used. At the beginning of the experiment, all cats had an ideal body condition score of 5. To reach this the GO cats had to pass a weight-loss program. Energy requirements of the cats were determined using respiration chambers, whereas the amount and frequency of food intake was measured with a feeding station recording the data automatically. Energy requirement at weight constancy of the GO cats was even on fat-free mass (FFM) significantly (P = 0.02) lower (162.6 kJ/kg FFM/d) than that of the “normal” lean cats (246 kJ/kg FFM/d). The GO cats also showed a higher food intake 34.5 ± 1.5 g dry matter/kg body weight0.67 compared to the GL cats (24.0 ± 2.1 g dry matter/kg body weight0.67)(P = 0.001). In conclusion quantifiable differences in food intake and behaviour in cats predisposed to overweight compared to “normal” lean cats were found. PMID:22623906

  14. Intact vinculin protein is required for control of cell shape, cell mechanics, and rac-dependent lamellipodia formation

    NASA Technical Reports Server (NTRS)

    Goldmann, Wolfgang H.; Ingber, Donald E.

    2002-01-01

    Studies were carried out using vinculin-deficient F9 embryonic carcinoma (gamma229) cells to analyze the relationship between structure and function within the focal adhesion protein vinculin, in the context of control of cell shape, cell mechanics, and movement. Atomic force microscopy studies revealed that transfection of the head (aa 1-821) or tail (aa 811-1066) domain of vinculin, alone or together, was unable to fully reverse the decrease in cell stiffness, spreading, and lamellipodia formation caused by vinculin deficiency. In contrast, replacement with intact vinculin completely restored normal cell mechanics and spreading regardless of whether its tyrosine phosphorylation site was deleted. Constitutively active rac also only induced extension of lamellipodia when microinjected into cells that expressed intact vinculin protein. These data indicate that vinculin's ability to physically couple integrins to the cytoskeleton, to mechanically stabilize cell shape, and to support rac-dependent lamellipodia formation all appear to depend on its intact three-dimensional structure.

  15. Intact vinculin protein is required for control of cell shape, cell mechanics, and rac-dependent lamellipodia formation

    NASA Technical Reports Server (NTRS)

    Goldmann, Wolfgang H.; Ingber, Donald E.

    2002-01-01

    Studies were carried out using vinculin-deficient F9 embryonic carcinoma (gamma229) cells to analyze the relationship between structure and function within the focal adhesion protein vinculin, in the context of control of cell shape, cell mechanics, and movement. Atomic force microscopy studies revealed that transfection of the head (aa 1-821) or tail (aa 811-1066) domain of vinculin, alone or together, was unable to fully reverse the decrease in cell stiffness, spreading, and lamellipodia formation caused by vinculin deficiency. In contrast, replacement with intact vinculin completely restored normal cell mechanics and spreading regardless of whether its tyrosine phosphorylation site was deleted. Constitutively active rac also only induced extension of lamellipodia when microinjected into cells that expressed intact vinculin protein. These data indicate that vinculin's ability to physically couple integrins to the cytoskeleton, to mechanically stabilize cell shape, and to support rac-dependent lamellipodia formation all appear to depend on its intact three-dimensional structure.

  16. Binge Alcohol Drinking by Mice Requires Intact Group1 Metabotropic Glutamate Receptor Signaling Within the Central Nucleus of the Amygdale

    PubMed Central

    Cozzoli, Debra K; Courson, Justin; Wroten, Melissa G; Greentree, Daniel I; Lum, Emily N; Campbell, Rianne R; Thompson, Andrew B; Maliniak, Dan; Worley, Paul F; Jonquieres, Georg; Klugmann, Matthias; Finn, Deborah A; Szumlinski, Karen K

    2014-01-01

    Despite the fact that binge alcohol drinking (intake resulting in blood alcohol concentrations (BACs) ⩾80 mg% within a 2-h period) is the most prevalent form of alcohol-use disorders (AUD), a large knowledge gap exists regarding how this form of AUD influences neural circuits mediating alcohol reinforcement. The present study employed integrative approaches to examine the functional relevance of binge drinking-induced changes in glutamate receptors, their associated scaffolding proteins and certain signaling molecules within the central nucleus of the amygdala (CeA). A 30-day history of binge alcohol drinking (for example, 4–5 g kg−1 per 2 h−1) elevated CeA levels of mGluR1, GluN2B, Homer2a/b and phospholipase C (PLC) β3, without significantly altering protein expression within the adjacent basolateral amygdala. An intra-CeA infusion of mGluR1, mGluR5 and PLC inhibitors all dose-dependently reduced binge intake, without influencing sucrose drinking. The effects of co-infusing mGluR1 and PLC inhibitors were additive, whereas those of coinhibiting mGluR5 and PLC were not, indicating that the efficacy of mGluR1 blockade to lower binge intake involves a pathway independent of PLC activation. The efficacy of mGluR1, mGluR5 and PLC inhibitors to reduce binge intake depended upon intact Homer2 expression as revealed through neuropharmacological studies of Homer2 null mutant mice. Collectively, these data indicate binge alcohol-induced increases in Group1 mGluR signaling within the CeA as a neuroadaptation maintaining excessive alcohol intake, which may contribute to the propensity to binge drink. PMID:23966068

  17. cAMP Response Element-Binding Protein Is Required for Dopamine-Dependent Gene Expression in the Intact But Not the Dopamine-Denervated Striatum

    PubMed Central

    Andersson, Malin; Konradi, Christine; Cenci, M. Angela

    2014-01-01

    The cAMP response element-binding protein (CREB) is believed to play a pivotal role in dopamine (DA) receptor-mediated nuclear signaling and neuroplasticity. Here we demonstrate that the significance of CREB for gene expression depends on the experimental paradigm. We compared the role of CREB in two different but related models: L-DOPA administration to unilaterally 6-hydroxydopamine lesioned rats, and cocaine administration to neurologically intact animals. Antisense technology was used to produce a local knockdown of CREB in the lateral caudate–putamen, a region that mediates the dyskinetic or stereotypic manifestations associated with L-DOPA or cocaine treatment, respectively. In intact rats, CREB antisense reduced both basal and cocaine-induced expression of c-Fos, FosB/ΔFosB, and prodynorphin mRNA. In the DA-denervated striatum, CREB was not required for L-DOPA to induce these gene products, nor did CREB contribute considerably to DNA binding activity at cAMP responsive elements (CREs) and CRE-like enhancers. ΔFosB-related proteins and JunD were the main contributors to both CRE and AP-1 DNA–protein complexes in L-DOPA-treated animals. In behavioral studies, intrastriatal CREB knockdown caused enhanced activity scores in intact control animals and exacerbated the dyskinetic effects of acute L-DOPA treatment in 6-OHDA-lesioned animals. These data demonstrate that CREB is not required for the development of L-DOPA-induced dyskinesia in hemiparkinsonian rats. Moreover, our results reveal an unexpected alteration of nuclear signaling mechanisms in the parkinsonian striatum treated with L-DOPA, where AP-1 transcription factors appear to supersede CREB in the activation of CRE-containing genes. PMID:11739600

  18. Fertilization in C. elegans requires an intact C-terminal RING finger in sperm protein SPE-42

    PubMed Central

    2011-01-01

    Background The C. elegans sperm protein SPE-42, a membrane protein of unknown structure and molecular function, is required for fertilization. Sperm from worms with spe-42 mutations appear normal but are unable to fertilize eggs. Sequence analysis revealed the presence of 8 conserved cysteine residues in the C-terminal cytoplasmic domain of this protein suggesting these residues form a zinc-coordinating RING finger structure. Results We made an in silico structural model of the SPE-42 RING finger domain based on primary sequence analysis and previously reported RING structures. To test the model, we created spe-42 transgenes coding for mutations in each of the 8 cysteine residues predicted to coordinate Zn++ ions in the RING finger motif. Transgenes were crossed into a spe-42 null background and protein function was measured by counting progeny. We found that all 8 cysteines are required for protein function. We also showed that sequence differences between the C-terminal 29 and 30 amino acids in C. elegans and C. briggsae SPE-42 following the RING finger domain are not responsible for the failure of the C. briggsae SPE-42 homolog to rescue C. elegans spe-42 mutants. Conclusions The results suggest that a bona fide RING domain is present at the C-terminus of the SPE-42 protein and that this motif is required for sperm-egg interactions during C. elegans fertilization. Our structural model of the RING domain provides a starting point for further structure-function analysis of this critical region of the protein. The C-terminal domain swap experiment suggests that the incompatibility between the C. elegans and C. briggsae SPE-42 proteins is caused by small amino acid differences outside the C-terminal domain. PMID:21345212

  19. Intact neurogenesis is required for benefits of exercise on spatial memory but not motor performance or contextual fear conditioning in C57BL/6J mice.

    PubMed

    Clark, P J; Brzezinska, W J; Thomas, M W; Ryzhenko, N A; Toshkov, S A; Rhodes, J S

    2008-09-09

    The mammalian hippocampus continues to generate new neurons throughout life. Experiences such as exercise, anti-depressants, and stress regulate levels of neurogenesis. Exercise increases adult hippocampal neurogenesis and enhances behavioral performance on rotarod, contextual fear and water maze in rodents. To directly test whether intact neurogenesis is required for gains in behavioral performance from exercise in C57BL/6J mice, neurogenesis was reduced using focal gamma irradiation (3 sessions of 5 Gy). Two months after treatment, mice (total n=42 males and 42 females) (Irradiated or Sham), were placed with or without running wheels (Runner or Sedentary) for 54 days. The first 10 days mice received daily injections of bromodeoxyuridine (BrdU) to label dividing cells. The last 14 days mice were tested on water maze (two trials per day for 5 days, then 1 h later probe test), rotarod (four trials per day for 3 days), and contextual fear conditioning (2 days), then measured for neurogenesis using immunohistochemical detection of BrdU and neuronal nuclear protein (NeuN) mature neuronal marker. Consistent with previous studies, in Sham animals, running increased neurogenesis fourfold and gains in performance were observed for the water maze (spatial learning and memory), rotarod (motor performance), and contextual fear (conditioning). These positive results provided the reference to determine whether gains in performance were blocked by irradiation. Irradiation reduced neurogenesis by 50% in both groups, Runner and Sedentary. Irradiation did not affect running or baseline performance on any task. Minimal changes in microglia associated with inflammation (using immunohistochemical detection of cd68) were detected at the time of behavioral testing. Irradiation did not reduce gains in performance on rotarod or contextual fear, however it eliminated gain in performance on the water maze. Results support the hypothesis that intact exercise-induced hippocampal neurogenesis

  20. Intact pericellular matrix of articular cartilage is required for unactivated discoidin domain receptor 2 in the mouse model.

    PubMed

    Xu, Lin; Polur, Ilona; Servais, Jacqueline M; Hsieh, Sirena; Lee, Peter L; Goldring, Mary B; Li, Yefu

    2011-09-01

    Increased expression of the discoidin domain receptor 2 (DDR2) results from its interaction with collagen type II. This induces expression of matrix metalloproteinase (MMP)-13, leading to osteoarthritis (OA). To investigate the impact of the pericellular matrix of chondrocytes on DDR2, we generated a mouse model with inducible overexpression of DDR2 in cartilage. Conditional overexpression of DDR2 in mature mouse articular cartilage was controlled via the cartilage oligomeric matrix protein promoter using the Tet-Off-inducible system. Doxycycline was withdrawn at 1 month of age, and knee joints were examined at 2, 3, and 4 months of age. Microsurgery was performed on 3-month-old transgenic mice overexpressing DDR2 to destabilize the medial meniscus, and serial paraffin sections were examined at 2, 4, 8, and 12 weeks after surgery. DDR2 expression increased in the knee joints of transgenic mice. However, the increased DDR2 did not induce MMP-13 expression. No OA-like changes were observed in the transgenic mice at the age of 4 months. When transgenic mice were subjected to destabilizing of the medial meniscus, we observed accelerated progression to OA, which was associated with DDR2 activation. Therefore, conditionally overexpressing DDR2 in the mature articular cartilage of mouse knee joints requires activation to induce OA, and altered biomechanical stress can accelerate the onset of cartilage loss and progression to OA in transgenic mice.

  1. A peroxiredoxin, PRDX-2, is required for insulin secretion and insulin/IIS-dependent regulation of stress resistance and longevity

    PubMed Central

    Oláhová, Monika; Veal, Elizabeth A

    2015-01-01

    Peroxiredoxins (Prx) are abundant thiol peroxidases with a conserved anti-ageing role. In contrast to most animals, the nematode worm, Caenorhabditis elegans, encodes a single cytosolic 2-Cys Prx, PRDX-2, rendering it an excellent model for examining how peroxiredoxins affect animal physiology and ageing. Our previous work revealed that, although PRDX-2 protects against the toxicity of peroxides, enigmatically, prdx-2-mutant animals are hyper-resistant to other forms of oxidative stress. Here, we have investigated the basis for this increased resistance. Mammalian FOXO and Nrf2 transcription factors directly promote the expression of a range of detoxification enzymes. We show that the FOXO orthologue, DAF-16, and the Nrf2 orthologue, SKN-1, are required for the increased stress resistance of prdx-2-mutant worms. Our data suggest that PRDX-2 is required for normal levels of insulin secretion and hence the inhibition of DAF-16 and SKN-1 by insulin/IGF-1-like signalling (IIS) under nutrient-rich conditions. Intriguingly, loss of PRDX-2 increases DAF-16 and SKN-1 activities sufficiently to increase arsenite resistance without initiating other IIS-inhibited processes. Together, these data suggest that loss of peroxiredoxin function may increase stress resistance by reducing insulin secretion, but that further changes in insulin signalling are required for the reprogramming of development and fat metabolism. In addition, we reveal that the temperature-dependent prolongevity function of PRDX-2 is required for the extended lifespan associated with several pathways, including further reductions in IIS. PMID:25808059

  2. Vaccinia Virus Protein Synthesis Has a Low Requirement for the Intact Translation Initiation Factor eIF4F, the Cap-Binding Complex, within Infected Cells

    PubMed Central

    Mulder, Jacqueline; Robertson, Morwenna E. M.; Seamons, Rachael A.; Belsham, Graham J.

    1998-01-01

    The role of the cap-binding complex, eIF4F, in the translation of vaccinia virus mRNAs has been analyzed within infected cells. Plasmid DNAs, which express dicistronic mRNAs containing a picornavirus internal ribosome entry site, produced within vaccinia virus-infected cells both β-glucuronidase and a cell surface-targeted single-chain antibody (sFv). Cells expressing sFv were selected from nonexpressing cells, enabling analysis of protein synthesis specifically within the transfected cells. Coexpression of poliovirus 2A or foot-and-mouth disease virus Lb proteases, which cleaved translation initiation factor eIF4G, greatly inhibited cap-dependent protein (β-glucuronidase) synthesis. Under these conditions, internal ribosome entry site-directed expression of sFv continued and cell selection was maintained. Furthermore, vaccinia virus protein synthesis persisted in the selected cells containing cleaved eIF4G. Thus, late vaccinia virus protein synthesis has a low requirement for the intact cap-binding complex eIF4F. This may be attributed to the short unstructured 5′ noncoding regions of the vaccinia virus mRNAs, possibly aided by the presence of poly(A) at both 5′ and 3′ termini. PMID:9765426

  3. Calcium-regulation of mitochondrial respiration maintains ATP homeostasis and requires ARALAR/AGC1-malate aspartate shuttle in intact cortical neurons.

    PubMed

    Llorente-Folch, Irene; Rueda, Carlos B; Amigo, Ignacio; del Arco, Araceli; Saheki, Takeyori; Pardo, Beatriz; Satrústegui, Jorgina

    2013-08-28

    Neuronal respiration is controlled by ATP demand and Ca2+ but the roles played by each are unknown, as any Ca2+ signal also impacts on ATP demand. Ca2+ can control mitochondrial function through Ca2+-regulated mitochondrial carriers, the aspartate-glutamate and ATP-Mg/Pi carriers, ARALAR/AGC1 and SCaMC-3, respectively, or in the matrix after Ca2+ transport through the Ca2+ uniporter. We have studied the role of Ca2+ signaling in the regulation of mitochondrial respiration in intact mouse cortical neurons in basal conditions and in response to increased workload caused by increases in [Na+]cyt (veratridine, high-K+ depolarization) and/or [Ca2+]cyt (carbachol). Respiration in nonstimulated neurons on 2.5-5 mm glucose depends on ARALAR-malate aspartate shuttle (MAS), with a 46% drop in aralar KO neurons. All stimulation conditions induced increased OCR (oxygen consumption rate) in the presence of Ca2+, which was prevented by BAPTA-AM loading (to preserve the workload), or in Ca2+-free medium (which also lowers cell workload). SCaMC-3 limits respiration only in response to high workloads and robust Ca2+ signals. In every condition tested Ca2+ activation of ARALAR-MAS was required to fully stimulate coupled respiration by promoting pyruvate entry into mitochondria. In aralar KO neurons, respiration was stimulated by veratridine, but not by KCl or carbachol, indicating that the Ca2+ uniporter pathway played a role in the first, but not in the second condition, even though KCl caused an increase in [Ca2+]mit. The results suggest a requirement for ARALAR-MAS in priming pyruvate entry in mitochondria as a step needed to activate respiration by Ca2+ in response to moderate workloads.

  4. Intact capture of cosmic dust

    NASA Technical Reports Server (NTRS)

    Tsou, P.

    1991-01-01

    The focus of this development effort is to capture dust particles at hypervelocities intact and unmelted in order to preserve volatile organics. At the same time, the capture process must minimize any organic elemental or compound contamination to prevent any compromise of exobiological analyses. Inorganic silicate aerogel has been developed as a successful capture medium to satisfy both requirements of intact capture and minimal organic contamination. Up to 6 km/s, silicate projectiles from a few microns up to 100 microns have been captured intact without any melting and with minimal loss of mass. Carbon in silicate aerogel can be reduced to less than 1 part in 1000 and hydrogen 3 parts in 1000 when baked in air. Under controlled inert gas environments, additional hydrocarbon reduction can be achieved.

  5. Optical manipulation of Saccharomyces cerevisiae cells reveals that green light protection against UV irradiation is favored by low Ca2+ and requires intact UPR pathway.

    PubMed

    Farcasanu, Ileana C; Mitrica, Radu; Cristache, Ligia; Nicolau, Ioana; Ruta, Lavinia L; Paslaru, Liliana; Comorosan, Sorin

    2013-11-01

    Optical manipulation of Saccharomyces cerevisiae cells with high density green photons conferred protection against the deleterious effects of UV radiation. Combining chemical screening with UV irradiation of yeast cells, it was noted that the high density green photons relied on the presence of intact unfolded protein response (UPR) pathway to exert their protective effect and that the low Ca(2+) conditions boosted the effect. UPR chemical inducers tunicamycin, dithiotreitol and calcium chelators augmented the green light effect in a synergic action against UV-induced damage. Photo-manipulation of cells was a critical factor since the maximum protection was achieved only when cells were pre-exposed to green light. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  6. Toll-Like Receptor Ligand-Based Vaccine Adjuvants Require Intact MyD88 Signaling in Antigen-Presenting Cells for Germinal Center Formation and Antibody Production

    PubMed Central

    Mosaheb, Munir M.; Reiser, Michael L.; Wetzler, Lee M.

    2017-01-01

    Vaccines are critical in the fight against infectious diseases, and immune-stimulating adjuvants are essential for enhancing vaccine efficacy. However, the precise mechanisms of action of most adjuvants are unknown. There is an urgent need for customized and adjuvant formulated vaccines against immune evading pathogens that remain a risk today. Understanding the specific role of various cell types in adjuvant-induced protective immune responses is vital for an effective vaccine design. We have investigated the role of cell-specific MyD88 signaling in vaccine adjuvant activity in vivo, using Neisserial porin B (PorB), a TLR2 ligand-based adjuvant, compared with an endosomal TLR9 ligand (CpG) and toll-like receptor (TLR)-independent (alum, MF59) adjuvants. We found that intact MyD88 signaling is essential, separately, in all three antigen-presenting cell types [B cells, macrophages, and dendritic cells (DCs)] for optimal TLR ligand-based adjuvant activity. The role of MyD88 signaling in B cell and DC in vaccine adjuvant has been previously investigated. In this study, we now demonstrate that the immune response was also reduced in mice with macrophage-specific MyD88 deletion (Mac-MyD88−/−). We demonstrate that TLR-dependent adjuvants are potent inducers of germinal center (GC) responses, but GCs are nearly absent in Mac-MyD88−/− mice following immunization with TLR-dependent adjuvants PorB or CpG, but not with TLR-independent adjuvants MF59 or alum. Our findings reveal a unique and here-to-for unrecognized importance of intact MyD88 signaling in macrophages, to allow for a robust vaccine-induced immune responses when TLR ligand-based adjuvants are used. PMID:28316602

  7. An intact DNA-binding domain is not required for peroxisome proliferator-activated receptor gamma (PPARgamma) binding and activation on some PPAR response elements.

    PubMed

    Temple, Karla A; Cohen, Ronald N; Wondisford, Sarah R; Yu, Christine; Deplewski, Dianne; Wondisford, Fredric E

    2005-02-04

    Peroxisome proliferator-activated receptor gamma (PPARgamma) interacts with retinoid X receptor (RXR) on PPAR response elements (PPREs) to regulate transcription of PPAR-responsive genes. To investigate the binding of PPARgamma and RXR to PPREs, three mutations were constructed in the DNA-binding domains of PPARgamma; two of the mutants maintained the structure of zinc finger I (PPARgamma-GS and PPARgamma-AA), and a third mutation disrupted the protein structure of zinc finger I (PPARgamma-CS). Results indicated that the mutations of PPARgamma that maintained intact zinc fingers were capable of binding to a variety of PPREs in the presence of RXR and could activate transcription on several PPREs. In parallel, a mutation was created in the DNA-binding domain of RXRalpha that maintained the structure of the zinc fingers (RXR-GS) but did not bind DNA and was transcriptionally inactive. Examination of the 3' half-site of several PPREs revealed that variations from the consensus sequence reduced or abolished transcriptional activity, but conversion to consensus improved transcriptional activity with PPARgamma-GS and PPARgamma-AA. Examination of the 5' half-site indicated that the upstream three nucleotides were more important for transcriptional activity than the downstream three nucleotides. Our data demonstrated that stringent binding of RXR to the 3' half-site of a PPRE is more influential on the binding of the PPARgamma/RXR heterodimer than the ability of PPARgamma to bind DNA. Thus, unlike RXR, PPARgamma exhibits promiscuity in binding on a PPRE, suggesting that the definition of a PPRE for PPARgamma may need to be expanded.

  8. Monitoring Intact Viruses Using Aptamers.

    PubMed

    Kumar, Penmetcha K R

    2016-08-04

    Viral diagnosis and surveillance are necessary steps in containing the spread of viral diseases, and they help in the deployment of appropriate therapeutic interventions. In the past, the commonly employed viral detection methods were either cell-culture or molecule-level assays. Most of these assays are laborious and expensive, require special facilities, and provide a slow diagnosis. To circumvent these limitations, biosensor-based approaches are becoming attractive, especially after the successful commercialization of glucose and other biosensors. In the present article, I have reviewed the current progress using the biosensor approach for detecting intact viruses. At the time of writing this review, three types of bioreceptor surfaces (antibody-, glycan-, and aptamer-based) have been explored on different sensing platforms for detecting intact viruses. Among these bioreceptors, aptamer-based sensors have been increasingly explored for detecting intact viruses using surface plasmon resonance (SPR) and other platforms. Special emphasis is placed on the aptamer-based SPR platform in the present review.

  9. Monitoring Intact Viruses Using Aptamers

    PubMed Central

    Kumar, Penmetcha K. R.

    2016-01-01

    Viral diagnosis and surveillance are necessary steps in containing the spread of viral diseases, and they help in the deployment of appropriate therapeutic interventions. In the past, the commonly employed viral detection methods were either cell-culture or molecule-level assays. Most of these assays are laborious and expensive, require special facilities, and provide a slow diagnosis. To circumvent these limitations, biosensor-based approaches are becoming attractive, especially after the successful commercialization of glucose and other biosensors. In the present article, I have reviewed the current progress using the biosensor approach for detecting intact viruses. At the time of writing this review, three types of bioreceptor surfaces (antibody-, glycan-, and aptamer-based) have been explored on different sensing platforms for detecting intact viruses. Among these bioreceptors, aptamer-based sensors have been increasingly explored for detecting intact viruses using surface plasmon resonance (SPR) and other platforms. Special emphasis is placed on the aptamer-based SPR platform in the present review. PMID:27527230

  10. Cyclosporine-induced autoimmunity. Conditions for expressing disease, requirement for intact thymus, and potency estimates of autoimmune lymphocytes in drug-treated rats

    PubMed Central

    1986-01-01

    These studies explore the phenomenon of cyclosporine-induced autoimmunity in irradiated Lewis rats. We show that (a) the presence of a thymus is required, and autoimmune precursors develop in and exit from this organ to the peripheral lymphocyte pool within a 2-wk period after the initiation of cyclosporine treatment; (b) adoptive transfers of drug-induced autoimmunity to irradiated secondary recipients can be accomplished with relatively few cells of the Th subset, and these transfers of autoimmunity can be blocked by cotransfer of normal lymphoid cells; and (c) potency estimates, using popliteal lymph node assays in syngeneic and F1 recipients indicate similar levels of auto- and alloreactivity by cells from drug-induced autoimmune donors. These various findings indicate that this particular animal model may be useful for studies of the onset and control of autoimmunity, and they raise the possibility that the lack of autoimmunity in normal animals and its induction with cyclosporine may involve similar cellular mechanism as have been found to be operative in GVH reactions and specifically induced immunologic resistance to GVHD. PMID:3490534

  11. Leaf Abscission Induced by Ethylene in Water-Stressed Intact Seedlings of Cleopatra Mandarin Requires Previous Abscisic Acid Accumulation in Roots.

    PubMed

    Gomez-Cadenas, A.; Tadeo, F. R.; Talon, M.; Primo-Millo, E.

    1996-09-01

    The involvement of abscisic acid (ABA) in the process of leaf abscission induced by 1-aminocyclopropane-1-carboxylic acid (ACC) transported from roots to shoots in Cleopatra mandarin (Citrus reshni Hort. ex Tan.) seedlings grown under water stress was studied using norflurazon (NF). Water stress induced both ABA (24-fold) and ACC (16-fold) accumulation in roots and arrested xylem flow. Leaf bulk ABA also increased (8-fold), although leaf abscission did not occur. Shortly after rehydration, root ABA and ACC returned to their prestress levels, whereas sharp and transitory increases of ACC (17-fold) and ethylene (10-fold) in leaves and high percentages of abscission (up to 47%) were observed. NF suppressed the ABA and ACC accumulation induced by water stress in roots and the sharp increases of ACC and ethylene observed after rewatering in leaves. NF also reduced leaf abscission (7-10%). These results indicate that water stress induces root ABA accumulation and that this is required for the process of leaf abscission to occur. It was also shown that exogenous ABA increases ACC levels in roots but not in leaves. Collectively, the data suggest that ABA, the primary sensitive signal to water stress, modulates the levels of ethylene, which is the hormonal activator of leaf abscission. This assumption implies that root ACC levels are correlated with root ABA amounts in a dependent way, which eventually links water status to an adequate, protective response such as leaf abscission.

  12. P2Y6 Receptors Require an Intact Cysteinyl Leukotriene Synthetic and Signaling System to Induce Survival and Activation of Mast Cells1

    PubMed Central

    Jiang, Yongfeng; Borrelli, Laura; Bacskai, Brian J.; Kanaoka, Yoshihide; Boyce, Joshua A.

    2008-01-01

    Cysteinyl leukotrienes (cys-LTs) induce inflammatory responses through type 1 (CysLT1R) and type 2 (CysLT2R) cys-LT receptors, and activate mast cells (MCs) in vitro. We previously demonstrated that cys-LTs cross-desensitized interleukin (IL)-4-primed primary human MCs (hMCs) to stimulation with the nucleotide uridine diphosphate (UDP). We now report that hMCs, mouse bone marrow-derived mast cells (mBMMCs), and the human MC line LAD2 all express UDP-selective P2Y6 receptors that cooperate with CysLT1R to promote cell survival and chemokine generation by a pathway involving reciprocal ligand-mediated cross-talk. LTD4, the most potent CysLT1R ligand, and UDP both induced phosphorylation of extracellular signal regulated kinase (ERK) and prolonged the survival of cytokine-starved hMCs and mBMMCs. ERK activation and cytoprotection in response to either ligand were attenuated by treatment of the cells with a selective P2Y6 receptor antagonist (MRS2578), which did not interfere with signaling through recombinant CysLT1R. Surprisingly, both UDP and LTD4-mediated ERK activation and cytoprotection were absent in mBMMCs lacking CysLT1R and the biosynthetic enzyme LTC4 synthase (LTC4S), implying a requirement for a cys-LT-mediated autocrine loop. In IL-4-primed LAD2 cells, LTD4 induced the generation of macrophage inflammatory protein 1β (MIP-1β) by IL-4-primed LAD2 cells, a response blocked by short hairpin RNA (shRNA)-mediated knockdown of CysLT1R or of P2Y6 receptors, but not of CysLT2R. Thus, CysLT1R and P2Y6 receptors, which are co-expressed with on many cell types of innate immunity, reciprocally amplify one another’s function in MCs through endogenous ligands. PMID:19124756

  13. Coaxial atomizer liquid intact lengths

    NASA Technical Reports Server (NTRS)

    Eroglu, Hasan; Chigier, Norman; Farago, Zoltan

    1991-01-01

    Average intact lengths of round liquid jets generated by airblast coaxial atomizer were measured from over 1500 photographs. The intact lengths were studied over a jet Reynolds number range of 18,000 and Weber number range of 260. Results are presented for two different nozzle geometries. The intact lengths were found to be strongly dependent on Re and We numbers. An empirical equation was derived as a function of these parameters. A comparison of the intact lengths for round jets and flat sheets shows that round jets generate shorter intact lengths.

  14. (Photosynthesis in intact plants)

    SciTech Connect

    Not Available

    1990-01-01

    Progress in the two years since the last renewal application has been excellent. We have made substantial contributions on both main fronts of the projects, and are particularly happy with the progress of our research on intact plants. The approach of basing our field work on a sound foundation of laboratory studies has enabled is to use methods which provide unambiguous assays of well characterized reactions. We have also made excellent progress in several laboratory studies which will have direct applications in future field work, and have introduced to the laboratory a range of molecular genetics techniques which will allow us to explore new options in the attempt to understand function at the level of molecular structure.

  15. Autism Spectrum Disorder and intact executive functioning.

    PubMed

    Ferrara, R; Ansermet, F; Massoni, F; Petrone, L; Onofri, E; Ricci, P; Archer, T; Ricci, S

    2016-01-01

    Earliest notions concerning autism (Autism Spectrum Disorders, ASD) describe the disturbance in executive functioning. Despite altered definition, executive functioning, expressed as higher cognitive skills required complex behaviors linked to the prefrontal cortex, are defective in autism. Specific difficulties in children presenting autism or verbal disabilities at executive functioning levels have been identified. Nevertheless, the developmental deficit of executive functioning in autism is highly diversified with huge individual variation and may even be absent. The aim of the present study to examine the current standing of intact executive functioning intact in ASD.

  16. C. elegans lifespan extension by osmotic stress requires FUdR, base excision repair, FOXO, and sirtuins

    PubMed Central

    Anderson, Edward N; Corkins, Mark E; Li, Jia-Cheng; Singh, Komudi; Parsons, Sadé; Tucey, Tim M; Sorkaç, Altar; Huang, Huiyan; Dimitriadi, Maria; Sinclair, David A

    2016-01-01

    Moderate stress can increase lifespan by hormesis, a beneficial low-level induction of stress response pathways. 5’-fluorodeoxyuridine (FUdR) is commonly used to sterilize Caenorhabditis elegans in aging experiments. However, FUdR alters lifespan in some genotypes and induces resistance to thermal and proteotoxic stress. We report that hypertonic stress in combination with FUdR treatment or inhibition of the FUdR target thymidylate synthase, TYMS-1, extends C. elegans lifespan by up to 30%. By contrast, in the absence of FUdR, hypertonic stress decreases lifespan. Adaptation to hypertonic stress requires diminished Notch signaling and loss of Notch co-ligands leads to lifespan extension only in combination with FUdR. Either FUdR treatment or TYMS-1 loss induced resistance to acute hypertonic stress, anoxia, and thermal stress. FUdR treatment increased expression of DAF-16 FOXO and the osmolyte biosynthesis enzyme GPDH-1. FUdR-induced hypertonic stress resistance was partially dependent on sirtuins and base excision repair (BER) pathways, while FUdR-induced lifespan extension under hypertonic stress conditions requires DAF-16, BER, and sirtuin function. Combined, these results demonstrate that FUdR, through inhibition of TYMS-1, activates stress response pathways in somatic tissues to confer hormetic resistance to acute and chronic stress. C. elegans lifespan studies using FUdR may need re-interpretation in light of this work. PMID:26854551

  17. C. elegans lifespan extension by osmotic stress requires FUdR, base excision repair, FOXO, and sirtuins.

    PubMed

    Anderson, Edward N; Corkins, Mark E; Li, Jia-Cheng; Singh, Komudi; Parsons, Sadé; Tucey, Tim M; Sorkaç, Altar; Huang, Huiyan; Dimitriadi, Maria; Sinclair, David A; Hart, Anne C

    2016-03-01

    Moderate stress can increase lifespan by hormesis, a beneficial low-level induction of stress response pathways. 5'-fluorodeoxyuridine (FUdR) is commonly used to sterilize Caenorhabditis elegans in aging experiments. However, FUdR alters lifespan in some genotypes and induces resistance to thermal and proteotoxic stress. We report that hypertonic stress in combination with FUdR treatment or inhibition of the FUdR target thymidylate synthase, TYMS-1, extends C. elegans lifespan by up to 30%. By contrast, in the absence of FUdR, hypertonic stress decreases lifespan. Adaptation to hypertonic stress requires diminished Notch signaling and loss of Notch co-ligands leads to lifespan extension only in combination with FUdR. Either FUdR treatment or TYMS-1 loss induced resistance to acute hypertonic stress, anoxia, and thermal stress. FUdR treatment increased expression of DAF-16 FOXO and the osmolyte biosynthesis enzyme GPDH-1. FUdR-induced hypertonic stress resistance was partially dependent on sirtuins and base excision repair (BER) pathways, while FUdR-induced lifespan extension under hypertonic stress conditions requires DAF-16, BER, and sirtuin function. Combined, these results demonstrate that FUdR, through inhibition of TYMS-1, activates stress response pathways in somatic tissues to confer hormetic resistance to acute and chronic stress. C. elegans lifespan studies using FUdR may need re-interpretation in light of this work. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. MRI of intact plants.

    PubMed

    Van As, Henk; Scheenen, Tom; Vergeldt, Frank J

    2009-01-01

    Nuclear magnetic resonance imaging (MRI) is a non-destructive and non-invasive technique that can be used to acquire two- or even three-dimensional images of intact plants. The information within the images can be manipulated and used to study the dynamics of plant water relations and water transport in the stem, e.g., as a function of environmental (stress) conditions. Non-spatially resolved portable NMR is becoming available to study leaf water content and distribution of water in different (sub-cellular) compartments. These parameters directly relate to stomatal water conductance, CO(2) uptake, and photosynthesis. MRI applied on plants is not a straight forward extension of the methods discussed for (bio)medical MRI. This educational review explains the basic physical principles of plant MRI, with a focus on the spatial resolution, factors that determine the spatial resolution, and its unique information for applications in plant water relations that directly relate to plant photosynthetic activity. © Springer Science+Business Media B.V. 2009

  19. The physics of intact capture

    NASA Technical Reports Server (NTRS)

    Tsou, Peter; Griffiths, D. J.; Albee, A. L.

    1994-01-01

    The ability to capture projectiles intact at hypervelocities in underdense media open a new area of study in physics. Underdense material behaves markedly different than solid, liquid, or gas upon hypervelocity impact. This new phenomenon enables applications in science that would either not be possible or would be very costly by other means. This phenomenon has been fully demonstrated in the laboratory and validated in space. Even more interesting is the fact that this hypervelocity intact capture was accomplished passively. A better understanding of the physics of intact capture will lead to improvements in intact capture. A collection of physical observations of this phenomenon is presented here.

  20. The physics of intact capture

    NASA Technical Reports Server (NTRS)

    Tsou, Peter; Griffiths, D. J.; Albee, A. L.

    1994-01-01

    The ability to capture projectiles intact at hypervelocities in underdense media open a new area of study in physics. Underdense material behaves markedly different than solid, liquid, or gas upon hypervelocity impact. This new phenomenon enables applications in science that would either not be possible or would be very costly by other means. This phenomenon has been fully demonstrated in the laboratory and validated in space. Even more interesting is the fact that this hypervelocity intact capture was accomplished passively. A better understanding of the physics of intact capture will lead to improvements in intact capture. A collection of physical observations of this phenomenon is presented here.

  1. Two-photon laser scanning microscopy imaging of intact spinal cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in immune cell infiltration of cortical injury sites

    PubMed Central

    Kim, Jiyun V.; Jiang, Ning; Tadokoro, Carlos E.; Liu, Liping; Ransohoff, Richard M.; Lafaille, Juan J.; Dustin, Michael L.

    2009-01-01

    The mouse spinal cord is an important site for autoimmune and injury models. Skull thinning surgery provides a minimally invasive window for microscopy of the mouse cerebral cortex, but there are no parallel methods for the spinal cord. We introduce a novel, facile and inexpensive method for two-photon laser scanning microscopy of the intact spinal cord in the mouse by taking advantage of the naturally accessible intervertebral space. These are powerful methods when combined with gene-targeted mice in which endogenous immune cells are labeled with green fluorescent protein (GFP). We first demonstrate that generation of the intervertebral window does not elicit a reaction of GFP+ microglial cells in CX3CR1gfp/+ mice. We next demonstrate a distinct rostro-caudal migration of GFP+ immune cells in the spinal cord of CXCR6gfp/+ mice during active experimental autoimmune encephalomyelitis (EAE). Interestingly, infiltration of the cerebral cortex by GFP+ cells in these mice required three conditions: EAE induction, cortical injury and expression of CXCR6 on immune cells. PMID:19800886

  2. Defects in a quinol oxidase lead to loss of KatC catalase activity in Pseudomonas aeruginosa: KatC activity is temperature dependent and it requires an intact disulphide bond formation system.

    PubMed

    Mossialos, Dimitris; Tavankar, Gholam Reza; Zlosnik, James E A; Williams, Huw D

    2006-03-17

    Mutation or overexpression of the cyanide-insensitive terminal oxidase (CIO) of Pseudomonas aeruginosa leads to temperature-sensitivity, multiple antibiotic sensitivity, and abnormal cell division and failure to produce a temperature-inducible catalase [G.R. Tavankar, D. Mossialos, H.D. Williams, Mutation or overexpression of a terminal oxidase leads to a cell division defect and multiple antibiotic sensitivity in Pseudomonas aeruginosa, J. Biol. Chem. 278 (2003) 4524-4530]. We identify this enzyme as KatC, a newly described catalase from P. aeruginosa. Loss of KatC activity leads to temperature-dependent hydrogen peroxide sensitivity, which correlates with its temperature-inducible expression pattern. This is the first description, to our knowledge, of a temperature-inducible bacterial catalase. The transcription of katC is not affected in strains lacking or overexpressing the CIO, indicating that a post-transcriptional effect leads to loss of KatC activity. Disulphide bond formation is affected in strains lacking or overexpressing the CIO. This is shown by reduced activity of the extracellular enzymes lipase and elastase, and an altered pattern of redox states of DsbA, a key protein in disulphide bond formation in P. aeruginosa, in these strains. Moreover, a dsbA mutant had no detectable KatC activity, demonstrating that an intact disulphide bond formation system is required for KatC activity and thus explaining the loss of this catalase in the cio mutant and overexpressing strains.

  3. Space research with intact organisms

    NASA Technical Reports Server (NTRS)

    Phillips, Robert W.; Haddy, Francis J.

    1992-01-01

    Effects of space exposure on intact organisms are briefly reviewed, and examples of future experiments that might provide new information on the role of gravity in the evolution of life are suggested. It is noted that long term experiments with intact plant and animals for studying gravitational thresholds will provide important new insights.

  4. 46 CFR 170.165 - International Code on Intact Stability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false International Code on Intact Stability. 170.165 Section... STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Intact Stability Criteria § 170.165 International Code on...) through (4) of this section, must comply with the Introduction and Part A of the International Code on...

  5. 46 CFR 28.570 - Intact righting energy.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 1 2013-10-01 2013-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of...

  6. 46 CFR 28.570 - Intact righting energy.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of...

  7. 46 CFR 28.570 - Intact righting energy.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 1 2012-10-01 2012-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of...

  8. 46 CFR 28.570 - Intact righting energy.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of...

  9. 46 CFR 28.570 - Intact righting energy.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 1 2014-10-01 2014-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of...

  10. 46 CFR 170.165 - International Code on Intact Stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false International Code on Intact Stability. 170.165 Section 170.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Intact Stability Criteria § 170.165 International Code on...

  11. 46 CFR 170.165 - International Code on Intact Stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false International Code on Intact Stability. 170.165 Section 170.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Intact Stability Criteria § 170.165 International Code on...

  12. 46 CFR 170.165 - International Code on Intact Stability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false International Code on Intact Stability. 170.165 Section 170.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Intact Stability Criteria § 170.165 International Code on...

  13. Ultraviolet-A triggers photoaging in model nematode Caenorhabditis elegans in a DAF-16 dependent pathway.

    PubMed

    Prasanth, Mani Iyer; Santoshram, Gunasekaran Santhi; Bhaskar, James Prabhanand; Balamurugan, Krishnaswamy

    2016-02-01

    Ultraviolet radiations (UV) are the primary causative agent for skin aging (photoaging) and cancer, especially UV-A. The mode of action and the molecular mechanism behind the damages caused by UV-A is not well studied, in vivo. The current study was employed to investigate the impact of UV-A exposure using the model organism, Caenorhabditis elegans. Analysis of lifespan, healthspan, and other cognitive behaviors were done which was supported by the molecular mechanism. UV-A exposure on collagen damages the synthesis and functioning which has been monitored kinetically using engineered strain, col-19:: GFP. The study results suggested that UV-A accelerated the aging process in an insulin-like signaling pathway dependent manner. Mutant (daf-2)-based analysis concrete the observations of the current study. The UV-A exposure affected the usual behavior of the worms like pharyngeal movements and brood size. Quantitative PCR profile of the candidate genes during UV-A exposure suggested that continuous exposure has damaged the neural network of the worms, but the mitochondrial signaling and dietary restriction pathway remain unaffected. Western blot analysis of HSF-1 evidenced the alteration in protein homeostasis in UV-A exposed worms. Outcome of the current study supports our view that C. elegans can be used as a model to study photoaging, and the mode of action of UV-A-mediated damages can be elucidated which will pave the way for drug developments against photoaging.

  14. Erratum to: Ethosuximide ameliorates neurodegenerative disease phenotypes by modulating DAF-16/FOXO target gene expression.

    PubMed

    Chen, Xi; McCue, Hannah V; Wong, Shi Quan; Kashyap, Sudhanva S; Kraemer, Brian C; Barclay, Jeff W; Burgoyne, Robert D; Morgan, Alan

    2015-10-23

    The original version of this article [1] unfortunately contained a mistake. The author list contained a spelling error for the author Hannah V. McCue. The original article has been corrected for this error. The corrected author list is given below:Xi Chen, Hannah V. McCue, Shi Quan Wong, Sudhanva S. Kashyap, Brian C. Kraemer, Jeff W. Barclay, Robert D. Burgoyne and Alan Morgan

  15. Effectiveness of intact capture media

    SciTech Connect

    Tsou, P.; Aubert, J.; Brownlee, D.; Hrubesh, L.; Williams, J.; Albee, A.

    1989-01-01

    The possibility of capturing cosmic dust at hypervelocity has been demonstrated in the laboratory and in the unintended Solar Max spacecraft. This technology will enable a comet coma sample return mission and be important for the earth orbital cosmic dust collection mission, i.e., the Space Station Cosmic Dust Collection Facility. Since the only controllable factor in an intact capture of cosmic dust is the capturing medium, characterizing the effectiveness and properties of available capture media would be very important in the development of the technique for capturing hypervelocity cosmic dust intact. We have evaluated various capture underdense media for the relative effectiveness for intact capture. 2 refs., 2 figs.

  16. Intact Transition Epitope Mapping (ITEM)

    NASA Astrophysics Data System (ADS)

    Yefremova, Yelena; Opuni, Kwabena F. M.; Danquah, Bright D.; Thiesen, Hans-Juergen; Glocker, Michael O.

    2017-08-01

    Intact transition epitope mapping (ITEM) enables rapid and accurate determination of protein antigen-derived epitopes by either epitope extraction or epitope excision. Upon formation of the antigen peptide-containing immune complex in solution, the entire mixture is electrosprayed to translate all constituents as protonated ions into the gas phase. There, ions from antibody-peptide complexes are separated from unbound peptide ions according to their masses, charges, and shapes either by ion mobility drift or by quadrupole ion filtering. Subsequently, immune complexes are dissociated by collision induced fragmentation and the ion signals of the "complex-released peptides," which in effect are the epitope peptides, are recorded in the time-of-flight analyzer of the mass spectrometer. Mixing of an antibody solution with a solution in which antigens or antigen-derived peptides are dissolved is, together with antigen proteolysis, the only required in-solution handling step. Simplicity of sample handling and speed of analysis together with very low sample consumption makes ITEM faster and easier to perform than other experimental epitope mapping methods.

  17. 46 CFR 170.285 - Free surface correction for intact stability calculations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Free surface correction for intact stability...) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Free Surface § 170.285 Free surface correction for intact stability calculations. (a) When doing the intact stability calculations required by...

  18. 46 CFR 170.285 - Free surface correction for intact stability calculations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Free surface correction for intact stability...) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Free Surface § 170.285 Free surface correction for intact stability calculations. (a) When doing the intact stability calculations required by...

  19. 46 CFR 170.285 - Free surface correction for intact stability calculations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Free surface correction for intact stability...) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Free Surface § 170.285 Free surface correction for intact stability calculations. (a) When doing the intact stability calculations required by...

  20. Small Particles Intact Capture Experiment (SPICE)

    NASA Technical Reports Server (NTRS)

    Nishioka, Ken-Ji; Carle, G. C.; Bunch, T. E.; Mendez, David J.; Ryder, J. T.

    1994-01-01

    The Small Particles Intact Capture Experiment (SPICE) will develop technologies and engineering techniques necessary to capture nearly intact, uncontaminated cosmic and interplanetary dust particles (IDP's). Successful capture of such particles will benefit the exobiology and planetary science communities by providing particulate samples that may have survived unaltered since the formation of the solar system. Characterization of these particles may contribute fundamental data to our knowledge of how these particles could have formed into our planet Earth and, perhaps, contributed to the beginnings of life. The term 'uncontaminated' means that captured cosmic and IDP particles are free of organic contamination from the capture process and the term 'nearly intact capture' means that their chemical and elemental components are not materially altered during capture. The key to capturing cosmic and IDP particles that are organic-contamination free and nearly intact is the capture medium. Initial screening of capture media included organic foams, multiple thin foil layers, and aerogel (a silica gel); but, with the exception of aerogel, the requirements of no contamination or nearly intact capture were not met. To ensure no contamination of particles in the capture process, high-purity aerogel was chosen. High-purity aerogel results in high clarity (visual clearness), a useful quality in detection and recovery of embedded captured particles from the aerogel. P. Tsou at the Jet Propulsion Laboratory (JPL) originally described the use of aerogel for this purpose and reported laboratory test results. He has flown aerogel as a 'GAS-can Lid' payload on STS-47 and is evaluating the results. The Timeband Capture Cell Experiment (TICCE), a Eureca 1 experiment, is also flying aerogel and is scheduled for recovery in late April.

  1. Small Particles Intact Capture Experiment (SPICE)

    NASA Technical Reports Server (NTRS)

    Nishioka, Ken-Ji; Carle, G. C.; Bunch, T. E.; Mendez, David J.; Ryder, J. T.

    1994-01-01

    The Small Particles Intact Capture Experiment (SPICE) will develop technologies and engineering techniques necessary to capture nearly intact, uncontaminated cosmic and interplanetary dust particles (IDP's). Successful capture of such particles will benefit the exobiology and planetary science communities by providing particulate samples that may have survived unaltered since the formation of the solar system. Characterization of these particles may contribute fundamental data to our knowledge of how these particles could have formed into our planet Earth and, perhaps, contributed to the beginnings of life. The term 'uncontaminated' means that captured cosmic and IDP particles are free of organic contamination from the capture process and the term 'nearly intact capture' means that their chemical and elemental components are not materially altered during capture. The key to capturing cosmic and IDP particles that are organic-contamination free and nearly intact is the capture medium. Initial screening of capture media included organic foams, multiple thin foil layers, and aerogel (a silica gel); but, with the exception of aerogel, the requirements of no contamination or nearly intact capture were not met. To ensure no contamination of particles in the capture process, high-purity aerogel was chosen. High-purity aerogel results in high clarity (visual clearness), a useful quality in detection and recovery of embedded captured particles from the aerogel. P. Tsou at the Jet Propulsion Laboratory (JPL) originally described the use of aerogel for this purpose and reported laboratory test results. He has flown aerogel as a 'GAS-can Lid' payload on STS-47 and is evaluating the results. The Timeband Capture Cell Experiment (TICCE), a Eureca 1 experiment, is also flying aerogel and is scheduled for recovery in late April.

  2. Intact capture of hypervelocity particles

    NASA Technical Reports Server (NTRS)

    Tsou, P.; Brownlee, D. E.; Albee, A. L.

    1986-01-01

    Knowledge of the phase, structure, and crystallography of cosmic particles, as well as their elemental and isotopic compositions, would be very valuable information toward understanding the nature of our solar system. This information can be obtained from the intact capture of large mineral grains of cosmic particles from hypervelocity impacts. Hypervelocity experiments of intact capture in underdense media have indicated realistic potential in this endeaver. The recovery of the thermal blankets and louvers from the Solar Max spacecraft have independently verified this potential in the unintended capture of cosmic materials from hypervelocity impacts. Passive underdense media will permit relatively simple and inexpensive missions to capture cosmic particles intact, either by going to a planetary body or by waiting for the particles to come to the Shuttle or the Space Station. Experiments to explore the potential of using various underdense media for an intact comet sample capture up to 6.7 km/s were performed at NASA Ames Research Center Vertical Gun Range. Explorative hypervelocity experiments up to 7.9 km/s were also made at the Ernst Mach Institute. These experiments have proven that capturing intact particles at hypervelocity impacts is definitely possible. Further research is being conducted to achieve higher capture ratios at even higher hypervelocities for even smaller projectiles.

  3. Intact capture of hypervelocity particles

    NASA Astrophysics Data System (ADS)

    Tsou, P.; Brownlee, D. E.; Albee, A. L.

    Knowledge of the phase, structure, and crystallography of cosmic particles, as well as their elemental and isotopic compositions, would be very valuable information toward understanding the nature of our solar system. This information can be obtained from the intact capture of large mineral grains of cosmic particles from hypervelocity impacts. Hypervelocity experiments of intact capture in underdense media have indicated realistic potential in this endeaver. The recovery of the thermal blankets and louvers from the Solar Max spacecraft have independently verified this potential in the unintended capture of cosmic materials from hypervelocity impacts. Passive underdense media will permit relatively simple and inexpensive missions to capture cosmic particles intact, either by going to a planetary body or by waiting for the particles to come to the Shuttle or the Space Station. Experiments to explore the potential of using various underdense media for an intact comet sample capture up to 6.7 km/s were performed at NASA Ames Research Center Vertical Gun Range. Explorative hypervelocity experiments up to 7.9 km/s were also made at the Ernst Mach Institute. These experiments have proven that capturing intact particles at hypervelocity impacts is definitely possible. Further research is being conducted to achieve higher capture ratios at even higher hypervelocities for even smaller projectiles.

  4. Acoustic emissions during deformation of intact and jointed welded tuff

    NASA Astrophysics Data System (ADS)

    Holcomb, D. J.; Teufel, L. W.

    1982-07-01

    Monitoring of acoustic emissions (AE) has been widely used as a means of detecting failure in intact rock. For intact rock the technique is simple, because an increasing rate of AE is usually a sign of impending failure. However, most large rock masses contain numerous joints and the behavior of the joints controls the properties of the rock mass In particular, the failure mode often becomes stable or unstable slip (stick-slip) on a joint at stresses well below those required for failure of the intact rock. As an aid to understanding and monitoring the behavior of jointed rock masses, we have done a series of experiments on intact and artificially jointed samples of Grouse Canyon tuff. The tuff was selected because it is under consideration as a disposal medium for nuclear wastes. The samples were instrumented to measure axial and transverse displacements and AE rates.

  5. Preparation of Cardiac Extracellular Matrix from an Intact Porcine Heart

    PubMed Central

    Wainwright, John M.; Czajka, Caitlin A.; Patel, Urvi B.; Freytes, Donald O.; Tobita, Kimimasa; Gilbert, Thomas W.

    2010-01-01

    Whole organ engineering would benefit from a three-dimensional scaffold produced from intact organ-specific extracellular matrix (ECM). The microenvironment and architecture provided by such a scaffold would likely support site-appropriate cell differentiation and spatial organization. The methods to produce such scaffolds from intact organs require customized decellularization protocols. In the present study, intact adult porcine hearts were successfully decellularized in less than 10 h using pulsatile retrograde aortic perfusion. Serial perfusion of an enzymatic, nonionic detergent, ionic detergent, and acid solution with hypotonic and hypertonic rinses was used to systematically remove cellular content. The resultant cardiac ECM retained collagen, elastin, and glycosaminoglycans, and mechanical integrity. Cardiac ECM supported the formation of organized chicken cardiomyocyte sarcomere structure in vitro. The intact decellularized porcine heart provides a tissue engineering template that may be beneficial for future preclinical studies and eventual clinical applications. PMID:19702513

  6. Preparation of cardiac extracellular matrix from an intact porcine heart.

    PubMed

    Wainwright, John M; Czajka, Caitlin A; Patel, Urvi B; Freytes, Donald O; Tobita, Kimimasa; Gilbert, Thomas W; Badylak, Stephen F

    2010-06-01

    Whole organ engineering would benefit from a three-dimensional scaffold produced from intact organ-specific extracellular matrix (ECM). The microenvironment and architecture provided by such a scaffold would likely support site-appropriate cell differentiation and spatial organization. The methods to produce such scaffolds from intact organs require customized decellularization protocols. In the present study, intact adult porcine hearts were successfully decellularized in less than 10 h using pulsatile retrograde aortic perfusion. Serial perfusion of an enzymatic, nonionic detergent, ionic detergent, and acid solution with hypotonic and hypertonic rinses was used to systematically remove cellular content. The resultant cardiac ECM retained collagen, elastin, and glycosaminoglycans, and mechanical integrity. Cardiac ECM supported the formation of organized chicken cardiomyocyte sarcomere structure in vitro. The intact decellularized porcine heart provides a tissue engineering template that may be beneficial for future preclinical studies and eventual clinical applications.

  7. An intact RNA interference pathway is required for expression of the mutant wing phenotype of vg(21-3), a P-element-induced allele of the vestigial gene in Drosophila.

    PubMed

    Hodgetts, Ross B; O'Keefe, Sandra L; Anderson, Kyle J

    2012-04-01

    We have determined that two P elements, P[21-3] and P[21r36], residing in the 5'-UTR of the vestigial wing gene, encode functional repressors in eye tissue. However, neither element fits a previous categorization of repressor-making elements as Type I or II. Both elements encode polypeptides that are shorter than the canonical elements they most closely resemble. DNA sequencing reveals that P[21r36] encodes an intact THAP domain that is missing in the P[21] element, which does not encode a functional repressor. Recovery of P[21-3] at sites other than vestigial (where it causes the wing mutant, vg(21-3)) reveals that the element can make repressor in wing tissue of sufficient activity to repress the mutant phenotype of vg(21-3). Why the P[21-3] element fails to produce repressor when located at vestigial may be explained by our observation that three different mutants in the RNA interference pathway cause a partial reversion of vg(21-3). We speculate that the vg and P-initiated transcripts that arise at the vg locus in the vg(21-3) mutant trigger an RNA interference response that results in the mutual degradation of both transcripts.

  8. Stimulation of host centriolar antigen in TC7 cells by simian virus 40: requirement for RNA and protein syntheses and an intact simian virus 40 small-t gene function.

    PubMed

    Shyamala, M; Atcheson, C L; Kasamatsu, H

    1982-08-01

    Simian virus 40 (SV 40) stimulated a host cell antigen in the centriolar region after infection of African green monkey kidney (AGMK) cells. The addition of puromycin and actinomycin D to cells infected with SV40 within 5 h after infection inhibited the stimulation of the host cell antigen, indicating that de novo protein and RNA syntheses that occurred within the first 5 h after infection were essential for the stimulation. Early viable deletion mutants of SV40 with deletions mapping between 0.54 and 0.59 map units on the SV40 genome, dl2000, dl2001, dl2003, dl2004, dl2005, dl2006, and dl2007, did not stimulate the centriolar antigen above the level of uninfected cells. This indicated that an intact, functional small-t protein was essential for the SV40-mediated stimulation of the host cell antigen. Our studies, using cells infected with nondefective adenovirus-SV40 hybrid viruses that lack the small-t gene region of SV40 (Ad2+ND1, Ad2+ND2, Ad2+ND3, Ad2+ND4, and Ad2+ND5), revealed that the lack of small-t gene function of SV40 could be complemented by a gene function of the adenovirus-SV40 hybrid viruses for the centriolar antigen stimulation. Thus, adenovirus 2 has a gene(s) that is analogous to the small-t gene of SV40 for the stimulation of the host cell antigen in AGMK cells.

  9. Intact capture of hypervelocity projectiles

    NASA Technical Reports Server (NTRS)

    Tsou, P.

    1990-01-01

    The ability to capture projectiles intact at hypervelocities opens new applications in science and technology that would either not be possible or would be very costly by other means. This capability has been demonstrated in the laboratory for aluminum projectiles of 1.6 mm diameter, captured at 6 km/s, in one unmelted piece, and retaining up to 95% of the original mass. Furthermore, capture was accomplished passively using microcellular underdense polymer foam. Another advantage of capturing projectiles in an underdense medium is the ability of such a medium to preserve a record of the projectile's original velocity components of speed and direction. A survey of these experimental results is described in terms of a dozen parameters which characterize the amount of capture and the effect on the projectile due to different capture media.

  10. Intact capture of hypervelocity projectiles

    NASA Technical Reports Server (NTRS)

    Tsou, P.

    1990-01-01

    The ability to capture projectiles intact at hypervelocities opens new applications in science and technology that would either not be possible or would be very costly by other means. This capability has been demonstrated in the laboratory for aluminum projectiles of 1.6 mm diameter, captured at 6 km/s, in one unmelted piece, and retaining up to 95% of the original mass. Furthermore, capture was accomplished passively using microcellular underdense polymer foam. Another advantage of capturing projectiles in an underdense medium is the ability of such a medium to preserve a record of the projectile's original velocity components of speed and direction. A survey of these experimental results is described in terms of a dozen parameters which characterize the amount of capture and the effect on the projectile due to different capture media.

  11. Intact capture of hypervelocity projectiles.

    PubMed

    Tsou, P

    1990-01-01

    The ability to capture projectiles intact at hypervelocities opens new applications in science and technology that would either not be possible or would be very costly by other means. This capability has been demonstrated in the laboratory for aluminum projectiles of 1.6 mm diameter, captured at 6 km/s, in one unmelted piece, and retaining up to 95% of the original mass. Furthermore, capture was accomplished passively using microcellular underdense polymer foam. Another advantage of capturing projectiles in an underdense medium is the ability of such a medium to preserve a record of the projectile's original velocity components of speed and direction. A survey of these experimental results is described in terms of a dozen parameters which characterize the amount of capture and the effect on the projectile due to different capture media.

  12. Stimulation of host centriolar antigen in TC7 cells by simian virus 40: requirement for RNA and protein syntheses and an intact simian virus 40 small-t gene function.

    PubMed Central

    Shyamala, M; Atcheson, C L; Kasamatsu, H

    1982-01-01

    Simian virus 40 (SV 40) stimulated a host cell antigen in the centriolar region after infection of African green monkey kidney (AGMK) cells. The addition of puromycin and actinomycin D to cells infected with SV40 within 5 h after infection inhibited the stimulation of the host cell antigen, indicating that de novo protein and RNA syntheses that occurred within the first 5 h after infection were essential for the stimulation. Early viable deletion mutants of SV40 with deletions mapping between 0.54 and 0.59 map units on the SV40 genome, dl2000, dl2001, dl2003, dl2004, dl2005, dl2006, and dl2007, did not stimulate the centriolar antigen above the level of uninfected cells. This indicated that an intact, functional small-t protein was essential for the SV40-mediated stimulation of the host cell antigen. Our studies, using cells infected with nondefective adenovirus-SV40 hybrid viruses that lack the small-t gene region of SV40 (Ad2+ND1, Ad2+ND2, Ad2+ND3, Ad2+ND4, and Ad2+ND5), revealed that the lack of small-t gene function of SV40 could be complemented by a gene function of the adenovirus-SV40 hybrid viruses for the centriolar antigen stimulation. Thus, adenovirus 2 has a gene(s) that is analogous to the small-t gene of SV40 for the stimulation of the host cell antigen in AGMK cells. Images PMID:6180184

  13. A Functional Genomic Screen for Evolutionarily Conserved Genes Required for Lifespan and Immunity in Germline-Deficient C. elegans

    PubMed Central

    Sinha, Amit; Rae, Robbie

    2014-01-01

    The reproductive system regulates lifespan in insects, nematodes and vertebrates. In Caenorhabditis elegans removal of germline increases lifespan by 60% which is dependent upon insulin signaling, nuclear hormone signaling, autophagy and fat metabolism and their microRNA-regulators. Germline-deficient C. elegans are also more resistant to various bacterial pathogens but the underlying molecular mechanisms are largely unknown. Firstly, we demonstrate that previously identified genes that regulate the extended lifespan of germline-deficient C. elegans (daf-2, daf-16, daf-12, tcer-1, mir-7.1 and nhr-80) are also essential for resistance to the pathogenic bacterium Xenorhabdus nematophila. We then use a novel unbiased approach combining laser cell ablation, whole genome microarrays, RNAi screening and exposure to X. nematophila to generate a comprehensive genome-wide catalog of genes potentially required for increased lifespan and innate immunity in germline-deficient C. elegans. We find 3,440 genes to be upregulated in C. elegans germline-deficient animals in a gonad dependent manner, which are significantly enriched for genes involved in insulin signaling, fatty acid desaturation, translation elongation and proteasome complex function. Using RNAi against a subset of 150 candidate genes selected from the microarray results, we show that the upregulated genes such as transcription factor DAF-16/FOXO, the PTEN homolog lipid phosphatase DAF-18 and several components of the proteasome complex (rpn-6.1, rpn-7, rpn-9, rpn-10, rpt-6, pbs-3 and pbs-6) are essential for both lifespan and immunity of germline deficient animals. We also identify a novel role for genes including par-5 and T12G3.6 in both lifespan-extension and increased survival on X. nematophila. From an evolutionary perspective, most of the genes differentially expressed in germline deficient C. elegans also show a conserved expression pattern in germline deficient Pristionchus pacificus, a nematode species

  14. Triton shells of intact erythrocytes.

    PubMed

    Sheetz, M P; Sawyer, D

    1978-01-01

    About 40% of human erythrocyte membrane protein is resistant to solubilization in 0.5% Triton X-114. These components comprise a structure called a Triton shell roughly similar in size and shape to the original erythrocyte and thus constitute a cytoskeleton. With increasing concentrations of Triton the lipid content of the Triton shell decreases dramatically, whereas the majority of the protein components remain constant. Exceptions to this rule include proteins contained in band 3, the presumed anion channel, and in band 4 which decrease with increasing Triton concentration. The Triton-insoluble complex includes spectrin (bands 1 and 2), actin (band 5), and bands 3' and 7. Component 3' has an apparent molecular weight of 88,000 daltons as does 3; but unlike 3, it is insensitive to protease treatment of the intact cell, has a low extinction coefficient at 280 nm, and is solubilized from the shells in alkaline water solutions. Component 7 also has a low extinction coefficient at 280 nm. Spectrin alone is solubilized from the Triton shells in isotonic media. The solubilized spectrin contains no bound Triton and coelectrophoreses with spectrin eluted in hypotonic solutions from ghosts. Electron micrographs of fixed Triton shells stained with uranyl acetate show the presence of numerous filaments which appear beaded and are 80--120 A in diameter. The filaments cannot be composed mainly af actin, but enough spectrin is present to form the filaments. Triton shells may provide an excellent source of material useful in the investigation of the erythrocyte cytoskeleton.

  15. 46 CFR 174.185 - Intact stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability. 174.185 Section 174.185 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Offshore Supply Vessels § 174.185 Intact stability. (a...

  16. 46 CFR 172.070 - Intact stability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Intact stability. 172.070 Section 172.070 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO... § 172.070 Intact stability. All tank vessels of 5,000 DWT and above contracted after the effective date...

  17. 46 CFR 172.070 - Intact stability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact stability. 172.070 Section 172.070 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO... § 172.070 Intact stability. All tank vessels of 5,000 deadweight tons (DWT) and above, contracted after...

  18. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a) Except...

  19. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a) Except...

  20. 46 CFR 172.070 - Intact stability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Intact stability. 172.070 Section 172.070 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO... § 172.070 Intact stability. All tank vessels of 5,000 deadweight tons (DWT) and above, contracted after...

  1. 46 CFR 172.070 - Intact stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability. 172.070 Section 172.070 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO... § 172.070 Intact stability. All tank vessels of 5,000 deadweight tons (DWT) and above, contracted after...

  2. 46 CFR 174.185 - Intact stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability. 174.185 Section 174.185 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Offshore Supply Vessels § 174.185 Intact stability. (a...

  3. 46 CFR 172.070 - Intact stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability. 172.070 Section 172.070 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO... § 172.070 Intact stability. All tank vessels of 5,000 deadweight tons (DWT) and above, contracted after...

  4. 46 CFR 28.545 - Intact stability when using lifting gear.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Intact stability when using lifting gear. 28.545 Section 28.545 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.545 Intact stability when using lifting gear. (a) Each...

  5. Structure-activity relationships at the 5-position of thiolactomycin: an intact (5R)-isoprene unit is required for activity against the condensing enzymes from Mycobacterium tuberculosis and Escherichia coli

    PubMed Central

    Kim, Pilho; Zhang, Yong-Mei; Shenoy, Gautham; Nguyen, Quynh-Anh; Boshoff, Helena I.; Manjunatha, Ujjini H.; Goodwin, Michael; Lonsdale, John; Price, Allen C.; Miller, Darcie J.; Duncan, Ken; White, Stephen W.; Rock, Charles O.; Barry, Clifton E.; Dowd, Cynthia S.

    2006-01-01

    Thiolactomycin inhibits bacterial cell growth through inhibition of the β-ketoacyl-ACP synthase activity of type II fatty acid synthases. The effect of modifications of the 5-position isoprenoid side chain on both IC50 and MIC were determined. Synthesis and screening of a structurally diverse set of 5-position analogues revealed very little tolerance for substitution in purified enzyme assays but a few analogues retained MIC, presumably through another target. Even subtle modifications such as reducing one or both double bonds of the diene were not tolerated. The only permissible structural modifications were removal of the isoprene methyl group or addition of a methyl group to the terminus. Co-crystallization of these two inhibitors with the condensing enzyme from E. coli revealed that they retained the TLM binding mode at the active site with reduced affinity. These results suggest a strict requirement for a conjugated, planar side chain inserting within the condensing enzyme active site. PMID:16392800

  6. An intact sequence-specific DNA-binding domain is required for human cytomegalovirus-mediated sequestration of p53 and may promote in vivo binding to the viral genome during infection

    SciTech Connect

    Rosenke, Kyle; Samuel, Melanie A.; McDowell, Eric T.; Toerne, Melissa A.; Fortunato, Elizabeth A. . E-mail: lfort@uidaho.edu

    2006-04-25

    The p53 protein is stabilized during infection of primary human fibroblasts with human cytomegalovirus (HCMV). However, the p53 in HCMV-infected cells is unable to activate its downstream targets. HCMV accomplishes this inactivation, at least in part, by sequestering p53 into viral replication centers within the cell's nucleus soon after they are established. In order to better understand the interplay between HCMV and p53 and the mechanism of sequestration, we constructed a panel of mutant p53-GFP fusion constructs for use in transfection/infection experiments. These mutants affected several post-translational modification sites and several sites within the central sequence-specific DNA-binding domain of the protein. Two categories of p53 sequestration were observed when the mutant constructs were transfected into primary fibroblasts and then infected at either high or low multiplicity. The first category, including all of the post-translational modification mutants, showed sequestration comparable to a wild-type (wt) control, while the second category, mutants affecting the DNA-binding core, were not specifically sequestered above control GFP levels. This suggested that the DNA-binding ability of the protein was required for sequestration. When the HCMV genome was analyzed for p53 consensus binding sites, 21 matches were found, which localized either to the promoters or the coding regions of viral proteins involved in DNA replication and processing as well as structural proteins. An analysis of in vivo binding to these identified sites via chromatin immunoprecipitation assays revealed differential binding to several of the sites over the course of infection.

  7. Signal Transducer and Activator of  Transcription (STAT)5 Activation by BCR/ABL Is Dependent on Intact Src Homology (SH)3 and SH2 Domains of BCR/ABL and Is Required for Leukemogenesis

    PubMed Central

    Nieborowska-Skorska, Malgorzata; Wasik, Mariusz A.; Slupianek, Artur; Salomoni, Paolo; Kitamura, Toshio; Calabretta, Bruno; Skorski, Tomasz

    1999-01-01

    Signal transducer and activator of transcription (STAT)5 is constitutively activated in BCR/ ABL-expressing cells, but the mechanisms and functional consequences of such activation are unknown. We show here that BCR/ABL induces phosphorylation and activation of STAT5 by a mechanism that requires the BCR/ABL Src homology (SH)2 domain and the proline-rich binding site of the SH3 domain. Upon expression in 32Dcl3 growth factor–dependent myeloid precursor cells, STAT5 activation–deficient BCR/ABL SH3+SH2 domain mutants functioned as tyrosine kinase and activated Ras, but failed to protect from apoptosis induced by withdrawal of interleukin 3 and/or serum and did not induce leukemia in severe combined immunodeficiency mice. In complementation assays, expression of a dominant-active STAT5B mutant (STAT5B-DAM), but not wild-type STAT5B (STAT5B-WT), in 32Dcl3 cells transfected with STAT5 activation–deficient BCR/ABL SH3+SH2 mutants restored protection from apoptosis, stimulated growth factor–independent cell cycle progression, and rescued the leukemogenic potential in mice. Moreover, expression of a dominant-negative STAT5B mutant (STAT5B-DNM) in 32Dcl3 cells transfected with wild-type BCR/ABL inhibited apoptosis resistance, growth factor–independent proliferation, and the leukemogenic potential of these cells. In retrovirally infected mouse bone marrow cells, expression of STAT5B-DNM inhibited BCR/ABL-dependent transformation. Moreover, STAT5B-DAM, but not STAT5B-WT, markedly enhanced the ability of STAT5 activation–defective BCR/ABL SH3+SH2 mutants to induce growth factor–independent colony formation of primary mouse bone marrow progenitor cells. However, STAT5B-DAM did not rescue the growth factor–independent colony formation of kinase-deficient K1172R BCR/ABL or the triple mutant Y177F+R522L+ Y793F BCR/ABL, both of which also fail to activate STAT5. Together, these data demonstrate that STAT5 activation by BCR/ABL is dependent on signaling from more

  8. FOXO/DAF-16 Activation Slows Down Turnover of the Majority of Proteins in C. elegans

    DOE PAGES

    Dhondt, Ineke; Petyuk, Vladislav A.; Cai, Huaihan; ...

    2016-09-13

    Most aging hypotheses assume the accumulation of damage, resulting in gradual physiological decline and, ultimately, death. Avoiding protein damage accumulation by enhanced turnover should slow down the aging process and extend the lifespan. But, lowering translational efficiency extends rather than shortens the lifespan in C. elegans. We studied turnover of individual proteins in the long-lived daf-2 mutant by combining SILeNCe (stable isotope labeling by nitrogen in Caenorhabditiselegans) and mass spectrometry. Intriguingly, the majority of proteins displayed prolonged half-lives in daf-2, whereas others remained unchanged, signifying that longevity is not supported by high protein turnover. We found that this slowdown wasmore » most prominent for translation-related and mitochondrial proteins. Conversely, the high turnover of lysosomal hydrolases and very low turnover of cytoskeletal proteins remained largely unchanged. The slowdown of protein dynamics and decreased abundance of the translational machinery may point to the importance of anabolic attenuation in lifespan extension, as suggested by the hyperfunction theory.« less

  9. FOXO/DAF-16 Activation Slows Down Turnover of the Majority of Proteins in C. elegans

    SciTech Connect

    Dhondt, Ineke; Petyuk, Vladislav A.; Cai, Huaihan; Vandemeulebroucke, Lieselot; Vierstraete, Andy; Smith, Richard D.; Depuydt, Geert; Braeckman, Bart P.

    2016-09-01

    Cellular protein quality can be maintained by proteolytic elimination of damaged proteins and replacing them with newly synthesized copies, a process called protein turnover (Ward, 2000). Protein turnover rates have been estimated using SILAC (stable isotope labeling by amino acids in cell culture) in prokaryotes and eukaryotes. The last decade has witnessed a growing interest in the analysis of whole-organism proteome dynamics in metazoans using the same approach (Claydon and Beynon, 2012). In recent work, SILAC was applied to monitor protein synthesis throughout life in adult Caenorhabditis elegans (Vukoti et al., 2015) and to investigate food intake (Gomez-Amaro et al., 2015

  10. Neuron-specific proteotoxicity of mutant ataxin-3 in C. elegans: rescue by the DAF-16 and HSF-1 pathways.

    PubMed

    Teixeira-Castro, Andreia; Ailion, Michael; Jalles, Ana; Brignull, Heather R; Vilaça, João L; Dias, Nuno; Rodrigues, Pedro; Oliveira, João F; Neves-Carvalho, Andreia; Morimoto, Richard I; Maciel, Patrícia

    2011-08-01

    The risk of developing neurodegenerative diseases increases with age. Although many of the molecular pathways regulating proteotoxic stress and longevity are well characterized, their contribution to disease susceptibility remains unclear. In this study, we describe a new Caenorhabditis elegans model of Machado-Joseph disease pathogenesis. Pan-neuronal expression of mutant ATXN3 leads to a polyQ-length dependent, neuron subtype-specific aggregation and neuronal dysfunction. Analysis of different neurons revealed a pattern of dorsal nerve cord and sensory neuron susceptibility to mutant ataxin-3 that was distinct from the aggregation and toxicity profiles of polyQ-alone proteins. This reveals that the sequences flanking the polyQ-stretch in ATXN3 have a dominant influence on cell-intrinsic neuronal factors that modulate polyQ-mediated pathogenesis. Aging influences the ATXN3 phenotypes which can be suppressed by the downregulation of the insulin/insulin growth factor-1-like signaling pathway and activation of heat shock factor-1.

  11. FOXO/DAF-16 Activation Slows Down Turnover of the Majority of Proteins in C. elegans

    SciTech Connect

    Dhondt, Ineke; Petyuk, Vladislav A.; Cai, Huaihan; Vandemeulebroucke, Lieselot; Vierstraete, Andy; Smith, Richard D.; Depuydt, Geert; Braeckman, Bart  P.

    2016-09-13

    Most aging hypotheses assume the accumulation of damage, resulting in gradual physiological decline and, ultimately, death. Avoiding protein damage accumulation by enhanced turnover should slow down the aging process and extend the lifespan. But, lowering translational efficiency extends rather than shortens the lifespan in C. elegans. We studied turnover of individual proteins in the long-lived daf-2 mutant by combining SILeNCe (stable isotope labeling by nitrogen in Caenorhabditiselegans) and mass spectrometry. Intriguingly, the majority of proteins displayed prolonged half-lives in daf-2, whereas others remained unchanged, signifying that longevity is not supported by high protein turnover. We found that this slowdown was most prominent for translation-related and mitochondrial proteins. Conversely, the high turnover of lysosomal hydrolases and very low turnover of cytoskeletal proteins remained largely unchanged. The slowdown of protein dynamics and decreased abundance of the translational machinery may point to the importance of anabolic attenuation in lifespan extension, as suggested by the hyperfunction theory.

  12. [Pulmonary atresia with an intact interventricular septum].

    PubMed

    Godart, F; Fall, A L; Francart, C; Brevière, G M; Cajot, M A; Rey, C

    2005-05-01

    We report this centre's experience of the treatment and follow up of pulmonary atresia with an intact septum. 35 infants were seen during the neonatal period. Opening via catheterisation was attempted in 21 patients with initial success in 10, and 11 failures leading to urgent surgery (surgical opening, n=9) and isolated Blalock-Taussig anastomosis (n=2). Fourteen other patients underwent immediate surgery: surgical opening, n=3, and Blalock alone, n=11. Four patients died in the neonatal period: 1 after successful opening via catheterisation, 3 others after Blalock anastomosis. Five others with a Blalock anastomosis died suddenly later. By the end of follow up, 16 patients had undergone biventricular type repair, of which 7 required additional procedures. 10 others had undergone cavo-pulmonary type repair, including 4 infants in whom the initial strategy of biventricular repair had failed. The only predictive factors at birth for subsequent progression to biventricular type repair were: larger tricuspid diameter at echography (10.9 +/- 2.25 mm versus 6.34 +/- 1.74 mm, p = 0.0007) or at angiography (10.07 +/- 2.09 mm versus 8.04 +/- 2.42 mm, p = 0.039), and the right ventricular morphology (p = 0.0011) with more tripartite ventricles, and less bipartite or even unipartite ones in the biventricular group.

  13. Uranium migration through intact sandstone cores

    NASA Astrophysics Data System (ADS)

    Read, D.; Lawless, T. A.; Sims, R. J.; Butter, K. R.

    1993-06-01

    Uranium is often considered to be a mobile radioelement in the natural environment owing to its tendency to form stable complexes with a number of aqueous anions, particularly in oxidising milieu. A series of infiltration experiments were devised to investigate this migration behaviour under rigidly controlled laboratory conditions. Intact cores of Permo-Triassic Clashach Sandstone were pre-equilibrated with synthetic groundwater solutions and continuous flow-through of uranium monitored together with pH and concentrations of other ions. Prior to performing each experiment a simulation was carried out using a one-dimensional coupled chemical transport code, encompassing a thermodynamic description of the electrical double layer. These calculations together with electron microscopy indicated the potential role played by iron oxyhydroxide grain coatings in retarding the uranium plume. Thus, a second series of experiments was initiated on pre-acidified cores from which all surface exposed iron had been removed, allowing an assessment of the retention capacity of non-ferric components. Taken together, the data clearly illustrate the strong affinity of aqueous uranium species for natural surfaces even under strongly oxidising conditions. The success of the model in predicting a priori the dominant trends in uranium migration behaviour is encouraging and may aid in prioritising analytical requirements for investigations in more complex geochemical situations than those studied here.

  14. Learning fast accurate movements requires intact frontostriatal circuits

    PubMed Central

    Shabbott, Britne; Ravindran, Roshni; Schumacher, Joseph W.; Wasserman, Paula B.; Marder, Karen S.; Mazzoni, Pietro

    2013-01-01

    The basal ganglia are known to play a crucial role in movement execution, but their importance for motor skill learning remains unclear. Obstacles to our understanding include the lack of a universally accepted definition of motor skill learning (definition confound), and difficulties in distinguishing learning deficits from execution impairments (performance confound). We studied how healthy subjects and subjects with a basal ganglia disorder learn fast accurate reaching movements. We addressed the definition and performance confounds by: (1) focusing on an operationally defined core element of motor skill learning (speed-accuracy learning), and (2) using normal variation in initial performance to separate movement execution impairment from motor learning abnormalities. We measured motor skill learning as performance improvement in a reaching task with a speed-accuracy trade-off. We compared the performance of subjects with Huntington's disease (HD), a neurodegenerative basal ganglia disorder, to that of premanifest carriers of the HD mutation and of control subjects. The initial movements of HD subjects were less skilled (slower and/or less accurate) than those of control subjects. To factor out these differences in initial execution, we modeled the relationship between learning and baseline performance in control subjects. Subjects with HD exhibited a clear learning impairment that was not explained by differences in initial performance. These results support a role for the basal ganglia in both movement execution and motor skill learning. PMID:24312037

  15. 46 CFR 178.320 - Intact stability requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... stability proof test in accordance with § 178.330 of this part in the presence of a Coast Guard marine inspector. (b) A pontoon vessel operating on protected waters must undergo a simplified stability proof test... cognizant OCMI may dispense with the simplified stability proof test in § 178.330 for a vessel carrying...

  16. 46 CFR 178.310 - Intact stability requirements-general.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... approved by the Commanding Officer, Marine Safety Center. ... 178.310 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS... stability requirements—general. (a) Except as provided in paragraph (c) of this section, each vessel...

  17. 46 CFR 174.145 - Intact stability requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... righting arm curve must be at least 16.9 foot-degrees (5.15 meter-degrees) up to the smallest of the following angles: (1) The angle of maximum righting arm. (2) The downflooding angle. (3) 40 degrees. (c) The area under each righting arm curve must be at least 5.6 foot-degrees (1.72 meter-degrees) between the...

  18. 46 CFR 174.145 - Intact stability requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... righting arm curve must be at least 16.9 foot-degrees (5.15 meter-degrees) up to the smallest of the following angles: (1) The angle of maximum righting arm. (2) The downflooding angle. (3) 40 degrees. (c) The area under each righting arm curve must be at least 5.6 foot-degrees (1.72 meter-degrees) between the...

  19. 46 CFR 174.145 - Intact stability requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... righting arm curve must be at least 16.9 foot-degrees (5.15 meter-degrees) up to the smallest of the following angles: (1) The angle of maximum righting arm. (2) The downflooding angle. (3) 40 degrees. (c) The area under each righting arm curve must be at least 5.6 foot-degrees (1.72 meter-degrees) between the...

  20. 46 CFR 174.145 - Intact stability requirements.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... righting arm curve must be at least 16.9 foot-degrees (5.15 meter-degrees) up to the smallest of the following angles: (1) The angle of maximum righting arm. (2) The downflooding angle. (3) 40 degrees. (c) The area under each righting arm curve must be at least 5.6 foot-degrees (1.72 meter-degrees) between the...

  1. On the sensitivity of intact cells to perturbation by ethanol

    SciTech Connect

    Hitzemann, R.; Whitaker-Azmitia, P. ); Dains, K.; Lin, J. )

    1989-01-01

    A comparison was made of ethanol's effects on the order of plasma membranes in intact cells and some isolated membrane preparations. Order was assessed by steady-state fluorescence polarization techniques using the non-permeant probe, TMA-DPH. The data show that two cultured cells, rat neonatal astroglial and N2A neuroblastoma, were sensitive to significant ethanol-induced disordering within the anesthetically relevant range. Human erythrocytes, cultured fibroblasts and homogenized astroglial cells required higher ethanol concentrations to produce a similar effect. Intact erythrocytes were approximately twice as sensitive as erythrocyte ghost membranes to ethanol induced perturbation. The neonatal glial and N2A cells were approximately five times more sensitive than synaptic membranes to ethanol effects. DMPC and DMPC + cholesterol liposomes and myelin membranes were insensitive to ethanol's effects. The incorporation of 10 mole % ganglioside GM{sub 1} sensitized the liposomes to ethanol-induced perturbation.

  2. A transcription elongation factor that links signals from the reproductive system to lifespan extension in Caenorhabditis elegans.

    PubMed

    Ghazi, Arjumand; Henis-Korenblit, Sivan; Kenyon, Cynthia

    2009-09-01

    In Caenorhabditis elegans and Drosophila melanogaster, the aging of the soma is influenced by the germline. When germline-stem cells are removed, aging slows and lifespan is increased. The mechanism by which somatic tissues respond to loss of the germline is not well-understood. Surprisingly, we have found that a predicted transcription elongation factor, TCER-1, plays a key role in this process. TCER-1 is required for loss of the germ cells to increase C. elegans' lifespan, and it acts as a regulatory switch in the pathway. When the germ cells are removed, the levels of TCER-1 rise in somatic tissues. This increase is sufficient to trigger key downstream events, as overexpression of tcer-1 extends the lifespan of normal animals that have an intact reproductive system. Our findings suggest that TCER-1 extends lifespan by promoting the expression of a set of genes regulated by the conserved, life-extending transcription factor DAF-16/FOXO. Interestingly, TCER-1 is not required for DAF-16/FOXO to extend lifespan in animals with reduced insulin/IGF-1 signaling. Thus, TCER-1 specifically links the activity of a broadly deployed transcription factor, DAF-16/FOXO, to longevity signals from reproductive tissues.

  3. Calorie restriction and susceptibility to intact pathogens

    PubMed Central

    2008-01-01

    Long-term calorie restriction (CR) causes numerous physiological changes that ultimately increase mean and maximum lifespan of most species examined to date. One physiological change that occurs with CR is enhanced immune function, as tested using antigens and mitogens to stimulate an immune response. Fewer studies have used intact pathogen exposure to test whether the enhanced capacity of the immune response during CR actually decreases susceptibility of hosts to their pathogens. So far, studies using intact bacteria, virus, and helminth worm exposure indicate that, despite similar or enhanced immune system function, CR hosts are more susceptible to infection by intact pathogens than their fully fed counterparts. Long-term CR studies that examine susceptibility to a variety of parasite taxa will help determine if direct CR or CR mimetics will be beneficial to people living in pathogen-rich environments. PMID:19424864

  4. HYDROCARBON VAPOR DIFFUSION IN INTACT CORE SLEEVES

    EPA Science Inventory

    The diffusion of 2,2,4-trimethylpentane (TMP) and 2,2,5-trimethylhexane (TMH) vapors put of residually contaminated sandy soil from the U.S. Environmental Protection Agency (EPA) field research site at Traverse City, Michigan, was measured and modeled. The headspace of an intact ...

  5. HYDROCARBON VAPOR DIFFUSION IN INTACT CORE SLEEVES

    EPA Science Inventory

    The diffusion of 2,2,4-trimethylpentane (TMP) and 2,2,5-trimethylhexane (TMH) vapors put of residually contaminated sandy soil from the U.S. Environmental Protection Agency (EPA) field research site at Traverse City, Michigan, was measured and modeled. The headspace of an intact ...

  6. Acoustic emissions during deformation of intact and jointed welded tuff

    SciTech Connect

    Holcomb, D.J.; Teufel, L.W.

    1982-07-01

    Monitoring of acoustic emissions (AE) has been widely used as a means of detecting failure in intact rock. For intact rock the technique is simple, because an increasing rate of AE is usually a sign of impending failure. However, most large rock masses contain numerous joints and the behavior of the joints controls the properties of the rock mass. In particular, the failure mode often becomes stable or unstable slip (stick-slip) on a joint at stresses well below those required for failure of the intact rock. As an aid to understanding and monitoring the behavior of jointed rock masses, we have done a series of experiments on intact and artificially jointed samples of Grouse Canyon tuff. The tuff was selected because it is under consideration as a disposal medium for nuclear wastes. The samples were instrumented to measure axial and transverse displacements and AE rates. Testing was done in a servo-controlled machine at axial displacement rates of 5 x 10{sup -5} cm/sec, and confining pressures ranging from 10 to 40 MPa. For the jointed samples four modes of slip were identified. First, stable sliding accompanied by a steady rate of AE. Second, stick-slip with a sharp drop in load, large displacements but no premonitory AE or slip. Third, stick-slip, as in mode 2, but with premonitory AE and slip. Fourth, slow stick-slip where the load dropped and the displacements increased but the process was slow and culminated in stable sliding. Mode 4 exhibited premonitory AE and slip and after the event, a steady rate of AE during sliding. There seemed to be no way to predict which mode would occur at a given point in the test. In all cases where stable or unstable slip occurred there was a corresponding occurrence of AE. This indicates that slip is related to damage to the joint surfaces and adjacent material. Monitoring AE would be a useful method of detecting slip.

  7. Vesta Is Not an Intact Protoplanet

    NASA Astrophysics Data System (ADS)

    Consolmagno, Guy; Turrini, Diego; Golabeck, Gregor; Jutzi, Martin; Sirono, Sin-iti; Svetsov, Vladimir; Tsiganis, Kleomenis

    2014-11-01

    Asteroid 4 Vesta has been identified as the likely source of howardite, eucrite, and diogenite (HED) basaltic achondrite meteorites, whose parent body differentiated and started solidifying within 3 Ma after the condensation of the Ca-Al-rich inclusions (CAIs). The formation of Jupiter and the disk-driven migration of the giant planets also occurred during this period; thus it was expected that Vesta could provide an intact record of large-scale early episodes of planetary migration and bombardment as in the proposed Jovian Early Bombardment and the “Grand Tack” scenarios. However, the results of the Dawn mission detailing Vesta’s mass, volume, density, and surface characteristics provide challenges for modeling the structure and evolution of this asteroid. All proposed models for the generation of the HEDs require the presence of a substantial olivine-rich mantle. But recent work on the depth of excavation of the large basins at the south pole of Vesta suggests that because there is not abundant mantle olivine visible on Vesta or in the Vestoid family asteroids, the crust of Vesta must be at least 80 km thick. Such a thick crust is radically at odds with previous models; should it exist, it ought to manifest itself in other ways such as Vesta’s density structure and bulk chemical composition. However, we find that no Vesta model of iron core, olivine-rich mantle, and HED crust can match the joint constraints of (a) Vesta’s density as derived from the gravity field observed by Dawn; (b) the observed depletion of sodium and potassium and trace element enrichments of the HED meteorites; and (c) the absence of exposed olivine on Vesta’s surface, among Vestoid asteroids, or in our collection of basaltic meteorites. Either Vesta was subjected to a radical change in composition, presumably due to the intense collisional environment where and when it formed, or the asteroid we see today is in fact a reaccretion of material formed elsewhere from now

  8. Placenta Accreta in a Woman with Escherichia coli Chorioamnionitis with Intact Membranes

    PubMed Central

    Montelongo, Emma M.; Blue, Nathan R.; Lee, Richard H.

    2015-01-01

    Background. Escherichia coli (E. coli) associated intrauterine infections with intact membranes are extremely rare. Case. A 30-year-old multiparous female presented at 26 weeks' gestation with clinical signs of chorioamnionitis but physical examination suggested intact membranes. Her dietary history was concerned with Listeriosis. An amniocentesis was performed. Shortly thereafter, the mother developed septic shock and an urgent Cesarean delivery was performed. The patient required a peripartum hysterectomy for placenta accreta. Amniotic fluid cultures grew E. coli. PMID:26819787

  9. Xenopus egg cytoplasm with intact actin.

    PubMed

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.

  10. Silica Aerogel Captures Cosmic Dust Intact

    NASA Technical Reports Server (NTRS)

    Tsou, P.

    1994-01-01

    The mesostructure of silica aerogel resembles stings of grapes, ranging in size from 10 to 100 angstrom. This fine mesostructure transmits nearly 90 percent of incident light in the visible, while providing sufficiently gentle dissipation of the kinetric energy of hypervelocity cosmic dust particles to permit their intact capture. We introduced silica aerogel in 1987 as capture medium to take advantage of its low density, fine mesostruicture and most importantly, its transparency, allowing optical location of captured micron sized particles.

  11. Silica Aerogel Captures Cosmic Dust Intact

    NASA Technical Reports Server (NTRS)

    Tsou, P.

    1994-01-01

    The mesostructure of silica aerogel resembles stings of grapes, ranging in size from 10 to 100 angstrom. This fine mesostructure transmits nearly 90 percent of incident light in the visible, while providing sufficiently gentle dissipation of the kinetric energy of hypervelocity cosmic dust particles to permit their intact capture. We introduced silica aerogel in 1987 as capture medium to take advantage of its low density, fine mesostruicture and most importantly, its transparency, allowing optical location of captured micron sized particles.

  12. 50 CFR 622.493 - Landing Caribbean queen conch intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... conch intact. (a) A Caribbean queen conch in or from the Caribbean EEZ must be maintained with meat and shell intact. (b) The operator of a vessel that fishes in the EEZ is responsible for ensuring...

  13. 50 CFR 622.493 - Landing Caribbean queen conch intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... conch intact. (a) A Caribbean queen conch in or from the Caribbean EEZ must be maintained with meat and shell intact. (b) The operator of a vessel that fishes in the EEZ is responsible for ensuring...

  14. Feasible pickup from intact ossicular chain with floating piezoelectric microphone

    PubMed Central

    2012-01-01

    Objectives Many microphones have been developed to meet with the implantable requirement of totally implantable cochlear implant (TICI). However, a biocompatible one without destroying the intactness of the ossicular chain still remains under investigation. Such an implantable floating piezoelectric microphone (FPM) has been manufactured and shows an efficient electroacoustic performance in vitro test at our lab. We examined whether it pick up sensitively from the intact ossicular chain and postulated whether it be an optimal implantable one. Methods Animal controlled experiment: five adult cats (eight ears) were sacrificed as the model to test the electroacoustic performance of the FPM. Three groups were studied: (1) the experiment group (on malleus): the FPM glued onto the handle of the malleus of the intact ossicular chains; (2) negative control group (in vivo): the FPM only hung into the tympanic cavity; (3) positive control group (Hy-M30): a HiFi commercial microphone placed close to the site of the experiment ear. The testing speaker played pure tones orderly ranged from 0.25 to 8.0 kHz. The FPM inside the ear and the HiFi microphone simultaneously picked up acoustic vibration which recorded as .wav files to analyze. Results The FPM transformed acoustic vibration sensitively and flatly as did the in vitro test across the frequencies above 2.0 kHz, whereas inefficiently below 1.0 kHz for its overloading mass. Although the HiFi microphone presented more efficiently than the FPM did, there was no significant difference at 3.0 kHz and 8.0 kHz. Conclusions It is feasible to develop such an implantable FPM for future TICIs and TIHAs system on condition that the improvement of Micro Electromechanical System and piezoelectric ceramic material technology would be applied to reduce its weight and minimize its size. PMID:22353161

  15. How can we conserve intact tropical peatlands?

    NASA Astrophysics Data System (ADS)

    Lawson, Ian; Roucoux, Katherine

    2017-04-01

    The scientific community has, for more than three decades, been expressing increasing alarm about the fate of peatlands in parts of Indonesia and Malaysia, where extensive land-use conversion and drainage for rice and oil palm have greatly compromised peatland hydrology, ecology, biological richness, and carbon storage. The discourse in the literature on these peatlands is now moving on from attempts to preserve the last remaining fragments of peat-swamp forest, towards discussion of how best to restore damaged ecosystems, and whether it is possible to manage plantations more 'sustainably'. It is becoming increasingly clear, however, that peatlands occur quite widely in other parts of the lowland tropics, including parts of Amazonia and the Congo Basin, and many of these peatlands can reasonably be described as 'intact': although few if any parts of the tropics are totally unaffected by human actions, the hydrology and functional ecology of these systems appear to be close to a 'natural' state. The question then arises as to what should be done with the knowledge of their existence. Here we analyse the arguments in favour of protecting intact peatlands, and the potential conflicts with other priorities such as economic development and social justice. We evaluate alternative mechanisms for protecting intact peatlands, focusing on the particular issues raised by peatlands as opposed to other kinds of tropical ecosystem. We identify ways in which natural science agendas can help to inform these arguments, using our own contributions in palaeoecology and carbon mapping as examples. Finally, we argue for a radical reconsideration of research agendas in tropical peatlands, highlighting the potential contribution of methodologies borrowed from the social sciences and humanities.

  16. Calcium Signaling in Intact Dorsal Root Ganglia

    PubMed Central

    Gemes, Geza; Rigaud, Marcel; Koopmeiners, Andrew S.; Poroli, Mark J.; Zoga, Vasiliki; Hogan, Quinn H.

    2013-01-01

    Background Ca2+ is the dominant second messenger in primary sensory neurons. In addition, disrupted Ca2+ signaling is a prominent feature in pain models involving peripheral nerve injury. Standard cytoplasmic Ca2+ recording techniques use high K+ or field stimulation and dissociated neurons. To compare findings in intact dorsal root ganglia, we used a method of simultaneous electrophysiologic and microfluorimetric recording. Methods Dissociated neurons were loaded by bath-applied Fura-2-AM and subjected to field stimulation. Alternatively, we adapted a technique in which neuronal somata of intact ganglia were loaded with Fura-2 through an intracellular microelectrode that provided simultaneous membrane potential recording during activation by action potentials (APs) conducted from attached dorsal roots. Results Field stimulation at levels necessary to activate neurons generated bath pH changes through electrolysis and failed to predictably drive neurons with AP trains. In the intact ganglion technique, single APs produced measurable Ca2+ transients that were fourfold larger in presumed nociceptive C-type neurons than in nonnociceptive Aβ-type neurons. Unitary Ca2+ transients summated during AP trains, forming transients with amplitudes that were highly dependent on stimulation frequency. Each neuron was tuned to a preferred frequency at which transient amplitude was maximal. Transients predominantly exhibited monoexponential recovery and had sustained plateaus during recovery only with trains of more than 100 APs. Nerve injury decreased Ca2+ transients in C-type neurons, but increased transients in Aβ-type neurons. Conclusions Refined observation of Ca2+ signaling is possible through natural activation by conducted APs in undissociated sensory neurons and reveals features distinct to neuronal types and injury state. PMID:20526180

  17. Sodium Absorption by Intact Sugar Beet Plants

    PubMed Central

    El-Sheikh, Adel M; Ulrich, Albert

    1971-01-01

    Sodium absorption by intact sugar beet plants (Beta vulgaris) was found to be mediated by at least two distinct mechanisms when uptake was studied over a wide range of Na and K concentrations. The first mechanism operates at low Na concentrations (<1 milliequivalent per liter); presence of K completely blocks this mechanism for Na. The second mechanism operates at high Na concentrations (>1 milliequivalent per liter), transporting Na as well as K; but apparently this mechanism is not active for Na absorption in young sugar beet plants up to the 10-leaf stage. PMID:16657872

  18. Rapid isolation of intact chloroplasts from spinach leaves.

    PubMed

    Joly, David; Carpentier, Robert

    2011-01-01

    In this chapter, a rapid method to isolate intact chloroplasts from spinach leaves is described. Intact chloroplasts are isolated using two short centrifugation steps and avoiding the use of percoll gradient. Intactness of chloroplast is evaluated by the inability of potassium ferricyanide to enter inside the chloroplasts and to act as an electron acceptor for photosystem II.

  19. Neural activation differences in amputees during imitation of intact versus amputee movements

    PubMed Central

    Cusack, William F.; Cope, Michael; Nathanson, Sheryl; Pirouz, Nikta; Kistenberg, Robert; Wheaton, Lewis A.

    2012-01-01

    The mirror neuron system (MNS) has been attributed with increased activation in motor-related cortical areas upon viewing of another's actions. Recent work suggests that limb movements that are similar and dissimilar in appearance to that of the viewer equivalently activate the MNS. It is unclear if this result can be observed in the action encoding areas in amputees who use prosthetic devices. Intact subjects and upper extremity amputee prosthesis users were recruited to view video demonstrations of tools being used by an intact actor and a prosthetic device user. All subjects pantomimed the movements seen in the video while recording electroencephalography (EEG). Intact subjects showed equivalent left parietofrontal activity during imitation planning after watching the intact or prosthetic arm. Likewise, when prosthesis users imitated prosthesis demonstrations, typical left parietofrontal activation was observed. When prosthesis users imitated intact actors, an additional pattern was revealed which showed greater activity in right parietal and occipital regions that are associated with the mentalizing system. This change may be required for prosthesis users to plan imitation movements in which the limb states between the observed and the observer do not match. The finding that prosthesis users imitating other prosthesis users showed typical left parietofrontal activation suggests that these subjects engage normal planning related activity when they are able to imitate a limb matching their own. This result has significant implications on rehabilitation, as standard therapy involves training with an intact occupational therapist, which could necessitate atypical planning mechanisms in amputees when learning to use their prosthesis. PMID:22754516

  20. RNA aptamer delivery through intact human skin.

    PubMed

    Lenn, Jon D; Neil, Jessica; Donahue, Christine; Demock, Kellie; Tibbetts, Caitlin Vestal; Cote-Sierra, Javier; Smith, Susan H; Rubenstein, David; Therrien, Jean-Philippe; Pendergrast, P Shannon; Killough, Jason; Brown, Marc B; Williams, Adrian C

    2017-09-20

    It is generally recognised that only relatively small molecular weight (typically < ∼500 Da) drugs can effectively permeate through intact stratum corneum. Here, we challenge this orthodoxy using a 62-nucleotide (MW=20,395) RNA-based aptamer, highly specific to the human IL-23 cytokine, with picomolar activity. Results demonstrate penetration of the aptamer into freshly excised human skin using two different fluorescent labels. A dual hybridisation assay quantified aptamer from the epidermis and dermis giving levels far exceeding the cellular IC50 values (> 100,000-fold) and aptamer integrity was confirmed using an oligonucleotide precipitation assay. A Th17 response was stimulated in freshly excised human skin resulting in significantly upregulated IL-17f, and 22; topical application of the IL-23 aptamer decreased both IL-17f and IL-22 by approximately 45% but did not result in significant changes to IL-23 mRNA levels, confirming that the aptamer did not globally suppress mRNA levels. This study demonstrates that very large molecular weight RNA aptamers can permeate across the intact human skin barrier to therapeutically relevant levels into both the epidermis and dermis and that the skin penetrating aptamer retains its biologically active conformational structure capable of binding to endogenous IL-23. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  1. ISOLATION AND PROPERTIES OF INTACT MITOCHONDRIA FROM SPHEROPLASTS OF YEAST

    PubMed Central

    Duell, Elizabeth A.; Inoue, Sakae; Utter, Merton F.

    1964-01-01

    Duell, E. A. (Western Reserve University, Cleveland, Ohio), Sakae Inoue, and Merton F. Utter. Isolation and properties of intact mitochondria from spheroplasts of yeast. J. Bacteriol. 88:1762–1773. 1964.—Functionally intact mitochondria can be obtained in good yields by osmotic disruption of spheroplasts formed from yeast by treatment with an enzyme mixture from the snail digestive tract. The useful range of this method is extended greatly by pretreatment of the yeast cells with 2-mercaptoethylamine and ethylene-diaminetetraacetate. The concentration of the yeast suspension can be increased greatly, the incubation period can be shortened considerably, and the requirement for log-phase cells is obviated. Mitochondria prepared by this method have been compared with those obtained by mechanical disruption in terms of respiratory control, specific activity on a wide range of oxidizable substrates, heterogeneity during centrifugation, and structures observed by electron microscopy. In all cases, the mitochondria obtained from spheroplasts appear to be much less damaged by the preparative procedures. Images PMID:14240967

  2. Recollections of Parent Characteristics and Attachment Patterns for College Women of Intact vs. Non-Intact Families

    ERIC Educational Resources Information Center

    Kilmann, Peter R.; Carranza, Laura V.; Vendemia, Jennifer M. C.

    2006-01-01

    This study contrasted offsprings' attachment patterns and recollections of parent characteristics in two college samples: 147 females from intact biological parents and 157 females of parental divorce. Secure females from intact or non-intact families rated parents positively, while insecure females rated parents as absent, distant, and demanding.…

  3. Recollections of Parent Characteristics and Attachment Patterns for College Women of Intact vs. Non-Intact Families

    ERIC Educational Resources Information Center

    Kilmann, Peter R.; Carranza, Laura V.; Vendemia, Jennifer M. C.

    2006-01-01

    This study contrasted offsprings' attachment patterns and recollections of parent characteristics in two college samples: 147 females from intact biological parents and 157 females of parental divorce. Secure females from intact or non-intact families rated parents positively, while insecure females rated parents as absent, distant, and demanding.…

  4. Oligodeoxynucleotide Probes for Detecting Intact Cells

    NASA Technical Reports Server (NTRS)

    Rosson, Reinhardt A.; Maurina-Brunker, Julie; Langley, Kim; Pynnonen, Christine M.

    2004-01-01

    A rapid, sensitive test using chemiluminescent oligodeoxynucleotide probes has been developed for detecting, identifying, and enumerating intact cells. The test is intended especially for use in detecting and enumerating bacteria and yeasts in potable water. As in related tests that have been developed recently for similar purposes, the oligodeoxynucleotide probes used in this test are typically targeted at either singlecopy deoxyribonucleic acid (DNA) genes (such as virulence genes) or the multiple copies (10,000 to 50,000 copies per cell) of 16S ribosomal ribonucleic acids (rRNAs). Some of those tests involve radioisotope or fluorescent labeling of the probes for reporting hybridization of probes to target nucleic acids. Others of those tests involve labeling with enzymes plus the use of chemiluminescent or chromogenic substrates to report hybridization via color or the emission of light, respectively. The present test is of the last-mentioned type. The chemiluminescence in the present test can be detected easily with relatively simple instrumentation. In developing the present test, the hybridization approach was chosen because hybridization techniques are very specific. Hybridization detects stable, inheritable genetic targets within microorganisms. These targets are not dependent on products of gene expression that can vary with growth conditions or physiological states of organisms in test samples. Therefore, unique probes can be designed to detect and identify specific genera or species of bacteria or yeast (in terms of rRNA target sequences) or can be designed to detect and identify virulence genes (genomic target sequences). Because of the inherent specificity of this system, there are few problems of cross-reactivity. Hybridization tests are rapid, but hybridization tests now available commercially lack sensitivity; typically, between 10(exp 6) and 10(exp 7) cells of the target organism are needed to ensure a reliable test. Consequently, the numbers of

  5. The IntAct molecular interaction database in 2012

    PubMed Central

    Kerrien, Samuel; Aranda, Bruno; Breuza, Lionel; Bridge, Alan; Broackes-Carter, Fiona; Chen, Carol; Duesbury, Margaret; Dumousseau, Marine; Feuermann, Marc; Hinz, Ursula; Jandrasits, Christine; Jimenez, Rafael C.; Khadake, Jyoti; Mahadevan, Usha; Masson, Patrick; Pedruzzi, Ivo; Pfeiffenberger, Eric; Porras, Pablo; Raghunath, Arathi; Roechert, Bernd; Orchard, Sandra; Hermjakob, Henning

    2012-01-01

    IntAct is an open-source, open data molecular interaction database populated by data either curated from the literature or from direct data depositions. Two levels of curation are now available within the database, with both IMEx-level annotation and less detailed MIMIx-compatible entries currently supported. As from September 2011, IntAct contains approximately 275 000 curated binary interaction evidences from over 5000 publications. The IntAct website has been improved to enhance the search process and in particular the graphical display of the results. New data download formats are also available, which will facilitate the inclusion of IntAct's data in the Semantic Web. IntAct is an active contributor to the IMEx consortium (http://www.imexconsortium.org). IntAct source code and data are freely available at http://www.ebi.ac.uk/intact. PMID:22121220

  6. Mass Spectrometry of Intact Membrane Protein Complexes

    PubMed Central

    Laganowsky, Arthur; Reading, Eamonn; Hopper, Jonathan T.S.; Robinson, Carol V.

    2014-01-01

    Mass spectrometry of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here, we describe a protocol for mass spectrometry of membrane protein complexes. The protocol begins with preparation of the membrane protein complex enabling not only the direct assessment of stoichiometry, delipidation, and quality of the target complex, but also evaluation of the purification strategy. A detailed list of compatible non-ionic detergents is included, along with a protocol for screening detergents to find an optimal one for mass spectrometry, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a Q-ToF mass spectrometer after introduction of complexes from gold-coated nanoflow capillaries. PMID:23471109

  7. Protein methylation reactions in intact pea chloroplasts

    SciTech Connect

    Niemi, K.J. )

    1989-04-01

    Post-translational protein methylation was investigated in Pisum sativum chloroplasts. Intact pea chloroplasts were incubated with ({sup 3}H-methyl)-S-adenosylmethionine under various conditions. The chloroplasts were then separated into stromal and thylakoid fractions and analyzed for radioactivity transferred to protein. Light enhanced the magnitude of labeling in both fractions. One thylakoid polypeptide with an apparent molecular mass of 43 kDa was labeled only in the light. Several other thylakoid and stromal proteins were labeled in both light and dark-labeling conditions. Both base-labile methylation, carboxy-methylesters and base-stable groups, N-methylations were found. Further characterization of the methyl-transfer reactions will be presented.

  8. Drilling to gabbro in intact ocean crust.

    PubMed

    Wilson, Douglas S; Teagle, Damon A H; Alt, Jeffrey C; Banerjee, Neil R; Umino, Susumu; Miyashita, Sumio; Acton, Gary D; Anma, Ryo; Barr, Samantha R; Belghoul, Akram; Carlut, Julie; Christie, David M; Coggon, Rosalind M; Cooper, Kari M; Cordier, Carole; Crispini, Laura; Durand, Sedelia Rodriguez; Einaudi, Florence; Galli, Laura; Gao, Yongjun; Geldmacher, Jörg; Gilbert, Lisa A; Hayman, Nicholas W; Herrero-Bervera, Emilio; Hirano, Nobuo; Holter, Sara; Ingle, Stephanie; Jiang, Shijun; Kalberkamp, Ulrich; Kerneklian, Marcie; Koepke, Jürgen; Laverne, Christine; Vasquez, Haroldo L Lledo; Maclennan, John; Morgan, Sally; Neo, Natsuki; Nichols, Holly J; Park, Sung-Hyun; Reichow, Marc K; Sakuyama, Tetsuya; Sano, Takashi; Sandwell, Rachel; Scheibner, Birgit; Smith-Duque, Chris E; Swift, Stephen A; Tartarotti, Paola; Tikku, Anahita A; Tominaga, Masako; Veloso, Eugenio A; Yamasaki, Toru; Yamazaki, Shusaku; Ziegler, Christa

    2006-05-19

    Sampling an intact sequence of oceanic crust through lavas, dikes, and gabbros is necessary to advance the understanding of the formation and evolution of crust formed at mid-ocean ridges, but it has been an elusive goal of scientific ocean drilling for decades. Recent drilling in the eastern Pacific Ocean in Hole 1256D reached gabbro within seismic layer 2, 1157 meters into crust formed at a superfast spreading rate. The gabbros are the crystallized melt lenses that formed beneath a mid-ocean ridge. The depth at which gabbro was reached confirms predictions extrapolated from seismic experiments at modern mid-ocean ridges: Melt lenses occur at shallower depths at faster spreading rates. The gabbros intrude metamorphosed sheeted dikes and have compositions similar to the overlying lavas, precluding formation of the cumulate lower oceanic crust from melt lenses so far penetrated by Hole 1256D.

  9. Label-free volumetric optical imaging of intact murine brains

    NASA Astrophysics Data System (ADS)

    Ren, Jian; Choi, Heejin; Chung, Kwanghun; Bouma, Brett E.

    2017-04-01

    A central effort of today’s neuroscience is to study the brain’s ’wiring diagram’. The nervous system is believed to be a network of neurons interacting with each other through synaptic connection between axons and dendrites, therefore the neuronal connectivity map not only depicts the underlying anatomy, but also has important behavioral implications. Different approaches have been utilized to decipher neuronal circuits, including electron microscopy (EM) and light microscopy (LM). However, these approaches typically demand extensive sectioning and reconstruction for a brain sample. Recently, tissue clearing methods have enabled the investigation of a fully assembled biological system with greatly improved light penetration. Yet, most of these implementations, still require either genetic or exogenous contrast labeling for light microscopy. Here we demonstrate a high-speed approach, termed as Clearing Assisted Scattering Tomography (CAST), where intact brains can be imaged at optical resolution without labeling by leveraging tissue clearing and the scattering contrast of optical frequency domain imaging (OFDI).

  10. 46 CFR 170.285 - Free surface correction for intact stability calculations.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Free surface correction for intact stability...) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Free Surface § 170.285 Free surface... tanks must be determined by calculating— (1) For each type of consumable liquid, the maximum...

  11. Population Studies of Intact Vitamin D Binding Protein by Affinity Capture ESI-TOF-MS

    PubMed Central

    Borges, Chad R.; Jarvis, Jason W.; Oran, Paul E.; Rogers, Stephen P.; Nelson, Randall W.

    2008-01-01

    Blood plasma proteins with molecular weights greater than approximately 30 kDa are refractory to comprehensive, high-throughput qualitative characterization of microheterogeneity across human populations. Analytical techniques for obtaining high mass resolution for targeted, intact protein characterization and, separately, high sample throughput exist, but efficient means of coupling these assay characteristics remain rather limited. This article discusses the impetus for analyzing intact proteins in a targeted manner across populations and describes the methodology required to couple mass spectrometric immunoassay with electrospray ionization mass spectrometry for the purpose of qualitatively characterizing a prototypical large plasma protein, vitamin D binding protein, across populations. PMID:19137103

  12. Ionic liquid matrices for MALDI-TOF-MS analysis of intact glycoproteins.

    PubMed

    Giménez, Estela; Benavente, Fernando; Barbosa, José; Sanz-Nebot, Victoria

    2010-09-01

    2,5-Dihydroxybenzoic acid (DHB) has been demonstrated to be a more suitable matrix than 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA) to obtain reliable molecular mass values of intact glycoproteins because it prevents sugar fragmentation. Lack of spot homogeneity during the crystallization step was prevented by drying the sample-matrix mixture under vacuum conditions. Nevertheless, this sample-matrix preparation procedure requires a specific experimental setup and may be time-consuming. In this work, we investigated the effectiveness of different ionic liquid matrices (ILMs) with SA and DHB on the ionization of a set of intact glycoproteins with several degrees of glycosylation. The obtained results demonstrate that some of the tested ILMs allow detection of the studied intact glycoproteins. Furthermore, the selected optimum conditions solve the reproducibility issue of using the DHB as a solid matrix without the vacuum drying method and, surprisingly, avoid sugar fragmentation when both SA and DHB were used as ILMs.

  13. NASA Hubble Sees Comet ISON Intact

    NASA Image and Video Library

    2013-10-09

    This image from NASA Hubble Space Telescope of the sunward plunging comet ISON suggests that the comet is intact despite some predictions that the fragile icy nucleus might disintegrate as the sun warms it. In this NASA Hubble Space Telescope image taken on October 9, 2013 the comet's solid nucleus is unresolved because it is so small. If the nucleus broke apart then Hubble would have likely seen evidence for multiple fragments. Moreover, the coma or head surrounding the comet's nucleus is symmetric and smooth. This would probably not be the case if clusters of smaller fragments were flying along. What's more, a polar jet of dust first seen in Hubble images taken in April is no longer visible and may have turned off. This color composite image was assembled using two filters. The comet's coma appears cyan, a greenish-blue color due to gas, while the tail is reddish due to dust streaming off the nucleus. The tail forms as dust particles are pushed away from the nucleus by the pressure of sunlight. The comet was inside Mars' orbit and 177 million miles from Earth when photographed. Comet ISON is predicted to make its closest approach to Earth on 26 December, at a distance of 39.9 million miles. http://photojournal.jpl.nasa.gov/catalog/PIA18153

  14. Ureide degradation pathways in intact soybean leaves.

    PubMed

    Vadez, V; Sinclair, T R

    2000-08-01

    Ureides dramatically accumulate in shoots of N(2)-fixing soybean (Glycine max L. Merr.) under water deficit and this accumulation is higher in cultivars that have N(2) fixation that is sensitive to water deficit. One possible explanation is that ureide accumulation is associated with a feedback inhibition of nitrogenase activity. A critical factor involved in ureide accumulation is likely to be the rate of ureide degradation in the leaves. There exists, however, a controversy concerning the pathway of allantoic acid degradation in soybean. Allantoate amidinohydrolase was reported to be the pathway of degradation in studies using the cultivar Maple Arrow and allantoate amidohydrolase was the pathway that existed in the cultivar Williams. This investigation was undertaken to resolve the existence of these two pathways. An in situ technique was developed to examine the response of ureide degradation in leaf tissue to various treatments. In addition, the response of ureide accumulation and N(2) fixation activity was measured for intact plants in response to treatments that differentially influenced the two degradation pathways. The results from these studies confirmed that Maple Arrow and Williams degraded allantoic acid by different pathways as originally reported. The existence of two degradation pathways within the soybean germplasm opens the possibility of modifying ureide degradation to minimize the influence of soil water deficits on N(2) fixation activity.

  15. Intact implicit verbal relational memory in medial temporal lobe amnesia

    PubMed Central

    Verfaelllie, Mieke; LaRocque, Karen F.; Keane, Margaret M.

    2012-01-01

    To elucidate the role of the hippocampus in unaware relational memory, the present study examined the performance of amnesic patients with medial temporal lobe (MTL) lesions on a cued category-exemplar generation task. In contrast to a prior study in which amnesic patients showed impaired performance (Verfaellie et al., Cognitive, Affective, and Behavioral Neuroscience, 2006, 6, 91–101), the current study employed a task that required active processing of the context word at test. In this version of the task, amnesic patients, like control participants, showed enhanced category exemplar priming when the context word associated with the target at study was reinstated at test. The finding of intact implicit memory for novel associations following hippocampal lesions in a task that requires flexible use of retrieval cues is inconsistent with a relational memory view that suggests that the hippocampus is critical for all forms of relational memory, regardless of awareness. Instead, it suggests that unaware memory for within-domain associations does not require MTL mediation. PMID:22609574

  16. Intact implicit verbal relational memory in medial temporal lobe amnesia.

    PubMed

    Verfaelllie, Mieke; LaRocque, Karen F; Keane, Margaret M

    2012-07-01

    To elucidate the role of the hippocampus in unaware relational memory, the present study examined the performance of amnesic patients with medial temporal lobe (MTL) lesions on a cued category-exemplar generation task. In contrast to a prior study in which amnesic patients showed impaired performance (Verfaellie et al., Cognitive, Affective, and Behavioral Neuroscience, 2006, 6, 91-101), the current study employed a task that required active processing of the context word at test. In this version of the task, amnesic patients, like control participants, showed enhanced category exemplar priming when the context word associated with the target at study was reinstated at test. The finding of intact implicit memory for novel associations following hippocampal lesions in a task that requires flexible use of retrieval cues is inconsistent with a relational memory view that suggests that the hippocampus is critical for all forms of relational memory, regardless of awareness. Instead, it suggests that unaware memory for within-domain associations does not require MTL mediation. Published by Elsevier Ltd.

  17. A MAP Kinase pathway in Caenorhabditis elegans is required for defense against infection by opportunistic Proteus species.

    PubMed

    JebaMercy, Gnanasekaran; Vigneshwari, Loganathan; Balamurugan, Krishnaswamy

    2013-01-01

    Caenorhabditis elegans innate immunity requires a conserved mitogen activated protein kinase (MAPK) pathway that regulates the basal and pathogen-induced expression of immune effectors. Being in the group of opportunistic pathogens, Proteus spp. cause large number of nosocomial infections. Since, Proteus spp. do not cause death in wild type C. elegans, to understand the role and contribution of MAP Kinase pathway, the mutants (sek-1 and pmk-1) of this pathway were employed. Physiological experiments revealed that the Proteus spp. were able to kill MAP Kinase pathway mutant's C. elegans significantly. To understand the involvement of innate immune pathways specific players at the mRNA level, the regulation of few candidate antimicrobial genes were kinetically investigated during Proteus spp. infections. Real-time PCR analysis indicated a regulation of few candidate immune regulatory genes (F08G5.6, lys-7, nlp-29, ATF-7 and daf-16) during the course of Proteus spp. infections. In addition, the lipopolysaccharides (LPS) isolated from Proteus mirabilis upon exposure to mutant C. elegans showed modifications at their functional regions suggesting that the pathogen modifies its internal machinery according to the specific host for effective pathogenesis.

  18. Positive Youth Development, Life Satisfaction, and Problem Behaviors of Adolescents in Intact and Non-Intact Families in Hong Kong

    PubMed Central

    Shek, Daniel T. L.; Leung, Hildie

    2013-01-01

    This study investigated whether Chinese adolescents living in intact and non-intact families differed in their positive development, life satisfaction, and risk behavior. A total of 3,328 Secondary 1 students responded to measures of positive youth development (such as resilience and psychosocial competencies), life satisfaction, and risk behavior (substance abuse, delinquency, Internet addiction, consumption of pornographic materials, self-harm, and behavioral intention to engage in problem behavior). Findings revealed that adolescents growing up in intact families reported higher levels of positive developmental outcomes and life satisfaction as compared with adolescents from non-intact families. Adolescents in non-intact families also reported higher levels of risk behaviors than those growing up in intact families. PMID:24400264

  19. The pH Requirement for in Vivo Activity of the Iron-Deficiency-Induced "Turbo" Ferric Chelate Reductase (A Comparison of the Iron-Deficiency-Induced Iron Reductase Activities of Intact Plants and Isolated Plasma Membrane Fractions in Sugar Beet).

    PubMed Central

    Susin, S.; Abadia, A.; Gonzalez-Reyes, J. A.; Lucena, J. J.; Abadia, J.

    1996-01-01

    The characteristics of the Fe reduction mechanisms induced by Fe deficiency have been studied in intact plants of Beta vulgaris and in purified plasma membrane vesicles from the same plants. In Fe-deficient plants the in vivo Fe(III)-ethylenediaminetetraacetic complex [Fe(III)-EDTA] reductase activity increased over the control values 10 to 20 times when assayed at a pH of 6.0 or below ("turbo" reductase) but increased only 2 to 4 times when assayed at a pH of 6.5 or above. The Fe(III)-EDTA reductase activity of root plasma membrane preparations increased 2 and 3.5 times over the controls, irrespective of the assay pH. The Km for Fe(III)-EDTA of the in vivo ferric chelate reductase in Fe-deficient plants was approximately 510 and 240 [mu]M in the pH ranges 4.5 to 6.0 and 6.5 to 8.0, respectively. The Km for Fe(III)-EDTA of the ferric chelate reductase in intact control plants and in plasma membrane preparations isolated from Fe-deficient and control plants was approximately 200 to 240 [mu]M. Therefore, the turbo ferric chelate reductase activity of Fe-deficient plants at low pH appears to be different from the constitutive ferric chelate reductase. PMID:12226175

  20. Experimental serpentinization of intact dunite cores

    NASA Astrophysics Data System (ADS)

    Luhmann, A. J.; Tutolo, B. M.; Kong, X. Z.; Bagley, B. C.; Schaen, A. T.; Saar, M. O.; Seyfried, W. E., Jr.

    2014-12-01

    Serpentinization in ultramafic-hosted hydrothermal systems, such as Lost City, produces relatively cool and alkaline fluids that support diverse ecosystems. To simulate serpentinization in such systems, we conducted single-pass, flow-through experiments on dunite cores cut out of a sample from Jackson County, North Carolina. Experimental seawater prepared using laboratory-grade reagents and standards was pumped through a core at 150ºC and 150 bar pore-fluid outlet pressure at a flow rate of 0.01 ml/min. An additional experiment will be conducted at 200ºC. At 150ºC, permeability decreased by 2.3 times with reaction progress over the course of the 36 day experiment. Fluid-rock reaction generally produced CO2, H2, CH4, and CO concentrations of 100 μmol/kg, up to 40 μmol/kg, 2 μmol/kg, and less than 1 μmol/kg, respectively. Outlet fluid chemistry was relatively stable, except for initial peaks in Al, Ba, Fe, Mn, and Si. pH of outlet fluids increased with reaction progress, but it was always lower (6.9-7.4) than the initial seawater (7.8). X-ray computed tomography scans were/will be collected for both pre- and post-experimental cores. The combination of flow-through experiments on solid, intact rock cores cut out of natural samples and X-ray tomography permits visualization and quantification of mineralogical changes and flow path evolution during serpentinization. This approach further permits physical and chemical processes to be documented on a fine scale to better understand feedbacks between chemical reactions and flow fields, with implications for ultramafic-hosted hydrothermal systems.

  1. 50 CFR 622.247 - Landing golden crab intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing golden crab intact. 622.247... ATLANTIC Golden Crab Fishery of the South Atlantic Region § 622.247 Landing golden crab intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that golden crab on that vessel in...

  2. 50 CFR 622.247 - Landing golden crab intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing golden crab intact. 622.247... ATLANTIC Golden Crab Fishery of the South Atlantic Region § 622.247 Landing golden crab intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that golden crab on that vessel in...

  3. 50 CFR 622.10 - Landing fish intact--general.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE FISHERIES OF THE CARIBBEAN, GULF OF MEXICO, AND SOUTH... landing fish intact that are broadly applicable to finfish in the Gulf EEZ and Caribbean EEZ, as specified... intact. (a) Finfish in or from the Gulf EEZ or Caribbean EEZ, except as specified in paragraphs (b)...

  4. 50 CFR 622.10 - Landing fish intact--general.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE FISHERIES OF THE CARIBBEAN, GULF OF MEXICO, AND SOUTH... landing fish intact that are broadly applicable to finfish in the Gulf EEZ and Caribbean EEZ, as specified... intact. (a) Finfish in or from the Gulf EEZ or Caribbean EEZ, except as specified in paragraphs (b)...

  5. 50 CFR 622.455 - Landing spiny lobster intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing spiny lobster intact. 622.455... ATLANTIC Spiny Lobster Fishery of Puerto Rico and the U.S. Virgin Islands § 622.455 Landing spiny lobster intact. (a) A Caribbean spiny lobster in or from the Caribbean EEZ must be maintained with head...

  6. 50 CFR 622.455 - Landing spiny lobster intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing spiny lobster intact. 622.455... ATLANTIC Spiny Lobster Fishery of Puerto Rico and the U.S. Virgin Islands § 622.455 Landing spiny lobster intact. (a) A Caribbean spiny lobster in or from the Caribbean EEZ must be maintained with head...

  7. 50 CFR 622.186 - Landing fish intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing fish intact. 622.186 Section 622...-Grouper Fishery of the South Atlantic Region § 622.186 Landing fish intact. (a) South Atlantic snapper... specified in paragraph (b) of this section. Such fish may be eviscerated, gilled, and scaled, but must...

  8. 50 CFR 622.186 - Landing fish intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing fish intact. 622.186 Section 622...-Grouper Fishery of the South Atlantic Region § 622.186 Landing fish intact. (a) South Atlantic snapper... specified in paragraph (b) of this section. Such fish may be eviscerated, gilled, and scaled, but must...

  9. 46 CFR 172.095 - Intact longitudinal stability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Intact longitudinal stability. 172.095 Section 172.095 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.095 Intact longitudinal stability. Each tank barge must be...

  10. 46 CFR 174.020 - Alternate intact stability criterion.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Alternate intact stability criterion. 174.020 Section 174.020 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY... Alternate intact stability criterion. A barge need not comply with § 174.015 and subparts C and E of part...

  11. 46 CFR 172.095 - Intact longitudinal stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact longitudinal stability. 172.095 Section 172.095 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.095 Intact longitudinal stability. Each tank barge must be...

  12. 46 CFR 172.095 - Intact longitudinal stability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Intact longitudinal stability. 172.095 Section 172.095 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.095 Intact longitudinal stability. Each tank barge must be...

  13. 46 CFR 172.165 - Intact stability calculations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability calculations. 172.165 Section 172.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.165 Intact stability calculations. (a) Design calculations must...

  14. 46 CFR 174.020 - Alternate intact stability criterion.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Alternate intact stability criterion. 174.020 Section 174.020 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY... Alternate intact stability criterion. A barge need not comply with § 174.015 and subparts C and E of part...

  15. 46 CFR 172.095 - Intact longitudinal stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact longitudinal stability. 172.095 Section 172.095 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.095 Intact longitudinal stability. Each tank barge must be...

  16. 46 CFR 172.165 - Intact stability calculations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability calculations. 172.165 Section 172.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.165 Intact stability calculations. (a) Design calculations must...

  17. 46 CFR 172.165 - Intact stability calculations.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Intact stability calculations. 172.165 Section 172.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.165 Intact stability calculations. (a) Design calculations must...

  18. 46 CFR 172.165 - Intact stability calculations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Intact stability calculations. 172.165 Section 172.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.165 Intact stability calculations. (a) Design calculations must...

  19. 46 CFR 172.165 - Intact stability calculations.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact stability calculations. 172.165 Section 172.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.165 Intact stability calculations. (a) Design calculations must...

  20. 46 CFR 172.095 - Intact longitudinal stability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact longitudinal stability. 172.095 Section 172.095 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.095 Intact longitudinal stability. Each tank barge must be...

  1. 50 CFR 622.38 - Landing fish intact.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... maintained with head and fins intact: cobia, king mackerel, and Spanish mackerel in or from the Gulf, Mid-Atlantic, or South Atlantic EEZ, except as specified for king mackerel in paragraph (g) of this section... must be maintained with meat and shell intact. (g) Cut-off (damaged) king or Spanish mackerel...

  2. 50 CFR 622.38 - Landing fish intact.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... and fins intact: cobia, king mackerel, and Spanish mackerel in or from the Gulf, Mid-Atlantic, or South Atlantic EEZ, except as specified for king mackerel in paragraph (g) of this section; dolphin and... shell intact. (g) Cut-off (damaged) king or Spanish mackerel that comply with the minimum size limits...

  3. 50 CFR 622.38 - Landing fish intact.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... maintained with head and fins intact: cobia, king mackerel, and Spanish mackerel in or from the Gulf, Mid-Atlantic, or South Atlantic EEZ, except as specified for king mackerel in paragraph (g) of this section... must be maintained with meat and shell intact. (g) Cut-off (damaged) king or Spanish mackerel...

  4. Development of spatial database on intact forest landscapes of India

    NASA Astrophysics Data System (ADS)

    Sudhakar Reddy, C.; Singh, Jyoti; Jha, C. S.; Diwakar, P. G.; Dadhwal, V. K.

    2017-01-01

    There is an increased interest in identifying the status of biodiversity in different spatial and temporal scales. The objective of the current research is to prepare a consistent spatial database of intact forest landscapes of India. The intact forest landscapes are located in the Himalayas, Andaman & Nicobar Islands, Western Ghats and Sunderbans. At national level 237 forest landscapes and 5.4% of the total natural forest remained as intact in India. Current intact forest landscapes of India consists of blocks larger than 10 km2 covering an area of 34,061 km2. Of the total area under intact forest landscapes, Eastern Himalayas represent 76.7% of the area, followed by Western Himalayas (8.8%), Andaman & Nicobar Islands (6.2%) and Western Ghats (5.7%). The largest intact forest landscape block occupies an area of 3342.9 km2 (9.8%) is found in western Arunachal Pradesh. Temperate forest zone represents the highest intactness (56.6%), followed by subtropical zone (19.2%), tropical zone (18.6%) and alpine zone (5.6%). Himalayan moist temperate forests represent the highest area (39.1%) of intact forest landscapes followed by subtropical broad-leaved hill forests, wet evergreen forests, and montane wet temperate forests. It is estimated that 4.4% of the area of intact forest landscapes fall inside the existing 47 protected areas. The results of the analysis best suited as input for the process of identification of new protected areas. The study recommends fine-scale mapping of biodiversity within the intact forest landscapes and to prepare efficient conservation plans.

  5. Optimized planning target volume for intact cervical cancer.

    PubMed

    Khan, Alvin; Jensen, Lindsay G; Sun, Shuai; Song, William Y; Yashar, Catheryn M; Mundt, Arno J; Zhang, Fu-Quan; Jiang, Steve B; Mell, Loren K

    2012-08-01

    To model interfraction clinical target volume (CTV) variation in patients with intact cervical cancer and design a planning target volume (PTV) that minimizes normal tissue dose while maximizing CTV coverage. We analyzed 50 patients undergoing external-beam radiotherapy for intact cervical cancer using daily online cone-beam computed tomography (CBCT). The CBCTs (n = 972) for each patient were rigidly registered to the planning CT. The CTV was delineated on the planning CT (CTV(0)) and the set of CBCTs ({CTV(1)-CTV(25)}). Manual (n = 98) and automated (n = 668) landmarks were placed over the surface of CTV(0) with reference to defined anatomic structures. Normal vectors were extended from each landmark, and the minimum length required for a given probability of encompassing CTV(1)-CTV(25) was computed. The resulting expansions were used to generate an optimized PTV. The mean (SD; range) normal vector length to ensure 95% coverage was 4.3 mm (2.7 mm; 1-16 mm). The uniform expansion required to ensure 95% probability of CTV coverage was 13 mm. An anisotropic margin of 20 mm anteriorly and posteriorly and 10 mm superiorly, inferiorly, and laterally also would have ensured a 95% probability of CTV coverage. The volume of the 95% optimized PTV (1470 cm(3)) was significantly lower than both the anisotropic PTV (2220 cm(3)) and the uniformly expanded PTV (2110 cm(3)) (p < 0.001). For a 95% probability of CTV coverage, normal lengths of 1-3 mm were found along the superior and lateral regions of CTV(0), 5-10 mm along the interfaces of CTV(0) with the bladder and rectum, and 10-14 mm along the anterior surface of CTV(0) at the level of the uterus. Optimizing PTV definition according to surface landmarking resulted in a high probability of CTV coverage with reduced PTV volumes. Our results provide data justifying planning margins to use in practice and clinical trials. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Optimized Planning Target Volume for Intact Cervical Cancer

    SciTech Connect

    Khan, Alvin; Jensen, Lindsay G.; Sun Shuai; Song, William Y.; Yashar, Catheryn M.; Mundt, Arno J.; Zhang Fuquan; Jiang, Steve B.; Mell, Loren K.

    2012-08-01

    Purpose: To model interfraction clinical target volume (CTV) variation in patients with intact cervical cancer and design a planning target volume (PTV) that minimizes normal tissue dose while maximizing CTV coverage. Methods and Materials: We analyzed 50 patients undergoing external-beam radiotherapy for intact cervical cancer using daily online cone-beam computed tomography (CBCT). The CBCTs (n = 972) for each patient were rigidly registered to the planning CT. The CTV was delineated on the planning CT (CTV{sub 0}) and the set of CBCTs ({l_brace}CTV{sub 1}-CTV{sub 25}{r_brace}). Manual (n = 98) and automated (n = 668) landmarks were placed over the surface of CTV{sub 0} with reference to defined anatomic structures. Normal vectors were extended from each landmark, and the minimum length required for a given probability of encompassing CTV{sub 1}-CTV{sub 25} was computed. The resulting expansions were used to generate an optimized PTV. Results: The mean (SD; range) normal vector length to ensure 95% coverage was 4.3 mm (2.7 mm; 1-16 mm). The uniform expansion required to ensure 95% probability of CTV coverage was 13 mm. An anisotropic margin of 20 mm anteriorly and posteriorly and 10 mm superiorly, inferiorly, and laterally also would have ensured a 95% probability of CTV coverage. The volume of the 95% optimized PTV (1470 cm{sup 3}) was significantly lower than both the anisotropic PTV (2220 cm{sup 3}) and the uniformly expanded PTV (2110 cm{sup 3}) (p < 0.001). For a 95% probability of CTV coverage, normal lengths of 1-3 mm were found along the superior and lateral regions of CTV{sub 0}, 5-10 mm along the interfaces of CTV{sub 0} with the bladder and rectum, and 10-14 mm along the anterior surface of CTV{sub 0} at the level of the uterus. Conclusion: Optimizing PTV definition according to surface landmarking resulted in a high probability of CTV coverage with reduced PTV volumes. Our results provide data justifying planning margins to use in practice and

  7. Reflectance Spectra Comparison of Orbital Debris, Intact Spacecraft, and Intact Rocket Bodies in the GEO Regime

    NASA Astrophysics Data System (ADS)

    Albercromby, Kira J.; Abell, Paul; Barker, Ed

    2009-03-01

    A key objective of NASA's Orbital Debris program office at Johnson Space Center (JSC) is to characterize the debris environment by way of assessing the physical properties (type, mass, density, and size) of objects in orbit. Knowledge of the geosynchronous orbit (GEO) debris environment in particular can be used to determine the hazard probability at specific GEO altitudes and aid predictions of the future environment. To calculate an optical size from an intensity measurement of an object in the GEO regime, a 0.175 albedo is assumed currently. However, identification of specific material type or types could improve albedo accuracy and yield a more accurate size estimate for the debris piece. Using spectroscopy, it is possible to determine the surface materials of space objects. The study described herein used the NASA Infrared Telescope Facility (IRTF) to record spectral data in the ~ 0.65 to 2.5 micron regime on eight catalogued space objects. For comparison, all of the objects observed were in GEO or near-GEO. The eight objects consisted of two intact spacecraft, three rocket bodies, and three catalogued debris pieces. Two of the debris pieces stemmed from Titan 3C transtage breakup and the third is from COSMOS 2054. The reflectance spectra of the Titan 3C pieces share similar slopes (increasing with wavelength) and lack any strong absorption features. The COSMOS debris spectrum has a slight slope and has no absorption features. In contrast, the intact spacecraft show classic absorption features due to solar cells with a strong band gap feature near 1 micron. The two spacecraft were spin-stabilized objects and therefore have solar panels surrounding the outer surface. Two of the three rocket bodies are inertial upper stage (IUS) rocket bodies and have similar looking spectra. The slopes flatten out near 1.5 microns with absorption features in the near-infrared that are similar to that of white paint. The third rocket body has a similar flattening of slope but

  8. Reflectance Spectra Comparison of Orbital Debris, Intact Spacecraft, and Intact Rocket Bodies in the GEO Regime

    NASA Technical Reports Server (NTRS)

    Barker, Ed; Abercromby, Kira J.; Abell, Paul

    2009-01-01

    A key objective of NASA s Orbital Debris program office at Johnson Space Center (JSC) is to characterize the debris environment by way of assessing the physical properties (type, mass, density, and size) of objects in orbit. Knowledge of the geosynchronous orbit (GEO) debris environment in particular can be used to determine the hazard probability at specific GEO altitudes and aid predictions of the future environment. To calculate an optical size from an intensity measurement of an object in the GEO regime, a 0.175 albedo is assumed currently. However, identification of specific material type or types could improve albedo accuracy and yield a more accurate size estimate for the debris piece. Using spectroscopy, it is possible to determine the surface materials of space objects. The study described herein used the NASA Infrared Telescope Facility (IRTF) to record spectral data in the 0.6 to 2.5 micron regime on eight catalogued space objects. For comparison, all of the objects observed were in GEO or near-GEO. The eight objects consisted of two intact spacecraft, three rocket bodies, and three catalogued debris pieces. Two of the debris pieces stemmed from Titan 3C transtage breakup and the third is from COSMOS 2054. The reflectance spectra of the Titan 3C pieces share similar slopes (increasing with wavelength) and lack any strong absorption features. The COSMOS debris spectra is flat and has no absorption features. In contrast, the intact spacecraft show classic absorption features due to solar panels with a strong band gap feature near 1 micron. The two spacecraft are spin-stabilized objects and therefore have solar panels surrounding the outer surface. Two of the three rocket bodies are inertial upper stage (IUS) rocket bodies and have similar looking spectra. The slopes flatten out near 1.5 microns with absorption features in the near-infrared that are similar to that of white paint. The third rocket body has a similar flattening of slope but with fewer

  9. Treatment for burn blisters: debride or leave intact?

    PubMed

    Murphy, Faye; Amblum, Jeshi

    2014-05-01

    This article presents findings from a systematic literature review of whether blisters arising from minor burns should be de-roofed or left intact. It discusses the risks of infection, healing outcomes, discomfort, choice of dressings and costs associated with each method, and reveals that debriding blisters larger than the patient's little fingernail while leaving smaller ones intact is generally agreed to be the best option. The article also explains external factors that influence the choice of whether to debride or leave blisters intact, reviews policy at the trust where one of the authors works in the context of the research and makes recommendations for practice.

  10. Transcatheter pulmonary valve perforation and balloon dilatation in neonates with pulmonary atresia and intact ventricular septum

    PubMed Central

    Gerestein, C.G.; Berger, R.M.F.; Dalinghaus, M.; Bogers, A.J.J.C.; Witsenburg, M.

    2003-01-01

    Background Pulmonary atresia and intact ventricular septum is characterised by a great morphological variety. Treatment is not uniform. Objective To evaluate our experience with transcatheter valvotomy and balloon dilatation in neonates with pulmonary atresia and intact ventricular septum. Design Retrospective. Methods Between January 1997 and September 2000 five neonates with pulmonary atresia and intact ventricular septum underwent transcatheter valvotomy and balloon dilatation. Results The catheter intervention was performed at a mean age of 27 days (range 3-95 days). The atretic pulmonary valve was successfully perforated in all neonates. Subsequent balloon dilatation was successful in four neonates. Balloon dilatation was unsuccessful in one patient, who underwent an elective surgical valvotomy of the pulmonary valve after five days. Three patients needed a modified Blalock-Taussig shunt after a mean of 23 days. Four patients required repeated balloon dilatation after a mean of 227 days. Mean follow-up was 2.7 years (range 1-5 years). Conclusions Transcatheter perforation of the pulmonary valve membrane and balloon dilatation is a good, safe initial therapy in selected neonates with pulmonary atresia and intact ventricular septum. This procedure can prevent open-heart surgery in these patients in the first months of life. ImagesFigure 1Figure 2 PMID:25696158

  11. Intactness of cell wall structure controls the in vitro digestion of starch in legumes.

    PubMed

    Dhital, Sushil; Bhattarai, Rewati R; Gorham, John; Gidley, Michael J

    2016-03-01

    Increasing the level of starch that is not digested by the end of the small intestine and therefore enters the colon ('resistant starch') is a major opportunity for improving the nutritional profile of foods. One mechanism that has been shown to be successful is entrapment of starch within an intact plant tissue structure. However, the level of tissue intactness required for resistance to amylase digestion has not been defined. In this study, intact cells were isolated from a range of legumes after thermal treatment at 60 °C (starch not gelatinised) or 95 °C (starch gelatinised) followed by hydrolysis using pancreatic alpha amylase. It was found that intact cells, isolated at either temperature, were impervious to amylase. However, application of mechanical force damaged the cell wall and made starch accessible to digestive enzymes. This shows that the access of enzymes to the entrapped swollen starch is the rate limiting step controlling hydrolysis of starch in cooked legumes. The results suggest that a single cell wall could be sufficient to provide an effective delivery of starch to the large intestine with consequent nutritional benefits, provided that mechanical damage during digestion is avoided.

  12. Prophylactic antibiotics for inhibiting preterm labour with intact membranes.

    PubMed

    Flenady, Vicki; Hawley, Glenda; Stock, Owen M; Kenyon, Sara; Badawi, Nadia

    2013-12-05

    benefit in important neonatal outcomes with the use of prophylactic antibiotics for women in preterm labour with intact membranes, although maternal infection may be reduced. Of concern, is the finding of short- and longer-term harm for children of mothers exposed to antibiotics. The evidence supports not giving antibiotics routinely to women in preterm labour with intact membranes in the absence of overt signs of infection.Further research is required to develop sensitive markers of subclinical infection for women in preterm labour with intact membranes, as this is a group that might benefit from future novel interventions, including new modalities of antibiotic therapy. The results of this review demonstrate the need for future trials in the area of preterm birth to include assessment of long-term neurodevelopmental outcome.

  13. Rosmarinus officinalis L. increases Caenorhabditis elegans stress resistance and longevity in a DAF-16, HSF-1 and SKN-1-dependent manner.

    PubMed

    Zamberlan, D C; Amaral, G P; Arantes, L P; Machado, M L; Mizdal, C R; Campos, M M A; Soares, F A A

    2016-08-01

    Improving overall health and quality of life, preventing diseases and increasing life expectancy are key concerns in the field of public health. The search for antioxidants that can inhibit oxidative damage in cells has received a lot of attention. Rosmarinus officinalis L. represents an exceptionally rich source of bioactive compounds with pharmacological properties. In the present study, we explored the effects of the ethanolic extract of R. officinalis (eeRo) on stress resistance and longevity using the non-parasitic nematode Caenorhabditis elegans as a model. We report for the first time that eeRo increased resistance against oxidative and thermal stress and extended C. elegans longevity in an insulin/IGF signaling pathway-dependent manner. These data emphasize the eeRo beneficial effects on C. elegans under stress.

  14. SIRT3 interacts with the daf-16 homolog FOXO3a in the Mitochondria, as well as increases FOXO3a Dependent Gene expression

    PubMed Central

    Jacobs, Kristi Muldoon; Pennington, J. Daniel; Bisht, Kheem S.; Aykin-Burns, Nukhet; Kim, Hyun-Seok; Mishra, Mark; Sun, Lunching; Nguyen, Phuongmai; Ahn, Bong-Hyun; Leclerc, Jaime; Deng, Chu-Xia; Spitz, Douglas R.; Gius, David

    2008-01-01

    Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway. PMID:18781224

  15. Rosmarinus officinalis L. increases Caenorhabditis elegans stress resistance and longevity in a DAF-16, HSF-1 and SKN-1-dependent manner

    PubMed Central

    Zamberlan, D.C.; Amaral, G.P.; Arantes, L.P.; Machado, M.L.; Mizdal, C.R.; Campos, M.M.A.; Soares, F.A.A.

    2016-01-01

    Improving overall health and quality of life, preventing diseases and increasing life expectancy are key concerns in the field of public health. The search for antioxidants that can inhibit oxidative damage in cells has received a lot of attention. Rosmarinus officinalis L. represents an exceptionally rich source of bioactive compounds with pharmacological properties. In the present study, we explored the effects of the ethanolic extract of R. officinalis (eeRo) on stress resistance and longevity using the non-parasitic nematode Caenorhabditis elegans as a model. We report for the first time that eeRo increased resistance against oxidative and thermal stress and extended C. elegans longevity in an insulin/IGF signaling pathway-dependent manner. These data emphasize the eeRo beneficial effects on C. elegans under stress. PMID:27533765

  16. 56. POWDER MAGAZINE, VIEW OF INTACT WOOD SHEATHING ON THE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    56. POWDER MAGAZINE, VIEW OF INTACT WOOD SHEATHING ON THE SOUTHWEST REAR VENTILATION PASSAGE. (SHEATHING HELP CONTROL HUMIDITY AND DECREASE DANGER OF MAETAL STRIKING STONE AND SPARKING.) - Fort Monroe, Fortress, Hampton, Hampton, VA

  17. 10. VIEW SHOWING FRAGMENTED AND INTACT ORIGINAL SIDEWALK ALONG SOUTH ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    10. VIEW SHOWING FRAGMENTED AND INTACT ORIGINAL SIDEWALK ALONG SOUTH SIDE OF SHERMAN CANAL, BETWEEN DELL AVENUE VEHICULAR BRIDGE AND GRAND CANAL COURT PEDESTRIAN BRIDGE, LOOKING EAST - Venice Canals, Community of Venice, Los Angeles, Los Angeles County, CA

  18. 50 CFR 622.276 - Landing fish intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... ADMINISTRATION, DEPARTMENT OF COMMERCE FISHERIES OF THE CARIBBEAN, GULF OF MEXICO, AND SOUTH ATLANTIC Dolphin and Wahoo Fishery Off the Atlantic States § 622.276 Landing fish intact. (a) Dolphin and wahoo in or...

  19. 50 CFR 622.276 - Landing fish intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... ADMINISTRATION, DEPARTMENT OF COMMERCE FISHERIES OF THE CARIBBEAN, GULF OF MEXICO, AND SOUTH ATLANTIC Dolphin and Wahoo Fishery Off the Atlantic States § 622.276 Landing fish intact. (a) Dolphin and wahoo in or...

  20. Proton nuclear magnetic resonance of intact friend leukemia cells: phosphorylcholine increase during differentiation

    SciTech Connect

    Agris, P.F.; Campbell, I.D.

    1982-06-18

    Proton nuclear magnetic resonance of intact Friend leukemia cells was used to analyze their erythroid-like differentiation. The technique, which requires only 10/sup 8/ to 10/sup 9/ cells and approximately 2 minutes for acquisition of each spectrum, demonstrated the occurrence of many signal changes during differentiation. With cell extracts, 64 signals were assigned to 12 amino acids and 19 other intermediary metabolites, and a dramatic signal change was attributed to a fourfrease in cytoplasmic phosphorylcholines.

  1. Genistein stimulates jejunum chloride secretion via an Akt-mediated pathway in intact female mice.

    PubMed

    Leung, Lana; Bhakta, Ashesh; Cotangco, Katherine; Al-Nakkash, Layla

    2015-01-01

    We have previously shown that daily subcutaneous injections with the naturally occurring phytoestrogen genistein (600 mg genistein/kg body weight/day, 600G) results in a significantly increased basal intestinal chloride, Cl(-), secretion (Isc, a measure of transepithelial secretion) in intact C57BL/6J female mice after 1-week of treatment, compared to controls (DMSO vehicle injected). Removal of endogenous estrogen via ovariectomy (OVX) had no effect on the 600G-mediated increase in basal Isc. Given the estrogen-like characteristics of genistein, we compared the effects of daily estradiol (E2) injections (10 mg E2/kg body weight/day, 10E2) on basal Isc in intact and OVX mice. In intact mice, 10E2 was without effect on basal Isc, however, in OVX mice, 10E2 significantly increased basal Isc (mimicked 600G). The goal of the current study was to characterize the intracellular signaling pathways responsible for mediating 600G- or 10E2-stimulated increases in basal Isc in intact female or OVX mice. We measured total protein expression in isolated segments of jejunum using western blot from the following six groups of mice; intact or OVX with; 600G, 10E2 or control. The proteins of interest were: Akt, p-Akt, p-PDK1, p-PTEN, p-c-Raf, p-GSK-3β, rap-1 and ERK1/2. All blots were normalized to GAPDH levels (n = 6-18/group). These data suggest that the presence of the endogenous sex steroid, estrogen, modifies the intracellular signaling pathway required to mediate Cl(-) secretion when the intestine is exposed to exogenous 600G or E2. These studies may have relevance for designing pharmacological tools for women with intestinal chloride secretory dysfunctions. © 2015 S. Karger AG, Basel.

  2. Comparison of Intact PTH and Bio-Intact PTH Assays Among Non-Dialysis Dependent Chronic Kidney Disease Patients

    PubMed Central

    Einbinder, Yael; Benchetrit, Sydney; Golan, Eliezer

    2017-01-01

    Background The third-generation bio-intact parathyroid hormone (PTH) (1–84) assay was designed to overcome problems associated with the detection of C-terminal fragments by the second-generation intact PTH assay. The two assays have been compared primarily among dialysis populations. The present study evaluated the correlations and differences between these two PTH assays among patients with chronic kidney disease (CKD) stages 3 to 5 not yet on dialysis. Methods Blood samples were collected from 98 patients with CKD stages 3 to 5. PTH concentrations were measured simultaneously by using the second-generation - PTH intact-STAT and third-generation bio-intact 1–84 PTH assays. Other serum biomarkers of bone mineral disorders were also assessed. CKD stage was calculated by using the CKD-Epidemiology Collaboration (EPI) formula. Results Serum bio-intact PTH concentrations were strongly correlated but significantly lower than the intact PTH concentrations (r=0.963, P<0.0001). This finding was consistent among CKD stages 3 to 5. PTH concentrations by both assays (intact and bio-intact PTH) positively correlated with urea (r=0.523, r=0.504; P=0.002, respectively), phosphorus (r=0.532, r=0.521; P<0.0001, respectively) and negatively correlated with blood calcium (r=−0.435, r=−0.476; P<0.0001, respectively), 25(OH) vitamin D, (r=−0.319, r=−0.353; respectively, P<0.0001) and the estimated glomerular filtration rate (r=−0.717, r=−0.688; P<0.0001, respectively). Conclusions Among patients with CKD stages 3 to 5 not on dialysis, the bio-intact PTH assay detected significantly lower PTH concentrations compared with intact PTH assay. Additional studies that correlate the diagnosis and management of CKD mineral and bone disorders with bone histomorphometric findings are needed to determine whether bio-intact PTH assay results are better surrogate markers in these early stages of CKD. PMID:28643486

  3. Comparison of Intact PTH and Bio-Intact PTH Assays Among Non-Dialysis Dependent Chronic Kidney Disease Patients.

    PubMed

    Einbinder, Yael; Benchetrit, Sydney; Golan, Eliezer; Zitman-Gal, Tali

    2017-09-01

    The third-generation bio-intact parathyroid hormone (PTH) (1-84) assay was designed to overcome problems associated with the detection of C-terminal fragments by the second-generation intact PTH assay. The two assays have been compared primarily among dialysis populations. The present study evaluated the correlations and differences between these two PTH assays among patients with chronic kidney disease (CKD) stages 3 to 5 not yet on dialysis. Blood samples were collected from 98 patients with CKD stages 3 to 5. PTH concentrations were measured simultaneously by using the second-generation - PTH intact-STAT and third-generation bio-intact 1-84 PTH assays. Other serum biomarkers of bone mineral disorders were also assessed. CKD stage was calculated by using the CKD-Epidemiology Collaboration (EPI) formula. Serum bio-intact PTH concentrations were strongly correlated but significantly lower than the intact PTH concentrations (r=0.963, P<0.0001). This finding was consistent among CKD stages 3 to 5. PTH concentrations by both assays (intact and bio-intact PTH) positively correlated with urea (r=0.523, r=0.504; P=0.002, respectively), phosphorus (r=0.532, r=0.521; P<0.0001, respectively) and negatively correlated with blood calcium (r=-0.435, r=-0.476; P<0.0001, respectively), 25(OH) vitamin D, (r=-0.319, r=-0.353; respectively, P<0.0001) and the estimated glomerular filtration rate (r=-0.717, r=-0.688; P<0.0001, respectively). Among patients with CKD stages 3 to 5 not on dialysis, the bio-intact PTH assay detected significantly lower PTH concentrations compared with intact PTH assay. Additional studies that correlate the diagnosis and management of CKD mineral and bone disorders with bone histomorphometric findings are needed to determine whether bio-intact PTH assay results are better surrogate markers in these early stages of CKD.

  4. Dead or Alive? Probing Microbial Ecosystems With Intact Polar Lipids.

    NASA Astrophysics Data System (ADS)

    Sturt, H. F.; Smith, K. J.; Xu, L.; Molyneaux, S. J.; Teske, A.; Orphan, V. J.; Summons, R. E.; Hinrichs, K.

    2002-12-01

    Multi-proxy approaches consisting of isotopic information derived from diagnostic lipid biomarkers together with phylogenetic techniques have advanced our understanding of naturally occurring microbial communities. Examples include the significant advances in our understanding of the ecology of archaeal methanotrophs and planktonic crenarchaeota. However, this approach has to be modified for application to microbial ecosystems in the deep subsurface in order to distinguish live matter from dead. Polar lipids, i.e., phospho- and glycolipids, fulfill that requirement and potentially provide information on the structure, size and function of the microbial community. We utilized a high-performance-liquid-chromatograph coupled to a mass-spectrometer (HPLC-MS) to examine the distribution of intact polar lipids from microbial isolates and surface sediments that are relevant for the study of deep subsurface biospheres. Structural features of bacterial and archaeal-derived polar lipids are identified on the basis of their sequential fragmentation in an ion-trap mass spectrometer upon electrospray ionization. Additional details are obtained from a series of chemical degradations designed to release alkyl units of the polar lipids rendering them analyzable by conventional gas-chromatography-mass-spectrometry (GC-MS). Fingerprints of polar lipids from microbial isolates allow distinction of taxonomically closely related prokaryotes. Moreover, sediments from seep environments in Eel River Basin and Guaymas Basin that host anaerobic methanotrophic communities are both dominated by similar types of glycosyl biphytanyl tetraethers suggestive of a predominant low-diversity archaeal assemblage. First applications of this technique to sediments from the deep subsurface will be presented.

  5. Structural and molecular interrogation of intact biological systems

    PubMed Central

    Chung, Kwanghun; Wallace, Jenelle; Kim, Sung-Yon; Kalyanasundaram, Sandhiya; Andalman, Aaron S.; Davidson, Thomas J.; Mirzabekov, Julie J.; Zalocusky, Kelly A.; Mattis, Joanna; Denisin, Aleksandra K.; Pak, Sally; Bernstein, Hannah; Ramakrishnan, Charu; Grosenick, Logan; Gradinaru, Viviana; Deisseroth, Karl

    2014-01-01

    Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease. PMID:23575631

  6. Vulnerability of ecosystems to climate change moderated by habitat intactness.

    PubMed

    Eigenbrod, Felix; Gonzalez, Patrick; Dash, Jadunandan; Steyl, Ilse

    2015-01-01

    The combined effects of climate change and habitat loss represent a major threat to species and ecosystems around the world. Here, we analyse the vulnerability of ecosystems to climate change based on current levels of habitat intactness and vulnerability to biome shifts, using multiple measures of habitat intactness at two spatial scales. We show that the global extent of refugia depends highly on the definition of habitat intactness and spatial scale of the analysis of intactness. Globally, 28% of terrestrial vegetated area can be considered refugia if all natural vegetated land cover is considered. This, however, drops to 17% if only areas that are at least 50% wilderness at a scale of 48×48 km are considered and to 10% if only areas that are at least 50% wilderness at a scale of 4.8×4.8 km are considered. Our results suggest that, in regions where relatively large, intact wilderness areas remain (e.g. Africa, Australia, boreal regions, South America), conservation of the remaining large-scale refugia is the priority. In human-dominated landscapes, (e.g. most of Europe, much of North America and Southeast Asia), focusing on finer scale refugia is a priority because large-scale wilderness refugia simply no longer exist. Action to conserve such refugia is particularly urgent since only 1 to 2% of global terrestrial vegetated area is classified as refugia and at least 50% covered by the global protected area network. © 2014 John Wiley & Sons Ltd.

  7. Tracking tagged molecules in single neurons in intact zebrafish.

    PubMed

    Armisen, Ricardo; Gleason, Michelle R; Fetcho, Joseph R; Mandel, Gail

    2007-08-01

    INTRODUCTIONThis protocol describes an approach for monitoring the movement of tagged molecules in single neurons in intact embryonic and larval zebrafish. The intact preparation provides a meaningful context for the physiological event being studied. Other advantages offered by the young zebrafish include direct in vivo imaging, the ability to produce large numbers of labeled embryos easily using microinjection, and the existence of identified sensory circuits that can be exploited to activate a particular cell type. One limitation of this system is the fragility of 2- to 3-d-old embryos, which demands delicate physical manipulation of the fish during all stages preceding and during the experiment. In contrast to brain slices or isolated cells, nearly all original neural connections and sensory components are maintained in the intact preparation, so the occurrence of a downstream event may be precluded (or its manifestation enhanced) by some complex interplay of biological processes that are not fully understood.

  8. Enhanced Dissociation of Intact Proteins with High Capacity Electron Transfer Dissociation

    PubMed Central

    Riley, Nicholas M.; Mullen, Christopher; Weisbrod, Chad R.; Sharma, Seema; Senko, Michael W.; Zabrouskov, Vlad; Westphall, Michael S.; Syka, John E.P.; Coon, Joshua J.

    2015-01-01

    Electron transfer dissociation (ETD) is a valuable tool for protein sequence analysis, especially for the fragmentation of intact proteins. However, low product ion signal-to-noise often requires some degree of signal averaging to achieve high quality MS/MS spectra of intact proteins. Here we describe a new implementation of ETD on the newest generation of quadrupole-Orbitrap-linear ion trap Tribrid, the Orbitrap Fusion Lumos, for improved product ion signal-to-noise via ETD reactions on larger precursor populations. In this new high precursor capacity ETD implementation, precursor cations are accumulated in the center section of the high pressure cell in the dual pressure linear ion trap prior to charge-sign independent trapping, rather than precursor ion sequestration in only the back section as is done for standard ETD. This new scheme increases the charge capacity of the precursor accumulation event, enabling storage of approximately three fold more precursor charges. High capacity ETD boosts the number of matching fragments identified in a single MS/MS event, reducing the need for spectral averaging. These improvements in intra-scan dynamic range via reaction of larger precursor populations, which have been previously demonstrated through custom modified hardware, are now available on a commercial platform, offering considerable benefits for intact protein analysis and top down proteomics. In this work, we characterize the advantages of high precursor capacity ETD through studies with myoglobin and carbonic anhydrase. PMID:26589699

  9. En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries.

    PubMed

    Yu, Yi; Xiong, Yuyan; Montani, Jean-Pierre; Yang, Zhihong; Ming, Xiu-Fen

    2016-02-25

    Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2(.-)) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2(.-) levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2(.-), it is difficult to measure NO and O2(.-) directly in a blood vessel. Numerous methods have been developed to measure NO and O2(.-) production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2(.-) using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2(.-) levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2(.-) in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

  10. Platelet Activation by Low Concentrations of Intact Oxidized LDL Particles Involves the PAF Receptor

    PubMed Central

    Chen, Rui; Chen, Xi; Salomon, Robert G.; McIntyre, Thomas M.

    2008-01-01

    Objective Mitochondrial depolarization aids platelet activation. Oxidized LDL (oxLDL) contains the medium length oxidatively truncated phospholipid hexadecyl azelaoyl-lysoPAF (HAz-LPAF) that disrupts mitochondrial function in nucleated cells, so oxLDL may augment platelet activation. Methods and Results Flow cytometry showed intact oxLDL particles synergized with sub-threshold amounts of soluble agonists to increase intracellular Ca++, and initiate platelet aggregation and surface expression of activated gpIIb/IIIa and P-selectin. oxLDL also induced aggregation and increased intracellular Ca++ in FURA2-labeled cells by itself at low, although not higher, concentrations. HAz-LPAF, alone and in combination with sub-stimulatory amounts of thrombin, rapidly increased cytoplasmic Ca++ and initiated aggregation. HAz-LPAF depolarized mitochondria in intact platelets, but this required concentrations beyond those that directly activated platelets. An unexpectedly large series of chemically pure truncated phospholipids generated by oxidative fragmentation of arachidonoyl-, docosahexaneoyl-, or linoleoyl alkyl phospholipids were platelet agonists. The PAF receptor, thought to effectively recognize only phospholipids with very short sn-2 residues, was essential for platelet activation because PAF receptor agonists blocked signaling by all these medium length phospholipids and oxLDL. Conclusions Intact oxLDL particles activate platelets through the PAF receptor, and the PAF receptor responds to a far wider range of oxidized phospholipids in oxLDL than anticipated. PMID:19112165

  11. The intact structural form of LLO in endosomes cannot protect against listeriosis

    PubMed Central

    Rodriguez-Del Rio, Estela; Frande-Cabanes, Elisabet; Tobes, Raquel; Pareja, Eduardo; Lecea-Cuello, M. Jesús; Ruiz-Sáez, Marta; Carrasco-Marín, Eugenio; Alvarez-Dominguez, Carmen

    2011-01-01

    LLO is the major immuno-dominant antigen in listeriosis and is also required for protective immunity. Two forms of LLO can be observed in endosomal membranes, a LLO intact form and a Ctsd-processed LLO1-491 form. Endosomes obtained from resting macrophages contained only LLO intact forms, while endosomes obtained from IFN-activated macrophages contained both forms. Both types of endosomes elicited LLO90-91/CD8+ and LLO189-201/CD4+ specific immune responses. However, only endosomes containing the Ctsd-processed LLO1-491 form showed significant CD4+ and CD8+ T cell responses similar to LM infected bone marrow derived macrophages and characteristic of protective Listeria immunity. Moreover, endosomes with intact LLO could not confer protection as vaccine carriers against murine listeriosis. While endosomes with Ctsd-processed LLO1-491 form showed a moderate ability, slightly lower than high efficiency vaccine vectors as MØ infected with LM. These studies argue that all cell-free membrane vesicles might serve as valid vaccine carriers against infectious agents. Exclusively those cell-free vesicles MIIC competent for LLO processing are protective vaccines vectors since they recruit significant numbers of mature dendritic cells to the vaccination sites and contain a LLO1-491 form that might be accessible for MHC class I and class II antigen presentation. PMID:22003433

  12. Calculation of ion currents across the inner membrane of functionally intact mitochondria

    PubMed Central

    Kane, Daniel A; Pavlov, Evgeny V

    2013-01-01

    Mitochondrial ion transport systems play a central role in cell physiology. Rates of Ca2+ and K+ transport across the inner mitochondrial membrane have been derived from the measurement of ion accumulation over time within functional isolated mitochondria or mitochondria of cultured cells. Alternatively, the electrical currents generated by ionic flux have been directly measured in purified and swollen mitochondrial samples (mitoplasts) or reconstituted channels, and typically range from 1 pA to several 100s pA. However, the direct electrophysiological approach necessarily requires extensive processing of the mitochondria prior to measurement, which can only be performed on isolated mitoplasts. To compare rates of mitochondrial ion transport measured in electrophysiological experiments to those measured in intact mitochondria and cells, we converted published rates of mitochondrial ion uptake into units of ionic current. We estimate that for monovalent ions, uptake by intact mitochondria at the rate of 1 nmol ∙ mg−1 protein ∙ min−1 is equivalent to 0.2 fA of current per whole single mitochondrion (0.4 fA for divalent ions). In intact mitochondria, estimated rates of electrogenic cation uptake are limited to 1–100 fA of integral current per single mitochondrion. These estimates are orders of magnitude lower than the currents through mitochondrial channels directly measured via patch-clamp or artificial lipid bilayer approaches. PMID:24037064

  13. Perceived parental control processes, parent-child relational qualities and psychological well-being of Chinese adolescents in intact and non-intact families in Hong Kong.

    PubMed

    Shek, Daniel T L; Lee, Tak Yan

    2007-01-01

    This paper examines whether Chinese adolescents' perceptions (N = 3,017) of parental behavioral control (parental knowledge, expectation, monitoring, discipline, and demandingness as well as parental control based on indigenous Chinese concepts), parental psychological control, parent-child relational qualities (perceived parental trust, child's trust of the parents, child's readiness to communicate with the parents, and child's satisfaction with parental control), and adolescent psychological well-being (hopelessness, mastery, life satisfaction and self-esteem) differed in intact and non-intact families. Results showed that relative to non-intact families, parental behavioral control processes were higher and parent-child relational qualities were better in intact families. In contrast, parental psychological control was higher in non-intact families than in intact families. Finally, the psychological well-being of adolescents in non-intact families was poorer than that of adolescents in intact families.

  14. Phosphorylation of intact erythrocytes in human muscular dystrophy

    SciTech Connect

    Johnson, R.M.; Nigro, M.

    1986-04-01

    The uptake of exogenous /sup 32/Pi into the membrane proteins of intact erythrocytes was measured in 8 patients with Duchenne muscular dystrophy. No abnormalities were noted after autoradiographic analysis. This contrasts with earlier results obtained when isolated membranes were phosphorylated with gamma-(/sup 32/P)ATP, and suggests a possible reinterpretation of those experiments.

  15. Operative balloon dilatation for pulmonary atresia with intact ventricular septum.

    PubMed Central

    Hamilton, J R; Fonseka, S F; Wilson, N; Dickinson, D F; Walker, D R

    1987-01-01

    In six infants with pulmonary atresia and intact ventricular septum operative balloon dilatation was used to achieve continuity between the right ventricle and the main pulmonary artery as the initial procedure. Two of the six subsequently needed an aortico pulmonary shunt. All six are alive and well. Images Fig PMID:3676024

  16. Interleukin 6 in intact and injured mouse peripheral nerves.

    PubMed

    Reichert, F; Levitzky, R; Rotshenker, S

    1996-03-01

    The multifunctional cytokine interleukin 6 (IL-6) has direct growth, survival and differentiation effects on peripheral and central neurons. Furthermore, it can modulate the production by non-neuronal cells of other cytokines and growth factors, and thereby affect nerve cells indirectly. We have studied IL-6 expression and production in intact and injured peripheral nerves of C57/BL/6NHSD mice, which display the normal rapid progression of Wallerian degeneration. The IL-6 mRNA was detected in nerves degenerating in vitro or in vivo, but not in intact nerves. In vitro- and in vivo-degenerating nerve segments and neuroma nerve segments synthesized and secreted IL-6. The onset of IL-6 production was rapid and prolonged. It was detected as early as 2 h after injury and persisted for the entire period of 21 days tested after the injury. Of the non-neuronal cells that reside in intact and injured nerves, macrophages and fibroblasts were the major contributors to IL-6 production. We also studied IL-6 production in intact and injured nerves of mutant C57BL/6-WLD/OLA/NHSD mice, which display very slow progression of Wallerian degeneration. Injured nerves of C57BL/6-WLD/OLA/NHSD mice produced significantly lower amounts of IL-6 than did rapidly degenerating nerves of C57/BL/6NHSD mice.

  17. Locomotion in intact and in brain cortex-ablated cats.

    PubMed

    López Ruiz, José Roberto; Castillo Hernández, Luis; De la Torre Valdovinos, Braniff; Franco Rodríguez, Nancy Elizabeth; Dueñas Jiménez, Judith Marcela; Dueñas Jiménez, Alejandro; Rivas-Carrillo, Jorge David; Dueñas Jiménez, Sergio Horacio

    2017-09-01

    The current decerebration procedures discard the role of the thalamus in the motor control and decortication only rules out the brain cortex part, leaving a gap between the brain cortex and the subthalamic motor regions. In here we define a new preparation denominated Brain Cortex-Ablated Cat (BCAC), in which the frontal and parietal brain cortices as well as the central white matter beneath them were removed, this decerebration process may be considered as suprathalamic, since the thalamus remained intact. To characterize this preparation cat hindlimb electromyograms (EMG), kinematics and cutaneous reflexes (CR) produced by electrical stimulation of sural (SU) or saphenous (SAPH) nerves were analyzed during locomotion in intact and in BCAC. In cortex-ablated cats compared to intact cats, the hindlimb EMG amplitude was increased in the flexors, whereas in most extensors the amplitude was decreased. Bifunctional muscle EMGs presented complex and speed-dependent amplitude changes. In intact cats CR produced an inhibition of extensors, as well as excitation and inhibition of flexors, and a complex pattern of withdrawal responses in bifunctional muscles. The same stimuli applied to BCAC produced no detectable responses, but in some cats cutaneous reflexes produced by electrical stimulation of saphenous nerve reappeared when the locomotion speed increased. In BCAC, EMG and kinematic changes, as well as the absence of CR, imply that for this cat preparation there is a partial compensation due to the subcortical locomotor apparatus generating close to normal locomotion. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  18. Fostering Activities of Daily Living by Intact Nursing Home Residents

    ERIC Educational Resources Information Center

    Blair, Charles E.; Glaister, Judy; Brown, Alston; Phillips, Carolyn

    2007-01-01

    We assessed effectiveness of four education programs in providing nursing assistants with ability to produce a therapeutic milieu supportive of intact residents' activities of daily living, positive self-esteem and mood: (1) a combination of Orem's Systems of Nursing Care and Skinner's Applied Behavioral Analysis, (2) Applied Behavioral Analysis,…

  19. Fostering Activities of Daily Living by Intact Nursing Home Residents

    ERIC Educational Resources Information Center

    Blair, Charles E.; Glaister, Judy; Brown, Alston; Phillips, Carolyn

    2007-01-01

    We assessed effectiveness of four education programs in providing nursing assistants with ability to produce a therapeutic milieu supportive of intact residents' activities of daily living, positive self-esteem and mood: (1) a combination of Orem's Systems of Nursing Care and Skinner's Applied Behavioral Analysis, (2) Applied Behavioral Analysis,…

  20. Factors mediating cheatgrass invasion of intact salt desert shrubland

    Treesearch

    Susan E. Meyer; Susan C. Garvin; Julie Beckstead

    2001-01-01

    Cheatgrass (Bromus tectorum) has recently displaced salt desert shrubland in many areas of the Great Basin. We studied the dynamics of cheatgrass invasion into an intact shadscale-gray molly community in Dugway Valley, Utah, by adding seeds and manipulating disturbance regime and resource availability. Shrub clipping or cryptobiotic crust trampling on large plots...

  1. Tympanoplasty in chronic otitis media patients with an intact, but severely retracted malleus: a treatment challenge.

    PubMed

    Hol, Myrthe K S; Nguyen, Dai Q; Schlegel-Wagner, Christoph; Pabst, Gunther; Linder, Thomas E

    2010-12-01

    To analyze the outcome of patients with chronic otitis media (COM) with an intact, but markedly medialized ossicular chain, treated by removing the malleus head and interposing an autologous incus and then an underlay myringoplasty. Retrospective clinical study. Tertiary referral center. The search criteria within the prospective surgical database was COM with a central perforation (without cholesteatoma) with a markedly medialized malleus handle (the umbo adherent to the promontory) with an intact ossicular chain (study, n = 15) or an incus necrosis at the lenticular process (incus, n = 23). Only primary surgeries performed at our otorhinolaryngology department were included. All patients underwent the same surgical procedure consisting of an autologous incus interposition and underlay myringoplasty with temporalis fascia. The patients' audiological and follow-up data were retrieved from the database. The postoperative audiogram (0.5-3 kHz) with the longest follow-up was used. The preoperative air-conduction thresholds were less impaired in the study group than in the incus group. After their surgery, all, except 3 patients, improved their hearing, and 97% had an intact tympanic membrane at a mean follow-up of 2 years. The air-bone gap was closed within 20 dB in 80% (study) and in 87% (incus), in one third of all patients even within 10 dB. Although the largest improvement was seen in the lower frequencies, closure of the air-bone gap at 4 kHz was difficult to achieve. Patients presenting with COM, a (central) perforation, a medially rotated malleus and intact ossicular chain are a treatment challenge. Lateralizing the malleus handle may require disconnection of the ossicular chain and an autologous incus interposition to bring back the reconstructed tympanic membrane in its original position and improve the hearing.

  2. Threats to intact tropical peatlands and opportunities for their conservation.

    PubMed

    Roucoux, K H; Lawson, I T; Baker, T R; Del Castillo Torres, D; Draper, F C; Lähteenoja, O; Gilmore, M P; Honorio Coronado, E N; Kelly, T J; Mitchard, E T A; Vriesendorp, C F

    2017-03-08

    Large, intact areas of tropical peatland are highly threatened at a global scale by the expansion of commercial agriculture and other forms of economic development. Conserving peatlands on a landscape scale, with their hydrology intact, is of international conservation importance to preserve their distinctive biodiversity and ecosystem services and maintain their resilience to future environmental change. We explored threats to and opportunities for conserving remaining intact tropical peatlands; thus, we excluded peatlands of Indonesia and Malaysia, where extensive deforestation, drainage, and conversion to plantations means conservation in this region can protect only small fragments of the original ecosystem. We focused on a case study, the Pastaza-Marañón Foreland Basin (PMFB) in Peru, which is among the largest known intact tropical peatland landscapes in the world and is representative of peatland vulnerability. Maintenance of the hydrological conditions critical for carbon storage and ecosystem function of peatlands is, in the PMFB, primarily threatened by expansion of commercial agriculture linked to new transport infrastructure that is facilitating access to remote areas. There remain opportunities in the PMFB and elsewhere to develop alternative, more sustainable land-use practices. Although some of the peatlands in the PMFB fall within existing legally protected areas, this protection does not include the most carbon-dense (domed pole forest) areas. New carbon-based conservation instruments (e.g., REDD+, Green Climate Fund), developing markets for sustainable peatland products, transferring land title to local communities, and expanding protected areas offer pathways to increased protection for intact tropical peatlands in Amazonia and elsewhere, such as those in New Guinea and Central Africa which remain, for the moment, broadly beyond the frontier of commercial development. © 2017 The Authors. Conservation Biology published by Wiley Periodicals, Inc

  3. The Multilevel Mixed Intact Group Analysis: A Mixed Method to Seek, Detect, Describe, and Explain Differences Among Intact Groups

    ERIC Educational Resources Information Center

    Schoonenboom, Judith

    2016-01-01

    Educational innovations often involve intact subgroups, such as school classes or university departments. In small-scale educational evaluation research, typically involving 1 to 20 subgroups, differences among these subgroups are often neglected. This article presents a mixed method from a qualitative perspective, in which differences among…

  4. The Multilevel Mixed Intact Group Analysis: A Mixed Method to Seek, Detect, Describe, and Explain Differences Among Intact Groups

    ERIC Educational Resources Information Center

    Schoonenboom, Judith

    2016-01-01

    Educational innovations often involve intact subgroups, such as school classes or university departments. In small-scale educational evaluation research, typically involving 1 to 20 subgroups, differences among these subgroups are often neglected. This article presents a mixed method from a qualitative perspective, in which differences among…

  5. Structural determination of intact proteins using mass spectrometry

    DOEpatents

    Kruppa, Gary; Schoeniger, Joseph S.; Young, Malin M.

    2008-05-06

    The present invention relates to novel methods of determining the sequence and structure of proteins. Specifically, the present invention allows for the analysis of intact proteins within a mass spectrometer. Therefore, preparatory separations need not be performed prior to introducing a protein sample into the mass spectrometer. Also disclosed herein are new instrumental developments for enhancing the signal from the desired modified proteins, methods for producing controlled protein fragments in the mass spectrometer, eliminating complex microseparations, and protein preparatory chemical steps necessary for cross-linking based protein structure determination.Additionally, the preferred method of the present invention involves the determination of protein structures utilizing a top-down analysis of protein structures to search for covalent modifications. In the preferred method, intact proteins are ionized and fragmented within the mass spectrometer.

  6. Radioactive satellites - Intact reentry and breakup by debris impact

    NASA Technical Reports Server (NTRS)

    Anz-Meador, P. D.; Potter, A. E., Jr.

    1991-01-01

    There is a substantial mass of radioactive material in nuclear reactors or radioisotope thermal generators (RTGs) in orbit about the earth. This paper examines the reentry of intact nuclear fuel cores and RTGs and the fragmentation and subsequent radioactive debris cloud deposition and evolution resulting from the impact of orbital debris upon an orbiting reactor, fuel core, or RTG. To assess the intact reentry, decay rates and a predicted decay date using historical and projected orbital decay data, are estimated. The current NASA debris environment model is utilized to estimate impact rates and debris cloud evolution of a fragmentation event. Results of these analyses are compared and concepts are tendered which would tend to minimize the radiological debris hazard to personnel and structures both on the earth's surface and in low earth orbit.

  7. Radioactive satellites - Intact reentry and breakup by debris impact

    NASA Technical Reports Server (NTRS)

    Anz-Meador, P. D.; Potter, A. E., Jr.

    1991-01-01

    There is a substantial mass of radioactive material in nuclear reactors or radioisotope thermal generators (RTGs) in orbit about the earth. This paper examines the reentry of intact nuclear fuel cores and RTGs and the fragmentation and subsequent radioactive debris cloud deposition and evolution resulting from the impact of orbital debris upon an orbiting reactor, fuel core, or RTG. To assess the intact reentry, decay rates and a predicted decay date using historical and projected orbital decay data, are estimated. The current NASA debris environment model is utilized to estimate impact rates and debris cloud evolution of a fragmentation event. Results of these analyses are compared and concepts are tendered which would tend to minimize the radiological debris hazard to personnel and structures both on the earth's surface and in low earth orbit.

  8. Intrathecal application of cyproheptadine impairs locomotion in intact rats.

    PubMed

    Majczyński, Henryk; Cabaj, Anna; Górska, Teresa

    In intact adult rats, cyproheptadine, a 5-HT2 antagonist, administered intrathecally at the midlumbar segments was found to impair hindlimb locomotor movements during overground locomotion. These effects were dose-dependent; they varied from transient complete hindlimb paraplegia seen at doses of 300 microg/20 microl, to short-lasting trunk instability at doses of 100 microg/20 microl. After the return of overground locomotion, transient abduction of one of the hindlimbs was observed in some animals. These findings demonstrate that the blockade of 5-HT2 receptors affects locomotion in intact rats. Our results provide support for the hypothesis of serotonergic involvement in rat locomotion, which, so far, has been based mainly on the effects of 5-HT2 agonists on the recovery of locomotion in spinal rats.

  9. Spelling-stress regularity effects are intact in developmental dyslexia.

    PubMed

    Mundy, Ian R; Carroll, Julia M

    2013-01-01

    The current experiment investigated conflicting predictions regarding the effects of spelling-stress regularity on the lexical decision performance of skilled adult readers and adults with developmental dyslexia. In both reading groups, lexical decision responses were significantly faster and significantly more accurate when the orthographic structure of a word ending was a reliable as opposed to an unreliable predictor of lexical stress assignment. Furthermore, the magnitude of this spelling-stress regularity effect was found to be equivalent across reading groups. These findings are consistent with intact phoneme-level regularity effects also observed in dyslexia. The paper discusses how findings of intact spelling-sound regularity effects at both prosodic and phonemic levels, as well as other similar results, can be reconciled with the obvious difficulties that people with dyslexia experience in other domains of phonological processing.

  10. Papain-gel degrades intact nonmineralized type I collagen fibrils.

    PubMed

    Bertassoni, L E; Marshall, G W

    2009-01-01

    Papain-gel has been utilized as a chemo-mechanical material for caries removal due to its ability to preserve underlying sound dentin. However, little is known about the effect of the papain enzyme on intact type I collagen fibrils that compose the dentin matrix. Here we sought to define structural changes that occur in intact type I collagen fibrils after an enzymatic treatment with a papain-gel. Intact and nonmineralized type I collagen fibrils from rat tail were obtained and treated with a papain-gel (Papacarie) for 30 s, rinsed with water and imaged using an atomic force microscope (AFM). Additionally, polished healthy dentin specimens were also treated using the same protocol described above and had their elastic modulus (E) and hardness (H) measured by means of AFM-based nanoindentation. AFM images showed that the papain-gel induced partial degradation of the fibrils surface, yet no rupture of fibrils was noticed. The distinction between gap and overlap zones of fibrils vanished in most regions after treatment, and overlap zones appeared to be generally more affected. Mechanical data suggested a gradual decrease in E and H after treatments. A significant two-fold drop from the values of normal dentin (E=20+/-1.9, H=0.8+/-0.08 GPa) was found after four applications (E=9.7+/-3.2, H=0.24+/-0.1 GPa) (P<0.001), which may be attributed to the degradation of proteoglycans of the matrix. In summary, this study provided novel evidence that intact nonmineralized type I collagen fibrils are partially degraded by a papain-gel. (c) 2010 Wiley Periodicals, Inc.

  11. Papain-gel Degrades Intact Nonmineralized Type I Collagen Fibrils

    PubMed Central

    BERTASSONI, L. E.; MARSHALL, G. W.

    2010-01-01

    Summary Papain-gel has been utilized as a chemomechanical material for caries removal due to its ability to preserve underlying sound dentin. However, little is known about the effect of the papain enzyme on intact type I collagen fibrils that compose the dentin matrix. Here we sought to define structural changes that occur in intact type I collagen fibrils after an enzymatic treatment with a papaingel. Intact and nonmineralized type I collagen fibrils from rat tail were obtained and treated with a papain-gel (Papacarie) for 30 s, rinsed with water and imaged using an atomic force microscope (AFM). Additionally, polished healthy dentin specimens were also treated using the same protocol described above and had their elastic modulus (E) and hardness (H) measured by means of AFM-based nanoindentation. AFM images showed that the papain-gel induced partial degradation of the fibrils surface, yet no rupture of fibrils was noticed. The distinction between gap and overlap zones of fibrils vanished in most regions after treatment, and overlap zones appeared to be generally more affected. Mechanical data suggested a gradual decrease in E and H after treatments. A significant two-fold drop from the values of normal dentin (E= 20 +/− 1.9, H = 0.8 +/− 0.08 GPa) was found after four applications (E = 9.7 +/− 3.2, H = 0.24 +/− 0.1 GPa) ( P<0.001), which may be attributed to the degradation of proteoglycans of the matrix. In summary, this study provided novel evidence that intact nonmineralized type I collagen fibrils are partially degraded by a papain-gel. PMID:20205185

  12. Chlorophyll Determination in Intact Tissues Using N,N-Dimethylformamide

    PubMed Central

    Moran, Rami; Porath, Dan

    1980-01-01

    Photosynthetic pigments from etiolated cucumber (Cucumis sativus var. Beit Alpha improved, Hazera Co., Gedera) cotyledons were extracted by direct immersion of the intact cotyledons into the solvent N,N-dimethylformamide (DMF). The solvent is especially efficient when pigment concentration is low; time and tools are saved and the loss of pigment that usually occurs in more complicated extraction procedures is prevented. The specific absorption coefficient of chlorophyll a in DMF was also determined. PMID:16661217

  13. Crisscross heart with dextrocardia and intact interventricular septum.

    PubMed

    Muneer, P Kader; Kalathingathodika, Sajeer; Chakanalil, Govindan Sajeev; Sony, Manuel M

    2014-01-01

    Crisscross heart is a rare congenital heart disease characterized by a twisted atrioventricular connection, as a result of rotation of the ventricular mass along its long axis. We report an asymptomatic 48-year-old woman referred to us for evaluation of a cardiac murmur. Further evaluation showed situs solitus, dextrocardia with normal atrioventricular and ventriculoarterial connection, and a crisscross relation of the atrioventricular valves. Unlike the usual case of crisscross heart, our patient had an intact ventricular septum.

  14. Binding of Clostridium botulinum C3 exoenzyme to intact cells.

    PubMed

    Rohrbeck, Astrid; von Elsner, Leonie; Hagemann, Sandra; Just, Ingo

    2014-06-01

    C3 from Clostridium botulinum (C3) specifically modifies Rho GTPases RhoA, RhoB, and RhoC by mono-ADP-ribosylation. The confined substrate profile of C3 is the basis for its use as pharmacological tool in cell biology to study cellular functions of Rho GTPases. Although C3 exoenzyme does not possess a cell-binding/-translocation domain, C3 is taken up by intact cells via an unknown mechanism. In the present work, binding of C3 to the hippocampus-derived HT22 cells and J774A.1 macrophages was characterized. C3 bound concentration-dependent to HT22 and J774A.1 cells. Pronase treatment of intact cells significantly reduced both C3 binding and C3 cell entry. Removal of sugar residues by glycosidase F treatment resulted in an increased binding of C3, but a reduced cell entry. To explore the involvement of phosphorylation in the binding process of C3, intact HT22 and J774A.1 cells were pre-treated with vanadate prior to incubation with C3. Inhibition of de-phosphorylation by vanadate resulted in an increased binding of C3. To differentiate between intracellular and extracellular phosphorylation, intact cells were treated with CIP (calf intestine phosphatase) to remove extracellular phosphate residues. The removal of phosphate residues resulted in a strong reduction in binding of C3 to cells. In sum, the C3 membranous binding partner is proteinaceous, and the glycosylation as well as the phosphorylation state is critical for efficient binding of C3.

  15. Dental abscess in a tooth with intact dens evaginatus.

    PubMed

    Cho, S Y

    2006-03-01

    This article reports a case of dental abscess in a mandibular premolar with intact dens evaginatus. Dentists are advised to critically evaluate those teeth with dens evaginatus, both clinically and radiographically, before attempting prophylactic treatments. This is particularly important medicolegally in case the tooth develops symptoms shortly after the prophylactic treatment. Dentists practising in Western countries should also be aware of this dental anomaly because of the increasing global migration of people from Asia.

  16. Antimicrobial activity of a new intact skin antisepsis formulation.

    PubMed

    Russo, Antonello; Viotti, Pier Luigi; Vitali, Matteo; Clementi, Massimo

    2003-04-01

    Different antiseptic formulations have shown limitations when applied to disinfecting intact skin, notably short-term tolerability and/or efficacy. The purpose of this study was optimizing a new antiseptic formulation specifically targeted at intact skin disinfection and evaluating its in vitro microbicidal activity and in vivo efficacy. The biocidal properties of the antiseptic solution containing 0.5% chloramine-T diluted in 50% isopropyl alcohol (Cloral; Eurospital SpA Trieste, Italy) were measured in vitro versus gram-positive-, gram-negative-, and acid-alcohol-resistant germs and fungi with standard suspension tests in the presence of fetal bovine serum. Virus-inhibiting activity was evaluated in vitro against human cytomegalovirus, herpes simplex virus, poliovirus, hepatitis B virus, and hepatitis C virus. Tests used different methods for the different biologic and in vitro replication capacity of these human viruses. Lastly, Cloral tolerability and skin colonization retardation efficacy after disinfection were studied in vivo. The antiseptic under review showed fast and sustained antimicrobial activity. The efficacy of Cloral against clinically important bacterial and viral pathogens and fungi was highlighted under the experimental conditions described in this article. Finally, microbial regrowth lag and no side effects were documented in vivo after disinfection of 11 volunteers. A stable chloramine-T solution in isopropyl alcohol may be suggested for intact skin antisepsis.

  17. Global forest loss disproportionately erodes biodiversity in intact landscapes.

    PubMed

    Betts, Matthew G; Wolf, Christopher; Ripple, William J; Phalan, Ben; Millers, Kimberley A; Duarte, Adam; Butchart, Stuart H M; Levi, Taal

    2017-07-27

    Global biodiversity loss is a critical environmental crisis, yet the lack of spatial data on biodiversity threats has hindered conservation strategies. Theory predicts that abrupt biodiversity declines are most likely to occur when habitat availability is reduced to very low levels in the landscape (10-30%). Alternatively, recent evidence indicates that biodiversity is best conserved by minimizing human intrusion into intact and relatively unfragmented landscapes. Here we use recently available forest loss data to test deforestation effects on International Union for Conservation of Nature Red List categories of extinction risk for 19,432 vertebrate species worldwide. As expected, deforestation substantially increased the odds of a species being listed as threatened, undergoing recent upgrading to a higher threat category and exhibiting declining populations. More importantly, we show that these risks were disproportionately high in relatively intact landscapes; even minimal deforestation has had severe consequences for vertebrate biodiversity. We found little support for the alternative hypothesis that forest loss is most detrimental in already fragmented landscapes. Spatial analysis revealed high-risk hot spots in Borneo, the central Amazon and the Congo Basin. In these regions, our model predicts that 121-219 species will become threatened under current rates of forest loss over the next 30 years. Given that only 17.9% of these high-risk areas are formally protected and only 8.9% have strict protection, new large-scale conservation efforts to protect intact forests are necessary to slow deforestation rates and to avert a new wave of global extinctions.

  18. Posttranslational protein modification by polyamines in intact and regenerating nerves.

    PubMed

    Chakraborty, G; Leach, T; Zanakis, M F; Sturman, J A; Ingoglia, N A

    1987-03-01

    A 150,000-g supernatant from axoplasm of the giant axon of the stellate nerve of the squid and from rat sciatic and goldfish optic nerves was found to be able to incorporate covalently [3H]putrescine and [3H]spermidine into an exogenous protein (N,N'-dimethylcasein). Incorporation of radioactivity was inhibited by CuSO4, a specific inhibitor of transglutaminases, the enzymes mediating these reactions in other tissues. Analysis of pH and temperature range and enzyme kinetics displayed characteristics predicted for transglutaminase-mediated reactions. Transglutaminase activity increased during regeneration of both vertebrate nerves, but greater activity was found in segments of nerve containing no intact axons than in either intact segments or in segments containing regenerating axons. Polyacrylamide gel electrophoresis of endogenous modified proteins (in the absence of N,N'-dimethylcasein) showed labeling of 18-, 46- and 200-kilodalton proteins by both [3H]putrescine and [3H]spermidine. Analysis of the protein-bound radioactivity from intact and regenerating rat sciatic nerves demonstrated it to be predominantly in the form of the parent radioactive polyamine. These experiments demonstrate the covalent modification of proteins by polyamines at low levels in squid axoplasm and at relatively higher levels in rat sciatic and goldfish optic nerves. In the latter two cases, the activity of these modification reactions may be due in part to the modification of axonal proteins, but the majority of the activity occurs in nonneuronal cells of the nerve.

  19. Vehicle influence on permeation through intact and compromised skin.

    PubMed

    Gujjar, Meera; Banga, Ajay K

    2014-09-10

    The purpose of this study was to compare the transdermal permeation of a model compound, diclofenac diethylamine, from a hydrophilic and lipophilic vehicle across in vitro models simulating compromised skin. Mineral oil served as a lipophilic vehicle while 10mM phosphate buffered saline served as a hydrophilic vehicle. Compromised skin was simulated by tape stripping, delipidization, or microneedle application and compared with intact skin as a control. Transepidermal water loss was measured to assess barrier function. Skin compromised with tape stripping and delipidization significantly (p<0.05) increased permeation of diclofenac diethylamine compared to intact and microneedle treated skin with phosphate buffered saline vehicle. A similar trend in permeation was observed with mineral oil as the vehicle. For both vehicles, permeation across skin increased in the same order and correlated with degree of barrier impairment as indicated by transepidermal water loss values: intact

  20. Does human leukocyte elastase degrade intact skin elastin?

    PubMed

    Schmelzer, Christian E H; Jung, Michael C; Wohlrab, Johannes; Neubert, Reinhard H H; Heinz, Andrea

    2012-11-01

    This study aimed to investigate the susceptibility of intact fibrillar human elastin to human leukocyte elastase and cathepsin G. Elastin is a vital protein of the extracellular matrix of vertebrates, and provides exceptional properties including elasticity and tensile strength to many tissues and organs, including the aorta, lung, cartilage, elastic ligaments and skin, and is thus critical for their long-term function. Mature elastin is an insoluble and extremely durable protein that undergoes very little turnover, but sustained exposure to proteases may lead to irreversible and severe damage, and thus to functional loss of the elastic fiber network. Hence, it is a key issue to understand which enzymes actually initiate elastolysis under certain pathological conditions or during intrinsic aging. In this paper, we provide a complete workflow for isolation of pure and intact elastin from very small tissue samples to test enzymes for their elastolytic potential. This workflow was applied to skin samples from variously aged individuals, and it was found that strong differences exist in the degradability of the elastins investigated. In summary, human leukocyte elastase was unable to degrade intact elastin fibers but hydrolyzed elastin derived from the skin of old people. However, cathepsin G cleaved all elastin samples, even those derived from younger individuals. These results indicate that human leukocyte elastase is not a driving force for elastolysis, but may nevertheless promote further breakdown of elastic fibers after the action of other enzymes such as cathepsin G. © 2012 The Authors Journal compilation © 2012 FEBS.

  1. On plant detection of intact tomato fruits using image analysis and machine learning methods.

    PubMed

    Yamamoto, Kyosuke; Guo, Wei; Yoshioka, Yosuke; Ninomiya, Seishi

    2014-07-09

    Fully automated yield estimation of intact fruits prior to harvesting provides various benefits to farmers. Until now, several studies have been conducted to estimate fruit yield using image-processing technologies. However, most of these techniques require thresholds for features such as color, shape and size. In addition, their performance strongly depends on the thresholds used, although optimal thresholds tend to vary with images. Furthermore, most of these techniques have attempted to detect only mature and immature fruits, although the number of young fruits is more important for the prediction of long-term fluctuations in yield. In this study, we aimed to develop a method to accurately detect individual intact tomato fruits including mature, immature and young fruits on a plant using a conventional RGB digital camera in conjunction with machine learning approaches. The developed method did not require an adjustment of threshold values for fruit detection from each image because image segmentation was conducted based on classification models generated in accordance with the color, shape, texture and size of the images. The results of fruit detection in the test images showed that the developed method achieved a recall of 0.80, while the precision was 0.88. The recall values of mature, immature and young fruits were 1.00, 0.80 and 0.78, respectively.

  2. A Caenorhabditis elegans developmental decision requires insulin signaling-mediated neuron-intestine communication

    PubMed Central

    Hung, Wesley L.; Wang, Ying; Chitturi, Jyothsna; Zhen, Mei

    2014-01-01

    Adverse environmental conditions trigger C. elegans larvae to activate an alternative developmental program, termed dauer diapause, which renders them stress resistant. High-level insulin signaling prevents constitutive dauer formation. However, it is not fully understood how animals assess conditions to choose the optimal developmental program. Here, we show that insulin-like peptide (ILP)-mediated neuron-intestine communication plays a role in this developmental decision. Consistent with, and extending, previous findings, we show that the simultaneous removal of INS-4, INS-6 and DAF-28 leads to fully penetrant constitutive dauer formation, whereas the removal of INS-1 and INS-18 significantly inhibits constitutive dauer formation. These ligands are processed by the proprotein convertases PC1/KPC-1 and/or PC2/EGL-3. The agonistic and antagonistic ligands are expressed by, and function in, neurons to prevent or promote dauer formation. By contrast, the insulin receptor DAF-2 and its effector, the FOXO transcription factor DAF-16, function solely in the intestine to regulate the decision to enter diapause. These results suggest that the nervous system normally establishes an agonistic ILP-dominant paradigm to inhibit intestinal DAF-16 activation and allow reproductive development. Under adverse conditions, a switch in the agonistic-antagonistic ILP balance activates intestinal DAF-16, which commits animals to diapause. PMID:24671950

  3. Combination of SDS-PAGE and intact mass analysis for rapid determination of heterogeneities in monoclonal antibody therapeutics.

    PubMed

    Yamada, Hideaki; Matsumura, Chiemi; Yamada, Keita; Teshima, Koichiro; Hiroshima, Takashi; Kinoshita, Mitsuhiro; Suzuki, Shigeo; Kakehi, Kazuaki

    2017-05-01

    mAbs are currently mainstream in biopharmaceuticals, and their market has been growing due to their high target specificity. Characterization of heterogeneities in mAbs is performed to secure their quality and safety by physicochemical analyses. However, they require time-consuming task, which often strain the resources of drug development in pharmaceuticals. Rapid and direct method to determine the heterogeneities should be a powerful tool for pharmaceutical analysis. Considering the advantages of electrophoresis and MS, this study addresses the combination of SDS-PAGE and intact mass analysis, which provides direct, rapid, and orthogonal determination of heterogeneities in mAb therapeutics. mAb therapeutics that migrated in SDS-PAGE were recovered from gel by treatment with SDC-containing buffer. Usage of SDC-containing buffer as extraction solvent and ethanol-based staining solution enhanced the recovery of intact IgG from SDS-PAGE gels. Recovery of mAbs reached more than 86% with 0.2% SD. The heterogeneities, especially N-glycan variants in the recovered mAb therapeutics, were clearly determined by intact mass analysis. We believe that the study is important in pharmaceuticals‧ perspective since orthogonal combination of gel electrophoresis and intact mass analysis should be pivotal role for rapid and precise characterization of mAbs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Release of proteins from intact chloroplasts induced by reactive oxygen species during biotic and abiotic stress.

    PubMed

    Kwon, Kwang-Chul; Verma, Dheeraj; Jin, Shuangxia; Singh, Nameirakpam D; Daniell, Henry

    2013-01-01

    Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress) or paraquat (abiotic stress), GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II) made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide), which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These results suggest a

  5. Release of Proteins from Intact Chloroplasts Induced by Reactive Oxygen Species during Biotic and Abiotic Stress

    PubMed Central

    Singh, Nameirakpam D.; Daniell, Henry

    2013-01-01

    Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress) or paraquat (abiotic stress), GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II) made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide), which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These results suggest a

  6. An integrated workflow for characterizing intact phosphoproteins from complex mixtures

    PubMed Central

    Wu, Si; Yang, Feng; Zhao, Rui; Tolić, Nikola; Robinson, Errol W.; Camp, David; Smith, Richard D.; Paša-Tolić, Ljiljana

    2014-01-01

    The phosphorylation of any site on a given protein can affect its activity, degradation rate, ability to dock with other proteins or bind divalent cations, and/or its localization. These effects can operate within the same protein; in fact, multisite phosphorylation is a key mechanism for achieving signal integration in cells. Hence, knowing the overall phosphorylation signature of a protein is essential for understanding the "state" of a cell. However, current technologies to monitor the phosphorylation status of proteins are inefficient at determining the relative stoichiometries of phosphorylation at multiple sites. Here we report a new capability for comprehensive liquid chromatography mass spectrometry (LC/MS) analysis of intact phosphoproteins. The technology platform built upon integrated bottom-up and top-down approach that is facilitated by intact protein reversed-phase (RP)LC concurrently coupled with Fourier transform ion cyclotron resonance (FTICR) MS and fraction collection. As the use of conventional RPLC systems for phosphopeptide identification has proven challenging due to the formation of metal ion complexes at various metal surfaces during LC/MS and ESI-MS analysis, we have developed a “metal-free” RPLC-ESI-MS platform for phosphoprotein characterization. This platform demonstrated a significant sensitivity enhancement for phosphorylated casein proteins enriched from a standard protein mixture and revealed the presence of over 20 casein isoforms arising from genetic variants with varying numbers of phosphorylation sites. The integrated workflow was also applied to an enriched yeast phosphoproteome to evaluate the feasibility of this strategy for characterizing complex biological systems, and revealed ~16% of the detected yeast proteins to have multiple phosphorylation isoforms. Intact protein LC/MS platform for characterization of combinatorial posttranslational modifications (PTMs), with special emphasis on multisite phosphorylation, holds

  7. A case of mixed transcortical aphasia with intact naming.

    PubMed

    Heilman, K M; Tucker, D M; Valenstein, E

    1976-09-01

    Altholgh Lichtheim recognized that Wernicke's 'reflex arch' (primary auditory area, to Wernicke's area, to Broca's area, to primary motor area) was important for repetition, he recognized that other areas of the brain (for example, area of concepts or semantic area) must be important in comprehension and voluntary speech. He suggested that Wernicke's area (phonemic area) not only projected to Broca's area (as Wernicke suggested) but that it also projected to the area of concepts. A lesion of this latter pathway or in the area of concepts would produce a syndrome where repetition was intact but comprehension was impaired (e.g. transcortical sensory aphasia). Lichtheim also thought that the area of concepts projected directly to Broca's area and that voluntary speech was mediated by this pathway. Although Lichtheim's model could explain the mechanism underlying transcortical aphasia, his schema could not explain anomic aphasia. Unlike Lichtheim's schema, Kussmaul's schema suggested that the area of concepts projects back to Wernicke's area before projecting to Broca's area. With this schema, a patient with a hypothetical lesion which interrupted the pathway from the area of concepts to Wernicke's area (but did not interrupt the pathway from Wernicke's area to the area of concepts) should be anomic, with normal comprehension and repetition. In order for this latter schema to be plausible there should also be a lesion which interrupts the pathway from Wernicke's area to the area of concepts but does not interrupt the pathway which goes from the area of concepts to Wernicke's area. A patient with this hypothetical lesion should comprehend poorly; however, in spite of poor comprehension, naming and repetition should be intact. We report a patient who demonstrates poor comprehension with intact naming and repetition. This patient could also read aloud but could not comprehend written language. Not only could this patient name objects but he could demonstrate their use

  8. 46 CFR 178.325 - Intact stability requirements-monohull sailing vessels.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... only operates during the daylight hours; (3) It is of the usual type, rig, and hull form, excluding vessels without a weathertight deck, such as open boats; (4) It carries not more than 49 passengers; (5... ballast outside the hull. ...

  9. 46 CFR 178.325 - Intact stability requirements for a sailing vessel.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... non-daylight hours; (3) A vessel of unusual type, rig, or hull form, including vessels without a weathertight deck, such as open boats; (4) A vessel that carries more than 49 passengers; (5) A sailing school... broad, shallow draft vessel with little or no ballast outside the hull. ...

  10. 46 CFR 178.325 - Intact stability requirements-monohull sailing vessels.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... only operates during the daylight hours; (3) It is of the usual type, rig, and hull form, excluding vessels without a weathertight deck, such as open boats; (4) It carries not more than 49 passengers; (5... ballast outside the hull. ...

  11. 46 CFR 178.325 - Intact stability requirements-monohull sailing vessels.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... only operates during the daylight hours; (3) It is of the usual type, rig, and hull form, excluding vessels without a weathertight deck, such as open boats; (4) It carries not more than 49 passengers; (5... ballast outside the hull. ...

  12. 46 CFR 178.325 - Intact stability requirements-monohull sailing vessels.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... only operates during the daylight hours; (3) It is of the usual type, rig, and hull form, excluding vessels without a weathertight deck, such as open boats; (4) It carries not more than 49 passengers; (5... ballast outside the hull. ...

  13. 46 CFR 171.057 - Intact stability requirements for a sailing catamaran.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... hull centerlines in meters (feet). As=the maximum sail area in square meters (square feet). Hc=the height of the center of effort of the sail area above the deck, in meters (feet). W=the total displacement of the vessel, in kilograms (pounds). X=4.88 kilograms/square meter (1.0 pounds/square foot)....

  14. 46 CFR 171.057 - Intact stability requirements for a sailing catamaran.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... centerlines in meters (feet). As=the maximum sail area in square meters (square feet). Hc=the height of the center of effort of the sail area above the deck, in meters (feet). W=the total displacement of the vessel, in kilograms (pounds). X=4.88 kilograms/square meter (1.0 pounds/square foot). (b) A...

  15. 46 CFR 171.057 - Intact stability requirements for a sailing catamaran.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... hull centerlines in meters (feet). As=the maximum sail area in square meters (square feet). Hc=the height of the center of effort of the sail area above the deck, in meters (feet). W=the total displacement of the vessel, in kilograms (pounds). X=4.88 kilograms/square meter (1.0 pounds/square foot)....

  16. 46 CFR 171.057 - Intact stability requirements for a sailing catamaran.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... hull centerlines in meters (feet). As=the maximum sail area in square meters (square feet). Hc=the height of the center of effort of the sail area above the deck, in meters (feet). W=the total...). As=the maximum sail area in square meters (square feet). Hc=the height of the center of effort of...

  17. Intact brown adipose tissue thermogenesis is required for restorative sleep responses after sleep loss.

    PubMed

    Szentirmai, Éva; Kapás, Levente

    2014-03-01

    Metabolic signals related to feeding and body temperature regulation have profound effects on vigilance. Brown adipose tissue (BAT) is a key effector organ in the regulation of metabolism in several species, including rats and mice. Significant amounts of active BAT are also present throughout adulthood in humans. The metabolic activity of BAT is due to the tissue-specific presence of the uncoupling protein-1 (UCP-1). To test the involvement of BAT thermogenesis in sleep regulation, we investigated the effects of two sleep-promoting stimuli in UCP-1-deficient mice. Sleep deprivation by gentle handling increased UCP-1 mRNA expression in BAT and elicited rebound increases in non-rapid-eye-movement sleep and rapid-eye-movement sleep accompanied by elevated slow-wave activity of the electroencephalogram. The rebound sleep increases were significantly attenuated, by ~ 35-45%, in UCP-1-knockout (KO) mice. Wild-type (WT) mice with capsaicin-induced sensory denervation of the interscapular BAT pads showed similar impairments in restorative sleep responses after sleep deprivation, suggesting a role of neuronal sleep-promoting signaling from the BAT. Exposure of WT mice to 35 °C ambient temperature for 5 days led to increased sleep and body temperature and suppressed feeding and energy expenditure. Sleep increases in the warm environment were significantly suppressed, by ~ 50%, in UCP-1-KO animals while their food intake and energy expenditure did not differ from those of the WTs. These results suggest that the metabolic activity of the BAT plays a role in generating a metabolic environment that is permissive for optimal sleep. Impaired BAT function may be a common underlying cause of sleep insufficiency and metabolic disorders.

  18. 46 CFR 171.057 - Intact stability requirements for a sailing catamaran.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... the following equation: ER10JA96.008 Where— B=the distance between hull centerlines in meters (feet... waters must be designed to satisfy the following equation: ER10JA96.007 Where— B=the distance between hull centerlines in meters (feet). As=the maximum sail area in square meters (square feet)....

  19. Tomato susceptibility to root-knot nematodes requires an intact jasmonic Acid signaling pathway

    USDA-ARS?s Scientific Manuscript database

    Response of tomato (Solanum lycopersicum) to root-knot nematode (RKN; Meloidogyne spp.) infection was monitored using TOM1 cDNA microarray with resistant (‘Motelle’; Mi-1) and susceptible (‘Moneymaker’; mi) tomato at 24 h after RKN infection. The array analysis identified 1497 genes and 750 genes d...

  20. Cellular trafficking of the IL-1RI-associated kinase-1 requires intact kinase activity

    SciTech Connect

    Boel, Gaby-Fleur . E-mail: boel@mail.dife.de; Jurrmann, Nadine; Brigelius-Flohe, Regina

    2005-06-24

    Upon stimulation of cells with interleukin-1 (IL-1) the IL-1 receptor type I (IL-1RI) associated kinase-1 (IRAK-1) transiently associates to and dissociates from the IL-1RI and thereafter translocates into the nucleus. Here we show that nuclear translocation of IRAK-1 depends on its kinase activity since translocation was not observed in EL-4 cells overexpressing a kinase negative IRAK-1 mutant (EL-4{sup IRAK-1-K239S}). IRAK-1 itself, an endogenous substrate with an apparent molecular weight of 24 kDa (p24), and exogenous substrates like histone and myelin basic protein are phosphorylated by nuclear located IRAK-1. Phosphorylation of p24 cannot be detected in EL-4{sup IRAK-1-K239S} cells. IL-1-dependent recruitment of IRAK-1 to the IL-1RI and subsequent phosphorylation of IRAK-1 is a prerequisite for nuclear translocation of IRAK-1. It is therefore concluded that intracellular localization of IRAK-1 depends on its kinase activity and that IRAK-1 may also function as a kinase in the nucleus as shown by a new putative endogenous substrate.

  1. Activation of the exchange factor Ras-GRF by calcium requires an intact Dbl homology domain.

    PubMed

    Freshney, N W; Goonesekera, S D; Feig, L A

    1997-04-21

    Ras-GRF is a guanine nucleotide exchange factor that activates Ras proteins. Its activity on Ras in cells is enhanced upon calcium influx. Activation follows calcium-induced binding of calmodulin to an IQ motif near the N-terminus of Ras-GRF. Ras-GRF also contains a Dbl homology (DH) domain C-terminal to the IQ motif. In many proteins, DH domains act as exchange factors for Rho-GTPase family members. However, we failed to detect exchange activity of this domain on well characterized Rho family members. Instead, we found that mutations analogous to those that block exchange activity of Dbl prevented Ras-GRF activation by calcium/ calmodulin in vivo. All DH domains are followed immediately by a pleckstrin homology (PH) domain. We found that a mutation at a conserved site within the PH domain following the DH domain also prevented Ras-GRF activation by calcium in vivo. These results suggest that in addition to playing a role as activators of Rho proteins, DH domains can also contribute to the coupling of cellular signals to Ras activation.

  2. Intact LKB1 activity is required for survival of dormant ovarian cancer spheroids

    PubMed Central

    Peart, Teresa; Valdes, Yudith Ramos; Correa, Rohann J. M.; Fazio, Elena; Bertrand, Monique; McGee, Jacob; Préfontaine, Michel; Sugimoto, Akira; DiMattia, Gabriel E.; Shepherd, Trevor G.

    2015-01-01

    Metastatic epithelial ovarian cancer (EOC) cells can form multicellular spheroids while in suspension and disperse directly throughout the peritoneum to seed secondary lesions. There is growing evidence that EOC spheroids are key mediators of metastasis, and they use specific intracellular signalling pathways to control cancer cell growth and metabolism for increased survival. Our laboratory discovered that AKT signalling is reduced during spheroid formation leading to cellular quiescence and autophagy, and these may be defining features of tumour cell dormancy. To further define the phenotype of EOC spheroids, we have initiated studies of the Liver kinase B1 (LKB1)-5′-AMP-activated protein kinase (AMPK) pathway as a master controller of the metabolic stress response. We demonstrate that activity of AMPK and its upstream kinase LKB1 are increased in quiescent EOC spheroids as compared with proliferating adherent EOC cells. We also show elevated AMPK activity in spheroids isolated directly from patient ascites. Functional studies reveal that treatment with the AMP mimetic AICAR or allosteric AMPK activator A-769662 led to a cytostatic response in proliferative adherent ovarian cancer cells, but they fail to elicit an effect in spheroids. Targeted knockdown of STK11 by RNAi to reduce LKB1 expression led to reduced viability and increased sensitivity to carboplatin treatment in spheroids only, a phenomenon which was AMPK-independent. Thus, our results demonstrate a direct impact of altered LKB1-AMPK signalling function in EOC. In addition, this is the first evidence in cancer cells demonstrating a pro-survival function for LKB1, a kinase traditionally thought to act as a tumour suppressor. PMID:26068970

  3. Numerical modeling of heat transfer and pasteurizing value during thermal processing of intact egg.

    PubMed

    Abbasnezhad, Behzad; Hamdami, Nasser; Monteau, Jean-Yves; Vatankhah, Hamed

    2016-01-01

    Thermal Pasteurization of Eggs, as a widely used nutritive food, has been simulated. A three-dimensional numerical model, computational fluid dynamics codes of heat transfer equations using heat natural convection, and conduction mechanisms, based on finite element method, was developed to study the effect of air cell size and eggshell thickness. The model, confirmed by comparing experimental and numerical results, was able to predict the temperature profiles, the slowest heating zone, and the required heating time during pasteurization of intact eggs. The results showed that the air cell acted as a heat insulator. Increasing the air cell volume resulted in decreasing of the heat transfer rate, and the increasing the required time of pasteurization (up to 14%). The findings show that the effect on thermal pasteurization of the eggshell thickness was not considerable in comparison to the air cell volume.

  4. Measurement of electrical conduction properties of intact embryonic murine hearts by extracellular microelectrode arrays.

    PubMed

    Taylor, David G; Natarajan, Anupama

    2012-01-01

    The study of the embryonic development of the cardiac conduction system and its congenital and toxicological defects requires protocols to measure electrical conduction through the myocardium. However, available methods either lack spatial information, necessitate the hearts to be sliced and mounted, or require specialized equipment. Microelectrode arrays (MEAs) are plates with embedded surface electrodes to measure localized extracellular ionic currents (field potentials) created by the depolarization and repolarization of cultured cells and tissue slices. Here we describe a protocol using MEAs to examine electrical conduction through intact and beating cultured hearts isolated from mouse embryos at 10.5 days postcoitus. This method allows measurements of conduction time, estimates of conduction velocity, atrioventricular conduction delay and block, and heart rate and rhythmicity.

  5. Imaging of Intact Tissue Sections: Moving beyond the Microscope*

    PubMed Central

    Seeley, Erin H.; Schwamborn, Kristina; Caprioli, Richard M.

    2011-01-01

    MALDI-imaging MS is a new molecular imaging technology for direct in situ analysis of thin tissue sections. Multiple analytes can be monitored simultaneously without prior knowledge of their identities and without the need for target-specific reagents such as antibodies. Imaging MS provides important insights into biological processes because the native distributions of molecules are minimally disturbed, and histological features remain intact throughout the analysis. A wide variety of molecules can be imaged, including proteins, peptides, lipids, drugs, and metabolites. Several specific examples are presented to highlight the utility of the technology. PMID:21632549

  6. Fast imaging of intact and shattered cryogenic neon pellets.

    PubMed

    Wang, Zhehui; Combs, S K; Baylor, L R; Foust, C R; Lyttle, M S; Meitner, S J; Rasmussen, D A

    2014-11-01

    Compact condensed-matter injection technologies are increasingly used in magnetic fusion. One recent application is in disruption mitigation. An imaging system with less-than-100-µm- and sub-µs-resolution is described and used to characterize intact and shattered cryogenic neon pellets. Shattered pellets contain fine particles ranging from tens of µm to about 7 mm. Time-of-flight analyses indicate that pellets could slow down if hitting the wall of the guide tube. Fast high-resolution imaging systems are thus useful to neon and other condensed-matter injector development.

  7. Controlled therapy by imaging of functional structures of intact liver

    NASA Astrophysics Data System (ADS)

    Wang, W.; Zhuang, Feng Y.; Ruan, G.; Kakihana, Yasuyuki; Krug, A.; Kessler, Manfred D.

    2000-04-01

    Ligustrazine, a Chinese herb medicine has been used to treat the diseases of cardiovascular and cerebral vascular diseases in China by Chinese traditional physicians or many years. Recently, results showed that ligustrazine is a powerful hepatic vasodilator. It can greatly change the blood supply of the tissues. Due to micro-optical tissue sensor developed recently it became possible to image functional structures of tissue on the level of intact blood capillaries. In our experiment we used the Oxyscan in order to study the effect of Ligustrazine on the oxygen supply of rat liver.

  8. Physiological characterization of motor unit properties in intact cats.

    PubMed

    O'Donovan, M J; Hoffer, J A; Loeb, G E

    1983-02-01

    Single motor units were isolated in intact cats, by microstimulation through chronically implanted microwires in the L5 ventral roots. Motor unit axonal and mechanical properties were obtained by stimulus-triggered averaging the signals from an implanted femoral nerve recording cuff and patellar tendon force transducer. All unit types were sampled with this technique, and it was also possible to stimulate in isolation an axon whose ventral root spike was recorded during treadmill locomotion. A new technique was described, spike-triggered microstimulation, for verifying the identity of a stimulated and a recorded axon.

  9. Fast Imaging of Intact and Shattered Cryogenic Neon Pellets

    SciTech Connect

    Wang, Zhehui; Combs, Stephen Kirk; Baylor, Larry R; Foust, Charles R; Lyttle, Mark S; Meitner, Steven J; Rasmussen, David A

    2014-01-01

    Compact condensed-matter injection technologies are increasingly used in magnetic fusion. One recent application is in disruption mitigation. An imaging system with less-than-100- m- and sub- s-resolution is described and used to characterize intact and shattered cryogenic neon pellets. Shattered pellets contain fine particles ranging from tens of m to about 7 mm. Time-of-flight analyses indicate that pellets could slow down if hitting the wall of the guide tube. Fast high-resolution imaging systems are thus useful to neon and other condensed-matter injector development.

  10. Fast imaging of intact and shattered cryogenic neon pellets

    SciTech Connect

    Wang, Zhehui; Combs, S. K.; Baylor, L. R.; Foust, C. R.; Lyttle, M. S.; Meitner, S. J.; Rasmussen, D. A.

    2014-11-15

    Compact condensed-matter injection technologies are increasingly used in magnetic fusion. One recent application is in disruption mitigation. An imaging system with less-than-100-µm- and sub-µs-resolution is described and used to characterize intact and shattered cryogenic neon pellets. Shattered pellets contain fine particles ranging from tens of µm to about 7 mm. Time-of-flight analyses indicate that pellets could slow down if hitting the wall of the guide tube. Fast high-resolution imaging systems are thus useful to neon and other condensed-matter injector development.

  11. Elemental and isotopic imaging to study biogeochemical functioning of intact soil micro-environments

    NASA Astrophysics Data System (ADS)

    Mueller, Carsten W.

    2017-04-01

    / inorganic N in the rhizosphere). However, especially the elemental mapping requires more sophisticated computational approaches to evaluate (and quantify) the spatial heterogeneities of biogeochemical properties in intact soil systems.

  12. Microarray Noninvasive Neuronal Seizure Recordings from Intact Larval Zebrafish

    PubMed Central

    Meyer, Michaela; Dhamne, Sameer C.; LaCoursiere, Christopher M.; Tambunan, Dimira; Poduri, Annapurna; Rotenberg, Alexander

    2016-01-01

    Zebrafish epilepsy models are emerging tools in experimental epilepsy. Zebrafish larvae, in particular, are advantageous because they can be easily genetically altered and used for developmental and drug studies since agents applied to the bath penetrate the organism easily. Methods for electrophysiological recordings in zebrafish are new and evolving. We present a novel multi-electrode array method to non-invasively record electrical activity from up to 61 locations of an intact larval zebrafish head. This method enables transcranial noninvasive recording of extracellular field potentials (which include multi-unit activity and EEG) to identify epileptic seizures. To record from the brains of zebrafish larvae, the dorsum of the head of an intact larva was secured onto a multi-electrode array. We recorded from individual electrodes for at least three hours and quantified neuronal firing frequency, spike patterns (continuous or bursting), and synchrony of neuronal firing. Following 15 mM potassium chloride- or pentylenetetrazole-infusion into the bath, spike and burst rate increased significantly. Additionally, synchrony of neuronal firing across channels, a hallmark of epileptic seizures, also increased. Notably, the fish survived the experiment. This non-invasive method complements present invasive zebrafish neurophysiological techniques: it affords the advantages of high spatial and temporal resolution, a capacity to measure multiregional activity and neuronal synchrony in seizures, and fish survival for future experiments, such as studies of epileptogenesis and development. PMID:27281339

  13. Enrichment and Analysis of Intact Phosphoproteins in Arabidopsis Seedlings.

    PubMed

    Aryal, Uma K; Ross, Andrew R S; Krochko, Joan E

    2015-01-01

    Protein phosphorylation regulates diverse cellular functions and plays a key role in the early development of plants. To complement and expand upon previous investigations of protein phosphorylation in Arabidopsis seedlings we used an alternative approach that combines protein extraction under non-denaturing conditions with immobilized metal-ion affinity chromatography (IMAC) enrichment of intact phosphoproteins in Rubisco-depleted extracts, followed by identification using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In-gel trypsin digestion and analysis of selected gel spots identified 144 phosphorylated peptides and residues, of which only 18 phosphopeptides and 8 phosphosites were found in the PhosPhAt 4.0 and P3DB Arabidopsis thaliana phosphorylation site databases. More than half of the 82 identified phosphoproteins were involved in carbohydrate metabolism, photosynthesis/respiration or oxidative stress response mechanisms. Enrichment of intact phosphoproteins prior to 2-DE and LC-MS/MS appears to enhance detection of phosphorylated threonine and tyrosine residues compared with methods that utilize peptide-level enrichment, suggesting that the two approaches are somewhat complementary in terms of phosphorylation site coverage. Comparing results for young seedlings with those obtained previously for mature Arabidopsis leaves identified five proteins that are differentially phosphorylated in these tissues, demonstrating the potential of this technique for investigating the dynamics of protein phosphorylation during plant development.

  14. Endogenous norepinephrine regulates blood flow to the intact rat tibia.

    PubMed

    Feitelson, J B A; Kulenovic, E; Beck, D J; Harris, P D; Passmore, J C; Malkani, A L; Fleming, J T

    2002-03-01

    The goal of our study was to determine if endogenous norepinephrine (NE) has a role in the regulation of basal blood flow to intact bone. The experimental plan was to measure bone blood flow before and after pharmacological blockade of alpha-adrenergic receptors. A significant increase in blood flow after receptor blockade would suggest that endogenous norepinephrine exerts a tonic constrictor effect on the vessels supplying blood to the bone. Mature, male rats were anesthetized with Inactin. Arterial blood pressure and left tibia blood flow (laser Doppler flowmetry) were measured. A cannula was inserted into the right iliac artery and advanced to the aortic bifurcation to deliver drugs into the left hindlimb circulation, including the left tibia vasculature. Bolus injection of norepinephrine caused a dose-dependent decrease in bone blood flow (30-40%). Blockade of alpha-adrenergic receptors with phentolamine or phenoxybenzamine attenuated by more than 50% the norepinephrine-induced decrease in bone blood flow. In separate rats that had not received exogenous norepinephrine, injection of phentolamine alone decreased bone vascular resistance by 34+/-3%. Similarly, phenoxybenzamine decreased resistance by 25+/-4%. These results are consistent with the conclusion that alpha-adrenergic receptors mediate a significant constriction of blood vessels which participate in the partial control of basal blood flow to the intact rat tibia.

  15. Nickel in plants: I. Uptake kinetics using intact soybean seedlings.

    PubMed

    Cataldo, D A; Garland, T R; Wildung, R E

    1978-10-01

    The absorption of Ni(2+) by 21-day-old soybean plants (Glycine max cv. Williams) was investigated with respect to its concentration dependence, transport kinetics, and interactions with various nutrient cations. Nickel absorption, measured as a function of concentration (0.02 to 100 mum), demonstrated the presence of multiple absorption isotherms. Each of the three isotherms conforms to Michaelis-Menten kinetics; kinetic constants are reported for uptake by the intact plant and for transfer from root to shoot tissues. The absorption of Ni(2+) by the intact plant and its transfer from root to shoot were inhibited by the presence of Cu(2+), Zn(2+), Fe(2+), and Co(2+). Competition kinetic studies showed Cu(2+) and Zn(2+) to inhibit Ni(2+) absorption competitively, suggesting that Ni(2+), Cu(2+), and Zn(2+) are absorbed using the same carrier site. Calculated K(m) and K(i) constants for Ni(2+) in the presence and absence of Cu(2+) were 6.1 and 9.2 mum, respectively, whereas K(m) and K(i) constants were calculated to be 6.7 and 24.4 mum, respectively, for Ni(2+) in the presence and absence of Zn(2+). The mechanism of inhibition of Ni(2+) in the presence of Fe(2+) and Co(2+) was not resolved by classical kinetic relationships.

  16. Intact memory for implicit contextual information in Korsakoff's amnesia.

    PubMed

    Oudman, Erik; Van der Stigchel, Stefan; Wester, Arie J; Kessels, Roy P C; Postma, Albert

    2011-08-01

    Implicit contextual learning is the ability to acquire contextual information from our surroundings without conscious awareness. Such contextual information facilitates the localization of objects in space. In a typical implicit contextual learning paradigm, subjects need to find a target among a number of distractors during visual search. Some of the configurations of stimuli are repeated during the experiment resulting in faster responses than for novel configurations, without subjects being aware of their repetition. Patients with Korsakoff's syndrome (KS) have been found to show devastating explicit spatial amnesia. Less is know about their implicit spatial memory abilities. The aim of the present research was to examine whether implicit contextual learning is intact in KS. Therefore, eighteen KS patients and twenty-two age-IQ- and education-matched controls performed the Implicit Contextual Learning task and a paradigm intended to assess explicit, spatial working memory, i.e. the Box task. Intact implicit contextual learning was observed in both the control group and the KS patients. In turn KS patients did have markedly lower explicit spatial working memory scores. The implicit learning effect was not related to the spatial working memory scores. Together these results clearly suggest that implicit and explicit spatial memory have a different neurocognitive basis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Analytical strategies for the global quantification of intact proteins.

    PubMed

    Collier, Timothy S; Muddiman, David Charles

    2012-09-01

    The quantification of intact proteins is a relatively recent development in proteomics. In eukaryotic organisms, proteins are present as multiple isoforms as the result of variations in genetic code, alternative splicing, post-translational modification and other processing events. Understanding the identities and biological functions of these isoforms and how their concentrations vary across different states is the central goal of proteomics. To date, the bulk of proteomics research utilizes a "bottom-up" approach, digesting proteins into their more manageable constitutive peptides, but sacrificing information about the specific isoform and combinations of post-translational modifications present on the protein. Very specific strategies for protein quantification such as the enzyme-linked immunosorbent assay and Western blot are commonplace in laboratories and clinics, but impractical for the study of global biological changes. Herein, we describe strategies for the quantification of intact proteins, their distinct advantages, and challenges to their employment. Techniques contained in this review include the more traditional and widely employed methodology of differential gel electrophoresis and more recently developed mass spectrometry-based techniques including metabolic labeling, chemical labeling, and label-free methodologies.

  18. Bacterial clearance in the intact and regenerating liver

    SciTech Connect

    Gross, K.; Katz, S.; Dunn, S.P.; Cikrit, D.; Rosenthal, R.; Grosfeld, J.L.

    1985-08-01

    The Kupffer cells in the liver play an important role in reticuloendothelial system (RES) function by clearing particulate matter and bacteria from the blood stream. While hepatocyte regeneration and function have been extensively studied following partial hepatectomy, little information is available concerning RES function in the regenerating liver. This study investigates hepatic RES function by evaluating bacterial clearance (live E. coli) in the intact and regenerating liver. Thirty-four young male Sprague Dawley rats were studied. Twenty-two animals underwent a standard 70% partial hepatectomy using ligature technique and 12 had a sham operation. Both groups of rats received 10(9) organism of TVS labeled E coli, intravenously at 24 hours, 72 hours, 2 1/2 weeks, and 6 weeks postoperatively. Rats were killed 10 minutes following injection and liver, lung, spleen, and kidney harvested, fixed, and radioactivity was determined using a scintillation spectrometer interfaced with a micro-computer counting the TVS radiolabel. The total organ count of trapped bacteria in liver in partially hepatectomized rats was lower than intact controls at 24 hours, but was similar at 72 hours, 2 1/2 weeks, and 6 weeks. Partial hepatectomy increased the amount of bacterial trapping in the lung at 24 hours and 72 hours and returned to normal at 2 1/2 weeks and 6 weeks. Splenic activity was increased following hepatectomy at 2 1/2 weeks. Renal clearance was increased at 72 hours and 2 1/2 weeks.

  19. Trehalose inhibits ethanol effects on intact yeast cells and liposomes.

    PubMed

    Mansure, J J; Panek, A D; Crowe, L M; Crowe, J H

    1994-05-11

    The effect of ethanol on stability of intact yeast cells has been investigated. Several strains with differences in trehalose metabolism were examined for their ability to survive in the presence of 10% (v/v) ethanol. A positive correlation was observed between cell viability and trehalose concentration. When leakage of electrolytes from the cells was recorded by observing changes in conductivity of the medium, we found that ethanol increases leakage, but the presence of trehalose reverses that effect. Similar studies were done with liposomes of similar composition to those seen in intact cells in log and stationary phases. In the presence of ethanol, carboxyfluorescein trapped in the liposomes leaked to the medium. When trehalose was added inside, outside or on both sides of the membrane, the ethanol-induced leakage was strongly inhibited. More leakage was observed in liposomes in gel phase state than in liquid-crystalline phase, suggesting that the thermotropic behavior of the lipids in the plasma membrane, together with trehalose, plays a role in enhancing ethanol tolerance.

  20. Multiplexed Intact-Tissue Transcriptional Analysis at Cellular Resolution.

    PubMed

    Sylwestrak, Emily Lauren; Rajasethupathy, Priyamvada; Wright, Matthew Arnot; Jaffe, Anna; Deisseroth, Karl

    2016-02-11

    In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization over large volumes has been largely restricted to labeling of proteins. But volumetric nucleic acid labeling may represent a far greater scientific and clinical opportunity, enabling detection of not only diverse coding RNA variants but also non-coding RNAs. Moreover, scaling immunohistochemical detection to large tissue volumes has limitations due to high cost, limited renewability/availability, and restricted multiplexing capability of antibody labels. With the goal of versatile, high-content, and scalable molecular phenotyping of intact tissues, we developed a method using carbodiimide-based chemistry to stably retain RNAs in clarified tissue, coupled with amplification tools for multiplexed detection. The resulting technology enables robust measurement of activity-dependent transcriptional signatures, cell-identity markers, and diverse non-coding RNAs in rodent and human tissue volumes. The growing set of validated probes is deposited in an online resource for nucleating related developments from across the scientific community.

  1. STREPTOMYCIN FORMATION BY INTACT MYCELIUM OF STREPTOMYCES GRISEUS

    PubMed Central

    Nomi, Ryosaku

    1963-01-01

    Nomi, Ryosaku (Rutgers, The State University, New Brunswick, N.J.). Streptomycin formation by intact mycelium of Streptomyces griseus. J. Bacteriol. 86:1220–1230. 1963.—A study was made of streptomycin formation by intact mycelium of Streptomyces griseus 107 grown in glucose-yeast extract medium. When mycelium harvested after 24, 48, and 72 hr was compared, the earliest growth showed the highest activity in producing streptomycin from glucose. The concentration of streptomycin in the mycelium was higher in the older growth. Calcium chloride had a remarkable effect in increasing streptomycin production from the precursors in the mycelium, especially when the mycelium was grown for 48 hr or longer. The effect of calcium chloride cannot be attributed to the precipitation of an excess of inorganic phosphate in the medium. Glucose, fructose, glycerol, lactic acid, glucosamine, streptidine, and inositol stimulated streptomycin formation, whereas gluconic acid, glucuronic acid, streptamine, and strepturea did not. When 24-hr-old mycelium was suspended and shaken in 0.5% glucose solution, the antibiotic precursors necessary to produce streptomycin were found mainly in the supernatant of the culture rather than in the mycelium. The supernatant included some substance which had a weak antibiotic activity. This substance was less basic than streptomycin and was transformed to streptomycin with a remarkable increase in antibiotic activity. PMID:14086093

  2. Nucleotidyl cyclase activity of soluble guanylyl cyclase in intact cells.

    PubMed

    Bähre, Heike; Danker, Kerstin Y; Stasch, Johannes-Peter; Kaever, Volkhard; Seifert, Roland

    2014-01-24

    Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α1β1 has been shown to additionally generate the cyclic pyrimidine nucleotides cCMP and cUMP. However, since cyclic pyrimidine nucleotide formation occurred only the presence of Mn(2+) but not Mg(2+), the physiological relevance of these in vitro findings remained unclear. Therefore, we studied cyclic nucleotide formation in intact cells. We observed NO-dependent cCMP- and cUMP formation in intact HEK293 cells overexpressing sGC α1β1 and in RFL-6 rat fibroblasts endogenously expressing sGC, using HPLC-tandem mass spectrometry. The identity of cCMP and cUMP was unambiguously confirmed by HPLC-time-of-flight mass spectrometry. Our data indicate that cCMP and cUMP play second messenger roles and that Mn(2+) is a physiological sGC cofactor. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Optimizing the analysis of dental enamel microstructure in intact teeth.

    PubMed

    Arrieta, Zulieth López; Line, Sergio Roberto Peres

    2017-07-01

    In most mammalian species enamel prisms are regularly arranged in layers of alternating directions forming an angle of approximately 90°. These successive layers of prisms are known as Hunter-Schreger bands (HSBs). The analysis of HSBs may provide valuable information regarding the species life history, taxon and personal identification, with evident applicability in physical anthropology and forensics. Obtaining good quality digital images of HSBs in intact specimens is not always a feasible task. The major problems are the low contrast of images; the reflection of incident light, which may create areas of intense shine in digital images; and the abrupt decrease in the degree of illumination that occurs after light crosses the vertical cracks, frequently present in enamel. We show here that the area of intense shine can be minimized by a polarizing filter coupled to the camera objective, and the filling of enamel cracks with corn oil can reduce refraction of light in enamel cracks. These procedures can significantly increase the quality and the area of HSBs that can be recorded in intact teeth. © 2017 Wiley Periodicals, Inc.

  4. Effects of Triclocarban on Intact Immature Male Rat

    PubMed Central

    Duleba, Antoni J.; Ahmed, Mohamed I.; Sun, Meng; Gao, Allen C.; Villanueva, Jesus; Conley, Alan J.; Turgeon, Judith L.; Benirschke, Kurt; Gee, Nancy A.; Chen, Jiangang; Green, Peter G.; Lasley, Bill L.

    2011-01-01

    Triclocarban (TCC; 3,4,4′-trichlorocarbanilide) is an antimicrobial agent used widely in various personal hygiene products including soaps. Recently, TCC has been shown to enhance testosterone-induced effects in vitro and to enlarge accessory sex organs in castrated male rats. This study was designed to evaluate the effects of TCC on intact age-matched male rats and on human prostate LNCaP and C4–2B cells. Seven-week-old male Sprague-Dawley rats received either a normal diet or a diet supplemented with TCC (0.25% in diet) for 10 days. Triclocarban induced hyperplasia of accessory sex organs in the absence of significant qualitative histological changes. Serum luteinizing hormone (LH) and testosterone were not significantly altered by TCC treatment. In prostate cancer-derived LNCaP and C4–2B cells, TCC potentiated androgen actions via androgen receptor-dependent actions. In conclusion, TCC significantly affects intact male reproductive organs and potentiates androgen effects in prostate cancer cells. PMID:20889956

  5. Mobility of the herbicide pyrithiobac through intact soil columns.

    PubMed

    Matocha, C J; Hossner, L R

    1999-04-01

    The relative mobility of pyrithiobac [sodium 2-chloro-6-(4, 6-dimethoxypyrimidin-2-ylthio)benzoate], a new herbicide used for postemergence control of broadleaf weeds in cotton (Gossypium hirsutum), was evaluated and compared against that of bromide (Br(-)) tracer on four soils representative of cotton-growing regions using intact soil columns under saturated flow conditions. Pyrithiobac breakthrough curves were asymmetrical in shape with significant tailing and displaced to the left of 1 pore volume in the Houston Black clay (fine, montmorillonitic, thermic Udic Pellustert), Orelia fine sandy clay loam (fine-loamy, mixed, hyperthermic Typic Ochraqualfs), and Ships silty clay (very-fine, mixed, thermic Udic Chromustert) soils. Breakthrough of pyrithiobac in the Hidalgo sandy loam soil (fine-loamy, mixed, hyperthermic Typic Calciustoll) was delayed and more symmetrical, with peak pyrithiobac concentration reached after 1.2 pore volumes. The immobile pore water (IPW) fractions estimated from the Br(-) breakthrough curves ranged from 20 to 87% of total pore water. The IPW values demonstrated that soils with the greatest amount of IPW (Ships with IPW = 87.3%) exhibited the most rapid movement of pyrithiobac (peak concentration after 0.04 pore volume). The experimentally determined pyrithiobac breakthrough curves confirmed the high mobility of this herbicide in these alkaline and predominantly smectitic soils. These results indicate that pyrithiobac mobility was influenced by soil type and preferential flow processes when leached through intact soil columns.

  6. Selective ion changes during spontaneous mitochondrial transients in intact astrocytes.

    PubMed

    Azarias, Guillaume; Chatton, Jean-Yves

    2011-01-01

    The bioenergetic status of cells is tightly regulated by the activity of cytosolic enzymes and mitochondrial ATP production. To adapt their metabolism to cellular energy needs, mitochondria have been shown to exhibit changes in their ionic composition as the result of changes in cytosolic ion concentrations. Individual mitochondria also exhibit spontaneous changes in their electrical potential without altering those of neighboring mitochondria. We recently reported that individual mitochondria of intact astrocytes exhibit spontaneous transient increases in their Na(+) concentration. Here, we investigated whether the concentration of other ionic species were involved during mitochondrial transients. By combining fluorescence imaging methods, we performed a multiparameter study of spontaneous mitochondrial transients in intact resting astrocytes. We show that mitochondria exhibit coincident changes in their Na(+) concentration, electrical potential, matrix pH and mitochondrial reactive oxygen species production during a mitochondrial transient without involving detectable changes in their Ca(2+) concentration. Using widefield and total internal reflection fluorescence imaging, we found evidence for localized transient decreases in the free Mg(2+) concentration accompanying mitochondrial Na(+) spikes that could indicate an associated local and transient enrichment in the ATP concentration. Therefore, we propose a sequential model for mitochondrial transients involving a localized ATP microdomain that triggers a Na(+)-mediated mitochondrial depolarization, transiently enhancing the activity of the mitochondrial respiratory chain. Our work provides a model describing ionic changes that could support a bidirectional cytosol-to-mitochondria ionic communication.

  7. An effective manual deboning method to prepare intact mouse nasal tissue with preserved anatomical organization.

    PubMed

    Dunston, David; Ashby, Sarah; Krosnowski, Kurt; Ogura, Tatsuya; Lin, Weihong

    2013-08-10

    The mammalian nose is a multi-functional organ with intricate internal structures. The nasal cavity is lined with various epithelia such as olfactory, respiratory, and squamous epithelia which differ markedly in anatomical locations, morphology, and functions. In adult mice, the nose is covered with various skull bones, limiting experimental access to internal structures, especially those in the posterior such as the main olfactory epithelium (MOE). Here we describe an effective method for obtaining almost the entire and intact nasal tissues with preserved anatomical organization. Using surgical tools under a dissecting microscope, we sequentially remove the skull bones surrounding the nasal tissue. This procedure can be performed on both paraformaldehyde-fixed and freshly dissected, skinned mouse heads. The entire deboning procedure takes about 20-30 min, which is significantly shorter than the experimental time required for conventional chemical-based decalcification. In addition, we present an easy method to remove air bubbles trapped between turbinates, which is critical for obtaining intact thin horizontal or coronal or sagittal sections from the nasal tissue preparation. Nasal tissue prepared using our method can be used for whole mount observation of the entire epithelia, as well as morphological, immunocytochemical, RNA in situ hybridization, and physiological studies, especially in studies where region-specific examination and comparison are of interest.

  8. Intact protein separation by chromatographic and/or electrophoretic techniques for top-down proteomics.

    PubMed

    Capriotti, Anna Laura; Cavaliere, Chiara; Foglia, Patrizia; Samperi, Roberto; Laganà, Aldo

    2011-12-09

    Mass spectrometry used in combination with a wide variety of separation methods is the principal methodology for proteomics. In bottom-up approach, proteins are cleaved with a specific proteolytic enzyme, followed by peptide separation and MS identification. In top-down approach intact proteins are introduced into the mass spectrometer. The ions generated by electrospray ionization are then subjected to gas-phase separation, fragmentation, fragment separation, and automated interpretation of mass spectrometric and chromatographic data yielding both the molecular weight of the intact protein and the protein fragmentation pattern. This approach requires high accuracy mass measurement analysers capable of separating the multi-charged isotopic cluster of proteins, such as hybrid ion trap-Fourier transform instruments (LTQ-FTICR, LTQ-Orbitrap). Front-end separation technologies tailored for proteins are of primary importance to implement top-down proteomics. This review intends to provide the state of art of protein chromatographic and electrophoretic separation methods suitable for MS coupling, and to illustrate both monodimensional and multidimensional approaches used for LC-MS top-down proteomics. In addition, some recent progresses in protein chromatography that may provide an alternative to those currently employed are also discussed.

  9. In vitro study to compare impact fracture resistance of intact root-treated teeth.

    PubMed

    McDonald, A V; King, P A; Setchell, D J

    1990-11-01

    The aim of this in vitro study was to determine the effect on impact fracture resistance of three methods for restoration of root-treated lower incisor teeth with otherwise intact natural crowns. In a control group (Group I) laterally condensed gutta-percha filled the entire root canal, whereas part of the root filling was removed to enable insertion of a 1 mm diameter post in the other two groups. Stainless steel posts were placed in Group II and experimental carbon fibre reinforced carbon (CFRC) in Group III. A composite resin luting agent was used to lute the posts, and standardized composite resin restorations were placed in each access cavity. Fifteen specimens of each group were tested to failure with a single impact force applied at 90 degrees to the mid-point inciso-cervically on the labial surface. The peak force, peak energy, and first peak total energy required to fracture each specimen were recorded. The results showed no significant difference between the three groups, nor was a difference in the mode or site of fracture observed. The results suggested that there is no advantage from the point of view of fracture mechanics in 'restoring' intact root-treated teeth with either stainless steel or carbon fibre reinforced carbon rods.

  10. Feasibility in multispectral imaging for predicting the content of bioactive compounds in intact tomato fruit.

    PubMed

    Liu, Changhong; Liu, Wei; Chen, Wei; Yang, Jianbo; Zheng, Lei

    2015-04-15

    Tomato is an important health-stimulating fruit because of the antioxidant properties of its main bioactive compounds, dominantly lycopene and phenolic compounds. Nowadays, product differentiation in the fruit market requires an accurate evaluation of these value-added compounds. An experiment was conducted to simultaneously and non-destructively measure lycopene and phenolic compounds content in intact tomatoes using multispectral imaging combined with chemometric methods. Partial least squares (PLS), least squares-support vector machines (LS-SVM) and back propagation neural network (BPNN) were applied to develop quantitative models. Compared with PLS and LS-SVM, BPNN model considerably improved the performance with coefficient of determination in prediction (RP(2))=0.938 and 0.965, residual predictive deviation (RPD)=4.590 and 9.335 for lycopene and total phenolics content prediction, respectively. It is concluded that multispectral imaging is an attractive alternative to the standard methods for determination of bioactive compounds content in intact tomatoes, providing a useful platform for infield fruit sorting/grading.

  11. Intact skull chronic windows for mesoscopic wide-field imaging in awake mice

    PubMed Central

    Silasi, Gergely; Xiao, Dongsheng; Vanni, Matthieu P.; Chen, Andrew C. N.; Murphy, Timothy H.

    2016-01-01

    Background Craniotomy-based window implants are commonly used for microscopic imaging, in head-fixed rodents, however their field of view is typically small and incompatible with mesoscopic functional mapping of cortex. New Method We describe a reproducible and simple procedure for chronic through-bone wide-field imaging in awake head-fixed mice providing stable optical access for chronic imaging over large areas of the cortex for months. Results The preparation is produced by applying clear-drying dental cement to the intact mouse skull, followed by a glass coverslip to create a partially transparent imaging surface. Surgery time takes about 30 minutes. A single set-screw provides a stable means of attachment for mesoscale assessment without obscuring the cortical field of view. Comparison with Existing Methods We demonstrate the utility of this method by showing seed-pixel functional connectivity maps generated from spontaneous cortical activity of GCAMP6 signals in both awake and anesthetized mice. Conclusions We propose that the intact skull preparation described here may be used for most longitudinal studies that do not require micron scale resolution and where cortical neural or vascular signals are recorded with intrinsic sensors. PMID:27102043

  12. Glucose Inhibition of Adenylate Cyclase in Intact Cells of Escherichia coli B

    PubMed Central

    Peterkofsky, Alan; Gazdar, Celia

    1974-01-01

    Previous studies in E. coli B have demonstrated an inverse correlation between the presence of glucose in the medium and the accumulation of cyclic AMP in the medium. This observation could not be explained by the action of glucose as a repressor of adenylate cyclase (EC 4.6.1.1) synthesis, as a stabilizer of cyclic AMP phosphodiesterase (EC 3.1.4.17) activity, or as a direct inhibitor of adenylate cyclase activity in cell-free preparations. The recent development of an in vivo assay for adenylate cyclase has provided a basis for further exploring the inhibitory action of glucose in intact cells. With this assay it has been possible to show that, while glucose does not affect adenylate cyclase in vitro, it rapidly inhibits the enzyme activity in intact cells. Extensive metabolism of glucose is not required, since α-methylglucoside also inhibits adenylate cyclase in vivo. When cells are grown on glucose as carbon source, some sugars (mannose, glucosamine) substitute for glucose as adenylate cyclase inhibitors while others (e.g., fructose) do not. Dose-response studies indicate that low concentrations of glucose lead to essentially complete inhibition of adenylate cyclase activity while only moderately decreasing intracellular cyclic AMP concentrations. The evidence presented suggests that the decreased cellular cyclic AMP levels resulting from glucose addition can be accounted for by inhibition of adenylate cyclase without any significant effect on cyclic AMP phosphodiesterase or the transport of cyclic AMP from the cells to the medium. PMID:4366761

  13. Gradients in intact polar diacylglycerolipids across the Mediterranean Sea are related to phosphate availability

    NASA Astrophysics Data System (ADS)

    Popendorf, K. J.; Tanaka, T.; Pujo-Pay, M.; Lagaria, A.; Courties, C.; Conan, P.; Oriol, L.; Sofen, L. E.; Moutin, T.; van Mooy, B. A. S.

    2011-12-01

    Intact polar membrane lipids compose a significant fraction of cellular material in plankton and their synthesis imposes a substantial constraint on planktonic nutrient requirements. As a part of the Biogeochemistry from the Oligotrophic to the Ultraoligotrophic Mediterranean (BOUM) cruise we examined the distribution of several classes of intact polar diacylglycerolipids (IP-DAGs) across the Mediterranean, and found that phospholipid concentration as a percent of total lipids correlated with phosphate concentration. In addition, the ratios of non-phosphorus lipids to phospholipids - sulfoquinovosyldiacylglycerol (SQDG) to phosphatidylglycerol (PG), and betaine lipids to phosphatidylcholine (PC) - were also found to increase from west to east across the Mediterranean. Additionally, microcosm incubations from across the Mediterranean were amended with phosphate and ammonium, and in the course of several days nutrient amendments elicited a shift in the ratios of IP-DAGs. These experiments were used to assess the relative contribution of community shifts and physiological response to the observed change in IP-DAGs across the Mediterranean. The ratio of SQDG to chlorophyll-a was also explored as an indicator of phytoplankton response to nitrogen availability. This study is the first to demonstrate the dynamic response of membrane lipid composition to changes in nutrients in a natural, mixed planktonic community.

  14. Gradients in intact polar diacylglycerolipids across the Mediterranean Sea are related to phosphate availability

    NASA Astrophysics Data System (ADS)

    Popendorf, K. J.; Tanaka, T.; Pujo-Pay, M.; Lagaria, A.; Courties, C.; Conan, P.; Oriol, L.; Sofen, L. E.; Moutin, T.; van Mooy, B. A. S.

    2011-08-01

    Intact polar membrane lipids compose a significant fraction of cellular material in plankton and their synthesis imposes a substantial constraint on planktonic nutrient requirements. As a part of the Biogeochemistry from the Oligotrophic to the Ultraoligtrophic Mediterranean (BOUM) cruise we examined the distribution of several classes of intact polar diacylglycerolipids (IP-DAGs) across the Mediterranean, and found that phospholipid concentration as a percent of total lipids correlated with phosphate concentration. In addition, the ratios of non-phosphorus lipids to phospholipids - sulfoquinovosyldiacylglycerol (SQDG) to phosphatidylglycerol (PG), and betaine lipids to phosphatidylcholine (PC) - were also found to increase from west to east across the Mediterranean. Additionally, microcosm incubations from across the Mediterranean were amended with phosphate and ammonium, and in the course of several days nutrient amendments elicited a shift in the ratios of IP-DAGs. These experiments were used to assess the relative contribution of community shifts and physiological response to the observed change in IP-DAGs across the Mediterranean. The ratio of SQDG to chlorophyll a was also explored as an indicator of phytoplankton response to nitrogen availability. This study is the first to demonstrate the dynamic response of membrane lipid composition to changes in nutrients in a natural, mixed planktonic community.

  15. Paralysis and killing of Caenorhabditis elegans by enteropathogenic Escherichia coli requires the bacterial tryptophanase gene.

    PubMed

    Anyanful, Akwasi; Dolan-Livengood, Jennifer M; Lewis, Taiesha; Sheth, Seema; Dezalia, Mark N; Sherman, Melanie A; Kalman, Lisa V; Benian, Guy M; Kalman, Daniel

    2005-08-01

    susceptible to EPEC effects when mutated, suggesting their involvement in protecting the worms. Moreover we have found that C. elegans genes controlling lifespan (daf-2, age-1 and daf-16), also mediate susceptibility to EPEC. Together, these data suggest that this C. elegans/EPEC system will be valuable in elucidating novel factors relevant to human disease that regulate virulence in the pathogen or susceptibility to infection in the host.

  16. The phosphorylation of coated membrane proteins in intact neurons

    PubMed Central

    1986-01-01

    To complement studies that have demonstrated the prominent phosphorylation of a 50-kD coated vesicle polypeptide in vitro, we have evaluated the phosphorylation of coated membrane proteins in intact cells. A co-assembly assay has been devised in which extracts of cultured rat sympathetic neurons labeled with [32P]-Pi were combined with unlabeled carrier bovine brain coat proteins and reassembled coat structures were isolated by gradient centrifugation. Two groups of phosphorylated polypeptides, of 100-110 kD (pp100-110) and 155 kD (pp155) apparent molecular mass, were incorporated into reassembled coats. The neuronal pp100-110 are structurally and functionally related to the 100-110-kD component of the bovine brain assembly protein (AP), a protein complex that also contains 50-kD and 16.5-kD components and is characterized by its ability to promote the reassembly of clathrin coat structures under physiological conditions of pH and ionic strength (Zaremba, S. and J. H. Keen, 1983, J. Cell Biol., 97:1337-1348). The neuronal pp155 detected in reassembled coat structures was readily observable in total extracts of [32P]-Pi-labeled neurons dissolved in SDS-containing buffer. A bovine brain counterpart to the neuronal pp155 was also observed when brain coated vesicles were subjected to two- dimensional gel electrophoresis. Phosphoserine was the predominant phosphoaminoacid found in both the pp100 and pp155. A structural and functional counterpart to the 50-kD brain assembly polypeptide (AP50) was also identified in these neurons. Although the brain AP50 is prominently phosphorylated by an endogenous protein kinase in isolated coated vesicle preparations, the neuronal AP50 was not detectably phosphorylated in intact cells as assessed by two-dimensional non- equilibrium pH gradient gel electrophoresis of labeled cells dissolved directly in SDS-containing buffers. These results demonstrate that the bovine brain assembly polypeptides of 50 kD and 100-110 kD that we have

  17. Rhesus monkey brain imaging through intact skull with thermoacoustic tomography.

    PubMed

    Xu, Yuan; Wang, Lihong V

    2006-03-01

    Two-dimensional microwave-induced thermoacoustic tomography (TAT) is applied to imaging the Rhesus monkey brain through the intact skull. To reduce the wavefront distortion caused by the skull, only the low-frequency components of the thermoacoustic signals (< 1 MHz) are used to reconstruct the TAT images. The methods of signal processing and image reconstruction are validated by imaging a lamb kidney. The resolution of the system is found to be 4 mm when we image a 1-month-old monkey head containing inserted needles. We also image the coronal and axial sections of a 7-month-old monkey head. Brain features that are 3 cm deep in the head are imaged clearly. Our results demonstrate that TAT has potential for use in portable, cost-effective imagers for pediatric brains.

  18. Nitrate-Dependent O2 Evolution in Intact Leaves 1

    PubMed Central

    de la Torre, Angel; Delgado, Begoña; Lara, Catalina

    1991-01-01

    Evolution of O2 by illuminated intact detached leaves from barley (Hordeum vulgare L. cv Athos) and pea (Pisum sativum L. cv Lincoln) in a CO2-saturating atmosphere was enhanced when KNO3 (1-2.5 millimolar) had been previously supplied through the transpiration stream. The extra O2 evolution observed after feeding KNO3 increased with the light intensity, being maximal at near saturating photon flux densities and resulting in no changes in the initial slope of the O2 versus light-intensity curve. No stimulation of O2 evolution was otherwise observed after feeding KCl or NH4Cl. The data indicate that nitrate assimilation uses photosynthetically generated reductant and stimulates the rate of non-cyclic electron flow by acting as a second electron-accepting assimilatory process in addition to CO2 fixation. PMID:16668272

  19. Citrate synthesis in intact rat-liver mitochondria is irreversible.

    PubMed

    Greksák, M; Lopes-Cardozo, M; van den Bergh, S G

    1982-02-01

    Rat-liver mitochondria were incubated with [1,5-14C]citrate in the presence of fluorocitrate to block its oxidation in the Krebs cycle. The reaction products were analysed enzymatically and by anion-exchange chromatography. Incorporation of 14C into acetyl-L-carnitine or ketone bodies via a backward action of citrate synthase was not observed. The optimal rate of citrate synthesis from pyruvate and malate in the presence of fluorocitrate was 15 nmol . mg-1 min-1. In the absence of fluorocitrate, but in the presence of malonate, citrate was oxidized to succinate at a rate of 4 nmol . mg-1 . min-1. We conclude that the synthesis of citrate by intact rat liver mitochondria is an irreversible process. The possible mechanism underlying this phenomenon and the consequence for metabolic regulation are discussed.

  20. Intact Conceptual Priming in the Absence of Declarative Memory

    PubMed Central

    Levy, D.A.; Stark, C.E.L.; Squire, L.R.

    2009-01-01

    Priming is an unconscious (nondeclarative) form of memory whereby identification or production of an item is improved by an earlier encounter. It has been proposed that declarative memory and priming might be related—for example, that conceptual priming results in more fluent processing, thereby providing a basis for familiarity judgments. In two experiments, we assessed conceptual priming and recognition memory across a 5-min interval in 5 memory-impaired patients. All patients exhibited fully intact priming in tests of both free association (study tent; at test, provide an association to canvas) and category verification (study lemon; at test, decide: Is lemon a type of fruit?). Yet the 2 most severely amnesic patients performed at chance on matched tests of recognition memory. These findings count against the notion that conceptual priming provides feelings of familiarity that can support accurate recognition judgments. We suggest that priming is inaccessible to conscious awareness and does not influence declarative memory. PMID:15447639

  1. Bursty gene expression in the intact mammalian liver

    PubMed Central

    Halpern, Keren Bahar; Tanami, Sivan; Landen, Shanie; Chapal, Michal; Szlak, Liran; Hutzler, Anat; Nizhberg, Anna; Itzkovitz, Shalev

    2015-01-01

    Summary Bursts of nascent mRNA have been shown to lead to substantial cell-cell variation in unicellular organisms, facilitating diverse responses to environmental challenges. It is unknown whether similar bursts and gene-expression noise occur in mammalian tissues. To address this, we combine single molecule transcript counting with dual-color labeling and quantification of nascent mRNA to characterize promoter states, transcription rates and transcript lifetimes in the intact mouse liver. We find that liver gene expression is highly bursty, with promoters stochastically switching between transcriptionally active and inactive states. Promoters of genes with short mRNA lifetimes are active longer, facilitating rapid response while reducing burst-associated noise. Moreover, polyploid hepatocytes exhibit less noise than diploid hepatocytes, suggesting a possible benefit to liver polyploidy. Thus temporal averaging and liver polyploidy dampen the intrinsic variability associated with transcriptional bursts. Our approach can be used to study transcriptional bursting in diverse mammalian tissues. PMID:25728770

  2. Temperature dependence of the zeta potential in intact natural carbonates

    NASA Astrophysics Data System (ADS)

    Al Mahrouqi, Dawoud; Vinogradov, Jan; Jackson, Matthew D.

    2016-11-01

    The zeta potential is a measure of the electrical charge on mineral surfaces and is an important control on subsurface geophysical monitoring, adsorption of polar species in aquifers, and rock wettability. We report the first measurements of zeta potential in intact, water-saturated, natural carbonate samples at temperatures up to 120°C. The zeta potential is negative and decreases in magnitude with increasing temperature at low ionic strength (0.01 M NaCl, comparable to potable water) but is independent of temperature at high ionic strength (0.5 M NaCl, comparable to seawater). The equilibrium calcium concentration resulting from carbonate dissolution also increases with increasing temperature at low ionic strength but is independent of temperature at high ionic strength. The temperature dependence of the zeta potential is correlated with the temperature dependence of the equilibrium calcium concentration and shows a Nernstian linear relationship. Our findings are applicable to many subsurface carbonate rocks at elevated temperature.

  3. Calcium Activation Profile In Electrically Stimulated Intact Rat Heart Cells

    NASA Astrophysics Data System (ADS)

    Geerts, Hugo; Nuydens, Rony; Ver Donck, Luc; Nuyens, Roger; De Brabander, Marc; Borgers, Marcel

    1988-06-01

    Recent advances in fluorescent probe technology and image processing equipment have made available the measurement of calcium in living systems on a real-time basis. We present the use of the calcium indicator Fura-2 in intact normally stimulated rat heart cells for the spatial and dynamic measurement of the calcium excitation profile. After electric stimulation (1 Hz), the activation proceeds from the center of the myocyte toward the periphery. Within two frame times (80 ms), the whole cell is activated. The activation is slightly faster in the center of the cell than in the periphery. The mean recovery time is 200-400 ms. There is no difference along the cell's long axis. The effect of a beta-agonist and of a calcium antagonist is described.

  4. Chemical Probes for Visualizing Intact Animal and Human Brain Tissue.

    PubMed

    Lai, Hei Ming; Ng, Wai-Lung; Gentleman, Steve M; Wu, Wutian

    2017-06-22

    Newly developed tissue clearing techniques can be used to render intact tissues transparent. When combined with fluorescent labeling technologies and optical sectioning microscopy, this allows visualization of fine structure in three dimensions. Gene-transfection techniques have proved very useful in visualizing cellular structures in animal models, but they are not applicable to human brain tissue. Here, we discuss the characteristics of an ideal chemical fluorescent probe for use in brain and other cleared tissues, and offer a comprehensive overview of currently available chemical probes. We describe their working principles and compare their performance with the goal of simplifying probe selection for neuropathologists and stimulating probe development by chemists. We propose several approaches for the development of innovative chemical labeling methods which, when combined with tissue clearing, have the potential to revolutionize how we study the structure and function of the human brain. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Probing Metabolism in the Intact Retina Using Stable Isotope Tracers.

    PubMed

    Du, Jianhai; Linton, Jonathan D; Hurley, James B

    2015-01-01

    Vertebrate retinas have several characteristics that make them particularly interesting from a metabolic perspective. The retinas have a highly laminated structure, high energy demands, and they share several metabolic features with tumors, such as a strong Warburg effect and abundant pyruvate kinase M2 isoform expression. The energy demands of retinas are both qualitatively and quantitatively different in light and darkness and metabolic dysfunction could cause retinal degeneration. Stable isotope-based metabolic analysis with mass spectrometry is a powerful tool to trace the dynamic metabolic reactions and reveal novel metabolic pathways within cells and between cells in retina. Here, we describe methods to quantify retinal metabolism in intact retinas and discuss applications of these methods to the understanding of neuron-glia interaction, light and dark adaptation, and retinal degenerative diseases.

  6. Perforation of Meckel's diverticulum by an intact fish bone

    PubMed Central

    Mouawad, Nicolas J; Hammond, Stephen; Kaoutzanis, Christodoulos

    2013-01-01

    Meckel's diverticulum is the most common congenital anomaly of the gastrointestinal tract, with an overall incidence of approximately 2.2%. It is generally noted incidentally during laparotomy for management of other abdominal pathology. Complications are infrequent, developing in 4% of individuals with this abnormality, and are usually seen in childhood. Herein, we discuss the case of a 52-year-old Caucasian man presenting with a 1-day history of worsening central and right-sided abdominal pain. Initial evaluation posed a broad differential however, following conservative measures and unremarkable plain films, the patient developed peritoneal signs necessitating operative intervention. During diagnostic laparoscopy, a Meckel's diverticulum was noted to be inflamed and perforated by an intact fish bone. The patient was treated successfully with a segmental resection and primary anastomosis, and had an uneventful postoperative recovery. PMID:23429021

  7. Static cerebrovascular pressure autoregulation remains intact during deep hypothermia.

    PubMed

    Goswami, Dheeraj; McLeod, Katherine; Leonard, Samantha; Kibler, Kathleen; Easley, Ronald Blaine; Fraser, Charles D; Andropoulos, Dean; Brady, Ken

    2017-09-01

    Clinical studies measuring cerebral blood flow in infants during deep hypothermia have demonstrated diminished cerebrovascular pressure autoregulation. The coexistence of hypotension in these cohorts confounds the conclusion that deep hypothermia impairs cerebrovascular pressure autoregulation. We sought to compare the lower limit of autoregulation and the static rate of autoregulation between normothermic and hypothermic piglets. Twenty anesthetized neonatal piglets (5-7 days old; 10 normothermic and 10 hypothermic to 20°C) had continuous measurements of cortical red cell flux using laser Doppler flowmetry, while hemorrhagic hypotension was induced without cardiopulmonary bypass. Lower limit of autoregulation was determined for each subject using piecewise regression and SRoR was determined above and below each lower limit of autoregulation as (%change cerebrovascular resistance/%change cerebral perfusion pressure). The estimated difference in lower limit of autoregulation was 1.4 mm Hg (lower in the hypothermic piglets; 95% C.I. -10 to 14 mm Hg; P=0.6). The median lower limit of autoregulation in the normothermic group was 39 mm Hg [IQR 38-51] vs 35 mm Hg [31-50] in the hypothermic group. Intact steady-state pressure autoregulation was defined as static rate of autoregulation >0.5 and was demonstrated in all normothermic subjects (static rate of autoregulation=0.72 [0.65-0.87]) and in 9/10 of the hypothermic subjects (static rate of autoregulation=0.65 [0.52-0.87]). This difference in static rate of autoregulation of 0.06 (95% C.I. -0.3 to 0.1) was not significant (P=0.4). Intact steady-state cerebrovascular pressure autoregulation is demonstrated in a swine model of profound hypothermia. Lower limit of autoregulation and static rate of autoregulation were similar in hypothermic and normothermic subjects. © 2017 John Wiley & Sons Ltd.

  8. Measuring mitochondrial respiration in intact single muscle fibers

    PubMed Central

    Schuh, Rosemary A.; Jackson, Kathryn C.; Khairallah, Ramzi J.; Ward, Christopher W.

    2012-01-01

    Measurement of mitochondrial function in skeletal muscle is a vital tool for understanding regulation of cellular bioenergetics. Currently, a number of different experimental approaches are employed to quantify mitochondrial function, with each involving either mechanically or chemically induced disruption of cellular membranes. Here, we describe a novel approach that allows for the quantification of substrate-induced mitochondria-driven oxygen consumption in intact single skeletal muscle fibers isolated from adult mice. Specifically, we isolated intact muscle fibers from the flexor digitorum brevis muscle and placed the fibers in culture conditions overnight. We then quantified oxygen consumption rates using a highly sensitive microplate format. Peak oxygen consumption rates were significantly increased by 3.4-fold and 2.9-fold by simultaneous stimulation with the uncoupling agent, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), and/or pyruvate or palmitate exposure, respectively. However, when calculating the total oxygen consumed over the entire treatment, palmitate exposure resulted in significantly more oxygen consumption compared with pyruvate. Further, as proof of principle for the procedure, we isolated fibers from the mdx mouse model, which has known mitochondrial deficits. We found significant reductions in initial and peak oxygen consumption of 51% and 61% compared with fibers isolated from the wild-type (WT) animals, respectively. In addition, we determined that fibers isolated from mdx mice exhibited less total oxygen consumption in response to the FCCP + pyruvate stimulation compared with the WT mice. This novel approach allows the user to make mitochondria-specific measures in a nondisrupted muscle fiber that has been isolated from a whole muscle. PMID:22160545

  9. Isolation of intact sub-dermal secretory cavities from Eucalyptus

    PubMed Central

    2010-01-01

    Background The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils. Results Leaves were digested using a variety of commercially available enzymes. A pectinase from Aspergillus niger was found to allow isolation of intact cavities after a relatively short incubation (12 h), with no visible artifacts from digestion and no loss of cellular integrity or cavity contents. Several measurements indicated the potential of the isolated cavities for further functional studies. First, the cavities were found to consume oxygen at a rate that is comparable to that estimated from leaf respiratory rates. Second, mRNA was extracted from cavities, and it was used to amplify a cDNA fragment with high similarity to that of a monoterpene synthase. Third, the contents of the cavity lumen were extracted, showing an unexpectedly low abundance of volatile essential oils and a sizeable amount of non-volatile material, which is contrary to the widely accepted role of secretory cavities as predominantly essential oil repositories. Conclusions The protocol described herein is likely to be adaptable to a range of Eucalyptus species with sub-dermal secretory cavities, and should find wide application in studies of the developmental and functional biology of these structures, and the biosynthesis of the plant natural products they contain. PMID:20807444

  10. Regulatory effects of phospholamban on cardiac function in intact mice.

    PubMed

    Lorenz, J N; Kranias, E G

    1997-12-01

    Phospholamban (PLB) regulates Ca(2+)-adenosinetriphosphatase activity in cardiac sarcoplasmic reticulum and participates in the regulation of myocardial performance. Animal models with altered levels of PLB permit in vivo evaluation of the physiological role of PLB. This study examined left ventricular (LV) performance in intact PLB heterozygous and homozygous mice under basal and stimulated conditions. A Millar Mikro-Tip transducer was inserted into the right carotid artery and advanced into the LV for direct measurement of ventricular pressure and the first derivative of intraventricular pressure (dP/dt). Baseline blood pressures were increased in PLB heterozygotes and even more so in PLB homozygotes compared with wild types (WT), and there were no differences in heart rate or LV end-diastolic pressure. The increase in pressure was primarily caused by an increase in systolic pressure. Baseline values for positive and negative dP/dt were linearly correlated with PLB levels. In PLB heterozygotes, contractile response to isoproterenol (Iso) was blunted compared with WT, but maximum rates of contraction were similar between the two groups. Contractile performance in PLB homozygous mice, which under baseline conditions was similar to maximum levels seen in WT, showed a blunted response to Iso, and maximum rates of contraction were significantly greater than in either of the other groups, indicating an essential but perhaps not exclusive role for PLB in mediating the inotropic effects of beta-adrenergic agonists. The effects of Iso on negative dP/dt were also blunted in both PLB heterozygous and PLB homozygous animals. Our results demonstrate that myocardial function in highly dependent on PLB level and suggest that the cardiovascular effects of PLB perturbations are largely uncompensated for in the intact mouse.

  11. Isolation and characterization of intact mitochondria from neonatal rat brain.

    PubMed

    Rajapakse, N; Shimizu, K; Payne, M; Busija, D

    2001-12-01

    Poor outcome after neonatal brain injury may be associated with alterations in mitochondrial function. Thus, isolated mitochondria have been a useful tool in understanding the underlying mechanisms of mitochondrial dysfunction. However, isolation and characterization of mitochondria from neonatal rat brain are not fully described. Thus, the aim of this study was to develop a rapid method for the isolation and characterization of functional mitochondria from neonatal rat brain. Mitochondria were isolated from 7-day-old rat brain weighing approximately 500 mg using a discontinuous Percoll density gradient. Brains were homogenized in 12% Percoll/sucrose buffer and layered onto a 26% Percoll/40% Percoll gradient followed by centrifugation. Four methods were used for assessing mitochondrial integrity and function: (1) electron microscopy to assess the morphology of the mitochondria and to determine the relative purity of the preparation; (2) fluorescence of chloromethyl-X-rosamine (Mito Tracker Red) in mitochondria as an indicator of mitochondrial membrane potential (Delta psi(m)); (3) state 3 and 4 respiration; and (4) protein import into mitochondria using an in vitro-synthesized mitochondrial malate dehydrogenase (mMDH). These studies demonstrated that the morphology of mitochondria is maintained with intact outer membranes and well-developed cristae, and Delta psi(m) is preserved. Respiration measurements revealed tightly coupled mitochondria with a respiration control ratio (RCR) of 4.1+/-0.18 (n=6). Import of precursor mMDH into mitochondria increased in a time-dependent manner maximizing at 15 min. The results indicate that neonatal brain mitochondria isolated using this method are well coupled, morphologically intact and are capable of protein import across the outer and inner mitochondrial membranes.

  12. Measuring mitochondrial respiration in intact single muscle fibers.

    PubMed

    Schuh, Rosemary A; Jackson, Kathryn C; Khairallah, Ramzi J; Ward, Christopher W; Spangenburg, Espen E

    2012-03-15

    Measurement of mitochondrial function in skeletal muscle is a vital tool for understanding regulation of cellular bioenergetics. Currently, a number of different experimental approaches are employed to quantify mitochondrial function, with each involving either mechanically or chemically induced disruption of cellular membranes. Here, we describe a novel approach that allows for the quantification of substrate-induced mitochondria-driven oxygen consumption in intact single skeletal muscle fibers isolated from adult mice. Specifically, we isolated intact muscle fibers from the flexor digitorum brevis muscle and placed the fibers in culture conditions overnight. We then quantified oxygen consumption rates using a highly sensitive microplate format. Peak oxygen consumption rates were significantly increased by 3.4-fold and 2.9-fold by simultaneous stimulation with the uncoupling agent, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), and/or pyruvate or palmitate exposure, respectively. However, when calculating the total oxygen consumed over the entire treatment, palmitate exposure resulted in significantly more oxygen consumption compared with pyruvate. Further, as proof of principle for the procedure, we isolated fibers from the mdx mouse model, which has known mitochondrial deficits. We found significant reductions in initial and peak oxygen consumption of 51% and 61% compared with fibers isolated from the wild-type (WT) animals, respectively. In addition, we determined that fibers isolated from mdx mice exhibited less total oxygen consumption in response to the FCCP + pyruvate stimulation compared with the WT mice. This novel approach allows the user to make mitochondria-specific measures in a nondisrupted muscle fiber that has been isolated from a whole muscle.

  13. Zeta Potential in Intact Natural Carbonates at Elevated Temperatures

    NASA Astrophysics Data System (ADS)

    Al-Mahrouqi, D.; Vinogradov, J.; Jackson, M.

    2015-12-01

    Measurements of zeta potential have been used to monitor subsurface flows in many natural brine systems. Numerous studies report zeta potentials in carbonates using crushed samples at low ionic strength and laboratory temperatures. However, natural brines have much higher salinity; moreover, temperatures are considerably higher in many subsurface settings. The variation of zeta potentials with temperature has not been examined in natural carbonates. We report zeta potential values interpreted from streaming potential measurements in two intact carbonate rock samples, saturated with artificial brines at elevated temperatures. We measure streaming potential using an experimental set-up that incorporates in-situ measurements of saturated rock conductivity, brine temperature, brine pH, brine electrical conductivity, pressure difference and voltage at temperatures up to 120oC. The streaming potential measurements are complemented with brine effluent studies. We find that the interpreted zeta potential is negative and decreases in magnitude with increasing temperature at low ionic strength (0.01M) and independent of temperature at high ionic strength (0.5M); consistent with published zeta potential in intact natural sandstones. The concentration of Ca2+ (main potential determining ion) also decreases with temperature at low ionic strength, but remains constant at high ionic strength. The temperature dependence of the zeta potential is consistent between two different natural carbonate samples and can be explained by the temperature dependence of pCa2+. We suggest that zeta potential of carbonate is independent of temperature or pH when pCa2+ remains constant. A linear variation of pH vs. pCa2+ is exhibited, at ambient and elevated temperatures, when pCa2+ is allowed to change with pH. This linear variation explains the numerous published data that shows apparent relationship between zeta potential of carbonates and pH.

  14. Three-dimensional Imaging of Crystalline Inclusions Embedded in Intact Maize Stalks

    PubMed Central

    Badger, John; Lal, Jyotsana; Harder, Ross; Inouye, Hideyo; Gleber, S. Charlotte; Vogt, Stefan; Robinson, Ian; Makowski, Lee

    2013-01-01

    Mineral inclusions in biomass are attracting increased scrutiny due to their potential impact on processing methods designed to provide renewable feedstocks for the production of chemicals and fuels. These inclusions are often sculpted by the plant into shapes required to support functional roles that include the storage of specific elements, strengthening of the plant structure, and providing a defense against pathogens and herbivores. In situ characterization of these inclusions faces substantial challenges since they are embedded in an opaque, complex polymeric matrix. Here we describe the use of Bragg coherent diffraction imaging (BCDI) to study mineral inclusions within intact maize stalks. Three-dimensional BCDI data sets were collected and used to reconstruct images of mineral inclusions at 50–100 nm resolution. Asymmetries in the intensity distributions around the Bragg peaks provided detailed information about the deformation fields within these crystal particles revealing lattice defects that result in distinct internal crystal domains. PMID:24091898

  15. Intact-protein analysis system for discovery of serum-based disease biomarkers.

    PubMed

    Wang, Hong; Hanash, Samir

    2011-01-01

    Profiling of serum and plasma proteins has substantial relevance to the discovery of circulating disease biomarkers. However, the extreme complexity and vast dynamic range of protein abundance in serum and plasma present a formidable challenge for protein analysis. Thus, integration of multiple technologies is required to achieve high-resolution and high-sensitivity proteomic analysis of serum or plasma. In this chapter, we describe an orthogonal multidimensional intact-protein analysis system (IPAS) (Wang et al., Mol Cell Proteomics 4:618-625, 2005) coupled with protein tagging (Faca et al., J Proteome Res 5:2009-2018, 2006) to profile the serum and plasma proteomes quantitatively, which we have applied in our biomarker discovery studies (Katayama et al., Genome Med 1:47, 2009; Faca et al., PLoS Med 5:e123, 2008; Zhang et al. Genome Biol 9:R93, 2008).

  16. Intact-Protein Analysis System for Discovery of Serum-Based Disease Biomarkers

    PubMed Central

    Wang, Hong; Hanash, Samir

    2015-01-01

    Profiling of serum and plasma proteins has substantial relevance to the discovery of circulating disease biomarkers. However, the extreme complexity and vast dynamic range of protein abundance in serum and plasma present a formidable challenge for protein analysis. Thus, integration of multiple technologies is required to achieve high-resolution and high-sensitivity proteomic analysis of serum or plasma. In this chapter, we describe an orthogonal multidimensional intact-protein analysis system (IPAS) (Wang et al., Mol Cell Proteomics 4:618–625, 2005) coupled with protein tagging (Faca et al., J Proteome Res 5:2009–2018, 2006) to profile the serum and plasma proteomes quantitatively, which we have applied in our biomarker discovery studies (Katayama et al., Genome Med 1:47, 2009; Faca et al., PLoS Med 5:e123, 2008; Zhang et al. Genome Biol 9:R93, 2008). PMID:21468941

  17. In vivo three-photon microscopy of subcortical structures within an intact mouse brain

    NASA Astrophysics Data System (ADS)

    Horton, Nicholas G.; Wang, Ke; Kobat, Demirhan; Clark, Catharine G.; Wise, Frank W.; Schaffer, Chris B.; Xu, Chris

    2013-03-01

    Two-photon fluorescence microscopy enables scientists in various fields including neuroscience, embryology and oncology to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of two-photon fluorescence microscopy to the cortical layer within mouse brain, and imaging subcortical structures currently requires the removal of overlying brain tissue or the insertion of optical probes. Here, we demonstrate non-invasive, high-resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein-labelled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher-order nonlinear excitation overcomes the limitations of two-photon fluorescence microscopy, enabling biological investigations to take place at a greater depth within tissue.

  18. A proximity-dependent assay for specific RNA-protein interactions in intact cells.

    PubMed

    Zhang, Wei; Xie, Mingyi; Shu, Mei-Di; Steitz, Joan A; DiMaio, Daniel

    2016-11-01

    The proximity ligation assay (PLA) is an immune staining method that detects protein-protein interactions in fixed cells. We describe here RNA-PLA, a simple adaptation of this technology that allows the detection of specific RNA-protein interactions in fixed cells by using a DNA oligonucleotide that hybridizes to a target RNA in combination with an antibody that recognizes the protein bound to the target RNA. Stable and transient RNA-protein interactions can be readily detected by generation of a fluorescent signal in discrete compartments in intact fixed cells with high specificity. We demonstrate that this approach requires the colocalization of the binding protein and its RNA target in the same cellular compartment, use of an oligonucleotide complementary to the target RNA, and the presence of a binding site for the protein in the target RNA. © 2016 Zhang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  19. [Laser Doppler vibrometry: a new tool for diagnosing hearing loss with an intact eardrum].

    PubMed

    Turcanu, Diana; Mârţu, D; Dalhoff, E; Gummer, A W

    2006-01-01

    The diagnosis of hearing loss with an intact eardrum frequently requires an entire battery of hearing tests, without the guarantee of an exact diagnosis. The techniques frequently provide only orientation for it, without establishing the site of the lesion and the etiology of the hearing loss. Laser Doppler vibrometry is a new technique, which has recently proved capable, of partially resolving this problem. The method is based on the study of the sound-induced vibration of the eardrum in humans in vivo, using a laser Doppler vibrometer. The method proved to be useful in the diagnosis of the pathology of the middle ear sound transmission system, avoiding the need for exploratory tympanotomy. Called "laser-audiometry", the method promises to become a new diagnostic tool for hearing impairment.

  20. In vivo robotics: the automation of neuroscience and other intact-system biological fields

    PubMed Central

    Kodandaramaiah, Suhasa B.; Boyden, Edward S.; Forest, Craig R.

    2013-01-01

    Robotic and automation technologies have played a huge role in in vitro biological science, having proved critical for scientific endeavors such as genome sequencing and high-throughput screening. Robotic and automation strategies are beginning to play a greater role in in vivo and in situ sciences, especially when it comes to the difficult in vivo experiments required for understanding the neural mechanisms of behavior and disease. In this perspective, we discuss the prospects for robotics and automation to impact neuroscientific and intact-system biology fields. We discuss how robotic innovations might be created to open up new frontiers in basic and applied neuroscience, and present a concrete example with our recent automation of in vivo whole cell patch clamp electrophysiology of neurons in the living mouse brain. PMID:23841584

  1. Structure, domain organization, and different conformational states of stem cell factor-induced intact KIT dimers

    PubMed Central

    Opatowsky, Yarden; Lax, Irit; Tomé, Francisco; Bleichert, Franziska; Unger, Vinzenz M.; Schlessinger, Joseph

    2014-01-01

    Using electron microscopy and fitting of crystal structures, we present the 3D reconstruction of ligand-induced dimers of intact receptor tyrosine kinase, KIT. We observe that KIT protomers form close contacts throughout the entire structure of ligand-bound receptor dimers, and that the dimeric receptors adopt multiple, defined conformational states. Interestingly, the homotypic interactions in the membrane proximal Ig-like domain of the extracellular region differ from those observed in the crystal structure of the unconstrained extracellular regions. We observe two prevalent conformations in which the tyrosine kinase domains interact asymmetrically. The asymmetric arrangement of the cytoplasmic regions may represent snapshots of molecular interactions occurring during trans autophosphorylation. Moreover, the asymmetric arrangements may facilitate specific intermolecular interactions necessary for trans phosphorylation of different KIT autophosphorylation sites that are required for stimulation of kinase activity and recruitment of signaling proteins by activated KIT. PMID:24449920

  2. In vivo robotics: the automation of neuroscience and other intact-system biological fields.

    PubMed

    Kodandaramaiah, Suhasa B; Boyden, Edward S; Forest, Craig R

    2013-12-01

    Robotic and automation technologies have played a huge role in in vitro biological science, having proved critical for scientific endeavors such as genome sequencing and high-throughput screening. Robotic and automation strategies are beginning to play a greater role in in vivo and in situ sciences, especially when it comes to the difficult in vivo experiments required for understanding the neural mechanisms of behavior and disease. In this perspective, we discuss the prospects for robotics and automation to influence neuroscientific and intact-system biology fields. We discuss how robotic innovations might be created to open up new frontiers in basic and applied neuroscience and present a concrete example with our recent automation of in vivo whole-cell patch clamp electrophysiology of neurons in the living mouse brain. © 2013 New York Academy of Sciences.

  3. Improved Methods for Estimating Microbial Activity and Moisture Characteristic Curves in Intact Unsaturated Soil Cores

    NASA Astrophysics Data System (ADS)

    Thompson, D. N.; Baker, K. E.

    2001-12-01

    Estimation of microbial activity in soils is a complex and often difficult process. In this work, we describe several new and innovative methods we have developed to measure microbial respiration in intact cores of unsaturated soils. The ultimate goal of this work is to predict the effect of microbial activity on contaminant mobility via CO2 generation in variably saturated vadose zone soils. This goal requires estimation of the effect of available water (i.e. in pores accessible to the microbes) on the microbial activity, and thus a homogeneous distribution of substrate throughout the soil water. Prior studies have added substrate solution drop wise to the soil, and then distributed the substrate throughout the soil by mixing. While this method distributes the substrate well, it alters the in situ pore volume distribution and has been shown to result in an anomalously high degree of microbial activity shortly after mixing. Traditional methods for uniformly distributing substrate in intact unsaturated soils require days to weeks to reach equilibrium. Since the substrate would be completely consumed in this time frame, an innovative approach is being used in this study to drain intact soil cores to the desired moisture contents in a matter of hours. This approach involves the use of the Unsaturated Flow Apparatus (UFAT). In the method, the samples are vacuum saturated under refrigeration to uniformly distribute a 14C-labeled substrate throughout the soil water, drained to various pressures in the UFA, and transferred to a sealed container and incubated. The labeled 14CO2 is then trapped and counted after incubation to determine microbial activity. Since the soil used in this study contains a high percentage of swelling clays, the cores tend to compact in the UFA, altering the macropore volume distribution. To address this alteration, we developed a correction function to correct the UFA-measured pore volume distribution at each rotational speed. Finally, the high

  4. Dynamic Mechanical Properties of Intact Human Cervical Spine Ligaments

    PubMed Central

    Ivancic, Paul C.; Coe, Marcus P.; Ndu, Anthony B.; Tominaga, Yasuhiro; Carlson, Erik J.; Rubin, Wolfgang; (FH), Dipl-Ing; Panjabi, Manohar M.

    2009-01-01

    BACKGROUND CONTEXT Most previous studies have investigated ligaments mechanical properties at slow elongation rates of less than 25 mm/s. PURPOSE To determine the tensile mechanical properties, at a fast elongation rate, of intact human cervical anterior and posterior longitudinal, capsular, and interspinous and supraspinous ligaments, middle-third disc, and ligamentum flavum. STUDY DESIGN/SETTING In vitro biomechanical study. METHODS A total of 97 intact bone-ligament-bone specimens (C2–C3 to C7-T1) were prepared from six cervical spines (average age: 80.6 years, range, 71 to 92 years) and were elongated to complete rupture at an average (SD) peak rate of 723 (106) mm/s using a custom-built apparatus. Non-linear force vs. elongation curves were plotted and peak force, peak elongation, peak energy, and stiffness were statistically compared (P<0.05) among ligament. A mathematical model was developed to determine the quasi-static physiological ligament elongation. RESULTS Highest average peak force, up to 244.4 and 220.0 N in the ligamentum flavum and capsular ligament, respectively, were significantly greater than in the anterior longitudinal ligament and middle-third disc. Highest peak elongation reached 5.9 mm in the intraspinous and supraspinous ligaments, significantly greater than in the middle-third disc. Highest peak energy of 0.57 J was attained in the capsular ligament, significantly greater than in the anterior longitudinal ligament and middle-third disc. Average stiffness was generally greatest in the ligamentum flavum and least in the intraspinous and supraspinous ligaments. For all ligaments, peak elongation was greater than average physiological elongation computed using the mathematical model. CONCLUSIONS Comparison of the present results with previously reported data indicated that high speed elongation may cause cervical ligaments to fail at a higher peak force and smaller peak elongation and may be stiffer and absorb less energy, as compared to a

  5. Cell proliferation in type C gastritis affecting the intact stomach

    PubMed Central

    Mac, D; Willis, P; Prescott, R; Lamonby, S; Lynch, D

    2000-01-01

    Aims—Type C gastritis caused by bile reflux has a characteristic appearance, similar to that seen in other forms of chemical gastritis, such as those associated with NSAIDs or alcohol. An increase in mucosal cell proliferation increases the likelihood of a neoplastic clone of epithelial cells emerging, particularly where there is chronic epithelial injury associated with bile reflux. It has been shown previously that type C gastritis is associated with increased cell proliferation in the postsurgical stomach. The aim of this study was to determine cell proliferation in type C gastritis caused by bile reflux affecting the intact stomach. Methods—Specimens from 15 patients with a histological diagnosis of type C gastritis on antral biopsy were obtained from the pathology archives between 1994 and 1997. A control group of nine normal antral biopsies was also selected and all underwent MIB-1 immunostaining. The gastric glands were divided into three zones (zone 1, gastric pit; zone 2, isthmus; and zone 3, gland base) and the numbers of positively staining nuclei for 500 epithelial cell nuclei were counted in each zone to determine the percentage labelling index (LI%). Results—Cell proliferation was significantly higher in all three zones of the gastric glands with type C gastritis compared with controls as follows: zone 1, median LI% in type C gastritis 64.7 (range, 7.8–99.2), controls 4.7 (range, 2.0–11.3); zone 2, median LI% in type C gastritis 94.7 (range, 28.8–98.7), controls 40.2 (range, 23.1–70.3); and zone 3, median LI% in type C gastritis 20.0 (range, 1.3–96.0), controls 2.6 (range, 0.9–8.7). Conclusions—Bile reflux is thought to act as a promoter of gastric carcinogenesis in the postsurgical stomach. The same may be true in the intact stomach. Key Words: cell proliferation • epithelial kinetics • chemical gastritis PMID:11064674

  6. Cationic Polymer Intercalation into the Lipid Membrane Enables Intact Polyplex DNA Escape from Endosomes for Gene Delivery.

    PubMed

    Vaidyanathan, Sriram; Chen, Junjie; Orr, Bradford G; Banaszak Holl, Mark M

    2016-06-06

    Developing improved cationic polymer-DNA polyplexes for gene delivery requires improved understanding of DNA transport from endosomes into the nucleus. Using a FRET-capable oligonucleotide molecular beacon (OMB), we monitored the transport of intact DNA to cell organelles. We observed that for effective (jetPEI) and ineffective (G5 PAMAM) vectors, the fraction of cells displaying intact OMB in the cytosol (jetPEI ≫ G5 PAMAM) quantitatively predicted the fraction expressing transgene (jetPEI ≫ G5 PAMAM). Intact OMB delivered with PAMAM and confined to endosomes could be released to the cytosol by the subsequent addition of L-PEI, with a corresponding 10-fold increase in transgene expression. These results suggest that future vector development should optimize vectors for intercalation into, and destabilization of, the endosomal membrane. Finally, the study highlights a two-step strategy in which the pDNA is loaded in cells using one vector and endosomal release is mediated by a second agent.

  7. Effect of extracorporeal shock wave therapy on fracture healing in rat femural fractures with intact and excised periosteum.

    PubMed

    Oktaş, Birhan; Orhan, Zafer; Erbil, Barış; Değirmenci, Erdem; Ustündağ, Nil

    2014-01-01

    The aim of this study is to compare the effect of extracorporeal shock wave therapy (ESWT) on fractures with intact periosteum and excised periosteum. Thirty-seven Wistar albino rats were randomized into four groups. Osteotomy and intramedullary Kirschner wire fixation were performed on all right femurs under ketamin anesthesia. The first group (n=10) was identified as control group. In the second group (n=10), periosteum located at the osteotomy site was excised circumferentially during surgery. In the third group (n=9), periosteum was left intact and ESWT was applied. In the forth group (n=8), periosteums of all rats were excised and ESWT was applied. All fracture lines were evaluated radiographically each two weeks and histologically at the sixth week. Results were evaluated statistically. In periosteum excised group which represents a model of open fractures with soft tissue defect, ESWT application had a significantly positive histologic effect on bone healing. However, radiological evaluation did not reveal any statistically significant difference between groups with intact and excised periosteums. According to our findings, ESWT can be used to improve fracture healing and prevent pseudoarthrosis in the treatment of open fractures with accompanying soft tissue and periosteum damage. However, further clinical studies are required to include ESWT in routine practice.

  8. Mechanisms of the Frank-Starling phenomena studied in intact hearts.

    PubMed

    Burkhoff, D; Stennett, R A; Ogino, K

    1995-01-01

    The impact of ventricular volume on the relationship between intracellular calcium and ventricular pressure under steady-state conditions was determined in intact ferret hearts. The results reveal major quantitative differences and minor qualitative differences between these relations and those previously measured in isolated intact and skinned cardiac muscle. The importance of these differences is discussed within the context of developing a comprehensive mechanistic theory to describe load-dependence of the intact ventricle.

  9. Changes in BETA-adrenoreceptor density in intact lymphocytes of hypertensive subjects

    SciTech Connect

    Krasnikova, T.L.; Il'inskii, O.B.; Parfenova, E.V.; Radyukhin, V.A.

    1985-05-01

    This paper determines the density of beta-adrenoreceptors of intact lymphocytes from patients with stable essential hypertension and compares it with that of normal subjects. Specific binding of tritium-dihydroalprenolol with intact lymphoctes under certain described conditions was characterized by saturation and high affinity. The results are evidence of a statistically significant increase (by 42%) in beta-adrenoreceptor density of intact lymphocytes in essential hypertension.

  10. Ladder Treadmill: A Method to Assess Locomotion in Cats with an Intact or Lesioned Spinal Cord.

    PubMed

    Escalona, Manuel; Delivet-Mongrain, Hugo; Kundu, Aritra; Gossard, Jean-Pierre; Rossignol, Serge

    2017-05-31

    After lesions of the CNS, locomotor abilities of animals (mainly cats) are often assessed on a simple flat treadmill (FTM), which imposes little demands on supraspinal structures as is the case when walking on targets. Therefore, the aims of the present work were as follows: (1) to develop a treadmill allowing the assessment of locomotion of intact cats required to place the paws on the rungs of a moving ladder treadmill (LTM); (2) to assess the capability of cats after a unilateral spinal hemisection at T10 to cope with such a demanding locomotor task; and (3) to regularly train cats for 6 weeks on the LTM to determine whether such regular training improves locomotor recovery on the FTM. A significant improvement would indicate that LTM training maximizes the contribution of spinal locomotor circuits as well as remnant supraspinal inputs. Together, we used 9 cats (7 females, 2 males). Six were used to compare the EMG and kinematic locomotor characteristics during walking on the FTM and LTM. We found that the swing phase during LTM walking was slightly enhanced as well as some specific activity of knee flexor muscles. Fore-hindlimb coupling favored a more stable diagonal coupling. These 6 cats were then hemispinalized and trained for 6 weeks on the LTM, whereas the 3 other cats were hemispinalized and trained solely on the FTM to compare the two training regimens. Intensive LTM training after hemisection was found to change features of locomotion, such as the foot trajectory as well as diminished paw drag often observed after hemisection.SIGNIFICANCE STATEMENT This paper introduces a method (ladder treadmill [LTM]) to study the locomotor ability of cats with an intact spinal cord or after a unilateral hemisection to walk with a precise foot placement on the rungs fixed to an ordinary flat treadmill (FTM). Because cats are compared in various conditions (intact or hemisected at different time points) in the same enclosure on the FTM and the LTM, the changes in

  11. Stress dependence of permeability of intact and fractured shale cores.

    NASA Astrophysics Data System (ADS)

    van Noort, Reinier; Yarushina, Viktoriya

    2016-04-01

    Whether a shale acts as a caprock, source rock, or reservoir, understanding fluid flow through shale is of major importance for understanding fluid flow in geological systems. Because of the low permeability of shale, flow is thought to be largely confined to fractures and similar features. In fracking operations, fractures are induced specifically to allow for hydrocarbon exploration. We have constructed an experimental setup to measure core permeabilities, using constant flow or a transient pulse. In this setup, we have measured the permeability of intact and fractured shale core samples, using either water or supercritical CO2 as the transporting fluid. Our measurements show decreasing permeability with increasing confining pressure, mainly due to time-dependent creep. Furthermore, our measurements show that for a simple splitting fracture, time-dependent creep will also eliminate any significant effect of this fracture on permeability. This effect of confinement on fracture permeability can have important implications regarding the effects of fracturing on shale permeability, and hence for operations depending on that.

  12. Adolescent Segond fracture with an intact anterior cruciate ligament.

    PubMed

    Reddy, Deepak; Alexander, Reginald; Hussain, Waqas M; Leland, J Martin

    2012-07-01

    Segond fractures, or avulsion fractures of the proximal lateral tibial plateau, have been well documented and studied since their original description in 1878. Segond fractures have a widely recognized pathognomonic association with anterior cruciate ligament (ACL) injuries and often prompt orthopedic surgeons to consider reconstruction following radiographic and clinical evaluation. Adolescent patients are particularly vulnerable to these fractures due to the relative weakness of their physeal growth plates compared with the strength of their accompanying ligamentous structures. This article describes a case of a 13-year-old boy who sustained a Segond fracture that was not coupled with an ACL avulsion or tear. The patient sustained a twisting injury to his knee. He presented to the emergency room with an effusion and radiographic findings consistent with a Segond fracture. On follow-up examination 1 week after injury, the ACL was intact. The patient was followed for 5 months of conservative treatment. At final follow-up, the patient had reestablished his previous level of activity. This article describes the history, physical examination, and radiographic findings necessary to care for patients who present with a Segond fracture. Although considered pathognomonic for an associated ACL injury, this article describes a Segond fracture that occurred in isolation.

  13. Measurement of the Mechanical Properties of Intact Collagen Fibrils

    NASA Astrophysics Data System (ADS)

    Mercedes, H.; Heim, A.; Matthews, W. G.; Koob, T.

    2006-03-01

    Motivated by the genetic disorder Ehlers-Danlos syndrome (EDS), in which proper collagen synthesis is interrupted, we are investigating the structural and mechanical properties of collagen fibrils. The fibrous glycoprotein collagen is the most abundant protein found in the human body and plays a key role in the extracellular matrix of the connective tissue, the properties of which are altered in EDS. We have selected as our model system the collagen fibrils of the sea cucumber dermis, a naturally mutable tissue. This system allows us to work with native fibrils which have their proteoglycan complement intact, something that is not possible with reconstituted mammalian collagen fibrils. Using atomic force microscopy, we measure, as a function of the concentration of divalent cations, the fibril diameter, its response to force loading, and the changes in its rigidity. Through these experiments, we will shed light on the mechanisms which control the properties of the sea cucumber dermis and hope to help explain the altered connective tissue extracellular matrix properties associated with EDS.

  14. Intact implicit processing of facial threat cues in schizophrenia.

    PubMed

    Shasteen, Jonathon R; Pinkham, Amy E; Kelsven, Skylar; Ludwig, Kelsey; Payne, B Keith; Penn, David L

    2016-01-01

    An emerging body of research suggests that people with schizophrenia retain the ability to implicitly perceive facial affect, despite well-documented difficulty explicitly identifying emotional expressions. It remains unclear, however, whether such functional implicit processing extends beyond emotion to other socially relevant facial cues. Here, we constructed two novel versions of the Affect Misattribution Procedure, a paradigm in which affective responses to primes are projected onto neutral targets. The first version included three face primes previously validated to elicit varying inferences of threat from healthy individuals via emotion-independent structural modification (e.g., nose and eye size). The second version included the threat-relevant emotional primes of angry, neutral, and happy faces. Data from 126 participants with schizophrenia and 84 healthy controls revealed that although performing more poorly on an assessment of explicit emotion recognition, patients showed normative implicit threat processing for both non-emotional and emotional facial cues. Collectively, these results support recent hypotheses postulating that the initial perception of salient facial information remains intact in schizophrenia, but that deficits arise at subsequent stages of contextual integration and appraisal. Such a breakdown in the stream of face processing has important implications for mechanistic models of social cognitive impairment in schizophrenia and treatment strategies aiming to improve functional outcome. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Activation and deactivation of vibronic channels in intact phycocyanin rods

    NASA Astrophysics Data System (ADS)

    Nganou, C.; David, L.; Meinke, R.; Adir, N.; Maultzsch, J.; Mkandawire, M.; Pouhè, D.; Thomsen, C.

    2014-02-01

    We investigated the excitation modes of the light-harvesting protein phycocyanin (PC) from Thermosynechococcus vulcanus in the crystalline state using UV and near-infrared Raman spectroscopy. The spectra revealed the absence of a hydrogen out-of-plane wagging (HOOP) mode in the PC trimer, which suggests that the HOOP mode is activated in the intact PC rod, while it is not active in the PC trimer. Furthermore, in the PC trimer an intense mode at 984 cm-1 is assigned to the C-C stretching vibration while the mode at 454 cm-1 is likely due to ethyl group torsion. In contrast, in the similar chromophore phytochromobilin the C5,10,15-D wag mode at 622 cm-1 does not come from a downshift of the HOOP. Additionally, the absence of modes between 1200 and 1300 cm-1 rules out functional monomerization. A correlation between phycocyanobilin (PCB) and phycoerythrobilin (PEB) suggests that the PCB cofactors of the PC trimer appear in a conformation similar to that of PEB. The conformation of the PC rod is consistent with that of the allophycocyanin (APC) trimer, and thus excitonic flow is facilitated between these two independent light-harvesting compounds. This excitonic flow from the PC rod to APC appears to be modulated by the vibration channels during HOOP wagging, C = C stretching, and the N-H rocking in-plan vibration.

  16. Activation and deactivation of vibronic channels in intact phycocyanin rods.

    PubMed

    Nganou, C; David, L; Meinke, R; Adir, N; Maultzsch, J; Mkandawire, M; Pouhè, D; Thomsen, C

    2014-02-28

    We investigated the excitation modes of the light-harvesting protein phycocyanin (PC) from Thermosynechococcus vulcanus in the crystalline state using UV and near-infrared Raman spectroscopy. The spectra revealed the absence of a hydrogen out-of-plane wagging (HOOP) mode in the PC trimer, which suggests that the HOOP mode is activated in the intact PC rod, while it is not active in the PC trimer. Furthermore, in the PC trimer an intense mode at 984 cm(-1) is assigned to the C-C stretching vibration while the mode at 454 cm(-1) is likely due to ethyl group torsion. In contrast, in the similar chromophore phytochromobilin the C5,10,15-D wag mode at 622 cm(-1) does not come from a downshift of the HOOP. Additionally, the absence of modes between 1200 and 1300 cm(-1) rules out functional monomerization. A correlation between phycocyanobilin (PCB) and phycoerythrobilin (PEB) suggests that the PCB cofactors of the PC trimer appear in a conformation similar to that of PEB. The conformation of the PC rod is consistent with that of the allophycocyanin (APC) trimer, and thus excitonic flow is facilitated between these two independent light-harvesting compounds. This excitonic flow from the PC rod to APC appears to be modulated by the vibration channels during HOOP wagging, C = C stretching, and the N-H rocking in-plan vibration.

  17. Intact amino acid uptake by northern hardwood and conifer trees.

    PubMed

    Gallet-Budynek, Anne; Brzostek, Edward; Rodgers, Vikki L; Talbot, Jennifer M; Hyzy, Sharon; Finzi, Adrien C

    2009-05-01

    Empirical and modeling studies of the N cycle in temperate forests of eastern North America have focused on the mechanisms regulating the production of inorganic N, and assumed that only inorganic forms of N are available for plant growth. Recent isotope studies in field conditions suggest that amino acid capture is a widespread ecological phenomenon, although northern temperate forests have yet to be studied. We quantified fine root biomass and applied tracer-level quantities of U-(13)C(2)-(15)N-glycine, (15)NH(4) (+) and (15)NO(3) (-) in two stands, one dominated by sugar maple and white ash, the other dominated by red oak, beech, and hemlock, to assess the importance of amino acids to the N nutrition of northeastern US forests. Significant enrichment of (13)C in fine roots 2 and 5 h following tracer application indicated intact glycine uptake in both stands. Glycine accounted for up to 77% of total N uptake in the oak-beech-hemlock stand, a stand that produces recalcitrant litter, cycles N slowly and has a thick, amino acid-rich organic horizon. By contrast, glycine accounted for only 20% of total N uptake in the sugar maple and white ash stand, a stand characterized by labile litter and rapid rates of amino acid production and turnover resulting in high rates of mineralization and nitrification. This study shows that amino acid uptake is an important process occurring in two widespread, northeastern US temperate forest types with widely differing rates of N cycling.

  18. Intact implicit statistical learning in borderline personality disorder.

    PubMed

    Unoka, Zsolt; Vizin, Gabriella; Bjelik, Anna; Radics, Dóra; Nemeth, Dezso; Janacsek, Karolina

    2017-09-01

    Wide-spread neuropsychological deficits have been identified in borderline personality disorder (BPD). Previous research found impairments in decision making, declarative memory, working memory and executive functions; however, no studies have focused on implicit learning in BPD yet. The aim of our study was to investigate implicit statistical learning by comparing learning performance of 19 BPD patients and 19 healthy, age-, education- and gender-matched controls on a probabilistic sequence learning task. Moreover, we also tested whether participants retain the acquired knowledge after a delay period. To this end, participants were retested on a shorter version of the same task 24h after the learning phase. We found intact implicit statistical learning as well as retention of the acquired knowledge in this personality disorder. BPD patients seem to be able to extract and represent regularities implicitly, which is in line with the notion that implicit learning is less susceptible to illness compared to the more explicit processes. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  19. Covert spatial attention is functionally intact in amblyopic human adults

    PubMed Central

    Roberts, Mariel; Cymerman, Rachel; Smith, R. Theodore; Kiorpes, Lynne; Carrasco, Marisa

    2016-01-01

    Certain abnormalities in behavioral performance and neural signaling have been attributed to a deficit of visual attention in amblyopia, a neurodevelopmental disorder characterized by a diverse array of visual deficits following abnormal binocular childhood experience. Critically, most have inferred attention's role in their task without explicitly manipulating and measuring its effects against a baseline condition. Here, we directly investigate whether human amblyopic adults benefit from covert spatial attention—the selective processing of visual information in the absence of eye movements—to the same degree as neurotypical observers. We manipulated both involuntary (Experiment 1) and voluntary (Experiment 2) attention during an orientation discrimination task for which the effects of covert spatial attention have been well established in neurotypical and special populations. In both experiments, attention significantly improved accuracy and decreased reaction times to a similar extent (a) between the eyes of the amblyopic adults and (b) between the amblyopes and their age- and gender-matched controls. Moreover, deployment of voluntary attention away from the target location significantly impaired task performance (Experiment 2). The magnitudes of the involuntary and voluntary attention benefits did not correlate with amblyopic depth or severity. Both groups of observers showed canonical performance fields (better performance along the horizontal than vertical meridian and at the lower than upper vertical meridian) and similar effects of attention across locations. Despite their characteristic low-level vision impairments, covert spatial attention remains functionally intact in human amblyopic adults. PMID:28033433

  20. Right ventricular outflow obstruction with intact ventricular septum in adults.

    PubMed Central

    Werner, A M; Darrell, J C; Pallegrini, R V; Woelfel, G F; Grant, K; Marrangoni, A G

    1997-01-01

    Cardiothoracic surgeons whose practice is limited to adults rarely see patients with right ventricular outflow obstruction and an intact ventricular septum. Of more than 10,000 open-heart procedures performed at our institution from 1983 to 1993 (in patients 18 to 75 years old), only 5 procedures were for correction of this problem. Both the pulmonary valve and the subvalvular area were abnormal in these 5 patients, and 4 of the 5 had subvalvular stenosis. The gradient across the right ventricular outflow tract was measured by cardiac catheterization before repair in all patients and averaged 118 mmHg. Various surgical approaches were used for repair. In the 2 patients whose pressures were measured postoperatively, the gradients were 25 mmHg and 45 mmHg, respectively. There were no operative deaths. At follow-up (range, 2 months to 5 years after surgery), all patients were in New York Heart Association functional class I and all had murmurs. Those who underwent echocardiography were found to have minimal gradients across the right ventricular outflow tract. Images PMID:9205983

  1. Follicle dynamics and global organization in the intact mouse ovary

    PubMed Central

    Faire, Mehlika; Skillern, Amanda; Arora, Ripla; Nguyen, Daniel H.; Wong, Jason; Chamberlain, Chester; German, Michael S.; Fung, Jennifer C.; Laird, Diana J.

    2015-01-01

    Quantitative analysis of tissues and organs can reveal large-scale patterning as well as the impact of perturbations and aging on biological architecture. Here we develop tools for imaging of single cells in intact organs and computational approaches to assess spatial relationships in 3D. In the mouse ovary, we use nuclear volume of the oocyte to read out quiescence or growth of oocyte-somatic cell units known as follicles. This in-ovary quantification of non-growing follicle dynamics from neonate to adult fits a mathematical function, which corroborates the model of fixed oocyte reserve. Mapping approaches show that radial organization of folliculogenesis established in the newborn ovary is preserved through adulthood. By contrast, inter-follicle clustering increases during aging with different dynamics depending on size. These broadly applicable tools can reveal high dimensional phenotypes and age-related architectural changes in other organs. In the adult mouse pancreas, we find stochastic radial organization of the islets of Langerhans but evidence for localized interactions among the smallest islets. PMID:25889274

  2. Basic Timing Abilities Stay Intact in Patients with Musician's Dystonia

    PubMed Central

    van der Steen, M. C.; van Vugt, Floris T.; Keller, Peter E.; Altenmüller, Eckart

    2014-01-01

    Task-specific focal dystonia is a movement disorder that is characterized by the loss of voluntary motor control in extensively trained movements. Musician's dystonia is a type of task-specific dystonia that is elicited in professional musicians during instrumental playing. The disorder has been associated with deficits in timing. In order to test the hypothesis that basic timing abilities are affected by musician's dystonia, we investigated a group of patients (N = 15) and a matched control group (N = 15) on a battery of sensory and sensorimotor synchronization tasks. Results did not show any deficits in auditory-motor processing for patients relative to controls. Both groups benefited from a pacing sequence that adapted to their timing (in a sensorimotor synchronization task at a stable tempo). In a purely perceptual task, both groups were able to detect a misaligned metronome when it was late rather than early relative to a musical beat. Overall, the results suggest that basic timing abilities stay intact in patients with musician's dystonia. This supports the idea that musician's dystonia is a highly task-specific movement disorder in which patients are mostly impaired in tasks closely related to the demands of actually playing their instrument. PMID:24667273

  3. Action Perception Is Intact in Autism Spectrum Disorder

    PubMed Central

    Williams, Justin H.G.; Neri, Peter

    2015-01-01

    Autistic traits span a wide spectrum of behavioral departures from typical function. Despite the heterogeneous nature of autism spectrum disorder (ASD), there have been attempts at formulating unified theoretical accounts of the associated impairments in social cognition. A class of prominent theories capitalizes on the link between social interaction and visual perception: effective interaction with others often relies on discrimination of subtle nonverbal cues. It has been proposed that individuals with ASD may rely on poorer perceptual representations of other people's actions as returned by dysfunctional visual circuitry and that this, in turn, may lead to less effective interpretation of those actions for social behavior. It remains unclear whether such perceptual deficits exist in ASD: the evidence currently available is limited to specific aspects of action recognition, and the reported deficits are often attributable to cognitive factors that may not be strictly visual (e.g., attention). We present results from an exhaustive set of measurements spanning the entire action processing hierarchy, from motion detection to action interpretation, designed to factor out effects that are not selectively relevant to this function. Our results demonstrate that the ASD perceptual system returns functionally intact signals for interpreting other people's actions adequately; these signals can be accessed effectively when autistic individuals are prompted and motivated to do so under controlled conditions. However, they may fail to exploit them adequately during real-life social interactions. PMID:25653346

  4. Basic timing abilities stay intact in patients with musician's dystonia.

    PubMed

    van der Steen, M C; van Vugt, Floris T; Keller, Peter E; Altenmüller, Eckart

    2014-01-01

    Task-specific focal dystonia is a movement disorder that is characterized by the loss of voluntary motor control in extensively trained movements. Musician's dystonia is a type of task-specific dystonia that is elicited in professional musicians during instrumental playing. The disorder has been associated with deficits in timing. In order to test the hypothesis that basic timing abilities are affected by musician's dystonia, we investigated a group of patients (N = 15) and a matched control group (N = 15) on a battery of sensory and sensorimotor synchronization tasks. Results did not show any deficits in auditory-motor processing for patients relative to controls. Both groups benefited from a pacing sequence that adapted to their timing (in a sensorimotor synchronization task at a stable tempo). In a purely perceptual task, both groups were able to detect a misaligned metronome when it was late rather than early relative to a musical beat. Overall, the results suggest that basic timing abilities stay intact in patients with musician's dystonia. This supports the idea that musician's dystonia is a highly task-specific movement disorder in which patients are mostly impaired in tasks closely related to the demands of actually playing their instrument.

  5. Covert spatial attention is functionally intact in amblyopic human adults.

    PubMed

    Roberts, Mariel; Cymerman, Rachel; Smith, R Theodore; Kiorpes, Lynne; Carrasco, Marisa

    2016-12-01

    Certain abnormalities in behavioral performance and neural signaling have been attributed to a deficit of visual attention in amblyopia, a neurodevelopmental disorder characterized by a diverse array of visual deficits following abnormal binocular childhood experience. Critically, most have inferred attention's role in their task without explicitly manipulating and measuring its effects against a baseline condition. Here, we directly investigate whether human amblyopic adults benefit from covert spatial attention-the selective processing of visual information in the absence of eye movements-to the same degree as neurotypical observers. We manipulated both involuntary (Experiment 1) and voluntary (Experiment 2) attention during an orientation discrimination task for which the effects of covert spatial attention have been well established in neurotypical and special populations. In both experiments, attention significantly improved accuracy and decreased reaction times to a similar extent (a) between the eyes of the amblyopic adults and (b) between the amblyopes and their age- and gender-matched controls. Moreover, deployment of voluntary attention away from the target location significantly impaired task performance (Experiment 2). The magnitudes of the involuntary and voluntary attention benefits did not correlate with amblyopic depth or severity. Both groups of observers showed canonical performance fields (better performance along the horizontal than vertical meridian and at the lower than upper vertical meridian) and similar effects of attention across locations. Despite their characteristic low-level vision impairments, covert spatial attention remains functionally intact in human amblyopic adults.

  6. Coronary arterial abnormalities in pulmonary atresia with intact ventricular septum.

    PubMed

    Calder, A L; Co, E E; Sage, M D

    1987-02-15

    The incidence and severity of abnormalities of the coronary arteries were evaluated in 35 necropsy patients with pulmonary atresia and an intact ventricular septum. Right ventricular to coronary artery fistulous connections were found in more than 60% of the cases. All patients with fistulous connections had histologic abnormalities of the coronary arteries. In 50% the lesions were mild, with medial and intimal thickening producing up to moderate luminal stenosis. In 50% there was loss of normal arterial wall structure and severe narrowing or obliteration of the arterial lumen. The coronary arterial fistulas and histologic abnormalities were prevalent in those with underdevelopment of the tricuspid valve and right ventricular cavity but were not found in patients with a normal or dilated tricuspid valve anulus. The coronary arterial abnormalities were found in more than 80% of patients with a tricuspid valve/mitral valve ratio less than 1. A single coronary artery occurred in 6 patients (17%) of this series. In 80% of those with 1 coronary artery arising from the other, the aberrantly arising coronary artery crossed anteriorly to the pulmonary artery and could be at risk in surgical attempts to reconstruct the right ventricular outflow. Aortography is recommended if the coronary arteries are not clearly delineated on ventricular cineangiocardiography.

  7. Spontaneous Postpartum Rupture of an Intact Uterus: A Case Report

    PubMed Central

    Mavromatidis, George; Karavas, George; Margioula-Siarkou, Chrysoula; Petousis, Stamatios; Kalogiannidis, Ioannis; Mamopoulos, Apostolos; Rousso, David

    2015-01-01

    Rupture of uterus is an obstetrical complication characterized by a breach in the uterine wall and the overlying serosa. We report an unusual case of spontaneous rupture of an unscarred uterus in a 33-year-old woman, a day after her third successful vaginal delivery. A 33-year-old pregnant woman, gravid 3, para 3, was referred to our department at 39 gestational week because of rupture of membranes. Despite tocolysis administration, her pregnancy was delivered vaginally after 2 days, giving birth to a male neonate of 3,020 g with normal Apgar scores at first and fifth minute. Her uterus was intact and gynecological examination after delivery was normal without any potential signs or symptoms of pathology. However, the day following her labor, patient complained of left iliac fossa pain. Her blood tests revealed a CRP value at 27.6 mg/L, whereas the X-rays revealed an extensive impacted fecal mass in the colon. MRI revealed that the left lower myometrial part of the uterus was depicted abrupt, with simultaneous presence of hemorrhagic stuff. The decision of laparotomy was therefore made in order to further evaluate rupture of uterus and properly treat patient. And subtotal hysterectomy was performed. Postoperative follow-up period was not characterized by any complications and patient was finally discharged 4 days after hysterectomy. PMID:25368704

  8. Amniotic Fluid Infection in Preterm Pregnancies with Intact Membranes

    PubMed Central

    Rahkonen, Leena; Nupponen, Irmeli; Pätäri-Sampo, Anu; Tikkanen, Minna; Sorsa, Timo; Juhila, Juuso; Andersson, Sture; Paavonen, Jorma; Stefanovic, Vedran

    2017-01-01

    Introduction. Intra-amniotic infection (IAI) is a major cause of preterm labor and adverse neonatal outcome. We evaluated amniotic fluid (AF) proteolytic cascade forming biomarkers in relation to microbial invasion of the amniotic cavity (MIAC) and IAI in preterm pregnancies with intact membranes. Material and Methods. Amniocentesis was made to 73 women with singleton pregnancies; 27 with suspected IAI; and 46 controls. AF biomarkers were divided into three cascades: Cascade 1: matrix metalloproteinase-8 (MMP-8), MMP-9, myeloperoxidase (MPO), and interleukin-6; Cascade 2: neutrophil elastase (HNE), elafin, and MMP-9; Cascade 3: MMP-2, tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), MMP-8/TIMP-1 molar ratio, and C-reactive protein (CRP). MMP-8 was measured by an immunoenzymometric assay and the others were measured by ELISA. Standard biochemical methods, molecular microbiology, and culture techniques were used. Results. MMP-8, MMP-9, MPO, elafin, and TIMP-1 concentrations were higher in IAI suspected cases compared to controls and also in IAI suspected cases with MIAC compared to those without MIAC when adjusted by gestational age at amniocentesis. All biomarkers except elafin and MMP-2 had the sensitivity of 100% with thresholds based on ROC-curve. Odd ratios of biomarkers for MIAC were 1.2-38 and 95% confidential intervals 1.0-353.6. Conclusions. Neutrophil based AF biomarkers were associated with IAI and MIAC. PMID:28167848

  9. Rapid isolation of intact, viable fetal cartilage models

    SciTech Connect

    Schmidt, R.R.; Chepenik, K.P.; Paynton, B.V.; Cotler, J.M.

    1982-04-01

    A rapid procedure is described for the isolation of viable, intact, femoral cartilage models (humeri and femora) obtained from pregnant rats on the 18th day of gestation. Viability of these models is demonstrated in an in vitro system where the incorporation of /sup 35/S-sulfate was linear with time of incubation and with numbers of cartilage models utilized. Treatment of cartilage models with ice-cold trichloroacetic acid and a boiling water bath prior to incubation with radiolabel, reduced the amount of radioactivity incorporated to 1.3% of that observed for models incubated by routine procedures. Furthermore, digestion of cartilage model homogenates with protease yielded a supernatant from which 51% to 57% of the radioactivity was precipitated as GAG. This method may also be used to isolate fetal cartilage models as early as the 16th day of gestation. with this system, specific biochemical parameters of mammalian fetal chondrogenesis may be surveyed in normally and abnormally developing fetal cartilage free of surrounding soft tissue.

  10. The prototype effect in recognition memory: intact in autism?

    PubMed

    Molesworth, Catherine J; Bowler, Dermot M; Hampton, James A

    2005-06-01

    There are two accounts of categorization performance in autism: that there is an impairment in prototype formation (Klinger & Dawson, 2001) and that there is an impairment in processing features held in common between stimuli (Plaisted, O'Riordan, & Baron-Cohen, 1998). These accounts, together with central coherence theory (Frith, 1989; Frith & Happé, 1994), imply a reduced or absent prototype effect in autism. Children with autism or Asperger syndrome (n = 15) matched on age, gender, and verbal mental age with typically developing children (n = 15) completed a picture recognition task (Experiment 1). These participants also studied categories of cartoon animals possessing either an average prototype structure (Experiment 2) based on (Younger's 1985) stimuli or a modal structure (Experiment 3) based on (Hayes and Taplin's 1993b) stimuli. Following the study phases, participants completed recognition tests comprising prototypes and other exemplars with varying degrees of similarity to the prototypes. For both participant groups, recognition memory appeared intact (Experiment 1) and a full prototype effect in recognition memory was observed in both Experiment 2 and Experiment 3. The present studies fail to support predictions of impaired prototype effects in autism. The discussion focuses on key methodological differences between these studies and those that support claims that central coherence, prototype formation, and common feature processing are impaired in autism.

  11. Compensatory mechanisms underlie intact task-switching performance in schizophrenia.

    PubMed

    Jamadar, S; Michie, P; Karayanidis, F

    2010-04-01

    Individuals with schizophrenia tend to perform poorly on many measures of cognitive control. However, recent task-switching studies suggest that they show intact task-switching performance, despite the fact that the regions involved in task-switching are known to be structurally and functionally impaired in the disorder. Behavioral, event-related potential (ERP) and functional magnetic resonance imaging (fMRI) measures were used to compare the temporal and spatial dynamics of task-switching performance in individuals with schizophrenia and controls. Consistent with previous studies, reaction time (RT) switch cost and its reduction with anticipatory preparation did not differ between groups. There were also no group differences on cue-locked ERP components associated with anticipatory preparation processes. However, both stimulus- and response-locked ERPs were significantly disrupted in schizophrenia, suggesting difficulty with task-set implementation. fMRI analyses indicated that individuals with schizophrenia showed hyperactivity in the dorsolateral prefrontal cortex and posterior parietal cortex. RT-fMRI and ERP-fMRI associations suggested that individuals with schizophrenia employ compensatory mechanisms to overcome difficulties in task-set implementation and thereby achieve the same behavioral outcomes as controls.

  12. Structure of the intact ATM/Tel1 kinase.

    PubMed

    Wang, Xuejuan; Chu, Huanyu; Lv, Mengjuan; Zhang, Zhihui; Qiu, Shuwan; Liu, Haiyan; Shen, Xuetong; Wang, Weiwu; Cai, Gang

    2016-05-27

    The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents.

  13. Structure of the intact ATM/Tel1 kinase

    NASA Astrophysics Data System (ADS)

    Wang, Xuejuan; Chu, Huanyu; Lv, Mengjuan; Zhang, Zhihui; Qiu, Shuwan; Liu, Haiyan; Shen, Xuetong; Wang, Weiwu; Cai, Gang

    2016-05-01

    The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents.

  14. Control of Respiration by Cytochrome c Oxidase in Intact Cells

    PubMed Central

    Dalmonte, Maria Elena; Forte, Elena; Genova, Maria Luisa; Giuffrè, Alessandro; Sarti, Paolo; Lenaz, Giorgio

    2009-01-01

    Metabolic control analysis was applied to intact HepG2 cells. The effect on the control coefficient of cytochrome c oxidase (CcOX) over cell respiration of both the electrical (Δψ) and chemical (ΔpH) component of the mitochondrial transmembrane proton electrochemical gradient (ΔμH+) was investigated. The overall O2 consumption and specific CcOX activity of actively phosphorylating cells were titrated with cyanide under conditions in which Δψ and ΔpH were selectively modulated by addition of ionophores. In the absence of ionophores, CcOX displayed a high control coefficient (CIV = 0.73), thus representing an important site of regulation of mitochondrial oxidative phosphorylation. A high control coefficient value (CIV = 0.85) was also measured in the presence of nigericin, i.e. when Δψ is maximal, and in the presence of nigericin and valinomycin (CIV = 0.77), when ΔμH+ is abolished. In contrast, CcOX displayed a markedly lower control coefficient (CIV = 0.30) upon addition of valinomycin, when Δψ is converted into ΔpH. These results show that Δψ is responsible for the tight control of CcOX over respiration in actively phosphorylating cells. PMID:19776013

  15. Hydrocarbon biodegradation kinetics in an intact unsaturated zone soil core

    SciTech Connect

    Moyer, E.E.; Ostendorf, D.W.; Richards, R.J.; Goodwin, S.

    1995-12-31

    Aerobic biodegradation of vapor-phase petroleum hydrocarbons was evaluated in an intact soil core from the site of an aviation gasoline release. A mid-depth unsaturated zone soil core was subjected to a flow of nitrogen gas, oxygen, water vapor, and vapor-phase hydrocarbons in a configuration analogous to a biofilter or an in situ bioventing or sparging situation. The vertical profiles of vapor-phase hydrocarbon concentration in the soil core were determined by gas chromatography of vapor samples. Steady-state concentrations were input to a simple analytical model balancing advection and first-order biodegradation of hydrocarbons. First-order rate constants for each major hydrocarbon compound were used to calibrate the model to the concentration profiles. Compounds with lower molecular weights, fewer methyl groups, and no quaternary carbons tended to have higher rate constants. The first-order rate constants were consistent with kinetic parameters determined from microcosm studies at the same field site, suggesting that both estimation methods were effective.

  16. Structure of the intact ATM/Tel1 kinase

    PubMed Central

    Wang, Xuejuan; Chu, Huanyu; Lv, Mengjuan; Zhang, Zhihui; Qiu, Shuwan; Liu, Haiyan; Shen, Xuetong; Wang, Weiwu; Cai, Gang

    2016-01-01

    The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents. PMID:27229179

  17. Kinetics of (-) SVIodocyanopindolol binding to intact human mononuclear cells

    SciTech Connect

    Graafsma, S.J.; van Tits, L.J.; Rodrigues de Miranda, J.F.; Thien, T.

    1988-01-01

    In association experiments of (-) SVIodocyanopindolol ( SVICYP) with human mononuclear cells (MNC) at 70 pM and a temperature of 37 degrees C equilibrium was reached within 30 min. However, when the same experiments were performed at a concentration of 4 pM SVICYP, equilibrium was only reached after 3 hours. The consequences of incomplete equilibrium for the interpretation of binding experiments under the incorrect assumption that equilibrium has been reached, was investigated at equilibration times of one, two and three hours. The dissociation constant, Kd, decreased from 7.4 +/- 0.2 pM after one hour to 2.5 +/- 0.4 pM after three hours of incubation while the receptor density, RO, decreased from 970 +/- 170 to 713 +/- 58 sites/cell. Analysis of computer simulated binding curves confirmed the decrease in Kd and RO at prolonged incubations. We conclude that in SVICYP binding in intact MNC one hour of incubation is not sufficient to obtain equilibrium at the lower concentrations. This leads to an overestimation of Kd- and to a lesser extent of RO-values. Extending the incubation time to three hours on the other hand may lead to a loss of cells and therefore to an underestimation of RO.

  18. Spontaneous NA+ transients in individual mitochondria of intact astrocytes.

    PubMed

    Azarias, Guillaume; Van de Ville, Dimitri; Unser, Michael; Chatton, Jean-Yves

    2008-02-01

    Mitochondria in intact cells maintain low Na(+) levels despite the large electrochemical gradient favoring cation influx into the matrix. In addition, they display individual spontaneous transient depolarizations. The authors report here that individual mitochondria in living astrocytes exhibit spontaneous increases in their Na(+) concentration (Na(mit)(+) spiking), as measured using the mitochondrial probe CoroNa Red. In a field of view with approximately 30 astrocytes, up to 1,400 transients per minute were typically detected under resting conditions. Na(mit)(+) spiking was also observed in neurons, but was scarce in two nonneural cell types tested. Astrocytic Na(mit)(+) spikes averaged 12.2 +/- 0.8 s in duration and 35.5 +/- 3.2 mM in amplitude and coincided with brief mitochondrial depolarizations; they were impaired by mitochondrial depolarization and ruthenium red pointing to the involvement of a cation uniporter. Na(mit)(+) spiking activity was significantly inhibited by mitochondrial Na(+)/H(+) exchanger inhibition and sensitive to cellular pH and Na(+) concentration. Ca(2+) played a permissive role on Na(mit)(+) spiking activity. Finally, the authors present evidence suggesting that Na(mit)(+) spiking frequency was correlated with cellular ATP levels. This study shows that, under physiological conditions, individual mitochondria in living astrocytes exhibit fast Na(+) exchange across their inner membrane, which reveals a new form of highly dynamic and localized functional regulation.

  19. Plasma concentrations of lidocaine in dogs following lidocaine patch application over an incision compared to intact skin.

    PubMed

    Joudrey, S D; Robinson, D A; Kearney, M T; Papich, M G; da Cunha, A F

    2015-12-01

    The objective was to compare plasma lidocaine concentrations when a commercially available 5% lidocaine patch was placed on intact skin vs. an incision. Our hypothesis was that greater absorption of lidocaine would occur from the incision site compared to intact skin. Ten dogs were used in a crossover design. A patch was placed over an incision, and then after a washout period, a patch was placed over intact skin. Plasma lidocaine concentrations were measured at patch placement; 20, 40 and 60 min; and 2, 4, 6, 12, 24, 36, 48, 72 and 96 h after patch placement. After patch removal, the skin was graded using a subjective skin reaction system. No dogs required rescue analgesia, and no toxicity or skin reaction was noted. Mean ± SD AUC and CMAX were 3054.29 ± 1095.93 ng·h/mL and 54.1 ± 15.84 ng/mL in the Incision Group, and 2269.9 ± 1037.08 ng·h/mL and 44.5 ± 16.34 ng/mL in the No-Incision Group, respectively. The AUC was significantly higher in the Incision Group. The results of the study demonstrate that the actual body exposure to lidocaine was significantly higher when an incision was present compared to intact skin. No adverse effects were observed from either treatment. Efficacy was not evaluated.

  20. Motor timing intraindividual variability in amnestic mild cognitive impairment and cognitively intact elders at genetic risk for Alzheimer's disease.

    PubMed

    Kay, Christina D; Seidenberg, Michael; Durgerian, Sally; Nielson, Kristy A; Smith, J Carson; Woodard, John L; Rao, Stephen M

    2017-11-01

    Intraindividual variability (IIV) in motor performance has been shown to predict future cognitive decline. The apolipoprotein E-epsilon 4 (APOE-ε4) allele is also a well-established risk factor for memory decline. Here, we present novel findings examining the influence of the APOE-ε4 allele on the performance of asymptomatic healthy elders in comparison to individuals with amnestic MCI (aMCI) on a fine motor synchronization, paced finger-tapping task (PFTT). Two Alzheimer's disease (AD) risk groups, individuals with aMCI (n = 24) and cognitively intact APOE-ε4 carriers (n = 41), and a control group consisting of cognitively intact APOE-ε4 noncarriers (n = 65) completed the Rey Auditory Verbal Learning Test and the PFTT, which requires index finger tapping in synchrony with a visual stimulus (interstimulus interval = 333 ms). Motor timing IIV, as reflected by the standard deviation of the intertap interval (ITI), was greater in the aMCI group than in the two groups of cognitively intact elders; in contrast, all three groups had statistically equivalent mean ITI. No significant IIV differences were observed between the asymptomatic APOE-ε4 carriers and noncarriers. Poorer episodic memory performance was associated with greater IIV, particularly in the aMCI group. Results suggest that increased IIV on a fine motor synchronization task is apparent in aMCI. This IIV measure was not sensitive in discriminating older asymptomatic individuals at genetic risk for AD from those without such a genetic risk. In contrast, episodic memory performance, a well-established predictor of cognitive decline in preclinical AD, was able to distinguish between the two cognitively intact groups based on genetic risk.

  1. Transcatheter pulmonary valve perforation using chronic total occlusion wire in pulmonary atresia with intact ventricular septum

    PubMed Central

    Bakhru, Shweta; Marathe, Shilpa; Saxena, Manish; Verma, Sudeep; Saileela, Rajan; Dash, Tapan K; Koneti, Nageswara Rao

    2017-01-01

    Background: Perforation of pulmonary valve using radiofrequency ablation in pulmonary atresia with intact ventricular septum (PA IVS) is a treatment of choice. However, significant cost of the equipment limits its utility, especially in the developing economies. Objective: To assess the feasibility, safety, and efficacy of perforation of pulmonary valve using chronic total occlusion (CTO) wires in patients with PA IVS as an alternative to radiofrequency ablation. Methods: This is a single-center, nonrandomized, retrospective study conducted during June 2008 to September 2015. Twenty-four patients with PA IVS were selected for the procedure during the study period. The median age and weight of the study population were 8. days and 2.65 kg, respectively. Four patients were excluded after right ventricular angiogram as they showed right ventricular-dependent coronary circulation. The pulmonary valve perforation was attempted using various types of CTO wires based on the tip load with variable penetrating characteristics. Results: The procedure was successful in 16 of twenty patients using CTO wires: Shinobi in nine, Miracle in four, CROSS-IT in two, and Conquest Pro in one. Two patients had perforation of right ventricular outflow tract (RVOT). Pericardiocentesis was required in one patient to relieve cardiac tamponade. Later, the same patient underwent successful hybrid pulmonary valvotomy. The other patient underwent ductus arteriosus (DA) stenting. Balloon atrial septostomy was needed in three cases with systemic venous congestion. Desaturation was persistent in five cases necessitating DA or RVOT stenting to augment pulmonary blood flow. There were two early and two late deaths. The mean follow-up was 22.66 ± 16 months. Three patients underwent one and half ventricle repair and one Blalock–Taussig shunt during follow-up. Conclusion: Perforation of the pulmonary valve can be done successfully using CTO wires in selected cases of pulmonary atresia with intact

  2. Proteome Dynamics: Tissue Variation in the Kinetics of Proteostasis in Intact Animals*

    PubMed Central

    Hammond, Dean E.; Claydon, Amy J.; Simpson, Deborah M.; Edward, Dominic; Stockley, Paula; Hurst, Jane L.

    2016-01-01

    Understanding the role of protein turnover in the maintenance of proteostasis requires accurate measurements of the rates of replacement of proteins in complex systems, such as intact animals. Moreover, any investigation of allometric scaling of protein turnover is likely to include species for which fully annotated proteomes are not available. We have used dietary administration of stable isotope labeled lysine to assess protein turnover rates for proteins from four tissues in the bank vole, Myodes glareolus. The annotated genome for this species is not available, so protein identification was attained through cross-species matching to the mouse. For proteins for which confident identifications were derived, the pattern of lysine incorporation over 40 days was used to define the rate of synthesis of individual proteins in the four tissues. The data were heavily filtered to retain a very high quality dataset of turnover rates for 1088 proteins. Comparative analysis of the four tissues revealed different median rates of degradation (kidney: 0.099 days−1; liver 0.136 days−1; heart, 0.054 days−1, and skeletal muscle, 0.035 days−1). These data were compared with protein degradation rates from other studies on intact animals or from cells in culture and indicate that both cell type and analytical methodology may contribute to variance in turnover data between different studies. These differences were not only due to tissue-specific proteins but were reflected in gene products common to all tissues. All data are available via ProteomeXchange with identifier PXD002054. PMID:26839000

  3. Cuticle and subsurface ornamentation of intact plant leaf epidermis under confocal and superresolution microscopy.

    PubMed

    Urban, Michael A; Barclay, Richard S; Sivaguru, Mayandi; Punyasena, Surangi W

    2016-04-25

    Plant cuticle micromorphology is an invaluable tool in modern ecology and paleoecology. It has expanded our knowledge of systematic relationships among diverse plant groups and can be used to identify fossil plants. Furthermore, fossil plant leaf micromorphology is used for reconstructing past environments, most notably for estimating atmospheric CO2 concentration. Here we outline a new protocol for imaging plant cuticle for archival and paleoecological applications. Traditionally, both modern reference and fossil samples undergo maceration with subsequent imaging via environmental SEM, widefield fluorescence, or light microscopy. In this paper, we demonstrate the capabilities of alternative preparation and imaging methods using confocal and superresolution microscopy with intact leaf samples. This method produces detailed three-dimensional images of surficial and subsurface structures of the intact leaf. Multiple layers are captured simultaneously, which previously required independent maceration and microtome steps. We compared clearing agents (chloral hydrate, KOH, and Visikol); mounting media (Eukitt and Hoyer's); fluorescent stains (periodic acid Schiff, propidium iodide); and confocal vs. superresolution microscopes. We conclude that Eukitt is the best medium for long-term preservation and imaging. Because of nontoxicity and ease of procurement, Visikol made for the best clearing agent. Staining improves contrast and under most circumstances PAS provided the clearest images. Supperresolution produced higher clarity images than traditional confocal, but the information gained was minimal. This new protocol provides the botanical and paleobotanical community an alternative to traditional techniques. Our proposed workflow has the net benefit of being more efficient than traditional methods, which only capture the surface of the plant epidermis. Microsc. Res. Tech., 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. The effect of coracoacromial ligament excision and acromioplasty on the amount of rotator cuff force production necessary to restore intact glenohumeral biomechanics.

    PubMed

    Budoff, Jeffrey E; Lin, Cheng-Li; Hong, Chih-Kai; Chiang, Florence L; Su, Wei-Ren

    2016-06-01

    Coracoacromial ligament (CAL) excision and acromioplasty increase superior and anterosuperior glenohumeral translation. It is unknown how much of an increase in rotator cuff force production is required to re-establish intact glenohumeral biomechanics after these surgical procedures. We hypothesized that, after CAL excision and acromioplasty, an increase in rotator cuff force production would not be necessary to reproduce the anterosuperior and superior translations of the intact specimens. Nine cadaveric shoulders were subjected to loading in the superior and anterosuperior directions in the intact state after CAL excision, acromioplasty, and recording of the translations. The rotator cuff force was then increased to normalize glenohumeral biomechanics. After CAL excision at 150 and 200 N of loading, an increase in the rotator cuff force by 25% decreased anterosuperior translation to the point where there was no significant difference from the intact specimen's translation. After acromioplasty (and CAL excision) at 150 and 200 N, an increase in the rotator cuff force of 25% and 30%, respectively, decreased superior translation to the point where there was no significant difference from the intact specimen's translation. At 150 to 200 N of loading, CAL excision and acromioplasty increase the rotator cuff force required to maintain normal glenohumeral biomechanics by 25% to 30%. After a subacromial decompression, the rotator cuff has an increased force production requirement to maintain baseline glenohumeral mechanics. Under many circumstances, in vivo force requirements may be even greater after surgical attenuation of the coracoacromial arch. Basic Science Study; Biomechanics. Copyright © 2016 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

  5. The intact capture of hypervelocity dust particles using underdense foams

    NASA Astrophysics Data System (ADS)

    Maag, Carl R.; Borg, J.; Tanner, William G.; Stevenson, T. J.; Bibring, J.-P.

    probability of survival for the impacting particle. The primary objectives of the experiment are to (1) Examine the morphology of primary and secondary hypervelocity impact craters. Primary attention will be paid to craters caused by ejecta during hypervelocity impacts of different substrates. (2) Determine the size distribution of ejecta by means of witness plates and collect fragments of ejecta from craters by means of momentum-sensitive mcropore foam. (3) Assess the directionality of the flux by means of penetration-hole alignment of thin films placed above the cells. (4) Capture intact the particles that perforated the thin film and entered the cell. Capture media consisted of both previously flight-tested micropore foams and aerogel. The foams had different latent heats of fusion and, accordingly, will capture particles over a range of momenta. intact.

  6. The intact capture of hypervelocity dust particles using underdense foams

    NASA Technical Reports Server (NTRS)

    Maag, Carl R.; Borg, J.; Tanner, William G.; Stevenson, T. J.; Bibring, J.-P.

    1994-01-01

    probability of survival for the impacting particle. The primary objectives of the experiment are to (1) Examine the morphology of primary and secondary hypervelocity impact craters. Primary attention will be paid to craters caused by ejecta during hypervelocity impacts of different substrates. (2) Determine the size distribution of ejecta by means of witness plates and collect fragments of ejecta from craters by means of momentum-sensitive mcropore foam. (3) Assess the directionality of the flux by means of penetration-hole alignment of thin films placed above the cells. (4) Capture intact the particles that perforated the thin film and entered the cell. Capture media consisted of both previously flight-tested micropore foams and aerogel. The foams had different latent heats of fusion and, accordingly, will capture particles over a range of momenta. Aerogel was incorporated into the cells to determine the minimum diameter than can be captured intact.

  7. The intact capture of hypervelocity dust particles using underdense foams

    NASA Technical Reports Server (NTRS)

    Maag, Carl R.; Borg, J.; Tanner, William G.; Stevenson, T. J.; Bibring, J.-P.

    1994-01-01

    probability of survival for the impacting particle. The primary objectives of the experiment are to (1) Examine the morphology of primary and secondary hypervelocity impact craters. Primary attention will be paid to craters caused by ejecta during hypervelocity impacts of different substrates. (2) Determine the size distribution of ejecta by means of witness plates and collect fragments of ejecta from craters by means of momentum-sensitive mcropore foam. (3) Assess the directionality of the flux by means of penetration-hole alignment of thin films placed above the cells. (4) Capture intact the particles that perforated the thin film and entered the cell. Capture media consisted of both previously flight-tested micropore foams and aerogel. The foams had different latent heats of fusion and, accordingly, will capture particles over a range of momenta. Aerogel was incorporated into the cells to determine the minimum diameter than can be captured intact.

  8. Intact reading in patients with profound early visual dysfunction.

    PubMed

    Yong, Keir X X; Warren, Jason D; Warrington, Elizabeth K; Crutch, Sebastian J

    2013-10-01

    Despite substantial neuroscientific evidence for a region of visual cortex dedicated to the processing of written words, many studies continue to reject explanations of letter-by-letter (LBL) reading in terms of impaired word form representations or parallel letter processing in favour of more general deficits of visual function. In the current paper, we demonstrate that whilst LBL reading is often associated with general visual deficits, these deficits are not necessarily sufficient to cause reading impairment and have led to accounts of LBL reading which are based largely on evidence of association rather than causation. We describe two patients with posterior cortical atrophy (PCA) who exhibit remarkably preserved whole word and letter reading despite profound visual dysfunction. Relative to controls, both patients demonstrated impaired performance on tests of early visual, visuoperceptual and visuospatial processing; visual acuity was the only skill preserved in both individuals. By contrast, both patients were able to read aloud words with perfect to near-perfect accuracy. Reading performance was also rapid with no overall significant difference in response latencies relative to age- and education-matched controls. Furthermore, the patients violated a key prediction of general visual accounts of LBL reading - that pre-lexical impairments should result in prominent word length effects; in the two reported patients, evidence for abnormal word length effects was equivocal or absent, and certainly an order of magnitude different to that reported for LBL readers. We argue that general visual accounts cannot explain the pattern of reading data reported, and attribute the preserved reading performance to preserved direct access to intact word form representations and/or parallel letter processing mechanisms. The current data emphasise the need for much clearer evidence of causality when attempting to draw connections between specific aspects of visual processing and

  9. Optical properties of intact leaves for estimating chlorophyll concentration

    NASA Technical Reports Server (NTRS)

    Carter, Gregory A.; Spiering, Bruce A.

    2002-01-01

    Changes in leaf chlorophyll content can serve as relative indicators of plant vigor and environmental quality. This study identified reflectance, transmittance, and absorptance wavebands and band ratios within the 400- to 850-nm range for intact leaves that could be used to estimate extracted leaf chlorophyll per unit leaf area (areal concentration) with minimal error. Leaf optical properties along with chlorophyll a, b, and a + b concentrations were measured for the planar-leaved sweetgum (Liquidambar styraciflua L.), red maple (Acer rubrum L.), wild grape (Vitis rotundifolia Michx.), and switchcane [Arundinaria gigantea (Walter) Muhl.], and for needles of longleaf pine (Pinus palustris Miller). Generally, reflectance, transmittance, and absorptance corresponded most precisely with chlorophyll concentrations at wavelengths near 700 nm, although regressions were also strong in the 550- to 625-nm range. A power function was superior to a simple linear function in yielding low standard deviations of the estimate (s). When data were combined among the planar-leaved species, s values were low at approximately 50 mumol/m2 out of a 940 mumol/m2 range in chlorophyll a + b at best-fit wavelengths of 707 to 709 nm. Minimal s values for chlorophyll a + b ranged from 32 to 62 mumol/m2 across species when band ratios having numerator wavelengths of 693 to 720 nm were used with the application of a power function. Optimal denominator wavelengths for the band ratios were 850 nm for reflectance and transmittance and 400 nm for absorptance. This information can be applied in designing field portable chlorophyll meters and in the landscape-scale remote sensing of plant responses to the environment.

  10. Polyacrylamide gel electrophoresis of intact bacteriophage T4D particles.

    PubMed Central

    Childs, J D; Birnboim, H C

    1975-01-01

    A method for the electrophoresis of intact bacteriophage T4D particles through polyacrylamide gels has been developed. It was found that phage particles will migrate through dilute polyacrylamide gels (less than 2.1%) in the presence of a low concentration of MgCl2. As few as 5 x 10(9) phage particles can be seen directly as a light-scattering band during the course of electrophoresis. The band can also be detected by scanning gels at 260 to 265 nm or by eluting viable phage particles from gel slices. A new mutant (eph1) has been identified on the basis of its decreased electrophoretic mobility compared with that of the wild type; mutant particles migrated 14% slower than the wild type particles at pH 8.3 and 35% slower at pH 5.0. The isoelectric points of both the wild type and eph1 mutant were found to be between pH 4.0 and 5.0. Particles of T4 with different head lengths were also studied. Petite particles (heads 20% shorter than normal) migrated at the same rate as normal-size particles. Giant particles, heterogenous with respect to head length (two to nine times normal), migrated faster than normal-size particles as a diffuse band. This diffuseness was due to separation within the band of particles having mobilities ranging from 8 to 35% faster than those of normal-size particles. These observations extend the useful range of polyacrylamide gel electrophoresis to include much larger particles than have previously been studied, including most viruses. Images PMID:240037

  11. Optical properties of intact leaves for estimating chlorophyll concentration.

    PubMed

    Carter, Gregory A; Spiering, Bruce A

    2002-01-01

    Changes in leaf chlorophyll content can serve as relative indicators of plant vigor and environmental quality. This study identified reflectance, transmittance, and absorptance wavebands and band ratios within the 400- to 850-nm range for intact leaves that could be used to estimate extracted leaf chlorophyll per unit leaf area (areal concentration) with minimal error. Leaf optical properties along with chlorophyll a, b, and a + b concentrations were measured for the planar-leaved sweetgum (Liquidambar styraciflua L.), red maple (Acer rubrum L.), wild grape (Vitis rotundifolia Michx.), and switchcane [Arundinaria gigantea (Walter) Muhl.], and for needles of longleaf pine (Pinus palustris Miller). Generally, reflectance, transmittance, and absorptance corresponded most precisely with chlorophyll concentrations at wavelengths near 700 nm, although regressions were also strong in the 550- to 625-nm range. A power function was superior to a simple linear function in yielding low standard deviations of the estimate (s). When data were combined among the planar-leaved species, s values were low at approximately 50 mumol/m2 out of a 940 mumol/m2 range in chlorophyll a + b at best-fit wavelengths of 707 to 709 nm. Minimal s values for chlorophyll a + b ranged from 32 to 62 mumol/m2 across species when band ratios having numerator wavelengths of 693 to 720 nm were used with the application of a power function. Optimal denominator wavelengths for the band ratios were 850 nm for reflectance and transmittance and 400 nm for absorptance. This information can be applied in designing field portable chlorophyll meters and in the landscape-scale remote sensing of plant responses to the environment.

  12. Raman mapping of intact biofilms on stainless steel surfaces

    NASA Astrophysics Data System (ADS)

    Nguyen, Julie K.; Heighton, Lynne; Xu, Yunfeng; Nou, Xiangwu; Schmidt, Walter F.

    2016-05-01

    Many issues occur when microbial bacteria contaminates human food or water; it can be dangerous to the public. Determining how the microbial are growing, it can help experts determine how to prevent the outbreaks. Biofilms are a tightly group of microbial cells that grow on living surfaces or surrounding themselves. Though biofilms are not necessarily uniform; when there are more than one type of microbial bacteria that are grown, Raman mapping is performed to determine the growth patterns. Depending on the type of microbial bacteria, they can grow in various patterns such as symmetrical or scattered on the surface. The biofilms need to be intact in order to preclude and potentially figuring out the relative intensity of different components in a biofilm mixture. In addition, it is important to determine whether one biofilms is a substrate for another biofilm to be detected. For example, it is possible if layer B appears above layer A, but layer A doesn't appear above layer B. In this case, three types of biofilms that are grown includes Listeria(L), Ralstonia(R), and a mixture of the two (LR). Since microbe deposits on metal surfaces are quite suitable, biofilms were grown on stainless steel surface slides. Each slide was viewed under a Raman Microscope at 100X and using a 532nm laser to provide great results and sharp peaks. The mapping of the laser helps determine how the bacteria growth, at which intensity the bacteria appeared in order to identify specific microbes to signature markers on biofilms.

  13. Optical properties of intact leaves for estimating chlorophyll concentration

    NASA Technical Reports Server (NTRS)

    Carter, Gregory A.; Spiering, Bruce A.

    2002-01-01

    Changes in leaf chlorophyll content can serve as relative indicators of plant vigor and environmental quality. This study identified reflectance, transmittance, and absorptance wavebands and band ratios within the 400- to 850-nm range for intact leaves that could be used to estimate extracted leaf chlorophyll per unit leaf area (areal concentration) with minimal error. Leaf optical properties along with chlorophyll a, b, and a + b concentrations were measured for the planar-leaved sweetgum (Liquidambar styraciflua L.), red maple (Acer rubrum L.), wild grape (Vitis rotundifolia Michx.), and switchcane [Arundinaria gigantea (Walter) Muhl.], and for needles of longleaf pine (Pinus palustris Miller). Generally, reflectance, transmittance, and absorptance corresponded most precisely with chlorophyll concentrations at wavelengths near 700 nm, although regressions were also strong in the 550- to 625-nm range. A power function was superior to a simple linear function in yielding low standard deviations of the estimate (s). When data were combined among the planar-leaved species, s values were low at approximately 50 mumol/m2 out of a 940 mumol/m2 range in chlorophyll a + b at best-fit wavelengths of 707 to 709 nm. Minimal s values for chlorophyll a + b ranged from 32 to 62 mumol/m2 across species when band ratios having numerator wavelengths of 693 to 720 nm were used with the application of a power function. Optimal denominator wavelengths for the band ratios were 850 nm for reflectance and transmittance and 400 nm for absorptance. This information can be applied in designing field portable chlorophyll meters and in the landscape-scale remote sensing of plant responses to the environment.

  14. Pointing in visual periphery: is DF's dorsal stream intact?

    PubMed

    Hesse, Constanze; Ball, Keira; Schenk, Thomas

    2014-01-01

    Observations of the visual form agnosic patient DF have been highly influential in establishing the hypothesis that separate processing streams deal with vision for perception (ventral stream) and vision for action (dorsal stream). In this context, DF's preserved ability to perform visually-guided actions has been contrasted with the selective impairment of visuomotor performance in optic ataxia patients suffering from damage to dorsal stream areas. However, the recent finding that DF shows a thinning of the grey matter in the dorsal stream regions of both hemispheres in combination with the observation that her right-handed movements are impaired when they are performed in visual periphery has opened up the possibility that patient DF may potentially also be suffering from optic ataxia. If lesions to the posterior parietal cortex (dorsal stream) are bilateral, pointing and reaching deficits should be observed in both visual hemifields and for both hands when targets are viewed in visual periphery. Here, we tested DF's visuomotor performance when pointing with her left and her right hand toward targets presented in the left and the right visual field at three different visual eccentricities. Our results indicate that DF shows large and consistent impairments in all conditions. These findings imply that DF's dorsal stream atrophies are functionally relevant and hence challenge the idea that patient DF's seemingly normal visuomotor behaviour can be attributed to her intact dorsal stream. Instead, DF seems to be a patient who suffers from combined ventral and dorsal stream damage meaning that a new account is needed to explain why she shows such remarkably normal visuomotor behaviour in a number of tasks and conditions.

  15. Secretion of acid and base equivalents by intact distal airways.

    PubMed

    Inglis, S K; Wilson, S M; Olver, R E

    2003-05-01

    Secretion of HCO(3)(-) by airway submucosal glands is essential for normal liquid and mucus secretion. Because the liquid bathing the airway surface (ASL) is acidic, it has been proposed that the surface epithelium may acidify HCO(3)(-)-rich glandular fluid. The aim of this study was to investigate the mechanisms by which intact distal bronchi, which contain both surface and glandular epithelium, modify pH of luminal fluid. Distal bronchi were isolated from pig lungs, cannulated in a bath containing HCO(3)(-)-buffered solution, and perfused continually with an aliquot of similar, lightly buffered solution (LBS) in which NaCl replaced NaHCO(3)(-) (pH 7 with NaOH). The pH of this circulating LBS initially acidified (by 0.053 +/- 0.0053 pH units) and transepithelial potential difference (PD) depolarized. The magnitude of acidification was increased when pH(LBS) was higher. This acidification was unaffected by luminal dimethylamiloride (DMA, 100 microM) but was inhibited by 100 nM bafilomycin A(1) (by 76 +/- 13%), suggesting involvement of vacuolar-H(+) ATPase. Addition of ACh (10 microM) evoked alkalinization of luminal LBS and hyperpolarization of transepithelial PD. The alkalinization was inhibited in HCO(3)(-)-free solutions containing acetazolamide (1 mM) and by DMA and was enhanced by bumetanide (100 microM), an inhibitor of Cl(-) secretion. The hyperpolarization was unaffected by these maneuvers. The anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate (300 microM) and combined treatment with DMA and bumetanide blocked both the alkalinization and hyperpolarization responses to ACh. These results are consistent with earlier studies showing that ACh evokes glandular secretion of HCO(3)(-) and Cl(-). Isolated distal airways thus secrete both acid and base equivalents.

  16. Uptake of intact nucleoside monophosphates by Bdellovibrio bacteriovorus 109J.

    PubMed Central

    Ruby, E G; McCabe, J B; Barke, J I

    1985-01-01

    The degraded nucleic acids and ribosomes of its prey cell provide Bdellovibrio bacteriovorus 109J with a source of ribonucleoside monophosphates and deoxyribonucleoside monophosphates for biosynthesis and respiration. We demonstrate that bdellovibrios, in contrast to almost all other bacteria, take up these nucleoside monophosphates into the cell in an intact, phosphorylated form. In this way they are able to assimilate more effectively the cellular contents of their prey. Studies with UMP and dTMP demonstrate that they are transported and accumulated against a concentration gradient, achieving internal levels at least 10 times the external levels. Treatment of the bdellovibrios with azide or carbonyl cyanide m-chlorophenylhydrazone eliminates their ability to either transport or maintain accumulated UMP and suggests the presence of a freely reversible exchange mechanism. There are at least two separate classes of transport systems for nucleoside monophosphates, each exhibiting partial specificity for either ribonucleoside monophosphates or deoxyribonucleoside monophosphates. Kinetic analyses of UMP transport in different developmental stages of strain 109J indicate that each stage expresses a single, saturable uptake system with a distinct apparent substrate affinity constant (Kt) of 104 microM in attack phase cells and 35 microM in prematurely released growth phase filaments. The capacity for transport of UMP by the growth phase filaments was 2.4 times that of the attack phase cells. These data, in addition to the apparent lack of environmental control of UMP transport capacity in attack phase cells, suggest that there are two transport systems for UMP in bdellovibrios and that the high-affinity, high-capacity growth phase system is developmentally regulated. PMID:4030692

  17. Intact reading in patients with profound early visual dysfunction

    PubMed Central

    Yong, Keir X.X.; Warren, Jason D.; Warrington, Elizabeth K.; Crutch, Sebastian J.

    2013-01-01

    Despite substantial neuroscientific evidence for a region of visual cortex dedicated to the processing of written words, many studies continue to reject explanations of letter-by-letter (LBL) reading in terms of impaired word form representations or parallel letter processing in favour of more general deficits of visual function. In the current paper, we demonstrate that whilst LBL reading is often associated with general visual deficits, these deficits are not necessarily sufficient to cause reading impairment and have led to accounts of LBL reading which are based largely on evidence of association rather than causation. We describe two patients with posterior cortical atrophy (PCA) who exhibit remarkably preserved whole word and letter reading despite profound visual dysfunction. Relative to controls, both patients demonstrated impaired performance on tests of early visual, visuoperceptual and visuospatial processing; visual acuity was the only skill preserved in both individuals. By contrast, both patients were able to read aloud words with perfect to near-perfect accuracy. Reading performance was also rapid with no overall significant difference in response latencies relative to age- and education-matched controls. Furthermore, the patients violated a key prediction of general visual accounts of LBL reading – that pre-lexical impairments should result in prominent word length effects; in the two reported patients, evidence for abnormal word length effects was equivocal or absent, and certainly an order of magnitude different to that reported for LBL readers. We argue that general visual accounts cannot explain the pattern of reading data reported, and attribute the preserved reading performance to preserved direct access to intact word form representations and/or parallel letter processing mechanisms. The current data emphasise the need for much clearer evidence of causality when attempting to draw connections between specific aspects of visual processing

  18. Multipurpose dissociation cell for enhanced ETD of intact protein species.

    PubMed

    Rose, Christopher M; Russell, Jason D; Ledvina, Aaron R; McAlister, Graeme C; Westphall, Michael S; Griep-Raming, Jens; Schwartz, Jae C; Coon, Joshua J; Syka, John E P

    2013-06-01

    We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section rf ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion-ion reactions. The approximately 2-fold higher quadrupole field frequency of this cell relative to that of the A-QLT enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell's longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation.

  19. Multipurpose Dissociation Cell for Enhanced ETD of Intact Protein Species

    PubMed Central

    Rose, Christopher M.; Russell, Jason D.; Ledvina, Aaron R.; McAlister, Graeme C.; Westphall, Michael S.; Griep-Raming, Jens; Schwartz, Jae C.; Coon, Joshua J.; Syka, John E.P.

    2013-01-01

    We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section RF ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion-ion reactions. The approximately two-fold higher quadrupole field frequency of this cell relative to that of the A-QLT, enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell’s longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation. PMID:23609185

  20. Ultrastructure of the intact skeleton of the human erythrocyte membrane.

    PubMed

    Shen, B W; Josephs, R; Steck, T L

    1986-03-01

    Filamentous skeletons were liberated from isolated human erythrocyte membranes in Triton X-100, spread on fenestrated carbon films, negatively stained, and viewed intact and unfixed in the transmission electron microscope. Two forms of the skeleton were examined: (a) basic skeletons, stripped of accessory proteins with 1.5 M NaCl so that they contain predominantly polypeptide bands 1, 2, 4.1, and 5; and (b) unstripped skeletons, which also bore accessory proteins such as ankyrin and band 3 and small plaques of residual lipid. Freshly prepared skeletons were highly condensed. Incubation at low ionic strength and in the presence of dithiothreitol for an hour or more caused an expansion of the skeletons, which greatly increased the visibility of their elements. The expansion may reflect the opening of spectrin from a compact to an elongated disposition. Expanded skeletons appeared to be organized as networks of short actin filaments joined by multiple (5-8) spectrin tetramers. In unstripped preparations, globular masses were observed near the centers of the spectrin filaments, probably corresponding to complexes of ankyrin with band 3 oligomers. Some of these globules linked pairs of spectrin filaments. Skeletons prepared with a minimum of perturbation had thickened actin protofilaments, presumably reflecting the presence of accessory proteins. The length of these actin filaments was highly uniform, averaging 33 +/- 5 nm. This is the length of nonmuscle tropomyosin. Since there is almost enough tropomyosin present to saturate the F-actin, our data support the hypothesis that tropomyosin may determine the length of actin protofilaments in the red cell membrane.

  1. Whole-Cell Patch-Clamp Recording of Mouse and Rat Inner Hair Cells in the Intact Organ of Corti.

    PubMed

    Goutman, Juan D; Pyott, Sonja J

    2016-01-01

    Whole-cell patch clamping is a widely applied method to record currents across the entire membrane of a cell. This protocol describes application of this method to record currents from the sensory inner hair cells in the intact auditory sensory epithelium, the organ of Corti, isolated from rats or mice. This protocol particularly outlines the basic equipment required, provides instructions for the preparation of solutions and small equipment items, and methodology for recording voltage-activated and evoked synaptic currents from the inner hair cells.

  2. 46 CFR 28.545 - Intact stability when using lifting gear.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 1 2012-10-01 2012-10-01 false Intact stability when using lifting gear. 28.545 Section... COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.545 Intact stability when using lifting gear. (a) Each vessel which lifts a weight over the side, or that uses fishing gear that can impose an...

  3. 46 CFR 28.545 - Intact stability when using lifting gear.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false Intact stability when using lifting gear. 28.545 Section... COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.545 Intact stability when using lifting gear. (a) Each vessel which lifts a weight over the side, or that uses fishing gear that can impose an...

  4. An improved procedure for the isolation of intact chloroplasts of high photosynthetic capacity.

    PubMed Central

    Cerović, Z G; Plesnicar, M

    1984-01-01

    An improved procedure for the mechanical isolation of chloroplasts of high degree of intactness (90-95%) and photosynthetic capacity (25-50 mmol of O2/s per mol of chlorophyll) is described. The combination of pea plants (Pisum sativum L.) as starting material and the high reproducibility of the procedure readily and cheaply yields reliable intact chloroplasts for photosynthetic studies. PMID:6497861

  5. Relationship between Size of Broken and Intact Families and Academic Achievement.

    ERIC Educational Resources Information Center

    Cherian, Varghese I.

    1991-01-01

    Compares relationship between family size and academic achievement of children from broken and intact families among Black African Xhosa-speaking children between the ages of 13 and 17. Results indicate a negative relationship between family size and academic achievement, regardless of broken or intact families. (Author/NL)

  6. Quantification of paracetamol in intact tablets using near-infrared transmittance spectroscopy.

    PubMed

    Eustaquio, A; Graham, P; Jee, R D; Moffatt, A C; Trafford, A D

    1998-11-01

    Production batch samples of paracetamol tablets and specially prepared out-of-specification batches covering the range 90-110% of the stated amount (500 mg) were analysed by the BP official UV assay and by NIR transmittance spectroscopy. NIR measurements were made on 20 intact tablets from each batch, scanned five times each (10 min measurement time per batch) over the spectral range 6000-11,520 cm-1. An average spectrum was calculated for each batch. Partial least squares (PLS) regression models were set up using a calibration set (20 batches) between the NIR response and the reference tablet paracetamol content (UV). Various pre-treatments of the spectra were examined; the smallest relative standard error of prediction (0.73%) was obtained using the first derivative of the absorbance over the full spectrum. Only two principal components were required for the PLS model to give a good relationship between the spectral information and paracetamol content. Applying this model to the validation set (15 batches) gave a mean bias of -0.08% and a mean accuracy of 0.59% with relative standard deviations of 0.75 and 0.44%, respectively. The proposed method is non-destructive and therefore lends itself to on-line/at-line production control purposes. The method is easy to use and does not require a knowledge of the mass of the tablets.

  7. Intact Automatic Imitation and Typical Spatial Compatibility in Autism Spectrum Disorder: Challenging the Broken Mirror Theory.

    PubMed

    Sowden, Sophie; Koehne, Svenja; Catmur, Caroline; Dziobek, Isabel; Bird, Geoffrey

    2016-02-01

    A lack of imitative behavior is frequently described as a core feature of Autism Spectrum Disorder (ASD), and is consistent with claims of mirror neuron system dysfunction in these individuals. Previous research has questioned this characterization of ASD however, arguing that when tests of automatic imitation are used--which do not require higher-level cognitive processing--imitative behavior is intact or even enhanced in individuals with ASD. In Experiment 1, 60 adult individuals with ASD and a matched Control group completed an automatic imitation task in which they were required to perform an index or a middle finger lift while observing a hand making either the same, or the alternate, finger movement. Both groups demonstrated a significant imitation effect whereby actions were executed faster when preceded by observation of the same action, than when preceded by the alternate action. The magnitude of this "imitation effect" was statistically indistinguishable in the ASD and Control groups. Experiment 2 utilized an improved automatic imitation paradigm to demonstrate that, when automatic imitation effects are isolated from those due to spatial compatibility, increasing autism symptom severity is associated with an increased tendency to imitate. Notably, there was no association between autism symptom severity and spatial compatibility, demonstrating the specificity of the link between ASD symptoms and increased imitation. These results provide evidence against claims of a lack of imitative behavior in ASD, and challenge the "Broken Mirror Theory of Autism." © 2015 International Society for Autism Research, Wiley Periodicals, Inc.

  8. After rotator cuff tears, the remaining (intact) tendons are mechanically altered.

    PubMed

    Perry, Stephanie M; Getz, Charles L; Soslowsky, Louis J

    2009-01-01

    Although presumed, damage in the remaining (intact) rotator cuff tendons in the presence of an isolated supraspinatus tendon tear or multiple tendon tear has not been well studied. This study used an animal model of multiple rotator cuff tendon tears to investigate alterations in the remaining (intact) tendon mechanical properties at 4 and 8 weeks after injury. Twenty-four rats served as uninjured controls, whereas 72 were divided among 3 tendon detachment groups: supraspinatus tendon detachment, supraspinatus + infraspinatus tendon detachment, and supraspinatus + subscapularis tendon detachment. The remaining (intact) rotator cuff tendons had decreased mechanical properties in the presence of rotator cuff tears. The remaining (intact) subscapularis and infraspinatus tendon cross-sectional areas increased, whereas tendon modulus decreased after tears of both 1 and 2 tendons. The remaining (intact) tendon cross-sectional areas continued to increase with time after injury. These alterations could potentially lead to further tendon damage and tear progression.

  9. Measurements and interpretations of light scattering from intact biological cells

    NASA Astrophysics Data System (ADS)

    Wilson, Jeremy D.

    Visible light interacts with biological cells primarily through elastic scattering. The details of how cells scatter light depend on their morphology and their substructures. In this thesis we first present a series of experiments and models to discern the specific contributions of certain sub-cellular constituents to whole-cell scattering. Exploiting the findings of those studies, we report on experiments within model systems of cell death that demonstrate the potential of light scattering measurements as a tool in modern biology. Instrumentation capable of exploiting the findings of this thesis from a biology-relevant microscopy platform is designed and developed. A Mie theory based interpretation of light scattering signals originating from a collection of particles with a broad size distribution is developed. Upon applying this model to scattering data from intact cells, we find that it robustly extracts the size scale of dominant light scattering particles, suggests that scattering measurements are sensitive primarily to mitochondrial and lysosomal morphology, and unites conflicting results in the literature. Using this model as a basis, we present a collection of studies in which we use various strategies of photodynamic therapy (PDT) as a biophysical tool to perturb mitochondria and lysosomes, and observe the effects of these perturbations on whole-cell scattering. Through these experiments, we are able to discern the individual contributions of mitochondria and lysosomes to whole-cell light scattering, and demonstrate that mitochondria are responsible for roughly 80% of the scattering signal. Results of experiments aimed at demonstrating the potential role that light scattering measurements have to play in future studies of cell death biology are presented. We first show that mitochondrial-PDT-induced morphology changes measured with light scattering map into the cell killing efficacy of the therapy. We next demonstrate that mitochondrial

  10. Cytoplasmic calcium buffers in intact human red cells.

    PubMed Central

    Tiffert, T; Lew, V L

    1997-01-01

    1. Precise knowledge of the cytoplasmic Ca2+ buffering behaviour in intact human red cells is essential for the characterization of their [Ca2+]i-dependent functions. This was investigated by using a refined method and experimental protocols which allowed continuity in the estimates of [Ca2+]i, from nanomolar to millimolar concentrations, in the presence and absence of external Ca2+ chelators. 2. The study was carried out in human red cells whose plasma membrane Ca2+ pump was inhibited either by depleting the cells of ATP or by adding vanadate to the cell suspension. Cytoplasmic Ca2+ buffering was analysed from plots of total cell calcium content vs. ionized cytoplasmic Ca2+ concentration ([CaT]i vs. [Ca2+]i) obtained from measurements of the equilibrium distribution of 45Ca2+ at different external Ca2+ concentrations ([Ca2+]o), in conditions known to clamp cell volume and pH. The equilibrium distribution of 45Ca2+ was induced by the divalent cation ionophore A23187. 3. The results showed the following. (i) The known red cell Ca2+ buffer represented by alpha, with a large capacity and low Ca2+ affinity, was the main cytoplasmic Ca2+ binding agent. (ii) The value of alpha was remarkably constant; the means for each of four donors ranged from 0.33 to 0.35, with a combined value of all independent measurements of 0.34 +/- 0.01 (mean +/- S.E.M., n = 16). This contrasts with the variability previously reported. (iii) There was an additional Ca2+ buffering complex with a low capacity (approximately 80 micromol (340 g Hb)(-1)) and intermediate Ca2+ affinity (apparent dissociation constant, K(D,app) approximately 4-50 microM) whose possible identity is discussed. (iv) The cell content of putative Ca2+ buffers with submicromolar Ca2+ dissociation constants was below the detection limit of the methods used here (less than 2 micromol (340 g Hb)(-1)). 4. Vanadate (1 mM) inhibited the Vmax of the Ca2+ pump in inosine-fed cells by 99.7%. The cytoplasmic Ca2+ buffering behaviour

  11. Daily online cone beam computed tomography to assess interfractional motion in patients with intact cervical cancer.

    PubMed

    Tyagi, Neelam; Lewis, John H; Yashar, Catheryn M; Vo, Daniel; Jiang, Steve B; Mundt, Arno J; Mell, Loren K

    2011-05-01

    To quantify interfraction motion in patients with intact cervical cancer and assess implications for clinical target volume (CTV) coverage and required planning margins. We analyzed 10 patients undergoing external beam radiotherapy using online cone beam computed tomography (CBCT) before each fraction. CTVs were contoured on the planning CT and on each CBCT. Each CBCT was rigidly registered to the planning CT with respect to bony anatomy. The CTV from each CBCT was projected onto the planning CT and compared to the CTV from the planning CT. Uniform three-dimensional expansions were applied to the planning CTV to assess required planning margins. For each fraction, the minimum margin required to encompass the CTV was calculated, and the volume of CTV (on the CBCT) encompassed by the PTV was determined as a function of margin size. A uniform CTV planning treatment volume margin of 15 mm would have failed to encompass the CTV in 32% of fractions. The mean volume of CTV missed, however, was small (4 cc). The mean planning margin (across patients and fractions) required to encompass the CTV was 15 mm. Variation in margin estimates was high, with interpatient variation being the predominant component. Increased rectal volume was associated with posterior (p < 0.0001) and superior (p = 0.0004) shifts in the CTV, whereas increased bladder volume was associated with superior shifts (p < 0.0001). Interfraction motion results in a high probability of missing the CTV using conventional planning margins, but the volume of CTV missed is small. Adaptive radiotherapy approaches are needed to improve treatment accuracy. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Daily Online Cone Beam Computed Tomography to Assess Interfractional Motion in Patients With Intact Cervical Cancer

    SciTech Connect

    Tyagi, Neelam; Lewis, John H.; Yashar, Catheryn M.; Vo, Daniel; Jiang, Steve B.; Mundt, Arno J.; Mell, Loren K.

    2011-05-01

    Purpose: To quantify interfraction motion in patients with intact cervical cancer and assess implications for clinical target volume (CTV) coverage and required planning margins. Methods and Materials: We analyzed 10 patients undergoing external beam radiotherapy using online cone beam computed tomography (CBCT) before each fraction. CTVs were contoured on the planning CT and on each CBCT. Each CBCT was rigidly registered to the planning CT with respect to bony anatomy. The CTV from each CBCT was projected onto the planning CT and compared to the CTV from the planning CT. Uniform three-dimensional expansions were applied to the planning CTV to assess required planning margins. For each fraction, the minimum margin required to encompass the CTV was calculated, and the volume of CTV (on the CBCT) encompassed by the PTV was determined as a function of margin size. Results: A uniform CTV planning treatment volume margin of 15 mm would have failed to encompass the CTV in 32% of fractions. The mean volume of CTV missed, however, was small (4 cc). The mean planning margin (across patients and fractions) required to encompass the CTV was 15 mm. Variation in margin estimates was high, with interpatient variation being the predominant component. Increased rectal volume was associated with posterior (p < 0.0001) and superior (p = 0.0004) shifts in the CTV, whereas increased bladder volume was associated with superior shifts (p < 0.0001). Conclusions: Interfraction motion results in a high probability of missing the CTV using conventional planning margins, but the volume of CTV missed is small. Adaptive radiotherapy approaches are needed to improve treatment accuracy.

  13. Brine Inclusions Migration in Intact Salt Crystals under Thermal Gradient

    NASA Astrophysics Data System (ADS)

    Caporuscio, F.; Boukhalfa, H.

    2013-12-01

    The behavior of water contained in rock salt under the influence of thermal gradients is critical to the performance of salt as a medium for the disposal of nuclear waste. Water contained in salt can be present as discrete inclusions within intact salt crystals, at the interface between salt crystals and aggregates, and also as hydration water and structural water present in accessory minerals present in salt. Water content in pure halite salt usually rages from 0.1 to 0.5 wt. % but is significantly higher in clay rich salt, for which water content can be up to several wt. %. Under the influence of thermal gradients brine inclusions and water associated to the accessory mineral is mobilized. Previous investigations have shown brine inclusions tend to move towards the heat source through a mechanism that involves the dissolution of salt at the hot face of the brine inclusion and its precipitation at the colder side of the inclusion. Uncertainties remain on the exact parameters that define the rate of brine migration and whether it truly migrates to towards the heat source. We performed studies under controlled thermal gradients to examine the behavior of brine inclusions in single salt crystals obtained from the underground salt mine at the Waste Isolation Power Plant (WIPP). We found that the behavior of the brine inclusions under thermal gradients is dependent on the thermal gradient magnitude and the nature of the inclusion. Full inclusions (liquid only) migrate predominantly towards the heat source, but when the inclusions are large and close to the surface they fracture the salt and release water near the surface. Inclusions that migrate towards the heat source migrate through a mechanism that involves the dissolution of salt at the hot side of the inclusion and its deposition along the migration path. SEM analysis of the migration pathways shows that brine migrates through the creation of a network of square shaped hollow channels of about 10 micron diameter

  14. DNA synthesis in periportal and perivenous hepatocytes of intact and hepatectomized young mice.

    PubMed

    Fernández-Blanco, A; Inda, A M; Errecalde, A L

    2015-01-01

    DNA synthesis of hepatocytes in two areas of Intact and Hepatectomized young mice liver along a circadian period was studied. DNA synthesis was significantly different at all analyzed time points in Intact and Hepatectomized animals. Differences between periportal and perivenous hepatocytes were found in hepatectomized animals at 04/42 and 08/46 hr of day/hour post-hepatectomy. DNAs peak in periportal hepatocytes regenerating liver occurs 4 hr earlier than in perivenous hepatocytes, probably reflecting their shorter G1 phase. Besides, daily mean values of regenerating livers were higher than those observed in Intact animals, as a consequence of surgical removal.

  15. Red blood cell ghosts and intact red blood cells as complementary models in photodynamic cell research.

    PubMed

    Kaestner, Lars

    2004-05-01

    Recent research on erythrocytes as model cells for photodynamic therapy showed differing behaviour of certain photosensitisers in erythrocytes compared to other cells. Differences of dye accumulation in the cell membrane were proposed to be the reason for the distinct photodynamic effects. Using pheophorbide a as an example, the combination of erythrocyte ghosts as models to follow the dye accumulation in the cell membrane and intact erythrocytes as model cells to show the photodynamic damage is provided. Evidence for the correctness of the combination of erythrocyte ghosts and intact erythrocytes as a functioning model system in photodynamic cell research is provided using the confocal laser scanning microscopy on intact, pheophorbide a loaded erythrocytes.

  16. Intact Imitation of Emotional Facial Actions in Autism Spectrum Conditions

    ERIC Educational Resources Information Center

    Press, Clare; Richardson, Daniel; Bird, Geoffrey

    2010-01-01

    It has been proposed that there is a core impairment in autism spectrum conditions (ASC) to the mirror neuron system (MNS): If observed actions cannot be mapped onto the motor commands required for performance, higher order sociocognitive functions that involve understanding another person's perspective, such as theory of mind, may be impaired.…

  17. Intact Imitation of Emotional Facial Actions in Autism Spectrum Conditions

    ERIC Educational Resources Information Center

    Press, Clare; Richardson, Daniel; Bird, Geoffrey

    2010-01-01

    It has been proposed that there is a core impairment in autism spectrum conditions (ASC) to the mirror neuron system (MNS): If observed actions cannot be mapped onto the motor commands required for performance, higher order sociocognitive functions that involve understanding another person's perspective, such as theory of mind, may be impaired.…

  18. Direct observation of local xylem embolisms induced by soil drying in intact Zea mays leaves

    PubMed Central

    Ryu, Jeongeun; Hwang, Bae Geun; Kim, Yangmin X.; Lee, Sang Joon

    2016-01-01

    The vulnerability of vascular plants to xylem embolism is closely related to their stable long-distance water transport, growth, and survival. Direct measurements of xylem embolism are required to understand what causes embolism and what strategies plants employ against it. In this study, synchrotron X-ray microscopy was used to non-destructively investigate both the anatomical structures of xylem vessels and embolism occurrence in the leaves of intact Zea mays (maize) plants. Xylem embolism was induced by water stress at various soil drying periods and soil water contents. X-ray images of dehydrated maize leaves showed that the ratio of gas-filled vessels to all xylem vessels increased with decreased soil water content and reached approximately 30% under severe water stress. Embolism occurred in some but not all vessels. Embolism in maize leaves was not strongly correlated with xylem diameter but was more likely to occur in the peripheral veins. The rate of embolism formation in metaxylem vessels was higher than in protoxylem vessels. This work has demonstrated that xylem embolism remains low in maize leaves under water stress and that there xylem has characteristic spatial traits of vulnerability to embolism. PMID:26946123

  19. Impaired reward learning and intact motivation after serotonin depletion in rats

    PubMed Central

    Izquierdo, Alicia; Carlos, Kathleen; Ostrander, Serena; Rodriguez, Danilo; McCall-Craddolph, Aaron; Yagnik, Gargey; Zhou, Feimeng

    2012-01-01

    Aside from the well-known influence of serotonin (5-hydroxytryptamine, 5-HT) on emotional regulation, more recent investigations have revealed the importance of this monoamine in modulating cognition. Parachlorophenylalanine (PCPA) depletes 5-HT by inhibiting tryptophan hydroxylase, the enzyme required for 5-HT synthesis and, if administered at sufficiently high doses, can result in a depletion of at least 90% of the brain s 5-HT levels. The present study assessed the long-lasting effects of widespread 5-HT depletions on two tasks of cognitive flexibility in Long Evans rats: effort discounting and reversal learning. We assessed performance on these tasks after administration of either 250 or 500 mg/kg PCPA or saline (SAL) on two consecutive days. Consistent with a previous report investigating the role of 5-HT on effort discounting, pretreatment with either dose of PCPA resulted in normal effortful choice: All rats continued to climb tall barriers to obtain large rewards and were not work-averse. Additionally, rats receiving the lower dose of PCPA displayed normal reversal learning. However, despite intact motivation to work for food rewards, rats receiving the largest dose of PCPA were unexpectedly impaired relative to SAL rats on the pretraining stages leading up to reversal learning, ultimately failing to approach and respond to the stimuli associated with reward. High performance liquid chromatography (HPLC) with electrochemical detection confirmed 5-HT, and not dopamine, levels in the ventromedial frontal cortex were correlated with this measure of associative reward learning. PMID:22652392

  20. In vivo three-photon microscopy of subcortical structures within an intact mouse brain

    PubMed Central

    Horton, Nicholas G.; Wang, Ke; Kobat, Demirhan; Clark, Catharine G.; Wise, Frank W.; Schaffer, Chris B.; Xu, Chris

    2013-01-01

    Two-photon fluorescence microscopy (2PM)1 enables scientists in various fields including neuroscience2,3, embryology4, and oncology5 to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of 2PM within the mouse brain to the cortical layer, and imaging subcortical structures currently requires the removal of overlying brain tissue3 or the insertion of optical probes6,7. Here we demonstrate non-invasive, high resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy (3PM) at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein (RFP)-labeled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher order nonlinear excitation overcomes the limitations of 2PM, enabling biological investigations to take place at greater depth within tissue. PMID:24353743

  1. Dissolution of Intact, Divided and Crushed Circadin Tablets: Prolonged vs. Immediate Release of Melatonin.

    PubMed

    Chua, Hui Ming; Hauet Richer, Nathalie; Swedrowska, Magda; Ingham, Stephen; Tomlin, Stephen; Forbes, Ben

    2016-01-07

    Circadin 2 mg prolonged-release tablet is the only licensed melatonin product available in the UK. Circadin is indicated for patients with primary insomnia aged 55 and over, but is more widely used "off-label" to treat sleep disorders especially in the paediatric population. Children and older people often have difficulty swallowing tablets and dividing the tablet is sometimes required to ease administration. The aim of this study was to measure the release profile of melatonin from Circadin tablets when divided or crushed, and compare this with release from intact tablets. Dissolution testing was also performed for unlicensed melatonin products for comparison. Dissolution tests were performed using the pharmacopoeial paddle apparatus, with melatonin release analyzed by high performance liquid chromatography. Melatonin content, hardness, friability, and disintegration of the products were also evaluated. The prolonged release of melatonin from Circadin tablets was unlike that of any other product tested. When divided into halves, Circadin preserved most of the prolonged-release characteristic (f2 = 58), whereas quarter-cut and crushed tablet had a more immediate melatonin release profile. Circadin is significantly less expensive and should be preferred to unlicensed medicines which are not pharmaceutically equivalent and offer less quality assurance.

  2. Dissolution of Intact, Divided and Crushed Circadin Tablets: Prolonged vs. Immediate Release of Melatonin

    PubMed Central

    Chua, Hui Ming; Hauet Richer, Nathalie; Swedrowska, Magda; Ingham, Stephen; Tomlin, Stephen; Forbes, Ben

    2016-01-01

    Circadin 2 mg prolonged-release tablet is the only licensed melatonin product available in the UK. Circadin is indicated for patients with primary insomnia aged 55 and over, but is more widely used “off-label” to treat sleep disorders especially in the paediatric population. Children and older people often have difficulty swallowing tablets and dividing the tablet is sometimes required to ease administration. The aim of this study was to measure the release profile of melatonin from Circadin tablets when divided or crushed, and compare this with release from intact tablets. Dissolution testing was also performed for unlicensed melatonin products for comparison. Dissolution tests were performed using the pharmacopoeial paddle apparatus, with melatonin release analyzed by high performance liquid chromatography. Melatonin content, hardness, friability, and disintegration of the products were also evaluated. The prolonged release of melatonin from Circadin tablets was unlike that of any other product tested. When divided into halves, Circadin preserved most of the prolonged-release characteristic (f2 = 58), whereas quarter-cut and crushed tablet had a more immediate melatonin release profile. Circadin is significantly less expensive and should be preferred to unlicensed medicines which are not pharmaceutically equivalent and offer less quality assurance. PMID:26751472

  3. Intact Rapid Facial Mimicry as well as Generally Reduced Mimic Responses in Stable Schizophrenia Patients

    PubMed Central

    Chechko, Natalya; Pagel, Alena; Otte, Ellen; Koch, Iring; Habel, Ute

    2016-01-01

    Spontaneous emotional expressions (rapid facial mimicry) perform both emotional and social functions. In the current study, we sought to test whether there were deficits in automatic mimic responses to emotional facial expressions in patients (15 of them) with stable schizophrenia compared to 15 controls. In a perception-action interference paradigm (the Simon task; first experiment), and in the context of a dual-task paradigm (second experiment), the task-relevant stimulus feature was the gender of a face, which, however, displayed a smiling or frowning expression (task-irrelevant stimulus feature). We measured the electromyographical activity in the corrugator supercilii and zygomaticus major muscle regions in response to either compatible or incompatible stimuli (i.e., when the required response did or did not correspond to the depicted facial expression). The compatibility effect based on interactions between the implicit processing of a task-irrelevant emotional facial expression and the conscious production of an emotional facial expression did not differ between the groups. In stable patients (in spite of a reduced mimic reaction), we observed an intact capacity to respond spontaneously to facial emotional stimuli. PMID:27303335

  4. Femtosecond Optoinjection of Intact Tobacco BY-2 Cells Using a Reconfigurable Photoporation Platform

    PubMed Central

    Mitchell, Claire A.; Kalies, Stefan; Cizmár, Tomás; Heisterkamp, Alexander; Torrance, Lesley; Roberts, Alison G.; Gunn-Moore, Frank J.; Dholakia, Kishan

    2013-01-01

    A tightly-focused ultrashort pulsed laser beam incident upon a cell membrane has previously been shown to transiently increase cell membrane permeability while maintaining the viability of the cell, a technique known as photoporation. This permeability can be used to aid the passage of membrane-impermeable biologically-relevant substances such as dyes, proteins and nucleic acids into the cell. Ultrashort-pulsed lasers have proven to be indispensable for photoporating mammalian cells but they have rarely been applied to plant cells due to their larger sizes and rigid and thick cell walls, which significantly hinders the intracellular delivery of exogenous substances. Here we demonstrate and quantify femtosecond optical injection of membrane impermeable dyes into intact BY-2 tobacco plant cells growing in culture, investigating both optical and biological parameters. Specifically, we show that the long axial extent of a propagation invariant (“diffraction-free”) Bessel beam, which relaxes the requirements for tight focusing on the cell membrane, outperforms a standard Gaussian photoporation beam, achieving up to 70% optoinjection efficiency. Studies on the osmotic effects of culture media show that a hypertonic extracellular medium was found to be necessary to reduce turgor pressure and facilitate molecular entry into the cells. PMID:24244456

  5. High surface area of porous silicon drives desorption of intact molecules.

    PubMed

    Northen, Trent R; Woo, Hin-Koon; Northen, Michael T; Nordström, Anders; Uritboonthail, Winnie; Turner, Kimberly L; Siuzdak, Gary

    2007-11-01

    The surface structure of porous silicon used in desorption/ionization on porous silicon (DIOS) mass analysis is known to play a primary role in the desorption/ionization (D/I) process. In this study, mass spectrometry and scanning electron microscopy (SEM) are used to examine the correlation between intact ion generation with surface ablation and surface morphology. The DIOS process is found to be highly laser energy dependent and correlates directly with the appearance of surface ions (Si(n)(+) and OSiH(+)). A threshold laser energy for DIOS is observed (10 mJ/cm(2)), which supports that DIOS is driven by surface restructuring and is not a strictly thermal process. In addition, three DIOS regimes are observed that correspond to surface restructuring and melting. These results suggest that higher surface area silicon substrates may enhance DIOS performance. A recent example that fits into this mechanism is the surface of silicon nanowires, which has a high surface energy and concomitantly requires lower laser energy for analyte desorption.

  6. High Surface Area of Porous Silicon Drives Desorption of Intact Molecules

    PubMed Central

    Northen, Trent R.; Woo, Hin-Koon; Northen, Michael T.; Nordström, Anders; Uritboonthail, Winnie; Turner, Kimberly L.; Siuzdak, Gary

    2007-01-01

    The surface structure of porous silicon used in desorption/ionization on porous silicon (DIOS) mass analysis is known to play a primary role in the desorption/ionization (D/I) process. In this study, mass spectrometry and scanning electron microscopy (SEM) are used to examine the correlation between intact ion generation with surface ablation, and surface morphology. The DIOS process is found to be highly laser energy dependent and correlates directly with the appearance of surface ions (Sin+ and OSiH+). A threshold laser energy for DIOS is observed (10 mJ/cm2), which supports that DIOS is driven by surface restructuring and is not a strictly thermal process. In addition, three DIOS regimes are observed which correspond to surface restructuring and melting. These results suggest that higher surface area silicon substrates may enhance DIOS performance. A recent example which fits into this mechanism is silicon nanowires surface which have a high surface energy and concomitantly requires lower laser energy for analyte desorpton. PMID:17881245

  7. Effect of Prostaglandin Duration on Outcomes in Transposition of the Great Arteries with Intact Ventricular Septum

    PubMed Central

    Butts, Ryan J.; Ellis, Alexander R.; Bradley, Scott M.; Hulsey, Thomas C.; Atz, Andrew M.

    2013-01-01

    Objective To study the effects of duration of preoperative prostaglandin E1 (PGE) exposure on perioperative outcomes of the arterial switch operation in patients with transposition of the great arteries with an intact ventricular septum (TGA-IVS). Design Retrospective chart review Setting Pediatric cardiac intensive care unit in a tertiary care children’s hospital. Patients All patients with TGA-IVS from 1995 to 2008 Outcome Measures Inotropic score was calculated for all patients in the first 5 postoperative days and maximum inotropic score was recorded. Length of postoperative mechanical ventilation, fluid balance, mechanical ventilation time, as well as ICU and hospital stay were recorded for all patients. Results Study population included 59 patients, 41(69%) underwent balloon atrial septostomy. PGE was used in 52 patients, median exposure of 59 hours, range 0 to 272 hours. Longer preoperative PGE exposure was associated with longer preoperative mechanical ventilation (p<.001). There was no association between preoperative PGE duration and cardiopulmonary bypass time, cross-clamp time, or total hospital stay. Patients with longer preoperative PGE exposure had a lower postoperative inotrope score (10 v. 15 p=.02). Conclusion Greater preoperative PGE exposure was associated with prolonged preoperative mechanical ventilation. Longer PGE exposure was associated wtih lower postoperative inotrope requirements. Aggressive efforts to avoid or shorten PGE infusion duration may not be warranted in this population. PMID:22360739

  8. DNA damage action spectroscopy and DNA repair in intact organisms: Alfalfa seedlings

    SciTech Connect

    Sutherland, B.M.; Quaite, F.E.; Sutherland, J.C.

    1993-12-31

    Understanding the effects of UV, and increased levels of UV, on DNA in living organisms requires knowledge of both the frequency of damages induced by the quantities and quality (wavelength composition) of the damaging radiation, and of the capacity of the organisms to carry out efficient and accurate repair. The major levels of uncertainty in understanding the responses of intact organisms, both plant and animal, to UV indicates that we cannot assess accurately the impact of stratospheric ozone depletion without major increases in knowledge of DNA damage and repair. What repair paths does alfalfa use for dealing with UV damages? The rate of pyrimidine dimers induced at a low exposure of 280 nm radiation to alfalfa seedlings, was observed to be about 8 dimers/million bases. After UV exposure, the seedlings were kept in the dark or exposed to blue light filtered by a yellow. filter which excluded wavelengths shorter than about 405 nm. Seedlings so exposed carry out photorepair, but do not seem to remove dimers by excision.

  9. An improved protocol for intact chloroplasts and cpDNA isolation in conifers.

    PubMed

    Vieira, Leila do Nascimento; Faoro, Helisson; Fraga, Hugo Pacheco de Freitas; Rogalski, Marcelo; de Souza, Emanuel Maltempi; de Oliveira Pedrosa, Fábio; Nodari, Rubens Onofre; Guerra, Miguel Pedro

    2014-01-01

    Performing chloroplast DNA (cpDNA) isolation is considered a major challenge among different plant groups, especially conifers. Isolating chloroplasts in conifers by such conventional methods as sucrose gradient and high salt has not been successful. So far, plastid genome sequencing protocols for conifer species have been based mainly on long-range PCR, which is known to be time-consuming and difficult to implement. We developed a protocol for cpDNA isolation using three different conifer families: Araucaria angustifolia and Araucaria bidwilli (Araucariaceae), Podocarpus lambertii (Podocarpaceae) and Pinus patula (Pinaceae). The present protocol is based on high salt isolation buffer followed by saline Percoll gradient. Combining these two strategies allowed enhanced chloroplast isolation, along with decreased contamination caused by polysaccharides, polyphenols, proteins, and nuclear DNA in cpDNA. Microscopy images confirmed the presence of intact chloroplasts in high abundance. This method was applied to cpDNA isolation and subsequent sequencing by Illumina MiSeq (2×250 bp), using only 50 ng of cpDNA. Reference-guided chloroplast genome mapping showed that high average coverage was achieved for all evaluated species: 24.63 for A. angustifolia, 135.97 for A. bidwilli, 1196.10 for P. lambertii, and 64.68 for P. patula. Results show that this improved protocol is suitable for enhanced quality and yield of chloroplasts and cpDNA isolation from conifers, providing a useful tool for studies that require isolated chloroplasts and/or whole cpDNA sequences.

  10. Patch clamped responses from outer hair cells in the intact adult organ of Corti.

    PubMed

    Mammano, F; Kros, C J; Ashmore, J F

    1995-09-01

    Outer hair cells (OHCs) from the mammalian cochlea act as both sensory cells and motor cells. We report here whole-cell tight seal recordings of OHC activity in their natural embedding tissue, the intact organ of Corti, using a temporal bone preparation. The mean cell resting potential, -76 +/- 4 mV (n = 19) and input conductance (10 +/- 3 nS at -70 mV) of third turn hair cells were significantly lower than have been found in isolated cells. Two main K+ currents in the cell were identified. One current, activated positive to -100 mV, was reduced by 5 mM BaCl2. The other current, activated above -40 mV, was reduced by 100 microM 4-aminopyridine (4-AP) and by 30 mM tetraethylammonium (TEA). Both of these currents have been also identified in recordings reported from isolated cells. On stepping to different membrane potentials, cells imaged in the organ of Corti changed length by an amount large enough to cause visible distortions in neighbouring cells. By quantifying such distortions we estimate that the forces generated by OHCs can account for the enhanced response to sound required by the cochlear amplifier.

  11. Scalable electrophysiology in intact small animals with nanoscale suspended electrode arrays

    NASA Astrophysics Data System (ADS)

    Gonzales, Daniel L.; Badhiwala, Krishna N.; Vercosa, Daniel G.; Avants, Benjamin W.; Liu, Zheng; Zhong, Weiwei; Robinson, Jacob T.

    2017-07-01

    Electrical measurements from large populations of animals would help reveal fundamental properties of the nervous system and neurological diseases. Small invertebrates are ideal for these large-scale studies; however, patch-clamp electrophysiology in microscopic animals typically requires invasive dissections and is low-throughput. To overcome these limitations, we present nano-SPEARs: suspended electrodes integrated into a scalable microfluidic device. Using this technology, we have made the first extracellular recordings of body-wall muscle electrophysiology inside an intact roundworm, Caenorhabditis elegans. We can also use nano-SPEARs to record from multiple animals in parallel and even from other species, such as Hydra littoralis. Furthermore, we use nano-SPEARs to establish the first electrophysiological phenotypes for C. elegans models for amyotrophic lateral sclerosis and Parkinson's disease, and show a partial rescue of the Parkinson's phenotype through drug treatment. These results demonstrate that nano-SPEARs provide the core technology for microchips that enable scalable, in vivo studies of neurobiology and neurological diseases.

  12. High-resolution visualization of airspace structures in intact mice via synchrotron phase-contrast X-ray imaging (PCXI).

    PubMed

    Parsons, David W; Morgan, Kaye; Donnelley, Martin; Fouras, Andreas; Crosbie, Jeffrey; Williams, Ivan; Boucher, Richard C; Uesugi, Kentaro; Yagi, Naoto; Siu, Karen K W

    2008-08-01

    Anatomical visualization of airspace-containing organs in intact small animals has been limited by the resolution and contrast available from current imaging methods such as X-ray, micro-computed tomography and magnetic resonance imaging. Determining structural relationships and detailed anatomy has therefore relied on suitable fixation, sectioning and histological processing. More complex and informative analyses such as orthogonal views of an organ and three-dimensional structure visualizations have required different animals and image sets, laboriously processed to gather this complementary structural information. Precise three-dimensional anatomical views have always been difficult to achieve in small animals. Here we report the ability of phase-contrast synchrotron X-ray imaging to provide detailed two- and three-dimensional visualization of airspace organ structures in intact animals. Using sub-micrometre square pixel charge-coupled device array detectors, the structure and anatomy of hard and soft tissues, and of airspaces, is readily available using phase-contrast synchrotron X-ray imaging. Moreover, software-controlled volume-reconstructions of tomographic images not only provide unsurpassed image clarity and detail, but also selectable anatomical views that cannot be obtained with established histological techniques. The morphology and structure of nasal and lung airways and the middle ear are illustrated in intact mice, using two- and three-dimensional representations. The utility of phase-contrast synchrotron X-ray imaging for noninvasively localizing objects implanted within airspaces, and the detection of gas bubbles transiting live airways, are other novel features of this visualization methodology. The coupling of phase-contrast synchrotron X-ray imaging technology with software-based reconstruction techniques holds promise for novel and high-resolution non-invasive examination of airspace anatomy in small animal models.

  13. Hydrophilic interaction liquid chromatography-mass spectrometry as a new tool for the characterization of intact semi-synthetic glycoproteins.

    PubMed

    Tengattini, Sara; Domínguez-Vega, Elena; Temporini, Caterina; Bavaro, Teodora; Rinaldi, Francesca; Piubelli, Luciano; Pollegioni, Loredano; Massolini, Gabriella; Somsen, Govert W

    2017-08-15

    Improved methods for detailed characterization of complex glycoproteins are required in the growing sector of biopharmaceuticals. Hydrophilic interaction liquid chromatography (HILIC) coupled to high resolution (HR) time-of-flight mass spectrometric (TOF-MS) detection was examined for the characterization of intact neo-glycoproteins prepared by chemical conjugation of synthetic saccharides to the lysine residues of selected recombinant proteins. The separation performances of three different amide HILIC columns (TSKgel Amide-80, XBridge BEH and AdvanceBio Glycan Mapping) were tested. Water-acetonitrile gradients and volatile eluent additives have been explored. Addition of 0.05% (v/v) trifluoroacetic acid to the mobile phase appeared to be essential for achieving optimum resolution of intact glycoforms and minimal ion suppression effects. Gradient elution conditions were optimized for each protein on every column. HILIC stationary phases were evaluated for the analysis of highly heterogeneous semi-synthetic derivatives of the same protein (ribonuclease A), and in the enhanced characterization of TB10.4 and Ag85B glycoconjugates, selected antigens from Mycobacterium tuberculosis (MTB). HILIC-MS results indicated that the HILIC selectivity is predominantly governed by size of the conjugated glycans and number of glycans attached, providing efficient glycoform separation. Moreover, HILIC separation prior to HRMS detection allowed assignment of several product impurities. Additional top-down MS/MS experiments confirmed conjugation at the N-terminus of TB10.4 next to its lysine residue. Overall, the obtained results demonstrate that amide-stationary-phase based HILIC coupled to MS is highly useful for the characterization of intact neo-glycoproteins allowing assessment of the number, identity and relative abundance of glycoforms present in the semi-synthetic products. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. High-resolution visualization of airspace structures in intact mice via synchrotron phase-contrast X-ray imaging (PCXI)

    PubMed Central

    Parsons, David W; Morgan, Kaye; Donnelley, Martin; Fouras, Andreas; Crosbie, Jeffrey; Williams, Ivan; Boucher, Richard C; Uesugi, Kentaro; Yagi, Naoto; Siu, Karen K W

    2008-01-01

    Anatomical visualization of airspace-containing organs in intact small animals has been limited by the resolution and contrast available from current imaging methods such as X-ray, micro-computed tomography and magnetic resonance imaging. Determining structural relationships and detailed anatomy has therefore relied on suitable fixation, sectioning and histological processing. More complex and informative analyses such as orthogonal views of an organ and three-dimensional structure visualizations have required different animals and image sets, laboriously processed to gather this complementary structural information. Precise three-dimensional anatomical views have always been difficult to achieve in small animals. Here we report the ability of phase-contrast synchrotron X-ray imaging to provide detailed two- and three-dimensional visualization of airspace organ structures in intact animals. Using sub-micrometre square pixel charge-coupled device array detectors, the structure and anatomy of hard and soft tissues, and of airspaces, is readily available using phase-contrast synchrotron X-ray imaging. Moreover, software-controlled volume-reconstructions of tomographic images not only provide unsurpassed image clarity and detail, but also selectable anatomical views that cannot be obtained with established histological techniques. The morphology and structure of nasal and lung airways and the middle ear are illustrated in intact mice, using two- and three-dimensional representations. The utility of phase-contrast synchrotron X-ray imaging for non-invasively localizing objects implanted within airspaces, and the detection of gas bubbles transiting live airways, are other novel features of this visualization methodology. The coupling of phase-contrast synchrotron X-ray imaging technology with software-based reconstruction techniques holds promise for novel and high-resolution non-invasive examination of airspace anatomy in small animal models. PMID:19172736

  15. Estimating dead organic matter carbon dynamics of an intact mixed dipterocarp forest in Brunei with a forest carbon model

    NASA Astrophysics Data System (ADS)

    Lee, J.; Lee, S.; Han, S. H.; Kim, S.; Roh, Y.; Abu Salim, K.; Davies, S. J.; Son, Y.

    2015-12-01

    Intact tropical forests contain substantial amount of carbon (C) and play an important role in global carbon cycles. Field measurement is needed to quantify dead organic matter (DOM) C dynamics in tropical forests while it requires much labor and cost. In contrast, forest carbon models can simulate C dynamics, overcoming the limitation of field measurement. In this study, we simulated the DOM C dynamics of an intact mixed dipterocarp forest at Kuala Belalong in Brunei, by using a forest carbon model. In order to estimate the annual changes in the C stocks of litter layer, dead root, coarse woody debris (CWD), and soils, a forest carbon model, KFSC model, was parameterized to the study site. In order to initialize the KFSC model, the C stocks in biomass, litter layer, CWD, and soils were measured in twenty seven 20 m x 20 m plots in 2014. The measured C stocks (Mg C ha-1) in litter layer, dead root, CWD, and soils were 3.2 ± 0.5, 4.0 ± 1.9, 32.5 ± 38.9, and 75.2 ± 9.2, respectively. The simulation results showed that the annual changes in the C stocks (Mg C ha-1 yr-1) of litter layer, dead root, CWD, and soils were 2.7 ± 2.0, 1.4 ± 1.8, -1.5 ± 7.7, and 0.5 ± 1.8, respectively. The total DOM C stocks tended to increase at the rate of 3.3 ± 9.6 Mg C ha-1 yr-1, but it exhibited the high variation. This might be attributed to the high variation of the annual changes in the CWD C stocks. Our results exhibited the applicability of forest carbon models to quantify DOM C dynamics in intact tropical rain forests. This study was supported by Korea Ministry of Environment (2014001310008) and Korea Forest Service (S121314L130110).

  16. Double-outlet right ventricle with intact ventricular septum in a foetus with trisomy-18.

    PubMed

    Patel, C R; Muise, K L; Redline, R W

    1999-07-01

    A rare case of double-outlet right ventricle with intact ventricular septum diagnosed by foetal echocardiography at 21 weeks of gestation is described. Amniocentesis revealed trisomy-18. The cardiac diagnosis was confirmed at autopsy.

  17. Isolation of Intact Chloroplasts of Euglena gracilis by Isopycnic Sedimentation in Gradients of Silica 12

    PubMed Central

    Salisbury, Jeffrey L.; Vasconcelos, Aurea C.; Floyd, Gary L.

    1975-01-01

    A technique is described for the isolation of structurally intact and partially active chloroplasts from photoheterotrophically grown Euglena gracilis. The separation of intact chloroplasts from stripped chloroplast membranes and other subcellular particles was achieved by sedimentation in continuous, isosmotic density gradients of Ludox AM, a silica sol. The final preparations contained an average of 93% intact chloroplasts and corresponded to approximately 10% of the the chlorophyll of the original cell suspension and 20 to 30% of the chlorophyll layered on the gradients. The chloroplasts obtained were intact by the criteria of ultrastructure, their content of ribulose diphosphate carboxylase, and their activity in a modified Hill reaction assay (U. Heber and K. A. Santarius. 1970. Z. Naturforsch. 25b: 718-727). In addition, the isolated chloroplasts were capable of incorporating amino acids into protein in the light. Images PMID:16659312

  18. High resolution TOF MS coupled to CE for the analysis of isotopically resolved intact proteins.

    PubMed

    Taichrib, Angelina; Pelzing, Matthias; Pellegrino, Cristoforo; Rossi, Mara; Neusüss, Christian

    2011-06-10

    Intact protein analysis by mass spectrometry is of great interest for the characterisation of biotechnological products. Exact mass measurement in combination with isotopic resolution allows the detection of modifications leading to small mass changes like deamidation or reduction of disulfide bonds directly on the level of the intact protein. Here, a concept is presented based on time-of-flight mass spectrometry. A bench top TOF MS and a high resolution TOF MS were used to resolve the isotopes of intact recombinant human growth hormone and intact human erythropoietin, respectively. Thus, these 22 and around 30kDa large proteins can be characterised sensitively in great detail and along with capillary electrophoretic separation unambiguous identification of minor protein modifications like deamidation is possible. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. Interrelationship between haemodynamic state and serum intact parathyroid hormone levels in patients with chronic heart failure.

    PubMed

    Sugimoto, Tadafumi; Dohi, Kaoru; Onishi, Katsuya; Watanabe, Kiyotaka; Sato, Yuichi; Sugiura, Emiyo; Nakamori, Shiro; Nakajima, Hiroshi; Nakamura, Mashio; Ito, Masaaki

    2013-01-01

    To assess the impact of serum intact parathyroid hormone (PTH) levels on haemodynamic state and their relations by comparing plasma B-type natriuretic peptide (BNP) levels. Cross-sectional study in molecular epidemiology. Mie University Hospital, Tsu, Japan. Consecutive 105 patients with chronic heart failure (CHF). Serum intact PTH and plasma BNP levels were assessed simultaneously with right heart catheterisation. Although serum intact PTH levels (46±25 pg/ml) were within the normal range (<65 pg/ml) in 87% of patients, log-transformed intact PTH levels significantly correlated with pulmonary capillary wedge pressure (PCWP: 15±9 mm Hg, r=0.55, p<0.05) and heart rate (73±14/min, r=0.40, p<0.05), whereas log-transformed intact PTH levels were inversely correlated with stroke volume index (SVI: 38±11 ml/m(2), r=-0.52, p<0.05) and cardiac index (2.6±0.7 l/min/m(2), r=-0.41, p<0.05) in all patients. PCWP and SVI were independent determinants of log-transformed intact PTH levels (β=0.40 and -0.37, p<0.05, respectively) after adjusting for variables associated with PTH. Conversely, after adjusting for variables associated with CHF, log-transformed intact PTH levels were an independent determinant of PCWP, SVI, heart rate and cardiac index (β=0.38, -0.33, 0.32, and -0.25, p<0.05, respectively), and might be defined as a superior determinant of SVI and cardiac index compared with log-transformed BNP levels using stepwise multivariate regression analyses. Increased PCWP and decreased SVI independently contribute to elevated intact PTH in patients with CHF.

  20. Impulse radar scanning of intact salt at the Avery Island Mine

    SciTech Connect

    Cook, C.W.

    1980-05-01

    A series of experiments was run in the Avery Island Mine to evaluate the capability of an impulse radar to locate anomalies and simulated waste targets in intact dome salt. Voids in salt were difficult to detect. On the positive side, metal targets and simulated waste (glass) were easily located in intact salt. Radar scanning at ranges of greater than 25 meters and short-range resolution of target positions to within a few centimeters were achieved.

  1. Imaging cerebral blood flow through the intact rat skull with temporal laser speckle imaging.

    PubMed

    Li, Pengcheng; Ni, Songlin; Zhang, Li; Zeng, Shaoqun; Luo, Qingming

    2006-06-15

    We discovered that laser speckle temporal contrast analysis (LSTCA) is able to access the two-dimensional (2D) cerebral blood flow velocity and vessel structure through the intact rat skull. It is demonstrated that LSTCA can significantly suppress the influence of the laser speckle from the stationary structure, such as the skull, and thus reveal the blood flow and morphology of blood vessels through the laser speckle images recorded from the intact rat skull.

  2. Imaging cerebral blood flow through the intact rat skull with temporal laser speckle imaging

    NASA Astrophysics Data System (ADS)

    Li, Pengcheng; Ni, Songlin; Zhang, Li; Zeng, Shaoqun; Luo, Qingming

    2006-06-01

    We discovered that laser speckle temporal contrast analysis (LSTCA) is able to access the two-dimensional (2D) cerebral blood flow velocity and vessel structure through the intact rat skull. It is demonstrated that LSTCA can significantly suppress the influence of the laser speckle from the stationary structure, such as the skull, and thus reveal the blood flow and morphology of blood vessels through the laser speckle images recorded from the intact rat skull.

  3. 40 CFR 261.41 - Notification and Recordkeeping for Used, Intact Cathode Ray Tubes (CRTs) Exported for Reuse.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Used, Intact Cathode Ray Tubes (CRTs) Exported for Reuse. 261.41 Section 261.41 Protection of... Tubes (CRTs) Exported for Reuse. (a) Persons who export used, intact CRTs for reuse must send a one-time... forth as follows: § 261.41 Notification and Recordkeeping for Used, Intact Cathode Ray Tubes...

  4. Risks for Conduct Disorder Symptoms Associated with Parental Alcoholism in Stepfather Families versus Intact Families from a Community Sample

    ERIC Educational Resources Information Center

    Foley, Debra L.; Pickles, Andrew; Rutter, Michael; Gardner, Charles O.; Maes, Hermine H.; Silberg, Judy L.; Eaves, Lindon J.

    2004-01-01

    Background: It is not known if the prevalence of parental psychiatric disorders is higher in stepfather than intact families, or if parental alcoholism is differentially associated with risk for conduct disorder (CD) symptoms in stepfather families versus intact families. Method: The sample comprised 839 girls and 741 boys from 792 intact families…

  5. Risks for Conduct Disorder Symptoms Associated with Parental Alcoholism in Stepfather Families versus Intact Families from a Community Sample

    ERIC Educational Resources Information Center

    Foley, Debra L.; Pickles, Andrew; Rutter, Michael; Gardner, Charles O.; Maes, Hermine H.; Silberg, Judy L.; Eaves, Lindon J.

    2004-01-01

    Background: It is not known if the prevalence of parental psychiatric disorders is higher in stepfather than intact families, or if parental alcoholism is differentially associated with risk for conduct disorder (CD) symptoms in stepfather families versus intact families. Method: The sample comprised 839 girls and 741 boys from 792 intact families…

  6. 40 CFR 261.41 - Notification and Recordkeeping for Used, Intact Cathode Ray Tubes (CRTs) Exported for Reuse.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Used, Intact Cathode Ray Tubes (CRTs) Exported for Reuse. 261.41 Section 261.41 Protection of... Tubes (CRTs) Exported for Reuse. (a) Persons who export used, intact CRTs for reuse must send a one-time... to export used, intact CRTs for reuse, the notifier's name, address, and EPA ID number (if...

  7. Investigation of mitochondrial dysfunction by sequential microplate-based respiration measurements from intact and permeabilized neurons.

    PubMed

    Clerc, Pascaline; Polster, Brian M

    2012-01-01

    Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria.

  8. Investigation of Mitochondrial Dysfunction by Sequential Microplate-Based Respiration Measurements from Intact and Permeabilized Neurons

    PubMed Central

    Clerc, Pascaline; Polster, Brian M.

    2012-01-01

    Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria. PMID:22496810

  9. Identification of Intrinsic Axon Growth Modulators for Intact CNS Neurons after Injury.

    PubMed

    Fink, Kathren L; López-Giráldez, Francesc; Kim, In-Jung; Strittmatter, Stephen M; Cafferty, William B J

    2017-03-14

    Functional deficits persist after spinal cord injury (SCI) because axons in the adult mammalian central nervous system (CNS) fail to regenerate. However, modest levels of spontaneous functional recovery are typically observed after trauma and are thought to be mediated by the plasticity of intact circuitry. The mechanisms underlying intact circuit plasticity are not delineated. Here, we characterize the in vivo transcriptome of sprouting intact neurons from Ngr1 null mice after partial SCI. We identify the lysophosphatidic acid signaling modulators LPPR1 and LPAR1 as intrinsic axon growth modulators for intact corticospinal motor neurons after adjacent injury. Furthermore, in vivo LPAR1 inhibition or LPPR1 overexpression enhances sprouting of intact corticospinal tract axons and yields greater functional recovery after unilateral brainstem lesion in wild-type mice. Thus, the transcriptional profile of injury-induced sprouting of intact neurons reveals targets for therapeutic enhancement of axon growth initiation and new synapse formation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  10. In Vitro Biosynthesis of Phosphorylated Starch in Intact Potato Amyloplasts1

    PubMed Central

    Wischmann, Bente; Hamborg Nielsen, Tom; Lindberg Møller, Birger

    1999-01-01

    Intact amyloplasts from potato (Solanum tuberosum L.) were used to study starch biosynthesis and phosphorylation. Assessed by the degree of intactness and by the level of cytosolic and vacuolar contamination, the best preparations were selected by searching for amyloplasts containing small starch grains. The isolated, small amyloplasts were 80% intact and were free from cytosolic and vacuolar contamination. Biosynthetic studies of the amyloplasts showed that [1-14C]glucose-6-phosphate (Glc-6-P) was an efficient precursor for starch synthesis in a manner highly dependent on amyloplast integrity. Starch biosynthesis from [1-14C]Glc-1-P in small, intact amyloplasts was 5-fold lower and largely independent of amyloplast intactness. When [33P]Glc-6-P was administered to the amyloplasts, radiophosphorylated starch was produced. Isoamylase treatment of the starch followed by high-performance anion-exchange chromatography with pulsed amperometric detection revealed the separated phosphorylated α-glucans. Acid hydrolysis of the phosphorylated α-glucans and high-performance anion-exchange chromatography analyses showed that the incorporated phosphate was preferentially positioned at C-6 of the Glc moiety. The incorporation of radiolabel from Glc-1-P into starch in preparations of amyloplasts containing large grains was independent of intactness and most likely catalyzed by starch phosphorylase bound to naked starch grains. PMID:9952440

  11. Comparative evaluation of impact strength of fragment bonded teeth and intact teeth: an in vitro study.

    PubMed

    Venugopal, L; Lakshmi, M Narasimha; Babu, Devatha Ashok; Kiran, V Ravi

    2014-06-01

    To test and compare the impact strength of fragment bonded teeth with that of intact teeth by using impact testing machine (pendulum type) as a mode of load. Forty extracted, maxillary, central incisors selected for this study (20 control group and 20 experimental group). In experimental group, teeth crowns were fractured with a microtome at 2.5 mm from mesioincisal angle cervically, fractured portion is attached to original crown portion with 3 M single bond dentin bonding agent and 3 M Z '100', composite resin. Impact strength of fragment bonded teeth and intact teeth tested with impact testing machine and compared. Mean impact strength of fragment bonded teeth (30.76 KJ/M(2) ) is not statistically significant deferent from mean impact strength of intact teeth (31.11 KJ/M(2) ). Mean impact strength of fragment bonded teeth is not statistically different with that of intact teeth. Hence, after fracture of teeth if it is restored with fragment reattachment by using 3 M single bond dentin bonding agent and 3 M Z '100' composite resin is having impact strength like that of intact teeth. How to cite the article: Venugopal L, Lakshmi MN, Babu DA, Kiran VR. Comparative evaluation of impact strength of fragment bonded teeth and intact teeth: An in vitro study. J Int Oral Health 2014;6(3):73-6.

  12. Electrophysiological Recording in the Brain of Intact Adult Zebrafish

    PubMed Central

    Johnston, Lindsey; Ball, Rebecca E.; Acuff, Seth; Gaudet, John; Sornborger, Andrew; Lauderdale, James D.

    2013-01-01

    Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages. PMID:24300281

  13. Choline oxidation by intact spinach chloroplasts. [Spinacia oleracea L

    SciTech Connect

    Weigel, P.; Lerma, C.; Hanson, A.D.

    1988-01-01

    Plants synthesize betaine by a two-step oxidation of choline (choline ..-->.. betaine aldehyde ..-->.. betaine). Protoplast-derived chloroplasts of spinach (Spinacia oleracea L.) carry out both reactions, more rapidly in light than in darkness. We investigated the light-stimulated oxidation of choline, using spinach chloroplasts isolated directly from leaves. The rates of choline oxidation obtained (dark and light rates: 10-50 and 100-300 nanomoles per hour per milligram chlorophyll, respectively) were approximately 20-fold higher than for protoplast-derived chloroplasts. Betaine aldehyde was the main product. Choline oxidation in darkness and light was suppressed by hypoxia. Neither uncouplers not the Calvin cycle inhibitor glyceraldehyde greatly affected choline oxidation in the light, and maximal choline oxidation was attained far below light saturation of CO/sub 2/ fixation. The light stimulation of choline oxidation was abolished by the PSII inhibitors DCMU and dibromothymoquinone, and was partially restored by adding reduced diaminodurene, an electron donor to PSI. Both methyl viologen and phenazine methosulfate prevented choline oxidation. Adding dihydroxyacetone phosphate, which can generate NADPH in organello, doubled the dark rate of choline oxidation. These results indicate that choline oxidation in chloroplasts requires oxygen, and reducing power generated from PSI. Enzymic reactions consistent with these requirements are discussed.

  14. Open Repair of Intact Thoracoabdominal Aortic Aneurysms in the ACS-NSQIP

    PubMed Central

    Bensley, Rodney P.; Curran, Thomas; Hurks, Rob; Lo, Ruby C.; Wyers, Mark C.; Hamdan, Allen D.; Chaikof, Elliot L.; Schermerhorn, Marc L.

    2013-01-01

    Objective Repair of thoracoabdominal aortic aneurysms (TAAA) is uncommon. Studies using national data report mortality rates of 20% while single institution studies report 5-8% mortality. Clinical trials are currently evaluating branched and fenestrated endografts. The purpose of this study is to establish a benchmark for future comparisons with endovascular trials using open repair of TAAA in the National Surgical Quality Improvement Program (NSQIP) database. Methods We identified all patients undergoing open surgical repair of intact TAAA (elective and emergent) in NSQIP 2005-2010 using CPT and ICD-9 codes. We analyzed demographics, comorbidities, 30-day mortality, postoperative complications, and length of stay. Multivariable logistic regression was used to identify predictors of mortality. Results We identified 450 patients (418 elective, 32 emergent) that underwent open surgical repair of an intact TAAA. Mean age was 69.4 years, 60.7% were male, and 85.6% were white. Comorbidities included hypertension (87.1%), COPD (27.3%), prior stroke/TIA (16.7%), diabetes (11.6%), and peripheral vascular disease (9.6%). Thirty-day mortality was 10.0%. Pulmonary complications were the most common: failure to wean from ventilator (39.1%), pneumonia (23.1%), and reintubation (13.8%). Acute renal failure requiring dialysis occurred in 10.7% of patients. On multivariable analysis, emergent repair [OR 3.3, 95% CI (1.03-10.83), P=.04], age > 70 years [OR 3.5, 95% CI (1.03-7.56), P=.001], preoperative dialysis [OR 8.4, 95% CI (1.90-37.29), P= .005], cardiac complication [OR 2.9, 95% CI (1.05-8.21), P=.04] and renal complication [OR 8.4, 95% CI (3.41-20.56), P<.001] were predictive of mortality. Conclusions In this study of NSQIP hospitals, the first to analyze open surgical repair of TAAA, the 30-day mortality rate of 10.0% is similar to single institution reports. However, morbidity and mortality after open TAAA repair remain high confirming the need for less invasive procedures

  15. A system and methodology for high-content visual screening of individual intact living cells in suspension

    NASA Astrophysics Data System (ADS)

    Renaud, Olivier; Heintzmann, Rainer; Sáez-Cirión, Asier; Schnelle, Thomas; Mueller, Torsten; Shorte, Spencer

    2007-02-01

    Three dimensional imaging provides high-content information from living intact biology, and can serve as a visual screening cue. In the case of single cell imaging the current state of the art uses so-called "axial through-stacking". However, three-dimensional axial through-stacking requires that the object (i.e. a living cell) be adherently stabilized on an optically transparent surface, usually glass; evidently precluding use of cells in suspension. Aiming to overcome this limitation we present here the utility of dielectric field trapping of single cells in three-dimensional electrode cages. Our approach allows gentle and precise spatial orientation and vectored rotation of living, non-adherent cells in fluid suspension. Using various modes of widefield, and confocal microscope imaging we show how so-called "microrotation" can provide a unique and powerful method for multiple point-of-view (three-dimensional) interrogation of intact living biological micro-objects (e.g. single-cells, cell aggregates, and embryos). Further, we show how visual screening by micro-rotation imaging can be combined with micro-fluidic sorting, allowing selection of rare phenotype targets from small populations of cells in suspension, and subsequent one-step single cell cloning (with high-viability). Our methodology combining high-content 3D visual screening with one-step single cell cloning, will impact diverse paradigms, for example cytological and cytogenetic analysis on haematopoietic stem cells, blood cells including lymphocytes, and cancer cells.

  16. Perforation forces of the intact porcine anterior lens capsule.

    PubMed

    Ullrich, Franziska; Lussi, Jonas; Felekis, Dimitrios; Michels, Stephan; Petruska, Andrew J; Nelson, Bradley J

    2016-09-01

    During the first step of cataract surgery, the lens capsule is perforated and a circular hole is created with a sharp instrument, a procedure called capsulorhexis. To develop automated systems that can assist ophthalmologists during capsulorhexis, the forces required must be quantified. This study investigates perforation forces of the central anterior lens capsule in porcine eyes, which are used as a conservative model for the human eye. A micro-mechanical characterisation method is presented that measures capsular bag perforation forces with a high precision positioning and high-resolution force sensing system. The force during perforation of the anterior lens capsule was measured with various sized needles and indentation speeds and is found to be 15-35mN. A bio-mechanical model is identified that describes an exponential correlation between indentation force and depth, indicating strain hardening behaviour of the porcine anterior lens capsule.

  17. Neural circuit recording from an intact cockroach nervous system.

    PubMed

    Titlow, Josh S; Majeed, Zana R; Hartman, H Bernard; Burns, Ellen; Cooper, Robin L

    2013-11-04

    The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

  18. Double-bundle reconstruction cannot restore intact knee kinematics in the ACL/LCL-deficient knee.

    PubMed

    Zantop, Thore; Schumacher, Tobias; Schanz, Steffen; Raschke, Michael J; Petersen, Wolf

    2010-08-01

    The aim of this study was to evaluate the effect of single-bundle (SB) and anatomic double-bundle (DB) anterior cruciate ligament (ACL) reconstruction on the resulting knee kinematics in a simulated clinical setting with ACL rupture and associated extra-articular damage to the lateral structures. It was hypothesized that anatomic DB ACL reconstruction restores the intact knee kinematics in ACL/LCL-deficient knees, whereas SB ACL reconstruction fails to restore the intact knee kinematics. Ten fresh-frozen human cadaver knees were subjected to anterior tibial load of 134 N (simulated KT 1000) and combined rotatory load of 10-Nm valgus and 4-Nm internal tibial torque (simulated pivot shift) using a robotic/UFS testing system. The resulting knee kinematics was determined for intact, ACL/LCL-deficient, SB ACL-reconstructed/LCL-deficient, and DB ACL-reconstructed/LCL-deficient knee. Statistical analysis was performed using a two-way ANOVA test with the level of significance set at P < 0.05. Under a simulated KT 1000 test, anterior tibial translation (ATT) following SB ACL reconstruction was statistically significant at 0 degrees , 30 degrees and 60 degrees of knee flexion when compared to the intact knee. ATT after DB ACL reconstruction showed no statistically significant difference from the intact knee; however, there was a significant difference in SB reconstruction at 0 degrees and 30 degrees of knee flexion. Under a simulated pivot shift test, both SB and DB ACL reconstruction failed to restore the intact knee kinematics. The results of the study did not support our initial hypothesis. Though DB reconstructions were significantly superior to SB reconstruction under simulated KT 1000 test, SB as well as DB reconstruction failed to restore the intact kinematics under simulated pivot shift loads. The clinical relevance of this study is that caution and precise preoperative diagnostics are needed to avoid failure of intra-articular ACL reconstruction if the extra

  19. The relationship between intact parathyroid hormone levels and daily physical activity in hemodialysis patients.

    PubMed

    Afsar, Baris

    2014-05-01

    Poor physical activity and decreased daily activities are commonly seen in hemodialysis (HD) patients. Along with the progression of chronic kidney disease (CKD), various abnormalities of mineral and bone metabolism develop, such as osteitis fibrosa and adynamic bone disease, which are related with intact parathyroid hormone (intact-PTH). Surprisingly, scarce data exists regarding the relationship between intact-PTH and daily physical activity in HD patients. Demographics, clinical parameters, laboratory data were recorded for all patients. Depressive symtoms, quality of life and daily activities of HD patients were measured by Beck Depression Inventory, SF-36, and Nottingham Extended Activities of Daily Living Scale (NEADLS), respectively. In total 114 patients (male/female: 63/51, aged: 53.0 ± 13.8 years) were enrolled. The value of intact-PTH for <25th (Group 1), < 25th-50th (Group 2), 50th-75th (Group 3) and >75th (Group 4) quartiles were <132.5 pg/ml, ≥132.5 <261.0, ≥261.0 <510.4 and ≥510.4, respectively. The NEADLS scores were 25.3 ± 10.8, 35.0 ± 9.4, 27.2 ± 13.9 and 26.4 ± 12.9 as going from Group 1 to Group 4 (p = 0.009). Post-hoc analysis of these four groups revealed that only Group 1 and Group 2 (p = 0.012), and Group 2 and Group 4 (p = 0.034) were different with respect to NEADLS scores. Intact-PTH levels were inversely associated with daily activities in whole group. However, the post hoc analysis demonstrated that the association between intact PTH and daily activity is not linear and daily physical activity was lower only in patients with lowest and highest quartiles of intact-PTH.

  20. Comparison of ripening processes in intact tomato fruit and excised pericarp discs.

    PubMed

    Campbell, A D; Huysamer, M; Stotz, H U; Greve, L C; Labavitch, J M

    1990-12-01

    Physiological processes characteristic of ripening in tissues of intact tomato fruit (Lycopersicon esculentum Mill.) were examined in excised pericarp discs. Pericarp discs were prepared from mature-green tomato fruit and stored in 24-well culture plates, in which individual discs could be monitored for color change, ethylene biosynthesis, and respiration, and selected for cell wall analysis. Within the context of these preparation and handling procedures, most whole fruit ripening processes were maintained in pericarp discs. Pericarp discs and matched intact fruit passed through the same skin color stages at similar rates, as expressed in the L(*)a(*)b(*) color space, changing from green (a(*) < -5) to red (a(*) > 15) in about 6 days. Individual tissues of the pericarp discs changed color in the same sequence seen in intact fruit (exocarp, endocarp, then vascular parenchyma). Discs from different areas changed in the same spatial sequence seen in intact fruit (bottom, middle, top). Pericarp discs exhibited climacteric increases in ethylene biosynthesis and CO(2) production comparable with those seen in intact fruit, but these were more tightly linked to rate of color change, reaching a peak around a(*) = 5. Tomato pericarp discs decreased in firmness as color changed. Cell wall carbohydrate composition changed with color as in intact fruit: the quantity of water-soluble pectin eluted from the starch-free alcohol insoluble substances steadily increased and more tightly bound, water-insoluble, pectin decreased in inverse relationship. The cell wall content of the neutral sugars arabinose, rhamnose, and galactose steadily decreased as color changed. The extractable activity of specific cell wall hydrolases changed as in intact fruit: polygalacturonase activity, not detectable in green discs (a(*) = -5), appeared as discs turned yellow-red (a(*) = 5), and increased another eight-fold as discs became full red (a(*) value +20). Carboxymethyl-cellulase activity, low in